3 1761 04900103 5 —UNIVOF Toronto LIBRARY j , ' val i “, al be i be - (Ge ¥ hf sh) es in on i ne ® as, wy _ Digitizedby the Internet Archive in 2008 with funding from ie a ail Microsoft Corporation aa: ee +o , a) fi Cie m 4 ti : owt Oy Rall Te : : : si ar y ¥ o> a . A My : i / er i V ect S /www.archive.org/det ¥ Me iy hg A ‘i j * ne iy as Bi om /experimentalanal07stocuoft fi Wy he I i a a RR COME bite Pel VL) t SaMAL, Piles Ft reas vt 7 7 NUS ay ae 1m Mite G) ua MEMOIRS OF THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY No. 7 AN EXPERIMENTAL ANALYSIS OF THE ORIGIN OF BLOOD AND VASCULAR ENDOTHELIUM I. THE ORIGIN OF BLOOD AND VASCULAR ENDOTHE- LIUM IN EMBRYOS WITHOUT A CIRCULATION OF THE BLOOD AND IN THE NORMAL EMBRYO FORTY-NINE FIGURES II. ASTUDY OF WANDERING MESENCHYMAL CELLS ON THE LIVING YOLK-SAC AND THEIR DEVELOPMEN- TAL PRODUCTS: CHROMATOPHORES, VASCULAR ENDOTHELIUM AND BLOOD CELLS THIRTY-FIVE FIGURES CHARLES R. STOCKARD DEPARTMENT OF ANATOMY CORNELL UNIVERSITY MEDICAL SCHOOL NEW YORK CITY PHILADELPHIA, PA. Reprinted from THE AMERICAN JOURNAL OF ANATOMY, Volume 18, Nos. 2 and 3, September and November, 1915 CONTENTS MPL ATEOOU CULO an ci. 5 «ote halts AMM: aorio ew che, Stent sala bine viele See aera howe « 4 i -eMethods o1experiment and material: ........ nas ~Sa) 63 64 CHARLES R. STOCKARD this individual the heart is a solid string and the blood had never circulated. The liver presents a dense appearance, no blood vessels are seen and blood corpuscles are entirely absent. The general differentiation and condition of the tissues are, how- ever, fairly normal and not at all degenerate. The intestinal epithelium is typical in structure. Above the intestine the well differentiated dorsal aorta is shown with connective tissue fibers abundantly present in its wall and a definite endothelial lining. The lumen of this aorta, however, has never contained any type of blood cells and the only solid particles within it are a slight coagulum near the center of the vessel. Above the dor- sal aorta are the two Wolffian ducts and between them under the notochord are a few mesenchymal cells which represent more posteriorly the remains of the intermediate cell mass. Almost all of the erythrocytes in this mass have completely de- generated or have been destroyed by mesenchymal cells. The embryos without a circulation thus furnish a definite means of establishing the actual haematopoetic value of any organ. They demonstrate that unless the blood current reaches the organ and thereby introduces embryonic blood cells into it the organ iself is incapable of giving rise to blood cells. This experiment also demonstrates with equal force the inabil- ity of vascular endothelium to form blood cells in the fish. I can see no reason if vascular endothelium possesses a blood forming power why the aorta and other interior vessels of these embryos are invariably empty and never contain any type of blood cell. It cannot reasonably be claimed that this inability is due to the abnormal condition of the embryo having taken away the power of the endothelium to form blood cells, since it is so absolutely demonstrated that real blood forming material in other portions of the embryo possesses its perfectly normal capacity to produce blood and does produce it in a very abundant fashion. These embryos furnish no evidence to indicate that there is any connection or association between the mesenchymal cells which are to form the connective tissue and those destined to form blood cells. There is no instance of a tendency for con- nective tissue cells to change into blood cells or of blood cells to give rise to any type of connective tissue cells. ; ORIGIN OF BLOOD AND ENDOTHELIUM ~ 65 Finally, one may conclude that the blood cells like many other specific tissues and organs have a definite localized specific anlage and that this anlage is distinct and separate in most cases from that of the vessel linings. In some cases, however, the blood and endothelial anlagen may come into intimate associa- tion, yet even here the two are probably of different mesenchymal origins. A CONSIDERATION OF THE EXPERIMENTAL STUDY ON THE ORIGIN OF BLOOD IN TELEOSTS IN RELATION TO THE MORE RECENT STUDIES ON THE ORIGIN AND DEVELOP- MENT OF VESSELS AND BLOOD CELLS 1. Introduction This experimental study of the origin and development of blood and vessels relates itself to three more or less separate fields of investigation. In the first place, the manner in which the blood anlage in Teleosts has separated itself as a unique intermediate cell mass has caused it to be studied as a special subject somewhat isolated from the more general literature on the development of blood in other vertebrates. Yet one very soon appreciates the mis- take of this isolation since contributions such as those of Felix (97) and Swaen and Brachet (’99, ’01, 04), in particular, on the Teleosts are of more general importance than most investi- gations dealing with the broad subject of blood development in the vertebrates. The very fact that in this group the blood anlage is so peculiarly localized in the embryo lends itself as a great aid to the solutions of many questions of haematopoesis or blood genesis. Secondly, a consideration of the origin ane formation of the heart lining or endocardium and the vascular endothelium, in these embryos which have developed without having had plasma or fluids to circulate within their vessels, may furnish much important data towards a final solution of the origin and signifi- cance of endothelial lining cells, and the manner of spread and distribution of such cells through the embryonic body and the yolk-sac. 66 CHARLES R. STOCKARD Lastly, such an experimental study bears closely upon the general questions of relationship between different blood cell types. The time and place of origin of the different cells, and the developmental relationship and powers of transmutability existing between various sorts of blood corpuscles as well as the endothelial lining cells of the vessel walls are all problems upon which the experimental results discussed above may throw light. Each of these three divisions of the problem embraces an extensive and often cumbersome literature which it would be quite out of place to consider in detail at the present time. We shall, therefore, only consider the bearing of the facts recorded in the previous pages upon the opinions and positions maintained by the more recent investigators of the origin and development of blood and endothelium. 2. The specific problems of blood and vessel formation in the bony jish It may be well to review first the special problems and ques- tions involved in the development of the blood in Teleosts as a group. According to Swaen and Brachet (’01), the meso- blast in the middle and posterior regions of the trout embryo is arranged in two parts, a median primary somite portion and an outer primary lateral plate part. The lateral plate in the mid- body region then divides off a portion immediately adjacent to the somites to constitute the intermediate cell mass. Lateral to this a second part of the lateral plate is separated off to form the primary nephric duct. In the mid-region of the body the somites become separated from the primary lateral plate and the lateral plate pushes or grows towards the median plane and gives off a keel shaped mass between the somites and hypo- blast. This mass unites in the median plane with a similar mass from the other side and here forms a large cell group triangular in cross-section, the intermediate cell mass. In the posterior region of the embryo a similar mass pinches away from the primary lateral plate and becomes the posterior continuation of the intermediate cell mass. ORIGIN OF BLOOD AND ENDOTHELIUM 67 Anterior of the first somite in the unsegmented mesoblast of the head this division or pinching away also takes place. Thus the intermediate cell mass of the body becomes continuous with a definite lamella of the head. This well definied topographical portion of the embryonic mesoblast, the intermediate cell mass and cell lamella, is, according to Swaen and Brachet, the only material which gives rise to the heart, the chief vessels and the blood in the embryo. This description by Swaen and Brachet (01) agrees very closely with that formerly given by Felix (97), except that Felix disagrees in not deriving the aorta from the intermediate cell mass but from the sclerotoms. The observations made upon the intermediate cell mass in Fundulus are in close accord with this summary. But no at- tempt has been made to solve the detailed question as to whether the aorta is derived from the intermediate mass or from the sclerotoms. It would seem that this vessel might arise from either source and still be formed from practically identical cells. Since in the separation of the primary lateral plate from the somite it is easily conceivable that some cells which generally — accompany the primary lateral plate might be left as part of the lateral portion of the somite. This lateral portion of the somite is the part which later separates as the sclerotom so that the cells destined to form the aortic endothelium might occur equally well within the intermediate cell mass or within the sclerotom. Their location might vary among different species or even among individuals, and yet these aortic cells would be derived from the same genetic source. Swaen and Brachet also indicate the head mesoblast as sepa- rated into three portions: the intermediate cell mass close to the top of the pharynx, the lateral plate split into two lamellae and the general head mesoblast close around the brain. The intermediate cell mass is more intimately connected with the splanchnic layer of the lateral plate. The pharynx widens in forming the gill pouches which continue to grow dorsally and finally separate the intermediate cell mass into two portions, one part thus comes to lie ventral of the pharynx and the other part dorsal. The ventral portions, at first solid masses below 68 CHARLES R. STOCKARD - either side of the pharynx, begin to migrate towards the mid- dle line. The two masses fuse into one, spaces are developed in the mass and finally the endothelial lining of the heart is differ- entiated out of this group of cells. The lamellae of the side plate become separated and the space between them gives rise to the pericardial cavity. Oellacher (’73), Wenckebach (86), Henneguy (’88), and Sobotta (94) have all described the origin of the heart in Teleosts in much the same way. Several of these investigators, Wenckebach, Swaen and Brachet and others, have called attention to a small mass of cells derived from the heart anlage which comes to lie beneath and outside the heart endothelium. This mass of cells has been claimed to wander away from below the pericardium and later to give rise to vessels and blood on the yolk-sac. In the non- circulating Fundulus embryos, however, neither vessels nor blood are formed on the extreme anterior portions of the yolk-sac. I have seen nothing in my studies which would indicate that any cells left over from the heart formation had wandered upon the ‘ yolk or given rise to blood cells or vascular endothelium. Swaen and Brachet are alone in showing that the heart cells are definitely continuous with the intermediate cell mass of the the trunk mesoderm. Many early workers on the fish embryo have claimed, as has been done for most vertebrae classes, that the heart lining arises from endoderm. The weight of evidence at the present time is so overwhelmingly against such a view that it warrants only a passing mention. Again, however, it must be realized that in the separation of the mesoderm from the endoderm it is possible that some future mesoderm cells may be left behind not cleanly separated. These cells might later isolate themselves from the endoderm to form vessels or blood. It nevertheless seems generally true that all blood forming cells are at one time in development contained within the mesodermal portion of the embryo. Gregory (’02) came to the conclusion that the endo- derm and mesoderm could be traced to an indifferent cell mass mesentoderm in certain Teleosts, and according to his view, there is no way to say from which germ layer the heart endothe- ’ ORIGIN OF BLOOD AND ENDOTHELIUM 69 lium actually arises. A mixture of endoderm and mesoderm cells gave rise to endocardium. The later development of the heart of the bony fish proceeds much as in the case of other vertebrates, as has been carefully described in detail by Senior (09). The only point of interest in the present discussion is the origin and significance of its en- dothelial portions, and here Senior after a very thorough investi- gation confirms in all general points the previous findings of Swaen and Brachet. In Fundulus as in other Teleosts the heart endothelium par- tially forms in loco but is also added to by wandering cells or ingrowths of mesenchymal cells adjacently located. ‘The venous end of the heart leads directly down upon the yolk periblast, and as was shown in several figures, this periblastic material with huge amorphous nuclei may be at times drawn up into the cavity of the heart. This would indicate that the venous end was entirely free or not connected with any other vascular en- dothelium. This condition is, no doubt, due to the absence of the anterior yolk vessels which should in ordinary cases unite or fuse with the end of the heart so as to establish a closed circulation. According to Swaen and Brachet in the region of the third somite the intermediate cell mass forms only the aorta, while caudad the aorta arises from the dorsal cells of the mass and the great part of the mass forms the red blood corpuscles and the venae cardinales. The endothelium of the cardinal veins finally surrounds the blood cells, but before these cells are fully developed or free, plasma has begun to flow in the aorta and other arteries. In pelagic forms in which the egg is extremely small and devel- ops very rapidly, the intermediate cell mass in the forward body sections is very small, sometimes only seen between the somites. This portion gives rise to the aorta. The cells are somewhat more numerous in the middle and posterior sections, but they never form a mass to the extent found in the larger demersal eggs. At the time of hatching the posterior cell strings form two lateral longitudinal vessels from the beginning of the mesonephros caudad to the anus. These two vessels, Swaen and Brachet consider to be homologous to the unpaired median stem vein 70 CHARLES R. STOCKARD* of the trout and this is thought to represent the conjoined car- dinal veins. We have noticed that in Fundulus the intermediate cell mass is sometimes divided forming two lateral cardinals loaded with blood cells, while generally it exhibits a median un- paired condition. In the pelagic forms the vessels are all hol- low at the time of hatching and the blood cells have not appeared. Derjugin (’02) claims from a study of the pelagic egg of Lophius that the vessel cells of the aorta and cardinals are derived from the sclerotom. Felix (’97) like Ziegler (’87) differs with Swaen and Brachet (’01, 704) in that he derives the aorta not from the “Venenstrang”’ but from the sclerotom which under the noto- chord forms a mesenchymal aortic string. Felix states that no blood eells are to be seen in the aortic anlage, while the cardinal veins, of course, are loaded with the blood cells of the inter- mediate cell mass. Felix, therefore, derives the two chief ves- sels of the embryo from two different parts of the mesoderm, the somites or sclerotoms and the lateral plates. Sobotta (02) terms the intermediate cell mass ‘‘Blutstrange”’ and derives it from the lateral plate, though he had earlier claimed it to arise from the somites. He described it in the trout embryo in the region from the eighth to the thirty-third somite. The ‘Blutstriinge’ at first paired, are naked cellular strings without a true vessel covering. This they receive later as the cardinal vein anlagen. The endothelial cells of the cardinal veins he derives from the same source which produces the aorta, namely, the sclerotoms. Finally, then, Swaen and Brachet derive the blood and vascu- lar endothelium of the aorta and venae cardinales from the inter- mediate cell mass which arises from the lateral plate. Felix derives only the blood and vascular endothelium of the cardinals from the intermediate cell mass which is separated originally from the lateral plate. The aortic endothelium arises from the sclerotoms. Sobotta considers the intermediate cell mass an exclusive blood forming material, while all vascular endothelium, including the heart, is derived from the sclerotoms which are budded off from the somite system. This disagreement, as we have pointed out before, is not of primary importance and ORIGIN OF BLOOD AND ENDOTHELIUM 71 may result merely from the fact of the intimate connection of the sclerotom and intermediate cell mass before their original separation. The question now arises whether all the blood of the Teleost embryo is exclusively derived from the intermediate ‘Blutstrange.’ Felix admits that the endothelium of the glomerular vessels of the mesonephros arise in leco and at the same time blood cor-- puscles often occur in this region. Sobotta claims that in the vascular network in the tail of the trout embryo some of the blood corpuscle anlage exists. Both of these exotic positions of origin may be easily under- stood. In the first place, the nephric anlage is formed from cells in direct association with those constituting the early intermediate cell mass, and in the separation it probably happens that some future blood cells are held within the kidney anlage and these cells later develop in their proper fashion. The presence of blood corpuscles in the vascular network of the tail is due to the fact that the intermediate cell mass in many Teleosts, as Marcus (05) has pointed out and as Senior (’09) particularly emphasized extends far back into the caudal region. A similar consideration is the question of origin of vessels from material other than that of the intermediate cell mass and sclerotom. This is also important, and numerous observa- tions would indicate that in the early bony fish embryo vessels unquestionably arise in loco and not solely as outgrowths or sprouts from a central vessel anlage. Sobotta (02) on the con- trary imagines a gradual growing away of the vascular system from its local origin, the sclerotom. ‘The aorta is the primary vessel and, for example, the sub-intestinal vein arises from the aorta by vascular sprouts which grow around the gut, broaden out and fuse on its ventral side and finally give rise to the longi- tudinal vein. This theory of Sobotta is as unacceptable in the face of the great body of evidence to the contrary, Felix (’97), Rickert (88), Hahn (’09) and many others, as is the opposite ingrowth parablast theory of His (’75). The consideration has been confined so far to the intra-embry- onic blood vessels. We may now briefly discuss thedevelop- 72 CHARLES R. STOCKARD ment of vessels and blood upon the yolk. There are here two opposed or different views. The first derives the yolk vessels and blood directly from the yolk syncytium or periblast. The second derives blood cells exclusively from the intermediate cell mass in the embryo, but admits that cells may secondarily come to lie on the yolk by being pushed out from the intermediate -eell mass with which, however, they maintain a definite conti- nuity. The vascular endothelial cells are also derived from the embryo as mesoblastic wandering cells, but these are not to be compared directly with blood cells since their parent cells have a separate place of origin. Most of the earlier workers thought that the blood in the Teleosts arose on the yolk-sac, as it does in other meroblastic embryos. ‘The more recent workers have gone to the other extreme and deny the presence of blood islands upon the yolk-sac as separate from the intermediate cell mass. As mentioned in describing the heart formation, numerous investigators have recorded wandering mesenchymal cells upon the yolk-sac, but from a study of the literature no clear con- ception can be formed as to the origin of blood cells or the vas- cular endothelial cells upon the yolk from these wandering cells. Some authors claim that the majority of wandering cells become pigment cells, while the remainder form the yolk vessels. In Fundulus the pigment cells very soon present a different appear- ance from the mesenchymal cells which are to form the vascular endothelium. Both types of cells may be readily seen wandering over the yolk between the ectoderm and periblast. Before the yolk vessels are completely formed, the circulation of a cell free plasma has begun. The extent of the spaces in which this cir- culation takes place is very variable. The arrangement of the veins of the yolk circulation is also extremely different in the different groups of Teleosts. One must agree with Hochstetter (93) in stating that the yolk circulation in different forms is so different from the start that it is not possible clearly to summar- ize the condition in order to give satisfactory comparisons with the same vessels in Selachians, and Amphibians. ORIGIN OF BLOOD AND ENDOTHELIUM , 73 When the plasma is flowing in a closed system within the em- bryo, it is still running as a wandering stream through lacunae and sinuses on the yolk. This probably explains why the blood cells reproduce for so long a time on the yolk-sac while no such reproduction is taking place in the well formed vessels of the embryo. It is difficult to determine the exact moment, when, or place at which the first blood cells get into the circulation. This probably varies even among embryos of the same species. Zieg- ler (’88) thinks, however, that just beyond the lateral plates in the plasma filled spaces of the yolk-sac which lie between the periblast and ectoderm, the first blood cells project into the circulation. They are in the form of cell strings which later connect the cardinal veins with the vascular yolk net. Swaen and Brachet saw in trout embryos of eleven days in the region of the fourteenth somite and posterior that the intermediate cell mass spreads out laterally below the lateral plate and on to the yolk surface. The cells thus came to lie above the yolk syncytium and first attained their red color in this position. These authors thus claim that in the bony fish with a large yolk- sac the haemoglobin free early blood cells through continued contact with the yolk become transformed into erythrocytes. The experimental embryos considered in the present paper demonstrate, however, that it is not at all necessary in such a Teleost to have the erythroblasts reach the yolk-sac in order to acquire their red haemoblobin condition. The tightly packed erythroblasts within the intermediate cell mass of the embryo develop perfectly and readily attain a normal red haemoglobin color. Finally, comparing the processes of vessel and blood formation in Teleosts with these processes in other vertebrate embryos, we find no definite explanation for the formation of the inter- mediate cell mass. In other embryos the blood is largely formed upon the yolk. However, it must be recognized from recent contributions that the formation of intra-embryonal blood is much more extensive and important than has formerly been 74 CHARLES R. STOCKARD supposed. The relation of the blood anlage to the cardinal vein and the position of the blood forming cells dorsal of the gut are unique in the Teleosts. The late formation of the yolk vessels and their type of origin from wandering mesenchymal cells is also of special interest. It would seem as though the peripheral mesoblast which in other vertebrate types grows and develops outside the embryo, had in the Teleosts been peculiarly concentrated and drawn into the embryo during its phylogenetic history. Yet in this intra- embryonic position the peripheral mesoblast gives rise to the same cells which it would ordinarily produce on the yolk-sac. The different Teleosts probably show this drawing in of the peripheral mesoderm to various degrees so that in some cases only part of the mesoderm is incorporated in the interme- diate cell mass, while the remaining part may still be outspread upon the yolk and there differentiates extra-embryonically. The intermediate cell mass is connected caudally with the end bud, just as the peripheral mesoblast of the Selachians is with the blastopore lip. In its genesis the intermediate cell mass is split off from the lateral plate and localized along its median border. Marcus (’05) in his study on Gobius capito advanced the opinion that the intermediate cell mass in this embryo is compar- able to the peripheral blood forming mesoderm of other mero- blastic eggs. In an embryo of eleven somites, the intermediate cell mass passes without a break caudad to the end bud and there connects with both the ectoderm and entoderm, just as the peri- pheral mesoderm would meet the other two germ layers at the blastopore lip. He attempted to show by diagrams the rela- tionship between the intermediate cell mass in Teleosts and the blood forming mesoderm of Selachians. As the homologue of the peripheral mesoderm the intermediate cell mass has the power to form vessels and blood cells. Most authors admit this power and only Sobotta (’02) denies the ves- sel forming property, while others claim that only the cardinal veins arise from the intermediate cell mass, still others, as Swaen ORIGIN OF BLOOD AND ENDOTHELIUM res and Brachet, would derive the endocardium and aorta also from this common source. The important fact is that in the small pelagic embryos, where no blood formation takes place before hatching, the inter- mediate cell mass forms the aorta and the cardinal veins and is also derived from the lateral plate. The lateral plate thus con- tains cells capable of forming vascular endothelium, and this is the case in all vertebrates. At an early time in evolution the extra-embryonic blood form- ing mesoderm has been included within the body of the Teleost embryo and lies over the gut as the intermediate cell mass repre- senting the yolk vascular layer. Here it is important to note that the yolk-sac of the Teleosts contains no real mesodermal layer, only separate wandering mesenchymal cells are found be- tween the ectoderm and periblast, and these wandering cells have migrated out from the embryo. Finally, as Mollier (’06) states in his review of this subject, it is not a question of the formation of the intermediate cell mass in the individual bony fish, but the wider question of the behavior of the blood forming peripheral mesoderm in the bony fish. All of the results must be considered in this light in their application to other animal classes. The intra-embryonic blood formation in the bony fish does not represent the primitive type for vertebrates as Sobotta (’02) claims, but this is, no doubt, a modified secondary condition accompanying the various other modified and special develop- mental processes which bony fish embryo so frequently presents. Wilson (’91) states of the mesoderm of the Teleost that: ‘‘The ventral subvitelline mesoderm, having in this way losts it func- tion in the Teleost, must be regarded as a rudimentary organ of the gastrula. It always remains very small, and does not form any special organ or set of organs in the embryo.” The real fact is that the subvitelline mesoderm is misplaced, being within the embryo as the intermediate cell mass and here forms the blood of the individual and, therefore, the yolk-sac of the bony fish has no mesodermic layer. 