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PRENTICE-HALL FOUNDATIONS OF MODERN BIOLOGY SERIES

The Cell, by Carl P. Swanson

Cellular Physiology and Biochemistry, by William D. McElroy
Heredity, by David Bonner

Adaptation, by A.M. Srb and Bruce Wallace

Animal Growth and Development, by Maurice Sussman
Animal Physiology, by Knut Schmidt-Nielsen

Animal Diversity, by Earl Hanson

Animal Behavior, by Vincent Dethier and Eliot Stellar

The Life of the Green Plant, by Arthur Galston

The Plant Kingdom, by Harold Bold

Man In Nature, by Marston Bates

Animal Growth
and

Development

Animal Growth
and

Development

MAURICE SUSSMAN

Brandeis University

Prentice-Hall, Inc.
ENGLEWOOD CLIFFS, NEW JERSEY

1960

Animal Growth and Development

Maurice Sussman

© Copyright 1960
by PRENTICE-HALL, INC.,
Englewood Cliffs, New Jersey.

All rights reserved.

No part of this book

may be reproduced in any form,

by mimeograph

or any other means,

without permission from the publishers.
Printed in the

United States of America

Library of Congress

Catalog Card Number 60-8510

PRENTICE-HALL FOUNDATIONS OF MODERN BIOLOGY SERIES

William D. McElroy and Carl P. Swanson, Editors

Design by Walter Behnke

Drawings by Felix Cooper

For Raquel

The science of biology today is not the same science

Foundations
of Modern
Biology
Series

of fifty, twenty-five, or even ten years ago.
Today’s accelerated pace of research, aided
by new instruments, techniques, and points
of view, imparts to biology a rapidly chang-
ing character as discoveries pile one on top
of the other. All of us are aware, however,
that each new and important discovery is
not just a mere addition to our knowledge;
it also throws our established beliefs into
question, and forces us constantly to reap-
praise and often to reshape the foundations
upon which biology rests. An adequate pres-
entation of the dynamic state of modern
biology is, therefore, a formidable task and
a challenge worthy of our best teachers.

The authors of this series believe that
a new approach to the organization of the
subject matter of biology is urgently needed
to meet this challenge, an approach that in-
troduces the student to biology as a grow-
ing, active science, and that also permits
each teacher of biology to determine the
level and the structure of his own course. A
single textbook cannot provide such flexibil-
ity, and it is the authors’ strong conviction
that these student needs and teacher pre-
rogatives can best be met by a series of
short, inexpensive, well-written, and well-
illustrated books so planned as to encompass
those areas of study central to an under-
standing of the content, state, and direction
of modern biology. The FOUNDATIONS
OF MODERN BIOLOGY SERIES repre-
sents the translation of these ideas into
print, with each volume being complete in
itself yet at the same time serving as an
integral part of the series as a whole.

1x

I

What Is Development? Page 1

THE CENTRAL PROBLEMS
OF DEVELOPMENTAL BIOLOGY

THE RELATIONSHIP
OF DEVELOPMENTAL BIOLOGY
TO OTHER BIOLOGICAL DISCIPLINES

Z

The Life Cycle of the Single Cell Page 6

VARIETIES OF CELL DIVISION

BIOCHEMICAL EVENTS
IN THE CELL DIVISION CYCLE

THE LIFE CYCLE OF A TYPICAL
UNICELLULAR ORGANISM—YEAST

THE LIFE CYCLE OF PARAMECIUM AURELIA

PERSISTENT RHYTHMS IN
GONYAULAX POLYHEDRA:
HOW ORGANISMS TELL TIME

THE LIFE CYCLE OF ACETABULARIA:
MORPHOGENESIS IN A MICROORGANISM

é

The Beginnings
of Multicellular Organization Page 26

THE CELLULAR SLIME MOLDS
A WATER MOLD, ACHYLA BISEXUALIS

3 6749

The Development
of a Primitive Animal:
The Coelenterates Page 37

LIFE CYCLE OF THE GENUS OBELIA
POLYMORPHISM IN THE COELENTERATES
REGENERATION OF POLYPS AND MEDUSAE
MORPHOGENETIC FIELDS

The Development
of the Vertebrate Embryo Page 47

THE EARLY STAGES OF EMBRYOGENESIS
CLEAVAGE

GASTRULATION

ORGAN FORMATION

BIOCHEMICAL EMBRYOLOGY

Cellular Differentiation Page 69

THE GENETIC BASIS
OF CELLULAR DIFFERENTIATION

CONCLUSION

Contents

=,

Cell Interactions
during Growth and Morphogenesis Page 80

MANY CELLS DO WHAT FEW CANNOT DO
INDUCTIVE INTERACTIONS
SYNERGISTIC INDUCTIONS
INHIBITORY INTERACTIONS

KNOWN MECHANISMS
OF CELL INTERACTION

CONCLUSION

Growth and Form Page 92

NUTRITION
THE S-SHAPED CURVE OF GROWTH

GROWTH OF THE
MULTICELLULAR ORGANISM

THE LIMITATIONS OF SIZE AND FORM
EQUATIONS THAT DESCRIBE GROWTH

Selected Readings Page 107

Index Page 111

= 1.
Ap Jax

GW tae

Tee essence of life is change. The essence of

What
Is

Development?

death is inertia. All of us learn this at an
early age. We watch the waxing and
waning of the seasons and observe a reg-
ular succession of changes in the living
things about us. We watch the ant on a
blade of grass, the fish in a bowl, the dog
in our back yard, and find them in states
of ceaseless activity. Some are short-term
activities such as moving, eating, excret-
ing, mating; some are long-term activities
that become apparent only after the pas-
sage of weeks and months. Ultimately,
we recognize these changes in ourselves
as we grow and develop from babyhood
to adulthood.

The biologist takes a more pene-
trating view of biological activity and
distinguishes three classes of alterations:

I. Short-term physiological and
morphological alterations. Many of these
changes are quite familiar to us. For ex-
ample, our body temperature may vary
from hour to hour depending on whether
we are awake or asleep, at work or at
rest. The color, texture, and thickness of
an animal pelt can vary rather signifi-
cantly from winter to winter in the life
of an animal, depending on the severity
of each winter. A callus may form on a
hard-working hand and disappear when
the hand’s owner takes up a more seden-
tary occupation. Sudden anger may in-
crease the heart beat, divert blood from
intestines to muscles, and augment the
rate of breathing.

As still another example, if we ex-
pose a population of yeast cells, which

I

What Is Development?

have previously been grown in the presence of glucose as an energy
source, to a new sugar, maltose, the cells, in order to utilize it, must first
synthesize an entirely new enzyme that splits the maltose into usable
fragments. They can do this in a relatively short time. If the maltose
were removed and replaced by glucose, the new enzyme would disappear
from the cells and no record would be left of this interlude of physio-
logical change.

All such activities have common properties. They do not occur in
any regular rhythm but merely represent sporadic adjustments to specific
environmental stimuli. They are usually reversible; that is, the progression
of changes leaves the organism neither vastly different from what it was
before nor unable to return to its former state.

2. Long-term genetic and evolutionary changes. Alterations in the
genetic apparatus of an organism are called mutations and are inherited
by the offspring. A single mutation may be minute in itself, perhaps lead-
ing only to the loss or gain of the capacity to synthesize a single enzyme,
and its effect on the form or functioning of the organism may be corre-
spondingly slight. If by virtue of the change, the mutant is more fitted
to survive than its unaltered relatives (if it can breed faster, for example),
it will flourish and the following generations will probably include a
greater proportion of that mutant type. When successive mutations stand
the test of natural selection, they can, over many generations, produce a
variety of organism greatly different from its distant forebears. In the
aggregate, these progressions contribute to the evolution of species.

Note how different these evolutionary changes are from the short-
term changes mentioned before. First of all, a much longer time is in-
volved, for the appearance of a new variety of organism in large numbers
may require many generations of growth and reproduction. Second, the
biologist who studies these phenomena must examine populations, not
individual organisms, in order to follow the spread of genetic changes.

3. Developmental events. Developmental events are seen most dra-
matically in the growth patterns of higher animals. The fertilized egg
undergoes a very orderly series of changes to become an embryo. The
embryo develops further into the young animal, which in turn matures
into an adult. The adult reaches its peak and then experiences a series of
degenerative changes that ultimately lead to death. At maturity the ani-
mal releases eggs or sperm which originate yet another cycle of develop-
ment.

The tempo of these phenomena is neither fast enough to be physio-
logical nor slow enough to be evolutionary. Moreover, they are progres-
sive; that is, they occur in a regular sequence with little variation, each
leaving the organism different from its former state and unable to return
to it. They begin before birth and end shortly after death, and in toto
they represent the life cycle of the organism.

What Is Development?

Developmental phenomena are not restricted to higher animals.
Even the single cell goes through a corresponding progression of changes.
Its life cycle begins when it arises from the division of its parent into two
daughters and ends when it in turn divides in two. Between times, it
grows in size and protoplasmic content, synthesizes cellular constituents
in an orderly and well-regulated manner, and can, indeed, develop new
organelles (a term used to designate any of the parts of a cell), and
change its form and functioning drastically. Many higher protozoa and
algae display complicated changes in this respect, and a few of these
cycles will be described in succeeding sections.

Populations of cells also show developmental phenomena. For ex-
ample, a culture of microorganisms has a life cycle of its own, as con-
trasted with those of the individual cells of which it is composed. As will
be described in detail later, when we add an inoculum of bacteria to
nutrient broth, the culture experiences an orderly succession of well-
defined growth stages, reaches a stationary state and ultimately a period
of decline during which the cells degenerate and die. Thus the bacteria
en masse act in much the same fashion as the cells of a higher organism
except that they are loosely distributed and largely independent, whereas
the cells of an animal are dependent upon each other and are compressed
into a compact organized structure. In fact, some sophisticated micro-
organisms exist for part of their culture cycle as independent cells, and
during the remainder of it actually do come together and construct organ-
ized multicellular structures. A later chapter will describe these fascinat-
ing organisms.

A little higher on the scale, we come to the primitive animals, which
display, on a limited level, the complicated series of developmental events
that the higher animals show. One of the most interesting primitive groups
in this respect, the coelenterates, will be discussed in detail.

The Central Problems of Developmental Biology

As we consider the life cycles mentioned above, three central prob-
lems of development become apparent and will be taken up here.

GROWTH

The egg starts as a single microscopic cell with one nucleus and by
growth and replication produces an adult containing millions of cells.
Needless to say, this represents a many-millionfold increase in proto-
plasmic mass. A number of questions come to mind when we think about
this phenomenon. How is growth initiated? What raw materials are re-
quired for the synthesis of protoplasm? What specific chemical reactions

What Is Development?

lead to the formation of nuclei, enzymes, cell walls, etc.? From where does
the organism draw the energy needed to carry out these syntheses? Why
does growth stop?

Another significant attribute of growth is the fact that all the parts
of an organism increase in a carefully regulated manner. That is, if a cell
doubles its size, the size of the nucleus, cytoplasmic constituents, cell wall,
etc., all increase in proportion to one another. When a boy grows into a
man, his organs increase harmoniously. Thus the length of his arm bears
a fixed relation to his height, and within rather close limits this same rela-
tion holds in all other boys. There must, therefore, be regulatory mech-
anisms in cells and organisms that govern such relationships. What are
these mechanisms?

CELLULAR DIFFERENTIATION

The millions of cells descended from the original fertilized egg are
not all the same. Some are skin cells, others are nerve cells, still others are
muscle cells, etc. They look different and perform decidedly different
functions. When we think about the reproduction of, say, a bacterium or
a rabbit, we normally expect the descendants of each to look and act very
much like if not the same as the original bacterium or rabbit. Such is not
the case, however, for the descendants of developing cells in an embryo.
The processes by which these cells become specialized are collectively
called cellular differentiation. These processes are still largely unknown
and present a major challenge to developmental biologists.

Another mysterious aspect of cellular differentiation is the way in
which the numbers of each cell type are carefully regulated. For example,
you will never find one embryo with many brain cells and few liver cells
or vice versa. Here again, as in the case of the boy and his arm, the vari-
ous tissues and organs of the body bear a relatively fixed relation to one
another. The mechanisms that control the proportions of differentiated
cells are still to be discovered.

MORPHOGENESIS

A multicellular organism is not simply a bag of cells thrown together
helter skelter. It is composed of organs and tissues, and, as has been men-
tioned, the parts of the animal are arranged in a specific pattern and bear
definite relations to one another in terms of size and cellular content.
Morphological regulation extends to the individual cell and its organelles.
The establishment of this pattern and the processes whereby the adult
organism takes its final shape is termed morphogenesis and requires ex-
planation in physical and chemical terms.

What Is Development?

The Relationship of Developmental Biology
to Other Biological Disciplines

As the various aspects of developmental biology are considered in
detail in later chapters, it will become obvious that this subject does not
exist apart from the other biological disciplines, for development involves
changes in the basic structure of an organism. Thus a rigorous description
of developmental events requires excursions into gross and microscopic
anatomy and into histology (which deals with tissue composition) and
cytology (which deals with cell-structure). And, since changes in the
structure of an organism are brought about by new combinations of
biochemical reactions and the new structure leads in turn to further
changes in function, biochemistry and physiology must be included in
developmental study. Finally, because development does involve change,
it bears a direct relation to genetics. For example, when cellular differ-
entiation occurs—i.e., when a single egg cell gives rise to a galaxy of cell
types: nerve, muscle, skin, cartilage cells—we want to know if the genetic
constitutions of these cells had to change in order to permit them to look
and act this differently, and if so, what was the nature of the change.

As you see then, developmental studies are intimately involved with
the rest of biology, and scientists who deal with developmental problems
in their research must bring many different kinds of skills and training to
bear upon such problems.

A growing cell faces the problem of making

The

Life Cycle
of the
Single Cell

enough of everything to construct two or
more daughter cells. And it must divide
the proceeds among them in such a way
as to produce normal, well-functioning
progeny. Most important, it has to endow
each daughter cell with a complete set
of genetic material. This material is car-
ried in large part upon the chromosomes
within the nucleus. However, some prop-
erties of organisms have been shown to
be initiated through genetic determinants
that reside in the cytoplasm and these
too must be shared.

Varieties of Cell Division

Nature has found several good solu-
tions to the problem of sharing, enabling
different methods of cell division to be
employed in various types of organisms
(Fig. 1).

1. Fission. The parent cell grows to
approximately twice its original size and
then splits into two more or less equal
daughter cells. Protozoan and animal
cells divide in this fashion, simply by
pinching off two daughters at the middle
and pulling apart the two halves. Bac-
terial and plant cells also reproduce by
fission but have rigid walls and divide by
constructing a cross wall.

2. Budding. The mother cell forms
a little bleb at the surface which grows

1 See Chapter 6 for a more detailed dis-
cussion of these matters.
6

Plastic cells 2
| Fission

Rigid cells

2 Budding

3 Filamentous growth

4 Fragmentation

Fig. |. Asexual reproduc-
tion. (1) Fission as it occurs
in amoebae and bacteria.
(2) Budding in yeast. (3)
Filamentous growth in 5 Non-cellular growth
molds. (4) Fragmentation

in Plasmodium malariae. (5)

Noncellular growth in the

myxomycetes.

rapidly to the approximate size of the mother and finally constricts off. It
may separate completely from the mother or may remain attached. In the
latter case, both mother and daughter may bud and so produce a chain
of cells. This is the manner in which yeast cells divide.

3. Growth of filaments. The cells of fungi and some algae are linked
together in thin hair-like fibers. Since growth can occur only at the tip
of each filament, the tip elongates and a cross wall forms to yield a cell
with a growing tip on its far end. Branching often occurs, when the
growing tip bifurcates and both branches elongate in separate strands.

4, Fragmentation. The animal parasite, Plasmodium malariae, is one
of a number of microorganisms that reproduce by fragmentation. The
plasmodium grows inside a red blood cell. The parent nucleus divides or
fragments into as many as 24 daughter nuclei and the cytoplasm coalesces
about each of them. Separate walls are formed around the conglomerates
of nucleus and cytoplasm, which are now called merozoites. The host red
cell ruptures and releases the merozoites and each can now infect another
red blood cell. (Incidentally, the well-known chills and fever of malaria
are associated with the simultaneous release of merozoites from many
blood cells.) The term fragmentation is a bit misleading because the
parent organism does not break up into incomplete fragments, each of
which reconstitutes a whole new organism. Rather it is as if a bacterium
would synthesize enough protoplasm to create many new cells instead
of just two. Many kinds of molds, algae, and protozoa form large numbers
of spores! at certain stages of their life cycles. In principle, the process
resembles that of Plasmodium malariae.

5. Noncellular growth. A few simpler organisms can grow exten-
sively without cell division. A group called the mycetozoa consists of

1 Special cells with thick walls that resist drying, heat, and radiation and which
can lie dormant for long periods of time until permitted to germinate.

The Life Cycle of the Single Cell

amoeboid masses of protoplasm that contain many nuclei. The proto-
plasm increases in amount and the nuclei divide as the organisms grow.
Each protoplasmic mass can fragment (in the true sense) to yield two or
more smaller masses. It should be noted that some higher animals can
also fragment into small pieces, each of which can then reconstitute a
whole new organism. Flatworms, starfish, and many others do this.

Biochemical Events in the Cell Division Cycle

In recent years, biologists have begun to study biochemical events
during the cell division cycle. Some have refined analytical techniques
to the point where they can study a single cell, weigh it (despite the fact
that they must deal with weights of only millionths of a gram), and, by
microchemical analysis, determine the amounts of the cell constituents
(even though these are present in infinitesimal quantities ). Other biolo-
gists have devised conditions to make cell populations divide synchro-
nously. Cell populations are usually asynchronous; that is, at any instant
some will be preparing to divide, some will have just completed division,
and others will be in an intermediate stage. With synchronized cells, one
need not apply very delicate and sensitive techniques for the study of a
single cell, but instead can work with large samples of cells and thereby
examine more easily the biochemical and morphological events that ac-
company each stage of the division cycle. This work has only begun, but
ultimately we may hope to understand how the cell coordinates the syn-
thesis of all its constituents. It is already clear from such studies that vari-
ous cell constituents are synthesized during very specific phases of the
division cycle. Figure 2 illustrates schematically the formation of deoxy-
ribose nucleic acid (DNA), the primary component of chromosomes and
the bearer of the cell’s genetic constitution. As you can see, the DNA is
synthesized during a relatively short period between successive mitoses.
Contrary to what one might expect, no DNA is formed during the period
when one sees the chromosomes actually divide.

NUCLEAR DIVISION

The primary method of vegetative (as opposed to sexual) nuclear
division is that of mitosis. The particular stages need not concern us here.
The significance of mitosis is that it is an almost foolproof way of ensuring
that each daughter nucleus will possess a copy of each chromosome and,
therefore, a complete set of the genetic material contained therein.

However, not all nuclei divide by mitosis. In some cells the nucleus
does not assume the mitotic configuration, but merely stretches out into a

Period of chromosome duplication (mitosis)

Period of cell growth
Period of cell division

Units of DNA
per cell

@a.[4| I~ l.K-2~ [o-
of) aq tte :

Fig. 2. DNA synthesis during the cell division cycle.

3-2

dumbbell shape. The dumbbell constricts in the middle and the two
daughter nuclei pinch apart. This is called amitosis. Amitotic division is
restricted to a few microorganisms and some cells of higher animals. As
you may know, certain tissue cells grow only in the young animal and
cease dividing in the adult, and it is interesting to note that their last few
divisions are very often amitotic. Degenerating or abnormal tissue cells
(e.g., tumors) also appear to divide amitotically.

EXTRA-NUCLEAR ELEMENTS

Some organelles of the cell seem to be partly or completely inde-
pendent of the nucleus. For example, the long whip-like flagellum seen
in many protozoa is formed from a small granule at its base called a
blepharoblast or basal granule. When certain protozoa divide, the old
flagellum disappears and the basal granule splits in two. Each daughter
cell receives a daughter granule and at the completion of cell division

9

10

The Life Cycle of the Single Cell

each granule forms a new flagellum. If by mischance one of the daughter
cells does not receive a basal granule, it and its descendants will never
again form a flagellum. Thus the basal granule is independent of the
nucleus to the extent that the latter cannot replace the former after it
once has been lost.

Other organelles (the gullets of ciliate protozoa, the chloroplasts of
some algae, etc.) act in the same fashion, i.e., they duplicate at cell divi-
sion and if lost from a cell cannot be regained. A most interesting case
involves the mitochondria of yeast. You will recall that mitochondria are
small bodies enclosed in membranes and distributed in the cytoplasm.
They contain organized collections of enzymes involved in respiration.
Recently it has been found that normally large yeast cells (which can
respire) occasionally give rise by budding to dwarf cells (which cannot
respire). And the descendants of these so-called “petite” cells are in-
variably petite even after hundreds of generations of growth. Thus, once
lost, the capacity to respire can never be regained. During budding, the
mitochondria concerned with respiration are normally shared between
mother and daughter cell, but once in a while by mistake the bud re-
ceives none of them. Because these mitochondria seem to be independent,
in the same sense as are flagellar granules, a cell bereft of them cannot
respire nor can its descendants do so, the loss being irretrievable.

The nucleus itself, of course, is an autonomous organelle. That is,
a cell deprived of its nucleus cannot create a new one. The loss of the
nucleus is lethal for the cell; when anucleate, it can no longer reproduce.

In summary, the cell is not simply a bag of homogeneous material
parcelled out in imprecise fashion during cell division. It is an assembly
of highly structured organelles. Many of these (nucleus, basal granules,
etc.) are autonomous in the sense that they control their own duplication.
Thus, for cell division to leave the daughters with a fair share of all the
constituents, the cell must make coordinated preparations, and organelles
must be moved to the right places in the mother cell.

The Life Cycle
of a Typical Unicellular Organism—Yeast

We choose common Baker’s yeast, Saccharomyces cerevisiae, to ex-
emplify the life cycle of simple plants and animals because it displays
three features that are present in the cycles of most organisms within
these groups:

1. The single cell as the unit of existence: In contrast to higher ani-
mals (i.e., organized assemblies of specialized cells that are dependent

The Life Cycle of the Single Cell

upon each other for their collective existence), the single yeast cell is the
total organism.

2. Growth by vegetative (asexual) reproduction: Yeast cells mul-
tiply asexually, a single cell giving rise to two by budding. Sexual fusion
of gametes does occur under special conditions (see below) but its occur-
rence is not obligatory to growth.

3. Sexual reproduction: You will recall from your study of genetics
that the higher plant or animal, consisting mostly of diploid cells (two of
each kind of chromosome), contains a few haploid germ cells or gametes
(one of each kind of chromosome). Two haploid gametes of the appro-
priate sexes can come together and fuse to yield a new diploid organism.

This process is called sexual fusion and serves a very important
function. The diploid individual receives a set of chromosomes from each
parent. By meiosis, it in turn produces gametes with a single chromosomal
set which, by random assortment, is generally made up of some chromo-
somes from each of the original parents. Thus, the genes of the parents
are shuffled together in the offspring and are reshuffled in the gametes
produced by that offspring (Fig. 3). Sexual fusion, then, is a source of
genetic recombination, producing new varieties by reshuflling the old.
New varieties are important to a population, for they ensure that there
will always be types that can take advantage of new environments and
thereby make the species fit to survive under a variety of conditions.

