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=On the earliest Blood-vessels in the Anterior Limb-buds of Birds and their relation to the Primary Subclavian Artery=

By

Herbert M. Evans. 


From the Anatomical Laboratory of the Johns Hopkins University.

With 20 Figures.

==I. Introductory==

Our knowledge of the origin and method of formation of the vascular system in vertebrate embryos is still far from satisfactory, and, indeed, in many instances surprisingly inadequate. This is true, too, in spite of a long series of observations and a number of important contributions in this ﬁeld. We owe much, for instance, to Rathke, whose classical paper on the aortic arches and the vessels derived from them, dates from 1857, and to Hochstetter, whose persistent labors have yielded such a wealth of facts on the development of various vessels.

But the knowledge which these and other researches have given us consists chieﬂy in an idea of the position and connections of the main blood-vessels present in successive stages. They tell us little of the beginnings or of the method of development of any of these vessels.

* A portion of the observations recorded in this paper were made while occupying one of the research rooms endowed by The Wistar Institute of Anatomy at the Marine Biological Laboratory, Woods Hole, Mass. I would acknowledge here my obligations and thanks to The Wistar Institute for this privilege, and also to Professor Frank R. Lillie, Director of the Marine Biological Laboratory.


Signiﬁcant advances in anatomy have owed much to improvements in the method of investigation. Thus von Lenhossek has declared it probable that a complete nerve cell, with all its processes had never been seen before the introduction of Grolgi’s method of staining.


In almost all instances, the study of the development of the vascular system has been done merely by the usual methods of histological investigation, and, as a consequence, has suﬁered keenly the limitations this method entails. The shrinkage which dehydration always produces in tissues prepared for serial sectioning is unavoidable. In perfect technique, this shrinkage is never great and is so uniform that great distortion cannot result; but it is present, nevertheless, and on no system or tissues of the embryo does it impress itself, even though slightly, as it does on the embryonic vessels. These delicate endothelial tubes easily collapse and_ the finest of them almost invariably do so. Thus the observer is fortunate to trace clearly the chief vessels and their tributaries. If new vessels, in the embryo as in the adult, ar.ise by simply capillary sprouts and plexuses, these must imperfectly be seen, and so in the development of most of the vessels we have doubtless missed these earliest and important stages.


But more serious errors than this have arisen, for the employment of tissue prepared in the usual way has often led the observer to consider the larger uncollapsed vessels as constituting the entire system present, and thus led him to the conception of the outgrowth of vascular trunks as such. This, however, is never the case.

To recognize the origin and the method of growth of the early blood-vessels, we must have a complete picture of the vascular tree, of all its capillaries and. even of their endothelial sprouts. Can any method accomplish this much?

Fortunately the experience of this laboratory during the last ten years has shown the existence of such a method. I refer to the method of injection. 

The first publication from this laboratory, referring to the injection of embryos, was made in 1900, in the research by Flint} on the blood—vessels of the adrenal. Injections were used extensively by Sabinz in the study of the development of the lymphatic system, and in her first publication on this subject (1902), mention is made of the injection of the blood vascular, as Well as lymphatic systems, in young pig embryos.

In 1904, Professor Mall’s “Development of the Blood—Vessels of the Brain in the Human Embryo” 3 appeared, and in it he reported brieﬂy some of the methods and results of the injection of embryos, which he had been conducting for some time.‘ His paper outlined a method of obtaining splendid double injections. India ink was injected into the liver and being taken to the pulsating heart, driven by the latter through the arterial tree. The embryo was then cooled and the beat of the heart arrested, whereupon a second injection by way of the liver now filled the veins. At other times single injections of the arteries, or of the veins, were made by the same method, '6. (3., through the liver, and these were, in many cases, remarkably complete. Thus the feasibility of demonstrating all the chief vessels of the embryonic venous or arterial system became an established fact.

But until recently, though injections of most of the vascular tree had been secured, it was necessary to perfect the method so that an approximately complete ﬁlling of all the body’s capillaries could be secured, and so that even the very youngest embryos could be successfully treated in this way.

Many hundred trial injections, with living embryos under the best conditions obtainable, have evolved methods delicate, and yet effective enough, to successfully fulfil these two conditions. I need not refer here to the details of such methods, since they will be described adequately elsewhere. Recently Knowerf has described carefully a method of using glass bulbs in this connection, which is very useful.

* Flint J. M. “Tine Bloodvessels, Angiogenesis, Organogenesis, Reticulum, and Histology of the Adrenal.” Contributions to the Science of Medicine, dedicated by his pupils to William Henry Welch. Baltimore, 1000. Pp. 153-228.
* Sabin, F. R. “On the Origin of the Lymphatic System from the .Veins and the Development of the Lymph Hearts and Thoracic Duct in the Pig.” Amer. Jour. o-f Anat., Vol. I, 1902, pp. 367-391. '
* Mall, F. P. “On the Development of the B1ood—Vessels of the Brain in the Iluman Embryo.” Amer. Jour. of Ana_t., Vol. IV, 1904. 284 Herbert M. Evans.


Whatever the details of the procedure are, fine glass canulae, the helpful binocular microscope, and living embryos comprise the essen+ tials. I used every possible channel as a starting place from which to reach the general circulation. The veins, the liver, and the heart itself were successivly chosen only to be abandoned. The larger arteries, however, permitted the employment of great pressure without danger of rupture. Thus the entire vascular system could be ﬁlled with the minute carbon particles of the injection mass (India ink), which, passing through arteries and capillaries, and again streaming into the heart by the veins, are pumped out again into the circulation. The more perfect of these beautiful specimens were subjected to detailed study and at length convinced us that in some instances we had attained a complete injection. The endothelial sprouts themselves were everywhere ﬁlled to their tips. V/Ve beheld the growing vascular system!

The revelations which such injections have made are in no place more striking than in the case of the origin of the chief vessels to the limbs; the femoral and the ‘subclavian arteries. The results here, too, are of peculiar interest, inasmuch as the latter vessels have been studied with most painstaking care and, in the recent work of Muller and of Rabl, have already been traced to a very priinitive condition.

The present communication deals chieﬂy with the first bloodvessels which grow into the anterior-limb buds, and is almost entirely based on a long series of chick and duck embryos, though I have also studied the early mammalian limb and the arm bud in man in this connection.

Before presenting these observations, however, some account of the literature on the origin of the avitansubclavian artery will not be out of place.

“I{nower, II. Meﬁ. “A New and Sensitive Metliod of Injecting the Vessels of Small Embryos, etc.” Anat. Record, Vol. II, No. 5}, .i\u§.::ust, .1908. Earliest Blood Vessels in Anterior Limb Buds. 285

==II. Historical==

Five important investigations have been made on this subject— the researches of Mackay, Hochstetter, C. G. Sabin, Rabl, and of Miiller; each paper containing a signiﬁcant contribution and, with the exception of Mii1ler’s account, which does not include very young embryos, each successfully carrying the subject to still earlier. stages of development.

Twenty years ago Mackay5 published the ﬁrst valuable account of the origin of the carotid and subclavian arteries in birds, and pointed out that the latter vessel in this class was entirely diﬁerent from the subclavian trunk in most mammals.

It had been known for a long time that this vessel lay dorsal to the superior caval vein and the vagus nerve in the mammals but always ventral to thes.e structures in the birds, and if the subclavians of these two classes were regarded as identical this diﬁerence in relations was quite unexplained. Moreover, the opinions of embryologists here were not helpful, for they did not describe a ventral but a dorsal Origin for the bird’ S subclavian, Rathke ﬁguring it as a derivative of the dorsal end of the fourth aortic arch, and Sabatier, following the earlier account by von Baer, as a dorsal derivative of the third aortic arch. Embryology should have thrown some light on the subject and Mackay set about to rework the embryology. It is his chief contribution to have shown clearly that the bird’s subclavian had ample reason to be different in position and in all its. relations from the mammalian vessel, for it arose at an entirely different point embryologically and in its growth, must come into entirely different relations with the thoracic structures. Mackay deserves the credit of being the "rst to recognize that the deﬁnitive avian subclavian grows down from the ventral portion of the third aortic arch and is thus from the beginning, and during its entire development, a ventral vessel, in no sense homologous with the dorsal outgrowth of the aorta which becomes the subclavian artery of mammals.

* Mackay, J. Y. “The Development of the Branchial Arches in Birds, with Special Reference to the Origin of the Subclavians and C‘~arotids.” Phil. Trans. Roy. Soc, London. Vol. 179. 1888. 

