THE EMBRYOLOGY O F TRYPAN BLUE INDUCED ABNORMALITIES IN MICE 1,

MAX HAMBURGH Department of Zoology, Columbia Umiversity, New Pork, N . P

TWENTY-FOUR FIQURES

INTRODUCTION

Considerable interest centers around the discovery of tera- togenic agents and the investigation of their effects on dif- ferent organisms and their development. The subject of teratogeny may be approached from several viewpoints.

1. Pharmacologists are primarily interested in this field for reason of possible toxicity of drugs.

2. Embryologists, in studies of the role of metabolic gradi- ents in development, have accumulated considerable data on the effects of various metabolic poisons on embryonic tissue, e.g. tithium (Herbst, 1892, for review of later literature see Hall, '47), Mg salts (Stockard, '07). Thus the study of tera- togenic effects of different agents has become a tool of experi- mental embryology.

3. Physiological Geneticists have shown considerable in- terest in teratogenic agents particularly when the effects of these agents show resemblance to conditions known to arise as the result of gene mutation. The theoretical implications of this type of approach have been stressed by Goldschmidt ( '45) and Landauer ( '48, '52).

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University, New York City, 1953.

2This work has been aided by a predoctoral fellowship from the Division of Research Grants and Fellowships of the U. S . Public Health Service.

Present address : Department of Anatomy, Medical School, Univemity of Peiinsylvania, Philadelphin, Pa .

409 T H E AXATOMICAL RECORD. VOL. 119, hO. 4

AUCiUST 1954

410 MAX HAMBURGH

An agent whose teratogenic effect was discovered recently is the dye “trypan blue.’’ Gillmann, Gilbert, Gillmann and Spence (’48) described the effects of trypan blue in newborn rats after injecting the dye into the bloodstream of pregnant mothers. Such injections resulted in a whole spectrum of malformations in the newborn, such as spina bifida, hydro- cephalus, cleft palate, eyelessness, ear defects, short tails, kinky tails, and absence of tails.

Some of these abnormalities in rats, particularly of the tail resembled malformations reported in mice as the result of mutations. In an attempt to test the effect of this dye on mice, Hamburgh ( ’52) obtained similar abnormalities in Bragg albino mice by treating pregnant females with trypan blue. These results were confirmed by Waddington and Car- ter (’52) in a different strain (CBA) of mice.

The next question to be taken up, logically, was whether the developmental steps of the trypan blue induced qbnor- malities were identical with those of some of the mutations with similar end effects.

Studies undertaken to answer this question are reported in this paper.

Grateful acknowledgment is made to Dr. Salonie Glueck- sohn-Waelsch of the Department of Zoology of Columbia University at whose suggestion this research was begun and under whose guidance, supervision, constant help and en- couragement it was carried out. Grateful acknowledgment is made to Dr. L. C. Dunn of the Department of Zoology of Columbia University whose continuing interest and sugges- tions proved of invaluable aid.

MATERIALS AND METHODS

Mice of the Bagg albino strain, inbred for 80 generations and more, were used for all experiments and ob~ervations.~

The colony was started with a strain from the lahoratory of Dr. L. C. Dunn and Dr. 8. Gluechsohn-Waelseh of Columbia University, and one kindly given us by Dr. E. C. McDowell of the Institute of Genrtics, Cold Spring Harbor, N, Y.

INDUCED MALFORMATIONS I N MICE 411

Females were mated to albino males and examined twice daily for copulation plugs to assure accuracy of timing within 7 and 17 hours, respectively. I n the first series of experi- ments 20 pregnant females were injected with $ to &em3 of 5% trypan blue and permitted to carry to term in order to test the response of the embryos to trypan blue treatment ( Hamburgh, '52).

In the second series of experiments the dosage was changed to *em3 of 1% trypan blue solution at 7 days after the de- tection of the copulation plug. Instead of permitting the pregnant females to go to term, the embryos were dissected out at intervals of 84, 9, 10, 11, 12, 13, and 14 days after fertilization.

A total of 571 embryos was examined. Of these, 122 em- bryos were selected for histological study.

RESULTS

Of the 20 females injected, only 12 carried through to suc- cessful parturition, the others showing signs of resorption in utero. From these, 67 newborn mice were recovered, of which 16 showed different types and degrees of tail abnor- malities such as short, kinky, curled and constricted tails

After it had then been established that the trypan blue treatment was effective in inducing abnormalities in mice, preliminary embryological examinations were performed. Bagg albino females were injected once in the manner de- scribed above, killed at 8-14 days after fertilization, and their embryos removed for gross examination and histologi- cal study.

Sixty-three injected mice yielded a total of 571 embryos varying in age from 83 to 14 days, r+ 3-8 hours in each case. Observation of these embryos gave the following results :

(figs. 1-2).

1. Seventy-five embryos were resorbed. 2. Three hundred and thirty-two embryos showed mal-

formations of varying degrees.