To clarify this point, consider a population of yeast cells living in a
moderately cool bit of dirt and plentifully supplied with glucose to grow
on. With the coming of summer, the soil becomes hot and the glucose is
replaced by a different sugar, galactose. Now suppose that the population
had originally consisted of two types: cells that were resistant to heat but
could grow only on glucose, and cells that were sensitive to heat but
could grow equally well on galactose or glucose. Under the new condi-
tions, type A could stand the heat but not the new sugar; type B could
grow on the new sugar but could not stand the heat. Of course, type A
might produce a mutant that could grow on galactose and type B a mutant
that could resist heat, but these would be exceedingly rare events that
might not come to pass, in which case the yeast population would be
wiped out. But if sexual reproduction could occur, the chromosomes
bearing the genes for heat resistance and the ability to grow on galactose
would be shuffled together in the zygote, and the recombinant type would
be able to survive. Figure 3 illustrates this.

Microorganisms such as yeasts and molds, protozoa, algae, and some
bacteria are capable of sexual fusion. That is, diploid cells can by meiosis
produce haploid gametes. These can be of different sexes (or mating types
as they are called). Haploid gametes of the appropriate sexes can fuse to
yield new diploid individuals.

11

Haploid cells

Sexual fusion

Diploid zygote

Formation of tetrads

Meiosis |

Meiosis |!

Fig. 3. Genetic recombination through sexual fusion. The gene for tem-
perature sensitivity is indicated by the symbol t*; +" is its allele and de-
notes temperature resistance. We use gal* to denote ability to utilize galac-
tose, gal” to denote inability. Thus, cell A is heat sensitive and can utilize
galactose; cell B is heat resistant but cannot utilize galactose. The chromo-
somes bearing these genes are shuffled together when cell A and cell B
fuse to form the zygote. Each pair of chromosomes then duplicates to
yield a quartet or tetrad. The nucleus then divides twice (meiosis | and II),
and in the process reduces the quartets to pairs and the pairs to singles.
The random assortment of chromosomes during meiosis | can produce a
situation in which the same cell will receive both the gene for temperature
resistance and the gene for galactose utilization; that is, a recombination
of the parental traits will take place.

12

The Life Cycle of the Single Cell

In higher animals, reproduction is tied to the sexual fusion of gam-
etes. Human beings cannot bud or fragment. They must mate to reproduce
their kind. In yeast cells, as in other microorganisms, sexual fusion is
separate from reproduction. Yeast grow by asexual means, but under
special conditions do employ sexual fusion to produce genetic recom-
binants (i.e., new varieties ).

The life cycle of yeast is illustrated schematically in Fig. 4. The
diploid individual is an elliptical cell. It reproduces asexually by budding
and can do so indefinitely as long as it is supplied with nutrient materials
and given conditions beneficial for growth. Under adverse conditions
(when starved and unable to bud), the diploid cell undergoes meiosis to
produce four haploid individuals. These are surrounded by thick walls
and are resistant to drying, heat, ultraviolet radiation, etc. They are called

Fig. 4. The life cycle of yeast.

Life cycle of
Saccharomyces cerevisiae

Two haploid cells of
different sexes

DO Asexual budding Sexual fusion eS
of haploid cells Diploid zygote

Asexual saling OO
Gannination of diploid cells
(or See esti Mature ascus /
with 4 ascospores sy Starved
- 2. diploid cells

Young ascus

Schizosaccharomyces Hansenula anomala Nematospora Spermophthora
octosporus phaseoli gossypii

The variety of asci and ascospores
seen in different species of yeast

13

14

The Life Cycle of the Single Cell

ascospores and are contained within a large sac or ascus. The ascus bursts,
the ascospores are released and then germinate, yielding small round
yeast cells, still haploid. Two haploid cells of the proper sexes 1 can come
together and fuse once again to yield a large diploid individual. The cycle
is complete at this point and can be repeated.

Many questions come to mind. For example, what specific environ-
mental conditions induce a diploid cell to undergo meiosis and produce
ascospores? What causes the ascospores to germinate into haploid cells?
What happens when the haploid cells are kept separate and not allowed
to meet other haploid gametes of the correct sexual type? Can they grow
by budding just like diploid cells? How is it that the haploid gametes can
be of different sexes and yet look alike? What is the physiological basis
of sex in these organisms? How is sex inherited in yeast? Some of these
questions have been answered through research. Many still remain un-
answered.

The Life Cycle of Paramecium Aurelia

Figure 5 is a drawing of Paramecium, a favorite subject of study in
elementary biology classes. Its large size (usually 150 microns x 50
microns?) and intricate structure attracted the attention of early biolo-
gists. In recent years its life cycle and genetics have been rigorously
defined. We can do no more here than briefly summarize these findings.

SOME FACTS ABOUT PARAMECIUM

P. aurelia is a fresh-water organism that preys upon bacteria and
algae. It is grown in the laboratory in association with a single bacterial
species. In recent years a bacteria-free nutrient medium has been devised
in which Paramecia can grow at a good rate. This medium is complex,
consisting as it does of proteins and many amino acids, eleven vitamins
including a derivative of vitamin D which is a requirement that is char-
acteristic of higher animals, and purines and pyrimidines (needed for the
synthesis of nucleic acids). P. aurelia possesses three nuclei. One is large
(macronucleus) and two are small (the micronuclei). It has a complex,
organized gut consisting of a mouth leading into a gullet lined with cilia

1 There are two sexes amongst yeast gametes, called + and —. Two + cells or
two — cells cannot fuse. Only a + and — cell.can. The two sexes look identical (in
contrast to those of higher organisms) and can only be distinguished by their mating
reaction. In each ascus two ascospores are of sex + and two are of sex —. From your
knowledge of genetics, can you deduce the manner in which sex is inherited by yeast?

“ A micron is 0.000001 meter. The average bacterium is about 2 x 0.5 microns.

The Life Cycle of the Single Cell

which waft the food down to the esophagus and thence into the interior
of the animal in the form of food vacuoles. The vacuoles circulate until
digestion is complete. The waste products are eliminated through the
anal pore. The surface of the animal is astonishingly intricate. It is cov-
ered by longitudinal rows of hexagons whose edges form distinct ridges.

Food vacuole
forming...

“.. circulating

Cilia
Trichocysts
(defense organelles)
Radiating canals

Contractile vacuole
forming

Contractile vacuole

Fig. 5. A schematic drawing of Paramecium, showing the many organelles.

Each hexagon contains a basal granule from which a cilium arises. There
are about 2,500 cilia and the ciliary pattern is inherited in a precise
manner.

BINARY FISSION

Well-fed Paramecia reproduce about five times per day. The cell
simply constricts at the middle and separates into anterior and posterior
daughter cells. At first these are readily distinguishable as the front and
rear ends, but they soon change into two identical and small but otherwise
normal animals. Prior to the constriction, the three nuclei divide. The
macronucleus elongates and the two halves pull apart. The micronuclei
divide by normal mitosis. One product of each of the three nuclei passes
to each daughter cell. A number of other cell structures duplicate during
cell division. Buds protrude from the mouth and gullet of the dividing
animal. The buds pass to the anterior daughter cell and produce a new
mouth and gullet. The posterior daughter retains the old ones. The ciliary
basal granules also bud off duplicates of themselves and these produce
new cilia. The granules, cilia, and hexagonal ridges are then rearranged
upon the daughter cells in their usual precise geometric pattern.

15

16 The Life Cycle of the Single Cell

CONJUGATION

When culture conditions are adverse (notably, when the Paramecia
are starved) these animals come together in pairs and undergo sexual
union or conjugation. The steps in conjugation are outlined below (see

Fig. 6).

Fig. 6. Conjugation in Paramecium aurelia.

O
O
———— Fn —— - ae
O
Animals pair Macronuclei Micronuclei sotoee 8 haploid
and become attached disintegrate

undergo meiosis

nuclei

O
7 of 8 haploid nuclei disappear;

the 8th moves into area of the bridge
between the animals

tb o3

The bridge ‘
between the animals

The nuclei Each gamete nucleus

migrate divides mitotically

Nuclear fusion

Mitotic division to
to yield diploids

yield 4 nuclei

se 8 96

Micronuclei divide;
cells begin to divide

dissolves

2 nuclei become macronuclei;
is complete

2 become micronuclei

The Life Cycle of the Single Cell

1. Pairing. The animals collide anteriorly and also become attached
at the region of the mouth. A bridge then forms between them. Mouths
and gullets regress into very tiny rudiments and much of the cell contents
becomes radically reorganized.

2. Nuclear changes. First, the macronuclei disintegrate into many
small fragments which eventually disappear. Second, the diploid micro-
nuclei undergo meiosis (i.e., each pair of chromosomes in the micronu-
cleus duplicates to yield a quartet; the micronucleus then divides twice,
reducing the quartets to pairs and thence to singles ). In this manner, each
micronucleus gives rise to 4 haploid nuclei, and the two micronuclei of
each conjugating cell together produce eight. Seven of the eight disappear.
Only the one nearest the bridge between the conjugant cells remains. This
nucleus, still haploid, divides once more and now each cell has two
gamete nuclei. One of these stays where it is. The other migrates through
the bridge to the partner cell. The result is that the conjugant Paramecia
have identical pairs of nuclei, each with one of its own and one of its
partner’s. The two haploid nuclei in each cell come together and fuse to
yield a single diploid nucleus.

3. Post-conjugative changes. The newly formed diploid nucleus di-
vides mitotically into two, and the two in turn produce four. One pair
takes a position at the anterior end of the cell and one pair at the posterior
end. The cell now constricts in the middle just as it does during asexual
fission and produces two daughter cells, each with two nuclei. One of the
two enlarges to form the new macronucleus. The other divides to yield the
two micronuclei. Thus, four post-conjugate daughter cells eventually
emerge from the two Paramecia that originally conjugated.

AUTOGAMY

Many organisms, particularly in the plant kingdom, are self-fertile,
which means that they can undergo sexual congress alone and need no
partner, i.e., they produce gametes that can fuse with each other in pairs
to yield fertile zygotes. Paramecium is capable of self-fertilization and
the process is designated autogamy.

The nuclear changes in an autogamous Paramecium are the same
as those involved during conjugation. The macronucleus disappears and
the micronuclei undergo meiosis to produce a total of eight haploid nuclei.
Seven disappear. The eighth divides mitotically into two gamete nuclei
and these fuse to yield a diploid zygote. The subsequent nuclear changes
follow the pattern of those displayed by cells after conjugation. Figure 7
summarizes autogamy.

17

$48 e

Macronuclear Meiosis to yield 8 7 of the 8
disintegration haploid nuclei disappear
2 nuclei become macronuclei: — Mitotic division Fusion of the nuclei — The remaining nucleus
2 become micronuclei to yield 4 nuclei to yield a diploid divides mitotically
Micronuclei divide; Autogamy
cell begins to divide is complete

Fig. 7. Autogamy in Paro-
mecium aurelia.

Persistent Rhythms in Gonyaulax Polyhedra:
How Crganisms Tell Time

Gonyaulax is a marine microorganism. It is single-celled, built like a
protozoan, but contains chlorophyll and is capable of photosynthesis. It
normally grows at the surface of the sea and can also be cultivated in the
laboratory. In addition to being photosynthetic, Gonyaulax can luminesce
(i.e., like fireflies and other insects, crustaceae, some molds, bacteria, and
algae, it can operate biochemical reactions that release energy in the form
of light instead of the more usual heat). It is one of the organisms respon-
sible for the brilliant displays of “phosphorescence” seen at night on ocean
waves, At least three important functions—photosynthesis, luminescence,
and cell division—are carried out in a regular rhythm keyed to the marine
habitat of the organism. Gonyaulax photosynthesizes by day, luminesces
by night, and reproduces just before dawn.

18

The Life Cycle of the Single Cell

For example, if a growing culture of Gonyaulax is exposed alter-
nately to 12-hour periods of light and dark (ie., a regular day-night
cycle), cell division is confined to a very short time just before the end
of the dark period. Thus the population increases like a staircase rather
than steadily and continuously with time as do most microbial cultures.
If now the culture is placed in constant dim light or total darkness, the
population continues to mark off 24-hour cycles of rhythmic cell division.
Thus the rhythm is persistent even though light, temperature, and other
environmental conditions may be constant. (See Fig. 8. )

The same was found to be true of its luminescence. A culture of
Gonyaulax was incubated during alternating 12-hour periods of light and
dark. At frequent intervals, measurements were made of the intensity at
which the cells luminesced, with an instrument somewhat like an ordinary

5000
Period of cell growth

Period of cell division

Period of cell division

4000

3000

Number of organisms per milliliter

2000

Fig. 8. Rhythmic cell
division in Gonyau- 1000
lax. The upper graph

shows the growth 0 Dark 12 Light 24 Dark 36 Light 48
curve of Gonyaulax

as a staircase rather

than as a continuous

line, because the

cells do not divide at 100
all times of the day
and night but only
during a short period
just before ''dawn."
The lower graph
shows that even
when the organisms
are removed from
the ordinary day-
night cycle and
placed in constant
dim light, the 24-
hour rhythm of cell
division persists.

Time in hours

Percentage of organisms undergoing cell division

6s es aS Se
Dark Light = Dark Light Constant dim light

Time in hours

Period of cell growth

Dark

19

60

20

The Life Cycle of the Single Cell

photographic light meter. It was found that the organisms luminesced
only at night, reaching a peak of brightness at midnight and a low at
noon (the upper part of Fig. 9). Then the animals were placed in con-
stant dim light, and they still continued to mark off 24-hour cycles of
luminescence over the course of many days. Finally, photosynthesis was

Intensity of luminescence

: Z a. z, - Soe
Light Dark Light Dark Light © -—— Constant dim light ——]

Intensity of luminescence

Oo 6 [2>” i8' 24 30, 36-42 54 66 78 90

Light Dark Light Dark Light Dark ight -=———== Constant dim light

Fig. 9. Rhythmic luminescence in Gonyaulax. The upper curve shows that
the rhythmic luminescence displayed during the usual day-night cycle per-
sists even when the organisms are placed in constant dim light. The lower
curve shows that although the luminescent rhythm can be altered by ex-
posing the organisms to a different day-night cycle (in this case 6 hours of
light, 6 of dark), it returns to the usual 24-hour rhythm as soon as the
organisms are placed in constant dim light, and no memory of the unusual
rhythm is retained.

also shown to be rhythmic even in constant dim light. The cells photo-
synthesized only during the daylight hours, reaching a peak at noon. Con-
sider how remarkable must be the control mechanism that could force
cells incubated in constant light to cease photosynthesis during the night
hours even though night was as bright as day!

The Life Cycle of the Single Cell

It was found that the rhythms could be altered in three ways. First,
the frequency, normally 24 hours, could be changed over a wide range.
For example, the organisms were exposed to alternating periods of 6 hours
of light and 6 of dark and their rhythms speeded up until they repeated
every 12 hours instead of 24. Other periods were attained by appropriate
changes in the duration of light and dark periods. However, no matter
how long the different regimen was imposed, as soon as they were re-
turned to constant dim light, the cells resumed their 24-hour cycle (lower
curve of Fig. 9). Second, the rhythm could be wiped out entirely by in-
cubating the cells in constant bright light. In this condition, the cells
photosynthesized all the time, reproduced asynchronously at any time,
and luminesced at no time. Again if placed back in constant dim light or
total darkness, they promptly resumed the normal rhythmic pattern.

Third, the rhythm could be reset. That is, the “clock” could be
pushed ahead or set back. This happened when cells were incubated in
constant darkness and suddenly were exposed to strong light for a minute
or two. Before exposure the cells had marked off regular 24-hour cycles.
After exposure they continued to do so but in a different time sequence, as
if one suddenly moved the hands of a watch forward or backward. The
light signal acted as the “reset” mechanism.

We have stressed these rhythmic activities because many living
organisms are rhythmic, and the rhythms profoundly affect their develop-
ment, indeed, are a part of it. Not all organisms operate a rhythmic cycle
of 24 hours’ duration. Some have a tidal rhythm, some a lunar (28-day)
rhythm. Others have an annual rhythm, and a few have several different
rhythms at the same time. Consider the following examples.

The fiddler crab, a common seashore denizen, changes color mark-
edly. The change is rhythmic and keyed to the tide. The same is true of
oysters and clams, who open and shut their shells according to a tidal
rhythm. Some insects pupate according to a 24-hour rhythm. That is, they
emerge from the pupal case at a certain time of day and only at that time.
The mating habits of certain fish are rhythmic according to a lunar cycle.
The hamster and rat show precise daily cycles of activity when placed
in special running cages (with automatic tachometers) for long periods
of time. The growth rates of some microorganisms show a similar daily
rhythm. Finally, the menstrual cycle of the human female has a lunar
rhythm. All of these, save the last, have been studied in the laboratory and
show the same characteristics as the rhythms of Gonyaulax. Let us sum-
marize these characteristics:

1. The rhythm is keyed to the physical environment of the organism
(day, tide, lunar month, year, etc.) and probably enables the organism to
feed or mate or protect itself more efficiently.

21

22

The Life Cycle of the Single Cell

2. The rhythm is persistent and is repeated by the organism even
under constant environmental conditions.

3. The rhythm can be temporarily wiped out or changed to a differ-
ent time schedule by altering the environment. The change is only tem-
porary, however, for the organism reverts to its original rhythmic habit
once it returns to a normal environment.

4, The rhythm can be reset (i.e., pushed forward or backward as a
clock is reset by appropriate signals in the environment).

Biologists interested in these problems are presently inquiring into
the causes of rhythms. Some are proceeding on the assumption that organ-
isms possess an internal “clock” which serves as the master control of all
rhythmic action and are busily at work trying to find out the properties of
the alleged clock. Others assume that there is no internal clock, that some-
thing in the physical environment (perhaps the fall of cosmic rays upon
the earth) varies in a rhythmic manner which directly affects each me-
tabolic reaction and so serves as the master control of rhythmic processes.
A number of crucial experiments indicate that the first explanation is
probably correct.

The Life Cycle of Acetabularia:
Morphogenesis in a Microorganism

Acetabularia illustrates the capacity of some unicellular organisms
to change their form drastically during their life cycle and to create very
elaborate structures in doing so. Because of its relatively enormous size,
it can reach a higher degree of complexity than most microorganisms.

Acetabulariae are green algae that live in tropical and subtropical
marine waters. As shown in Fig. 10, the cell consists of (a) a large cap with
leaf-like members arranged like the petals of a daisy, (b) a long stalk,
(c) a root-like process at the bottom called the rhizoid. The stalk can be
4-6 cm long and the cap up to 1 cm in diameter in a mature individual.
The growing cell contains a single, enormous nucleus usually situated in
the rhizoid but sometimes found in the lower stalk.

The complete life cycle of Acetabularia mediterranea takes about 3
years. After one year of growth the cell consists of a rhizoid, which has
sent out rootlets that hold the alga fast to the substratum, and a cylindrical
stalk without a cap. In the autumn, the stalk dries up and falls off leaving
the rhizoid which subsists on stored food reserves through the winter. The
following spring a stalk grows out and forms a rudimentary cap before
regressing once more. In the third year a fully-formed stalk and cap
appear. At this time the single, huge, diploid nucleus undergoes meiosis

Adult cap

Resting cyst

Rhizoid

Fig. 10. Life cycle of Ace-
tabularia mediterranea
(after Brachet, Biochemical
Cytology, p. 303). In the
laboratory, unlike in the
natural habitat, develop-
ment of the adult structure
from the zygote is direct,
without regression, and
takes only one year.

to yield haploid daughter nuclei. Subsequent mitotic divisions produce a
great number of tiny haploid nuclei that stream away from the rhizoid
into the cap where they are gathered into large bodies with thick walls
called cysts. The cysts are eventually shed from the cap and lie dormant.
By this time, each nucleus within the cyst is surrounded by cytoplasm and
a cell membrane. They look like flagellate protozoa, being pear-shaped
with two large flagella protruding from the front end. Eventually the lid
of the cyst flips off, freeing the flagellate cells which swim about for a
time and then conjugate in pairs. The two cells become one, the two
haploid nuclei fuse to yield a diploid, and the zygote can then give rise
after 3 years to an adult alga with cap, stalk, and rhizoid to complete the
cycle.

In nature, Acetabularia can regenerate lost parts. For example, the
seasonal disappearance of the stalk is followed by the synthesis of a new
stalk, and the loss of the adult cap by mechanical abrasion is followed by
the formation of a new cap. The regenerative powers of Acetabularia have
been employed to study its morphogenesis and in particular the role
played by the nucleus in morphogenetic processes. One can ask whether
or not the nucleus has to be present at all for regeneration to occur. It can
in fact be shown that pieces of stalk lacking a nucleus (anucleate) can
produce new cell membranes, chloroplasts, a perfectly normal new cap,
and, in a few cases, even a new rhizoid—in short, everything but another
nucleus. After a few months all these parts die. In contrast, pieces con-
taining a nucleus can regenerate lost parts over and over again. These
activities are accompanied by extensive synthesis of proteins and nucleic
acids. Thus, we can conclude that an anucleate segment of Acetabularia

has stored reserves of material and energy sufficient to construct a com-
23

Conjugation
and nuclear fusion

Nucleus

Zygote
2 days

24

The Life Cycle of the Single Cell

plete adult at least once. However, to do so again and again requires a
continual supply of energy and materials, and for this to occur, the nucleus
must be present.

One can also ask whether the ability of a segment to form a new cap
depends on the size or position of the segment. It turns out that long
anucleate pieces of stalk can construct caps better, faster, and with greater
frequency than short pieces, and pieces of stalk removed from near the
cap have greater competence than pieces of equal length taken from posi-
tions near the rhizoid. These data have been given several interpretations.
As an exercise, see if you can explain these phenomena and can design an
experiment that would test the validity of your explanation.

The various species of Acetabularia display wide differences in the
form of the cap (number, shape, and size of the leaves, etc.). This specific-
ity, i.e., the power to control cap design, appears to reside in the nucleus.
Grafts have been made between two species, Acetabularia mediterranea
(med) and A. crenulata (cren). The following results have been obtained
and are summarized in Fig. 11:

Fig. 11. Regeneration in Acetabularia. Experiment | shows that a piece of
one species, containing a nucleus, when joined to a piece of another species,
without a nucleus, at first forms an intermediate cap, but, in subsequent
regenerations, forms a cap characteristic of the species that contributed the
nucleus. Experiment 2 shows that two nucleated pieces not only form an
intermediate cap at first but also in subsequent regenerations. Experiment 3
shows that when two pieces of one species are joined to a piece of the
other species, all containing nuclei, the cap is intermediate but resembles
more closely the species that contributed the two nuclei.

Type of cap Type of cap

Cren

Cren ; ! re) :
First: cren-med intermediate Me First: cren-med intermediate
|. Later: only med Later: only eren
——> —
Med Med

Type of cap

First: cren-med intermediate
Later: cren-med intermediate

Type of cap

Cren

Type of cap
First: more med than cren

First: more cren than med
Cren Later: more cren than med

Later: more med than cren

The Life Cycle of the Single Cell

1. A rhizoid containing a nucleus (med) was joined to an anucleate
segment of stalk (cren). After a few months of incubation a new cap was
formed. Its appearance was intermediate between med and cren. If, how-
ever, the newly formed cap were cut off, a second cap was produced and
this was a med-type cap with no remnant of cren characteristics. Further-
more, additional removals of the cap yielded only med-type caps. The
reverse experiment (cren rhizoid with nucleus and a med anucleate stalk
segment) also yielded at first a cap which was intermediate between the
cren and med types. When this cap was removed, all subsequently formed
caps were purely cren.

2. Two nucleate rhizoids, one of each species, were joined, cut end
to cut end. At the junction a stalk grew out which formed a cap inter-
mediate between med and cren. Any subsequent removals of the cap
yielded regenerates still intermediate between the two species.