76 CHARLES R. STCCKARD 3. Vascular endothelium, and vascular growth and development Mollier (06) concludes in his review regarding the origin of vessels as follows. As to the genesis of embryonal vessels we may pass the judgment that the theory of the local origin of the vascular endothelium is val- uable. The notion of His (’75) and Vialleton (92) that the vessel strands of the embryo grow in as sprouts from the extra-embryonal anlage (vascular anlage) is not nearly so probable as that the individual vessel cells arise in loco and thus form the vascular nets. This statement agrees in every way with the contentions so fully presented by Huntington (710, 714), McClure (10, 712) and others, regarding the origin of lymph vessels. Lately it receives additional substantiation from the experimental results recorded by Miller and McWhorter (14) on the origin of blood vessels in the chick embryo. Such a position is further strength- ened by the still more recent experimental evidence, presented by Reagan (’15) which shows the origin in loco of vessels in isolated parts of chick embryos. All of these experiments con- firm the earlier results of Hahn (’09) on the origin of vessels in the chick. In the Teleost embryos studied during the present investi- gation there can be no doubt that the heart endothelium and aortae arise in loco within the embryo, and here there are no vessels, or even mesoderm, present on the yolk-sac in the an- terior portion. Certain vessels do partially grow from the em- bryo out on to the yolk-sac and other smaller vessels arise in many separate regions of the yolk-sac as the products of wan- dering mesenchyme cells which become arranged to form the tubular vessels. All of these vessels after they have arisen may grow by budding or sprouting off new vessels or may increase in length by a forward growth so well described in living embryos by E. R. Clark (’09, 712) in his careful studies of this subject. Felix (97) describes the origin of the aorta as follows: The ‘mesenchymaortenstrang’ arises from the two lines of sclerotoms after they are finally pinched away from the somites. No fusion of cell material occurs between this and the ‘venenstrang,’ the inter- mediate cell mass. This ‘mesenchymaortenstrang’ comes from that ¢ ORIGIN OF BLOOD AND ENDOTHELIUM _ i part of the somites that was immediately in contact with the inter- mediate cell mass portion of the primary seitenplatte. As the forward somites bud off sclerotoms, these also are added to the ‘mesenchy- maortenstrang.’ The median part of the ‘strang’ forms the aorta, ‘aortenstrang,’ the lateral the ‘mesenchymgewebe’ (mesenchymestrang). The ‘aorten- stranq’ is at first solid and does not obtain a continuous lumen to begin with, but here and there develops a space, and these spaces become confluent to form the tubes and build the paired aortae. Certain portions of the strang remain solid much longer than others. The association of the paired aortae to form an unpaired single vessel soon follows. While the aorta is being so formed, one never finds blood cells within its lumen. Blood cells occur only in the ‘venenstrang’ and in certain vessels of the nephric glomeruli. Occasionally certain of the glomerular vessels contain blood corpuscles at a time when the blood circulation is not yet closed. Felix (97) cites the observation of P. Mayer (’94) on very young Selachian embryos in which the medulary tube was still open. It was found in such embryos that the aorta is segmental and derived from the somites and subsequently the longitudinal tube is formed by the fusion of these isolated points. Felix agrees with P. Meyer’s observations from his study on the Teleost. There has been great diversity of opinion regarding the germ layer from which the vascular endothelium and blood corpuscles arise. In the literature it may be found that certain competent investigators have in each vertebrate class claimed the vascular endothelium and blood cells to be derived from the endoderm, while other workers of equal authority have found the vessels and corpuscles to arise from the mesoderm. The consistency of the disagreement which one finds in a review of this literature is most peculiar. These disagreements have their foundation in the extreme difficulty of the problem on fixed material. It is interesting to note that in no case has the same author derived the blood and vessel endothelium from different germ layers. Each author always takes the position that blood and vascular endothelium arise from either the mesoderm or the endoderm. We have here much to do with wandering cells which become lost from their epithelial layer, and it is impossible to state their: 78 CHARLES R. STOCKARD origin. This is left to the imagination of the individual investi- gator and further possibilities of error are open. Wenckebach’s (’86) observations of living embryos are most important in this connection. He noted that not only the layers but that independent mesoblast cells with-amoeboid processes wander out of the embryo and over the yolk. These wandering cells play a great part in the formation of the anlage of the heart endothelium and great vessels. In the Teleost embryo one may readily observe these wandering cells in the yolk-sac, and they doubtless give rise to the yolk vessels and blood islands as well as the pigment cells so abundantly present. Ziegler (87) has suggested that it may be that the blood anlage in phylogeny has been passed to the mesoderm from the endoderm, and for this reason the endodermal origin may sometimes occur in coenogenetic development. Goette (’90) also held that the endodermal origin of the blood was the more primitive one. This point of view overlooks the fact that in the invertebrates generally the blood and vessel walls are de- rived from the mesoderm. In discussing the question of the place of origin of the vessels, Felix (97) points out that Rickert (’88) claimed in Selachians, that the aorta arose in loco. P. Mayer and Strahl (95), have also stated that the great vessels are late in appearing and arise in loco in the embryo’s body. Felix states that the glomerulus of the bird mesonephros originates 7n loco independently of the aorta. Further that the stammvene, venenplexus of the mesone- phros, certain vessels of the glomerulus, and also the mesenteric artery along with the aorta in the Salmoniden arise in loco. Regarding the anlage of the heart and vena sub-intestinales, Felix is not certain but thinks that these hkewise arise 2 loco. All of these observations are directly opposed to the theory of ingrowth of vessels from the yolk-sac, the parablast theory of His (’75) as well as the outgrowth of vessels in the sense ad- vocated by Sobotta (’02). Ziegler (’89) and Felix (797) have both speculated considerably as to the relationship of the cavity of the circulatory system with the primary body cavity and the coelom. Ziegler pomted out ORIGIN OF BLOOD AND ENDOTHELIUM . 79 that in the phylogenetic origin of the blood vascular system we have the following changes: The primitive condition is repre- sented by the development of a space between the body wall, the ectoderm, and the gut wall, the endoderm, that is, the primary body cavity or protocoel. Embryologically the blasto- coel of the blastular or after gastrulation, the space between the invaginated endoderm and the ectoderm, the schizocoel, repre- sents the primitive vascular space. The body cavity in rotifers, nematodes, bryozoa and arthropods is a primary body cavity and is filled with a fluid, the haemolymph. In the arthropods on the dorsal side of the body is the pulsating heart which sets the fluid in circulation and this fluid contains corpuscles similar to the white blood corpuscles of vertebrates. Tn the arthropods the vessels and heart are often highly devel- oped but all communicate with lacunae and spaces between the gut wall and body wall. The heart is surrounded by a pericardial space (not truly coelomic) which is full of haemolymph, and as the heart pulsates this haemolymph is drawn in through ostia along its walls and then propelled out through the aorta and its arches to the vessels and spaces of the body. These body spaces, or the haemocoele, are thought by some to be a secondary or specialized cavity. Yet it is not coelomic and has no definite lining and resembles very closely the primary body cavity of the rotifers, nematodes, and other invertebrate forms which it most probably represents. In some of the higher Crustacea a secondary body cavity or coelomic space of limited extent is present enclosing the ophthalmic artery in Paelamonetes. The cavities surrounding the gonads are also coelomic, and since these are well developed species the coelomic space here prob- ably represents a progressive rather than a regressive condition. The second step in Ziegler’s evolution of blood vessels is illus- trated by the conditions in the molluscs. In these animals between the gut and body wall lacunae and interstitial spaces exist which occupy the position of the primary body cavity and these are filled with blood. Vessels lead into the lacunae and the cavities of these vessels as well as the cavity of the well formed heart are also considered to be part of the primary body 80 CHARLES R. STOCKARD cavity with which they arecontinuous. The pericardial cavity in the molluscs is true coelom and not a part of the primary body cavity and contains no blood. In almost all of the molluscs the pericardium is in communication with the nephridia and the nephric duct usually leads from the pericardium to the outer body- wall. The pericardial cavity in contrast to the primary body cavity is designated as secondary body cavity or true coelom. The final step in the phylogeny of the blood vascular system is characterized by an important expansion of the secondary body cavity or coelom as is the case in the echinoderms, annelids and vertebrates. As a result of the expansion of the secondary body cavity, the primary cavity is reduced merely to a system of channels or vessels and small interstitial lacunae. In the vertebrates, therefore, according to Ziegler, the blood and lymph vascular system represents the persistent part of the pri- mary body cavity. Ziegler considersthe blood vascular system and lymph vascular system to have had a common origin. The blood vessel endothelium is closely similar in all respects to the lymphatic endothelium. He thus agrees with Biitschli (82) that in all metazoa the blood vascular system has its origin from the blastocoel. Felix (97) holds that his studies on the Salmoniden will not fit into Ziegler’s scheme. He claims that the origin of the stamm- vene in the cranial portion is the same as that of the primary mesenephros in the caudal region, and is also of the same origin as that of the primary nephric duct. Cells of the splanchnic as well as cells of the parietal layer of the mesoderm enter into the structural material of the stammvene. The cavity of the venenstrang is the same as the cavity between the lamellae of the secondary lateral plate, that is, true coelomic cavity. The three structures referred to are all portions of the same base, the lateral plate mesoderm, the primary seitenplatte. Felix states, as there is little doubt that the cavity of the primary nephric duct is homologous with the secondary body cavity, so there is little doubt that the cavity of the venenstrang is also. The development of the aorta shows similar relations. It arises, according to Felix, from the sclerotomes which come from the ORIGIN OF BLOOD AND ENDOTHELIUM 81 somites and contains both the somatic and splanchnic layers of mesoderm. The origin of the aorta from the ‘mesenchymaor- tenstrang’ is from the same cell material as the mesodermal layers. The cavity of the myotom is secondary body cavity, coelom, and so also is the aortic cavity. Neither is in any way primary body cavity. The formation of the aortic cavity is a similar process to the canalization of the stammvene. Felix in this way arrives at a conclusion diametrically opposed to Ziegler. These conclusions he recognizes are not facts but are based on facts obtained from a study of Teleosts which are a side branch of the vertebrate stem, but from which one may still generalize to some extent. Felix calls attention to the fact that in the selachians Zeigler (92), and in the reptiles Strahl (’83, ’85), and in the birds KOlliker (’84) and Ziegler (’92), and in the mammals Kolliker (’84), all claim that the first vessel anlagen are found in the mesoderm and not between the mesoderm and endoderm. Only in the mesoderm the secondary body cavity arises by splitting, and since the solid vascular anlagen are formed within the mesoderm their cavities should not be considered primary body cavity. The writer is entirely unable to agree with such an analysis of the origin of vessels, particularly yolk vessels, as well as of the primary and secondary body cavities for reasons given below. Felix (97) now goes further and assumes that the lymph vessels arise in mesenchyme and their cavity is primary body cavity and their wall cells are modified connective tissue cells. This position is difficult to appreciate since it must be admitted that mesenchyme is a direct product of the mesoderm, and, according to Felix, any definitely formed cavity arising between such cells would seem to be coelom. I question, however, whether any other morphologist would put the same interpreta- tion on all the spaces cited by Felix as being in the coelomic category. Felix states, for example, that the aorta arises from a mesenchymaortenstrang derived from the sclerotom. The sclerotom is more or less mesenchymal in nature and certainly contains many cells which will later give rise to types of con- 82 CHARLES R. STOCKARD nective tissue. If the aorta did arise from this group of cells its cavity is scarcely of an origin comparable to that of the coelom. Its endothelial wall is certainly much the same as that of the lymph vessels. The cavities of the nephric duct, ovarian duct, kidney tubules and other tubules derived from the mesoderm are not usually considered to be parts of the coelomic cavity. The blood and lymph vessels do arise from the mesoderm but not in such a way that their cavity can be readily homologized with the coelomic space originating between the lamellae of the mesoderm. The vessels on the yolk-sac of the Teleosts are formed from discon- nected wandering mesenchyme cells which are easily demon- strated. The cavity of these vessels surely cannot be interpreted to arise between mesenchyme cells some of which are derived from the somatic and some from the splanchnic mesodermal layers. The yolk vessels in Teleosts arise by arrangement of mesenchyme cells and so apparently do other vessels within the embryo. Thus these blood vessels are similar in origin to the lymphatics according to Felix’s notion of the mesenchymal origin of lymphatics. The numerous recent investigators of the origin of the lymphatics, athough to some extent divided into two schools, all treat the lymph vessels and blood vessels as being of the same general genetic type Sabin (713) and Hunt- ington (714). Finally, the most damaging evidence against Felix’s notion that the blood vascular spaces are derived from the coelom, and that these spaces are actually now comparable to the coelo- mic space is the following: Before a true coelom, such as that to which Felix refers in the vertebrates, has arisen in the animal series blood vessels are already present and these vessels often communicate with or are actually a part of the primary body cavity. When the true coelom does arise in the invertebrate series blood vessels never open into its cavity or communicate with it. Felix has therefore derived an older and more general- ized animal system from a newer or later formation. This of course is contrary to any principle of phylogenetic calcula- tions. ORIGIN OF BLOOD AND ENDOTHELIUM 83. The weight of evidence at the present time is then in favor of the earlier notion of Ziegler. The blood vascular system if it is associated with, or phylogenetically derived from any other body cavity, that cavity is really the primary body cavity or embryologically the blastocoel. 4. Haematopoesis, the monophyletic and polyphyletic news, etc. The experiments recorded above are of particular value in the solution of that very complex problem, the origin and relation- ship of the different types of blood corpuscles. We may here then briefly consider the evidence they furnish in connection with the various theories and points of view recently advanced in explanation of the origin of blood cells. The vertebrate animals present two entirely different types of cells floating in their blood fluid. The white blood corpuscles are cells of primitive type and are not only found within the ves- sels but they also wander through the interstitial spaces of all the tissues of the body. These wandering white blood cells, amoebo- cytes, are almost universally distributed throughout the animal kingdom being found in all the invertebrate groups above the one or two very lowest as well as in all the vertebrate classes. In no animal do these cells contain haemoglobin, haemocyanin or any compound that would particularly qualify them as oxy- gen carriers, or give to them any function as an organ of respira- tion. These white blood cells found outside of the blood currents as well as in the blood are to be looked upon as cells which are not particularly associated with any specific blood function. They merely find the blood current a ready or rapid means of being carried from place to place within the body. The red blood corpuscles, erythrocytes, are in contrast to the white cells a very highly specialized type of cell and specifically a blood cell. In fact, this is one of the most specialized cells within the body. In mammals, for example, it is specialized to such a degree that its functional perfection is actually accom- panied by the loss of its nucleus and necessarily, therefore, the loss of its own future existence after a short period of time. 84 CHARLES R. STOCKARD Contrasted with the almost universal distribution of the white cells within the animal kingdom the erythrocyte is confined to the vertebrates phylum and to certain particular cases among the invertebrates. The respiratory function of the invertebrate blood is often claimed to be confined to the fluid or plasma mass, and only among certain members of the higher groups is a cell developed with the function of carrying oxygen to the body tissues and even this cell can not be said to possess the regular typical characters of the vertebrate erythrocyte. The vertebrate erythrocyte along with the typical verte- brate mouth, the pharyngeal gills, the dorsal nerve cord, the notochord, and bony skeleton and the many other possessions characterizing the vertebrate group, separates it in gulf-like fashion from the invertebrates. The white blood cells bridge this gulf but the red blood corpuscle differs from that of the invertébrate in a way comparable to the difference between the vertebrate mouth and that of the invertebrate, both serve the same function but are structurally unlike. Just as the mouth and pharyngeal gills and vertebral column have no invertebrate forerunner, so no cell within the invertebrate animals can at the present moment be sought out or designated as the certain an- cestor of the red blood cell. The cells of the vascular walls are closely similar in both vertebrates and invertebrates, as pointed out above. In both animal divisions they probably arise and develop in the same fashion. The white blood corpuscles probably do also. Yet the red cells, although they too originate from the mesenchyme in the vertebrates, are not in any way certainly descended from the invertebrate oxygen carrying cell or the wandering leuco- blast or amoebocyte. There is certainly no phylogenetic or comparative morphologi- cal evidence to warrant one in deriving vascular endothelium, leucocytes, and erythrocytes from a common cell ancestry ex- cept, of course, they are all derived from the mesenchyme or same germ-layer. The fundamental histological study of the early developmental stages of the blood elements in vertebrates was contributed by ORIGIN OF BLOOD AND ENDOTHELIUM 85 Van der Stricht (’94). His studies were especially confined to the mammals. As has often been the case the conclusions reached from this pioneer study are largely correct in the light of recent investigations. Van der Stricht held that the first blood cells arising within the area vasculosa are entirely young red cells, erythroblasts. When one surveys the literature of this subject, it is found that all authors with three or four recent exceptions (Bryce (’05), Dantschakoff (’07) and Maximow 09), hold that the blood islands give rise exclusively to red, haemoglobin bearing corpuscles, erythroblasts or finally erythro- eytes. This is true for the Fundulus embryos described in this paper and even though the cells are confined to their place of origin and never flow away, since there is no circulation, yet the groups always consistently contain only erythrocytes. Van der Stricht holds that the leycoblasts and leucocytes are independent of the erythroblasts and arise extra-vascularly in the mesenchyme and later wander into the vessels. Browning (’05) and Goodall (’07) have both recently claimed that the leucocytes have a different origin from the erythrocytes and arise at a later period. Goodall states: When leucocyte proliferation in the liver has begun, the islands of erythroblasts and leucoblasts are definitely separate in position, and the distinctness of their identity is obvious, and no transitions between them can be seen. These facts argue strongly against the view that the erythroblasts are derived from the primitive leucoblasts. Jolly and Acuna (05) have pointed out that in early stages only red cells are found in the blood. The first lymphocytes occur very late and still later the granulocytes, so that the guinea-pig embryo has attained a length of sixteen mm. before white blood corpuscles are present. Again all authors with few exceptions seem entirely agreed that the leucoblasts arise much later than the erythroblast. All without exception also agree that the leucoblasts arise extra- vascularly while the erythroblasts arise partially within the sinuses, and that the island groups of erythroblasts soon become surrounded by vascular endothelium while no vessel walls have ever been described to form around the groups of leucoblasts. These facts are no doubt of much genetic importance. 