Mackay went further and declared that in the Amniota generally there occur these two distinct subclavians—-the dorsal vessel, possessed by man and by most of the mammals, and by the Lacersilia, the ventral vessel occurring in birds, Chelonia, Crocidilia, and the Cetacea. Moreover in one form-in Chamaeleo vulgaris—both kinds of subclavians occurred, the ventral vessel, however, supplying chieﬂy the shoulder muscles.

The next advance was made by Hochstetter“ who, in 1890, published his paper on the “Origin of the Subclavian Artery in Birds.” Independently of Mackay, he also had reached the conclusion that the dorsal subclavian of mammals was not represented in the adult bird, butthat the deﬁnitive wing vessels in the latter class arose from the ventral segment of the carotid arch. In addition, Hoch— stetter announced the discovery that in still earlier embryos the birds possessed a subclavian which corresponded to that vessel in the mammals. The secondary subclavian, the adult vessel of the birds and the trunk which Mackay had discovered, did not arise, said Hochstetter, till the beginning of the sixth day in the chick. Preceding it, on the ﬁfth day, the primary subclavian vessel had arisen and extending into the early limb buds, had grown to trunks of considerable size, furnishing a splendid arterial supply to the growing extremities. This primary aortic subclavian was a branch of the "fteenth dorsal segmental artery on each side and Hochstetter was thus inclined to regard the subclavians as a modiﬁcation of the pair of segmental vessels. On the sixth day, the ﬁnal vessel began its downgrowth from the ventral portion of the third aortic arch and could be seen anastomosing with the primary vessel derived from the aorta. Thus, during the sixth and seventh days, the chick’s wing buds were supplied by a double source, a condition corresponding to that seen by Mackay in the adult Chamaeleo.

Hochstetter’s account established the fact that even in those amniotes where the deﬁnitive vessel is the ventral subclavian, there arise in development, nevertheless, typical dorsal subclavians which correspond to those in the mammals, and in fact to the subclavian of the Anamniota, since this is also the vessel which occurs in both the ﬁsh and amphibia. Thus this important paper emphasized the fact that in all vertebrates possessing fore limbs, the primary subclavian artery arises from the dorsal aorta.

* Hochstetter, F. “Ueber den Ursprung der Arteria Subclavia der Viigel.” Morph. J ahrb. 1890. 



In 1905, C. G. Sabin,7 working in Locy’s laboratory, reworked the subject and furnished us for the ﬁrst time with good illustrations of the origin and course of both the primary and secondary subclavians in the chick. Sabin not only pushed the time of origin of the primary vessel to a stage much earlier than Hochstetter had suspected, 13. 6., to the third, instead of the fifth, day of incubation, but he also made another observation which must be considered an important contribution to the subject. Sabin was the ﬁrst to show that the primary subclavian was not at first a branch of the dorsal segmental artery but instead was primarily an independent outgrowth of the aorta and only secondarily came to be included as a branch of the dorsal vessel. T

The admirable paper published in the summer of 1907, by Rabl,8 was by far the most complete account We possessed of the subclavian arteries in birds and it marked the discovery also of a new fact of great importance, namely that the single primary subclavian is itself preceded by a row of some three segmental subclavians. These early segmental subclavians had been entirely overlooked. T

Rabl’ s research was based on a careful study of sections of duck embryos. The account which he has given us is of the greatest interest, for it indicates that the metamerism of the limb is as distinctly expressed in its primitive vessels as it is in the nerves and myotomes that enter into its structure, a point which was first discovered by Erik Miiller“ in selachians and in reptiles, and which has been emphasized by the latter observer in several articles on the arm vessels.

* Sabin, C. G. “The Origin of the Subclavian Artery in the Chick.” Anat. A112,, Bd. XXVI, 1905, p. 317.
* Rabl, Hans. “Die Erste Aniage der Arterieu der vorderen Extreinitiiten bei den Viigeln.” Arch. f. mik. Anat, Bd. 69, pp. 340-387.
* Erik Miiller “3eitr‘.ige zur Morphologie des Gefiisssystems.” Anat. Hefte, June, 1903; Dec-., 1904; and 1+‘eb., 1908. 288 Herbert M. Evans.

It was from this standpoint, among others, that Professor Mall suggested that We study carefully the earliest arm vessels and it occurred to me that in the embryos of the bird we had a splendid opportunity for delicate injections. The greatly expanded extraembryonic vessels furnished a good channel of entrance into the circulation of the embryo proper.

A segmental blood supply to the early limbs mus‘; new be regarded as an established fact. Keibel and Elzem and the writer” have shown it to be a normal stage in man, and observations which I have recently been able to make on mammalian embryos show its occurrence there also.

However, even the segmental subclavians are not the earliest plan of blood supply to the limb buds, as my injections plainly show; for in the birds, at any rate, the capillaries which ﬁrst nourish the young limb bud, course at ﬁrst entirely regardless of any segmental alignment and in a profusion hitherto unsuspected.

==III. Observations on the Origin and Character of the First Blood-Vessiels in the Anterior Limb Buds of Chick Embryos==

Both chick and duck embryos were employed in the present study and it is for this reason highly probable that the results are generally applicable to the class Aves. Most of my description, however, will be conﬁned to the condition in chicks, inasmuch as it was easier to secure here a larger series of the embryos for study. Nevertheless, in view of Rabl’s study of the duck, it was important to see if the conclusions reached in the chick could apply here also, and I consequently incubated a considerable number of duck eggs and, at length, obtained successful injections of these embryos.

The drawings illustrating my ﬁndings have been done with the greatest care and ﬁdelity possible.

I shall report ﬁrst the conditions observed in the chick.

“Keibel 11. Elze. “Normentafeln zur Entwick. des Menschen.” Jena, 1908. “Evans, Herbert M. “On an Instance of two Subclavian Arteries to the

Early Arm Bud of Man and its Fundamental Signiﬁcance.” Anat. Record, II, 9, Dee., 1908. Earliest Blood Vessels in Anterior Limb Buds. 289

The injections gave apparently complete pictures of the entir.e capillary system throughout the body and showed a wealth of these

‘delicate vessels often where they had previously been poorly re-

vealed. It consequently became of great interest to know if the early body wall~—the somatopleure—=were s.upplied by capillaries before any portion of it was elevated to form the limb buds, and such was, indeed, found to be the case.

In embryos of some twenty somites, the capillaries which lie in the upper somatopleurc, and which form a small ple.\'us in the angle between the duct of Cuvier and the posterior cardinal vein, now begin to grow downward. Behaving like typical capillaries, these vessels frequently anastomose and so begin to form a narrow plexus filling most of the somatopleure. Often their endothelium meets and coalesces with that of the posterior cardinal vein. By the time this simple plexus has reached the position of the earliest wing bud, the latter structure begins to form and perhaps through some inﬂuence exerted by the mitoses of the limb cells, endothelial outgrowths from the aortic wall are now stimulated. These outgrowths are again typical capillaries in size and character, sometimes anastomosing almost immediately after their origin from the lateral wall of the aorta (producing the appearance called “insel—bildung”), but more commonly growing rapidly toward the lim.b cells where they meet the chain of capillaries previously mentioned. Thus there is established in the newly—formed limb its first circulation, a circulation merely of capillary character through the simple mesh work of these anastomosing vessels. Coincident with the establishment of a circulation, however, transformations occur in the capillary mesh, for there come into force now hydrodynamic laws which are at work everywhere in the circulation. The path from arm bud to Cuvier’s duct now receiving a good current of blood becomes an important drainage channel and this role almost immediately fashions from the capillary mesh a fairly direct and constantly enlarging path—the umbilical vein. No more striking instance of the applicability of those mechanical laws which Thoma discovered could be found, for it is of the greatest signiﬁcance that the mesh of capillaries in the somatopleure remains of this primitive character 290 Herbert M. Evans.

until the appearance of a good circulation. Not until then is a vein formed.

Portions of this system of capillaries constituting the later umbilical xreinmthe primary body-wall plexus, I have cal].ed them—~—ltave been seen before. Brouha mentioned them and Rabl has described them in some detail. However, no ﬁgures of them have ever been published, nor has the whole story of their origin and downgrowth ever been adequately described.

In order to facilitate a description of the various embryos stu-

died, the subjoined table can present a summary of the chief facts relating to the limb vessels.

TABLE SHOWING THE NUMBER or SUBCLAVIAN ARTERIES PRESENT IN CHICK Elusauvos or FROM 24 TO 48 SOMITES.

