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THE JOURNAL

OF

COMPARATIVE NEUROLOGY

EDITORIAL BOARD

Henry H. DonaLpson ADOLF MEYER The Wistar Institute Johns Hopkins University J. B. JOHNSTON OuiveR 8S. StRoNG . University of Minnesota Columbia University

C. Jupson HERRICK, University of Chicago Managing Editor

VOLUME 33

APRIL—DECEMBER 1921

PHILADELPHIA, PA.

THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY

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CONTENTS

No. 1. APRIL

F. L. Lanpacre. The fate of the neural crest in the head of the urodeles. Four plates

eleven fPUTES). «2066.0. - occ os oe ep RMB: 26s See eee dens con ete he Be Eh Joe 1 Marcaret H. Coox anp H. V. Neat. Are the taste-buds of elasmobranchs endodermal

in origin? Four plates (twenty-nine figures)............--- 0s eee eee eee eee eee es 45 H. V. Neat. Nerve and plasmodesma. One plate (five figures)..............-.++-5-+- 65 S. E. Jonnson anp M. L. Mason. The first thoracic white ramus communicans in man.

TDA GET Tee eee ene Adin ac ae S30 eats Oe roe acne aie aneee SIE TY RCA! Seen ac 77

Sypnry E. Jonnson. An experimental study of the sacral sympathetic trunk of the cat, with special reference to the occurrence of intrinsic commissural neurons. Seven

eee Anita MEH Dah eGo tafe wise cisrseaic, o's </aha,d eile alma sia/s nie wreeiw ohm eit e = Sinise sei sen eye 24 85 No. 2. JUNE O. Larsetu. Nerve terminations in the lung of the rabbit. Fifteen figures............ 105 E. C. Casz. On an endocronial cast from a reptile, Desmatosuchus spurensis, from the iaper briassre of western Vexas. Nine fsures.... 06.6.4 «ace se alas <a seas able es 5 133 J. M. D. Otmstep. Effect of cutting the lingual nerve of the dog. Six Porpee . 149 Howarp Ayers. Ventral spinal nerves in Amphioxus. Seven figures.................-- 155 Apa R. Hatu. Regeneration in the annelid nerve cord. Seventeen figures............. 163

No. 3. AUGUST

Epwarp Horne Craici£. The vascularity of the cerebral cortex of the albino rat. Five

BPRCOR REE Netes lie Pe 8). Oy fe Sprig Mats a Sate peter Shea ttat s Bla aces aces « Faden she 193 C. Jupson Herrick. The connections of the vomeronasal nerve, accessory olfactory bulb and amygdala in amphibia. Thirty-seven figures.................¢. cesses eens 213

Cut Pine. On the growth of the largest nerve cells in the superior cervical sympathetic ganglion of the albino rat—from birth to maturity. Six charts and one plate (seven HURAURRE ERE eT ON alti Agen aie 5 <i Re chem area ete ake Cai fo, cry 2 paral @igli th ele) Atoe ot WAT & > 281

No. 4. OCTOBER

Cut Pine. On the growth of the largest nerve cells in the superior cervical sympathetic ReanPiiencan THEUNOLWAY Tat, Bive CHATts, xc. 52.5 Sopcast: 5 -a/oncdsees ame Ma arines <4 313

Howarp Ayers. Vertebrate cephalogenesis. V. Origin of jaw apparatus and trigem-

inus complex—Amphioxus, Ammocoetes, Bdellostoma, Callorhynchus. Thirty-six

1V CONTENTS

No. 5. DECEMBER

Hatsert L. Dunn. The growth of the central nervous system in the human fetus as

expressed by graphic analysis and empirical formulae. Thirty-eight figures...... 405 S. R. DerwiterR anp Henry Laurens. Studies on the retina. Histogenesis of the visual cells. in Amblystoma, - (hirteen fieures! 2.5.7 .% 20s. os = + ee see ee 493

O. LarseLL AnD M. L. Mason. Experimental degeneration of the vagus nerve and its relation to the nerve terminations in the lung of the rabbit. Five figures.......... 509

Resumen por el autor, Francis L. Landacre. El destino de la cresta neural en la cabeza de los Urodelos.

La cresta neural de Plethodon se incorpora primeramente al tubo neural, y después emigra desde la superficie dorsal del tubo en direccién ventral, a lo largo de la superficie lateral del tubo, formando porciones de los ganglios de los nervios V, VII, IX y X, continuando después su emigracién ventral hacia las barras branquiales, arco mandibular y arco hioideo, en los cuales forma mesenquima y cartilagos. El mesenquima ectodérmico puede distinguirse del derivado del mesodermo (mesenquima endodérmico) por el tamafio de las células, ntimero y tamafio de los granos de vitelo, pigmentacién y por la continuidad de las lAminas de células emigrantes. El] mesodermo produce todos los musculos de la cabeza, mesenquima de la regién cefdlica dorsal, y la porcién posterior de las trabéculas, los paracordios, base del erdneo, arco occipital, cdpsula auditiva y segundo basibranquial. La cresta neural produce la porcién anterior de las trabéculas, cartilago de Meckel, palatocuadrado, y todos los cartilagos branquiales con la excepcién del segundo basi- branquial, asi como el mesenquima de la regién ventral de la cabeza.