3. Two nucleate rhizoids of cren were joined, cut end to cut end.
The cut end of a third nucleate rhizoid (med) was fused to the junction
point of the first two. A stalk grew out from this point whose cap was
intermediate in appearance but much more like cren then med. When the
experiment was reversed, i.e., the two med rhizoids joined with one cren
rhizoid, the cap was much more like med than cren.

From these experiments we conclude that it is the nucleus which
ultimately controls the morphogenetic activities of Acetabularia, pre-
sumably by directing the synthesis of substances that play specific roles
in the construction of the cap. These materials are to some extent stored
within the anucleate portions of the stalk but are not spread equally
throughout. Thus, pieces of stalk taken from the apex have more of them
than pieces taken from the base; longer stalk pieces have more of them
than shorter pieces.

With these experiments as a background, it now is possible to in-
quire into the nature of the biochemical reactions that mediate morpho-
genesis in Acetabularia and into how the nucleus exerts control over them.
Such problems represent an exciting challenge to young biologists and
may lead to a general understanding of morphogenetic events in many
organisms.

25

ne organisms are generally classified as uni-

The
Beginnings

of
Multicellular
Organization

cellular (the individual is a single cell)
or multicellular (the individual is an or-
ganized collection of specialized cells).
Although such categories imply rigid dis-
tinctions, we must remember that dis-
crete boundaries do not exist in nature
but only in the minds of biologists. In
reality, there is a middle ground oc-
cupied by organisms that are neither
wholly unicellular nor wholly multicellu-
lar. For example, many protozoa, algae,
fungi, and bacteria are transiently colo-
nial; they come together for a time as
loose clusters of quasi-independent cells
with no organization or specialization.
Others attain a primitive level of organ-
ization in which some cells of the colony
become specialized for feeding, others
for sexual reproduction, etc. A highly
complicated level is attained by one such
group, the cellular slime molds. During
growth, the cells exist as orthodox uni-
cellular forms, each living independently
of its neighbors. After growth has ceased,
the cells come together and cooperate to
produce a single, organized, multicellular
structure with well-differentiated tissues.

Many algae, fungi, and protozoa,
and also one group of bacteria, are per-
manently colonial, because the cells do
not separate after reproduction but in-
stead adhere to one another. In some
cases these become merely tangled amor-
phous masses of cells and we can see
nothing except the cells that might be
called individuals. In others a great deal
of organization is attained. The individ-

26

The Beginnings of Multicellular Organization 27

uality of these cells is clearly submerged within the colony and we can
begin to talk of the colony as the individual. Thus botanists are accus-
tomed to call a colony of a higher mold a “plant” as if it were—as indeed
it is—a single functional entity.

Multicellularity requires that nature must solve many problems re-
lating first to the formation of the cell community and second to its proper
functioning, problems that do not arise in isolated cells. Organisms that
display primitive multicellular organization are of great interest to biolo-
gists because they illustrate nature’s first efforts to develop an individual
beyond the single cell. Furthermore, by learning the genetic and bio-
chemical mechanisms of primitive morphogenesis, we hope to uncover
principles that are general to all multicellular organisms, including higher
plants and animals.

The Cellular Slime Molds

Figure 12 summarizes the life cycle of one species of cellular slime
mold called Dictyostelium discoideum. The cycle is divided into four
stages: growth, aggregation, migration, and fruiting-body construction.
It starts with the germination of spores to yield amoeboid cells termed
myxamoebae. The myxamoebae live in the soil or on the agar surface of
a Petri dish, feed upon bacteria, and reproduce by binary fission. When

G
Spore eae et

Amoeba

Mature
fruiting body

A f

Growth and
multiplicatio

Aggregation

ie

Fig. 12. The life cycle of Ns eG va
Dictyostelium discoideum. Migration

28

The Beginnings of Multicellular Organization

the supply of bacteria is exhausted, the cells stop growing and enter the
aggregation stage. The myxamoebae stream radially toward central col-
lecting points, and, as they reach the center, a conical mound of cells is
built up. When all the cells have aggregated, each conical mound falls
over on its side and is transformed into a worm-like slug up to a few
millimeters in length. The slug migrates over the substratum and finally
comes to rest. It then proceeds to construct a fruiting body with a round
mass of spores at the top, a stalk enclosed in a cellulose sheath below, and
a basal disc at the bottom. These constitute three clearly different cell
groups. Ultimately the spores are cast off to repeat the cycle while the
stalk cells and basal disc cells desiccate and die. Figure 13 shows photo-
graphs of an aggregation sequence, migrating slugs, and the construction
of a fruiting body.

The cellular slime molds display the same attributes that more com-
plicated developmental systems, i.e., embryos, do. These are categorized
as cellular differentiation, cell interactions during development, and
regulation.

CELLULAR DIFFERENTIATION

As we have mentioned, the fruiting body consists of three distinct
cell groups: spores, stalk cells, and basal disc cells. These have radically
different sizes, shapes, constitutions, and functions—as do the various
tissue cells of a frog embryo. We can ask: (a) whether cells normally
destined to become spores can develop instead into stalk cells? and (b) if
a stalk cell or basal disc cell were removed from the fruiting body before
desiccation and death and permitted to grow and reproduce, would its
progeny develop normally, i.e., could they aggregate and build a fruit
containing all three tissues?

The fate of a cell (i.e., whether it will become a spore or stalk cell
or basal disc cell) appears to depend on the order in which it enters the

Fig. 13. Slime mold morphogenesis. Left, an ag-
gregation sequence; top right, migrating slugs
(from J. T. Bonner, The Cellular Slime Molds.
Princeton: Princeton University Press, 1959, Plate
Il); bottom right, the stages of fruiting body
construction—the photographs were taken ap-
promixately |!/> hours apart (also from Bonner,
Plate III).

a

9.1MM

30

The Beginnings of Multicellular Organization

aggregate (Fig. 14). Cells that enter the aggregate first become the lead-
ing element of the migrating slug and ultimately the lower stalk of the
fruit. Later arrivals take up progressively more posterior positions in the
slug and become upper stalk cells and spores. Those entering last comprise
the tail end of the slug and ultimately the basal disc of the fruit. One can
tentatively imagine that the position of a cell in the aggregate and in the

Fig. 14. The fate of the cells depends on the order in which they enter
the fruit.

slug subjects it to a specific set of environmental conditions and of chem-
ical signals imposed by its neighbors that forces it to become a spore or
stalk cell or basal disc cell. These differences are reversible. Slugs have
been cut into head and tail segments. Both could construct fruits sepa-
rately. Although not completely normal, both contained all three types of
cells even though the head segment would ordinarily have given rise to
stalk only, while the tail segment would have yielded spores and basal
disc cells but not stalk. In addition, stalk cells have been separated from
the fruiting body before they desiccated and died and have been allowed
to feed on bacteria and reproduce. The progeny of each such stalk cell
were themselves capable of aggregating and constructing normal fruits
with viable spores.

Another example of cellular differentiation is seen in the migrating
slug. If the front end of the slug (about 10 per cent of the cells) is sepa-
rated from the rear end, it continues to migrate, while the rear end stops
in its tracks. If the front end is replaced, the two parts fuse, and migration
of the whole slug proceeds once again in normal fashion.

These and other experiments demonstrated that two cell types,
“leaders” and “followers,” exist and that the slug is an organized entity.
The biochemical mechanisms by which the leaders guide the migration
of the followers and the manner in which cells become leaders and fol-
lowers are still unknown.

The Beginnings of Multicellular Organization

Finally, there is a great deal of evidence to suggest that, in Dicty-
ostelium discoideum, the formation of an aggregate requires the presence
of a specially endowed cell, which has been called the “initiator cell.”
Thus at the time of aggregation, the population of myxamoebae is differ-
entiated into initiator cells, which provide the signal to start aggregating
(and in whose absence aggregation does not occur), and the responder
cells, which answer the signal.

CELL INTERACTIONS DURING DEVELOPMENT

The development of a multicellular system is accompanied, indeed
is largely directed, by a hierarchy of cell interactions. One cell or one
tissue may stimulate or inhibit the development of another by exchange of
appropriate chemical agents. It is this matrix of interactions that ensures
the harmonious organization of the whole, i.e., that the components will
develop at the right time, in the right place, and in the right amount. As
we will see in Chapter 7, the use of cell interactions reaches its highest
degree of complexity and refinement in vertebrate embryos. However,
the beginnings of these control mechanisms operate in primitive organ-
isms such as the slime molds.

A case in point is the way in which the cells aggregate. A large body
of evidence has been accumulated to show that outlying cells are attracted
and caused to aggregate by specific chemical agents produced at the
center. (Attraction of cells by a chemical compound is called chemotaxis. )
For example, if the myxamoebae are permitted to aggregate under a
flowing stream of water (Fig. 15), a center forms, but the cells upstream
are unaffected. Only those downstream are attracted by and move toward
the center. This is just what you would expect if the center were produc-
ing a chemical agent that was being carried downstream by the current.

o ° Co °
° © 6 =
o
°
o- oO °
°
Pe ore Za?
°
ae Bie. of
. * 8 o 7. oO c
ne
Direction of 2 Qasr. ae
current 2 0 SS re}

Fig. 15. Myxamoebae ag-
gregate under a_ flowing
stream.

Cells upstream from Cells downstream
aggregative center elongate and move
are unaffected toward the center

31

32

The Beginnings of Multicellular Organization

REGULATION

This term refers to the ability of a morphogenetic system to turn out
normal products of different absolute sizes. For example, individual frog
embryos or tadpoles or adult frogs of a given species can vary markedly
in size and yet be perfectly proportioned. That is, the sizes of the various
parts (arms, legs, head, etc.) at each developmental stage bear precise
relationships to the total length or total mass. Furthermore, if cells are
surgically removed from a very young frog embryo, the remainder will
develop into a very tiny but nonetheless normal tadpole, again demon-
strating the same relationship of parts to whole.

Slime molds also display regulatory capacities. For example, myx-
amoebae packed together in a solid mass produce relatively large migrat-
ing slugs and fruiting bodies; some are as much as 5 mm in length and
contain hundreds of thousands of cells. If sparsely distributed, the myx-
amoebae form tiny slugs and fruits a few tenths of a millimeter in length
that contain a few hundred cells. Yet the gross proportions and the rela-
tive amounts of cells comprising the various parts of the fruiting body
remain the same. This regulatory capacity is exhibited at a truly startling

Fig. 16. Two views of a
fruiting body formed by
the "fruity'’ mutant of D.
discoideum. This highly or-
ganized multicellular struc-
ture consists of 9 spores, 2
stalk cells, and | basal cell.

The Beginnings of Multicellular Organization

level by a mutant of Dictyostelium discoideum that can form normal
fruits containing hundreds of thousands of cells or as little as 10-12 cells.
Figure 16 shows an example of the latter, a fruiting body with 12 cells—9
spores, 2 stalk cells, and 1 basal disc cell. This exquisite miniature contains
all the elements of a morphogenetic system—cellular differentiation, cell
interactions, and regulation—but in a tiny packet of cells.

In summary, these are some of the lessons learned thus far from the
study of slime molds:

a. That just as in the development of higher forms (vertebrate em-
bryos, etc. ), slime-mold development involves the appearance of new cell
types (initiator and responder cells; leader and follower cells; spores, stalk
and basal disc cells) which play a causal role in the construction of the
multicellular whole. We would like to know how and when they arise and
in what numbers, and what physiological mechanisms are responsible
for the roles they play in morphogenesis.

b. That here as in other developmental systems, a matrix of cellular
interactions helps to regulate the process. Chemical signals are passed
between initiator and responder cells, between responder and responder
during aggregation, and between leader and follower cells during migra-
tion of the slug. Finally, chemical signals tell some cells to become spores,
others to become stalk cells, and still others to become basal disc cells in
the fruiting body. We want to know what are these signals and how they
act.

c. That normal development need not involve participation of huge
numbers of cells. Instead, even as few as 10 cells can provide the com-
ponents and the chemical interactions needed to construct a perfect fruit-

ing body.

A Water Mold, Achlya Bisexualis

Achlya is an example of a permanently colonial organism. The cells
grow in long, branched filaments which form a tangled mass. Some of
these filaments can penetrate the substratum (a floating seed, a dead in-
sect) to anchor the colony and absorb nutrient materials and thus serve
as a primitive root system. Others grow as aerial stalks and produce special
structures called sporangia that bear spores. The spores are cast off and
germinate to yield new filamentous colonies. Some of the aerial filaments
can also produce sexual structures, primitive counterparts of the male and
female structures of plants. A single Achlya colony produces either male
or female structures, not both. The fascinating aspect of this development
is that the formation of male and female structures is triggered by hor-
mones that pass between the colonies.

33

34

The Beginnings of Multicellular Organization

The sequence of events is shown in Fig. 17. When small pieces of
male and female colonies are innoculated at opposite ends of the Petri
dish, they grow toward each other as expanding mats of branched fila-
ments (vegetative stage). As they approach each other: (1) the tips of

Fig. 17. Sexual hormones in Achlya.

:

Male and female |. Female secretes Hormone A. Male reacts
vegetative filaments by producing antheridial initials.

2. Having reached this stage, male secretes 3. Female cogonial initial secretes Hormone C.
Hormone B. Female is induced to form This causes the antheridial initials to grow directly
the oogonial initial. toward the oogonial initial and plaster themselves

against it.

4. Male secretes Hormone D. That causes the 5. Finally, the antheridia, presumably directed by
cytoplasm to congeal about the oogonial nuclei a female hormone, put out germ tubes that
and produce functional female gametes. connect up with the female gametes and permit

entry of the male nuclei.

The Beginnings of Multicellular Organization

the male filaments send out huge numbers of gnarled, branched shoots;
(2) the female filaments expand at their tips into large sacs; (3) the male
shoots grow in a directed manner toward the female sacs and _ plaster
themselves around their surfaces; (4) cytoplasm congeals about the nuclei
inside the female sacs, each aggregate of nucleus and cytoplasm being
called an oosphere; (5) the male nuclei penetrate the sac through tiny
tubes and each unites with a female nucleus to produce a zygote.

Male and female colonies must be grown together to yield sexual
structures. Grown alone, they merely produce the usual filaments with
sporangia. Furthermore, sexual structures begin to form before the two
colonies actually come into physical contact. That is, they appear when
the colony edges are still separated by several millimeters, and develop
in a wave, back from the advancing edge of the colonies. Thus, we can
conclude that (1) the presence of a male colony is necessary for the de-
velopment of sex structures in a female colony and vice versa, and (2) the
effects are mediated by agents, i.e., “sex hormones,” that can diffuse
through agar over considerable distances.

In addition, it has been found that each male and female Achlya
colony produces several hormones that trigger successive stages in the
development of the sexual structures. That is, a hormone A produced by
the female vegetative colony induces the male to send out gnarled,
branched shoots called antheridial initials (stage 1 of Fig. 16). Having
done so, the male can now produce a hormone B that causes the female
vegetative colony to produce the large sacs called oogonial initials (stage
2). The female can at this point only produce a third hormone C that
causes the male shoots to grow in directed fashion toward the source of
this hormone and to plaster themselves around the sacs (stage 3). Upon
contact, the male produces a fourth hormone D that causes the female
sacs to develop oospheres, and so on.

Attempts have been made to isolate the first of these hormones (A).
Female Achlya was grown in 500 gallons of liquid culture medium, under
conditions which permitted the fungus to pour out maximal quantities
of hormone A. The medium was collected and concentrated. By exploit-
ing various means of chemical purification, the intrepid biologists ended
up with 0.0003 grams of material, still impure, but which could act
upon the male Achlya when diluted to 1 part in 10,000,000,000,000 parts of
water! Unfortunately, 0.0003 grams was too small an amount to permit
chemical identification, and this gallant attempt had to be abandoned.
Recently, however, the project has been resumed, this time starting with
10,000 gallons of Achlya culture! In addition, a similar hormone has re-
cently been isolated from another water mold distantly related to Achlya.
This hormone, given the romantic name sirenin, has been isolated, purified,
and crystallized. Now chemical studies are being done to characterize the

35

36

The Beginnings of Multicellular Organization

agent and yield a structural formula. The importance of this work lies in
the fact that, while hormone-triggered development is a common thing
in fungi, none of the agents has been chemically identified and nothing is
known about the biochemistry and physiology of these processes.

A tissue is defined as a group of cells having

The
Development
of a Primitive
Animal:

The
Coelenterates

similar structure and function and _ ar-
ranged in a compact, organized array.
Thus, we talk of connective tissue or bone
tissue or epidermal tissue. The revolu-
tionary transformation of multicellular
organisms from quasi-amorphous con-
glomerates of cells into structures with
well-defined tissues was a_ significant
step, for it enabled these tissues subse-
quently to combine into organs and or-
gan systems and thus permitted the
levels of complexity that higher animals
have since attained.

The most primitive group of organ-
isms still extant that displays a definite
tissue organization is the Phylum Coelen-
terata (or Cnidaria as it is now called).
This is a group of fresh-water and marine
animals that includes Hydra (a favorite
laboratory animal in elementary biology)
and its relations, the sea anemones that
abound on rocky coasts, the corals, the
jellyfish, and the awesome Portuguese
Man of War, whose toxin has paralyzed
many unwary swimmers and led to death
by drowning. A brief résumé of the gen-
eral properties of these organisms illus-
trates their primitive condition:

1. The body is composed of only
two tissues, the outer epidermis and the
inner gastrodermis. These arise early in
the development of the coelenterate em-
bryo. In contrast, the tissues of all higher
animals stem originally from three em-
bryonic cell layers. Within the two coe-
lenterate tissue layers are a number of cell
types, some of which appear in Fig. 18.

37

] ney Hf

ltl
mn
1 \ | \ \\ ("

Interstitial cell

Muscular cell :
Contractile

xe Interstitial cell process
Nerve

cell Muscle-like cells of Hydra Nerve-like cells in the sea anemone

Tissue arrangement (after Schechter) (after Schechter)

(after Storer and Usinger)

Fig. 18. Cell types found in coelenterates.

2. The body is essentially in the shape of a sack. That is, only one
opening exists, and this serves as both mouth and anus. The internal lining
of the sack (gastrodermis) serves to digest the food carried in through
the mouth, but there is no suggestion of a digestive organ system.

3. There is no blood or circulatory system, no excretory or respira-
tory organs.

4. There is a diffuse network of nerve cells, but no central nervous
system and only rudimentary sense organelles.

The adult coelenterates occur in two forms. One is the polyp, which
has a tubular body with a closed end that is attached to the substratum.
There it grows singly or in colonies. The second is the medusa, a free
swimming, gelatinous body with a shape like an umbrella; its mouth
hangs down from the concave undersurface. Figure 19 shows examples of
these. They are variants of the same basic sack-like structure. In the polyp,

Fig. 19. Body plans of adult coelenterates.

Tentacle

ss

SY
O
o
$
oO
g

\
2

Gastrodermis

a CX Epidermis

ZAMS
Polyp Medusa

The Development of a Primitive Animal

the sack (coelenteron) is elongated and narrow with its open end up. In
the medusa, the sack is squat and rounded with its open end down. Some
species appear both as polyps and medusae during their life cycles. In
other species, one or the other adult form may be rudimentary and very
short-lived or entirely absent.

In both forms, the mouth is surrounded by tentacles, and these con-
tain special stinging cells called cnidoblasts (hence the name Cnidaria).
Upon stimulation, they shoot out spear-like processes bearing powerful
toxins to paralyze their prey (small crustaceans, insect larvae). Very little
is known about the toxins. The few that have been studied appear to be
proteins. When injected into rabbits, they produce neural damage and
elicit the formation of antibodies, just as do certain snake venoms, tetanus
toxin from bacteria, and other poisonous proteins.

Life Cycle of the Genus Obelia

Both polypoid and medusoid stages figure prominently in the life
cycle of Obelia, which is summarized in Fig. 20. The polyps grow as colo-
nies upon rocks and shells in shallow marine waters. The colony is
fastened to the substratum by a mass of root-like runners that bear slender,
branched stems, and from these extend polyps of two kinds: the feeding
polyps equipped with mouth and tentacles, and the reproductive polyps,
which lack all feeding mechanisms and are designed for one purpose—to
produce medusae that bud off from the central cylinder and escape
through the hole in the vase-shaped outer covering.

Fig. 20. The life cycle of Obelia (after Storer and Usinger).
POLYP MEDUSAE

Expanded .. ; g Contracted JOR) WS 7 ite on

Me... fee

Medusa pee

ge
Ue!

uy ASEXUAL

ea eea

—— Part of a i Sid
Entire colony, mature colony Starts new colony Settles
life size

39

40

The Development of a Primitive Animal

The roots and stems of the colony are hollow cylinders composed of
the same two tissue layers, epidermis and gastrodermis. The internal
space, called the coelenteron, is filled with fluid, is continuous, and extends
into the polyps. This means that the feeding and reproductive polyps and
the cells in the walls of runners and stems are all in intimate contact, and
all parts of the colony can be supplied with the food materials that enter
through the feeding polyps.

Both kinds of polyp are initiated as buds on the stems. At first the
bud is simply a protuberance of the stem wall that contains both tissue
layers. The protuberance lengthens rapidly by cell division and by migra-
tion from other areas. Infoldings and constrictions of the tissue ultimately
give rise to the organs that are characteristic of the feeding and reproduc-
tive polyps.

As we have mentioned, the medusae also are formed by budding
from the central cylinder of the reproductive polyps. Fully formed, they
are minute jellyfish, shaped like an umbrella and rimmed with tentacles.
The mouth hangs down from the concave side and leads upward into the
digestive cavity in the middle of the umbrella. They move by jet propul-
sion, water being forced out of the mouth, and feed upon crustacea and
protozoa. They are of two sexes, male and female. Their gonads develop
inside the digestive cavity, and eggs or sperm are propelled through the
mouth. Fertilization takes place in the sea.

The fertilized egg promptly cleaves into two, the two into four, and
so on until a hollow ball of cells is produced. This is called the blastula
stage and corresponds to a similar stage in the development of vertebrate
embryos (see Chapter 5). Some of the cells are forced into the interior of
the ball and become the gastrodermis, while the cells that remain outside
become the epidermis. This corresponds to the gastrula stage of embryo-
genesis, encountered in higher invertebrates and vertebrates, during
which their three basic tissue layers appear. After tissue separation, the
Obelia embryo elongates, and cilia appear over the outer surface. The
constituent cells now begin to transform into sensory, gland, and muscle
cells and cnidoblasts. At this point, embryogenesis is concluded, and the
animal is called a planula larva (i.e., the immature, larval form of the
adult polyp). The planula swims about for a period of hours to days and
then attaches to a rock or shell. The attached end develops into a root-
like runner, and the free end acquires a mouth and tentacles. Buds appear,
the polyp colony is initiated, and the cycle is complete.

Polymorphism in the Coelenterates

Polymorphism (a variety of forms) occurs at two levels in the coe-
lenterates. First, there is the basic difference in the adult structures (i.e.,

The Development of a Primitive Animal

polyp and medusa ). One can ask why it is that in an Obelia colony a bud
on the stem of a feeding polyp gives rise to another polyp, while a bud
on the cylinder of a reproductive polyp gives rise to a medusa. Are the
constituent cells different, or is it merely that they inhabit different areas
of the colony and so are subject to different environmental conditions?
Actually the same sort of question can be asked about our own bodies;
namely, why is it that the cells in one part of a human embryo develop
into brain cells and in another part develop into liver cells? These ques-
tions will be taken up in detail in the chapter on cellular differentiation.