E " E | >1 . jg *" E: I 5 23 § Number of Aer-! magi Egg go tCap1lltaﬁ'y i

Q  U. grow I S. ‘G 1 R '
5%: g 33 §,‘§’{,‘1'f§r Hours of 11 Somites “sis (éfbcliivlan ediergriginegion ,3 S3 ‘3mbI.y0_ Incubation: Present. 4_., ‘-7 g memes’) 3 Subclavian o 33;; ' I gﬁ 1;’ Arteries. §--* 0 +1 $1, 5'3. gig ‘ éi gag Left. Right. :_ _ |______,,__.. ._, i. __ __ T_-_ 1 -r . . 24 Opposite 0 10 . . . . . . . . 5 9311 i . 2 _! 30 14th 0 1 113th mtersomitic space 3 60 32 20th 4 10 14th-17th “ Period of pri4 60 . . “ 5 11 14th-18th “ mary subclar < 5 65 31 21st 5 6 15th-—19th “ vian capillary 6 60 = . . “ 4 6 15th#19th “ plexus. _ 7 66 I 33 “ 3 4 15th~18th “ Period of muI8 72 . . 4 4 “ “ “ tiple segmen— { 9 72 . . Completely 3 4 16th~18th “ t a 1 subc1a.10 72 34 “ 2 4 18t.h——19th “ vians. ~ 11 78 36 “ 2 2 18th-—19th “ Period of p r i' 12 80 I; 45 “ 1 2 18th-19th mary subc1a13 84 . . “ 1 1 18th “ “ vian artery. ,1 48  % ll _ 16 116 ii “ 1 1 18th “ “ 17 70 I 38 21813 5 6 16th*20th "

1 I 1

It will be seen by referring to the table that the deﬁnitive primary subclavian artery is at the level of the eighteenth inter-somitic space. It is a branch of the dorsal segmental vessels of that interspace. My injections, controlled by careful study of serial sections, show that in the embryos embraced by the table, '5. 6., in chicks possessing from twenty-four to forty—eight somites, the ﬁrst interspace e., Earliest Blood Vessels in Anterior Limb Buds. 291

that between the ﬁrst and second myotomes) is not occupied by a dorsal segmental ar.tery. The series of dorsal segimerttal a/rteries in these embryos begins with the vessel present in the intersp-doe between the second and third somites. Consequently the vessels present in the eighteenth interspace are really the seventeenth pair of the series actually present, and I have so labelled them in all the drawings. It is to be borne in mind, then, that the seventeenth segmental vessels of the ﬁgures are in the eighteenth inter—somitic septum. Inasmuch as the ﬁrst four somites of the chick are to be considered cephalic, rather than cervical, in their ultimate fate, and as the third actual segmental artery later courses near the ﬁrst cervical nerve, and is hence the ﬁrst cervical artery, the primary subclavian artery arises from the ﬁfteenth cervical segmental artery, a vessel occurring in the eighteenth interspace a11d the seventeenth of the series actually present.

FD

There are ve periods in the history of the bird’ s subclavian and not four as Rabl maintained. These may be brieﬂy enumerated as

follows :

I. Period of capillary outgrowth from the aorta forming a primary—limb plexus, no‘; influenced in its arrangement by metamerism.

II. Period of multiple segmental subclavians, a condition resulting from the atrophy of all capillaries in the preexisting plexus not at segmental points.

III. Period of the establishment of the primary subclavian artery from the persistence of one of the pairs of segmental subclavians—i. e., that of the eighteenth segment.

IV. Period of double arterial supply through contemporary existence of dorsal and newly arisen ventral subclavians.

V. Period of enlargement of the permanent channel, the secondary subclavian, and coincident atrophy and disappearance of the primary vessel.

The last three periods or phases were described by Hochstetter and Sabin, the second period, in which segmental subclavians exist, by Rabl and Muller, the earliest or first period for the ﬁrst time in the present study. 292 Herbert M. Evans.

It will be seen that the embryos included in the table presented comprise only those in the first three periods or stages of the development of the subclavian artery

Embryos 1 and 2 show some early s.teps in the downgrowth of the primary body-wall plexus, the system forming the latter. umbilical vein.

Embryo 1, with twenty—four somites, shows this mesh of capillaries extended caudally in the somatopleure to the level of the ninth inter—somitic space.

In embryo 2, with thirty sornites, these capillaries have reached a point opposite the fourteenth interspace. In addition, from the aorta itself several capillaries have now grown out, one of which l1as joined the main plexus at a point near the thirteenth interspace and its dorsal segmental vessels. These apparently unimportant endothelial sprouts are but the ﬁrst of a considerable series to grow from the lateral aortic wall, and though there is as yet no external indication of a limb bud, they are_probably to be regarded as the first limb capillaries.

In the next s.ucceeding stage, embryo 8, of thirty-two somites, a slight swelling of the somatopleure constitutes an inﬁnite, yet undoubted, limb bud, and we find a considerable row of these aortic or rather subclavian capillaries. A glance shows that these vessels are not segmentally arranged Figs. 2a and 2b).

In such injected specimens one may look down on the preparation as a whole and examine carefully any area. No reconstruction is necessary; no doubt about relations exists, for the entire picture is spread out before one. The dors.al segmental vessels stand out sharply and it is easy to determine the relation of these to the new subclavian capillaries. It is impossible to say that the latter vessels are determined in position by the former for the whole appcamnce given is of a prof/use irrcgu.Zar outgrowth of capillaries urhich form a simple plea"/uvs. On the right side, as the ﬁgure shows,

FIG. 1.—(L‘1'1ick embryo of 30 soinites (enibryo 2 of table). Sl'1o\vi1_1g downgrowth of pi-iinary wall plexus, >< 531/3. Ant. Card. V., anterior cardinal vein. Ext. Jug. V.._ external or inferior jugular vein (linguo—facia1 vein). Post Card. V., posterior cardinal vein. P. 1'). W. p., primary body wall plexus. EARLIEST BLOOD VESSELS IN ANTERIOR LIMB BUDS.

HERBERT M . EVANS.

THE AMERICAN JOURNAL on‘ ANATOMY.-—VOL. IX, No. 2.

FIG.

-‘

Earliest Blood Vessels in Anterior Limb Buds. 293

there are as many as ten of these vessels and on the left side, four. They are all remarkably delicate, being smaller, on the whole, than

Prim. __ C ap. Piex. OfAu1.' Limb.

""""" """"PosT.Card.Vein'_

FIG. 2b—Chicl: e111b1'yo -of 32 somites (e11'1b1'y0 3 of table) showing establishment of primary capillary plexus of limb bud by union of subelavian capillaries with primary body wall plexus, X 53%.

most of the capillaries in the somatopleure. One can see that under ordinary conditions such structures might be diﬂieult to trace, and 294 Herbert M. Evans.

especially so if empty and collapsed, as is most often the case with the capillaries generally.

The early subclavian capillaries are true lateral derivatives of the aorta, and at the present stage arise at considerable intervals laterally from the points of origin of the dorsal segmental vessels. In the ﬁgure (Fig. 2b}, the latter vessels are represented for convenience as if cut off, but they may be seen forming a typical plexus on the sides of the spinal cord. For a clearer picture of the behavior of these dorsal segmental vessels, I have drawn with care their

entire course and capillary bed in a slightly older embryo. (Embryo .

17 ; Fig. The cross section, shown in Fig. 4, is also helpful here. ‘With the aid of such preparations, one can determine accurately the extent of capillary growth in this region of the body, for the only vessels present at this stage, besides those in the early limb, are these capillaries surrounding the spinal cord and a few to the Wolfﬁan body. The injections show that the dorsal segmental vessels are concerned as yet solely in the supply of the lateral aspect of the cord. The segmental artery reaches the cord near its ventrolateral angle, and from this point there radiates the capillary plexus conﬁned as yet entirely to the lateral aspect of the cord. On the dorsal surface of the cord no vessels are yet present, the upper limit of extension of the lateral vessels is well marked by the level of emergence of the segmental veins. A few sprouts are pushing ventrally, but as a whole, that surface of the cord, like the dorsal, is as yet non—vascular. Thus it appears that the capillaries supply first those areas of the spinal cord where the greatest development or cell activity occurs, for it is well known that the lateral region of the cord is at this stage concerned in the formation of the spinal nerves, their ganglia and their roots. It is not improbable that the same correspondence is found in other tissues with an early blood supply, and that at the stage we are cliscussing, the appearance of the subclavian and nephric capillaries is similarly related to marked cell activity in these areas, activity which is responsible in the one region for the outgrowth of the limb bud and in the other, in the formation of the mesonephros.