86 CHARLES R. STOCKARD The question involved is then: Which is correct, the mono- phyletic or polyphyletic theory of haematopoesis? It is recog- nized by all that both propositions are classed only as theory. It must further be recognized that both theories are based at the present time only upon the interpretations of various ob- servers, these interpretations are not necessarily facts. I trust, therefore, that the experiments on Fundulus embryos may add a basis of unquestionable facts which may show the correctness of one or the other of these interpretations. With this point of view, we may undertake a critical examina- tion of the evidence so ably presented by Maximow (’09) in his study on the mammalian embryo. The observations he con- strues as strong argument in favor of the monophyletic origin of all types of blood cells and vascular endothelium. This contri- bution by Maximow (’09) has been accepted by many embryolo- gists a bras ouverts, and has been largely incorporated into several recent chapters on the development of the blood, for example, by Schaefer (12), and Minot (’12). In the primitive streak stage of the rabbit embryo Maximow states that the peripheral mesoderm in which the blood islands will later occur has in no sense the character of a connected epithelial layer, but consists merely of local accumulations of cells of mesenchymatous type. The cells of this mass are of long thin spindle shape or with star-like processes. These cells are probably much of the same type as the wandering cells seen on the yolk-sae of the Fundulus embryos. In this peripheral mesen- chymatous mesoblast the first blood islands arise in the caudal part of the area opaca, as originally described by Van der Stricht. The blood islands are formed from the spindle or branched mesen- chyme cells which become associated into groups. Maximow states that the first endothelial cells like the pri- mary blood cells are also derived from the mesoblast-mesenchyme. Vith this one may fully agree and several other tissues could be included in the statement as derived from mesenchyme. Maximow, however, goes further and thinks this general source a common specific source. Thus the endothelial cells and blood cells are closely related and arise from a common stem cell in ’ ORIGIN OF BLOOD AND ENDOTHELIUM 87 the blood islands and may continue to arise from such a cell during later development. Die ersten Endothelien und die ersten Blutzellen sind also beides Mesoblast- resp. Mesenchymzellen. In den Blutinseln sehen wir sie von unseren Augen aus einer gemeinsamen Quelle entstehen. Auch in der spiteren Entwicklung werden wir oft Gelegenheit haben, die enge Verwandtschaft dieser beiden Arten von Mesenchymzellen zu _ beo- bachten. This is merely a matter of interpretation and not at alla demonstrated fact. In reply to such a position we must call for an explanation of the demonstrated fact presented on pre- vious pages showing that vascular endothelium forms in a per- fectly normal fashion within the heart and head region of em- bryos without circulating blood, but in no ease in early or late stages was the endothelial lining of the aorta or other vessels capable of giving rise to any type of blood corpuscles. Yet the power to form blood corpuscles was abundantly present in the “same embryos as shown by the huge numbers of blood cells within the blood forming regions, the intermediate cell mass and yolk islands. Why do not the mesenchyme cells within the liver and all vascular endothelium form blood when no cir- culating blood reaches them? (If ever, there should then be the stimulus to give rise to its formation). The red blood cell anlage is a definite mesenchyme cell or group of cells and only members of this cell group possess the blood forming power. To cite a parallel case, the liver cells are de- rived from the common endodermal cell stock yet not all early endodermal cells, in fact only a few, have the power to develop into a liver, or a pancreas or a lung as the case may be. The embryological argument is indeed rather loose that on account of the fact of vascular endothelium and blood cells arising from mesenchyme would assume, therefore, that these very different cells had a common stem mother cell and later actually pos- sessed some powers of transmutability. Maximow advances the interpretation that the first blood cells in the area vasculosa are not all erythroblast or future red blood corpuscles. These cells he designates as ‘primitive blood 88 CHARLES R. STOCKARD cells’ since they may form either white or red corpuscles. Yet in the yolk islands of the Fundulus embryos without circulation only red blood cells, erythrocytes, are produced and they remain in this location to be observed throughout embryonic life. The evidence for Maximow’s position seems to me somewhat insuffi- cient. During the summer of 1914, I had the privilege of examining Mme. Dantschakofi’s preparations which both she and Maximow cite in support of the monophyletic theory of blood cell origin. One so inclined might interpret these specimens as showing that the red and white corpuscles do arise from the common stem mother cell. The youngest lymphocytes were invariably scat- tered among the mesenchymal cells while the erythroblasts were budded off into more or less well defined vessels. No one could emphatically state that the two classes of blood corpuscles had ever actually divided off from any one single mother cell. The more or less constant separation of the early leucoblasts and erythroblasts, as is also shown in Maximow’s figures and those’ of other workers, would seem to indicate their origins from two different mother cells. If one mother cell only forms or divides off cells which develop into lymphocytes or leucocytes and another mother cell gives rise to only erythroblasts, then there is no reason to say that the two mother cells were the same although they appeared to be two similar mesenchymal cells. They were potentially different, and this potential difference is all that the diphyletic notion of blood cell origin demands. A careful study of the embryos without a blood circulation will demonstrate the fact of this different origin of white and red corpuscles. Maximow then advocates the last clause in the monophyletic code, and states that the intravascular primitive blood cells are not only increased by mitosis but are also added to by the pro- duction of the same kind of cells from the fixed endothelial wall of the primitive vessels. Endothelial cells may wander away into the mesenchyme or may wander into the vessel lumen. One often sees according to Maximow a cell project into the vessel, its body assumes a rounded form and its protoplasm ORIGIN OF BLOOD AND ENDOTHELIUM 89 changes into that of an erythroblast. It must be distinctly remembered that these appearances are in dead stained speci- mens and many possibilities exist which might explain their occurrence. Granting that such a phenomenon actually appears to occur there is one very probable explanation without assuming that true vascular endothelium may form blood corpuscles. Let it be supposed, for example, that in the formation of the vascular wall around the yolk-sac blood islands that some of the peripheral cells of the island might lag behind in their differentiation re- taining their more or less mesenchymal type. Such a cell may come to be closely pressed against the vascular wall and really appear as though it were one of the vessel wall cells. This might readily happen, and probably does happen, and may ac- count for the occasional appearance of ‘vessel wall cell’ forming a — blood cell. Why do not the endothelial cells in the experimental embryos possess the power to form blood cells when the vessel is totally empty of blood cells? Even though it is clearly shown that other cells of the embryo do possess the normal blood building power. These specimens are exactly such as should supply definite proof of blood cells arising from endothelium, but the evidence they furnish really disproves the proposition. Schridde (’07, ’08) according to Maximow has gone so far as to claim that in young human embryos endothelium can directly form primitive erythroblasts. Maximow does not agree with this since in his specimens the endothelium gives rise only to indifferent colorless cells. Shridde’s claim is based upon misinterpretation and so, I believe, is any claim that blood cells arise from formed vascular endothelium. Most authors find that in the very early embryonic blood there are no white corpuscles but only red cells present. Bryce (05) however, describes in Lepidosiren the very early origin of leucoblasts from primitive blood cells, and later Dantschakoff and Maximow find lymphocytes not only in the vascular net of the area vasculosa but also, though at first very few, in the circulating blood. Maximow thinks that when these early 90 CHARLES R. STOCKARD lymphocytes are not seen it is due to poor technique or defective material. Maximow believes the red blood cells may finally arise from lymphoblasts as erythroblasts, then erythrocytes. This mode of development of the definite erythroblasts continues through- out life and is accomplished in the same manner in all erythro- poetic organs. Wherever such indifferent mesenchyme cells, lymphoblasts, are found this locality is eo ipso a new place of origin of erythroblasts out of these colorless stem cells. If this be actually true, why then do not red cells, erythroblasts, finally form all through the body of non-circulating fish embryos, since the wandering lymphocytes surely have the power to reach many places other than the normal sites of erythroblasts formation, _ the intermediate cell mass and yolk islands? Maximow claims that both types of blood cells red and white arise at one and the same period from one and the same source, the primitive blood cells in the area vasculosa. The experiments on Teleosts do not bear out such a position since the original or first blood cells from the intermediate cell mass are all erythro- blasts and show a characteristic type at a very early time. The blood origin on the area vasculosa is not so extensive, but here also first form only erythroblasts. Supporters of the polyphyletic origin of blood cells have been able to make equally strong observations in favor of their view on similar material to that studied by Maximow, Dantschakoff and other advocates of the monophyletic theory. Maximow suggests that since the “primitive blood cell’ has no haemoglobin it really stands nearer to the leucocyte than to the erythrocyte, and one might say that the leucocyte arises first in development and the haemoglobin cell later. This is most decidedly not the case in the Teleosts where the primitive mesenchymal blood cell passes directly into the erythroblast without ever showing a stage suggesting either lymphoblast or leucocyte. With Weidenreich (’05), Maximow takes the position which he had earlier manitained that all non-granular leucocytes and also the wandering cells of the tissues constitute one great cell e ORIGIN OF BLOOD AND ENDOTHELIUM 9] group. The position determines the direction of their develop- ment and only in certain-places does one find all types being formed, for example, in the embryonic liver and the adult bone marrow. In reply to the extreme monophyletic position it may be asked: Why are only erythrocytes present in the old blood islands on the yolk of non-circulating specimens? Why is no cellular blood element present in the aorta and other endothelial lined vessels in the anterior region of similar embryos? Why are wandering ‘“‘primitive blood cells’ unable to form blood in the liver and other positions while blood forming power is present to a vigorous extent in certain regions of the same embryo but from a definite anlage? Wandering cells in the Teleost embryos have to do with yolk-sac blood origin, but these wandering cells are the equivalent of a part of the peripheral mesoderm and always wander out on to comparable regions of the yolk and never wander to other places within the embryo. Maximow probably criticizes correctly the many artificial dis- tinetions between various leucocytes which are pointed out by some advocates of the polyphyletic theory. My experiments do not bear on this point up to the present time. Finally Maximow does not imply that a granular leucocyte . or red corpuscle can change into anything else, they cannot un- differentiate. He states that before there is any development of granulation or haemoglobin in two different cells, there is really a difference in the cells though we are unable to distinguish it. This invisible difference determines the destiny of the cell to form either a leucocyte or erythrocyte. This is certainly true, but we cannot stop just at this point; these differences must be carried a step further or really back to their actual beginning. Then it is found that although two wandering mesenchyme cells on the yolk-sac of the fish embryo are indistinguishable so far as our powers of observation go, yet they are fundamentally different since one is destined to form only an erythroblast while the other possesses no such power and can form only an endothelial lining cell or pigment cell as the case may be. 92 CHARLES R. STOCKARD This is all the diphyletic or polyphletic school would ask. That is, that certain definite mesenchymal cells are actually the red blood cell anlage and only from these particular mesenchy- mal cells do red blood cells arise. Here we logically stop, for this is what is conceived by embryologists to be an anlage, back of this we go to certain germ layers and still further back to the developmental potentialities of certain individual cells as fol- lowed in studies of cell-lineage and finally we reach the elemen- tary proposition of deriving everything from the original egg cell. But to stop with the tissue anlage we find strong evidence to indicate that certain special mesenchymal cells are designated to form erythroblasts, others leucoblasts, and still others, and these are much more universally scattered throughout the embryo, give rise to vascular endothelium. These latter may really be admitted to form endothelium largely as a response to physical conditions. SUMMARY AND CONCLUSIONS The present contribution attempts an experimental analysis of the origin and development of blood cells and the endothelial lining cells of the vascular system. Studies on the origin of blood and endothelium in the normal embryo are rendered peculiarly difficult on account of the important role that wander- ing mesenchyme cells play in this process as well as the perplexing mixture of cells of different origin brought about by the early established circulation. The origin of no other tissue is so con- fused by mechanical and physical conditions. The first difficulty has been met by a study of living Fundulus heterochtus embryos with the high power binocular microscope. The wandering mesenchyme cells may in this way be followed to a great extent. The disadvantages due to the intermixture of cells in the blood current have been overcome by the investi- gation of embryos in which a circulation of blood is prevented from taking place. When Fundulus eggs are treated during early developmental stages with weak solutions of alcohol the resulting embryos in ORIGIN OF BLOOD AND ENDOTHELIUM 93 many cases never establish a blood circulation. In other res- pects these embryos may be very nearly normal and the develop- ment and differentiation of their tissues and organs often pro- ceeds in the usual manner though at a somewhat slower rate. The heart and chief vessels are formed and the blood cells arise and develop in a vigorous fashion. The heart pulsates rythmi- cally but is unable to propel the body fluid since its venous end does not connect with the yolk vessels and in many cases its lumen is partially or completely oblitered by periblastic material and nuclei which seem to be sucked into the heart cavity from the surface of the yolk. In these embryos without a mension of the blood one is -enabled to study the complete development of the different types of blood corpuscles in the particular regions in which they originate. There is no contamination of the products of a given region through the introduction of foreign cells normally carried in the blood current. The actual haematopoetic value of the different organs and tissues may be determined in the experimental embryos, and clearly distinguished from the ordinary reproduction or multi- plication of blood cells which in the normal embryo would reach these organs through the circulation. The debated question as to the production of blood cells from vascular endothelial cells may be conclusively answered, at least for the species here studied. The results and conclusions derived from these experiments may be summarized as follows: 1. The Teleost embryo is capable of living and developing in an almost normal fashion without a circulation of its blood. Red blood cells may be seen to arise and differentiate in these living embryos in two definite localities, one within the posterior body region, and the other the blood islands on the yolk-sac. The blood cells remain confined to their places of origin, yet they attain a typical red color and may persist in an apparently functional condition on the yolk-sac for as long as sixteen or twenty days. The normal embryo becomes free swimming at from twelve to fifteen days, but these individuals without a 94 CHARLES R. STOCKARD circulation never hatch although they may often live for more than thirty days. All recent investigators have claimed that there are no blood islands present on the Teleostean yolk-sac. Yet the presence of such islands is readily demonstrated in living Fundulus embryos, in normal specimens as well as in those with no cir- culation. 2. The plasma or fluid in the embryos failing to developa circulation begins to collect at an early time in the body cavities. The pericardium becomes hugely distended with fluid as well as the lateral coelomic spaces and the Kupffer’s vesicle at the pos- terior end of the embryo. The great distension of the pericardium due to this fluid accumulation pushes the head end of the embryo unusually far away from the surface of the yolk. The heart is thus stretched into a long straight tube or string leading from the ventral surface of the head through the great pericardial cavity to the anterior yolk surface (compare figures 15 to 20). No blood vessels form on the extreme anterior portion of the yolk-sac, so that the venous end of the heart is never connected with veins, and does not draw fluid into its cavity to be pumped away through the aorta. When the heart cavity does contain fluid it is unable to escape and small floating particles may often be observed rising and falling with the feeble pulsations of the heart. 3. The hearts in embryos without a circulation are lined by a definite endocardium, but the myocardium is poorly developed, sometimes consisting of only a single cell layer. Chromatophores are not present in the wall of the normal heart but in the experi- mental hearts these large cells ladened with pigment granules are invariably found. The cavity in many of the heartsis almost if not entirely obliterated by the presence of periblastic material and large amorphous periblast nuclei. The conus end of such hearts leads directly to a more or less closed ventral aorta, portions of the aortic arches are seen in the sections as open spaces, and dorsal aortae are almost invari- bly seen as typical spaces lined by characteristic embryonic endothelium. ORIGIN OF BLOOD AND ENDOTHELIUM 95 A point of much importance is the fact that neither these hearts with their endothelial linings nor any portion of the aortae at any stage of development have ever been seen to contain an erythroblast or an erythrocyte. Cells of this type are completely absent from the anterior region of the embryo. 4. Pigment cells normally occur on the Fundulus yolk-sac and arrange themselves along the vascular net so as to map out the yolk-sac circulation in a striking manner. Loeb has thought that this arrangement along the vessel walls was due © to the presence of oxygen carried by the corpuscles within the vessels. In the embryos without a yolk-sac circulation the pigment cells arise but rarely become fully expanded so that the usual long branched processes are represented only by short projections, the chromatophore consequently seems much smaller than usual. The unexpanded pigment cells, however, wander over the yolk-sae and collect in numbers around the plasma filled spaces. The yolk surface of the pericardium and the periphery of the Kupffer’s vesicle are often almost covered with pigment. The hearts are during early stages full of plasma and the pigment cells form a sheath around them, while pigment cells are never present on the normal hearts during the embryonic period. These facts would seem to indicate that the plasma rather than the erythrocytes contain the substance which attracts the chromatophores and initiates their arrangement along the normal vascular net of the yolk-sac. 5. A definite mass of cells characteristic of the Teleost embryo is located in the posterior half of the body between the notochord and the gut and extends well into the tail region. This so- called ‘intermediate cell mass’ is the intra-embryonic red blood cell anlage in many of the species. The peripheral cells of the mass as claimed by Swaen and Brachet or the mesenchyme about the mass, Sobotta, forms a vascular endothelium which encloses the central early erythro- blasts. In individuals without a circulation the erythroblasts arise in a normal manner in this centrally located position, and be- 96 CHARLES R. STOCKARD come erythrocytes filled with haemoglobin. Typical vascular endothelium completely surrounds the erythrocytes which instead of being swept away as usual by the circulating current remain in their place of origin. All of the early blood forming cells of this intermediate mass give rise only to erythroblasts. 