The second level of polymorphism is exemplified in Obelia by the
specialization of the polyps. Another genus, Hydractinia, displays an even
greater range of differences in polyp structure, as Fig. 21 shows. Hy-
dractinia colonies live on the surface of a specific type of snail shell but
only when inhabited by hermit crabs. Three types of polyp develop:

1. Feeding polyps. These correspond in essential features to the
feeding polyps of Obelia.

2. Deferisive polyps. These are concentrated at the lip of the snail
shell. They lack a mouth and their tentacles are reduced to small knobs
arranged in two circular rows. The stems have considerable musculature
and can whip about fiercely, thereby permitting the cnidoblasts on the
tentacle knobs to discharge a concentrated volley upon contact with the
foe.

3. Reproductive polyps. In Hydractinia, the medusa stage is absent.
Instead, the reproductive polyps produce sex cells directly. There are two

Fig. 21. Polymorphism in Hydractinia.

Feeding Defensive Female reproductive Spiral polyp
polyp polyp polyp bearing eggs

41

42

The Development of a Primitive Animal

sexes (a single colony produces reproductive polyps of one sex only). The
male and female varieties are quite differently formed, as seen in Fig. 20.

Regeneration of Polyps and Medusae

The decapitation of a polyp is followed within a few days by the
appearance of a new head at the cut end complete with mouth and ten-
tacles. If a segment is cut from a polyp stem or from a root-like runner
and incubated in water, the isolated piece can give rise to a new polyp.
Similarly, a piece taken from a medusa, if large enough, can reorganize
itself into a complete functional medusa with all parts normal and intact.

The regeneration is specific. That is, a piece of tissue taken from a
polyp regenerates a polyp. A piece taken from a medusa regenerates
a medusa. In a recent study, it has been shown that this specificity ex-
tends even to the kind of polyp from which the piece of tissue was
taken. Cut stems taken from a Hydractinia colony regenerated true to
type. Stems of feeding polyps regenerated only feeding polyps. Those
from reproductive polyps regenerated only reproductive polyps. Two con-
clusions are permissible from these data:

1. A complete, functional, adult coelenterate contains many cell
types (i.e., cnidoblasts, epidermal cells, gland cells, muscle cells, nerve
cells, etc.). These results show that even a small piece of coelenterate
tissue either contains all the necessary types or can give rise to them by
appropriate transformation of one cell type to another.

2. The pieces of tissue appear to “know” from what kind of adult
they came and to be able to reorganize themselves accordingly.

Compare these results and conclusions with those gained from the
study of Acetabularia regeneration described in Chapter 2. Please note
that they are essentially identical, save that in one case we deal with a
structure whose parts are divided up into cellular packets, while in the
other we deal with one whose parts are not so divided but are nevertheless
organized in a specific manner.

Regeneration by stem segments has other interesting features that
are summarized in Fig, 22. First, the rate and extent of new head develop-
ment depends on the part of the stem from which the segment is taken.
If a polyp stem is cut into serial segments of equal size, the one from the
end nearest the previous head (distal end) develops fastest and most
completely, while that from the lowest (proximal) end is the most laggard.
In other words, along the stem there is a gradient of potency for the pro-
duction of new heads. In addition, one part of a stem in producing a new
head can prevent another part from so doing. For example, a stem segment

Cuts

Time

A. Distal segments develop faster and better than proximal segments.

Distal end a2 :
— eae
Distal end
Glass |
eee ine > -———— >

B, Short segments form new heads C. If the distal end of a short segment
only at the distal ends; long is prevented from forming a new head,
segments, both at distal and proximal. the proximal end will do so.

D. If the two ends of a short segment
are physiologically separated, both
will form a new head.

Fig. 22. Regeneration of polyps by stem segments.

of moderate length develops a new head at only one cut end, and this is
invariably at the distal end. However, if we prevent the distal end from
regenerating by slipping a glass cap over it, then the proximal end will
form the head (although slower than the distal end would have done it).
This interaction can be relieved in two ways: first, by making the stem
segment so long that the two ends are not in close contact; second, by
separating the two ends physiologically. The latter can be accomplished
by tying a string tightly around the middle of the segment so as to prevent
the flow of materials and cells between the ends. When these measures
are taken, heads form at both ends without any evidence of competition
between them.

This type of phenomenon is commonly encountered in develop-
mental systems. A case in point is the development of the hind limb in a
chick embryo. As described in Chapter 5, the limb originates as a small
protuberance from a specific point along the flank of the embryo. Areas
surrounding this point can also be shown to be capable of forming the

43

44

The Development of a Primitive Animal

protuberance and ultimately a complete limb. We can ask, therefore, why
it is that the limb forms where it does and why other areas that have the
potency for limb construction normally do not grow one.

A system in which many parts can develop in a certain way but
where only one part actually does so is called a morphogenetic field,
which will be discussed in detail in the next section. The dominance of
one part of the field over another maintains regularity of development.
It ensures that a coelenterate polyp will have one head and that it will be
where it ought to be, i.e., at the top of the stem. It ensures that a chicken
will have one tail, two wings, and two hind limbs in the right places.
Moreover, it makes for versatility, for if by accident the dominant area of
a morphogenetic field is destroyed, a neighboring area has the potency to
take its place. Thus the top of the coelenterate stem and only the top
normally forms a head. But if by accident the head is destroyed, a new
one can be constructed from underlying tissues.

Morphogenetic Fields

The concept of a morphogenetic field had its modern inception in
the work of Professor C. M. Child, a famous American embryologist. Dr.
Child was the first to emphasize the fact that in such fields one area will
gain dominance over another by its superior capacity to form a given
structure and by its ability to inhibit neighboring areas from doing so. He
and his students provided many examples of field development in the
regeneration of coelenterates, flatworms, and starfish and in the forma-
tion of embryos. Thus we speak of head fields, limb fields, tail fields, heart
fields, etc., to denote this kind of development. Attempts have been made
to describe morphogenetic fields in mathematical terms in an effort to
understand the processes by which dominance is established. A simplified
version of the mathematical argument is given below.

Consider a group of coelenterate stem cells that can form a head.
We divide this group into two separate areas. The speed at which the
stem cells in an area will construct a head should depend on their inherent
capacity (i.e., developmental potential) to be converted into head cells
and also on the number of cells available in the area for head construction.
This can be written formally as:

rate of head development = (developmental potential ) 1

< (number of stem cells)
The rate of development in area I might be greater, equal to, or less than
that of area II, depending on the relative developmental potential and on
the numbers of cells that comprise each area. However, a difference in
developmental potency is not enough to create a morphogenetic field,

The Development of a Primitive Animal

since, if the two areas were isolated from each other, each would con-
struct a head at its characteristic rate and neither would dominate the
other. It is necessary for the two areas to be able to interact upon one
another.

Suppose the developing head cells can pour out materials that inhibit
others from becoming head cells. First of all, the cells in area I that had
already been incorporated into the developing head would inhibit the
remaining stem cells in that area from changing into head cells. The
degree of inhibition would depend on the inherent sensitivity of the area I
stem cells toward being inhibited by their own head (autosensitivity ) and
on the number of head cells that are producing the inhibitory materials.
This inhibition would have to be subtracted from the rate of head de-
velopment defined in statement 1. Thus,

rate of head developmental number of
development = potential xX { stem cells

in area I of area I in area I

auto- number of
—| sensitivity } x { head cells
in area I in area I

According to this statement, the rate of development would be very high
at the beginning of head formation. Then, as the head grew larger and
more and more cells were incorporated into it, they would produce in-
hibitory materials and detract materially from the rate at which additional
stem cells would be incorporated into the head. Meanwhile, the number of
stem cells would decrease as they were converted into head, and the first
term on the right side of the above equation would decrease. Eventually,
the two terms on the right side would cancel out, and the rate of head
development would become zero (i.e., the head would now be complete).
In the case under consideration, the rate would fall to zero before all the
stem cells had been converted into head cells, since a coelenterate head
would look pretty silly without a stem to support it.

Now suppose the two areas are in contact with each other (for ex-
ample, if the two have a common coelenteron and are bathed by the same
body fluid). Then the head cells of area II would inhibit the stem cells
in area I in the same fashion as described above. That is, the rate of area I
development will be impeded by the inherent sensitivity of area I stem
cells to the inhibitory products ! of area II head cells (heterosensitivity )

1] have described the interaction between areas I and II as the result of the
production of inhibitory materials. We could equally well account for it by assuming
that the areas compete for a common pool of nutritive materials that serve to stimulate
development. Were this the case, area II would inhibit area I by depriving it of these
materials and vice versa. Actually, the mathematical argument turns out to be the
same whether we put it in terms of inhibition or deprivation.

45

46

The Development of a Primitive Animal

and by the number of head cells in area II. The complete description of
the rate of head development in area I would then become:

rate of head developmental number of autosensi-
development = potential x | stem cells }— | tivity in

in area I in area I in area I area I
number of heterosen- number of

x | head cells } — sitivity } x { head cells

in area I in area I in area II

The same sort of equation can be written for the rate of head development
in area II.

We can now ask how area I can gain dominance over area II and
prevent it from forming a head, and we see that this can be done in a
number of ways:

1. Area I could have a much higher developmental potential or a
much greater number of cells. In either case, it would form a head so fast
and begin pouring out inhibitory materials so soon that area II wouldn't
have a chance to get started.

2. Area I might be very resistant to auto-inhibition (i.e., the inhibi-
tory materials might be carried away by the body fluid circulation quickly,
whereas area II might be very sensitive (i.e., the inhibitory materials
might not be carried away ).

3. Area I might not be very heterosensitive, while area II might be
extremely so.

4, Even were developmental potentials, sensitivities, and cell num-
bers equal, area I could gain dominance by starting head formation first.
The head start (forgive me) would permit it to inhibit area II.

Any one of these conditions, or a combination of them, would serve to
establish the dominance of area I.

At present, none of the biochemical and genetic mechanisms that
create differences in developmental potential or that exert inhibitory ef-
fects have yet been identified for any morphogenetic field. Thus, this kind
of developmental study is in its infancy.

Nature's job in embryogenesis is to create a

The
Development
of the
Vertebrate
Embryo

young, functioning, multicellular organ-
ism. Her raw materials are (1) an egg, a
single cell, large with respect to sperm
but tiny with respect to the finished
product that is to come, and (2) a sperm
containing little more than a nucleus
which, when joined with the egg nucleus,
will provide the instructions needed to
produce the embryo. She solves the prob-
lems of embryogenesis by the following
strategems:

1. Cleavage: the fertilized egg is
first subdivided into a large number of
smaller cells, because little self-contained
packets are easier to control and alter
than a single, undivided mass of proto-
plasm.

2. Gastrulation: the cleaved cells
are moved about so as to create three
basic tissue layers. These become further
subdivided and give rise to the tissues
and organs of the functional adult.

3. Organ formation: each subdivi-
sion of the three original tissue layers be-
comes a semi-autonomous system within
which cells appear by division, are
grouped into tissues, and finally become
functional entities (kidney, spleen, brain,
etc.). Thus, many changes occur simul-
taneously within the subsystems of the
developing embryo, and they must be
linked in such a way as to operate har-
moniously, in the right place at the right
time. This is accomplished in part by
having chemical signals pass between de-
veloping subsystems so that one of them

47

48

The Development of the Vertebrate Embryo

can trigger or curtail the next and therefore provide chronological order
to what would otherwise be chaos.

In the rest of this chapter we shall consider these processes, as well
as the earlier phases of embryogenesis, i.e., the formation and union of
the gametes, in some detail.

The Early Stages of Embryogenesis

THE FORMATION OF GAMETES: THE SPERM

The first step in the recipe for roast chicken is: find your chicken.
Similarly, nature’s instructions to a sperm are: first, find your egg. To
accomplish this, the mature sperm is a stripped-down model equipped
with organs of locomotion and with a high rate of metabolism so that it
can generate energy to move rapidly over relatively great distances. Once
in contact with the egg, it must deliver a nucleus containing a haploid
set of chromosomes, one-half the complement of the future embryo. Fig-
ure 23 is a drawing of the sperm of several animals and includes a recon-
struction of what we see in the electron microscope when we look at the
midpiece of ram sperm. This should give you some idea of the sperm’s
structural complexity. Various sperm may look very different, but they all
contain these basic parts:

Fig. 23. Spermatozoa.

Fine structure of mid-
piece as revealed by
electron microscope
(after Waddington)

Human Fowl Salamander Crustacea

The Development of the Vertebrate Embryo

1. The head. This portion is taken up almost completely by the
sperm nucleus.

2. The midpiece. This part contains material which will be put to
use after the sperm penetrates the egg. It aids in the fusion of the egg and
sperm nuclei and in the cleavage of the fertilized egg into the first two
daughter cells.

3. The tail. This can be long or short, flexible or stiff. It may vibrate,
whip, or rotate in a screw-like motion depending on the species. It is the
locomotor organelle.

The ancestors of the sperm start out as ordinary diploid individuals.
The germ cells, as they are called (both in males and females), arise early
in the development of the embryo. They appear to be irreplaceable, be-
cause when embryos are treated in a manner that destroys or removes (by
ultraviolet irradiation or surgery) the germ cells, the resultant adult or-
ganisms are sterile.

Gametogenesis, the process by
which the diploid germ cells give
rise to haploid sperm or eggs, is
properly within the domains of cy-
tology and genetics but will be re-
Germ cell viewed briefly here. Figures 24 and

25 are schematic summaries of the
maturation of male and female
gametes. For simplicity, we imagine
that the diploid nucleus contains
only two pairs of chromosomes.
eeamchon Each chromosome duplicates, yield-
of tetrads ing a quartet or tetrad of chroma-
tids. The cell itself then divides into
two, each with a pair of sister chro-
Meiosis | matids. This is meiosis I. These cells
in turn divide without chromosomal
duplication to yield four (meiosis
IT) so that the daughter nuclei end
up with two chromosomes rather

Fig. 24. Maturation of the sperm.

See than the original four and are there-
o) fore haploid. In the case of sperm,
the daughter cells are changed from

Mature - . :
sperm ordinary-looking cells into func-

tional gametes after meiosis II.

49

50

The Development of the Vertebrate Embryo

THE FORMATION OF GAMETES: THE EGG

The egg has three functions to perform:

1. To supply a nucleus containing half the chromosomal comple-
ment of the future embryo.

2. To supply almost all the cytoplasm upon union with the sperm.

3. To supply food reserves that will enable the embryo to develop
to a stage where it can begin to feed upon exogenous materials.

Because it must do all these things, the animal egg is a cell of giant size,
Consider for example, the size of a hen’s egg. Even that of the mammal,
.05-.25 mm, is considerably larger than ordinary body cells.

The egg is covered by a protective envelope: in birds, by an in-
organic shell; in amphibians, by a jelly coat made up of polysaccharide
and protein of high molecular weight. In mammals, a single layer of very
small protective cells cover the egg surface. The egg proper is bounded
by one or more membranes. A single haploid nucleus resides within, to-
gether with cytoplasmic constituents whose fine structure, revealed by the
electron microscope, does not seem to be different in kind from the con-
stituents of other cell types. Finally, there is yolk, the food supply for
the embryo. This is a heterogeneous mixture of fat droplets, granules, and
small bodies, bounded by membranes. Some of these are highly pigmented
and, because the yolk is organized material, can be seen to occupy definite
regions of the egg and to be parceled out amongst the daughter cells in a
definite manner.

There are great differences among eggs of the animal species with
tespect to yolk content. For example, the eggs of marine animals contain
relatively little yolk because the embryo begins active feeding at a very
early stage. What yolk there is in marine eggs is distributed homogene-
ously and not concentrated in a particular region. In contrast, the eggs
of birds contain enormous amounts of yolk. For example, the hen’s egg
contains a tiny disc of cytoplasm sitting on top of an enormous mass of
yolk. Insect eggs also contain large amounts of yolk, which is situated at the
center of the egg and surrounded by a thin outside layer of cytoplasm. In
these forms, the embryo undergoes considerable developmental changes
before emerging as an actively feeding organism; hence the large supply
of food reserves. At the top of the evolutionary scale are the mammals. A
mammalian embryo develops in a sense as a parasite inside its mother
and receives a continuous supply of food materials from her through the
circulatory system, much as if it were just another of her internal organs.
A large yolk reserve is therefore unnecessary, and mammalian eggs re-
semble the primitive marine eggs in this respect.

The Development of the Vertebrate Embryo

The maturation of the egg,
like that of the sperm, proceeds via
two meiotic divisions from a small
diploid parent cell (Fig. 25). The
parent cell chromosomes duplicate,
and the cell then grows to enormous
size. The proteins, fats, nucleic

acids, and carbohydrates that com-
ines prise the yolk are synthesized within
the egg or are received from
without (contributed by so-called
“nurse” cells). When the final size
is attained, the egg nucleus goes
through meiosis I and two cells ap-
pear. The cell division is very un-
equal, however, and the lucky nu-
cleus finds itself with most of the
cytoplasm and yolk, the unlucky
nucleus with very little. Meiosis II
follows and now four haploid cells
are present. Again, because of a
very unequal division, one of the
daughter cells contains practically
Functional egg and all the yolk and cytoplasm. This,
three polar bodies : ;
then, is the mature egg, and its
three tiny sisters (called polar
bodies ) shortly degenerate and dis-
appear. Figure 24 is a schematic
Fig. 25. Maturation of the egg. summary of the Process.
In most cases, developing eggs
reach stage C and then lie dormant
until fertilization, at which time the process is completed and the sur-
viving female gamete nucleus unites with the sperm nucleus. In a few
cases the egg is arrested at stage B and very few reach stage D before
fertilization.

Germ cell

Meiosis II

THE LIFE SPAN OF GAMETES

A mature egg must be fertilized within a short time, if it is to be
fertilized at all. Thus, human eggs generally remain functional for about
12-24 hours after their release from the ovary. The eggs of sea urchins,
which are shed into the water, last for 48 hours, but the eggs of most
invertebrates as well as fish and amphibia must be fertilized within a few
minutes of being shed. The life spans of other animal eggs are of the same
order of magnitude as those of human eggs.

51

52

The Development of the Vertebrate Embryo

Sperm are generally just as short-lived as eggs. However, in some
cases (bees, bats) the sperm can remain viable for years within the female
genital tract. The study of sperm preservation has recently gained great
economic importance because of the use of artificial insemination for
livestock breeding. In the case of cattle, a stud bull is induced to service
an artificial vagina (a water-jacketed rubber tube with a collecting bottle
at one end and the bull at the other). About 4.5 ml of semen is collected
per ejaculation, and the bull can be used for service up to 10 times in a
two-hour period. The semen is cooled slowly to a temperature just above
freezing and can be stored for seven days at most. It is diluted before use
and injected into the uterus of a cow in heat, through a pump. A good
stud bull can be used to impregnate literally thousands of cows in a single
season. It should also be noted that many human females fail to conceive
by natural means even though the gametes of both husband and wife are
perfectly viable. In such cases, artificial insemination of the wife with her
mate’s semen is generally successful in inducing pregnancy.

FERTILIZATION

The act of fertilization can be divided arbitrarily into three phases:
(1) penetration of the egg by the sperm, (2) activation of the egg, and
(3) the union of egg and sperm nuclei.

PENETRATION. Contact between sperm and egg is generally the result
of random collision. The discharge of huge numbers of sperm by the male
increases the chance that at least one such collision will occur. Upon con-
tact, the sperm is bound tightly to the outer surface of the egg. This may
be accomplished by chemical reactions between proteins at the surfaces of
both gametes called fertilizins. Fertilizin, isolated from eggs, causes sperm
to become sticky and to clump together, while anti-fertilizin obtained
from the sperm does the same thing to eggs. The substances are specific
and act only upon gametes of the same species. Thus, fertilizin and anti-
fertilizin may increase the chance of an effective and lasting contact be-
tween egg and sperm. However, the evidence that the fertilizins actually
do operate during fertilization is still equivocal.

The sperm that is now bound to the egg must penetrate its mem-
brane. Material extracted from sperm has been shown capable of dis-
solving the egg membrane by enzymatic action. Presumably the intact
sperm employs the enzyme to dissolve a hole big enough to permit en-
trance. In mammals, the egg is surrounded by a layer of tiny follicle cells
cemented together by a substance called hyaluronic acid. Mammalian
semen contains an enzyme, hyaluronidase, that dissolves this cement and
permits the sperm to slip through. Many pathogenic bacteria use this same
device to slip through body tissues and invade all parts of the organism.

The Development of the Vertebrate Embryo

ACTIVATION. As mentioned previously, the egg as it develops in the
female stops at one or another stage of meiosis and lies dormant until
activated by contact with the sperm. A number of characteristic changes
then occur:

1. Completion of meiosis. The nuclear membrane breaks down and
the egg nucleus completes the meiotic process (in eggs where it is incom-
plete).

2. Fertilization cone. In some eggs a conical projection pushes up
through the surface jelly from the egg membrane. It “captures” the sperm
at the jelly surface and by contraction draws it down toward the egg.
Meanwhile, the sperm dissolves a portion of the membrane to permit
ingress.

3. Block to polyspermy. Penetration by the first sperm makes most
eggs almost instantaneously impervious to the entrance of any others.
The mechanism of this reaction is still unclear. In a few species, however
(some reptiles and insects), the entrance of more than one sperm is a
normal event. One of the sperm nuclei fuses with the egg nucleus and
the others degenerate and disappear.

4, Rearrangement in egg contents and physiology. Upon fertiliza-
tion the egg changes its shape, generally veering toward the spherical.
The permeability of the egg membrane to the ingress of salts and other
substances rises markedly. Cytoplasmic materials migrate to new areas.
The axes of the future embryo (dorsal-ventral, anterior-posterior) are

fixed.

Many eggs can be activated in the absence of sperm. Among treat-
ments found successful are: X-ray and ultraviolet irradiation, introduction
of foreign protein by a needle, heat shock, addition of certain salts and
acids. The activated egg proceeds through the changes noted above and
goes on to cleave and construct an embryo. The eggs of certain organisms
(sea urchins, starfish, frogs, bony fishes, rabbits) can develop perfectly
normally without fertilization by sperm and give rise to viable adults. The
process, called parthenogenesis, is carried out routinely in nature by colo-
nial insects including bees. Although only the egg nucleus is present, the
cells of a parthenogenetic embryo need not remain haploid. In some cases
the activated haploid egg duplicates its chromosomes without cell divi-
sion, thereby producing a diploid, homozygous individual.

FUSION OF NUCLEI. The sperm head and midpiece enter the egg,
membranes disappear, and the sperm chromosomes arrange themselves on
a spindle constructed by material in the midpiece (Fig. 26). Sperm and
egg nuclei migrate toward each other and the egg chromosomes arrange
themselves on the spindle. The pattern now resembles that seen in meta-

53

Fig. 26. Fertilization (after Wilson). (1) Sperm contacts egg. (2) Sperm
head and midpiece enter egg through fertilization cone; egg nucleus pro-
ceeds through meiosis I]. (3) Sperm nucleus enlarges; midpiece constructs
spindle and asters; meiosis Il of egg completes to yield polar body and
functional, haploid egg nucleus. (4) Sperm and egg nuclei migrate toward
each other across midpiece spindle; nuclear membranes break down. (5)
Chromosomes dispose themselves upon mitotic spindle. (6) Chromosomes
duplicate and mitosis draws to a close in preparation for first cleavage.

phase of mitosis. The chromosomes duplicate, the first cleavage of cells
occurs, and two daughter cells, now diploid, emerge. The egg has become
an embryo.

Cleavage

In all eggs, fertilization is followed by a period of extensive cell divi-
sion without an increase in size. The fertilized egg is cleaved repeatedly
into smaller and smaller cells. The early divisions are generally synchro-
nous; thus the egg yields two cells, the two produce four, the four, eight,
and so on. From this time on, synchrony may disappear and different parts
of the embryo may cleave at different rates. At the end of the cleavage
stage, the embryo, now called a blastula, is a ball of thousands of small
cells (blastomeres ).