The drainage of the early limb bud is interesting. “Then one EARLIEST BLOOD VESSELS IN ANTERIOR LIME BUDS.

HERBERT M. EVANS.



--—3%"Aa'ricArcl1.


_ ----Duct ofcuvier --Umbilical vim

FIG. 23.

THE AMERICAN JOURNAL or ANATOMY.-—VOL. IX, _.1\'u 2 EARLIEST BLOOD VESSELS IN ANTERIOR LIMB BUDS.

_IE[ERBERT M. EVANS.

THE AMERICAN JOURNAL or ANA.'].‘OMY.—VOL. IX. No. 2. FIG3FIG. 3.-—Chick embyro of 38 somites (embryo 17 of table) showing multiple subclavians and their relation to the dorsal segmental vessels, X 53174;. -

5th Dor. Seg. Ves., ﬁfth dorsal segmental vessels, occupying the sixth intersomitic septum.

Ot. Ves., otic vesicle. 4th Subcl. Art., fourth subclavian artery of right side, opposite the seven-

teenth dorsal segmental vessels (those of the eighteenth septum) and hence .

likely to become the chief primary subclayian artery of later stages.

U. V., umbilical vein, here subserving no function other than draining the limb bud. '

The ﬁgure shows clearly the dorsal segmental vessels and their distribution as a simple capillary plexus investing the lateral Walls of the spinal cord. Earliest Blood Vessels-in Anterior Limb Buds. 295

considers the story told in the earliest stages (embryos 1 an_d 2), it is evident that we must consider the umbilical vein as its earliest _drainage channel. This vessel grows rapidly in size and receives a constantly increasing number and caliber of venules from the limb. But a process of coalescence of the limb’s capillaries with the posterior cardinal vein opens up other avenues of drainage, so that there comes to be a row of venules opening from the capillary plexus of the limb bud into the posterior cardinal vein. In embryo

3, there are four of these venules on the right side but many others are soon to be formed.

FIG. 4.—Cross section of chick embryo of 33 son1ites_(embryo 7 of table), showing the ﬁrst left subclavian artery, X 531/_,. Sp. C-‘h., spinal cord. Dor. Seg. Vein, dorsal segmental vein. Dor. Seg. Art., dorsal segmental artery.

Subcl. Art, subelavian artery. One notes its origin from the mid-lateral aortic wall.

Nep. Art, nephric artery.

The next embryo, No. 4, is remarkable in possessing the greatest number of subclavian capillaries found in the series (Fig. 5). 5296 Herbert M. Evans. T

Eleven of these vessels spring from the lateral aortic wall cmcl omistomostng, form CL simple plexus in the limb. Six venules may be seen entering the posterior cardinal trunk. The umbilical vein, seen faintly in the somatopleure, is of considerable size and receives many tributaries from the limb (Fig.

It is Well to call attention here to a constant phenomenon observed in the spread of the capillaries. through the limb bud. They extend in every direction through the limb tissue, and ﬁll it with a uniform mesh of vessels, save in a deﬁnite b_order zone. This border zone or marginal nonyascular” area is never invaded by capillary sprouts and remains uninvaded till the time of establishment of the border vein. The latter structure, constructed out of the most peripheral portion of the limb’s plexus, thus marks the old boundary between the primary vascular and non-vascular zones.

Embryos 5 and 6 (Figs. 6 and 7) furnish other instances of the variations in the exact pattern of these capillaries forming the primary limb plexus. Both the anastomosis of the subclavian capillaries soon after their emergence from the aortic Wall, and the occasional division of these vessels soon after their origin, are to be expected from the usual behavior of capillaries. Instances of this are seen in both ﬁgures. Some Writers have seen ﬁt to especially remark

“The signiﬁcance of non_vascular areas is as yet unsolved. However, careful studies on a series of complete injections show them to be a deﬁnite feature in the circulation of every region of the early embryo. We have to do here perhaps with a matter of cell chemistry and tropisms, for endothelium apparently avoids certain are_as in the embryo—the non—vascu1ar areas. In the case of the_ arm buds, the early non—vascular area consists of a narrow strip o-t‘ denser mesenchyme adjoining the ectoderm.

FIG. 5.——Chic1; embryo of sixty hours incubation (embryo 4 of table), showing profuse outgrowth of primary subclavian capillaries into early limb bud, )( 531/2. '

14th D. I. V., fourteenth dorsal intersegmental vein, that of the ﬁfteenth inter-somitic space.

Pri.m. Cap. Plexus, primary capillary plexus of the limb bud.

Post. Card. Vein, posterior cardinal vein.

Prim. Snbcl. Art, primary subclavian artery of left side,_ the lowest one of four subclavians here, but opposite the seventeenth dorsal inter-segmental vessels and already the largest of the series. EARLIEST BLOOD VESSELS IN ANTERIOR LIMB BUDS.

HERBERT M. EVANS.

-Umbilical
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FIG. 5.

THE AMERICAN JOURNAL 012' ANA’.[‘OMY.—-VOL. IX, No. 2. Earliest Blood Vessels in Anterior Limb Buds. 297

on these occurrences, to call them “unusual divisions” and “island formation,” etc., yet this is all to be expected of capillaries tending to plexify.” It is, indeed, more remarkable that the subclavian capillaries usually cross the posterior cardinal vein before anastomosing to any great extent. The general appearances given by the limb vessels in these two embryos are quite signiﬁcant. They must


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FIG. 6.——Right Wing bud of chick embryo of 31 somites (embryo 5 of table), showing primary subclavian capillary plexus, )( 531/3. Instances of “inselu bildung” and early division of the subclavians are seen.

19th D. I. V., nineteenth dorsal intersegmental vein (that of the twentieth interspace) .

convince one of the existence here of a simple plexus——a plexus the first meshes of which are elongated, as a rule, but a true plexus, nevertheless, of true capillary vessels. Nor would any of the appear.ances lead one to believe that this primitive plexus in the limb is in any way arranged according to a segmental plan.

I have designated Embryo 7 as the last of those having the sub-

“’*Thus Rabl seems surprised at these appearances. “Die mittlere der drei Subclavien zeigt eine hesondere Eigentiiinlichkeit, indem sie sich unmittelbar nach ihrem Ursprung teiit.” P. 355, 10c. cit. See also his text ﬁgures. 298 Herbert M. Evans.

clavian vessels still in the first phase or period of development. It is", in every respect, slightly older than the embryos we have just been considering. Some of the primary subclavian capillaries have undoubtedly disappeared and there now remain but a total of seven of these vessels on both sides, four on the right and three on the left. I have considered it as belonging to the first general period


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in the development of the subclavian, however, for the majority of the vessels are at unsegmental points. With the existence of so large a proportion of the vessels. out of harmony with the segmental plan, I think we can hardly classify this embryo as in the period of segmental subclavians. Of the four suloclavians present on the right side, the ﬁrst arises opposite the sixteenth dorsal segmental vessels, the second midway between the sixteenth and seventeenth segmentals, the third exactly opposite the seventeenth, and the fourth Earliest Blood Vessels in Anterior Limb Buds. 299

about half—way between the seventeenth and eighteenth dorsal segmentals. Thus two of the snbclavian arteries on the right side are true segmental Vessels——those opposite the sixteenth and seventeenth dorsal segmental arteries—but an equal number, two, are completely out of harmony with the segmental plan. On the left side, the ﬁrst subclavian is opposite the seventeenth dorsal. segmental vessels, the second about midway between the seventeenth and eighteenth, and

FIG. 8.—Cross section of chick of 33 somites in region of anterior limbs and midway between the sixteenth and seventeenth intersegmental vessels. The section shows the fourth right and the second left subclavian. The section shows clearly the peripheral limit of extension of the limb capillaries and the nonvaseular, marginal zone.