6. Contrary to the opinion of most recent observers on blood development in Teleosts, the Fundulus embryos both with and without a circulation possess blood islands on the posterior and ventral portions of the yolk-sac. These blood islands are formed by wandering mesenchymal cells which migrate out from the posterior region of the embryo. They represent all that remains of the peripheral yolk-sac mesoderm in the Teleosts and probably wander way from mesoderm related to that of the intermediate cell mass. The intermediate cell mass may possibly represent the bulk of the peripheral mesoderm which is here included within the embryonic body, while in other meroblastic eggs it is spread out over the yolk. The only mesodermal portion of the yolk- sac in Fundulus is made up of the disconnected wandering mesenchyme cells some of which group themselves to form the blood islands, while others give rise to the yolk vessel endothelium, and still other wandering cells develop into the chromatophores. 7. The non-circulating red-blood corpuscles within the em- bryo remain in a fully developed condition for eight or ten days and then undergo degeneration. In an old embryo of sixteen days it is sometimes found that very few of the corpuscles in the intermediate mass are still present and these are degenerate. The vascular endothelium has been lost and numerous mesenchymal] cells have wandered in and lie among the corpuscles. On the yolk-sac the corpuscles no doubt have a better oxygen supply and here they maintain their color longer but finally also present a degenerate appearance with small densely staining nuclei and cell bodies much reduced in size. 8. Vascular endothelium arises in loco in many parts of the embryonic body in which blood cell anlagen are not present. This endothelium is in all cases utterly incapable of giving rise to any type of blood cell. This incapacity cannot be attributed to the abnormal condition of the embryo as true blood cell anla- gen in the same specimen produce blood corpuscles in abundance. ' ORIGIN OF BLOOD AND ENDOTHELIUM 97 Vascular endothelium in the fish embryo has no haemato- poetic function. 9. Neither lymphocytes nor leucocytes have been found to arise in the yolk-sae blood islands nor within the intermediate cell mass. The embryonic white blood cells are most abundant in the anterior body and head regions, and these cells occupy extra- vascular positions usually lying among the mesenchymal cells. The sources of origin of the white and red blood corpuscles in Fundulus embryos are distinct, and these two different types of cells cannot be considered to have a monophyletic origin except in so far as both arise from mesenchymal cells. The adult blood of Fundulus contains lymphocytes and several varieties of granular leucocytes. 10. There is evidence to indicate that definite environmental conditions are necessary for blood cell proliferation or multi- plication. Blood cells do not normally divide when completely enclosed by vascular endothelium. This is the key to the shifting series of so-called haematopoetic organs found during embryonic development. Erythroblasts lying about spaces unenclosed by vascular endothelium proliferate steadily and give off their products into the spaces from which they find their way into the embryonic vessels. Should such an erythroblast be carried by the circu- lation to another unlined space it may become arrested there and again undergo a series of divisions giving rise to other erythro- blasts. When, however, these spaces become lined by endo- thelium the blood cell reproduction stops. It most embryos the earliest blood cell formation occurs in the yolk-sac blood islands. The cells in these islands continue to divide until they become surrounded by endothelium, then the yolk-sae blood islands lose their haematopoetic function and become a vascular net through which the blood circulates. The liver now takes up the role of harboring dividing blood cells within its tissue spaces, when these spaces become vascularized by endothelium, here again the blood cells no longer multiply but merely circulate. 98 CHARLES R. STOCKARD Finally, in the mammalian embryo, one organ after another ceases to offer the necessary harbor for dividing blood cells until the red bone marrow is the only tissue presenting the proper relationship of spaces and vessels, and here alone the erythro- poetic function exists to supply the red blood cells for the entire body circulation. The red blood corpuscles are always produced so as to be delivered into the vessels and thus very soon occupy an intra-vascular position, while the white blood cells arise and remain for some time among the mesenchymal tissue cells in an extra-vascular position. 11. Lymphocytes and leucocytes along with the invertebrate amoebocytes are all generalized more or less primitive wander- ing cells, and are almost universally distributed throughout the metazoa. Erythrocytes are very highly specialized cells with a peculiar oxygen carrying function due to their haemoglobin content. - In contrast to the universal distribution of the leucocytes the erythrocytes are only found in the vertebrate phylum, except for a few cases existing in some of the higher invertebrate groups. Yet even in these particular cases the oxygen carrying blood cell never presents the typically uniform appearance of the verte- brate erythrocyte. The oxygen carrying function in inverte- brates is usually confined to the liquid plasma. Typical vascular endothelium is widely distributed in the animal kingdom and appears to be formed from a simple slightly modified mesenchymal cell. These three very different types of cells all seem to arise from mesoderm—the mesenchyme. Yet the present investigation would indicate that each arises from a distinetly different mesen- chymal anlage. The erythrocyte anlage is localized and perfectly consistent in the quality of its production. The lymphocyte and leucocyte anlage is more dimmeele ar- ranged and not definitely localized in any particular cell group. The vascular endothelium appears to be formed in loco in almost all parts of the embryonic body, and its formation is absolutely independent of a circulating fluid or the presence of blood cells. ORIGIN OF BLOOD AND ENDOTHELIUM 99 The facts presented seem to indicate that vascular endothelium, erythrocytes and leucocytes although all arise from mesenchyme are really polyphyletic in origin: that is, each has a different mesen- chymal anlage. To make the meaning absolutely clear, I con- sider the origin of the liver and pancreas cells a parallel case both arise from endoderm but each is formed by a distinctly different endodermal anlage, and if one of these two anlagen is destroyed, the other is powerless to replace its product. LITERATURE CITED Brownine, C. H. 1905 Observations on the development of the granular leucocytes in the human fetus. Jour. Path. and Bacter., vol. 10. Bryce, T. H. 1905 The histology of the blood of the larva of Lepidosiren paradoxa. Part II. Haematogenesis. Transact. Roy. Soc. Edin- burgh, vol. 41, Part II, no. 19. Biscuit, O. 1882 Uber eine Hypothese beziiglich der Phylogenetischen Herleitung des Blutgefiissapparates eines Theil der Metazoon. Mor- phol. Jahrbuch, Bd. 8. CuarK, E. R. 1909 Observations on living growing lymphatics in the tail of the frog larva. Anat. Rec., vol. 3. 1912 Further observations on living growing lymphatics: their rela- tion to the mesenchyme cells. Am. Jour. Anat., vol. 13. Cuark, E. R.,andE. L. 1912 Observations on the development of the earliest lymphatics in the region of the posterior lymph heart in living chick embryos. Anat. Rec., vol. 6. DantscHakorr, W. 1907 Uber das erste Auftreten der Blutelemente im Hiihnerembryo. Vorl. Mitt. Folia haematologica, 4. Jahrg., Suppl. no. 2. 1908 Untersuchungen iiber die Entwicklung des Blutes und Binde- gewebes bei den Voégeln. I. 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Rickert, J. 1887 Ueber die Anlage des mittleren Keimblattes die erste Blut- bildung bei Torpedo. Anat. Hefte. Bd. 2. 1888 Ueber die Entstehung der endothelialen Anlagen des Herzens und der ersten Gefissstiimme bei Selachierembryonen. Biol. Centralbl Bd. 8, nos. 138, 14. 1903 Ueber die Abstammung der bluthaltigen Gefabanlagen beim Huhn und iiber die Entstehung des Randsinus beim Huhn und bei Torpedo. Sitzungsber. der K. bayr. Akad. d. Wiss., Bd. 32. Sapin, F. R. 1913 The origin and development of the lymphatic system. Johns Hopkins Hospital Reports. Monographs, new series, no. 5. Scuarnrer, 2. A. 1912 Textbook of microscopic anatomy. Longmans, Green and Co., New York. 102 CHARLES R. STOCKARD Scumipt, M. B. 1892 Uber Blutzellenbildung in Leber und Milz, ete. Zeig- lers Beitrige, Bd. 11. ScHRIDDE, 1906 Uber Myeloblasten und Lymphoblasten. Verhandig. der Kongresses f. innere Medizin, Bd. 23, Vers., Miinchen. 1907-1908 Die Entstehung der ersten embryonalen Blutzellen des Menschen. Verhandl. d. deutsch. path. Gesellsch., 11. Tag. Dres- den, 16-19. Scouttr, H. von W. 1914 Early stages of vascologenesis in the cat (Felis domestica) with especial reference to the mesenchymal origin of en- dothelium. Mem. Wistar Inst. of Anat., no. 3. Senior, H. D. 1909 The development of the heart in shad (Alosa sapadissima) Am. Jour. Anat., vol. 9. Sosotra, J. 1894 Ueber Mesoderm-, Herz-, Gefiib- und Blutbildung bei Salmoniden. Verh. d. Anat. Ges. auf d. 8. Vers. zu Strassburg. 1902 Ueber die Entwickelung des Blutes, des Herzens und der grossen Gefiissstimme der Salmoniden nebst Mitteilungen iiber die Austildung der Herzform. Anat. Heft 63 (Bd. 19, Heft 3.) Stockarp, C. R. 1907 The influence of external factors, chemical and physical, on the development of Fundulus heteroclitus. Jour. Exp. Zool., vol. 4. 1915 An experimental study of the origin of blood and vascular en- dothelium in the Teleost embryo. Proc. Amer. Ass’n Anat., Anat. Rec., vol. 9. Srraut, H. 1883 Ueber die Anlage des Gefiisssystems in der Keimscheibe von Lacerta agilus. Marb. Sitzungsber. 1885 Der Parablast der Eidechse. Marb. Sitzungaber. 1888 Die Dottersackwand und der Parablast der Eidechse. Zeitschr. f. wiss. Zool., Bd. 45. Sumner, F. B. 1900 Kupffer’s vesicle and its relation to gastrulation and concrescence. Mem. N. Y. Acad. Sci., vol. 11. Swann, A., and Bracnet, A. 1899 and 1901 Etude sur les premiéres phases du développement des organs dévisés du mésoblaste chez les poissons Téléostéens. Arch. de Biol., T. 16 and 17. 1904 Etude sur la formation des feuillets et des organs dans le bour- geon terminal et dans la queue des embryons des poissons téléostéens. Arch. de Biol., T. 20. VAN DER Stricut, QO. 1892 Nouvelles recherches sur la genése des globules rouges et des globules blancs du sang. Arch. de Biol. T. 12. 1893 Sur l’existence d’jlots cellulaires 4 la périphérie du blastoderme de poulet. Anat. Anz. T. 8. 1895 De la premiére origine du sang et des capillaires dans l’aire vasculaire du lapin. Comptes rend. de la Soc. de biol. S. 10. T. 2. *1896 Origine des globules sanguins, de l’aorte et de |’endocarde chez les embryons de Sélachiens. Ibidem. ORIGIN OF BLOOD AND ENDOTHELIUM 103- VAN DER Strricut.O. 1899 L’origine des premiéres cellules sanguines et des pre- miers vaisseaux sanguins dans l’aire vasculaire, de chauves-souris. Bull. de l’Acad. Roy. de méd. Belgique. 8S. 4. T. 13. VIALLETON, L. 1892 Sur l’origine des germes vasculaires dans l’embryon de poulet. Anat. Anz., Bd. 7. WEIDENREICH, F. 1904-05 Die roten Blutkérperchen II. Ergebnisse der Anatomie und Entwicklunggeschichte von Merkel u. Bonnet, Bd. 14. 1905 Uber die Enstehung der weissen Blutkérperchen im postfetalen Leben. Verh. d. Anat. Gessellch., Bd. 19. Vers., Genf. WENCKEBACH, K. F. 1885 The development of the blood corpuscles in the embryo of Perea fluviat. Jour. Anat. and Physiol., vol. 19. 1886 Beitrage zur Entwickelungsgeschichte der Knochenfische. Arch. f. mik.. Anat., Bd. 28. Wiuson, H. V. 1891 The embryology of the sea-bass (Seranus atrarius). Bull. United States Fish Comm., vol. 9. ZEIGLER, H. E. 1882 Die Embryonalentwickelung von Salmo salar. Diss. inaug. Freiburg 1. B. 1887 Die Entstehung des Blutes dei Knochenfischenbryonem. Arch. f. mik. Anat., 30. 1888 Der Urspring der mesenchymatischen Gewebe bei Selachiern. Arch. f. mikr. Anatomie, Bd. 32. 1889 Die Entstehung des Blutes bei Wirbeltieren. Berichte d. natur- forsch Gesselschaft Freiburg i. B. Bd. 4. ‘ 1892 Uber die embryonale Anlage des Blutes dei Wirbeltieren. Ver- handl. der deutschen Zoolog. Gessellschaft. Zincupr, H. E., and Zircier, E. 1892 Beitrige zur Entwickelungsgeschichte von Torpedo. Arch. f. mik. Anat., Bd. 39. i ¥ ‘ , , u ‘A SS ae ey 2 to em iy nh AL ota Fa) oem y PARA 2 CONTENTS Is< ISAT NYO ACO) (tee eon e mictro Mo ctee era rama asa hncooo bOodoupeadaooder 105 ies VMiaterial andemethods of stuGyeo-s- cscs tee ae eee eee 108 HiileeRheveatly ra) 6) ExS 29.09 © S 0g08} * SO @) 05500 ase 50 0° » “o%o YQ 6) S04 | 50 re) alo Fig. 5 Camera lucida outline of the tail end and caudal yolk region of an embryo 48 hours old, fixed and cleared in glycerine-formalin. The germ ring just closing over the yolk pole, numerous mesenchyme cells beginning to wander away from the caudal end, huge periblast nuclei indicated in outline and stipple over yolk. Yk, polar bit of yolk just being covered by union of germ-ring border. Fig. 6 Portion of the caudal half of an embryo of 48 hours showing the first two pairs of somites recently formed, cells of the lateral plate mesoderm extend out upon the yolk as shown in outline, some beginning to wander away. Peri- blast nuclei outlined and stippled. Glycerine-formalin specimen. ‘ ‘DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 07 cell migration is taking place from this region. A few mesenchyme cells are found along the border of the head; these cells later take part in either the formation of the heart or pericardial wall. The tail end of the same embryo, figure 8, shows a remarkable contrast; here there is an enormous wandering out of cells from the mesoblast of the embryo. The two figures show the huge periblast nuclei to be widely distributed throughout the surface of the yolk sphere. These drawings are from cleared specimens and the cell outlines are more or less circular without the beauti- ful processes characteristic of the living. The tail end of a living embryo 72 hours old, some time before the blood began to circulate, is illustrated by figure 9. The circle beneath the tail reptesents Kupffer’s vesicle. The various shaped mesenchymal cells are represented in the act of wander- ing out over the nearby surface of the yolk. The embryo and yolk are beautifully transparent in life and the cells dre clearly seen as they move upon the surface of the periblast. An entire embryo, except the anterior portion of the head which extends beyond the curve of the yolk, is shown in figure 10 at a lower magnification. This specimen was 76 hours old when drawn. The heart had begun to contract slowly and feebly but no circulation of fluid had begun. Groups of mesenchymal cells are seen wandering away from the lateral and particularly the caudal regions of the embryo and are now scattered broadly over the yolk surface; there being very few, however, in the anterior region. The lateral plates of the mesoderm are seen at the sides of the head, and a circle at the caudal end indicates the Kupffer’s vesicle which is always clearly shown at this stage. In embryos of 72 hours, and somewhat earlier, there are wandering out from the tail region a number of cells slightly smaller than the two types mentioned above. These small cells tend to be more or less circular in outline but show slow amoeboid movement as they send out short blunt processes. They group themselves into small clumps and are to give rise to erythroblasts or future red blood corpuscles in the yolk-sae as shall be discussed beyond. Figure 31, page 569, shows six such cells from the living yolk-sae of an embryo 90 hours old; ; DEVELOPMENT OF WANDERING MESENCHYMAL CELLS’ 119 a circulation was partially established in this specimen but these cells had not yet been taken into the vessels. The various wandering cells then represent, the mesodermal layer of the yolk-sac in the teleost. They never assume a membranous layer-like arrangement, but finally differentiate into the characteristic structures of the yolk-sac. As is shown by the illustrations, these cells are very numerous and during their earlier stages are actively changing their shape and moy- ing over the yolk surface. We may now consider the further development of such cells the living embryos. Fig. 7 Outline of the head end of a 56 hour embryo, scarcely any wandering mesenchymal cells in this region. Large periblast nuclei scattered over yolk surface. Fig. 8 The caudal end of the same embryo; note the great contrast in the abundance of out-wandering mesenchymal cells. Glycerine-formalin specimen. Fig. 9 The caudal end of a living normal embryo of 72 hours, with beauti- fully delicate mesenchymal cells wandering away from the body; Kv., Kupffer’s vesicle (3b. 2/3 ob.). 120 CHARLES R. STOCKARD Fig. 10 A camera sketch of an entire embryo of 76 hours except the anterior end of the head. The mesenchymal cells wandering away from the tail region and to a less degree from the sides of the body. DEVELOPMENT AND DIFFERENTIATION OF THE WANDERING CELLS 1. Chromatophores a. The black type of chromatophore. The first to be considered of the four types of cells which develop on the yolk-sac are the black chromatophores. These are the largest and most con- spicuous cells of the yolk-sac. In the early stages discussed above, one notes even in embryos of 2 days that certain’ cells of the yolk mesenchyme are considerably larger than others. \ DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 12h These large cells may be followed through their development and they will be found to differentiate into one or the other of the two types of chromatophores. The amoeboid cell shown in different stages of movement already referred to as figure 4, from a 52 hour embryo, is of this large type, and concluding from my observations on great numbers of embryos, this is an early condition of the future black chromatophore before any pigment granules are deposited. Slightly older stages, figures 22 and 26, show the same cells containing a light amount of pigment granules. Between the second and third days the pigment granules appear and in an embryo 72 hours old, end of the third day, the chromatophores are already well differentiated freely moving huge cells. A black chromatophore from an embryo 72 hours old is shown in figure 11 with one of its processes overlying the body of a brown chromatophore, the type to be considered in the following section. The black cell is loaded with coarse granules. The nucleus occupies a central position and is clearly shown on ac- count of the displacement of the pigment granules by its trans- parent body. Several pseudopod-like processes project from the chromatophore which is actively moving. The clear cyto- plasmic tip of the pseudopod extends beyond the granular mass. Figures 12 and 13 are two other illustrations of the same cell after 15 minute intervals. Its shape is constantly changing and it is slowly moving in a direction towards the right side of the page. The brown pigment cell is also moving and their rates of progress are indicated by the increasing distance between them. This movement of the chromatophores continues until about the end of the fourth or middle of the fifth day in the normal embryos. By this time all of the black pigment cells of the yolk-sae with few exceptions have taken up more or less perma- nent positions along the walls of the blood vessels or around the surface of the pericardial space. The individual chromatophores have increased enormously in size as is seen by comparing figures 11, 12 and 13 with figure 14, all drawn at the same magnification, though figure 14 is one-third more reduced in reproduction. 22 CHARLES R. STOCKARD DEVELOPMENT OF WANDERING MESENCHYMAL CELLS ' 123 It must be appreciated, however, that some of the difference in extent is due to the flattening of the cells in figure 14. Figure 14 shows two huge pigment cells on the yolk-sac of a 5 day embryo in the act of arranging themselves along a vessel wall. The granules are not so densely arranged as in the younger stages, since the cell body is greatly thinned out in pressing around the vessel. A number of granules are often arranged in solid black lines and masses as indicated in the figure. The two cells are close together and a very peculiar phe- nomenon is taking place. Each cell sends out short processes to meet similar processes from its neighbor. The processes fuse, and finally the two cell bodies melt into one thus forming a pig- mented syncytium about the vessels of the yolk-sac. The syncytia continue to expand along the vessels as enclosing sheaths (fig. 15). The dense black of the young chromatophores becomes a steel grey as the granules are more thinly spread along the vessels. In order to test whether the cells had actually joined or fused to form a true syncytium, I attempted to contract them, think- ing that this should pull them apart unless they were actually united. The various solutions of KCl which Dr. Spaeth has found to contract the chromatophores within the embryo’s body failed entirely to produce any change in the chromatophores of the yolk-sac. Solutions of adrenalin of one to 1000, one to 10,000 and one to 100,000, which Dr. Spaeth so kindly supplied me, were then tried. These solutions contract the pigment cells on the brain of the embryo until they appear as small black dots, but neither the black nor brown chromatophores on the yolk-sac respond in the slightest degree. Such specimens were preserved to show the extreme contraction of the chromatophores over the brain of the embryo in contrast to the unchanged pigment cells of the yolk-sae. Fig. 11 A black and brown chromatophore lying in contact on a yolk-sac of 72 hours. The black cell is muchthe larger with broader pseudopod-like proc- esses; both are in active movement as shown by comparing figure 12, of the same two cells 15 minutes later and figure 13, the same cells 20 minutes after figure 125(3b: DDT ob»). 