412 MAX HAMBURGH

3. The majority of abnormal embryos showed two major types of malformations :

(a ) Abnormalities of the anterior region, e.g. failure of the head folds to close, or an everted brain sitting like a cap on top of the developing skull, resembling the mutation pseudencephaly (figs. 7-9). (b) Abnormalities of the posterior axis, such as kinky, curly, short and constricted tails (figs.

The results observed in the embryos are summarized in table 1.

Ten uninjectcd females were kept as controls. Embryos were dissected out a t 9-;, lo+, 11, 12, and 13 days. No mal- formations were recoveded, and litter size was unaffected.

10-12).

Abnorntalities of t he crnterior axis

h number of striking morphological peculiarities of the nervous system were noted: (1) The brain folds remained wide open throughout the entire head (figs. 4, 6) and neck region. (2) I n a large number of cases the neural folds at the point of closure formed an irregular zig-zag line dorsally (fig. 14). ( 3 ) A number of cases of microcephalia were noted in which the brain vesicles were either reduced in size or asymmetrical as the result of abnormal growth on one side of the brain (fig. 3). (4) Heavy thickening of the roof of the hindbrain and the anterior cord (fig. 9) both of which were everted. The neural fold eversion ran as far down as the level of the heart (fig. 13). (5) A brain sitting like a cap on top of the skull (fig. 7 ) . I n these cases the brain bulged out of the cranial cavity, and was thus left in free communication with the outside. Various degrees of flatten- ing and bulging could be observed. The brain itself seemed to have grown to abnormal proportions.

dbnorrnalities of the posterior axis Tail abnormalities did not appear in embryos before the

age of 10 days after fertilization. At that time 4 types of

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deviations from normal tail morphology could be recognized : kinky, curly, constricted and short tails (figs. 10-12).

About half of the tail abnormalities were designated as kinky tails. The tail, which under normal conditions follows a more or less straight line from base to tip, shows here a t least one or more kinks (fig. 10).

Next in frequency was the formation of curly tails. Here the tail axis, instead of changing its course abruptly, forms a smooth curve, describing in extreme cases a perfect circle or semicircle. In some cases it is difficult, however, t o draw a sharp line between so-called “kinky” and “curly” tails

Constricted tails were of relative low incidence. I n these cases the tail is sharply constricted a t its base, thinner in diameter than normal distal to the point of constriction (fig. 13).

Shortened tails were the least frequent group of tail abnor- malities. The outstanding feature was reduced length of the embryonic tail, usually accompanied by a blunting of the tip, giving the tail the appearaiice of a stump in extreme cases (fig. 11).

(fig. 7) .

In addition to the specific malformations listed so far mis- cellaneous abnormalities and some of a more general nature were observed.

The most obvious and the most frequently occurring patho- logical feature was the presence of large and transparent blisters distributed a t random over the body of the embryo (figs. 3, 4, 6). Through these transparent blisters destruction of the underlying regions could be observed. For example, somites covered by such transparent blisters showed a faded outline and mere smaller than enighboring normal somites.

Unlike the blisters distributed a t random over the body of the embryos, hematomata were coiicentrated most fre- quently in the tail region. There they were distributed a t

INDUCED MALFORMATIONS IN MICE 415

random, occurring just as often at the tip of the tail as anywhere along the axis (fig. 22).

A great number of cases (fig. 5) showed reduced growth and tissue degeneration, accompanied by blisters and hemato- mata. At the 8-10-day stage there was a large group of embryos which, at the time of dissection, were so necrotic that their morphological features could not be recognized. These embryos apparently had died before dissection, pos- sibly as a result of their abnormalities.

Histological analysis

One hundred and twenty-two embryos from mothers in- jected with trypan blue were sectioned and examined histo- logically. One hundred and seven of these were selected because of some major malformation of the types outlined above, visible a t gross inspection. The other 15 were normal- looking embryos from mothers subjected to trypan blue treat- ment. All the latter embryos came from litters where the injection had been effective in causing malformation in some of the sibs.

The histological sections of early embryonic states reveals the failure of the neural folds to close (figs. 13, 15). Hyper- trophy of neural tissue manifests itself in a number of ways: In the region of the midbrain the neural folds form a thick- ened plate. There is increased and irregular growth of all parts of the brain (fig. 15, 16, 17) and of the neural tube (figs. 18, 24). In addition diverticula and folds are found (figs. 16, 18) which push deep into the brain ventricles and neurococle causing the lumen to become much narrowed (fig. 16). A few cases (4) were observed in which the neural tube seemed doubled or to have formed several irregular out- growths (figs. 19, 20). The doubling effect was observed in the posterior trunk and tail region only and always was associated with gross morphological tail effects.

A study of the sections of embryos from trypan blue in- jected mothers revealed that the notochord was affected only

416 MAX HAMBURGH

in a limited number of cases. Eight cases were recorded in which the notochord appeared irregular and wavy at the level of the posterior trunk and tail region (figs. 19, 21). Three cases were found in which the notochord was forked.

Some of the somites of 8-10-day-old embryos were exposed to the destructive effects of blisters distributed widely and randomly. Under their influence somite tissue seemed to be subject to varying degrees of destruction. Histological ex- amination of early embryos (8-10 days old) showed no somite abnormalities other than those caused by blisters.