U. V., umbilical vein.

Post. Card. V., P0-steri=o-1' cardinal vein.

the third s.ubel.aVian considerabl y below the interspaee belonging to the eighteenth pair. Here, then, only one of the subclavians is a segmental Vessel, and of the total of seven vessels but three are segmentally placed. The umbilical Vein has been appreciably eX~ tended considerably below the region opposite the limb bud by a still further caudal downgrowth of the primary body wall capillaries. Opposite the limb, it receives on each side about ten distinct tributaries, the upper ones, especially, being no longer capillaries in size 300 Herbert M. Evans.

but small venules. Thus the earliest drainage channel for the limb does not lose this function during the next succeeding stage, but instead becomes increasingly important as the chief vein of the limb. '

I have presented two typical sections through this embryo in the arm region since these will answer well for the relations thus shown in all the embryos belonging to the "rst subclavian period. Fig. 8 shows a cross-section through the embryo at the region of origin of the fourth right and the second left subclavians. Both vessels are unsegmentally arranged and hence the dorsal segmental vessels do not appear in the section. While on the right side the subclavian arises from the dorso-lateral angle of the aorta, the left vessel emerges from the true lateral side of the aortic wall, and only a short distance above the origin of the nephric capillaries, one of which is shown in the section. Thus in the early stages the place of origin of the subclavians from the aortic circumference varies considerably, and Fig. 4, showing the "rst left subclavian in this embryo, indicates how far laterally the early vessels of the subclavian series may arise. In this instance the sabclavian -is almost a mid lateral derivative of the aorta. Such vessels must curve dorsally in crossing the posterior—cardinal vein to reach the tissue of the limb, but the early dorso-lateral branches all course in a straight transverse line.” The character of the dors-.al—segmental vessels and their capillaries has already been mentioned. .

A review of the table which has been presented, shows two features of interest in connection with these earliest stages in the vascularization of the limbs. I refer to the high position of the first sub~

“Rabl has emphasized this straight course of the early subclavians, pointing out that Sabin missed it, for the stages which the latter studied were all old enough to show the dorsal bend which the subclavians then take in reaching the limbs. This dorsal bending is assuredly a secondary bending of an original straight vessel; b'ut my own specimens have disclosed a number of the very earliest subclavians arising from so low a point on the lateral aortic wall that a primary arching course is necessary to reach the limb tissue. It is not unlikely that these subclavians disclose the more primitive place o-f origin of the subclavian series, for they do not occur in even slightly older embryos. G-o-od justiﬁcation thus exists i11 considering the subclavians as primarily true lateral branches of the aorta. Earliest Blood Vessels in Anterior Limb Buds. 301

clavian capillaries—-their origin in the neighborhood of the twelfth and thirteenth segments——-and to certain differences in the vascularization of the right and left limbs.

Rabl’s studies indicated that the subclavians of later stages arose at successively lower levels from the aortic wall. The injections here reported, however, indicate a more cephalic extension of the subclavians than had been previously suspected, and so extend even more the “wandering” of the upper limb and its vessels.

It appears that the right limb bud is the first to receive capillaries, and that this limb in the early stages always possesses a greater number of subclavian capillaries than the left limb. Apparently the two limbs are identical in their relations to the body and in the conditions with which they have to deal in their development save in one respect, namely, that at the time of origin and earliest stages of the limb buds, the embryo is always resting on the left side. This causes a slightly more flexed position of the under or left li1nb—bud with reference to the body wall and permits a somewhat extended, freer projection of the uppermost right limb. This may be related to the greater speed and profusion with which the first vessels grow into the right limb.

But in summing up the co-ndition found in the five embryos illustrating the first period in the development of the suhclaeian, nothing is more strihin g than that we have to deal here merely with an irregular plexus of true capillary vessels which are in no wa-y related to a segmental plan. Thus if the chance arrangement of any irregular capillary plexus obtains here, it should happen that as many of the vessels arise from non—scgmental as. from segmental points, a.nd this is actually the case.

EMBRYOS 01+‘ *r11.n SECOND PER-I01).

The first embryo classed in the stage of segmental subclavians (Embryo 8), has almost as high a proportion of non—s'egmental vessels as has Embryo 7, but two sigificant changes have occurred. These consist in the purely rudimentary character of the non—segmental suhclavians and the enlargement of that pair of segmental subcla/vians opposite the eighteenth pair of dorsal segmental ves302 Herbert M. Evans.

sels. The ﬁgure (Fig. 9) plainly indicates this. On the right side, the segmental subclavian opposite the nineteenth pair of dorsal segmentals is somewhat larger than the uppermost atrophying, nonsegmental subclavians of that side. Some in"uence, then, favors the subelavians at strictly segmental points e., opposite the intersomitic intervals) and is inimical to the growth of those not so situated. Of the segmental subclavians, one, doubtless for purely hydrodynamical reasons, begins to be the chief supply of the limb.

Embryo 9 of the series shows a mos‘; interesting condition (Fig. 10). Here all but one of the subelavian series persisting are approximately in harmony with the segmental plan. 011 the left side, excepting the main vessel, the only subclavians which have survived are those at true segmental points. Thus the dorsal segmental vessels have opposite them at the sixteenth and seventeenth interspaces, two delicate segmental subclavians. The main subclavian artery on this side does not arise at an exactly segmental point and it is fo-rniecl by two frequently anastomosing vessels, arising somewhat in front of the eighteenth interspace. Doubtless this channel is to be shifted by unequal growth and be incorporated with the eighteenth dorsal segmentals in a later stage. The right side has two vestigial vessels, which no longer reach the limb tissue, and a larger channel opposite the eighteenth segment and constructed here also not from one but from several preexisting capillaries.

Embryo 10 possesses thirty—four somites, and has some six subclavians, four on the right and two on the left side. On the former side, two of the persisting subclavians are segmental vessels, exactly opposite the eighteenth and nineteenth dorsal segmentals, but the remaining two are non-segmental and occur in the interspaoe be-

tween the former two. These non-segmental suhclavians are, strangely enough, large trunks, equally as large as the true segmental suhclavians, plunging into the core of the limb and being important

FIG. 9.——Dorsa1 view of anterior limb buds and their vessels in a chick of seventy-two hours incubation, X 53%. _

15th D. I. _V., ﬁfteenth dorsal intersegniental vein, /3. e., that of the sixteenth interspace.

Trans. Subcl. Aria, transitory Si1l'J€l€l\-'i£11]. artery.

Chief Prim. Subc]. Art, chief primary subciavian artery. EARLIEST BLOOD VESSELS IN ANTERIOR LIMB BUDS.

HERBERT M. EVANS.

THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. EARLIEST BLOOD VESSELS IN ANTEBIOR LIMB BUDS.

HERBERT M. EVANS.

FIG. 10.

THE AMERICAN JOURNAL OF ANATOMY.-——VOL. IX, N0. 2. Earliest Blood Vessels in Anterior Limb Buds. . 303

arterial sources in the limb’s circulation. On the left side, both of the subclavians existing are true segmental vessels, at the eighteenth and nineteenth segmental points.

The cross section (Fig. 11) shows the pair of segmental subclavians corresponding to the eighteenth segment, and the corresponding dorsal segmentals. The subelavians arise at the dorso-lateral angle of the aortic circumference and, indeed, in a slight local bulging of the aortic wall, from which the dorsal segmental vessels also take origin. The limbs do not project laterally as before but are bent in more, parallel with the main body axis. The aorta is elongated dorso-ventrally with a slight compensatory lateral narrowing more marked ventrally so that in section the whole vessel now appears triangular. The dorsal segmental vessels are still conﬁned in dis.tribu':.ion to the spinal cord and chie y to its lateral aspect. Neither the dorsal nor the ventral surface of the cord are yet supplied with capillaries though these vessels have begun to extend over both of these surfaces. The highest tributaries of the segmental veins are thus now somewhat above the dorso-lateral angle of the cord.

The remaining embryo of the second period—the period of segmental subclavians——-is Embryo 11, with thirty-six somites and two segmental subclavians on each side, those of the eighteenth and the nineteenth segments; but the latter vessels are now mere vestigial rudiments. The common origin of the dorsal segmental ves-

sels and the subclavians is. somewhat more pronounced. I need not dwell longer on the four embryos which belong to the period of multiple segmental subclavians. The accounts of

FIG. 10.——Dorsal View of anterior limb buds and their vessels in a chick of seventy-two hours incubation, )< 53% (embryo 9 of table).

Trans. S. A., transitory subclavian artery, here opposite the sixteenth dorsal intersegmental vessels. The ﬁgure shows an interesting stage in the evolution of the limb’s vessels. The original subclavian capillaries are now chieﬂy represented by those at intersegmental points, i. e., the so-called “segmental subclavians.” But even here there are atrophying and the chief primary subclavian arteries remain. The latter vessels happen to be constructed from several contiguous subolavian capillaries rather than from a single one as is usually the case. 3304 Herbert M. Evans.