124 CHARLES R. STOCKARD From this it would seem as though the material of the chro- matophore had lost its contractile or wandering power after once becoming arranged around the yolk vessels. Those black chromatophores which retain their cellular individuality along the borders of the pericardial space also fail to contract when treated with KC] or adrenalin. Although this physiological test failed to serve the purpose for which it was used I feel certain, after many observations, that the black chromatophores actually do form true syncytial masses as they surround the vessels. b. The brown type chromatophore. The brown chromato- phores differentiate on the yolk-sac at about the same time as the black. They are always somewhat smaller and more deli- cately formed cells than the black, and react in a slightly differ- ent manner. Figures 22 and 26 show several brown chromato- phores before the end of the third day. They are paler in appearance and more elongate in shape than the black cells. The two types of cells are well contrasted in figures 11, 12 and 13 referred to above. The brown cell is smaller, with more deli- cate processes and is the more rapidly moving of the two. The three figures indicate its condition in embryos of 72 hours. These pigment cells also wander to the vessel walls and yolk spaces and take on their permanent condition about the fifth day. Figure 16 illustrates one of the exquisite brown pigment cells in a yolk-sac of 5 days. The nucleus is still distinguishable in life while it is not in the black cells of this age. The mossy branched processes projecting from all sides give to this cell a most fascinating form. Fig. 14 A camera lucida drawing of two huge black chromatophores lying upon a yolk vessel of a5 day embryo. The adjacent sides of the chromatophores are beginning to fuse to form a syncytium. The direction of blood flow is indi- cated by the arrows. Fig. 15 A syncytial mass of black chromatophores forming a sheath about the vitelline vessels. The chromatophores become so thin that the pigment granules are spread apart giving a less intense color. The individual cells are completely lost in the syncytium (3b. DD. ob.). DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 125 126 CHARLES R. STOCKARD ff Fig. 16 A brown chromatophore on the yolk of adday embryo. The cell is coming in contact with two vessels shown in outline. The moss-like processes extend from all sides of the cell. Fig. 17 A similar cell 12 days old surrounding a yolk vessel. The complex processes from this cell are quite in contrast to the almost smooth border of the black chromatophores of figures 14and 15. The brown cells never fuse to form syncytia. { DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 127 Finally, in older embryos the cell body often surrounds a vessel, as shown in figure 17, but the processes persist and pro- ject from it in all directions, forming a striking contrast to the more or less smooth outlines fnally assumed by the black cells, figures 14 and 15, as they surround the vessels. The brown chromatophores do not group themselves together or form a syncytial mass as the black pigment cells are prone to do. They remain individually separated and many really never become associated with vessel walls, but lie scattered on the yolk surface. In early embryos, from 72 to 90 hours, the brown pigment cells may sometimes, though rarely, get into the blood stream. I have never been able to observe one in the act of entering the current. Yet in a quiescent state they might become sur- rounded by endothelial cells along with the erythroblasts, and finally be swept away. They might, on the other hand, actually migrate through the porous wall of an early vessel. The enormous brown pigment cell presents a smooth circular outline as it is carried along in the blood current. On account of its size the chromatophore often meets with difficulties in passing narrow portions of the vascular system. Several such cells were seen in the blood circulation of different embryos during the course of the observations, and when once located in the current the same cell could be seen periodically for a long time as it came around again and again through the vesssel within the field of study. There is no question of the identity of these cells, as their characteristic reddish brown color and coarse granular struc- ture is readily recognized. It is most improbable to think of them as becoming changed into any type of blood corpuscle, and it is doubtless entirely by accident that they occasionally become entrapped within the vessel wall and washed away by the current. c. Behavior of the chromatophores in specimens with no cir- culation. The behavior of both the black and brown types of pigmented cell is distinctly different in embryos without a circu- lation of the blood from that described in the two previous sec- tions for normal embryos. 128 CHARLES R. STOCKARD During the early stages, up to the beginning of the fourth day, the cells wander in amoeboid fashion much the same as in ordi- nary specimens. In other words, at this time the condition is the same in all embryos since the blood has not begun to circulate in any. At about 72 hours the blood circulation begins in the normal embryos and the pigment cells seem to be attracted to the vessel walls, as already pointed out. If the circulation does not begin at this age, the plasma accumulates in various spaces, chiefly the pericardial sac and Kupffer’s vesicle at the caudal end of the embryo. The excessive accumulation of plasma in these spaces causes them to be in many cases hugely distended. The heart in such specimens also becomes a sacular structure filled with plasma which it is unable to pump on account of one or another deficiency in the vascular system. Large numbers of chromatophores of both types tend to aggre- gate about these plasma filled spaces and partially cover their walls. The spaces are thus rendered more conspicuous. In some specimens this coating of the distended plasma sacs by pig- ment cells is most remarkable, but such an arrangement is not invariable and in a number of individuals the pigment cells are irregularly scattered over the yolk-sac with no recognizable pattern or system. The heart of embryos in which there is no blood circulation is almost without exception covered with chromatophores. ‘These cells often form a perfect sheath about such hearts whether the heart is a plasma filled sac or a mere string. The patterns of these arrangements are illustrated by numerous figures, partic- ularly figures 15 to 20 in the previous paper. A point of much interest in this connection is the fact that the heart of the normal embryo is entirely free of pigment cells. The behavior of the chromatophores of the yolk-sac in normal individuals where they tend so decidedly to arrange themselves along the blood vessel walls along with their affinity for the plasma filled spaces in the non-circulating condition would seem to indicate that the chromatophore was attracted by the plasma itself, or some element which it contains. The distended plasma filled heart in the non-circulating cases is covered with pigment ¢ DEVELOPMENT OF WANDERING MESENCHYMAL CELLS ' 129 as would be expected, yet the solid string-like heart present in many such specimens is also covered with pigment though it of course is entirely empty of plasma. In this last case, how- ever, the string-like heart actually stretches as an axis through the pericardial space which is distended with fluid. The cells arrange themselves around the wall of the pericardium and on reaching the venous end of the heart migrate along it and so cover the heart string or tube in their effort to come in close proximity to the plasma. The distended condition of the pericardium may in this way account for the pigment arrangement along the heart in the cases with experimentally arrested circulation. The normal heart is constantly pumping the plasma through itself, yet pigment cells are never present in its wall since they are all arranged along the vessels of the yolk-sac. In non-cir- culating cases many vessels form on the yolk-sac and some be- come quite well developed, while others actually surround the blood corpuscles of the yolk islands. Such vessels are at times covered with pigment but probably through accident as the pigment is irregularly scattered over the entire yolk-sac. Yet the pigment cells on such vessels never arrange themselves in the definite sheath-like fashion characteristic of the vessel pig- ment in the normal embryo. Figure 18 illustrates the lack of arrangement of the chromato- phores on the yolk-sac of a 16 day embryo without a circulation; compare figures 15 and 17 of pigment in the normal embryo. Figure 35, see beyond, also illustrates in a striking way the irregular grouping of black chromatophores in the neighborhood of a collection of stagnant blood islands in an embryo of 14 days that never had a circulation of its blood. All of these reactions cause one to wonder what is the actual function of the pigment cells upon the yolk-sac. The entire egg is rather transparent and their function might be to protect the vessels from the light, yet the vessels are never completely covered and the development of the eggs in the dark is normal but not in any way supernormal. The pigment cells form such a complete sheath about the vessels in some cases that one might be led to imagine that their 130 CHARLES R. STOCKARD expansion and contraction would serve as vaso dilator and con- tractor. Yet when they are along the vessel wall I have failed to see them contract or expand even when treated with sub- stances such as KCl and adrenalin that violently contract the chromatophores within the embryonic body. These experi- ments have not been carried sufficiently far since they were directed towards another point, still they indicate at least that the pigment sheath of the vessel wall does not respond as a deli- eate vaso-motor apparatus. ie? eS Fig. 18 A group of brown, indicated in outline only, and black chromato- phores on the yolk-sac of a 16 day embryo in which the blood has never circulated. There is no arrangement of the pigment cells on vessels and no real syncytium of black chromatophores as compared with the conditions in the normal embryo. Wenckebach (’86) found in certain pelagic eggs containing a number of oil drops which invariably floated up that pigment cells often completely surrounded the oil globules, and as he thought prevented these globules from focussing heat or light on parts of the embryonic body. The oil drops in the demersal Fundulus yolk do not particularly attract the pigment cells and they are rarely found to lie against the oil globule. The function of the pigment in the yolk-sae of Fundulus, if it has any function other than its own existence, is most difficult to determine. The same is true of the abundant pigment in the coelomic wall and other internal structures of many animals. ’ { DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 131 d. Relationship of chromatophores to blood and_ endothelial cells. There has been much discussion in the literature regarding the relationship of the chromatophores to blood corpuscles and to endothelial cells. The actual relationship of these cells is clearly brought out by a careful study of the living yolk-sac in Fundulus. The cells are completely different and their struc- tures when once established are consistent in their particular type. The black chromatophores, the brown chromatophores, the endothelial cells and blood corpuslees are all derived from mesen- chymal cells which wander away mainly from the caudal region of the embryo during early stages of development. These cells come to lie in the primary segmentation cavity of the yolk-sac beneath the ectoderm and over the periblast syncytium. The mesenchymal cells very soon begin to show certain differential characters in structure and behavior. When certain ones of the cells incline in a definite direction their development progresses continuously along this line. Observations on the normal living embryos and comparison with those individuals without a yolk-sac circulation lead one to conclude as follows regarding the wandering mesenchymal cells. At the time these cells leave the embryo proper to wander over the yolk, differentiation has proceeded to some extent in the embryo and the mesenchyme cells are probably somewhat limited in their potentialities. Certain of them are derived from the same portion of mesenchyme that gives rise to the intermediate cell mass or future red blood cell forming mass within the embryo. This mass is located towards the caudal end of the embryo and the wandering cells derived from it finally come to lie on the pos- terior and ventral surfaces of the yolk-sac and form islands of red blood corpuscles. Few if any of these cells reach the anterior regions of the yolk-sac before the circulation of the blood begins. In embryos that never have a circulation the blood islands lie beneath the tail of the embryo and on the ventral yolk surface. The future endothelial cells wander out from the caudal end and side of the embryonic body and finally line up to form vessels in a manner to be described beyond. The pigment cells also wander out from the lateral regions and differentiate into chromatophores of either the black or brown variety. P32 CHARLES R. STOCKARD It would seem that these cells must have some potential differences at the time they come to lie in the yolk-sac, since from that time on they all appear to be in an identical environ- ment. ‘Two cells lying side by side in the yolk-sac above the periblast and beneath the ectoderm would be expected to de- velop and grow in similar fashion unless there were some internal difference between them. I have thus concluded that the mesen- chymal cells which wander in the yolk-sac of the Fundulus embryo must be potentially of four different classes when they first wan- der out, although all have the ordinary appearance of embryonic mesenchymal cells. Otherwise, it is difficult to conceive why they should develop into four distinct types of cells while all are surrounded by an identical environment so far as is possible to discover. Differentiation in various directions must be due either in the first place to similar cells developing in different chemical or physical surroundings, or in the second place it may result from potentially different cells developing under identical conditions. The four types of cells are all derived from mesenchyme, just as the thyroid follicles and pulmonary epithelium are derived from endoderm but from different endodermal anlagen, and further than this there is no relationship. Pigment cells and blood corpuscles are perfectly separate and distinct types derived from different mesenchymal analgen and are not in any way transmutable. 2. History of the endothelial cells The endothelial cells on the living yolk-sac of Fundulus em- bryos are readily recognized. Their entire behavior in the for- mation of the earliest yolk vessels may be traced in a manner to fully repay the patient observations necessary in order to follow through the processes. Among the early wandering cells migrating away from the lateral borders and caudal end of the embryo it is noted that certain ones assume a delicate spindle shape with filamentous processes,.extending from their ends and occasionally projecting DEVELOPMENT OF WANDERING MESENCHYMAL CELLS'- 133 from their long sides. In a 48 hour embryo these cells already present the appearance of individual endothelial cells. They migrate indefinitely for a few hours and then tend to group them- selves in more or less irregular collections. Up to this time no one from mere observation could be abso- lutely certain that the cells of this rather characteristic ap- pearance are actually to become vascular endothelial cells in all cases. The possibility, of course, exists that the elongate spindle cells may at times round up and then assume the more amoeboid shape of the probable future chromatophore. Yet since the shape of these cells is so characteristic and such shapes are so constantly present, one is inclined to believe that the same cell may actually retain this character until it really becomes a com- ponent part of a vascular endothelial arrangement. Figures 19, 20 and 21 illustrate the region along the side of the embryo’s head at 48 hours old. It is in this region that the first large yolk vessel develops. This vessel carries blood from the body of the embryo around a short circuit to reach the venous end of the heart and thus in a way relieves the flow that other- wise would force itself through the small poorly formed vessels in the embryonic body. This vessel is, therefore, of necessity one of the earliest to develop. The three figures, 19, 20 and 21, show variations in the arrangement of the wandering mesen- chymal cells in the region of the future vessel. In figure 19 there is really no definite cell aggregation except ° along the edges of the head mesoblast as it spreads somewhat over the yolk, yet a few of the cells show the typical spindle shape. Figure 20 indicates a tendency of the mesenchymal cells to line themselves in a group exactly along the course of the coming vessel. Many of the cells in this group give the actual appearance of an endothelial cell after it is fully developed and forming one of the units in a vessel wall. The embryo illustrated by figure 21 shows much the same condition. Very few mesen- chymal cells occur between this cell aggregation and the side wall of the head. Lateral of the vessel group the cells are also not numerous and have no system of arrangement. 134 CHARLES R. STOCKARD 9° ie} BS Ke: IN Ss fF te) Oo 270 >, OF QQ, xe 0? Se OpoG se a8 PPA P| 1S: Q ao —- g Figs. 19, 20 and 21 Outlines of the head regions of three living embryos from 48 to 50 hours old, showing different conditions in the grouping of mesenchymal cells on the yolk which later give rise to the large vessel that short circuits blood from the side of the embryo around over the yolk to the venous end of the heart. The future vessel wall is now separate spindle shaped mesenchyme cells. This cellular aggregation may then be regarded as the actual anlage of the vascular endothelium of the future vessel. The anlage consists merely of a group of separate wandering mesenchyme cells, and not of a capillary net in any sense. — \ DEVELOPMENT OF WANDERING MESENCHYMAL CELLS’ 135 A slightly older embryo shows a still more definite alignment of the mesenchymal cells and still later presents the appearance of cellular strings or cords as illustrated inan embryo of 67 hours by figure 22. Here the wandering mesenchyme cells have differ- entiated to such an extent that they are readily distinguishable as black and red chromatophores and elongate endothelial cells. Fig. 22 A sketch of a 67 hour embryo showing the stage in the origin of yolk vessels in which the mesenchymal cells have a linear arrangement. Early black and brown chromatophores are also shown in the yolk-sac. Od, oil drops. Early erythroblasts are also seen on the caudal region of the yolk- sac in such an embryo, but are not shown in the aspect here illustrated. The endothelial cells are strung out in various directions and several linear groups are more or less isolated from the rest. The string to be the future large vitelline vessel is not clearly continu- ous posteriorly, but anteriorly it is well outlined extending to- wards the venous end of the now forming heart which has not yet 136 CHARLES R. STOCKARD begun to pulsate. Here there is no further doubt that these elongate spindle cells are the elements which will make up the endothelial lining of the vessel wall. There is considerable variation in the rate of development of the yolk vessels in embryos of the same number of hours. Some individuals may be in the condition just described, while others of this age may have already begun to establish a circulation of the blood. Figure 23 shows the yolk region lateral of the head in another embryo at 67 hours. In this specimen an incipient cir- culation has begun and the cord of cells illustrated in figure 22 has now become a small hollow tube sufficiently open to allow the passage of a single file of corpuscles from the side of the embryo around to the venous end of the heart. The individual cells composing the vessel are distinctly seen and their nature is clearly made out with a higher magnification. They retain the same general appearance presented before entering into the vascular arrangement. Near this vessel is shown in figure 23 a partially formed vas- cular plexus which is broken in several places and entirely dis- connected from the large vein through which the blood is flowing. There is of course no circulation of fluid in this partially formed plexus. Figure 23 was sketched with a camera lucida at 12 mM. and about three hours later at 2.45 p.m., figure 24 was sketched from the same field. The main vessel in accord with Thoma’s (’93 and 96) first law of vessel growth has increased in calibre on account of the increased flow and pressure of the circulation. It now permits the passage of three or more corpuscles abreast and is a strongly developed vessel. The former disconnected vascular plexus has grown towards the large vessel and two of the projections shown in figure 23 have now met the wall of the vein and joined with it. One of the first corpuscles from the circulation to enter the plexus is shown in the figure tightly held in the small vessel. Immediately opposite this vessel a sprout is Seen growing away from the wall of the large vein. Figure 25 illustrates the state of arrangement at 6.00 P.M., three hours older than figure 24. The third process from the ‘ DEVELOPMENT OF WANDERING MESENCHYMAL CELLS’ 137 Fig. 23 The large vitelline vein in an embryo of 67 hours just beginning to permit the passage of blood through its lumen. Corpuscles moving in single file. This becomes the largest vessel of the embryo and arose from the arrange- ment of the freely wandering mesenchymal cells of figures 19, 20 and 21. In the 138 CHARLES R. STOCKARD plexus has here joined the vein and corpuscles are freely passing into the vessels. The sprout from the vein wall is still seen oppo- site the entrance of the middle vessel. The small plexus arose in loco entirely independently and subsequently became connected with the larger vessel which also arose as we have seen from a group of mesenchymal cells. Figure 26 shows another lateral view of the head region of an embryo of 67 hours in which the large vein is filled with circulat- ing corpuscles and the beginning of the same plexus followed in figures 23, 24 and 25 is seen lateral of the vein. At this stage the plexus is entirely disconnected and separated from the vein. In the formation of the large yolk-sac vein, as well as all other vessels arising upon the yolk, there is nothing to be seen of the forerunning capillary plexus so strongly emphasized by Thoma in the yolk-sac of the chick. There is no selection and dilation of certain channels in the capillary plexus of the teleost’s yolk- sac to form the veins. Here the veins seem to arise in rather definite localities and soon expand into their full form after the circulation has become established. This method of the formation of vessels was beautifully brought out by Wenckebach (’86) in the early study already referred to so often. He concludes: ‘‘Aus diesen Beobachtungen geht hervor, _ dass Mesoblastzellen durch selbstaindige amoeboide Bewegungen die Winde der Blutgefiisse des Dotters bilden.’”’ Raffaele (92) later confirmed this observation and further was strongly of the opinion that in selachians and other vertebrates a similar process of vessel building from wandering cells also takes place. From my present studies on the normal and abnormal Fundulus embryos, I can see no way to doubt that the endothelial wall of the primary yolk vessels in the bony-fish is formed by arrange- ments of wandering mesenchymal cells. lower part of the camera sketch is shown an independent capillary plexus not yet connected with the vein. Ht, heart. Fig. 24 The same vessels 2 hours and 40 minutes later. The main vessel has increased in caliber and two branches of the capillary net have joined the vessel. Fig. 25. The same vessel three hours later, a sprout is given off opposite the union with the middle capillary and corpuscles now enter all three capillaries. The arrows indicate the direction of blood flow. DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 139 Wenckebach’s description cannot be fully agreed with in all detail. He thinks, for instance, that cells forming part of the vessel wall are brought by the blood stream. These cells have small protoplasmic processes but they are not in any way to be confounded with the ‘‘definitiven Blutkérperchen.”’ Such cells have never been observed in the Fundulus embryos and if they exist, which is very improbable, their part in vascular formation is extremely insignificant. Wenckebach observed the three primary vessels on the yolk to bud and give off sprouts forming other vessels. The wander- ing cells also formed small separate tubes which later became connected to form a portion of the vascular net. By these methods the complex vascular net of the yolk-sac was finally formed. This agrees closely with what may actually be seen to occur in the embryos of Fundulus. Considerable variation occurs in the position of vessels and a number of actual abnormalities are found in which the bilateral arrangement is completely disturbed. These abnormalities are frequently very instructive for a thorough understanding of the origin and development of the yolk-vessels. Occasionally, a group of cells will form a completely isolated endothelial space which may fail to associate itself with a vessel. Figure 27 shows such an isolated space in a yolk-sac of 90 hours old; solid endo- thelial tips project from the space, yet it is completely isolated so far as can be determined with the highest power, and at the same time every part of it is clearly and distinctly seen. When the early yolk vessels are studied under high magni- fication, the individual cells may be clearly observed and they are strikingly the same as before they became associated to form the vessel. The cells are not closely arranged but distinct inter- vals and spaces exist between them and filamentous processes often project far into the lumen and may actually at times fuse with a similar process from a cell on the opposite side of the wall. These filaments thus stretch across the vessel and may even persist after the blood has begun to flow. They are well seen by focussing so as to get an optical section through the cavity. Cor- 140 CHARLES R. STOCKARD as) 27 Bs Fig. 26 Outline of the yolk vascular condition in the head region of a 67 hour embryo. Blood is circulating freely in the large vessel but the yolk net of ves- sels is not yet connected with the current. Early black and brown chromato- phores are also shown. Fig. 27. An isolated endothelial cavity with solid projecting tips, no con- nection can be seen with any other vascular spaces. From an embryo of 90 hours. DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 141 puscles often strike against the cell processes and cause them to wave back and forth as the current flows past. The cells of the vessel wall thus maintain much of their indi- viduality and may actually separate themselves or loosen away from the small growing tip of a vessel. The tips of the vascular sprouts probably break up or disassociate in this manner to include small groups of corpuscles which may be seen to enter the vessels from the yolk surface. A most instructive specimen for a study of the cellular elements of the vascular endothelium is one in which the circulation has just begun. The vessels in such an embryo are still growing in length and sprouting off branches rather actively. Figure 28 illustrates such a vessel with its incipient branches. Corpuscles are passing through the vessel in the plasma current; one of these, X, is seen harbored behind an endothelial cell at the base of the outgrowth to the right. This corpuscle remained in position for more than one hour, being protected from the current by the projecting endothelial process. Such a condition is frequently seen and conveys some idea of the actual irregularity of the vascular wall. The cells constituting the walls of the outgrowths from the vessel are changeable in shape and doubtless move their positions to some extent. The cells at the tip of the growing sprout may be seen to send out processes as if they were actually creeping along. The behavior of these vessel walls is strikingly similar to that which Clark (’09 and 712) has so clearly described for the growing lymphatics in the tail of the tadpole. Figure 29 shows a similar vessel with a projecting bud. The cells of this bud are seen to exhibit a most indefinite arrangement; their processes project across the space and join the cells of the opposite side and none are completely elongated or flattened as are the cells of the main vessel wall. The tip cells might still be described as stellate mesenchymal cells. The appearance shown in figure 30 is much the same. The walls of these early vascular buds are extremely loose membranous arrangements with irregularities in their surfaces and openings and spaces between the cellular components. 142 CHARLES R. STOCKARD Figs. 28, 29 and 30. Portions of vessels from three yolk-sacs of 6 day embryos. The vessels show blind endothelial sacs projecting from their walls. The con- stituent cells of these sacs are distinctly seen, and still retain their wandering mesenchymal characters. Filamentous processes from these cells may extend entirely across the lumen and fuse with processes from the cells on the opposite side of the wall. Corpuscles are often entangled in the filaments as well as the spaces between the endothelial cells. X, a resting corpuscle harbored behind an endothelial projection. These porous or incomplete endothelial walls permit the blood cells to occasionally escape from the vessel cavity and become free within the space of the yolk-sac; or, on the other hand, a growing vascular tip may be observed at certain stages to come in contact with a group of erythroblasts, or actually a blood island unsurrounded by vascular endothelium. The tip of the ¢ DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 143 vessel seems to disorganize to some extent and its cellular ele- ments slowly surround the group of corpuscles which are later taken into the circulation as the current becomes established in the including vessel. Unfortunately, I have never been able to observe the con- secutive steps in any one case of this kind, so that an absolutely positive statement cannot be made at present. Yet numerous observations of the contact of vascular sprouts with groups of uncovered corpuscles and the ends of such sprouts containing corpuscles, as well as other arrangements, would indicate that the behavior of the endothelium in surrounding the naked groups of erythroblasts on the yolk-sae probably takes place about in the manner just outlined. Figure 32, page 568, illustrates a highly magnified field on the yolk-saec of a 90 hour embryo. ‘This field shows a very interest- ing composite of the vascular condition at such an age. The rapidly flowing blood current is freely passing through the vessel on the right. A short circuit is forming across below the curve of an arch in the vessel. This small vessel permits only a single line of corpuscles to pass. At this time only one cor- pusele has entered and it is caught in the narrow tube. This corpuscle remained fixed for a long time and so enabled a com- parison of its structure and appearance with the erythroblasts forming the group just below the huge black chromatophore. The cells of this group are uncovered by endothelium. On the left of the figure a portion of a vascular net not yet connected with the circulating current presents the typical appearance of an early blood vessel formation. The individual cells are loosely associated and the tip projecting towards the right slowly changes position. This tip later approached the group of erythroblasts and finally these cells were all included within the vessel by a process which, as stated above, I was not able to follow definitely. After closely studying these early vessels in a large number of living yolk-saes, the observer is able to establish very clearly the actual relationship between the vascular endothelial cells and the erythroblast or early blood corpuscles. ‘The corpuscles on the yolk are always of a distinctly different shape and size, and 144 CHARLES R. STOCKARD lie, as described below, in small groups with originally no endo- thelial cells around them. The groups are later either sur- rounded by endothelium or taken into the early vessels as already indicated. Nothing has been observed during a long study of these cells on the living-yolk sac of normal embryos with a free circulation, or on the yolk-sacs of embryos experimentally pre- vented from establishing a circulation, or finally in sections of embryos of various ages, that would indicate even a tendency of endothelial cells to change into any type of blood corpuscle. The endothelial cell, whether in the free and wandering mesen- chymal state or constituting a part of the vessel wall, presents a typical shape and clear appearance entirely distinct from the wandering erythroblasts. In observing the early yolk vessels certain things may be seen which are most important in interpreting sections supposedly showing the transition of vascular endothelial cells into erythro- blasts or primitive blood cells. Frequently, one or more cor- puscles become entangled within the spaces and filaments existing between the cells of the vessel wall. Other corpuscles flowing in the current strike these entangled ones and beat against them sometimes for hours before they become disentangled from the vascular pits and holes, to flow again in the current. This is an extremely common sight during the early hours of the circulation of blood in any of the yolk vessels. Tt may now readily be imagined that if the embryo was killed and fixed while the corpuscles were tangled in the spaces of the vascular endothelium, a study of sections might produce the impression that the cells of the vessel wall were “‘ protruding into the lumen and assuming the typical characters of primitive blood cells,”’ according to the description of many that imagine the occurrence of such things. I have previously offered another explanation of these appearances and many phenomena observed on the living yolk-sac bear out the point of view. It may some- times happen when the vascular endothelium encloses a group of primitive corpuscles that one or more of these future blood corpuscles come to lie in the plane of the vessel wall, or may actually seem to form one of the cellular components of the wall. ¢ DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 145 When the circulation begins, however, this cell becomes loosened away from the wall for mechanical reasons, the lack of long processes, etc., and projects into the lumen to be finally washed away. Any one may readily observe such occurrences who will study the living yolk-sac of Fundulus with a high power microscope and a strong condenser so as to use a darkened field. All of these observations lead one to conclude that the only connection between vascular endothelium and primitive blood cells is one of association. The endothelial cells never meta- morphose into blood cells. It is important here to recall the fact previously emphasized by the author that in those speci- mens in which there is never a circulation of the blood or plasma the vascular endothelium develops in a perfectly normal manner in the aorta and other intra-embryonic vessels, as well as in the vessels on the yolk-sac, yet in none of these does one find any appearance indicating a tendency of the lining cells of the vessel wall to change into any type of blood cell. Numerous other details from my notes might be enumerated which would bear on this question, but sufficient care has been taken to definitely establish the above crucial points as facts. This may not of course hold for all types of animals but it does for those studied. I have seen a number of sections on which other investigators have based their claim that vascular endothelial cells do change into primitive blood cells and although inclined towards the acceptance of such a view from a mere acquaintance with the literature, a study of such material has convinced me that the negative interpretation is equally plausible in all cases. 3. Blood corpuscles on the yolk-sac of teleost embryos In all meroblastic eggs except those of the teleost a great sheet of mesoblast is found extending over the yolk as the so- called peripheral or ventral mesoderm, or subvitelline mesoderm. It is this peripheral mesoderm that gives rise to the blood islands of the yolk-sac in selachians, reptiles, birds and mammals The teleost, however, presents a unique case in that the ventral mesoderm does not spread out over the yolk but is included 146 CHARLES R. STOCKARD within the embryonic body. The differentiation of this mesoderm within the embryo is much the same as that of the peripheral ventral mesoderm in the yolk-sac of other groups. Thus in the bony-fish the great bulk of blood formation takes place within the so-called intermediate cell mass, the probable homologue of a portion of the yolk-sac mesoderm of other vertebrates. The intermediate cell mass first described by Oellacher (73) and later fully studied by Ziegler (’87), Swaen and Brachet (’99, 01), and numerous others, is derived from the primary lateral plate mesoderm, separating away from the median border of this plate. The cell masses of the two sides remain apart in some species and form the future cardinal veins and red blood cor- puscles, while in others the two masses unite in the median line to form the conjoined cardinals or stem vein, which is loaded with the primitive erythroblasts—the red blood anlage. All recent workers on the development of the blood in the bony-fish have considered this intra-embryonic blood formation as being the only source of blood cells in these animals. Several authors, however, Swaen and Brachet among them, have recog- nized that the cells of the stem vein may become so packed and crowded within the embryo that masses of them are directly pushed out laterally upon the yolk. They have also thought it possible that a very few cells might wander upon the yolk- sac and there form blood, but this has been considered question- able in all cases. No one has recognized the actual occurrence of blood islands upon the teleostean yolk-sac. Even Wencke- bach in his study of living embryos, although he made so many important observations on the formation of the periblast and yolk vessels, entirely overlooked the early or primitive yolk-sac blood cells. This is probably due to the fact that he studied only normal embryos. In embryos without a circulation of the blood one observes the yolk islands much more readily as the cells finally become filled with haemoglobin and present a bright red color. When they are once located in these experimental embryos it becomes much easier to trace them in the younger normal individuals, and finally the observer readily locates these cells and may follow completely their migrations and association to form the yolk-sae blood islands. ' DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 147 The arrangement of these wandering cells in the yolk-sac, figures 7, 8 and 10, suggests in a way the regions of growth of the yolk-sac mesoderm in other meroblastic eggs. About the caudal region there is an ‘area opaca’ formed by the great number of wandering cells, while around the head end the scarcity of mesenchymal cells might be considered an area pallucida. All of the yolk-sac blood islands in the Fundulus embryos are formed from certain of the early wandering mesenchymal cells on the yolk. During the early wandering stages when the future endothelial cells have a spindle shape and the future chromato- phores are large amoeboid cells, other mesenchymal cells on the yolk are small and more or less circular in outline. These small circular cells move slower than the other types and throw out short thick pseudopod-like processes. Whereas the spindle shape cells wander away from the embryo along its entire lateral border as well as the caudal end, and the large amoeboid future chromatophores have almost an equally extensive place of origin, the small round cells wander out only from a limited region. The earliest ones of these to be seen are near the caudal end of the embryo before the tail fold has com- pletely separated the caudal end from the yolk surface. As the tail is moulded free from the yolk-sac, the point of outwandering of the circular cells follows the place of union between the ven- tral wall of the tail and the yolk-sac. Just at this place the mesenchyme of the embryonic body extends itself out on to the yolk as free wandering cells. In a study of sections this mesenchyme is found to lead directly to the end-bud, Endknospe, which may be considered to represent the blastopore lip. The cord of mesoblast which has been des- ignated as intermediate cell mass leads caudally to the end-bud which is well out in the tail. The ventral cells from this mass wander away into the yolk-sac from the extreme caudal position and other cells also wander away laterally from the intermediate cell mass. Figure 8, the tail end of an embryo 56 hours old, illustrates very well the place of outwandering of the round cells. Few, if any such cells wander out from the lateral borders of the embryo in regions more cephalad than this. 148 CHARLES R. STOCKARD This confined local origin of the round cells and the period at which they wander out, along with their general appearance, lead one to believe that these cells are actually derived from the same general mass or group of cells which goes to form the intermediate cell mass or red blood anlage within the embryo. All of these slowly wandering circular cells finally differentiate into red blood corpuscles, as described below, just as cells of the intermediate cell mass will finally do. In this connection a most instructive defect is frequently found among embryos developing in the stronger solutions of aleohol. Many such embryos are of the common short type with their tails split, cauda-bifida, figure 4 of the previous paper. This defect is due to the fact that the germ-ring descends over the yolk in a slow or arrested fashion and may never succeed in fully enclosing it. The caudal end of the embryo is thus divided and the two tail moieties remain spread apart laterally along the line of the germ-ring. This condition renders the caudal portion incapable of including all of its usual median tis- sues and such cells extend past the angle of the split and lie between the two parts of the bifid tail. The interesting thing is that the cells constituting the blood-forming intermediate cell mass lie in just this position. Figure 33, a diagram, illustrates the embryonic body with a bifid caudal end. The great lake of blood corpuscles is situated beyond the angle of the split tail. Such an abnormality liber- ates the future blood forming mass from the body of the embryo and the mass spreads posteriorly over the yolk surface, yet not in a diffuse manner since it maintains its densely packed cellular structure. We might consider that here the evolutionary events are reversed. The blood anlage in the primitive fishes, the se- lachians, is spread over the yolk in the area opaca. In the nor- mal teleost this primary yolk-sac blood anlage has been included within the embryonic body and localized in the intermediate cell mass. While in the abnormality here considered the inter- mediate cell mass is again outspread upon the yolk somewhat suggestive of the old ancestral selachian arrangement. This abnormality, in other words, may give some notion of the actual ‘ Fig. 31 A group of six early erythroblasts unsurrounded by endothelium on the yolk-sac of a 90 hour embryo. Short amoeboid processes project and the cells move very slowly. Fig. 32 A camera lucida sketch of a microscopic field on the yolk-sac of a 90 hour embryo. All four derivatives of the wandering mesenchymal cells are shown. The circulation is established in the vessel to the right and the current follows the direction of the arrows. To the left is a small vessel not yet connected with the current; its wandering endothelial tip is approaching a group of erythroblasts still uninclosed by endothelium as they lie near the huge black chromatophore. A brown chromatophore is seen on the large vessel. 149 150 CHARLES R. STOCKARD Fig. 33 An outline of a short 6 day embryo from a strong alcohol solution. The embryo presents a cauda-bifid condition and the normally intra-embryonic blood-forming mass is represented by a densely packed expanse of corpuscles out- side the body of the embryo and spread upon the yolk. incorporation of the primitive blood-forming mesoderm of the yolk-sac into the body of the teleost embryo. The situation may be pictured as follows. In the reptiles and birds, for example, the peripheral mesoderm is outspread over the yolk and in it differentiates the blood islands of the area vasculosa. The peripheral mesoderm in Fundulus and teleosts generally does not become outspread over the yolk, but is con- centrated into a median cord or mass within the caudal half of the embryo. Yet even here there is actually a tendency for the cells of this mass to be attracted to the regions of the yolk- sac, and during the early stages of development many cells sepa- e DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 151 rate from the mass and wander freely on the yolk. The extent of such wandering is probably variable in different species. Yet in all eggs with an extensive vitelline circulation the wandering of these future red blood cells probably takes place to a con- siderable extent in spite of the fact that the cells have been so generally overlooked. A fact easily accounted for when one realizes that most of the studies in blood origin in teleosts have been confined to sections of the embryos of the salmon and trout. Sections are extremely slow in revealing the significance or even existence of wandering cells in development. We may now consider the individual wandering cells and their subsequent differentiation. Figure 31 shows a group of six such cells. They are small when compared with the huge chromato- phores but are about as large as the endothelial cells, though completely different in shape, texture and behavior. Figure 32, a camera lucida sketch, serves well to illustrate the relative sizes of the different typecells on the yolk-sac. In this figure areshown all four types, the enormous black chromatophore, the very large brown chromatophore, the delicate elongate endothelial cells of the vascular wall with their filamentous processes, and the almost circular erythroblast, small when compared with the first two types, but as large or even larger than the endothelial cell. Figure 34 was drawn from an embryo that had been fixed in such a way as to render conspicuous the cell outlines of the ecto- dermal layer of the yolk-sac. Below the ectoderm groups of erythroblasts forming blood islands are shown, and the extremely small size of the erythroblasts in comparison with the enormous dimensions of the ectodermal cells is most striking. As mentioned before, the erythroblasts tend towards a cir- cular shape but send out short processes, while they move in a sluggish amoeboid fashion. The cytoplasm of these cells is slightly greyish and not so perfectly transparent as that of the spindle cells; this difference between the two types is not so marked during the early stages but is readily noticeable in embryos of 80 or 90 hours. The future erythroblasts very slowly wander away from the tail region of the embryo and down the posterior surface of the iy) CHARLES R. STOCKARD Fig. 34. A camera lucida outline of the caudal region of the yolk-sac in a 96 hour embryo with the ectodermal cell borders made visible by mercury fix- ation. An island of blood corpuscles is indicated by small circles which give some idea of the minute size of the erythrocytes as compared with the huge ectodermal cells. yolk to reach its ventral surface. At various places on the posterior and ventral yolk surfaces the cells collect into groups and become less active, although their movement does not entirely cease. These groups constitute early blood islands. They are at first not surrounded by endothelial cells but later become enclosed or taken into the ends of the incipient vessels as briefly described above. When the circulation first begins on the yolk-sae of a normal embryo a great many of these islands are present, but are more or less isolated or out of the channels through which the fluid is DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 153 flowing. The taking up of the islands by the circulation is most interesting to observe. At first those cells near the tail of the embryo, which are enclosed by endothelium, are taken. A few . of the corpuscles are shaken by the current and these strike against the other members of the group until all become loosened and move slightly to and fro; finally one or two are suddenly washed away, then others follow—few at first, until the entire group loses its stand and is swept away by the current. One and then another of the islands may be seen to perish in this manner before the irresistible force of the tiny stream. Yet even after the yolk circulation is fairly well established a number of islands of the round cells may still exist unsurrounded by the endothelial vessels. Figure 32 shows such a case. A ~ well established current flows through the vessel to the right, while to the left is an incipient vessel not yet connected with