El ectodermo lateral de la regién oral produce un collar ecto- dérmico, en el cual se desarrollan los dientes y tejidos adyacentes. Con la excepcién del collar ectodérmico oral, el ectodermo lateral no produce mesenquima, sino las placodas dorso-laterales, placodas epibranquiales, parte de los ganglios de la linea lateral, lineas laterales y ganglio del nervio profundo.

Translation by José F. Nonidez Cornell Medical College, New York

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, MARCH 14

THE FATE OF THE NEURAL CREST IN THE HEAD | OF THE URODELES

F. L. LANDACRE Department of Anatomy, Ohio State University

ELEVEN FIGURES

CONTENTS MAG rOCUCELOMI eee Noh tere Lt ET IST ESSE EM cae Wee e ela clotengs eroteleraetes 1 EISTORICATES KU CHirges ce ter esennes ote eae aes oes co Neuaraiolckal snares Sheie eueitionyaiecwrae was 3 INS SRCTON DY Geese, oes Sis ARS oe Leteoee Ia eRe t rre Pa 9 hevoriainromshesneuralaeresbers. ces ts otek. ne Fate 6 nae ate cee oe sae eeiele cies 11 The migration of the neural crest and its relation to the mesoderm....... 18 ahevderiwvativessomune) lateral egtouderm.. 1. ss ciaaeactec.esslscraciowe acl 4 ae oe 22 aM RO NA IME CUO ERIN eR Trina Wee vasa cues chav iopanel Bayshore tots. ofr evo) w et(arcemat onaTe ny hone evens se Beans 25 The differentiation of the mesoderm and neural crest in the head.......... 28 SUTIN yews API syn ew Pee CEN ahs: SEIU A 14h RAN YSN 2 Lights Mnaccihe ecg 32 ILS Aer CNL |v ee hes OA Mee Ot I ORE pee we he > an Rae Se rae ase oe 34 ‘INTRODUCTION

During the course of an investigation of the origin of the cerebral ganglia of the urodeles it became necessary to determine accurately the ventral limits to which the neural crest migrates. In the attempt to do this the distribution of the neural crest was found to be so extensive and to involve so much tissue of a non-nervous character that it was decided for the time being to discontinue the study of the cerebral ganglia and follow the history of that portion of the crest which is not concerned in the formation of these ganglia.

. The ventral limit of ganglia into the composition of which the neural crest enters, namely, V, VII, IX, and X, is at the level of the epibranchial placodes, approximately at the dorsal border of the corresponding gill slit. The neural crest in all vertebrate _types, apparently, migrates ventral to this level beyond its ganglion-forming region and further is distributed in the head of the embryo anterior to the level of the gasserian ganglion. 1

2 F. L. LANDACRE

Only a relatively small portion of the neural crest in the head is involved in the formation of cerebral ganglia.

In the urodeles the neural crest forms in the anterior head region an almost continuous sheet of mesenchyme, lying lateral to the axial mesoderm which is derived from the endoderm, and is interrupted for a time by the olfactory bulb and optic stalks only. In the region of the mandibular and branchial bars, the neural crest migrates ventrally into these bars to the extreme ventral level of the body and is prevented from forming a junc- tion with the crest of the opposite side by the presence of the heart and ventral aorta.

The possibility of determining the fate of neural crest cells that do not enter into the composition of cerebral ganglia depends upon conditions that exist in some types and are apparently absent in others. It is the almost unanimous opinion of workers that it is extremely difficult and often impossible to determine the fate of all neural crest cells in the anterior head region. In the branchial region of the lower vertebrates particularly, the situation is very different. A number of reliable workers have given detailed descriptions of the fate of the neural crest cells and of the manner in which they enter into the composition of structures usually considered as mesodermal in origin. As to the migration of the neural crest beyond ganglion-forming regions in all vertebrates, there is apparently little doubt. }

In the urodeles the determination of the extent of the migra- tion of the neural crest and the differentiation of the neural crest and its derivatives from those of the endoderm are com- paratively easy except in the anterior region. The contrast between the large, heavily yolk-laden, light staining endoderm cells and their derivatives on one hand and the smaller, dark staining, pigmented, slightly yolk-laden ectoderm cells and their derivatives on the other hand is very striking. In addition to these distinctions based upon size of cells, staining reaction, pigmentation, and number of granules, there is also an actual difference in the size of yolk granules carried by endoderm cells as compared with those carried by ectoderm cells, those carried by endoderm cells being larger. These distinctions, furthermore,

FATE OF NEURAL CREST IN HEAD OF URODELES 3

persist up to so late a stage in development in certain regions of the head that it can be determined accurately that head struc- tures, usually considered as of mesodermal origin, are derived from the ectoderm of the neural crest.