Rabl and of Miiller have already sufiiciently emphasized this interesting stage in the limb vessels.

VVe have seen that most of the subclavian capillaries arising from the aorta at non-segmental points. eventually atrophy, and there now remain only the vessels which stand opposite the segmental interspaces. Thus are produced the segmental subclavians, a truly metameric arrangement of the limb vessels.

Two features of some importance in these stages have been previously overlooked. These are:

1. Abundant traces of the earlier capillary plexus stage of subclavians occur in the period" of segmental subclavians. These consist in several smaller or atrophying vessels of the subclavian series which do not stand at segmental points. Such vessels are often present to complicate the picture of the segmental subclavians, especially early in this period. When the stage is reached in which the multiple segmental subclavians are carried up as common branches with the dorsal segmental vessels, these non-segmental rudiments rarely persist longer and we have at length a perfect picture of multiple true segmental subclavians.

Most of the non—segmental subclavians of this stage are delicate

vessels, but it occasionally happens that some of them are larger

sturdy channels of equal value with the segmentals. This was the case, for instance, in Embryo 10 of the series. It was thus surprising to me that Rabl had not found such vessels, but a careful rereading of his descriptions shows that he doubtless saw some instances of them. He attached a peculiar signiﬁcance to them, however, conceiving that they came about through a splitting of a preexisting single segmental vessel, thus forming a double vessel Whose roots wandered apart! It was quite impossible to him that the subclavians should arise at other than segmental points. Even the cases of “insel bildung” he would make come through a similar splitting of single vascular channels.

2. The second point which I wish to make is that it must occasionally happen that even the vessel most favored in the row of subclavians may not be at ﬁrst at an exactly segmental point as Fig. 10 plainly showed. Earliest Blood Vessels in Anterior Limb Buds. 305

The effect of the intrusion of a metameric inﬂuence in the plan of the lin1b’s vessels is as plainly marked in the ea.se of its veins as in the arteries, for of the row of venules entering the posterior cardinal vein, those at segmental points are often deﬁnitely larger than the remainder. Thus segmental veins as well as arteries exist.

F10. 11.———Gross section of injected chick embryo of 34 somites in the region of the anterior limb Duds (embryo 10 of table),

D. S. V., seventeenth dorsal intersegmental vein.

Sp. G., spinal ganglion; N. Gh., notoehord; P. C. V., posterior cardinal vein; S. C. V., subcardinal vein; U. V., umbilical vein; D. S. A., seventeenth dorsal

intersegmental artery ('2'. (3., that of the eighteenth interspace); Subcl. Art, subclavian artery; N. V., nephric vein.

The fact that the veins draining more ventrally into the umbilical vein are not effected by the segmental plan would indicate that a metamerism does not pervade the entire limb tissue. One feels that the segmental arrangement of the arteries and more dorsally placed Veins is the direct result of the inﬂuence of the adjoining myotomes.

I must comment here on the embryo listed as No. 17 in the series. 306 Herbert M. Evans.

FIG. 12.—Bight. wing bud of chick of 45 somites, X 53%.

. % 5 IA t. 552:5.)/i‘O«:t of? Bbéerg. «i58emgD»§«:t.

FIG. 12b.—Right and left wing buds of a chick of the ﬁfth day. Earliest Blood Vessels in Anterior Limb Buds. 307

I have placed it there since it is the only one which does not ﬁt well into the series, for though its age and number of somites would indicate a more advanced scheme in the vessels of the limb, I found here no less than six vessels of the subclavian series. The embryo may be viewed, as an instance, in which the limb and its vascular system has run slightly behind the normal for this age, or as a case of the persistence of multiple non—segmental subclavians. I have drawn the embryo, as a whole, since it shows splendidly the typical relations of the early limb capillaries and those belonging to the dorsal segmental series (Fig.

PERIOD on PRIMARY SUBCLAVIABT ARTiERY.

Embryos 12 to 16 all illustrate stages in the growth of the primary subclavian artery. The relation of this trunk to the preexisting segmental subclavians, has already been clearly indicated in Embryo 8; even there we saw the early exaggeration of one of the members of the subclavian series. The processes of vascular atrophy and death which early eliminate the original non—segmental subclavians, destroy also eventually the segmental subclavians with the single exception of that vessel destined to become the primary subclavian artery.

It is of interest that even in these late stages, there sometimes persists a 11on—seg1nental artery. Fig. 12 gives an instance of this. It is the Embryo 12 in the series and possesses some forty-ﬁve somites. The large primary subclavian artery has below it and near the middle of the adjoining somite, a narrow rudimentary vessel which has persisted from the primary subclavian series. True segmental vessels, other than the main one, may likewise persist in limbs of this age. This is possible through the early proximal anastomoses between subelavians. The use of several -of these paths will give several segmental roots of or.igin to the primary subclavian trunk. Fig. 12b, is a striking instance of this.

In embryos of eighty-four and ninety—six hours incubation, the primary subclavian artery has attained a large size. The dorsal segmental vessels have also increased in caliber. The capillaries belonging to the latter system have S1I1']°.O111’1(l(3(l the spinal cord com.308 1 Herbert M. Evans.

pletely and grown out as a loose plexus over the outer surface of the myotomes.

Fig. 13, from the embryo of 116 hours (No. 16) show the further growth and elaboration of these changes. The common trunks of the subclavian and dorsal segmental vessels, are themselves being shifted toward the mid-dorsal line, soon to arise from a single common trunk. The segmental arteries and veins have each two main systems of branches which alternately supply and drain the cord at successive points around its circumference. Penetrating arteries extend frorn the ventral arterial tract into the cord substance at the boundary zone of the ncuroblasts and ependyma. They are drained by delicate transverse venulcs.

The subclavian arteries have two small branches before supplying the limb proper, a dorsal branch which supplies the outer capillary plexus over the myotomes and a ventral twig to the \Volfﬁan duct.

The subclavians are large vessels and control the blood supply to the limb. They must be considered now at the height of functional activity, and with this stage in the history of the primary subclavian the present account closes.

IV. OBSERVATIONS on THE CONDITIOXS PRESENT AT SIMILAR STAGES IN Emnnros on THE DUCK.

Rabl’s research on the development of the subclavian artery was conducted '-entirely on ducks. In it he failed to ﬁnd stages earlier than the period of strictly segmental subclavians. It was consequently of some importance that these forms be investigated to see if the early subclavian capillary plexus which was present in chicks was not of fundamental value and hence present here also. Of a series of ten of these duck embryos I shall describe carefully only two typical ones, one, an embryo of thirty—three somites in the stage of an irregular subclavian capillary plexus, and the other,

FIG. 13.—Cross section of chick of one hundred and sixteen hours incubation, in region of fore limbs. M_v., niyotoine; D01‘. Vein, dorsal branch of the segmental vein; Post. Sp. Art, branches of the segmeiital artery which contribute to the formation of the posterior spinal artery; Pen. Art... penetrating artery; P. C. V., posterior cardinal vein; Se. V., Seitenrumpfvene, thoracoepigastric veins; Rad. A1-t.. radicular artery. EAR-LIEST BLOOD VESSELS IN Al\"1'I:‘.Rl0R L'Il\I}3 BEDS.

HIERBERT M. EVA NS.

Fm} 13.

THE Anmmcax JOURNAL or A2\’A'I‘OM}E.-—-VOL. IX, No. 2. EAELIEST BLOOD VESSELS IN ANTERIOR LIMB BUDS.

HERBERT M. EVANS.

THE AMERICAN JOURNAL OF ANA'I‘O.\-IY.—-VOL. IX, N0. 2. Earliest Blood Vessels in Anterior Limb Buds. 309

an embryo of thirty—eight somites, belonging to the period of segmental subclavians. Both embryos illustrate strikingly the facts observed in these stages for chicks.

DUCK El-MBRYOS.

Number of Subclavians present. No. Somites present. -—Left. Right. 1 33 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1| 6 2 38 . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . . . . . 5 , 6

The duck embryo of thirty-three somites was drawn carefullly from several aspects before being out into serial sections. A general view of the region of the a.nterior limb is shown in “Fig. 14. One sees clearly the irregular VGHOUS channel through the remains

of the primary body—wall plexus, leading now from the capillaries of the limb to the duct of Ouvier. The umbilical Vein in the duck has an exactly similar origin as in the chick, and is here also the primary drainage channel for the limb. Between the sixteenth and twentieth dorsal segmental vessels an outgrowth of limb capillaries from the aortic wall occurs. There are six of these V€SS(-3lS. They anastomose promptly and form a continuous irregular capillary plexus extending into the limb, which is as yet a mere swelling of the somatopleure. No one capillary of the subclavian series is larger than its neighbor. The series is in no way arranged in a segmental plan.