Although there is no size increase during this period, a considerable
amount of synthesis occurs. The cleavages are regular mitotic divisions so
that chromosomes and total nuclear content must be duplicated in the
daughter cells. This means extensive synthesis of deoxyribose nucleic
acid plus other nuclear constituents. In addition, a great deal of ribose
nucleic acid and proteins, including enzymes, are formed in the cytoplasm.
The energy and raw materials for these processes come, of course, from
the supply of yolk.

54

The Development of the Vertebrate Embryo

Cleavage performs at least three functions in embryonic develop-
ment:

1. It provides an adequate number of cells, i.e., building blocks for
the future organization of tissues and organs.

2. It lays the groundwork for the gross design of the embryo (dorsal-
ventral axis, anterior-posterior axis, etc.) by shifting around the material
(both cytoplasm and yolk) of the egg and compartmentalizing this ma-
terial within separate cells.

3. It brings nuclear and cytoplasmic materials into balance. The
egg starts as a cell with a single nucleus and an enormous amount of cyto-
plasm. During cleavage, total cytoplasmic content does not change mark-
edly but thousands of nuclei appear as cell division proceeds.

The pattern of cleavage is determined in large part by the amount
of yolk in the egg. In eggs that contain a little yolk that is homogeneously
distributed, cleavages are equal; that is, the daughter cells are always the
same size. Figure 27a shows this type of cleavage in Amphioxus. Eggs
with a large amount of inhomogeneously distributed yolk cleave un-
equally. Figure 27b illustrates this type of cleavage in the frog. The yolk
in the frog egg is concentrated in one hemisphere (the vegetal), and de-
creases in a sharp gradient toward the opposite (animal) hemisphere. The
first two cleavages split the frog egg longitudinally to produce four cells
shaped like the segments of an orange. The third cleavage is transverse
and separates the yolky (vegetal) half from the non-yolky (animal) half.
The split is unequal, since the four animal blastomeres are much smaller
than the four vegetal ones. Subsequently, the yolky cells cleave far more
slowly than the non-yolky ones. The result is that in the completed
blastula, the dorsal part contains many small cells while the ventral part
contains fewer but larger cells. In fish and birds, the extreme of yolkiness
is reached. The cytoplasm and nucleus of the egg sit atop a huge ball of
inert yolk. The yolky area does not cleave at all and only the little cap on
top divides into cells (Fig. 27c).

The specific appearance of cleaving cells is much the same in all
embryos. A groove (called the cleavage furrow) appears at one point of
the egg. For example, in the frog the first furrow appears at the animal
pole. The furrow then deepens and extends downward on both sides. The
two ends meet at the vegetal pole. The furrow then extends inward ra-
dially, finally constricting the egg into two sister blastomeres.

In most cases, as the cells cleave, a cavity appears in the middle of
the ball of cells. This is the blastular cavity. Thus, by the end of the cleav-
age stage, the embryo is a spherical or flattened hollow ball generally one
cell thick. The blastomeres can vary in size, yolk content, and cytoplasmic
organization, but no tissues and certainly no organs yet exist.

55

2-celled Before cleavage

4-celled

Blastula-exterior 8-celled

Blastula-vertical section

AMPHIOXUS AMPHIBIAN BIRD

"ae

: Caceres

5 ae aa
5 Maat © @: seers

i/

Fig. 27. Patterns of cleavage in Amphioxus, frogs, and birds (from Storer
and Usinger).
56

The Development of the Vertebrate Embryo

The early cleavages are regular phenomena that occur at specified
times after fertilization. Thus in the egg of the frog, Rana sylvatica, in-
cubated at 18°C, the first cleavage (longitudinal) occurs at 2.5 hours
after fertilization, the second (also longitudinal) occurs at 3 hours, the
third (transverse) at 4.5 hours, the fourth (again longitudinal) at 5.5
hours, leaving the embryo as a still solid ball consisting of two hemispheres
of eight cells each, the dorsal being smaller than the ventral. The blastular
cavity soon appears and produces a hollow ball with a thin roof and a
thick floor. By 21 hours the blastula is complete and gastrulation com-
mences.

Gastrulation

At the beginning of gastrulation, the embryo is a hollow ball of
cells. By the end of gastrulation, the embryo consists of three basic tissue
layers: an outer layer (ectoderm), an inner layer (endoderm), and an in-
termediate layer (mesoderm). Gastrulation, then, involves a series of
integrated cell movements that lead to the formation of these discrete
layers. Because the movements are quite complex, they are hard to follow.
Investigators have observed them by time-lapse movie photography and
by marking areas of the blastula with carbon particles or dyes and follow-
ing the path of motion. In this way they have been able to map the origins
of the three tissue layers back to cells of the early blastula stages. The
gastrular movements of many species have now been studied and show
that they have achieved a variety of solutions to the problem of establish-
ing three tissue layers. Three mechanisms of gastrulation will be dis-
cussed: in Amphioxus, in the frog, and in the chick.

AMPHIOXUS

Amphioxus or the Lancelet is a primitive chordate, a marine animal
resembling the fishes. As seen in Fig. 27a, cleavage is virtually equal and
produces a blastula whose vegetal-pole cells are only slightly larger than
those at the animal pole. Gastrulation begins when the cells at the vegetal
pole move to the interior (Fig. 28). As they press upward, the blastular
cavity grows progressively smaller and finally disappears when the vege-
tal-hemisphere cells press up against the animal-hemisphere cells. The
embryo is now a double-walled cup such as might be made if we pressed
upon one side of a hollow rubber ball. The external wall is the ectoderm
and will ultimately give rise to the epidermis, certain external organs, and
the nervous system. The internal wall is the endoderm, and the hollow
U-shaped cavity is called the archenteron. Surrounded by endodermal

57

wR rn.

ey

Anterior bPRe~aEe

Notochord

Inner.and outer
mesodermal
linings

Fig. 28. Gastrulation and later embryonic stages in Amphioxus (after Villee).

cells, it is the primitive digestive tract (gut). The embryo now lengthens
and the site of the old animal pole becomes the anterior end of the em-
bryo; the site of the old vegetal pole becomes the posterior.

The gastrula is still only two-layered and the third layer (mesoderm)
appears at this time. Three long tubes of cells are pinched off from the
endoderm. The middle one becomes the notochord, a flexible skeletal rod
which persists in amphioxus as its axial supporting member. (In higher
chordates, the notochord appears during embryogenesis but is later sur-
rounded or replaced by the vertebral column.) The lateral tubes grow by
cell division into large tissue masses that are divided into segments along
the length of the embryo and are called somites. These expand and plaster

58

The Development of the Vertebrate Embryo 59
themselves exteriorly up against the ectoderm to become the second layer
of skin and interiorly against the endoderm to become the outer lining of
the gut. Eventually internal organs such as the gonads, kidneys, etc. will
pinch off from the mesoderm and inhabit the internal body cavity that
lies between the mesodermal linings. With the formation of the third basal
layer, the gastrula is complete.

THE FROG

At the end of the cleavage stage, the frog embryo is a hollow ball
with a thin roof and a thick floor. A small dimple appears near the vegetal
pole when large yolky cells leave the surface of the egg and begin to
press inward. This process of inward movement is called invagination.
As more cells invaginate, the dimple becomes a crescent-shaped groove
curving downward toward the vegetal pole. The groove is called the
blastopore and has an upper (dorsal) and a lower (ventral) lip (Fig. 29).

Fig.
Ectoderm
(future epidermis)

29. Gastrulation stages in the frog.

Ectoderm
(future neural
plate)

Blastular
cavity

Archenteron

“~ Mesoderm
(future
notochord)

Blastopore
Mesoderm P

Endoderm

_ Notochord
KY

a
SEZ

Somites

Lateral
mesoderm

A
A
2)
2)
S;

Endodermal folds

coming together to

enclose archenteron
Endoderm

The sheet of cells from the upper surface of the blastula moves down-
ward and converges upon the blastopore. As the cells reach the dorsal lip,
they turn inward and enter the interior of the embryo. Similarly, the
ventral cells invaginate over the ventral lip of the blastopore. As the
presumptive (future) mesoderm folds inward over the dorsal lip, it sepa-

rates from the endoderm that entered before it. The mesoderm plasters
itself against the outer layer of ectoderm and continues to move away

60

The Development of the Vertebrate Embryo

from the blastopore and up toward the animal pole. The detached endo-
dermal cells move to the middle of the embryo and form a long trough-
shaped structure. The edges of the trough move up and around, finally
meet, and the trough is converted into a tube. The mesoderm breaks up
longitudinally into three masses: a central tube, the notochord (this later
disappears and is replaced by the vertebral column), and two lateral
masses which later segment and become somites.

THE CHICK

At the end of cleavage, the chick blastula is a small cap of cells sev-
eral layers thick that sits on top of the yolk. A thin cavity appears within
the blastoderm, so that at completion it is a flattened hollow ball. Despite
a great deal of descriptive study, it is not clear whether the lower layer
of cells simply splits off from the upper layer or whether cells at the edges
of the cap move underneath it and join at the center to form the lower
layer. In any case, the upper layer of cells is the source of both ectoderm
and mesoderm; the lower layer is the endoderm.

A long narrow groove called the primitive streak appears on the
surface of the blastodisc and runs from the approximate center back to-
ward one edge. This edge will become the posterior of the embryo and
its opposite will be the anterior. Cells stream toward the primitive streak
and then turn under to spread out in a sheet between the ectoderm and
the endoderm. This intermediate layer is mesoderm and when it is fully
formed, gastrulation is complete. Note that the embryo is still only three
isolated sheets of cells like a layer cake. Eventually the blastoderm in the
anterior and posterior regions and at the sides will get tucked under the
embryo. When the two infoldings meet in the middle, the ectoderm will
have been transformed from a single sheet of cells into a complete outer
covering. The endoderm will also have been tucked under to produce a
long hollow tube, the gut. The mesoderm at the same time gives rise to
notochord and somites. Figure 30 is a schematic representation of these
changes.

THE SUBSEQUENT DEVELOPMENT
OF ECTODERM, MESODERM, AND ENDODERM

An embryo develops in one sense much as a delta-choked river
travels to the sea. That is, it starts as one big channel and then splits up
into several smaller ones that in turn branch and rebranch. The original
embryonic channel is the fertilized egg. The three intermediate channels
are ectoderm, mesoderm, and endoderm. The diagram opposite sum-
marizes these ramifications.

Primitive
groove

Primitive
streak

Blastula”

“Bis

Ectoderm Invaginating cells

Beginning Notochord— Mesoderm

Loose mesodermal cells
Dorsal ectoderm

Mesoderm

Beginning of gut Endoderm

Headfold Notochond Ventral
D. Longitudinal section E. Cross section Je /Se LET!
Fig. 30. Gastrulation in the chick.
Blastula
Gastrula
aes
Embryonic gut
Epidermis and Brain and Inner lining of| Inner lining of
associated structures nervous system digestive tract | respiratory tract

(hair, nails, etc. )
Glands—including
liver, pancreas

Mesoderm
Notochord Somites
Muscle Outer covering
of internal
organs
Excretory
system
Mesenchyme
(loose, migratory cells )
Dermis (inner Bones and cartilage

skin layer )
Circulatory system—
including heart, blood vessels, etc.
61

62

The Development of the Vertebrate Embryo

Most of the research carried out so far on gastrulation has been de-
scriptive, consisting of efforts to follow the patterns of the cell move-
ments, to trace the origins of the three basic tissue layers back into the
blastula and even to the fertilized egg, and to account for the tissues and
organs that are produced in the later embryonic stages. Now, biologists
can begin to ask questions at the mechanistic level. For example, what is
the motive force for gastrular movements? That is, are the cells pushed
from behind or pulled from in front, or does each cell move as an individ-
ual at the same rate as its neighbors? Why is it that the cells move to
specific areas of the embryo? What motivates somite cells to plaster them-
selves against ectoderm and endoderm respectively? By what mechanism
do endoderm cells move up and around so as to produce a tube-like gut?
Are these simply random movements that are channeled into specific pat-
terns by the shape of the embryo, or are the cells attracted or repelled by
specific chemical stimuli and then move toward or away from the places
where these substances are produced?

As you can see, many problems of biological interest involving
gastrulation still remain unsolved and are open to investigation.

Organ Formation

Toward the end of gastrulation, ectodermal cells immediately in
front of the blastopore (or in front of the primitive streak of the chick
and mammal) begin to divide rapidly. The resultant crowding forces
some cells beneath the surface, and in this manner a thick plate of ecto-
derm appears. This is the neural plate. The wave of cell division proceeds
anteriorly and the neural plate finally extends along the entire dorsal line
of the embryo. Now the edges of the plate become elevated into folds
and the center of the plate is depressed. This produces a groove which
steadily deepens as the neural folds come together along the midline.
Finally the upper margins of the folds touch and fuse and the groove is
now formed into a tube, the neural tube, which is overlaid with a continu-
ous layer of ectoderm. Figure 31 shows these foldings in cross section, and
Fig. 32 shows the external appearance. As described below, the neural
tube is the rudiment of the central nervous system, including the brain
and the spinal cord. The embryo has reached the neurula stage.

While the neural system takes shape, other parts change as well.
Externally, the embryo elongates and becomes sculptured into head and
trunk regions. Limb buds, tail buds, and gill slits appear. Internally, the
mesoderm splits into notochord and somites; the endoderm takes a long
trough-like shape and rolls up into a tube, the primitive gut, which is open
at the rear through the anus and later at the front through the mouth.

Pressure caused by Continued thickening
thickening of neural plate forces greater folding

Neural
crest cells

Fig. 31. Neural tube formation.

Fig. 32. Changes in the external form of the amphibian embryo, from the
appearance of the neural fold to the completion of the neural tube.

DEVELOPMENT OF THE CENTRAL NERVOUS SYSTEM

Originally the neural tube is wider at the front end of the embryo.
This width is enhanced by local swellings that produce three distinct
bulges. These are the forebrain, midbrain, and hindbrain. Now the fore-
brain bulges laterally and two cup-like rudiments appear. Simultaneously
the surface ectoderm (the epidermis ) folds inward to meet the optic cups.
Folds are pinched off and ultimately develop into eye lenses. The ears
and nostrils also appear at first as epidermal infoldings. In the meantime
the spinal cord undergoes considerable growth and cell division. Meso-
dermal cells migrate toward the cord and aggregate around it and the un-
derlying notochord. They transform into cartilage and ultimately into bone
tissue, giving rise to a hollow vertebral column with associated ribs, etc.

The spinal-cord cells are at first large and rounded. They now as-
sume typical neuron shapes, spinning out long fibers. The cells in the
ventral part become motor neurons, and their fibers penetrate peripheral

63

es OT a>

WE
Na primordium

Forebrain
\ Lens primordium
G ‘al
( Optic cup
Midbrain

Hindbrain

Ear primordium Fig. 33. The development
of the brain and associated
sense organs (after Wad-
dington).

organs and tissues. Cells that had previously been crowded out of the
neural folds (the neural crest cells in Fig. 31) migrate down toward the
cord and aggregate into cell masses called ganglia. These cells become
sensory neurons and send dendrites to connect up with the dorsal part
of the spinal cord and axons into surrounding tissues. In this manner the
complete sensory-motor network is constructed.

The growth of the nerve fibers and their penetration into peripheral
tissues is a fascinating subject. The process has been studied: (a) in the
normally developing embryo, (b) by cutting functional nerves in order to
follow the regeneration of a fiber from the old stump, and (c) by separat-
ing embryonic nerve cells and growing them in an artificial medium (usu-
ally on a blood clot surrounded by blood serum and other constituents
found by experience to be delectable to a nerve cell). The presumptive
nerve cell body appears to send out an amoeboid process; the nucleus
remains within the cell body. Protoplasm flows into the process causing
it to move ahead while still connected to the cell body by a thin proto-
plasmic strand. This is the fiber. Special cells called Schwann cells then
flatten out and wrap themselves around the fiber in much the same way
that insulation is wrapped around an electrical conductor.

The first fibers to penetrate into surrounding tissues are called
pioneer fibers and these are followed by others which apply themselves
to the pioneer and form a cable. Many problems involving the mechanism
of nerve growth remain unsolved. For example, how does a fiber know
to what specific organ or tissue it must go, and how does it know when it
has gotten there? How do the follower fibers duplicate the precise route
of the pioneer fiber? Why do only fibers of similar function collect into
cables? What is the control system which ensures that certain tissues will
receive many sensory and motor fibers while others will be innervated
only sparsely?

64

The Development of the Vertebrate Embryo

DEVELOPMENT OF THE DIGESTIVE AND RESPIRATORY SYSTEM

At first the gut is a simple tube stretching from mouth to anus. At
the anterior end, five folds appear on each side which penetrate and
finally pierce the mesoderm and make contact and fuse with the outer
ectoderm. Slits appear where endoderm and ectoderm meet. These are
the gill slits. In fish they remain as functional organs, but in higher verte-
brates they disappear and the component tissues are transformed into
other organs. The lungs also originate from the anterior gut, first as a
simple outpocketing which soon branches into two bag-like structures.

Similar pocketings, extensions, and local swellings in the posterior
portion of the gut result in the formation of stomach, intestines, liver, etc.

DEVELOPMENT OF MESODERMAL STRUCTURES

By the end of gastrulation, the mesoderm makes up the bulk of em-
bryonic tissue. The notochord is the first mesodermal organ to appear,
and the remainder of the mesoderm is spread out as a thin sheet lying
between the ectoderm and endoderm. This sheet breaks up into longitu-
dinal strips. The thick strips lying on both sides of the notochord become

Fig. 34. Advanced em-
bryos: (A) the chick, (B)
the salamander, and (C)
the human.

65

66

The Development of the Vertebrate Embryo

segmented into blocks of tissue called somites. The somite cells migrate
toward the notochord and the spinal cord and form the vertebrae (as
described previously ) and also skeletal muscles. They also spread upward
to underlie the outer ectoderm and become part of the skin and inward
to become the outer covering of internal organs. Portions of the mesoderm
lateral to the somites pinch off as longitudinal strips of tissue and give rise
to the urogenital tract, including kidneys, gonads, etc. The heart and
circulatory system also originate from the non-somite mesoderm.

DEVELPOMENT OF LIMBS

The limbs first appear as slight external swellings (limb buds) which
rapidly elongate. The bud is simply a mass of loose mesodermal tissue
capped by an ectodermal cover. As the limb elongates, the mesodermal
cells grow rapidly in number and come together into a series of tight ag-
gregates corresponding to the skeletal elements of the adult limb. They
then are transformed into cartilage and finally into bone tissue. The re-
maining mesoderm forms muscles and blood vessels. Meanwhile, the
external sculpturing of the limb, joints, digits, etc. is accomplished.

Biochemical Embryology

We are accustomed to the fact that the specialized cells of a multi-
cellular organism have vastly different structures and functions. I have
mentioned before, but cannot stress too greatly, the fact that these are
simply outward manifestations of differences in metabolism, in the spectra
of chemical reactions which in turn reflect what enzymes and enzyme
concentrations these cells possess. Therefore, merely to describe the ac-
quisition of new structure by a developing embryo would be incomplete
without a concomitant catalog of biochemical changes.

As a result of biochemical research over the past thirty years, a mul-
titude of enzymes has been discovered, purified, and characterized. The
reactions that they catalyze have been studied in the test tube to the point
where we can specify the reacting molecules, the products, and the chem-
ical mechanisms by which reagents are transformed into products. These
reactions, each representing a relatively minor chemical alteration, have
been integrated into comprehensive reaction chains and cycles that ex-
plain the broad pathways of breakdown and synthesis of cell components
and of energy generation, transport, and utilization. This in turn has led
to a rational basis for understanding the nutritional requirements of or-
ganisms (i.e., the starting materials needed for synthesis and energy gen-
eration). In addition, the chemistry of the major cell components, pro-

The Development of the Vertebrate Embryo

teins, nucleic acids, polysaccharides, etc., has been elucidated, and the
relationships between the structure of cell organelles and their biochemi-
cal functions have begun to emerge.

Until recently, however, most biochemical studies have been done
with bacteria harvested after they had stopped growing or with adult
animal tissues and cells (liver or muscle slices, etc. ). Consequently, they
could tell us only about the biochemical machinery of a cell at one instant
in time, so to speak. But with our present biochemical understanding, it is
now possible to examine how the biochemical apparatus changes as the
organism proceeds upon its developmental course. At this early stage,
some of the directions taken by students of biochemical embryology are
outlined below:

1. Ghemical characterization of the egg. For example, we can ask:
do the components of an egg differ in kind or quantity from those of an
ordinary cell? Do the organelles (nucleus, mitochondria, ergastoplasm,
etc.) have the same chemical composition and biochemical activities as
they do in ordinary cells? What are the components of yolk? How are they
utilized?

2. Biochemistry of spermatozoa. What are the energy requirements
for sperm locomotion? What substrate is used (the sugar, fructose)? How
(via the glycolytic cycle, as explained in the monograph on cell physi-
ology, and therefore much like other cells)? A very interesting question is
how the sperm midpiece synthesizes the protein fibers that make up the
spindle apparatus for the first cleavage.

3. Cleavage. A great deal of work has already been done, partic-
ularly with the sea-urchin blastula. As mentioned previously, the egg starts
out with very disproportionate amounts of nuclear and cytoplasmic ma-
terials. As cleavage proceeds, the ratio of the two rises to a normal bal-
ance. We want to know about the chemical details of the shift and of the
controlling mechanisms. We want to know why the first cleavages are
synchronous and why later on some parts of the blastula cleave faster
than others.

4. Nutrition of the embryo. For example, early chick embryos have
been separated from their yolk supply and placed on agar medium con-
taining amino acids, vitamins, mineral salts, and glucose as an energy
source. The embryos developed in normal fashion under these conditions
and formed a full complement of organs. We can ask: what are the mini-
mum nutritional requirements for normal embryonic development? What
are the biochemical pathways of energy generation and how is the energy
employed for cellular differentiation, morphogenesis, and growth? If we
omit a specific essential nutrient, does all embryogenesis cease or are only
certain organs affected?

67

68

The Development of the Vertebrate Embryo

~

5. Enzymic changes during development. What enzymic changes
accompany the transformation of unspecialized cells into an organized
tissue or organ, i.e., which enzymes appear, which disappear, which in-
crease or decrease in concentration, what are the control mechanisms?
The cataloging of enzymes and enzyme concentrations is now a routine
procedure and can be accomplished with ease and precision.

6. The appearance of tissue-specific substances during embryogen-
esis. For example, we can follow the development of the heart in the early
embryo by looking for the presence of heart-muscle myosin, a contractile
protein unique to this muscle tissue. How to distinguish between heart
myosin and the myosins of other muscles? We can purify the heart myosin
and inject it into rabbits. Because chick heart myosin is a foreign sub-
stance to the rabbit, the animal can make antibodies that will react
specifically with chick heart myosin. Thus the rabbit serum containing
these antibodies can be employed as a specific indicator of the contractile
protein, and we can answer questions such as at what time the myosin
appears and from where, how much, and in what cells, at a stage long
before the actual heart is evident as a discrete organ.

You will notice that the above categories are heavily punctuated by
question marks. This is as it should be, for chemical embryology is in its
infancy and not nearly at the level of sophistication that one would like.
Here, perhaps more than any other, is an area open to fruitful investiga-
tion by physics- and chemistry-minded young students.

By fessor Curt Stern, one of the fathers of mod-

Cellular
Differentiation

ern genetics, in his article! entitled “Two
or Three Bristles,” has a superb discus-
sion of cellular differentiation that is de-
signed to be read by scientists who are
not biologists; it can easily be understood
and will be greatly appreciated by seri-
ous beginning students of biology.