HISTORICAL SKETCH

The idea that ectodermal cells, derived from either neural crest or from the lateral ectoderm, can be traced into permanent head: structures, other than ganglia, was first stated definitely by Miss Platt in 1893. It was claimed in a preliminary paper published by her at that time that branchial cartilages arose from ectodermal cells. Before the appearance of her paper, however, several papers appeared bearing more or less directly on the fate of the non-nervous portion of the neural crest. Mar- shall (78), working on the chick, and van Wijhe (’82), on the selachians, both called attention to the fact that the neural crest is present in the head anterior to the trigeminus ganglion, but could not determine the fate of that portion of the crest which does not enter into the formation of that ganglion.

Kastschenko (’88) went a step farther and stated that, in the head of the selachian embryo, other layers than the endoderm, particularly the ectoblast, take part in the formation of mesen- chyme, and that in the formation of the neural crest some of the cells detach themselves from the neural-crest mass, become loosely arranged, and form mesenchyme, while other cells from the same source are concerned in the formation of ganglia. The further fate of the mesenchymal cells is not discussed, neither is their relation to mesenchyme derived from endoderm treated.

Goronowitsch (’92) published a preliminary paper in which he determined for birds not only that the neural-crest cells give rise to mesenchyme in the head region, but that they fuse with the axial mesoderm derived from the endoderm to form a homo- geneous mesenchyme in which neural-crest cells can no longer be distinguished. He also noted a _ proliferation of mesoderm (mesenchyme?) cells from the ectoderm in the dorsal portion of

the gill.

4 . F. L. LANDACRE |

Miss Platt (’93), as noted above, published a preliminary notice in which she traced the branchial cartilages definitely to the ectoderm derived apparently from the lateral body wall rather than from the neural crest. Her statement is rather hard to follow and caused her work to be criticised and possibly to be misunderstood. She seems to derive the mesoderm from two longitudinal ridges which later break up into three vertical ridges. The median portion of each vertical ridge proliferates mesoderm into the gill arch to form the cartilage of the gill arch. Ventral to this point the lateral ectoderm proliferates cells into the branchial arch to form mesoderm. Miss Platt states that the neural cells (neural crest?) break up into stellate mesoderm, whose fate she is unable to follow. She does not in her pre- . liminary paper introduce the terms ‘mesectoderm’ and ‘mesento- derm’ for mesenchyme derived, respectively, from ectoderm and entoderm.

Later in the same year Goronowitsch (’93 a and b) published on the development of the neural crest in birds and fishes and asserted again the origin of mesenchyme from both the neural crest and the lateral ectoderm. He makes the surprising state- ment that the neural crest is concerned neither in the formation of ganglia nor nerves, but only in the formation of mesenchyme, a part of which latter forms the sheath of Schwann and deter- mines the course of growing nerves. The nerves.arise from neuroblasts. Goronowitsch derives mesenchyme from the ecto- derm in the region of the gill arch. The periaxial mesoderm comes from both neural crest and from the lateral ectoderm and is associated with the formation of the gill arch presumably, but he does not derive the cartilages specifically from the ecto- derm as Miss Platt had done.

In 1894 Miss Platt elaborated the idea contained in her pre- liminary paper introducing the terms mesectoderm and mesento- derm for mesenchyme derived from the ectoderm and endoderm, respectively. She further recognizes the neural crest as con- tributing to the formation of mesectoderm, though the lateral ectoderm is its chief source. The axial mesoderm is recognized in this paper as coming from the endoderm, but in later papers

FATE OF NEURAL CREST IN HEAD OF URODELES 5

(Platt, ’96, ’97) this is questioned, and as a consequence she discards the term mesentoderm. In these later papers she derives ganglia, nerves, mesenchyme, and branchial cartilages and the dentine of the teeth from the mesectoderm, but neither muscles nor embryonic nerve supporting tissue (sheath of Schwann). In Miss Platt’s second paper (’94) she follows the ectoderm cells into the gill bars, but not to their complete differ- - entiation into cartilage. The later history of these cells and their differentiation into cartilages was given in the paper pub- lished in 1897.

Later papers by Kupffer (95), by Lundborg (’99), by Dohrn (02), and by Brauer (’04) support in the main Miss Platt’s contention. However, Rabl (’94), Corning (99), Minot (01), and Buchs (’02) do not agree with her interpretation. The criticisms of Miss Platt’s theory will be given first.