Cross sections through the embryo show the topography of the limb region. I have drawn one which shows strikingly the hitherto undescribed origin of these earliest subclavian capillaries from the mid-lateral region of the aortic wall. They are compelled to bend dorsally in growing into the limb bud. The section (Fig. 15) shows the fourth left and the sixth right subclavians. Neither Vessel happens to arise at a segmental point.

FIG. 14.——Upper body wall of due]: embryo of 32 somites. Lettering as in previous ﬁgures. The wall of the aorta is concealed behind the vein. 310 Herbert M. Evans.

There are six snbclavians on either side in this embryo. The first subclavian on the right side occurs midway between the sixteenth and seventeenth dorsal segmental Vessels, the second subclaVian opposite the seventeenth segmentals, the third somewhat beyond this point, the fourth ust in front of the eighteenth dorsal segmentals, the ﬁfth somewhat beyond this point, and the sixth midway between the eighteenth and nineteenth segme-ntals.


FIG. 15.——Cross section of the duck embryo shown in Fig. 14 in the region of the anterior limb buds. One notes the midlateral origin of the subclavian capillaries from the aortic wall.

On the left side, the ﬁrst snbclavian arises opposite the seven-

‘teenth segmental Vessels, the second midway between these and the

eighteenth vessels, the third just in front of the eighteenth seg~ mentals, the fourth midway between the eighteenth and nineteenth segmentals, the ﬁfth at the level of the nineteenth segmentals, and the sixth midway between the nineteenth and twentieth segn'ie11— tale.

It is impossible, here also, to see in the arrangement of the subclavians any inﬂuence of nletainerism. There are as many vessels out of segmental alignment as are in accordance with it and this because, again, in the origin of the typical plexus here as many Earliest Blood Vessels in Anterior Limb Buds. 311

capillaries should chance to be opposite the intersomitic spaces as are opposite the somite masses and vice versa.

Duck embryo 2, possessing thirty—eight somites, happens to have almost as many subclavians as occurred in the younger embryo, but in the older stage, besides being larger, these vessels are almost all at segmental points, so that the embryo belongs clearly to the period of multiple segmental subclavians. T

On the right side, there are six subclavian vessels arising from the aorta. The first subclavian occurs just in front of the six~ teenth segmental vessel, the second and third at the level of the seventeenth segmental vessel, the fou_rth and ﬁfth at the level of the eighteenth segmentals, and the sixth halfway between the eighteenth and nineteenth segmentals.

On the left side, the first subclavian stands opposite the sixteenth segmentals, the second opposite the seventeenth vessels, the third opposite the eighteenth segmentals, and the ﬁfth opposite the nine teenth segmentals. Thus there are on the left side as many as four segments represented by subclavians.

However the study of ' even this embryo, with such a complete series. of segmental subclavians, shows that here also there persist some vessels not in segmental alignment. The last subclavian on the right side is such a vessel, for it occurs midway between the eighteenth and nineteenth dorsal segmental vessels. The cases of two subclavians existing opposite a segmental point are easily explained by the chance origin of two of the early capillaries opposite one of the inter—somitic clefts. In such cases both vessels are equally favored, and both persist to the stage of segmental subcla— vians, where they increase the number of vessels to be expected. 1 have no doubt but that the condition in this Embryo 2, was preceded by a stage of some ten or twelve subclavian capillaries, similar to those seen in chicks 3 and 4, but these interesting stages are so transitory in character that it is only rarely. that we have the good fortune to see them. Some capillaries, here as elsewhere in the developing vascular system, push out, function slightly and die in a surprisingly short time. 312 Herbert M. Evans.

V. COMPARISON WITH THE POSTERIOR LIMB BUD.

It was of great interest to ascertain Whether the leg bud in the embryo had a similar capillary plexus from the aorta in its earliest stages. Such was actually found to be the case. Fig. 16 shows the hind limb buds in a chick embryo of thirty—two somites, No. 3 of the series.

One may see distinctly the dorsal segmental vessels and in addition, independent lateral offshoots from the aorta. At this time in the leg, the posterior—cardinal vein has not yet extended there, and both the dorsal segmental Vessels and the lateral capillaries anastomose in the tissue of the limb and furnish its primary plexus. This plexus is dorsal to a more ventral plexus which arises Very early; in fact, with the formation of the lower aorta, and is not to be confused with the latter.

The injections demonstrate the later origin and growth of the sciatic artery from this mesh of capillaries, so that We have to do here with an exactly analogous condition as occurs in the upper limb’s vessels. In both cases, the chief axial Vessel of the limb is preceded by, or may be said to exist in the form of, a simple capillary plexus arising directly from the aortic Wall.

==VI. Observations on the Early Mammalian Arm Bud==

At the present time We have the most complete history of the earliest limb Vessels in the birds, but it is. naturally of the greatest interest to compare these ﬁndings with the conditions obtaining in mammalian embryos.

Little is known of stages preceding the single axial subclavian in the latter class, save the two human embryos listed by Keibel and Elze, and the instance of early segmental human subclavians de— scribed by the writer.

I accordingly under.took a series of injections of young mammalian embryos, choosing on account of the abundance of the material,

FIG. 16.-—The caudal end of a cliick en1br3"o of 32 somites (embryo 3 of table), showing the primary capillary plexus in the posterior limb buds.

26th Dor. Se;-'_.,'. Veiii. tWenty—si.\'tl1 dorsal segmental vein. '5. 0., that in the twe11t}'-seventli i11te1'spa(‘e. 

embryos of the pig. Embryos young enough to show the earliest conditions in the arm bud are not common, and to supplement this material I have been fortunate enough to examine several perfect series of rabbit embryos from the Harvard Embryological Collection through the kindness of Professor Minot. The latter embryos are all the more interesting since they were the types chosen in the compilation of the “Normal Plates on the Development of the Rabbit” by Minot and Taylor.“-5 Thus the stages of development may be accurately known from the various details listed opposite them in the latter. work. They comprised embryos designated as Nos. 562, 559 and 556 and in the “Normal Plates” are given the table numbers, 10, 11 and 12. I shall describe these very brieﬂy. Embryo 562 has the limb buds as mere svvellings of the somatopleure.' On the left side, one could not be certain of the existence of any subclavian capillaries, but on the right side, a subclavian is present midway between the sixth cervical and the seventh cervical segmental vessels.


FIG. 17.—Reconstruction of the position and course of the segmental subclavian arteries present in a rabbit enibryo of the tenth day, No. 559 Harvard Embryological Collection.


“Minot and Taylor. “Normal Plates on the Developinent of the Rabbit.” In the series edited by Keibel. 314 Herbert M. Evans.

Embryo 559 has three segmental subclavians on the right side and but one on the left. The right subclavians arise from the seventh and eighth cervical and the "rst thoracic segmental arteries. The left subclavian is a branch of the seventh cervical segmental vessel. Fig. 17 shows a reconstruction of the subclavians in this embryo.

Embryo 556, slightly older, possesses only a single subclavian on the right side, that of the seventh cervical segment, but two segmental subclavians on the left side, those of the seventh and eighth segments.

In both the latter cases (Embryos 559 and 556) the subclavian arteries are already b-ranches of the dorsal segmental vessels, but in the earlier case (No. 562), in which a single non-segmental subclavian existed, this was obviously not the case. There is every reason for believing that this youngest embryo is in the ﬁrst stage of development of the subclavians, and one feels that the study of more mammalian limb buds at this stage will show more segmental and non—segmental subclavian capillaries.”

Not only in its arterial but also in its venous system does the early mammalian arm bud agree strikingly with that of the bird. In mammals, also the ﬁrst and most important drainage channel for the arm is the umbilical vein. Figs. 18 and 19 show the position and character of the venules which drain the early mammalian arm bud into the umbilical vein. The uppermost or cephalic portion of the mammalian umbilical vein has long been known to persist for a considerable time as a much attenuated channel, still connecting with the duct of Cuvien after the main vessel has established connections through the liver. The reason for this persistence of the old upper portion of the umbilical vein is now clear, for it still furnishes an important drainage channel for the arm bud.