To illustrate the problems inherent
in cellular differentiation, Dr. Stern de-
scribes the formation of bristles upon the
abdomen of the fruit fly:

In some regions there arise short or
long outgrowths—the bristles—strong and
wide at the base and gently tapering to
a fine point. Narrow grooves, as in fluted
columns with a slightly baroque twist,
extend along their lengths. A short stalk
fits each bristle into a round socket within
the body armor so that the bristle can be
moved within this articulation . . . The
bristles are tiny sense organs, perhaps
sensitive to the fluctuations of air pressure
when the fly is in flight.

Dr. Stern goes on to describe the
cellular structure of the bristle organ. It
consists of three cells: the bristle cell it-
self, which secretes the tapered out-
growth; the socket cell that secretes a
socket-like ring of hard chitin into which
the base of the bristle fits; and, below
these two, a sensory nerve cell that is
linked to the bristle by a short nerve
fiber and whose other long nerve fiber
connects up with the central nervous sys-
tem, thereby communicating to it stimuli
felt by the bristle.

1 Published in American Scientist, April 1,
1954.
69

70

Cellular Differentiation

The three bristle organ cells come from a single ancestor. At first, the
abdominal epidermis is simply a continuous sheet of many identical cells.
At a fixed stage in the development of the young adult from the immature
larva, single cells within the sheet grow to giant size. Division occurs and
one of the daughter cells is transformed into a sensory nerve cell. The
other daughter divides once more and one of these becomes the bristle-
forming cell, the other the socket-forming cell. See Fig. 35 for the lineage.

It should be noted that a fixed number of giant cells appear in the
abdominal epidermis and always at specific points so as to yield a precise
pattern. There are mutant fruit flies, however, that display different num-
bers and patterns of bristles, and as one would expect in these organisms,
the number and positions of the giant cells differ from the norm. In addi-
tion, some mutants produce bristles of different shape and size or produce
none at all. These abnormalities, too, can be traced back to specific events
in the early development of the fly. For example, in one of the types that
produce no bristles, the giant cells arise at the right time and place and

Fig. 35. Development of the bristle organ (adapted from C. Stern).

|. Giant cell appears 2. Giant cell divides 3. Divisions are complete, yielding
pre-nerve cell below and
parent of socket
and bristle cells above

4. Upper cell proceeds through mitosis 5. Division of upper cell yields
the bristle and socket cells

Socket LE Bristle

, Cuticle

ae aD, = pity Bristle cell
Socket cell ioe {/
Fiber leading to
central nervous system

Fiber leading to bristle

Nerve cell

6. A semidiagrammatic drawing of the functional bristle apparatus

- Primitive =
Parenchyma cell spongioblast Medulloblast neuroblast Epithelial cell
6
Ependymal Eas | e O s B®,
spongioblast S Unipolar s S
ioblast
| | spengionlee Oligodendroglia

r-,

Astrocyte

Fig. 36. The main cell types that arise from neural epithelial tissue (after
P. A. Weiss).

each divides into two. One of the daughters as usual becomes a nerve cell
and the other divides as it should. But instead of yielding a bristle and a
socket cell, two socket cells appear!

This work raises many questions. What genetic and biochemical
processes cause one cell among many suddenly to become a giant? Why
does it appear here and not there? Why, having produced three daughter
cells, does cell division stop? What causes three cells coming from the
same parentage to be transformed into totally different individuals?

Cellular differentiation, as we see it in bristle formation, occurs in
every developmental system, whether it be slime mold, coelenterate, or
human embryo. Each stage of development is accompanied by the appear-
ance of new cell types which play a direct role in the formation of the
adult structure. Some of these cells, like the giant bristle cell in the fruit fly,
perform a specific act and then do one of three things: die, divide, or
simply become unimportant for further development, in which case they
can be ignored. Others, like the nerve cell in the bristle organ, persist and
play a role in the subsequent functioning of the adult. Figure 36 sum-
marizes a more complicated series of cellular differentiation in the verte-
brate embryo. All the cell types shown arise in the neural tube in the
region of the brain. They are descended from ectodermal cells that in-

71

72

Cellular Differentiation

habited the dorsal area of the embryo after gastrulation. Only those cells
that remain in the inner core of the neural tube continue to reproduce
actively and to resemble their parents. Those cells that are pushed toward
the periphery cease dividing and are transformed into the types illustrated
in Fig. 36. It should be realized that the drastic morphological changes
shown are reflections of equally drastic changes in the metabolic capabil-
ities of these cells.

The extent to which differentiated cells can differ from one another
can be appreciated from current work on the reaggregation of tissue cells.
Cartilage tissue was cut from a chick embryo and the cells were dispersed
by treatment with an enzyme that destroyed the material cementing them
together. A suspension of the loose cells in nutrient broth was then
pipetted into a glass chamber. The cells promptly reaggregated and
became cemented together so as to construct a tissue of uniform texture,
a perfectly normal cartilage tissue. When dispersed kidney cells and
cartilage cells from the chick were intermixed and pipetted into the cham-
ber, the two cell types ignored each other. They formed separate ag-
gregates, the one producing something approaching normal kidney tissue
and the other apparently normal cartilage. In other words, the kidney and
cartilage cells recognized their mutual differences and refused to become
cemented one to the other in a mixed tissue. Now, dispersed cartilage
cells from chick and mouse embryos were mixed. The two kinds of cells
could be distinguished by size and nuclear structure. The cells came to-
gether and were cemented into a single cartilage tissue in which chick
and mouse cells were homogeneously interspersed. Mouse kidney and
chick cartilage cells ignored each other, as did the homologous chick mix-
tures. Two conclusions are permissible:

1. With respect to at least one criterion (the ability to stick together
when they meet and so form a stable aggregate and later a homogeneous
tissue ), chick cartilage cells are very much like mouse cartilage cells and
very unlike chick kidney cells. Thus, in this instance, embryological differ-
ences within the same animal can transcend evolutionary differences be-
tween two animals as diverse as a bird and a mammal.

2. The ability of two cells to be cemented together is a very specific
process. While the mechanisms are still unknown, apparently the same
ones are used regardless of species (otherwise mouse and chick tissues
could not stick together ).

The Genetic Basis of Cellular Differentiation

When we say that parent cells give rise to progeny that display a
wide difference in structural and metabolic properties, it is the ears of the

Cellular Differentiation

geneticist that perk up most sharply. For the possibility arises that the
genetic endowment of the progeny has been altered. In actuality, cellular
differentiation can be explained in two ways from the geneticists’ view-
point. To understand the explanations, it is necessary to review the mod-
ern concept of heredity.

The genetic endowment of a cell is simply a collection of macro-
molecules that contains a set of instructions for making the remainder of
the cellular constituents. The instructions are provided in the form of a
code. The separate letters of the code are represented by the small build-
ing blocks of which the macromolecules are composed. The order in which
the building blocks are strung together determines the meaning of the
built-in message. A strand of deoxyribose nucleic acid (DNA) exemplifies
macromolecules of this type (Fig. 37). Its backbone is a chain of alternat-
ing sugar (deoxyribose) and phosphate molecules. Attached to each
sugar is one of four molecules: Adenine (A), Guanine (G), Thymine

(T), or Cytosine (C). Thus the se-

Fig. 37. The structure of quence, ATTCG ... , would have
Bape ane fostiersen a meaning different from TATCG.

. . . It is these sequences, biologists
think, that must constitute the mes-
sage. The nature of the code is one
Wes of the most exciting problems in
present-day biology, and we all
await with impatience the Rosetta
stone that will unravel the genetic
language.

Most of the genetic informa-
tion is in the nucleus, borne by the
chromosomes in the form of genes
or, if you will, separate sentences of
the message. But it is equally clear
that the cytoplasm also contains
macromolecules bearing informa-
tion not present in the nucleus (see
Chapter 2).

How is the genetic informa-
tion employed in making the rest of
the cellular constituents? The pres-
ent view, supported by strong but
not conclusive evidence, is that the
separate genetic sentences built into
genes or extra-nuclear elements de-
termine the order of the amino acids

73

74

Cellular Differentiation

in corresponding protein molecules. (A protein of molecular weight 10,000
contains about 75 amino acids linked together in chains. If a chain were
composed of a series, alanine-glycine-tryptophane-alanine-valine, it would
have unique properties directly attributable to the particular order of the
amino acids.) In other words, the gene controlling the hydrolysis of the
sugar, lactose, determines the order of amino acids in a particular protein.
The unique amino acid sequence permits this protein to act as an enzyme,
to combine specifically with lactose and to split it. Other genetic deter-
minants guide the synthesis of still other enzymes. Ultimately, the re-
mainder of the constituents and products of a cell arise through the action
of these enzymes.

To recapitulate, then, the genetic material of a cell has two jobs to
perform:

1. Each coded macromolecule must guide the synthesis of an exact
copy so that when the cell divides each daughter will have a complete set
of genetic instructions.

2. Each coded macromolecule must guide the synthesis of a specific
enzyme or enzymes.

Genetic information provides the cell with the potential for making
a variety of cell constituents and for performing many functions. How-
ever, the fact that a cell is genetically equipped to carry out a particular
activity does not guarantee that it will do so, for the external environment
in which it lives also tells the cell what it can and cannot do. Three ex-
amples are provided to show what we mean by this statement:

a. Euglena is a protozoan-like organism that is capable of photo-
synthesis and contains about 10 chloroplasts per cell. When grown in the
dark, where it generates energy not by photosynthesis but by oxidation of
organic nutrients, the chloroplasts disappear. The instructions for making
chloroplasts are still intact, but whether or not the cell does make them
is decided by the light; when Euglena is once again exposed to light,
chloroplasts are again formed.

b. The bacterium, Escherichia coli, can synthesize the amino acid,
arginine, from simple starting materials via a series of stepwise reactions.
It therefore does not require an external supply of arginine for growth.
When, however, arginine is supplied to the cell in high concentration, it
inhibits the formation of an enzyme, Ornithine transcarbamylase, which
catalyzes a step in the synthesis of arginine. The absence of the enzyme
causes the cell to stop making arginine, and it uses only the external
supply. Thus E. coli is told by its environment not to synthesize its own
arginine although it retains the genetic instructions that will enable it to

Cellular Differentiation

do so whenever the external supply of arginine is exhausted and it can
again synthesize the missing enzyme.

c. The lymphoid tissue of an animal exposed to diphtheria toxin will
form antibodies (proteins that specifically neutralize the toxin). Although
the genetic potential for making antibodies is ever present, only exposure
to the foreign toxin can trigger antibody synthesis.

In brief, then, what a cell can do is determined by its genetic endow-
ment, i.e., the instructions provided by its genetic material. The geneticist
terms this endowment the genotype. The cell’s actual activity is the result
of the interaction between the genetic potential of the cell and the environ-
ment in which the cell finds itself. What the cell does (i.e., its structure and
metabolic capacities ) is termed the phenotype.

Faced with the fact that during the development of a multicellular
organism, daughter cells can gain new capacities or lose old ones and so
become different both from their parents and from their sisters, we can
ascribe the change to one or both of two processes:

1. The daughter cell receives from the parent its genetic endowment
intact and unchanged. However, the environment in which it must live is
different from that which the parent cell faced. Consequently, it synthe-
sizes cell constituents that its parent did not and vice versa. The altered
metabolism leads to morphological differences as well.

2. The genetic endowment of the daughter cell is different from
that of the parent. Thus the act of cellular differentiation would involve
a change in the primary genetic material and the alteration would be
inherited by the progeny, if any, of the differentiated cell. The genetic
macromolecules being altered, the enzymes constructed under their direc-
tion would be altered in quantity or kind. In this manner, the altered
genotype becomes amplified into an altered phenotype.

This second process, though not given in chemical terms, was im-
plicitly contained in an early theory offered by A. Weissmann in 1900.
Weissmann proposed that, when a fertilized egg cleaved, daughter cells
did not receive identical sets of genetic determinants. Instead, these were
parceled out in a regular fashion depending on the location of the cell in
the embryo, so that different cells received different genetic endowments.
The difference in genetic content would account for cellular differentia-
tion. The parceling out according to location would insure normality of
form. Figure 38 is a summary of this scheme in which the original egg
cell contains all the genetic elements necessary to make a complete em-
bryo. After cleavage, its descendants not only contain different sets of
genetic elements, but the necessary cell types end up in the right places in

75

76

Cellular Differentiation

the embryo. Weissmann realized that the embryo would ultimately be-
come an adult animal, that it would produce eggs or sperm, and that these
would necessarily have to contain all the genetic elements to make the
next generation of embryos. He made provision for this by adding the
qualification that during cleavage a few cells would inherit a complete set
of genetic elements (see Fig. 38) and that these would become the
gametes of the future adult.

In order to test this hypothesis, biologists permitted egg cells to
cleave into 2, 4, 8, 16, or more cells and then removed some of them. If
all the cells had inherited a complete set of instructions for making an
embryo, the loss of a few cells would not be expected to matter. In con-
trast, if most of the cells received an incomplete set of instructions, the
removal of some cells would remove certain instructions that the other
cells lacked and an incomplete embryo would result. Experiments of this
kind have tended to group embryos into two categories. If we permit a
fertilized egg to cleave into two blastomeres and then separate them,
each will, it is true, produce a complete embryo. However, if the egg hap-
pens to be from a clam, starfish, sea urchin, or comb jelly, this capacity
disappears rapidly. That is, once the egg has cleaved into 8 or 16 or 32

Fig. 38. A schematic illustration of Weissmann's hypothesis (the segrega-
tion of genetic elements).

ABCDE a ABCDE ABCDE |. ———>

ABCDE
ABCDE

Germ cell

Cellular Differentiation

cells and some of these are removed, the remaining cells form an incom-
plete embryo, with specific parts missing according to which of the cells
were removed. (This is called mosaic development.) In contrast, the em-
bryos of frogs, salamanders, and chicks are more malleable. Portions of
the early frog embryo can be removed without reducing its capacity to
turn out a normal product. The remaining cells simply take over the func-
tions of the missing ones. (This is called regulative development.) But
even here, if we wait long enough and permit the embryo to develop far
enough, removal will result in incomplete development. We might con-
clude from the above that, during development, the cells remaining after
excision had lost the capacity to replace the missing contingent. But we
can equally well imagine that by the time the excision is made, the en-
vironmental conditions no longer permit the transformation of a cell from
one type to another.

In recent years an elegant set of experiments has shown that cell
nuclei taken from late embryonic stages are no longer equivalent to the
nucleus of the egg cell from which they were derived. Frog eggs were
enucleated; that is, the nucleus was removed with a microtool. It was re-
placed with a nucleus taken from a cell at the blastula or gastrula or
neurula stage of frog embryogenesis. In other words, the egg cytoplasm
was combined with a nucleus representative of cells that had already
become differentiated. This synthetic egg was then allowed to develop
in order to determine the degree of normality and completeness achieved.

When a blastula nucleus was added to the enucleated egg, a normal
embryo resulted. When nuclei from later stages were employed, the re-
sulting embryos were grossly abnormal. Most important, the kind of ab-
normality observed depended on the part of the embryo from which the
nucleus had been taken. Thus, an endoderm nucleus added to the enu-
cleated egg gave rise to an embryo whose structures, derived from endo-
derm, were normal, but whose other structures were abnormal. A meso-
derm nucleus yielded an embryo whose mesodermal development was
normal, but whose other parts were abnormal. For reasons we have not
space to deal with here, these experiments and the ones mentioned earlier
do not conclusively demonstrate that a change takes place in the genetic
elements of cells undergoing differentiation, but they certainly do not
argue against this thesis.

Conclusion
We can now define cellular differentiation in a general way. We

know that the development of an organized multicellular structure always
involves the appearance of new cell types, different from their parents

77

78 Cellular Differentiation

A. Procedure:
Nucleus from

Egg nucleus Oe Needle Egg nucleus embryonic cell

|. Needle is inserted 2. Egg nucleus is pushed 3. The nucleus of an 4. The "synthetic egg’,
into the egg outside and degenerates embryonic cell is consisting of the original
inserted by egg cytoplasm and the
a micropipette transplanted nucleus

B. Results of the development of the synthetic eggs:

|. Enucleated egg + nucleus from a ————» __ normal embryo
blastomere
2. Enucleated egg + nucleus from an ———» normal embryo
early gastrula cell
3. Enucleated egg + nucleus of an endoderm —» an embryo with
cell (taken from normal endodermal
the neurula stage) tissues (gut, etc.) but
with abnormal and
degenerate mesoderm
and ectoderm
4. Enucleated egg + nucleus of a mesoderm—» an embryo with
cell (taken from the normal mesodermal
late gastrula stage) tissues (notochord,
muscles, cartilage, etc.)
but abnormal endoderm
and ectoderm

Fig. 39. Transplantation of embryonic nuclei.

and from their sisters. If these new cell types can be shown to play a causal
role in the construction of the multicellular being and/or its subsequent
functioning, such cells are said to be differentiated, and the processes by
which they came to be different are collectively called cellular differentia-
tion.

The question arises, what kind of information do we have to provide
before we can be satisfied that a single act of cellular differentiation has
been adequately described and understood? The categories of necessary
information are outlined below.

I. Describe the new phenotype. It is not enough to say that the
differentiated cell differs from its parent or sister cells. We must show
how it differs. Which enzymes are present in the new cell type and are
absent or greatly diminished in the old, and vice versa? Which specific
structures in the cell have been altered? This is one of the points of de-

Cellular Differentiation

velopmental biology where cytology and cell physiology both enter.

2. Account for the function of the differentiated cell. As stated in
the definition, the new cell type is meaningful because it contributes in
some specific manner to the construction of the multicellular organism
(i.e., without the appearance of the new cell type, the normal develop-
ment of the multicellular structure or the normal functioning of the adult
derived from it would be impossible). We must show the specific role
played by the new cell type and how its altered metabolism and structure
control or trigger later developmental processes in the rest of the organism.

3. Account for the number of each type of cell. We must be able to
examine a very small sequence in the development of the multicellular
organism, pinpoint precisely the time at which a particular new cell type
appears, count the number of them in the cell population, and account
for the number that we see by studying the rate at which they appear and
the rate, if any, at which they disappear.

4. Determine the manner in which the new cell type arose from its
parent. Here we return to the question broached in an earlier section. We
must determine whether, in a specific act of cellular differentiation, the
new cell type possesses genetic instructions different from those of its
parent or whether, faced with a new environment, it does different things
from what its parent did but uses the same old set of instructions. Cell
genetics in recent years has provided ways of distinguishing between
these alternatives and also has provided methods to determine in each
case some of the specific mechanisms involved.

As you see, the description of even one step of cellular differentia-
tion is a tall order. At present no such step has been completely elucidated
in any organism and, in fact, even the most precise descriptions so far are
very primitive and not very quantitative. Consequently, the elucidation of
processes of cellular differentiation is one of the most exciting challenges
in modern biology and presents the hope that, by studying them, we can
uncover new mechanisms of cell variation.

79

W. have seen the complexities that attend the

Cell
Interactions
during Growth
and
Morphogenesis

construction of a multicellular organism,
whether it be slime mold fruiting body
or vertebrate embryo. Many new cell
types arise and must maintain fairly rigid
numerical proportions. Cells must mi-
grate to specific areas of the aggregate
and must be integrated into tissues and
organs. All these processes are underlain
by a bewildering array of biochemical
activities that must occur in strict chron-
ological order.

How does the system maintain a
balance of kind, number, place, and
time? Obviously, each subunit of the or-
ganism cannot be permitted to develop
autonomously, disregarding other sub-
units. Each, therefore, must receive in-
formation from other parts about what
these have been doing in the immediate
past and about what it may do and must
not do in the immediate future. We will
now concern ourselves with the exchange
between cells of chemical messengers
that trigger, assist, control, and inhibit
developmental events.

Many Cells Do What Few Cannot Do

The simplest example of this kind
of interaction is seen in cultures of micro-
organisms or dispersed animal or plant
cells growing in a liquid nutrient me-
dium, a topic we will take up in detail
in the next chapter and briefly describe
here. Such cells, when inoculated into
fresh medium, experience a lag of vari-

80

Cell Interactions during Growth and Morphogenesis

able duration before they can begin to reproduce actively, a lag that
results mainly from the fact that each cell must accumulate threshold
concentrations of key compounds (vitamins, amino acids, etc.) before
cell division can commence. Yet as fast as the cell produces these com-
pounds, they pour out into the medium by diffusion through the cell mem-
brane. As the external concentrations of these key substances rise, the
rate of diffusion out of the cell decreases, enabling the internal concentra-
tions to increase and finally reach the necessary thresholds. The time
needed to saturate the external environment with the critical substances
and so permit internal accumulation will depend on how fast the sub-
stances can be produced. Obviously, if each cell produces them at a
constant rate, two cells will produce twice as much per unit time as will
one and therefore do the job in half the time, i.e., many cells inoculated
into a given volume of fresh medium can begin growing very quickly,
whereas a few cells in the same medium would take a much longer time
or might never get started at all.

Cooperative interactions also occur between embryonic cells. For
example, if we cut out a large piece of dorsal ectoderm from a chick or
mouse embryo and cultivate it in a dish with appropriate nutrients, the
tissue will yield nerve cells just as it would if left in the embryo. If, how-
ever, the single large piece is cut up into many small pieces and each is
cultivated separately, no nerve cells appear. The mere act of cutting does
not affect the result, because if the pieces are not separated but are cul-
tivated together in a compact mass, they produce nerve cells as efficiently
as before. Thus, many cells can do what few cannot, in this case become
transformed into specialized nerve cells.

Inductive Interactions

We have already dealt with one very fine example of induction—
namely, the production of male and female sex organs in the water mold,
Achlya (see Chapter 3). The properties of this system reflect perfectly
the general features of all inductive morphogeneses.

1. The male sex organelle will not develop when the plant is grown
in isolation, but only when induced to do so by the presence of the female
plant. The development of the female sex organelle is similarly dependent
on the presence of the male (i.e., one cell group induces another to de-
velop along a specific path).

2. The induced cells must be at the correct stage of development in
order to be receptive to the inductive signal. For example, after the female
plant has produced the large sacs, oospheres are induced to form in re-

81

82

Cell Interactions during Growth and Morphogenesis

sponse to an inductive stimulus from the male. If too long a time elapses
before receiving the signal, the female ceases to be responsive and will
no longer produce oospheres.

3. The inducing cells must also be at the correct developmental
stage. For example, only after the gnarled, branched shoots of the male
Achlya have grown out toward the female sac and plastered themselves
around it can the male produce the hormone which induces oosphere for-
mation.

THE EMBRYONIC ORGANIZER

The most complex inductive mechanisms are found in vertebrate
embryos; one example is the embryonic organizer demonstrated by Dr. H.
Spemann, one of the founders of modern embryology. Spemann was in-
terested in the problem of whether fragments of an embryo could contain
or produce all the cell types necessary for the construction of a complete,
normal adult (see Chapter 6). He cut salamander embryos in half at
various stages of development and found that, before gastrulation, both
halves yielded normal adults but that after gastrulation both developed
aberrantly. To fix the point at which the capacity for normal development
was lost, Spemann cut embryos in half at various times during gastrula-
tion and, to his surprise, one of the halves invariably gave rise to a com-
plete, normal embryo, while the other yielded a degenerate, amorphous
cell mass. Upon closer examination, he found that the half yielding a
normal product was always the one that contained the dorsal lip of the
blastopore! and that the half that degenerated always lacked the dorsal
lip. It appeared from these results that: (1) cells from the dorsal lip have
the power to organize or initiate the construction of the embryo, and (2)
no other cells can do this (if the dorsal lip is absent, therefore, embryonic
organization does not occur).