Rabl (94) at the Strassburg meeting of the Anatomische Gesellschaft criticised Goronowitsch’s description of the mode of derivation of the mesenchyme from ectoderm in birds and teleosts as an assumption, because Goronowitsch admitted that after ectoderm cells fuse with mesoderm he could no longer follow them, and consequently he had no right to assume that they became a permanent part of the mesenchyme. ‘This objection seems to be valid so far as birds and bony fishes are concerned. His objection to Miss Platt’s mode of derivation of cartilage is based not on a study of Necturus, which he admits he has not examined, but on a study of Triton, salamander, and axolotl. He thinks that the appearance, which Miss Platt finds in her preparations, of.cells being proliferated from the ectoderm, can be explained best as due to faulty fixation. Rabl makes a vigor- ous defense of the idea of the integrity of the germ layers. Most of his criticism is devoted to Klaatch’s (94) conception of the origin of the skeleton of the fins of the fishes. Harrison (’95) has since shown that Klaatch was wrong in his interpretation.

Corning (’99) derives, in the Anura, from the neural crest, only ganglia and nerves, the ventral portions of which extend well down into the corresponding branchial arches. The neural crest is closely fused with the lateral ectoderm, deriving some of.

6 F, L. LANDACRE

its cells from that source, but in Rana it does not break down into mesenchyme, according to this author. The mesenchyme of the head is derived from the endoderm, and is consequently mesentoderm in Miss Platt’s sense of this word or mesoderm in the older sense.

Minot (’01), in an address before the New York Pathological Society, makes a vigorous defense of the doctrine of the integrity of the germ layers, in which he agrees with Rabl (’94), whose opinion was expressed under somewhat similar circumstances. He calls attention to Miss Platt’s work specifically and says, “‘an examination of a number of series and stages has not enabled me to find the slightest evidence in favor of Miss Platt’s con- clusions.”” He says further that ‘‘we may, therefore, I think safely regard this attempt to overthrow the morphological value of the germ layers as unsuccessful. I know of no other attempt of sufficient importance to be even mentioned.” He states in an earlier paragraph that ‘‘the efforts to upset the validity of this fundamental doctrine have failed to find support or recognition from any leading embryologist.”” These statements of Doctor Minot’s were made previous to the appearance of the work by (Dohrn (’02) and Brauer (’04), but after the appearance of that of Kupffer (95) which he must have overlooked. Both Minot and Rabl seem, in the opinion of the writer, to have given too much weight to the doctrine of the integrity of the germ layers in their estimate of a question, which is purely one of accuracy of observation and description.

Buchs (’02), after studying Necturus, disagrees with Miss Platt’s conclusions, taking exception particularly to her state- ments concerning the derivation of mesenchyme from the lateral ectoderm and to her distinction between mesectoderm and mesentoderm on the basis of the amount and size of the yolk granules. Buchs’ opposition to Miss Platt’s interpretation of the mesenchyme is based frequently on minor details and possible ambiguities in statements, although he has worked over the same type. He derives cartilage from mesenchyme which arises from endoderm by a folding process of the endoderm and not from ectoderm. He can find no evidence for the wandering of

FATE OF NEURAL‘CREST IN HEAD OF URODELES 7

the precartilage cells from the nerve anlagen or from the ecto- derm. He denies the contribution of cells from the ectoderm to the mesenchyme, but does not follow the fate of neural-crest cells.

Of the writers opposing Miss Platt’s hypothesis, two only, Corning (’99) and Buchs (’02), give sufficiently detailed descrip- tions and figures to enable one to estimate the value of their criticism. Corning certainly did not follow his stages far enough to determine that the mesoderm of the branchial bars is com- pletely surrounded by neural-crest cells. This can be seen on any good series of the frog, Rana pipiens. Buchs, on the other hand, contents himself with an effort at destructive criticism. His actual evidence is of a negative character, since he does not follow the fate of neural-crest cells that do not form ganglia.

Of the authors who support wholly or in part Miss Platt’s ~ contention, the earliest is Kupffer. Kupffer (95) described in detail and figured the cartilages in Ammocoetes as arising from the deeper layer of the ectoderm in the region of the branchial bars. He had previously (94) designated this layer as neuro- dermis, believing it to be concerned in the formation of the branchial nerves, but here designates it as branchiodermis and derives not only the cartilages, but goes a step farther than Miss Platt and derives muscles also from it. He agrees fully with Miss Platt’s interpretation after examining her preparations. He further derives mesenchyme in the dorsal anterior head regions from the neural crest.

Lundborg (99) derived the pterygopalatine cartilages in Salmo salar and the trabeculae in Rana temporaria from the ectoderm of the roof of the mouth. He also derived the ethmoid cartilages in Salmo in the same manner. The anterior end of these cartilages in Salmo are in process of formation in sixty- eight-day-old embryos.