the current. In the center of the figure is a group of corpuscles below a huge black chromatophore. These round cells con- stitute a blood island still unenclosed by vessel endothelium; in the course of a few hours, however, they too will become enclosed by a vessel and subsequently be included among the circulating blood cells. The early erythroblasts which are in this manner included within the circulation assume a circular outline as they float in the current. In the previous paper, figures 31 and 32 of cross sections through the intermediate cell mass, and figures 34 and 35 of cells in a yolk island, all from a young 72 hour embryo, illustrate the definite circular outline of these cells. In life they may be carefully observed.in the small vessels where a single cell passes with difficulty, and are here seen to be globular in shape and to retain their slight amoeboid movement. The form of the early erythroblast is readily changeable for the first one or two days after entering the current. After two or three days, that is, in embryos five or six days old, the cells in the blood current assume a typical erythrocyte appearance, becoming elongate and elliptical in shape when seen from one position, while they are thin in profile view. They are now ellipsoidal nucleated red blood corpuscles. At about the time they begin 154 CHARLES R. STOCKARD to change from the globular to the elipsoidal shape, the accumu- lation of haemoglobin takes place and the cells begin to show a typical straw color. It may then actually be seen in the living that the early or primitive erythroblast is really a more or less globular amoeboid cell without haemoglobin and resembling more closely a lympho- cyte or early leucocyte than a fully formed erythrocyte. This is probably true of the early stages in many cases of cytomor- phosis, yet the globular amoeboid cells of the yolk islands are not indifferent ‘primitive blood cells’ in the sense Maximow (’09) has concluded, but they are definitely future erythrocytes. This point is established by a study of these cell groups in the normal as well as in embryos without a circulation of the blood. In the latter individuals the islands arise by the forma- tion of local aggregations of the early wandering cells in an exactly similar manner to that described for the normal em- bryo. In fact, the observer cannot distinguish between the two specimens in many cases, yet one fails to establish a circu- lation and the islands are thus enabled to retain their positions on the yolk-sae. The constituent cells of such a permanent island may be observed from time to time or continuously, and will be found to pass through changes exactly identical with those which take place in the island cells that become swept into the blood stream of the normal embryo. They are for a few days globular in shape, but capable of slightly changing their form and sending out short pseudopod-like projections. When the embryos are five or six days old the cells in these islands then become flattened ellipsoidal corpuscles and attain a haemoglobin content exactly as in the normal embryo. The blood islands now appear bright red in color and are quite conspicuous on the yolk-sac where they permanently remain. The globular colorless cells are thus seen to differentiate directly into the typical ictheoid erythrocyte. From a study of the living embryos alone one could not, of course, be certain that all of the cells of these islands had differ- entiated into red blood corpuscles. However in the previous study of the non-circulating embryos, I have examined a large number of yolk islands completely and have never seen any DEVELOPMENT OF WANDERING MESENCHYMAL CELLS’ 155 type of blood cells other than erythrocytes in such a position. The same is true of the cellular products of the intermediate cell mass; in hundreds of sections studied with the oil immersion not one case has been found of a lymphocyte or leucocyte arising from a cell of the original intermediate cell mass. It is thus concluded that this mass is the red-blood anlage of the teleost and the wandering cells ofthe yolk-sae which are very probably derived from the same mesodermal stem that forms the inter- mediate cell mass are likewise a portion of the red blood cell anlage. The embryos that fail to develop a circulation are most im- portant material for the study of these questions of relationship among the different types of blood cells. The observations on the living show definitely the qualities of the early blood forming mesenchymal cell in its assumption of the globular slowly wander- ing type, which would fully satisfy the descriptions of Maximow’s ‘primitive blood cell’ as being closer in appearance to a lymph- ocyte than to a red corpuscle. When one follows these supposedly indifferent primitive blood cells which are claimed to possess the power of differentiating into either a leucocyte or an erythro- blast, he invariably observes them to differentiate only into erythrocytes. Although all cells of the early islands are dis- tinctly visible, they are not mixed in type, but are of one class. A long study of these early islands in sections also fails to reveal any other than red blood cells. The leucocytes are not very numerous in the blood, yet they should be seen if present in these islands, as they are found in other positions and are well represented in the blood of the adult Fundulus. The further history of the cells in the stagnant masses on the yolk-sac of an embryo in which the blood has never circulated is instructive in several ways. In the first place, all of these islands seem to become surrounded by endothelium, yet the endothelial arrangement cannot be completely traced in every case and it rarely extends much beyond the island mass so far as one can observe in life. Pigment cells are irregularly scattered in the neighborhood of the islands, as they are throughout the yolk region. The 156 CHARLES R. STOCKARD chromatophores do not, however, assume any arrangement with reference to the islands of cells and as mentioned above they retain their original condition as separate cells instead of fusing into syncytial masses, as is the case in specimens with a free blood circulation. 35 Fig. 35 The arrangement of red blood corpuscles in more or less connected endothelial sacs on the ventro-lateral surface of the yolk in an embryo 14 days old that had never had a circulation of fluid in its vessels. All of these blood cells wandered away from the caudal body regions of the embryo during early stages. The chromatophores are irregularly scattered among the old blood- islands. Od, oil drop. Figure 35 illustrates a group of blood masses on the yolk-sac of an embryo 14 days old. In this specimen there had been absolutely no circulation or movement of the blood fluids within the vessels at any time. This is most important to know in the case of all specimens without a circulation at the period they chance to be examined. I shall return to this point below. The cell groups in the specimen without a circulation are arranged somewhat like a vascular net in the region illustrated by figure 35, yet they present a deadly still appearance as contrasted with the lively movement of the corpuscles within the yolk vessels of a normal individual. The erythrocytes forming these islands ‘ DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 1g are brilliantly red in color and their shape and size are apparently normal. The blood corpuscles are thus found to differentiate in a typical fashion and to retain their haemoglobin reaction for a long period without having circulated in the vessels. The function of an erythro- cyte would thus seem to be entirely independent of its circulation so far as its capacity to form oxyhaemoglobin goes. These cells are also able to accumulate oxygen within the intermediate cell mass in its central position in the embryonic body. The embryo from which figure 35 was taken had lived 14 days without its blood having circulated, which is about the period required for the young fish to hatch and become free swimming. In older embryos the erythrocytes begin to degenerate and in many they lose their red color, the haemoglobin probably break- ing down. The islands on the yolk then become pale in color and finally almost white, as if the cells were dead. The color of the blood cells seems to fade within the embryo earlier than on the yolk-sae as a rule, probably due to the better chances of obtaining oxygen on the thin yolk-sac than in the thicker embry- onic body. The non-circulating specimens often continue to live for a long time even after the blood cells have lost their color. Some such specimens may exist for more than 40 days, which is a very long time considering that the normal embryo may hatch when from 11 to 20 days old. The specimens without a circulation are always weak and delayed in development and of course never succeed in hatching from the egg membrane. In a study of the embryos treated with weak alcohol solutions, one very frequently finds cases in which the circulation of the blood may start almost normally and finally stop permanently, although the embryo continues to live. Other embryos may fail to establish a circulation at the proper time and yet may develop a freely flowing circulation of their blood some days later. Again, an embryo may have a fairly normal circulation and for some reason lose it for a few hours, or for one or two days, and then regain it, at first slowly and finally in a fairly strong fashion. All three of these phenomena have also been observed in eggs developing in ordinary sea-water when they were not properly 158 CHARLES R. STOCKARD separated so as to allow free respiration. The egg membrane is covered with long hair-like filaments which become. entangled with those of neighboring eggs and in this way masses become closely packed. The central eggs of such a mass develop in a poor atmosphere and go more slowly than their neighbors on the outside of the mass. These centrally located eggs show many abnormal and arrested conditions of much the same type as may be obtained by treating the eggs with various injurious solutions. The changeable states in the circulation offer many pitfalls for one attempting to determine the sites of origin of blood cells in the non-circulating embryos. Old embryos are seen in which there are beautiful blood islands on the yolk-sae and great clots of blood in the head or other unusual position. The heart is very frequently completely loaded with corpuscles, and yet there is not the slightest movement of the blood cells or any sign of a circulation at this time. The heart itself may be pul- sating feebly or even practically stopped. Another source of blood movement which is slight, yet to be guarded against, is that due to the muscular twitching of the embryo’s body. This movement may frequently serve to push cells from the intermediate cell mass out on to the yolk-sac, but usually by way of the vessels. These dangers are to be taken seriously in experiments of this kind. Since one is able to be absolutely certain that the blood never circulates in a great num- ber of embryos, only such embryos should be considered in a study of blood origin. During a study of this exact problem now extending over four spawning seasons, I have seen blood in almost every conceivable position in embryos without a circu- lation at the time of the observation. The accumulation of blood is more frequent in certain positions and regions than in others. The venous end of the heart is a most common place for a clot, the sides of the head, the large vessels of the yolk just lateral to the body, and various places on the anterior and lateral yolk surfaces. : When, however, the experimenter collects a number of embryos that have really never experienced the slightest flow of their blood, ihe case is very definite. No blood clots ever occur in regions other ‘ DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 159 than the ‘intermediate cell mass,’ within the embryo, and the islands on the caudal and ventral yolk surfaces which have been formed as described by the early wandering cells that migrate away from the caudal region of the embryo. All embryos whose history for lack of circulation throughout their existence is actually known show the blood pattern most consistently, there being of course a certain amount of variation in the extent and position of the yolk-islands but not enough in any case to confuse the problem. These observations may readily be made by any observer, but can only be made in a reliable fashion with the high power microscope and strong condensers so that the light may be sufficiently regulated to observe the most transparent cells. The movements and differentiation of these cells should be carefully followed through every step in a number of cases, in order to fully appreciate the significance of their position and behavior. The cells may be seen even with an ordinary binocular micro- scope to some extent, but the arrangements for light regulation and the magnification are insufficient for determining the im- portant details. After the red blood cells have formed, they are readily located even with a low power yet such an examination could only determine their places of origin provided the embryo has been carefully watched with a high power magnification to make certain that it has had no blood flow. The condition of the yolk-sac mesenchyme must be fully under- stood and must always be considered in interpreting the origin of blood-islands and clots. For example, clots seen at the ven- ous end of the heart or on the extreme anterior surface of the yolk must be most cautiously considered, remembering the scarcity or even absence of the wandering mesenchyme in these regions. Clots in such places probably always result from a partial circulation of short duration and there is abundant evi- dence to support such a view: Although the future red blood cells migrate upon the yolk in their early mesenchymal stages, after they once group them- selves and differentiate into erythrocytes their powers of wander- ing become very much limited if they exist at all. I have never 160 CHARLES R. STOCKARD seen anything to indicate that a fully formed erythrocyte was capable of automatic migration. Yolk-sac blood islands of all ages have been examined in great numbers, but never has an erythrocyte appeared wandering away from such an island into neighboring regions. This fact is most important in the study of the blood-islands in the non-circulating specimens. When the slightest flow does exist for any length of time, there is a definite tendency, as mentioned above, for the blood to accumulate in certain sinuses and vessels. The positions of accumulation vary somewhat with the stages at which the circulation ceased, as well as the manner of stoppage of the flow, whether it was gradual or sudden. When the circulation stops during early stages, there is a great accumulation or massing of the blood over almost the entire ventral surface of the yolk. In other words, there is a hemorrhage or bleeding into these spaces or vessels until no more blood is left in other regions of the embryo, the heart gradually becomes empty of corpuscles and no longer passes them along. The packing of the yolk vessels probably clogs or blocks the circu- lation so that it ceases. Again, the circulation may stop more suddenly and the venous end of the heart or the entire heart may be seen packed with corpuscles while the vessels immediately entering and leaving it are comparatively or entirely empty. In older embryos there is the tendency to accumulate red cells in the vessels of the head so that brilliant red clots are frequently seen in these positions. In all cases it is interesting in these individuals in which the circulation has ceased at one or another period in development, and doubtless for different reasons in different specimens, to observe the way in which the blood sooner or later accumulates in one or another vascular space and does not remain uniformly distributed throughout the vascular system. Only when the heart is suddenly stopped and the blood quickly fixed by some strong killing fluid does one get a good pattern of the vascular system loaded with corpuscles throughout most of its extent. In rare cases, three during the present summer in some hundreds of embryos examined, will a specimen without a circulation at ’ DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 161 the time observed show almost all of its vessels loaded with blood cells, and this is probably due to a slowing down gradually of the circulation on account of the heart itself which finally stops with the vessels in a balanced state. The study of the yolk-sac in the living embryo enables one to observe every phase in the development of the red blood corpuscles from the early time when they wander as amoeboid mesenchyme cells to collect into groups of globular cells with short processes, the ‘“primi- tive blood cells’ of descriptive histologists, to be later surrounded by vascular endothelium, and then to change from the globular wander- ing cells into the flattened ellipsoidal erythrocyte loaded with haemoglobin, and finally freely floating in the current of the blood stream. The fully formed corpuscles apparently become incap- able of independently migrating even when not carried by the circulation. DISCUSSION AND CONCLUSIONS In the previous paper on the origin of blood and endothelium, a somewhat full discussion of the problems of blood formation in the teleosts and other vertebrates was entered into. A con- sideration of the questions of origin and development of vascular - endothelium was also undertaken in the light of the results there presented and the more recent general literature bearing on this subject. The experimental results then contributed seemed in the light of the past literature to render highly probable, if not to actually prove, the polyphyletic origin of the various types of blood cells, as opposed to the now extremely improbable monophyletic theory of origin of blood cells and vascular endo- thelium. For a general consideration, the reader is referred back to these discussions. A number of particularly significant points are brought out in the present study of the living normal and experimental embryos which bear directly on several of the past theories and specu- lations regarding the origin of vessels and blood. Only these special points will be briefly considered and analyzed at this time. 162 CHARLES R. STOCKARD In the first place, the writer cannot resist the impulse to highly recommend that all students of haematogenesis and vas- cular origin spend some time at least in a study of living mesenchymal cells and their cytomorphosis. Sucha study will soon convince one of the great disadvantages under which an investigator labors in attempting to solve the origin of blood from observations on dead material in serial sections. The problem becomes so simplified and devoid of laborious unin- structive technique that it seems almost superficial. One may learn as much from the living yolk-sac in an hour of careful study as in almost a week’s perusal of sections. Most important is the fact that certain things may actually be seen to occur that secticns could scarcely stimulate the mind toimagine. The only disadvantage is that the worker may be led to wonder whether so apparently simple a problem is actually of scientific impor- tance. Fortunately this mental state is soon passed over on realizing the necessary care and precaution which must be taken in following the movement and changes in the living cells. Each cell must be recognized as a living complex and the ob- server will realize the importance as well as the difficulties of thoroughly understanding and interpreting correctly its mani- fold changes and behavior. Material which to some extent allows such a study is often available. The Fundulus yolk-sac, however, is exceptionally adapted to this study on account of the beautiful simplicity of its structure, as well as the remarkable clearness with which each cell may be observed. An investigation of the Fundulus yolk-sac readily supplies a crucial answer to the old question regarding the relation of the blood vessel lumen to other body cavities and spaces. Ryder (84) was right in describing the blastoeoel of the bony-fish as remaining an extensive cavity for some time. ‘This is the space between the ectoderm and yolk and is identical and continuous with the cavity which arises very early beneath the blasto- derm and above the yolk periblast. Agassiz and Whitman (84), as well as Ryder (’84), Wilson (’90), and others, have identified this correctly as the cleavage cavity, the blastocoel. Later in development, the blastocoel extends over the yolk, forming the DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 163 space into which we have seen the free mesenchyme cells wander, and finally within this space groups of these cells form the yolk-sac vessels. Wenckebach (’86) described very clearly the origin of vessels from the free mesenchyme within the segmentation cavity. My study of a somewhat similar yolk-sac confirms the main points brought out by Wenckebach and all serve as crucial facts in support of the early theory advanced by Biitschli (’82) in his “Die phylogenetischen Herleitung des Blutgefissapparates der Metazoen.”’ Biitschli held that in the Metazoa the lumen of the blood vascular system was derived from the blastocoel. Later, Hubrecht (’86) supported the same standpoint from his studies on Nemertines. Hubrecht also found wandering cells playing an important role. Ziegler (’87) gives a most careful analysis of the continuity of the vascular lumen with the blasto- coel in his studies on the development of the bony-fish. The foregoing description and figures of the origin of vessels on the yolk-sae of Fundulus leaves no doubt that the vascular lumen in these animals, coenogenetically at any rate, is con- tinuous with the blastocoel or primary body cavity and is in no way related to the coelom. Almost twenty years ago, Felix (’97) advanced the opposing theory that the vascular lumen was really a localized or separate part of the secondary body cavity, or true coelom. The many decided objections to this theory from the standpoint of com- parative anatomy, the presence of blood vessels before the acquisition of a true coelom in the animal kindgom, and the numerous embryological contradictions in its path were pointed out in the discussion of this matter in the previous paper. Very recently Bremer (’14) has advocated the theory of the origin of vessels as parts separated from the coelomic cavity, or strands of cells from’the coelomic epithelium. In the first place, the material on which his investigation was based, early human embryos, will scarcely permit such generalizations. At least more suitable material could be found for the analysis of this problem. Further than this, his consideration of the questions involved does not lead one to form a definite idea of 164 CHARLES R. STOCKARD the exact direction he considers his evidence to lead. He credits Biitschli (82) with having originated the coelom theory that accords, so he thinks, with his evidence. This is entirely incor- rect, at Biitschli’s theory is exactly on the other side. The morphology of the yolk-sac of the chick, sheep and numerous other animals, as the literature of the subject readily shows, is entirely out of accord with such speculations. The yolk-sae . of the bony-fish shows this view to be really impossible and there should be no longer any doubt that vessels arise from loose and wandering mesenchymal cells in many animal species, and certainly not from ingrowths from the coelomic epithelium in any species. The formation of vessels on the yolk-sac of the teleost further limits the generalization of the origin of larger vessels from capillary nets. Thoma (’93) in his masterly study of the vas- culogenesis of the yolk-sac of the bird, held that “‘the first vas- cular spaces, the rudimentary capillaries, were formed by the secretory activity of the cells forming their wall.” These capillaries formed an extensive net and the arteries and veins arose secondarily and differentiated from the capillaries on account of the flow of blood set in motion by the beat of the heart. The anlage of the vascular system was the capillary. These principles of Thoma are not, however, applicable to the development of vessels in the embryonic bony-fish. The aorta arises as one or two vessels independent of any flow of blood or the existence of a capillary net. The first vessels on the tele- ostean yolk-sac are the large vitelline veins, as described by Wenckebach, and the median vitelline vein or the net of vessels in its place. These large important channels arise entirely inde- pendently and separated from the capillary net if such exists at the time. They also develop entirely independently of the blood flow, and not as a result of the pressure due to the heart beat. The capillaries and other vessels in many cases arise separately or away from these primary vessels and finally con- nect with them in a way similar to the connection formed be- tween the Randvene and the venous end of the heart. Other capillaries and small vessels arise as buds or sprouts from the first formed veins on the yolk-sac. : DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 165 More recently Evans (’09) with a very efficient and delicate method of injection has shown many of the larger intra-em- bryonic vessels of the chick embryo to develop from a foregoing capillary network. He found the same principles of develop- ment that Thoma had observed on the yolk-sac to hold for the development of certain vessels within the embryo. ‘These prin- ciples of vascular development Evans thought applied to verte- brates generally, but such is certainly not the case, the large vessels of the teleost embryo arise directly from associated mesenchymal cells and are not preceded by a capillary net. His evidence was derived from injected vessels and could not justify the statement, p. 512, of ““The presence always in the embryo of a united vascular system’’—‘‘a single branched en- dothelial tree.’ Such a united vascular system is rather late in its establishment in the fish embryo and there is no “‘single branched endothelial tree’? present when the first blood vessels are formed. ‘These facts may readily be demonstrated on the living embryo by direct observation. The vessels of the yolk-sac and several of the larger vessels within the body of the teleostean embryo form independently of any foregoing capillary network. In the teleost, then, the anlage of the vascular system is not the capillary but the mesenchymal cells which directly give rise to the chief arteries and veins, as well as to numerous groups of isolated capillaries. Other small vessels and capillaries grow as branches or sprouts from the arteries and veins. Thoma advanced three laws for the formation and growth of vessels. The first law was considered the most important, but rather destructive evidence is thrown against it by the present study. The law may be stated as follows: ‘‘The increase in the size of the lumen of the vessel, or what is the same thing, the increase in the surface of the vessel wall, depends upon the rate of the blood current.”’ The vessel increases in size when the rate is exceeded, becomes smaller when the rate is slowed, and disappears when the flow is finally arrested. Thoma (’96) states: “This law which brings the growth of the surface of the vessel into dependence upon the rate of the flow of the blood is, I con- 166 CHARLES R. STOCKARD sider, the first and most important histo-mechanical principle which determines the state of the lumen of the vessel under physiological and pathological conditions.” Thoma again states this principle thus: ‘‘In development the vessels in which the blood stagnates degenerate, and in those in which the rapidity is too great the lumen in enlarged.”’ No one could fail to admire the splendid manner in which Thoma attacked the problem of vasculogenesis in the yolk-sac of the bird, or the ingenious way in which he attempted to analyze the problem and deduce his three laws of histo-mechan- ical processes. Yet the ‘‘ First and most important histo-mechan- ical principle’? does not apply to the development of vessels in Fundulus embryos where there is no circulation of the blood. Many vessels grow in size or ‘‘what is. the same thing, show an in- crease in the surface of the vessel wall”’ without any “‘rate of the blood current.’’ The aorta in old embryos that never had their blood to circulate and in which the heart is actually a solid string of tissue, grows and attains a well developed lumen and a wali lined with endothelium and surrounded by concentric fibers of connective tissue as is shown in figure 49 in the previous paper, drawn from such aspecimen. This vessel is very slow to degener- ate, in fact, it shows no sign of degeneration and actually persists as long as the embryo is able to exist without a circulation, for 30 days or more. Vessels also develop upon the yolk-sac without ever having a fluid to circulate through their lumen. Other vessels are developed around the blood cells of the intermediate cell mass and the yolk-sac islands and in such vessels ‘the blood stagnates’ from the first yet the vessels degenerate very slowly, in some cases scarcely at all. In still other cases the blood may have circulated for a winte and then stopped for some tinre, but the vessels do not degenerate as is proven by the fact that the circulation through them may again be resumed. Such a sequence of events may be occasion- ally observed in the experimental embryos. The function of the vessel as a blood conductor, therefore, seems in these embryos of Fundulus, both the early normal and those without a circulation of the blood, to have little if anything to do with its early develop- ment and not much effect on tts ability to survive. ; DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 167 On the other hand, when Thoma has a straight normal case, the lumen may readily be seen to increase in size with the rate of flow. Yet in the entire absence of this action the vessel is still capable of increasing in size and it becomes question- able whether the rate of flow is ever an actual cause of size increase beyond mechanical stretching. These facts are most significant in a consideration of the influence of function on growth and development, auto-differ- entiation. Here it is seen that the structure both grows and develops in entire absence of its function. “In normal cases the function of the vessel as a blood conductor exerts more likely a physical rather than a biological effect on development. Thus the development of blood vessels on the yolk-sac of the living Fundulus embryo proves that the capillaries are not universally the anlage of the arteries and veins, but that these larger vessels may arise directly from wandering mesenchyme cells. Such arteries and veins may grow and persist without a circulation of the blood through their lumen and even though stagnant masses of corpuscles may crowd the vessel cavity. The development of vessels in Fundulus also directly disproves the claims made by Sobotta (’02) that the vessels in the teleost grow over the yolk entirely as branches from those near the embryo and without the wandering cells taking part. This assumption is probably due to the difficulty of estimating the part played by wandering cells from a study of serial sections. From a study of the living yolk-sac there is no question of the major part played by the wandering cells: in the origin and formation of vessels on the yolk. Sobotta also advances the opinion that the entire yolk vessels may sprout from the heart. This is much of the same nature as the ingrowth or parablast theory of His (’75), and is obviously defeated by the same array of facts which long ago relegated the parablast theory to a place of mere historic interest, in spite of the fact that it is so often revived for literary reasons. Finally, we may consider the study of the developmental products of the early wandering mesenchymal cells on the yolk- sac of the Fundulus embryo as a problem of cell lineage carried 2 168 CHARLES R. STOCKARD to its ultimate end. The primordial mesoderm cell or cells carry within their bodies all the potentialities of the mesoderm and may give rise to a series of cells which are capable of develop- ing muscle, cartilage, bone, connective tissue proper, blood cells, vessels, ete. Yet after a few cell generations the indi- viduals in the series derived from these early cells containing all the mesodermal potentialities no doubt become somewhat limited as to their potentialities. In a certain generation there may be definite cells more or less generally distributed which possess the capacity to give rise to muscle cells, but to no other type of mesodermal tissues. Still later in development these cells may come to be even more limited in their developmental capacities and thus have the power to produce only a certain type of muscle cell and no other type. Collections of such cells would then be designated embryo- logically as the anlage of striated muscle, smooth muscle, or heart muscle as the case might be Yet it is not to be forgotten that at this stage there might be really no means of distinguish- ing between the several different types of mesodermal cells. Limitization of potentialities in the individual cells has ap- parently reached a comparable stage just about the time when the mesenchymal cells begin to wander upon the yolk-sac of Fundulus. We have seen these cells as they wander out and have noted how very soon they may be separated into four distinctly different types, and following the development and behavior of these types it has seemed evident that they are entirely separate and do not intergrade or transmutate. The black chromatophore does not change its nature or divide off other cells which become different in type from the parent cell. Neither do the endothelial cells lining the vessel walls change into chromatophores or into erythroblasts, or vice versa. From all the observations on these yolk-sacs we must conclude that the four types of cells described above have developed from four different anlagen, although these anlagen were not neces- sarily localized groups of cells, but were diffusely scattered mesenchymal cells capable of developing into a definite product, either normal or abnormal, depending upon the nature of the é DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 169 developmental environment. Therefore, the four distinct mesen- chymal anlagen each give rise to a perfectly typical and distinct cell type although all develop in, as far as one can judge, an identical environment, the cavity of the yolk-sac between the ectoderm and the periblastic syncytium. The differences among the four cell types produced are from the standpoint of our present knowledge in all probability due tothe potential differ- ences among the apparently similar mesenchymal cells from which they arose. The four types including endothelial cells and erythrocytes we must consider from an embryological stand- point as arising from different mesenchymal anlagen. SUMMARY The yolk-sac of the teleost egg is a most beautiful object for observing the movements and migrations of cells in the develop- ing embryo. Such a yolk-sac has only one really definite con- tinuous membranous cell layer, the ectoderm; a true endodermal layer is absent, though a superficial syncytium, the periblast, fuses with the actual yolk surface. The mesodermal layer is represented by numerous separate wandering mesenchymal cells. These freely wandering mesenchymal cells may be clearly observed through the perfectly transparent ectoderm as they move over the surface of the periblast. The present contribution attempts to give a full account of the movements of the mesenchyme cells and their manner of development and differentiation in the yolk-sac. Observations have been made on the normal embryos from the earliest stages at which the mesenchyme wanders out upon the yolk up to the late embryo in which a complex vitelline circulation is fully established, and all of the products of the yolk mesenchyme completely differentiated. The study has been greatly facili- tated by a comparison of the normal embryos with specimens in which the circulation of the blood was experimentally pre- vented from taking place. In such specimens the cells on the yolk-sac never became confused or contaminated with other cellular elements introduced by the circulating blood. The 170 CHARLES R. STOCKARD wandering cells may thus be completely followed through all stages in their isolated position. The behavior of the migrating cells impresses one with the very important réle of such elements in the formation of tissues and organs, particularly the blood vessels and certain blood cells. The observer is also struck by the fact that such phenomena are extremely difficult if not actually impossible to interpret from a mere study of dead specimens cut in serial sections. Of course the study of sections greatly aids the observations on the living, and but for the fact of a long acquaintance with the Fundulus yolk-sac in sections, the writer would have found it much more difficult to identify many of the cells in hfe. The results of this investigation of wandering mesenchymal cells may be summarized as follows: 1. The wandering cells begin to migrate away from the embry- onic shield or line of the embryonic body at an early period, when the embryo is about 40 hours old, the germ ring having almost completely passed over the yolk sphere to enclose its vegetal pole. The cells migrate away chiefly from the caudal end of the embryo, only a few wandering out from the head region. The regions of the yolk-sac thus suggest an area opaca about the tail end and an area pallucida around the neighborhood of the head. All of the cells wander into the so-called subgerminal cavity, the space Wilson (’90) and others consider a late stage of the segmentation cavity, between the yolk-sac ectoderm and the periblast syneytium. When the cells first appear they are all closely similar in shape and about the same size. Very soon, however, they begin to exhibit certain differences. Many become elongate spindle cells with delicate filamentous processes, sometimes producing a stellate appearance. Others are more amoeboid in shape with conical ‘pseudopod-like processes which are constantly being thrown out at one place and withdrawn at another. Still a third class of cells appear somewhat later than the other two; these are more circular in outline with short thick pseudopods and are more slowly moving. DEVELOPMENT OF WANDERING MESENCHYMAL CELLS 171 The movements of these extremely numerous cells and their changes of position may be readily followed with a high magni- fication. In embryos of about 60 hours, still some time before the heart begins to beat or the blood to flow, four clearly distinct types of cells may be recognized among these originally similar mesenchymal cells, and the further history of the four types has been completely traced. 2. The amoeboid cells with conical pseudopod-like processes shortly after 60 hours begin to show an accumulation of pigment granules within their cytoplasm. Just at this time they are seen to be of two distinct varieties, one depositing a black and the other a brownish red pigment. The black chromatophore increases rapidly in size and by the end of the third day becomes an enormous amoeboid body wandering over the yolk. These cells are attracted to the walls of blood vessels and plasma filled spaces, such as the pericardial cavity becomes in specimens without a blood circulation. When the embryo is five days old the chromatophores are abundantly arranged along the walls of the vitelline vessels, but the pigmented cells are distinctly separate. After this time neighboring cells begin to fuse along their adjacent borders and large pigment syncytia are formed which completely surround and ensheath the vessels. A single syncytium is often of con- siderable extent, as shown in figure 15. The brown chromatophores have a somewhat different history. They never become so massive as the black, and their processes are more delicate and graceful in appearance. Yet these cells also attain a large size and in embryos of 72 hours are scattered over the entire yolk-surface. After the third day when the blood begins to flow in the yolk vessels, the brown chromatophores likewise become attracted to the vessel wall. These exquisitely branched cells apply themselves to the wall of the vessel and may often completely surround it, as shown in figure 17. This type of chromatophore, however, always maintains its cellular indi- viduality and never fuses with other cells to form a syncytium as is the case with the black type. The function of the chromatophores on the yolk-sac is most difficult to decide, but one thing is certain, they never become 72 CHARLES R. STOCKARD changed into any type of blood cell. The brown chromatophore in early stages may accidentally reach the blood current; it then becomes spherical and may be readily observed for a long time on account of its huge size as compared with the blood cells. It never, however, changes in type. In specimens without a circulation of the blood both types of chromatophores arise in a normal manner and differentiate normally. Their arrangement along the vessel walls fails to occur and they remain scattered over the yolk or collected about the plasma filled spaces. The heart in such embryos is sheathed with pigment, while the normal heart never has a chromatophore on it. 3. The elongate spindle cells with their delicate filamentous processes are small in comparison with the two chromatophore types. These spindle cells retain in general their original ap- pearance, but their behavior is most important. In embryos of about 48 hours such cells aggregate into certain rather definite groups; later, these groups become more linear in shape and finally these lines of cells arrange themselves so as to form tubular vessels. Several of the larger vessels arise independently upon the yolk, and certain ones of them later become connected with the venous end of the heart, while in all cases capillary nets which also arise independently become connected with the larger ves- sels. These processes may actually be followed through every step in the living yolk-sac. The wall of the early vessels is very irregular with spaces exist- ing between the component cells. Corpuscles are often caught in these spaces or entangled in the filamentous processes of the endothelial cells. Such conditions in sections would appear as though the corpuscles actually formed a part of the endothelial wall and might incorrectly be interpreted as endothelial cells changing into blood cells. Nothing has been seen in the living embryos to indicate that an endothelial cell has the power to produce a blood cell or to change into a blood cell of any type, but much has been seen to the contrary. The generalization particularly made by Thoma (793) that larger vessels arise from a net-work of capiliaries is not true for the large vitelline vessels on the fish yolk-sac. It is also found DEVELOPMENT OF WANDERING MESENCHYMAL CELLS iio in the specimens without a circulation of the blood that the vessels arise and increase in size and persist for a long time with- out ever experiencing any effect of the blood current upon their walls. In many embryos the circulation after having begun may stop for a time and then later be reestablished, the vessels having persisted in a normal condition. Thoma’s so-called laws of vessel formation are, therefore, rudely violated by the develop- ment of the vascular system in these embryos. The vessels arising from independent mesenchymal cells in the space of the blastocoel in the teleost yolk-sac entirely overthrow any notion that vessels arise ontogenetically as portions of the coelomic epithelium. The vascular lumen is originally continu- ous with the primary body cavity, the segmentation cavity, and never with the secondary body cavity or coelomic cavity. 4. The fourth class of cells wander out from the embryonic body somewhat later than the three former types. These are small globular cells with short pseudopod-like processes. They move very slowly, but finally collect into groups on the posterior and ventral regions of the yolk-sphere. The round cells wander away only from the caudal region of the embryo and probably are derived from the so-called inter- mediate cell mass which is the anlage of the red blood corpuscles in the fish embryo. The groups of round cells are slow in their differentiation but just before the circulation of the blood begins, they are seen to be circular erythroblasts. The observer may follow the dis- appearance of the islands of cells one by one as they are enclosed by the vessels and swept into the circulating stream. About the fifth day these circular erythroblasts become flattened ellipsoidal erythrocytes filled with haemoglobin, the typical red blood cor- pusele. The complete change from wandering more or less spherical mesenchymal cells into typical haemoglobin bearing corpuscles may be followed in the living yolk-sac. In several instances the entire body proper of the embryo failed to develop or else degenerated very early, yet the yolk-sac formed or persisted with numerous blood islands fully differ- entiated. 174 CHARLES R. STOCKARD The embryos in which there has been no circulation of the blood form the blood islands from the wandering cells on the yolk-sac, and the constituent elements of these islands differentiate per- fectly and may maintain their red color for many days. Yet they never leave the locality in which they have differentiated. The fully formed red blood corpuscles have little if any power of migrating. When the observer can be positive that the blood has never circulated, and this requires very consistent watching, the islands of the yolk are always limited to certain regions and never occur so far anteriorly on the ventral surface of the yolk as to reach the venous end of the heart. 5. On the yolk-sac of Fundulus embryos one thus finds four distinctly different products differentiating from the apparently similar wandering mesenchymal cells. The environment in which the four types differentiate is identical as far as is possible to determine, and the only explanation of their various modes of differentiation is that the original mesenchymal cells that wandered out were already of four potentially different classes. These differences in potentiality within the early cells gave rise to the four different directions of cytomorphosis in one and the same environment. The four resulting types of cells are then in an embryological sense derived from different mesenchymal anlagen. LITERATURE CITED (Only those titles not given in the previous paper) Acassiz, L. anp Wuitman, C. O. 1884 On the development of some pelagic fish eggs. Proc. Amer. Acad. Arts and Sci., xx. Bremer, J. L. 1914 The earliest blood-vessels in man. Amer. Journ. Anat., 16. Evans, H. M. 1909 On the development of the aortae, cardinal and umbilical veins and the other blood vessels of vertebrate embryos from capillaries. Anat. Record, 3. Ryper, J. A. 1884 A contribution to the embryology of osseous fishes. Report U. 8. Fish Comm. for 1882, Washington. 1887 On the development of osseous fishes. Report U.S. Fish Comm. for 1885, Washington. Toma, R. 1893 Untersuchungen ueber die Histogenese und Histomechanik des Gefiisssystems. Enke, Stuttgart. 1896 Text-book of general pathology and pathological anatomy. Translated by Bruce, London. ’ ih i : tial ot lip eon QL Stockard, Charles Rupert ee An experimental analysis S75 of the origin of blood and vascular endothelium BioMed PLEASE DO NOT REMOVE CARDS OR SLIPS FROM THIS POCKET UNIVERSITY OF TORONTO LIBRARY 2 ee Shire sae Cente mee Sot a te ae we oe piael 7. Poe wen lest tar ats 3 ; 4 : ; : % ; : . bas we aie anan Sat a goiter ast orate hah. fy on br : ; ne es paiesevenn eos