To confirm that dorsal-lip cells (called prospective chorda-meso-
derm) were the inducers of the embryonic organization, Spemann cut
out pieces of this tissue and implanted them in the ventral sides of other
gastrulas (in contrast to the position that chorda-mesoderm normally
occupies, i.e., under the dorsal ectoderm). Two results were observed:

1. At the site of implantation, a small but perfect embryo developed
as a sort of Siamese twin of the host embryo. Brain, spinal cord, and asso-
ciated organs were present.

under that part of the ectoderm that will later give rise to the central nervous system.
They become the central part of the mesoderm and give rise to notochord and somites,

Cell Interactions during Growth and Morphogenesis

2. The implanted chorda-mesoderm went ahead and developed pre-
cisely as it would have had it been in its normal position in the embryo;
that is, it constructed a notochord and somites. In contrast, any other part
of the gastrula, when implanted in this manner, did not develop as it
would have if it had not been moved, but instead developed in accordance
with its new locale.

The second result showed that indeed there was something special
about dorsal-lip tissue. The first result revealed that dorsal-lip cells could
induce overlying ectoderm to form brain, nerve cord, and associated
parts. When that part of the dorsal-lip tissue that normally underlies the
brain was implanted, it induced brain development very well and tail de-
velopment very poorly, and when posterior chorda-mesoderm was im-
planted, it induced tail very well and brain very poorly. Thus the chorda-
mesoderm is a specific inducer.

The main question, of course, is what compound or compounds does
the chorda-mesoderm supply to the overlying ectoderm to produce the
organization of the ectoderm tissue? Unfortunately, any of a wide variety
of substances from many sources has been found to do this, so wide in
fact that the induction appears to have very little specificity. It is almost
as if the ectoderm at this stage of development is a gun primed to shoot
in a fixed direction and needing only a touch on the trigger to set it off.
This “sensitivity” raises two other very interesting questions.

1. Although it is true that the embryologist can make ectoderm
organize itself by treating it with a wide variety of agents, in the actual
embryo only dorsal-lip cells can do this. What, then, passes between the
chorda-mesoderm and the overlying ectoderm? To answer this question,
we label the constituents of dorsal-lip cells with radioactive carbon or
sulfur or phosphorus and thus determine what materials pass from the
“hot” mesodermal cells to the “cold” ectodermal cells.

2. If it is true that the embryologist can cause ectoderm to organize
itself by applying many agents, why is it in the actual embryo that only
one part of the ectoderm normally becomes organized into brain and
spinal cord? Why aren’t secondary embryos caused to pop out all over the
place, having been induced by any of the multitude of materials that
normally leak out of cells? Here we return to the problem of the morpho-
genetic field discussed in Chapter 4.

SECONDARY INDUCERS IN EMBRYOS

As we just described, dorsal-lip tissue induces overlying ectoderm to
transform into brain and nerve cord, while it itself forms notochord and

83

84

Anterior NUCH Notochord Somites
endoderm mesoderm

Teeth

Gills

Mouth

Cell Interactions during Growth and Morphogenesis

somites. From this point on, many contiguous tissues provide each other
with inductive signals that trigger the formation of additional organs. In
general, the proof that such inductions operate has fallen into two cate-
gories: investigators have shown (a) that if tissue A is removed or physi-
cally separated from tissue B, tissue B will not construct a specific organ
as it usually does; and (b) that if tissue A is moved to a neighboring spot
occupied by cells closely allied to B, the organ in question will form at the
new site occupied by tissue A and not at the old.

Figure 40 is a schematic and partial summary of the manifold induc-
tive relationships uncovered in this manner. In some cases, these induc-
tions have, by and large, proved to be much more specific than that
supplied by the embryonic organizer. However, little of the rigorous
biochemistry needed to shed light on them has been performed so far.

Fig. 40. Inductive relationships between different parts of the developing
embryo (after Holtfreter). Arrows point from the inducing tissues to the
induced organ that ultimately appears. Bear in mind that this array of in-
ductions is far from complete.

Lateral
mesoderm

, Central
Forebrain nerve chord
Digestive tract

Neural crest cells
Dorsal fin
Ear

Hind-brain

Cell Interactions during Growth and Morphogenesis 85

Synergistic Inductions

Synergistic inductions are those in which two different tissues mu-
tually interact so that each causes the other to develop in a way that it
would not do when alone. For example, the developing kidney contains
many small S-shaped secretory tubules connected to a fan-like network
of branched collecting tubes which serve to transport material collected
by the secretory tubules into the ureter to be voided as urine. Figure 41
is a schematic diagram of kidney-tubule development. The tubes and
tubules originate from two different rudimentary tissues, starting at about
the eleventh day of embryonic life; these are the nephrogenic cord, a
loose collection of mesodermal cells, and the ureteric bud, a compact
tubular tissue also derived from mesoderm. The development of the secre-
tory tubules from the former and the collecting tubes from the latter has
been found to depend on the intimate association of both. Removal of
either from the embryo stops the development of the other.

It is possible to separate the ureteric bud and the associated nephro-
genic cord from a mouse embryo and cultivate the tissues on a blood clot
overlain with a nutrient solution. Under these conditions, tubes and
tubules develop as they do in the embryo. If the two tissues are separated
from each other and cultivated, tubes and tubules never develop. Many
unsuccessful attempts have been made to extract material from either
tissue, that, when added to a culture of the other, would make it develop
in normal fashion. If, however, the two tissues are separated by a very
thin membrane with tiny holes, the synergistic induction does occur. Al-
though the membrane is thick enough to prevent the two kinds of cells
from touching each other across the membrane, blobs of cytoplasm could
conceivably pass between the two. When opposed across thicker mem-
branes, synergistic development does not occur.

This type of induction, termed direct-contact induction, is a common
phenomenon in developmental systems. It requires the reacting tissues to

Fig. 41. Formation of kidney tubules (after Arey).

"9,

0D)

Cooawsanoaeen

COp/dlcfolobio)
NOLO

Collecting tubule

86

Cell Interactions during Growth and Morphogenesis

remain in intimate association for long periods of time, in contrast to
the “Achlya” type of induction, which occurs when the reacting tissues
are separated by a considerable distance. Many chemical explanations can
be imagined to account for induction by direct contact but, as in much
of what we have already described, the necessary biochemical analyses
are only just beginning.

Inhibitory Interactions

As we stressed in Chapter 5, all morphogenetic fields involve inhibi-
tion—i.e., cells in one part of the field develop in a certain manner and
simultaneously inhibit the surrounding cells from doing so. This is the
case in hydranth development in coelenterates, bud development in plants,
the construction of the head, heart, limbs, and eyes in vertebrate embryos,
etc. As we mentioned in Chapter 5, inhibition can conceivably exist at
two levels:

a. Where competition for a common store of materials in the en-
vironment enables only the cells that receive the greatest supply to de-
velop.

b. Where the production of material by one cell inhibits another.

Known Mechanisms of Cell Interaction

Space limits us to only a few examples of known mechanisms by
which one cell can influence the growth and development of another. We
have drawn these from experiments with microorganisms which, being
simpler to manipulate and easier to grow under defined conditions, permit
more rigorous analyses at present. Unfortunately, these experiments in-
volve only single celled organisms and thus their interactions can serve
merely as models for similar interactions among the cells of higher plants
and animals.

SELECTIVE INHIBITION DURING BACTERIAL GROWTH

The bacterium Brucella abortus causes aborting fever in cattle and
Brucellosis in man. When B. abortus is taken from the blood of a diseased
animal and grown on an agar medium, it forms a colony with a very even
outline and mucoid texture, because each cell in the colony is surrounded
by a polysaccharide capsule. This type of colony is called “smooth,” and
the constituent cells are called S-cells. During the growth of S-cells, rare

Cell Interactions during Growth and Morphogenesis

mutations occur that prevent the mutant and its offspring from synthe-
sizing a capsule. The mutants are called R-cells, and since the colonies
derived from them possess an uneven outline and wrinkled appearance,
they are called “rough.”

If we inoculate a flask of nutrient broth with S-cells, we find that
by the time growth has ceased, the population is no longer composed only
of S-cells but also contains a great majority of R-cells. Figure 42 sum-
marizes the results. The total population grows normally until the fourth
day of incubation, then it dips quickly, begins to grow again, and finally
reaches a stationary state. Until the fourth day, the population is made up
almost completely of S-type cells. After 4 days, these decline rapidly.
R-cells make their appearance coincidentally with the decline of S-cells, in-
crease rapidly, and by 20 days make up over 90 per cent of the population.

As the bacteria grow, intracellular substances leak into the broth;
one of these substances is the amino acid, alanine (CH3:CH*-NH2*COOH ).
The appearance of alanine coincides with the decline of the S-cells. The
S-cells are very sensitive to alanine and die in its presence, while R-cells
are resistant to alanine and can grow normally. Thus by the fourth day, the

Fig. 42. Selective inhibition during growth of Brucella abortus cells.

Total cells

108

S-cells
S
~ can
~S
~

~
~
=
/

107

~

Number of bacteria

10°

2 4 6 8 10 12 14 16 18 20 Days

87

Cell Interactions during Growth and Morphogenesis

S-cells have produced enough alanine to be poisoned by it. In the mean-
time, a few R-cells have arisen by mutation. Since the S-cells cannot grow,
the R-cells do and eventually dominate the population. The proportion
of R- and S-cells is controlled at any time by the levels of alanine.

The interaction between the two cell types, therefore, is not simply
the result of an exchange of materials between the two. The S-cells, by
acting upon themselves (i.e., committing suicide), permit the R-cells to
do something they ordinarily could not have done, namely grow. (Had
the S-cells not died out, there would have been no opportunity for the
R-cells to grow. )

TRANSFORMATION OF CELL TYPES DIPLOCOCCUS
PNEUMONIAE

The bacterium D. pneumoniae gives rise to mutant types that are
different in many ways from the parental stock. For example, we can
isolate mutants that are resistant to penicillin or streptomycin (when the
parent type is sensitive to these antibiotics), mutants that can degrade
sugars that the parent type is incapable of degrading, and mutants that
require various compounds for growth which the parent type can make
for itself.

Highly purified deoxyribose nucleic acid (DNA) has been pre-
pared from penicillin-resistant mutant cells. When the sensitive parental
type is exposed to this DNA, a large proportion of the cells are trans-
formed into the penicillin-resistant type, and what’s more, they pass on
this new capacity to their offspring. The DNA can only cause the exposed
cells to acquire capacities that are possessed by the cells from which the
DNA was obtained (i.e., DNA from penicillin-sensitive bacteria never
transforms the exposed cells to penicillin-resistant varieties ). In essence,
then, it is possible to extract genetic information from one cell and intro-
duce it into another. The macromolecule bearing this information can be
incorporated into the genetic apparatus of the treated cell and thus the
information is passed on to the progeny.

SYNERGISTIC GROWTH BY BIOCHEMICALLY DEFICIENT MUTANTS

We deal here indirectly with some of the most important genetic
experiments of our time, which were conducted upon the bread mold
Neurospora crassa. Among other things, they provided convincing proof
that genes act by controlling the synthesis of specific enzymes. Neurospora
crassa can be grown on a relatively simple medium containing glucose as
a source of carbon and energy, ammonium chloride as a source of nitrogen,
a few mineral salts, and two vitamins. Mutants were isolated, that, unlike

Cell Interactions during Growth and Morphogenesis

the parental type, had to be supplied with various compounds in order
to grow because they could not synthesize the compounds; some examples
are histidineless mutants (which cannot synthesize histidine and will not
grow unless supplied with it) and Thiamineless, Riboflavinless, and
Tryptophaneless mutants, and many others. These are called biochemi-
cally deficient mutants.

In all cases, the mutant lacks a single enzyme necessary for the
synthesis of a particular cell constituent. Amino acids, vitamins, etc., are
generally synthesized in stepwise fashion from a pool of common, simple
substances in a manner shown symbolically below:

gene 1 gene 2 gene 3 gene 4 gene 5

enzyme 1
enzyme 2
enzyme 3 <—
enzyme 4
enzyme 5

| pool |——> A ——— B ———> C ———> D ——> cel constituent

The absence of enzyme 2 would eliminate the reaction A —> B and
thus prevent the production of intermediates B, C, D, and the final prod-
uct. If gene 2 in the nucleus mutated to a non-functional form (i.e., one
unable to direct the synthesis of enzyme 2), the mutant cell would be
unable to synthesize the final product and would grow only when it was
supplied in the culture medium. Two predictions can be made for such
mutants and they turn out to be true. These predictions are:

1. That the mutant lacking enzyme 2 could still grow if supplied
with the final product, or with intermediates B, C, or D, since the cell can
convert these into the final product.

2. That intermediate A would accumulate in the mutant cell, since
the reaction A —-> B is blocked. Because these intermediates are gen-
erally of low molecular weight and are readily diffusible, they not only
accumulate inside the mutant cell but usually seep out of the cell into
the surrounding medium.

Synergistic growth of deficient mutants occurs because of this os-
motic property. As an example, we shall consider biochemically deficient
mutants of the bacterium Escherichia coli, which are all arginineless (un-
able to synthesize the essential amino acid, arginine). Arginine is nor-
mally synthesized via the following pathway:

pool of common 1 ) 3
substances ———— omithine ————— citrulline ————— arginine

89

90 Cell Interactions during Growth and Morphogenesis

Three kinds of mutants have been isolated:

Mutant 1 could grow if supplied with ornithine, citrulline, or ar-
ginine (i.e., the enzyme that catalyzes reaction 1 is miss-
ing).

Mutant 2 could grow if supplied with citrulline or arginine, but not
if supplied with ornithine. However, ornithine accumu-
lated in the cells and seeped out into the medium (i.e.,
the enzyme-catalyzing reaction 2 was missing ).

Mutant 3 could grow only if supplied with arginine, not with orni-
thine or citrulline. However, citrulline accumulated in the
cells and seeped out into the medium (i.e., the enzyme-
catalyzing reaction 3 was missing).

If, as shown in Fig. 43, the three mutant types are streaked on agar
containing a tiny amount of arginine, synergistic growth occurs. Mutant 3
grows very sparingly until the arginine in the medium is used up, and then
it stops. It continues, however, to generate energy by oxidation and to
carry out a wide variety of biochemical reactions, including the synthesis
of citrulline, which accumulates and seeps out into the medium. Mutant 2,
growing nearby, can convert the citrulline to arginine. Since mutant 3
makes a large quantity of citrulline, mutant 2 can grow very well. If the
mutant 2 cells do not lie next to mutant 3, they grow very sparingly, but
they still can metabolize and accumulate ornithine. Since mutant 1 can

Fig. 43. Synergistic growth
by three mutant types of
E. coli.

Cell Interactions during Growth and Morphogenesis

convert both citrulline or ornithine to the needed final product, it grows
very well if it is streaked next to either mutant 2 or mutant 3. In multi-
cellular organisms, the various tissues may well influence the growth of
neighboring cells in just this manner. There are numerous examples in
embryonic development of the effect of one organ or tissue upon the
growth of another.

Conclusion

All cells in nature interact with one another. Sister microbial cells
compete with each other for food and space, and sometimes exert direct
inhibitory effects on each other in order to survive the competition. They
can also assist each other by exchange of metabolites. Genetic information
can be transferred from one to another and produce new capacities in the
recipients. In multicellular forms, where the constituent cells are packed
together in a compact mass, opportunities for cell interaction are very
great and, since developmental activities must be timed perfectly, are
even more necessary than in single-celled organisms. The kinds of inter-
action can be classed as follows:

a. population-density effects—where many cells of the same kind co-
operate to do something that few together could not do.

b. inductive interactions.

c. synergistic inductions.

d. inhibitory interactions.

The vehicles of interaction seem to fall into these categories:

a. Interactions promoted by diffusible agents between cells sepa-
rated from one another. Such agents can be carried by simple diffusion or
transported through the circulatory system.

b. Direct-contact interactions between the effector and effectee cells.
Such systems probably require intimate contact because the material
transferred is very unstable and will not survive transport over great dis-
tances or because large amounts of cytoplasm are exchanged and several
substances must travel as a unit.

c. Contact interactions without exchange of material. The surface of
one cell reacts with that of another to create drastic alterations of the sur-
face, which are then reflected by secondary changes in the metabolism of

the cell.

91

H ow an organism grows and the processes, both in-

Growth
and
Form

ternal and external, that control and limit
growth are fundamental biological prob-
lems. A wide variety of living organisms
has served as objects of study. Much has
been learned by examining the growth
of populations of microorganisms—bac-
teria, protozoa, algae, and fungi. In re-
cent years, biologists have been able to
remove tissues of higher animals and
grow them outside the body (tissue cul-
ture). In some cases, it has been possible
to disaggregate such tissues and to cul-
tivate the cells as dispersed individuals
exactly as if they were microorganisms.
Even the cells of human beings (tumor
cells, kidney cells, etc.) can be so treated
and have been found to obey the same
rules of growth and nutrition as their
more primitive brethren. Intact higher
animals have been grown under con-
trolled conditions in which the natural
diet is replaced by mixtures of known
chemical compounds delivered in meas-
ured amounts. The rat is a favorite lab-
oratory animal for this purpose, but other
animals such as the fruit fly (Drosophila)
and the university undergraduate (vol-
untarily ) have been used.

The first lesson learned was that
the term “growth” has several meanings
and that an organism may grow in one
sense but not in another. Growth, under
one interpretation, is an increase in the
number of cells, which is found by count-
ing the total cell population or a meas-
ured sample from it. Growth can also be
described as an increase in protoplasmic

92

Growth and Form

material, which we find by determining the weight or volume changes in
an organism. Or we can pick out some constituent of protoplasm that is
always a constant proportion of the whole, such as the nitrogen content
or protein content. If either were to increase, we would know to what
extent the total protoplasmic mass had increased.

It should be noted that both cell number and protoplasmic content
do not always increase together. For instance, cell division can occur
without any increase in protoplasm; the result is a greater number of
smaller cells. Alternatively, protoplasm can be synthesized in the absence
of cell division, in which case the cells grow larger but not more numerous.
However, isolated growth of this kind can go on only under exceptional
conditions and for a relatively short time. Ultimately cell division must
cease without protoplasmic increase and vice versa.

Nutrition

The act of growth, when it involves an increase in protoplasm, re-
quires the synthesis of a wide variety of cell constituents—nuclei including
chromosomal material, centrioles, nucleoli, etc., mitochondria and other
cytoplasmic organelles, thousands of enzyme molecules, cell membranes
and other structural material. These require the synthesis of macromole-
cules such as proteins, nucleic acids, and polysaccharides in which many
subunits are linked together. The subunits themselves, amino acids,
sugars, fatty acids, vitamins, etc., must be synthesized from still simpler
compounds or must be assimilated from the environment.

Two basic needs must be met if these activities are to be accom-
plished—the need for energy and the need for raw materials. Cells obtain
energy by the oxidation of materials obtained from the environment.
Sugars are the prime fuels for this purpose, but many other oxidizable
compounds can be employed by one or another kind of cell. The energy
thus made available is employed for the synthetic reactions. At their
simplest levels, the raw materials for the synthesis of protoplasm are the
elements of which cells are composed. The major components are carbon,
hydrogen, oxygen, nitrogen, sulfur, phosphorus, magnesium, manganese,
iron, and traces of many other elements as well. The elements must, how-
ever, be supplied in usable form. For example, the amino acid, alanine,
CH;CH;—NH: COOH, is essential for the synthesis of proteins. No known
organism can synthesize alanine if it is merely supplied with elemental
carbon, hydrogen, oxygen, and nitrogen. But some cells can do so by
converting nitrogen from the air to ammonia and combining the ammonia
with pyruvic acid as follows:

NH, + 2H + CH,CO COOH —> CH;CHNH,:COOH + H,O

93

94

Growth and Form

Other cells cannot convert Nz to NHs3, but if given NH; and pyruvic acid
they can make alanine. Still other cells, which do not possess any of the
enzymes that catalyze these reactions, must be supplied with alanine itself
if they are to grow. The diversity of nutritional requirements results from
the inability of cells to synthesize one or another protoplasmic constitu-
ent.

The S-Shaped Curve of Growth

Figure 44 shows growth curves which, as you can see, are S-shaped.
The abscissa is time—minutes, days, hours, years, depending on the organ-
ism studied. The ordinate is growth and can describe the number of cells
in a bacterial culture, the number of human beings on earth, the size or
weight of a sunflower seedling or a rat, the size or weight of the heart or
the brain. In other words, the ordinate is a measure of the growth of a
population or a single organism or any of its parts and, when growing
under optimal conditions, all show precisely this sort of growth curve.
Many questions come to mind when we look at this curve. Why does
growth start? Why does it stop? Why is the curve S-shaped?

In this discussion of the factors affecting growth, we shall deal with

Fig. 44a. The growth curve of the human population of the world. The
circles indicate census totals, the solid line the mathematically fitted,
smooth, S-shaped curve (after Pearl and Gould, from Allee, et al., Prin-
ciples of Animal Ecology).

Upper asymptote = 2645.5

Population in millions

Lower asymptote =445.5

1650 1700 ‘50 1800 ‘50 1900 ‘50 2000 ‘50 2100

Years

Growth and Form 95

100

80

60

40

Percentage of adult weight

20
Hatching

Fig. 44b. Growth of the
chick during its embryonic
and post-hatching periods
(redrawn from Weiss and a ee 1s ee A
Kavanau). Days

Fig. 44c. Bacteria are introduced into fresh nutrient broth. At intervals, a
sample is abstracted to determine the number of bacteria per cubic cen-
timeter of broth. Three phases of the resultant growth curve are recognized:
I—lag phase; II—logarithmic phase; I||—stationary phase. If, now, bacteria
from the stationary phase are used to inoculate another flask of nutrient
broth, the growth pattern repeats itself.

100,000,000

50,000,000

Number of bacteria
per cubic centimeter of nutrient broth

0 24 48 72 24 48
Time in hours Time in hours

96

Growth and Form

a culture of bacteria because it is the easiest to understand, and conclu-
sions reached from studies of it are applicable to all organisms, plant and
animal alike.

Imagine that we introduce a few hundred bacterial cells into a
cotton-stoppered flask containing sterile, nutrient broth (called the “me-
dium”). Every hour or so we abstract one cubic centimeter of the broth
and determine the number of living bacterial cells. Figure 44c shows the
complete curve that would be obtained.

The curve is divided into three parts: I—the period called the lag
phase during which the cells prepare for growth; II—the period of actual
growth, called the exponential or logarithmic phase; I11—the period in
which growth ceases and the population enters the stationary phase. None
of these phases is of set duration. That is to say, the time a culture spends
in each phase depends on the particular species of bacteria and the condi-
tions under which they are grown.

LAG PHASE

The lag phase is a period of rapid protoplasmic growth and a period
of preparation for the cell division that is to come. Thus, it is a time when
the cells become larger but remain constant in number.

We may ask why cells must prepare for cell division. Why can they
not enter the exponential phase immediately? The answer is that a cell
in the exponential phase is somewhat like an assembly line operating full-
blast. Parts are fabricated on the line in consecutive stages or are received
from outside contractors and these are ultimately put together to yield
the finished product. The prime requirement for an efficient assembly line
is that all the operations mesh harmoniously. Supplies must arrive in the
right place, at the right time, and in the right amounts, and the operations
along the assembly line must proceed at equivalent speeds. If, then, we
start a culture with cells that are already in the exponential phase (i.e., the
“assembly lines” are already set up and operative) and supply them with
everything they need, the lag phase should be eliminated, and this is just
the result that is obtained. In contrast, if we start a culture with cells that
are not in the exponential phase, they require time to prepare for growth.
We will now take up the preparations that are necessary to generate the
exponential phase.