Koltzoff (02), in Petromyzon, derives mesenchyme in the head from both the neural crest and lateral ectoderm, but is unable to follow its fate beyond the point of the mingling of - ectodermal cells with those derived from endoderm.

8 F. L. LANDACRE

Dohrn (’02) gives a full description with numerous figures of the migration of the neural crest ventrally into the branchial region and its metamorphosis into mesenchyme and cartilage of the branchial bars in Torpedo ocillata. He agrees fully with Miss Platt’s interpretation of the origin of branchial cartilages from ectoderm, although he derives them from the neural crest rather than from the lateral ectoderm. He does not exclude the contribution of the cells from the lateral ectoderm in later stages, although neither his figures nor text include such a contribution. He adopts Miss Platt’s term mesectoderm and criticises the effort of Corning to maintain the integrity of the germ layers in the formation particularly of structures derived from the meso- derm. Dohrn’s evidence for the derivation of connective tissues and cartilage from ectoderm is more convincing, if possible, than that of Brauer in the Gymnophiona and of Kupffer in Petromyzon, both of whom give detailed descriptions. Dohrn’s figures taken with Neal’s (’98) reconstructions of Squalus (figs. 7 to 21, pls. 3 and 4) furnish convincing evidence for the continuous migra- tion ventrally (Neal) of the neural crest and its ultimate trans- formation into permanent mesenchyme and cartilage of the branchial bars (Dohrn).

Brauer (’04), working on the Gymnophiona, derives mesen- chyme of the anterior region of the head from neural-crest cells which later mingle with cells derived from endoderm to form mesenchyme in which the two derivatives cannot be recognized after this fusion. He can find no evidence for the disappearance of these neural-crest cells, however, before their fusion with cells derived from the endoderm. In the posterior head region neural-crest cells, when not involved in the formation of ganglia, grow ventrally into the branchial bars and surround the meso- derm of the bar at first lying on its lateral surface, but finally entirely surrounding it. He can find no evidence for the deriva- tion of mesenchyme in the gill bar from the adjacent lateral ectoderm, as Miss Platt had done, but derives it entirely from the neural crest. He does not deny the possible later mingling of cells derived from mesoderm with those derived from the neural crest, but insists that the chief part in the formation of

FATE OF NEURAL CREST IN HEAD OF URODELES 9

the gill bar is performed by cells derived from the neural crest. Brauer objects to Miss Platt’s terms mesectoderm and mesento- derm which had been accepted by Dohrn and Koltzoff. He reserves his description of the ultimate fate of the ectodermal mesenchyme of the gill bar for a later paper and consequently does not describe the origin of definite cartilage.

An impartial examination of the papers cited above furnishes strong evidence for the formation of the mesenchyme in the anterior head region in the embryo from both endodermal (mesentoderm) and ectodermal (mesectoderm) sources. In all cases the fate of these individual cells is lost and it is not possible to determine the extent to which either or both of them is con- cerned in the formation of adult mesenchyme in this region. However, evidence for the disappearance of mesectoderm cells in the anterior head region is conspicuously absent. The same statement holds in the main for the dorsal mesenchyme in the posterior portion of the head.

The fate of the ectodermal derivatives in the branchial regions is much more definitely stated. Platt (93), Kupffer (95), Brauer (’04), and Dohrn (’02), all derive either the cartilages and mesenchyme or both cartilages and muscles in addition to mesenchyme from the ectoderm. Platt (’93), Kupffer (95), and Koltzoff (’02) derive the ectodermal cells in the branchial region largely or even altogether from the lateral ectoderm, while Brauer (’04), Corning (’99), and Buchs (’12) can find no evidence for the proliferation of cells from the ectoderm, and Brauer (’04) and Dohrn (’02) derive the structures in the branchial bar entirely so far as they are ectodermic from the neural crest. _

MATERIAL

The material on which the work was‘done consists of three series of urodele embryos collected in the same pond, but repre- senting at least two different species. The youngest series (series I) consists of thirty-five stages taken from two egg clusters at intervals of five to five and one-half hours, reared at room temperature, and covers an interval of eight days. The first

10 F, L. LANDACRE

stage of this series was fixed immediately after collection and the medullary folds appear in stage 6. This series, judged by the size of the egg clusters and character of development, is evidently Plethodon glutinosus.

The second series is older and consists of thirty-two stages taken from four egg clusters reared at room temperature and fixed at intervals of about five hours. The total period covered by this series is about eight days. The youngest stage of this series corresponds closely to no. 32 of series I. The larvae were reared and identified as Plethodon glutinosus. These larvae hatched in the twenty-fourth stage, so that combining series I and II there are fifty-six stages taken previous to hatching.