“Since these -observations were made, Goppert has published an account of the early blood vessels in the arm buds in white mice and his reconstructions bear this out. He has shown striking instances of a segmental subclavian series, with segmental and non—segmental members, though he does not realize the signiﬁcance of the latter vessels. Goppert is disposed to view this merely as an evidence of variability in the embryonic arterial system. He has missed the key to the solution, however, for we are dealing here, as my injections show, with the persisting members of an early irregular, capillary plexus. In such ﬂeeting phenomena as the outgrowth and regression of many of these capillaries, we must expect to see embryos from the same uterus in slightly different stages of development. There is as good reason for this interpretation, surely as there is for his of variability.

‘ Goppert, E. “Variabilitiit im embryonalen Arteriensystem.” Verhandlungen der Anatomischen Gesellschaft, Anat. Anz., Bd. XXXII, 1908, pp. 92-103. Earliest Blood Vessels in Anterior Limb Buds. 315


Thus in pig embryos 71/2 millimeters long this cephalic part of the umbilical vein still receives some seven or eight tributaries from


FIG. 18.——Latera1 view of pig embryo G nnns. long, showing drainage of arm bud into umbilical vein, X 11.2. '

FIG. 19.——Detai1ed View of venules draining the left anterior limb bud of the pig embryo shown in Fig. 18, X 331/3.

the arm bud as shown in Fig. 20. The mammalian arm bud is also drained by a series of vcnules opening into the posterior cardinal vein.

SUMMARY of RESULTS.

The chief facts brought forward in the present investigation may be surnmarized as followed:

1. The first bloocl-‘vessels supplying the limb buds are capillaries which grow from multipleirregular points of the lateral aortic wall 316 Herbert M. Evans.

and auastomosiug, often even before they reach the root of the limb, form a simple and quite typical plexus. ‘In the arm hucl, this capillary plexus constitutes the earliest stage of the suhclavian artery, in the leg bud, of the femoral artery. The first subclavian capillaries, partaking of the character of any irregular capillary plexus, are thus never arranged in a truly segmental plan.

2. The subclavian capillaries join another plexus of capillaries, which has grown down in the b0dy—Wall from Cuvier’s du.ct—the


FIG. 20. Lateral view of pig embryo 71/; mm. long, showing the persisting cephalic portion of the umbilical vein still receiving tributaries from the arm

bud. The embryo is drawn at the same magniﬁcation as that ﬁgured in Fig. 18, 11. e., X 11.2.

primary body—-Wall plexus. The consequent establishment of a circulation from the aorta to Cuvier’s duct converts the subclavian capillaries into arterioles and certain of the primary body—wall capillaries into a vein- the umbilical vein. In the birds, the drainage of the early wing bud is thus the sole primary function of the urnbilical vein. In the mammals, although the development of the umbilical vein in connection with the chorionic circulation precedes the formation of the limb buds, nevertheless, when the arm buds arise, their capillaries establish, here also, a drainage into the umbilical vein. This drainage of the mammalian arm bud into the upper portion of the umbilical vein persists after the latter vessel has established its chief circulation through the liver and is doubtless one of the chief causes delaying the atrophy of the upper or cephalic portion of the umbilical vein.

3. The occurrence of a period of multiple segmental subclavians is brought about by processes of atrophy and doubtless slight shifting. Thus most of the primary subclavian capillaries which are not at segmental points, i. 9., opposite the interspaces between the somites, eventually atrophy, leaving as functioning vessels only those members of the early series which are fortunately situated in accordance with this plan.

4. Even during the period of true segmental subclavians, however, there often persist some members of the first s.ubclavian series which are out of segmental alignment. These may indeed get to be vessels of some size. The chief primary subclavian artery itself may not at first happen to lie at exactly a segmental point. The chief determining factors in the persistence of vessels are doubtless hydrodynamical and only secondarily the influence of metamerism.

5. A purely segmental character in the arm vessels is ﬁnally secured at the time of inclusion of the subclavian vessels as common trunks with the dorsal segmental vessels. This union is not a process of active fusion of the subclaviaii and dorsal trunks but is effected by processes of unequal growth which occur in the expansion of the aortic wall. Dorsal and subclavian arteries are carried out together by a local bulging of the aortic wall, which becomes a common trunk.

6. The primary subclavian artery, represents the exaggeration of one of the pairs of segmental subclavians, which is most favorably situated as the principal circulatory channel for the limb.

VIII. APPLICATION on rrrnsx Fxcrs TO THE GZENERAL EMBRYOLOGY or TI-Iii.‘ Vnscurxn SYSTEM.

Two conceptions have arisen regarding the method of development of the vascular system. According to the one, arteries and veins grow out to their end beds as development proceeds, but according to the other, vascular activity is always initiated by capillaries 318 Herbert M. Evans.

which tend everywhere to form a mesh—work or plexus; arteries and veins are always subsequent formations from such capillary plexuses due to the transforming inquence of the circulation.

The former conception would appear to be held by most of the workers in angiogenesis, though most of the descriptions of the development of vessels are so worded as to avoid a lucid statement on this fundamental point. I may refer, for instance, to the many admirable researches of Hochstetter, where, though many important facts concerning the chief embryonic vessels are clearly given, one may look in vain for anything bearing on this point. In the case of the limb vessels, for instance, we must imagine from his description that the single axial vessel grew out into the core of the limb. Very recently Curt Elze” has ranged himself with those who would recognize such a process as the means of development of all the body’s vessels and as deﬁnitely opposed to the idea of a capillary plexus anlage for any of them. On the other hand, Hans Rabl and Erik Miiller have supported vigorously the latter idea, the foundation for which had been laid in the great paper of Thoma on the origin of the chicks yolk vessels.

There is plenty of morphological evidence in the adult body for a preexisting plexus or net—like condition of all the vascular trunks. The remarkable number of variations in the position, system of branching and anastomoses cannot be explained as satisfactorily on any other basis. Thus, without knowledge of conditions in the embryo, Aebylg and Baader” promulgated such a plexus origin for blood—vessels many years ago.

Professor His did not hesitate to state that the main vessels in the embryo were derived from net—like anlagen, but it remained for Thoma” to make the meaning of all this very signiﬁcant.

Thoma observed that in its early stages the system of the vitelline vessels in the chick formed a strikingly uniform simple plexus of

”E1ze. “Besehreibung eines menschliclien Embryo V011 zirka 7 mm. griisster Ld.11g€‘.” Anat. Hefte, 1-35, 1907.

“Achy. “Der Ban des menschlichen Ktirpers.” 1871.

1°Baader. “Ueber die Varietiiten der Armarterien des Menschen.” Inaug. Diss. Bern, 1866.

"°’l‘ho1na, R. "U11ters11cI111nge11 ﬁber die Histogenese und Histo1nec11a11i1: des Gefiisssysteins.” 1893. Earliest Blood Vessels in Anterior Limb Bud. 319

irregular capillaries. He observed that the fortuitous position of some of these capillaries with respect to the aortae and venous ostia of the heart gave them a more constant and rapid circulation than occurred in other capillaries of the mesh. Later. stages showed these capillaries became arteries and veins respectively. As the vitelline vascular system grew, Thoma saw the same laws at work from center to periphery, that the further elab-oration of the arterial and venous trees was the result of successive incorporation of adjoining portions of the general capillary plexus. If these processes are at work everywhere in the development of the vascular system, they furnish us with a better understanding of angiogenesis, for the development of a given artery or vein to any portion of the body cannot be due to miraculous predestination but to the deﬁnite action of quite deﬁnite physical laws. Capillaries first invade a region and the relation of these capillaries to the nearest arterial and venous channels determines always the manner in which the new veins and arteries shall arise. Then elaboration of arteries and veins is always the result of hydrodynamical forces involved in the circulation.

The application of Thoma’s work to the development of the bloodvessels in the body of the embryo has never been adequately tested.

The method of injecting completely the embryonic vascular sys tem has furnished much evidence that the capillary plexus anlage can be demonstrated for. all the body’s vessels. The preceding account of earliest circulatory conditions in the limb bud gains much significance in this light, for before there can be said to be limb arteries or veins, a primitive plexus of capillaries grows into the limb tissue. From this pleccus i-n later stages, arteries and veins are formed ,

Rabl has shown the origin of several arteries in the fore-limb region from capillary nets, but one must leave this interesting story, the development of the later vessels, to another time. In the present study, we have been able to see that the limbs, the main vessels themselveswtihe femoral and the subclavian arterieswexist originally in the f0’)".’)?’L of a capillary plexus.


In conclusion I beg to speak with gratitude of the many suggestions and helpful interest in the present investigation which I owe to Professor Mall.