RESYNTHESIS OF ENZYME SYSTEMS REQUIRED FOR GROWTH. A cell that
has already passed the exponential phase is no longer faced with the prob-
lems of growing, only with those of survival; it must protect itself against
metabolic waste products and other toxic substances that may have built
up in the environment and must conserve whatever raw materials and
energy that it still may have left. In so doing, it may lose some enzymes
that are no longer needed and synthesize others that are required for its

Growth and Form

survival. If now such cells are exposed to a fresh nutrient medium in which
growth is once again possible and survival no longer a problem, they must
reverse the course of events mentioned above. But this involves extensive
protoplasmic reorganization and a further synthesis of enzymes and other
macromolecules. All this takes time; hence the lag phase.

THE SYNTHESIS OR ASSIMILATION OF RAW MATERIALS FOR GROWTH.
Each original cell must stock up on the chemical subunits (amino acids,
sugars, fatty acids, vitamins, etc.) that are needed to construct enough
protoplasm to make two cells. If the nutrient supply were rich and these
compounds already present in the environment, the job would be simple
and the lag phase correspondingly short. If the nutrient supply were poor,
the missing subunits would have to be synthesized from simpler materials
in the environment. The job, then, would be more difficult and the lag
phase correspondingly longer. The lag phase can also depend on how
many cells are actually present, for each is a little factory geared to the
production of the subunits, and the more factories there are, the faster
they can be synthesized and the shorter will be the preparatory period. In
the jargon of the bacteriologist, the cells “condition the medium.” More-
over, like a well-functioning assembly line, each original cell that has
stocked up on enough subunits to create two new cells also insures the
continuity of supply so that the two in turn will, without delay, be able to
collect or synthesize enough subunits to make four, and so on. Thus, no
further lag is apparent and growth can proceed apace. To summarize,
then, the lag phase is influenced by:

. The richness of the nutrient supply.
. The number of cells preparing for growth.
. The “physiological state” of the cells.

wWwr

THE EXPONENTIAL PHASE

We now come to the stage of active growth and reproduction and
the peculiar S-shaped curve. To understand it we need merely consider
the properties of populations that are growing actively under optimal
conditions:

1. The more organisms there are, the more there can be. In other
words, offspring must have parents and the more parents, the more off-
spring.

2. Most of the organisms in such a population reproduce during
their life cycles. Very few are barren.

3. The generation time is constant. In other words, the time for
each organism to emerge, mature, and reproduce is approximately the
same as long as the population continues to grow.

97

Number of organisms

98

Growth and Form

To return to our bacterial culture, the above statements mean (1)
that during the exponential phase each bacterial cell gives rise to two,
the two to four, the four to eight, etc., and (2) that it takes the same
amount of time for one to yield two as for two to yield four, etc. Suppose
the generation time were one hour. Then the population would, on the
average, double once every hour. If we draw a graph in which the number
of cells is plotted against time, we get the curve shown in Fig. 45. This is
the first part of the S-shaped curve. If now the period of active growth
draws gradually to a close—cells begin to die rather than reproduce and
those that can reproduce take longer than an hour to do so—the second
part of the S-shaped curve appears.

If growth were unlimited, the growth curve would simply go up
and up at an ever increasing rate, and it is interesting to speculate about
what would happen to our planet if the growth of, say, bacteria were
unlimited. One species of bacteria, Escherichia coli, can divide every 20
minutes in an optimal environment. Thus, if at time zero there were one
cell, at 20 minutes there would be 2, at 40 minutes 4, at 60 minutes 8, and
so on. The series 1, 2, 4, 8 can be expressed as 2° (=1); 2! (=2); 2?
(—4); 2? (=8); note that the exponents represent the number of genera-
tions of growth that have occurred. Therefore, the number of cells present
after n generations of growth would be 2”. Since the generation time of
Escherichia coli is 20 minutes, there could be 3 generations per hour, or
24 xX 3 = 72 generations per day. In other words, were growth not
limited, one bacterial cell would give rise in 24 hours to 27° = 40,000,000,-

Fig. 45. The first part of
the S-shaped curve is gen-
erated when every organ-
ism gives rise to progeny
(in this case by binary fis-
sion) and the generation
time is constant.

Time

Growth and Form 99

000,000,000,000,000 cells. A mass of bacterial cells this size would weigh
8 million pounds. We would quite literally be up to our ears in bacteria.

Time for Organisms To Double Their Mass

Escherichia coli 20 min
Fly larva 13 hr

Silk worm 68 hr

Rabbit at birth 6 days
Pig at birth 6-7 days
Sheep at birth 10 days
Guinea pig at birth 18 days
Horse at birth 60 days
Man at birth 180 days

The same arguments hold for higher animals. As anyone knows who
has had experience with rabbits, they may differ from bacteria to the
extent that you need at least two (of the appropriate sexes) to get more,
but they are the same as bacteria in that the more rabbits you have the
more rabbits you will surely get. Therefore, rabbit colonies or any other
animal population, when well fed and given enough space, display
S-shaped curves of growth. It should be noted that two rabbits do not
necessarily, and in fact hardly ever do, produce only four rabbits and
four only eight. The fact that with each generation the number of organ-
isms more than doubles would not change the shape of the curve but
would merely make it steeper.

THE STATIONARY PHASE

As already mentioned, toward the end of the exponential curve,
more and more cells begin to die and those that survive take longer and
longer to reproduce. Ultimately the rate of cell death increases so much
and the rate of cell growth decreases so much that cells die as fast as new
ones appear and the total number of living cells remains constant. This is
the stationary phase.

Why does the population stop growing? What limits growth? There
are at least two general causes:

1. The supply of an essential nutrient becomes exhausted. If an or-
ganism requires a particular nutrient for growth and the supply gives out,
the organism will cease growing no matter how much there remains of
any other nutrient. Whether the exhausted nutrient happens to be a
vitamin or an amino acid or a source of energy, or a metal ion or oxygen,
the result will be the same. The cessation of growth may be subtle, as in

1 See the section at the end of the chapter for a detailed discussion of growth
kinetics.

100

Growth and Form

a bacterial culture, or very dramatic, as when populations of Alaskan lem-
mings exhaust their food supply and in the course of frantic migration
commit mass suicide by leaping into the ocean.

2. The environment becomes too toxic for further growth. Organ-
isms, as they grow, pour out metabolic waste products and thus pollute
the environment. As long as the number of organisms is small, the con-
centration of wastes will be low and will not poison them. When the
number is large, the rate of waste production will rise and the concentra-
tion in the environment will increase to a toxic level and lower the vitality
of the organisms to the extent that they can no longer grow or perhaps
even survive.

We should mention a special case under this second category that
is particularly pertinent for animal populations, namely the stoppage of
growth not by toxic materials but by epidemics caused by parasites.
Epidemics are like nuclear chain reactions. That is, the host population
must rise above a critical density before the epidemic can take place. (Can
you construct an explanation of this?) Thus, when an animal population
does grow to great numbers, an epidemic can strike and reduce the pop-
ulation drastically. Then another cycle of growth will begin. Biologists
have charted such growth cycles for many wild animal populations.

Growth of the Multicellular Organism

A population of microorganisms is a collection of discrete cells that
are largely independent of each other for their existence. The higher ani-
mal is itself simply a collection of discrete cells that is organized into a
compact whole, but the cells are closely dependent upon each other for
their continued existence. To the degree that the animal is simply a collec-
tion of discrete cells, it grows according to the same rules as does a micro-
bial culture. But the interdependence of cells in the animal body brings
complications:

1. All parts of the animal do not grow at the same rate. Some tissues
do not grow at all once the embryo is formed. Others grow very slowly.
Still others grow very rapidly. The total organism, however, which is the
resultant of all the individual cells and tissue, does grow precisely as do
populations of microorganisms, i.e., in an S-shaped curve.

2. All parts of the organism do not stop growing simultaneously. In
fact, some tissues continue to grow during the entire lifetime of the ani-
mal. However, the total organismic mass does remain relatively constant
once maturity is reached (in the absence of pathological changes).

Growth and Form

3. The growth of one part of an animal can be and usually is con-
trolled by the activities of another part. A prime example of this is the
dependence of many tissues upon secretions of the pituitary gland.

The Limitations of Size and Form

There are obviously great variations in the sizes of living things,
particularly between different species but often even within them. Many
factors govern size. Among them are the following:

1. Genetic constitution. Mendel did some of his first experiments
with the dwarf and giant varieties of pea plants and showed that the two
differed by a single pair of genes. Such genetic determinants act by
affecting the rates of critical metabolic processes.

2. Nutrient supply and toxicity. As shown in the previous section,
the growth of populations is limited by these factors. The same is true of
the growth of a single cell or a single multicellular organism. Any environ-
mental condition that affects either factor can also affect size and form.

3. The ratio of surface to volume. Imagine an organism, perfectly
spherical in shape, which starts life with a radius of 1 centimeter and
grows to a radius of 10. Being spherical, its volume would be 442r° and
its surface 4xr?. At the beginning, the volume would have totaled 44 X
3.14 x (1)? = % x 3.14 x 1 = 4.2 cubic centimeters (cm). After the
growth period, the volume would be 44 < 3.14 * (10)? = % x 3.14 X
1000 or 4200 cm?. Thus, the volume of the organism would have increased
by 4200/4.2 = 1000 times. The surface at the beginning would have been
4 < 3.14 x (1)? or 12.56 square centimeters (cm?) and at the end 4 X
3.14 x (10)2 = 4 x 3.14 x 100 or 1256 cm?2. Thus, its surface would have
increased by 1256/12.56 or only 100 times. In other words, when an organ-
ism increases in size, the volume increases much faster than its available
surface area. The point of this is that if an organism experiences a thou-
sandfold increase in protoplasmic volume, it is going to require a thou-
sandfold more food to sustain itself and it is going to have to get rid of a
thousandfold more waste products. But food enters and wastes leave
through the surface of this organism which, as we have seen, increases
much less than its volume. Consequently, this limits the size that the or-
ganism can attain. Different species have overcome this limitation by:

1. Changing their shape in order to decrease the disparity between vol-
ume and surface as the two increase during growth.

2. Using their food supply more efficiently or by devising ways to make
its entrance more rapid.

3. Devising biochemical tricks for cutting down on the production of toxic
wastes or by speeding the exit of wastes out of the system.

101

102 Growth and Form

4, Structural limitations. Nature is an engineer and an architect. She
builds conservatively and according to sound engineering principles. She
pays attention to strength of materials and patterns of stress. She believes
that form should follow function. The man to realize these ideas most
strongly and to express them most convincingly in terms of mathematical
rigor was a great biologist named D’Arcy Thompson, who wrote a book on
the subject entitled Growth and Form.

Equations That Describe Growth

Many attempts have been made to describe mathematically the
growth of a population of cells or of multicellular individuals. It is not
difficult to arrive at an equation that can generate an S-shaped curve.
What is difficult is to invest the constants of this equation with biological
meaning, that is, to interpret them as being due to specific physiological
mechanisms that initiate, maintain, and terminate growth. Generally, we
start with the growth-affecting processes we know about (production of
diffusible subunits, production of toxic wastes, exhaustion of the nutrient
supply ), then describe them in mathematical terms, and derive the proper
equation relating the number or mass of organisms to the passage of
time. The extent to which the equation (called the logistic equation) ac-
curately describes growth, as measured in specific organisms, tells how
much we really know about growth processes. The extent to which it
does not, tells us how much is not known but, more important, provides a
testing ground for the inclusion of additional physiological processes that
we suspect may affect growth. The following paragraphs contain in ab-
breviated form the arguments that led to the derivation of the logistic
equation. It is not expected that you will understand the subject com-
pletely from this exposition, but I hope you will appreciate from it that a
rigorous, quantitative approach to biological phenomena is possible and
represents a fertile field of investigation for the mathematically-minded
student.

The first part of the S-shaped curve, the period of so-called exponen-
tial growth, is simple to derive. It is described by the equation:

N
log, No —Ikt 1

where N is the number of organisms at time t, No is the starting number, k
is a constant, and ¢ is time. As mentioned previously, the growth of a
population of cells that divide by binary fission follows the series 1, 2, 4,
8, 16, . . . and these can be stated as powers of 2, i.e., 2°, 21, 2?, 23,

in asia the exponents refer to the number of teres a Leesan that

Growth and Form

have occurred. Therefore, starting with one cell and after n generations
of growth, the number of cells would be 2”. If we started with 1000 cells
instead of 1, the series would be 1000, 2000, 4000, .. . ie., 1000 « 2°,
1000 « 21, 1000 x 2?, .. . and after n generations, No2", where No is
the starting number. The general statement of the number N of organisms
would be:

N = No2” 2

Let us assume that these cells reproduce once every two hours (a con-
stant generation time). Then the population would have undergone one
generation of growth in 2 hours, two in 4 hours, three in 6 hours, and so
on. In short, the number of generations would be proportional to time,
and this is stated algebraically as n = k’t where k’ is a constant. Equation
2 can be restated as:

N = N,2#"t <= Okt 3
Taking logarithms of both sides, we get:
log, xT = (log. 2) Kt
and if we put k’(log, 2) equal to a new constant, k, the equation becomes:
log, x = kt 4

which is identical with equation 1. Remember that the only two assump-
tions used to derive this relationship are that 1 organism gives rise to 2
and that the generation time is constant. Obviously, the shorter the gen-
eration time, the faster would the population grow and the higher would
be the value of k. Suppose the cells do not divide by binary fission but,
instead, each cell gives rise to 3. Then the growth would be 1, 3, 9, 27,
81, . . . a series in powers of 3, and the equation would become:

log, x = (log, 3)kt

Only the value of k in equation 4 would change, but the basic equation
would remain the same. In other words, whether 1 organism gives rise
to 2 or 3 or more, or whether 2 organisms must cooperate to yield 3 or 4
or more does not change the equation but only the value of the constant.

But equation 4 as written does not describe the entire growth se-
quence. Note that as time passes (t gets larger), N/No would get larger

103

104

Growth and Form

and never reach a limit, i.e., growth would never stop. To show how an
automatic brake can be added to this equation, we derive it in another
and more general way. Consider that growing organisms display two
properties:

a. To get any progeny at all, you must start with at least one in-
dividual or, in the case of higher animals, with at least two of the right
sexes (i.e., there is no spontaneous generation of life).

b. The more parents you have, the more progeny you will get. In
other words, the rate of increase in the number of organisms is propor-
tional to the number already present.

Now rate of increase is simply the change in the number of organ-
isms per unit time, or:

total change in number of organisms
total change in time

We symbolize this as AN/At. Since the rate of increase is proportional to
the number, N, of organisms already present, we can write:

AN
he = kN 5
This equation can be solved by the integral calculus. That is, the relation
between N and t can be deduced from the way in which N changes with
time. By these means (the mechanical manipulations are unimportant
here), we obtain the same equation as already has been derived, i.e.,
equation 4.

Equation 5 can be elaborated to account for the stoppage of growth.
What we want is to have the rate of growth start out high and give us
the first part of the growth curve and then to decline steadily to zero, at
which point the number of organisms would remain stationary. For some
organisms that do not remain stationary after cessation of growth but
actually die out, we would want the rate of growth to become negative
after a time (a negative rate of increase is itself a decrease ). These qual-
ifications are accomplished by making k not a constant but a variable.
For example, one way of doing this is to set k equal to (a — bN), where
a and b are constants. Equation 5 would then become:

—— = (a—bN)N 6

At the start, when the number of organisms was very small, bN would be
negligible, and the rate of growth would be substantially equal to aN

Growth and Form

(i.e., would be like equation 5) and would yield the first part of the
S-shaped curve. As growth proceeded and N became larger, bN would
become larger and begin to detract appreciably from a. Thus the rate of
growth would progressively decrease. When N became so large that bN
equalled a, the term (a — bN) would be zero, the rate of growth would
be zero, and growth would cease. The constant, b, could be used to de-
scribe the decrease in the food supply or accumulation of toxic wastes that
inhibit growth. In the case of the growth of organisms that serve as prey
for other organisms, b could be used to describe the way in which the pred-
ator cuts down the growth of the prey (by killing off potential parents).
In this case, b would not be a constant but would depend on the number
of predatory organisms. Finally, b might be used to account for epidemics
that kill substantial numbers of organisms and would be treated in much
the same way as if a predator were feeding upon the population. If we
were studying the growth of an organ, say the number of liver cells in a
human, b might also be used to describe the action of hormones from
other parts of the body upon the liver cells.

105

we ad = he

opie ee eae nae

Oy Aen Le e@alnit ut)

pi Roki) cow pda be tee
italy OES tye gangs fae. i, mAr a on? guiding
6 att eth) amie of hee tl. ale ated Wn :
= i¥ inet dndigiltes ba!

a

Selected Readings 107

Selected Readings

Popular discussions of many of the investigations described in these
chapters can be found in the back issues of Scientific American. Ask your
school librarian for a list of the articles.

Brachet, J., Biochemical Cytology. New York: Academic Press, 1958. A
very advanced study of cell development.

Brachet, J., Chemical Embryology, 2nd ed. New York: Interscience, 1950.
An advanced discussion of the subject.

Butler, E., and D. Rudnick, eds., The Symposia of the Society for the
Study of Growth and Development. Princeton: Princeton Univer-
sity Press, 1953 to present. Contains a wide variety of general re-
views of developmental biology. These discussions are at an ad-
vanced level.

Waddington, C. H., Principles of Embryology. London: Allen & Unwin,
1956. A good elementary embryology text.

Willier, B. H., P. A. Weiss, and V. Hamburger, Analysis of Development.
Philadelphia: Saunders, 1955. A comprehensive advanced treatise
on embryology.

Wilson, E. B., The Cell in Development and Heredity. New York: Mac-
millan, 1925. A true classic, as fresh and illuminating as if it had
been written yesterday. It should, however, be reserved by the
serious student until his senior year for full appreciation.

A

Acetabularia:
life cycle of, 22-23
morphogenetic specificity in, 25
regeneration of, 23-24
Achlya:
hormonal influences in, 35-36
induction in, 81
life cycle of, 33-34
sexual development of, 34
Activation of eggs, 53
Aggregation of slime molds, 28
Amitosis, 9
Amphioxus:
cleavage, 55, 56
gastrulation, 57-59
Animal hemisphere, 55
Antheridia, 35
Archenteron, 57
Artificial insemination, 52
Autogamy, 17

Basal granule, 9

Biochemical embryology, 66-68
Biochemically deficient mutants, 88-89
Binary fission, 6, 15

Biological clocks, 18-22
Blastomeres, 55

Blastopore, 59

Blastula, 40, 54-57

Blastular cavity, 55
Blepharoblast, 9

Brain development, 61

Bristle organ, 69-71

Brucella abortus, 86-87
Budding, 6

Cc

Cell division:
biochemical events in, 8
genetic aspects of, 8-10
varieties of, 6-8

111

112

Index

Cell interactions:
in Achlya, 35
inductive, 81—84
inhibitory, 86
in slime molds, 31
known mechanisms of, 86-91
mass interactions, 80-81
morphogenetic fields, 44-46
synergism, 85, 88-89
Cellular slime molds (see Slime molds)
Central nervous system, 61, 63-64
Chemotaxis, 31
Chick embryo:
cleavage, 55, 56
gastrulation, 60
Chloroplasts, 10
Chorda-mesoderm, 82
Cilia, 14-15
Circulatory system, 61
Cleavage, 47, 55-57, 67
Cnidoblasts, 39
Coelenterates (Cnidaria):
general properties of, 37-39
life cycle of, 39-40
polymorphism in, 40—42
regeneration of, 42-46
Collecting tubules, 85
Conjugation, 16-17
Cytology, 5

Deoxyribose nucleic acid (DNA), 8,
73-74, 88
Dermis, 61
Development:
mosaic, 77
regulative, 77
Developmental biology, 3
Dictyostelium discoideum, 27-33
Differentiation:
definition of, 4, 77-79
genetic basis of, 72-77
of bristle cells, 70-71
of coelenterates, 37-38
of neural tube cells, 71-72
of slime mold myxamoebae, 28-31
specificity of, 72
Diplococcus pneumoniae, 88
Diploid, 11, 17, 22

Ectoderm, 60, 61
Egg, 50-52, 67
Embryogenesis:
Amphioxus, 55, 56, 57-59
chick, 55, 56, 60
coelenterate, 40
frog, 55, 56, 59-60
Endoderm, 60, 61
Enzymes, 67, 74, 88-89, 96
Epidermis, 40, 61
Escherichia coli, 74-75, 89-90, 98
Euglena, 74
Exponential phase, 97-98
Extranuclear elements, 9

F

Fertilization, 52-54
Fission, 6, 15
Fragmentation, 7
Frog embryo:
cleavage, 55, 56
gastrulation, 59-60
later stages, 63
Fruiting body, 25

G

Gametes, 11, 48-52
Ganglia, 64
Gastrodermis, 40
Gastrula, 40, 57-62
Generation times, 99
Genetic changes, 2
Genetic information, 73-74
Genetic recombination, 11
Genotype, 75
Glands, 61
Gonyaulax, 18-21
Growth:
equations for, 102-105
exponential phase of, 97-99
filamentous, 7
fragmental, 7
lag phase of, 96-97
noncellular, 7

Growth (Cont.):
nutrition, 93-94
of multicellular organisms, 100
size limitations, 101-102
s-shaped curves, 94-95
stationary phase of, 99-100
Gut, 58, 62

H

Haploid, 11, 17, 23
Histology, 5
Hormones, 33
Hydractinia, 41-42

Induction, 81-86
Invagination, 59

L

Lag phase, 96
Log phase, 97-99

Macronucleus, 14
Medusa, 38
Mesoderm, 60
Mesenchyme, 61
Meiosis, 11, 49
Merozoite, 7

Micron, 14
Micronucleus, 14
Mitochondria, 10
Mitosis, 8
Morphogenesis, 4, 22
Morphogenetic fields, 44-46
Mutants, 2, 86, 88-89
Mycetozoa, 7

Nervous system, 61, 63-64
Neural plate, 62

Index

Neurula, 62

Neurospora, 88—90
Notochord, 58

Nuclear division, 8
Nuclear transplantation, 77
Nucleic acids, 8, 73, 88
Nutrition, 93-94

oO

Obelia, 39-40

Oogonia, 35

Organelles, 3, 9

Organ formation, 47, 62-66
Organizer, 82

P

Paramecium, 14-17
Parthenogenesis, 53
Penetration of sperm, 52
Phenotype, 75
Photosynthesis, 20
Physiological change, 1
Pioneer fibers, 64
Plasmodium malariae, 7
Planula larva, 40

Polar bodies, 51
Polymorphism, 40-41
Polyp, 38, 41, 42-48
Polyspermy, 53
Portuguese Man of War, 37
Primitive streak, 60

Regeneration, 42-44
Regulation, 32
Respiration, 10
Rhizoid, 24-25
Rhythms, 18-22

Ss

Secretory tubules, 85
Selective inhibition, 86

113

114 = Index

Sexual reproduction, 11 Tissue culture, 92
Siamese twinning, 82 Transformation, 88
Sirenin, 35

Slime molds:
cellular differentiation in, 28-31, 33
cellular interaction in, 31, 33 V
life cycle of, 27-28
regulation in, 32
Somites, 58, 61
Spemann, H., 82

Vegetal hemisphere, 55

Sperm, 48, 67
Spinal cord, 62 al
Sporangia, 33 :
Stationary phase, 99-100 GSE bio, Ue
Stern, C., 69
Surface-volume ratio, 101
Synergism: Y

growth, 88-90

inductive, 85 Yeast, 10-13

Yolk, 50
Lp
Z

Tetrads, 49

Tissue, 37 Zygote, 11, 35

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