The third series (series III) consists of fifty-one stages taken from five egg clusters reared at room temperature and fixed at four-hour intervals. The total period covered is again about eight days. ‘The first stage of series III corresponds closely to no. 10 of series I and the twenty-third stage of series III corresponds to no. 1 of series II. ‘The larvae of this series were reared up to metamorphosis, but escaped before an identification could be made. Seventeen stages of this series only were cut.

Including duplicates, there are 292 slides in all. All stages were cut transversely, and critical stages were cut in the sagittal and coronal planes also. The material was fixed in Zenker and stained in Delafield’s haematoxylin and counterstained in orange G. The younger stages were covered with a film of celloidin. to prevent the loss of yolk-laden cells which are likely to become detached. ‘The closeness of the series and their sequence are particularly important in the discussion of the problem involved in this paper. Where a series is taken from more than one cluster of eggs, care was used to insure that the series would be continuous by having the eggs taken from the two lots overlap in time. )

FATE OF NEURAL CREST IN HEAD OF URODELES iy

THE ORIGIN OF THE NEURAL CREST

There are in the vertebrates three modes in which the neural crest is related to the neural tube and overlying ectoderm. Har- rison (’01) has described and compared two of these. In the first type, the neural-crest cells—selachians and other types— represent the dorsal portion of the lateral walls of the neural tube which is at first continuous with the ectoderm. The neural crest is incorporated in the neural tube, forming a wedge- shaped mass in its dorsal portion. This wedge-shaped mass later becomes detached from the tube and migrates laterally and ventrally. In the second mode the neural crest (teleosts) lies between the dorsal border of the neural tube and the ecto- derm, not being included strictly in either, but forming later a cap over the dorsal border of the neural tube. In the third mode (Ameiurus and other types (Landacre, ’10)) the neural crest or cells homologous to the neural-crest cells remain in the ectoderm lateral to the neural canal and are later detached from this position to form parts of cranial ganglia and other structures.

The three series of urodeles studied correspond closely in the behavior of the neural crest to the first type mentioned above and do not require an extended description. The neural crest is completely incorporated in the dorsal border of the neural tube, but can be distinguished from the tube, usually, by its looser structure. The following description is based on the behavior of the crest at the level of the VII ganglion.

When the medullary plate can be identified first, it is very broad and its lateral borders include the two portions of the neural crest. Just before the closure of the neural canal (fig. 1) the superficial layer of the ectoderm and much of what will become neural crest are invaginated and included within the limits of the neural groove. The superficial heavily pigmented layer of the ectoderm forms the inner lining of the greater portion of the neural groove. The looser texture and greater pigmenta- tion of the dorsomesial portions of the walls of the neural groove indicate roughly the position of the neural-crest cells.

11 F, L. LANDACRE

In the closure of the neural groove (fig. 2) the superficial pigmented cells lining the dorsal two-thirds of the neural tube come into contact and obliterate that portion of the canal lined by flat cells. The line of juncture is indicated by the heavy pigmentation of the cells. The dorsomesial portion of the wall of the tube has a looser texture and less of a syncytial character than the lateral and ventral portions. The neural tube as a whole is well delimited from the ectoderm except at the dorsal border.

The outline of the neural tube (fig. 3) as distinct from the neural crest first becomes apparent when the cells of the tube assume a syncytial character with their nuclei arranged with their long axes toward the center of the neural canal. At the same time the neural crest while still forming a conspicuous wedge in the dorsal portion of the tube is evidently now largely outside the limits of the tube. The dorsal third of the tube becomes neural crest and presents the appearance of being erupted from the tube. The tube is horseshoe-shaped with the open dorsal portion filled with a wedge of neural-crest cells. The tube except at the ventral border is of uniform thickness.

The next step (fig. 4) involves the further exclusion of neural- crest cells from the dorsal wall of the tube. In this process many of the loosely arranged, heavily pigmented cells are left for a time in the position of the original wedge. In fact, after the neural crest is well defined and has begun to migrate ventrally (fig. 5) a few cells of this type still form the roof plate of the neural canal. In this stage (fig. 5) the greater portion of the - neural crest rests upon the dorsal portion of the neural tube, but there are two prominent lateral extensions lying between the dorsolateral border of the neural tube and the ectoderm. At a slightly later stage (fig. 6) the neural crest is represented almost exclusively by these lateral extensions. The large mass of cells previously lying over the tube is now represented by a few flat cells connecting the two lateral portions of the neural crest. These flat cells disappear later. In figure 6 the original wedge seems to be represented by a few irregularly arranged cells.

FATE OF NEURAL CREST IN HEAD OF URODELES 13

Up to the last stage described there is practically no indication that cells are added to the crest, as defined, from the lateral ectoderm. The crest is continuous on its dorsal border with the inner layer of the ectoderm, and undoubtedly receives cells from this source (figs. 1, 2, 3, and 5), and in several series at the age of that from which figure 3 is taken and the crest is continuous ventrally with the inner layer (fig. 3). This continuity is usually absent at the level under consideration as the lateral portions of the crest become better defined (figs. 5 and 6) and also in the earlier stages (fig. 4).

These conditions as described at the level of the VII sun are duplicated at the levels of ganglia V, IX, and X. In the intervals between these ganglia the neural-crest cells are less numerous and the lateral extensions contain fewer cells and do not reach so far ventrally. At the stage from which figure 5 was taken (fig. 7), the neural crest is continuous throughout the whole region. In the stage from which figure 6 was taken (fig. 8) the crest is interrupted, except for a few scattered cells on the dorsal portion of the cord, between the V and VII ganglia and again between the VII and IX ganglia.

THE MIGRATION OF THE NEURAL CREST AND ITS RELATION TO THE MESODERM

The accuracy with which the migration of the neural crest ean be followed depends upon histological differences between cells derived from the ectoderm as distinguished from those derived from the endoderm. ‘These distinctions have been stated already in the introduction (p. 2). They will be assumed for the present and the migration of the crest will be described from the reconstructions given in figures 7 to 11. The discussion of the basis of these distinctions together with the discussion of two other disputed points, namely, the question as to the con- tribution by the lateral ectoderm of cells to the mesenchyme and the question as to the fate of cells in the anterior head region, will be deferred to a later section.

As to the use of terms, Miss Platt rejected the term ‘mesento- derm’ presumably because the term implies a derm or germ layer

14 F, L. LANDACRE

derived from entoderm, and of course this is equivalent to meso- derm and therefore superfluous. There is in the head region, however, a good deal of loose tissue quite similar to mesenchyme found in the trunk and like that found in the trunk, derived from mesoderm, which is itself a derivative of endoderm. Now, since it is necessary, in describing the behavior of mesenchyme in the head, to distinguish between that derived from the neural crest, which retains most of its ectodermal characters, and that derived from mesoderm, which for a long time retains its endo- dermal characters, I have ventured to suggest the terms ‘ento- dermal mesenchyme’ and ‘ectodermal mesenchyme’ for mesen- chyme in the head where the two types of cells can be distin- guished. When head mesenchyme becomes homogeneous, that is when we can no longer distinguish two types of cells, it will be referred to as mesenchyme, with the implication, however, that it sometimes contains both ectodermal and entodermal cells. While both types of mesenchyme have passed through inter- mediate stages, the entoderm through a mesodermal stage and the ectoderm through a neural crest stage, each retains the char- acter of its more remote rather than of its immediate ancestor. This seems to justify the terms ectodermal mesenchyme and entodermal mesenchyme rather than neural-crest mesenchyme and mesodermal mesenchyme. The term ectodermal mesen- chyme is substituted for mesectoderm, which has become general in the literature. The tissue we are dealing with is not a derm or layer, but a true mesenchyme quite similar to mesenchyme in the trunk, but coming from ectoderm rather than from mesoderm as is in the trunk.

The use of the terms ectodermal mesenchyme and endodermal mesenchyme is further justified by the fact that in the head all branchial muscles come from the mesoderm, and show through- out their earlier stages definite somatic and splanchnic layers, indicating their relation to the lateral mesoderm of the body, while the mesenchyme of the head is more or less loose and of a syncytial character, like that derived in the body from the sclerotomes and the lateral and splanchnic mesoderm. As the mesoderm of the anterior trunk region grows forward into the

FATE OF NEURAL CREST IN HEAD OF URODELES 15

head, it gives rise to a) eye muscles from its dorsal or somitic portion; 6b) branchial muscles from its ventral portion, and, c) mesenchyme which retains it endodermal characters. That por- tion of head mesoderm which gives rise to branchial muscles does not become loose or syncytial in character as does the mesenchyme, but retains its integrity to such an extent that two layers, somatic and splanchnic, can for a long time be identified.

The neural crest, on the other hand, gives rise to specific cerebral ganglia such as V, VII, IX, and X and then migrates ventrally into the ventral head region and into the branchial bars and differentiates into cartilages and loose mesenchyme. The neural-crest ectoderm furnishes three specific derivatives, a) ganglia, b) cartilages, c) mesenchyme. Since the two types of mesenchyme overlap and are histologically distinct for a long time and since the ventral portion of the neural crest passes from a mesenchymal stage to a cartilaginous stage, the terms entodermal mesenchyme and ectodermal mesenchyme seem not only to be justified, but to be absolutely necessary to accurate description.

We shall first follow the migration of the neural crest and mesoderm.

In the first stage plotted, which is 3 mm. in length (fig. 7), the neural crest is continuous along its dorsal border throughout the whole head region, beginning anteriorly at the level of the middle of the eye horizontally and extending caudally into the spinalmneural crest. It presents three conspicuous enlargements. The anterior enlargement extends caudally to the vertical level of the posterior border of the eye. The gasserian ganglion differentiates out of the posterior portion of this enlargement and can be identified at this stage by the slight condensation of the cells. This enlargement is a ventral extension of the neural crest, but owing to the flexure of the head it seems to extend caudally.

_. The second enlargement or ventral extension is at the level of the VII ganglion, the third at the level of the IX ganglion, ‘and the fourth inconspicuous enlargement at the level of the

16 F. L. LANDACRE

anterior division of the X ganglion. Posterior to this level the neural crest gradually becomes narrower dorsoventrally and passes into the neural crest of the spinal cord. In referring to the V, VII, IX, and X ganglia it is to be understood that only the general cutaneous and general visceral portions of these ganglia are under consideration. These are homologous to spinal ganglia. The special somatic and special visceral ganglia are referable to other sources.

‘The endodermal derivatives at this stage fall into two regions. First the posterior head region, which extends cephalad to the level of the anterior end of the alimentary canal and is char- acterized by the presence of somites and a two-layered lateral mesoderm. The somites are not well defined at this stage and the lateral mesoderm forms a broad sheet extending from a level slightly dorsal to the notochord to the ventral limit of the body, being absent only in the heart region and the region of the stomodaeum.

The second, or anterior head region, lies anterior to the level of the anterior end of the alimentary canal and forms a loose mesial or axial mass of entodermal mesenchyme and two lateral extensions. These lateral extensions contain cavities which in the urodeles do not form definite head cavities or somites, but their dorsal portions are undoubtedly homologous to the head somites of selachians. The dorsal border of this entodermal mesenchyme maintains the same relative level as the dorsal border of the somites. It reaches cephalad to the vertical level of the middle of the optic vesicle and ventrally it forms a promi- nent extension, which is separated from the anterior border of the lateral mesoderm by an area free from mesoderm cells. This area’ corresponds roughly to the future position of the spiracular - gill cleft. The ventral border of this extension reaches nearly to the ventral limit of the optic vesicle. The whole of this anterior head mesoderm consists of the large endoderm cells containing large yolk granules and except those regions giving rise to eye and branchial muscle has a loose arrangement presag- ing its modification into typical mesenchyme. The neural crest overlaps the cells derived from endoderm at two points only—

FATE OF NEURAL CREST IN HEAD OF URODELES 17

in the region of the V ganglion and at the ventral border of the IX ganglion. In the overlapping areas mentioned the ento- dermal mesoderm lies mesial to the neural crest.

In the second stage plotted, 3 mm. in length (fig. 8), but six hours older than the last, the neural crest in the region of the gasserian ganglion and anterior to this ganglion has migrated ventrally and caudally and now forms two prominent extensions, the one anterior to the eye and mesial to the olfactory capsule, the other posterior to the eye and extending slightly into the mandibular bar ventral to its ganglion-forming region. This anterior neural crest is now completely detached from the neural crest in the region of the VII ganglion, except for a few scattered cells on the dorsal portion of the neural tube.

The neural crest at the level of the VII ganglion has grown ventrally to the region of the dorsal border of the alimentary canal, doubling its length as compared with the last stage. The IX ganglion has also grown ventrally to the same extent and the X is now represented by a conspicuous ventrocaudal extension of the same general neural-crest mass from which the IX ganglion forms. The neural crest of VII, IX, and X have not, as yet, grown ventrally beyond their ganglion-forming regions. The neural crest in the region of VII is connected dorsally by a well- defined strand of neural crest cells with that of ganglia [X and X.

The lateral mesoderm lying posterior to the anterior end of the alimentary canal at this stage is interrupted by the out- growth of the endoderm to form the spiracular and hyoid pha- ryngeal pouches. The interruption of the lateral mesoderm at the level of the spiracular cleft is not formed entirely. by the pharyngeal pocket. Its ventral portion represents the remains of the prominent notch shown just caudal to the eye in figure 7.

The head mesoderm lying anterior to the alimentary canal has extended in two directions, dorsally and anteriorly, until it has reached the dorsal wall of the brain at the vertical level of the anterior end of the optic vesicle. It has also extended ventrally and caudally posterior to the optic vesicle. The posterior boundary of this extension lies just ventral to the spiracular pharyngeal pocket. The ventral limit of this extension

18 F. L. LANDACRE

reaches almost to the ventral border of the optic vesicle. The region of the overlapping of neural crest and mesoderm is quite extensive, as indicated in figure 8. In the region just posterior to the eye the mesoderm is not completely covered by neural crest. In the region of the VII, IX, and X ganglion the ventral half of each ganglion overlaps the lateral mesoderm and lies lateral to it.

In figure 9 from a stage 4 mm. long and ten hours older than the stage from which figure 8 was taken, the neural crest has not altered its relation greatly except that posterior to the eye it has grown ventrally and caudally into the mandibular bar and now almost covers, on the lateral surface,