In later stages somite alignment seems to be disturbed as a result of the often severe irregularities of the course of the neural tube. This leads in extreme cases to disarrange- ment of the somites as they follow the tortuous outline of the neural tube (fig. 24).

Embryos of 10 or more days showed many large blood- filled blebs in the tail region. Histological examination con- firms this observation (figs. 22, 23) and shows the destruction effect of these hematomata on notochord, somites and under- lying mesoderm. Invasions of clumps of blood cells into notochord, somite and mesenchyme of the tail region have been observed (fig. 23). Pycnosis was also occasionally noted in the very tail tip, even though the invasion of hematomata did not reach all the way into that region.

DISCUSSION

Observation of the earliest embryonic stages revealed fail- ure of the anterior neural fold to close, and hypertrophy of the neural tissue to be the major deviation from normal development.

Consequently, one possible explanation for failure of the neural tube to close normally might be an increased tissue proliferation in the neural folds. On the other hand, mechani- cal interference by the blebs, described on page 413 might be responsible for failure of normal closure. Observations in support of either view are available.

INDUCED MALFORMATIONS I N MICE 417

The observations of the anterior abnormalities seem to indicate a direct teratogenic effect on the nervous system of the embryos of trypan blue treated mothers, not mediated through some other developmental system.

Our observations show that by far the greatest number of tail abnormalities in the embryos of trypan blue injected mothers are correlated with the presence of hematomata in the tail region. These hematomata and the invasion of clumps of blood cells into somite and mesenchymal tissue might have a destructive effect on these structures and their derivatives, leading through degenerative changes to shortening of the tail or to its bending and curling.

A significant number of tail malformations in our material, however, show no vascular abnormalities at all, but exhibit either notochord deviations and more frequently irregulari- ties of the neural tube in the posterior axis.

It appears from our data that the excessive growth of the neural folds may be a contributing factor not only to the brain malformations, but also to those of the tail region. This irregular growth, leading to the brain eversion at the anterior level of the body, may influence growth and differ- entiation of the tail in the posterior region of the body through interference of part of the twisted neural tube with normal arrangement of somites along either side of it. The neural tube may thus be responsible for some of the tail abnormalities observed in embryos and newborn. In this connection it is noteworthy that a dependency of normal spine development on the presence of a normal neural tube has been shown to exist in amphibians by Holtzer ('52).

The tail abnormalities in our material may therefore to a certain extent be the result of: (a ) the effect which the crooked and irregular neural tube may exert on somite align- ment in the tail region, in analogy with Holtzer's ('52) re- sults; and (b) the destruction of the tail tissue by hemato- mata. This does not seem unreasonable, since any teratogenic agent would be expected to create more than one type of disturbance. Mosley ( '47) demonstrated three different types

418 MAX HAMBURGH

of developmental pathways in insulin treated chickens, each of which in its own way lead to the rumpless condition.

When the experiments of injecting trypan blue into preg- nant females were begun, the possibility existed that this treatment might be able to reproduce the sequence of de- velopmental events known to occur in mice as the result of certain mutations. If this had actually been the case, the abnormalities observed as the result of trypan blue effect would deserve to be called true “phenocopies.”

It should be of interest therefore to compare abnormalities of the two types in more detail and to attempt an evaluation of their respective resemblances and differences.

Pseudencephaly in the house mouse has been described by Bonnevie (’36)’ as a single recessive mutation and as the result of the effect of a chromosomal translocation by Snell, Bodemann and Hollander ( ’34). It was also recorded as one of the pleiotropic effects of some tail mutations such as “Fused” (Gluecksohn-Waelsch, ’51) and “Crooked” (Mor- gan, in press).

The embryology of the genetically determined form of pseudencephaly is comparable to the trypan blue induced con- dition with respect to the following features: (1) Irregular growth, diverticula and curvature as well as hypertrophy of medullary tissue was reported in the embryos of the geneti- cally and the trypan blue induced form of pseudencephally. (2) Failure of the neural folds to close was observed in our own material as early as 8& days old embryos. This con- trasts with Bonnevie’s (’36) finding on the mutation pseud- encephaly. She asserted that neural fold closure is initiated at the normal time, but may be inhibited later. (3) The critical time designated by Bonnevie (’36) was the 8th day after fertilization. At that time the first deviation from the normal course of development begins to appear in em- bryos of pseudencephalic mutants in mice.

Our own experiments with trypan blue are in agreement with the findings of Kaven ( ’38)’ Russell ( ’50)’ Wilson, Jor- dan and Brent (’53) and Ingalls and coworkers (’51) that

INDUCED MALFORMATIONS IN MICE 419

the 8th day following fertilization is also critical for produc- tion of brain abnormalities by experimental means.

Genetic analysis of the house mouse furthermore has shown that the growth and form of the tail are subject to the control of a great number of genetic factors and that changes in these, i.e., mutations, produce abnormalities of the tail.

Chesley ( '35 ) described abnormalities of the neural tube in both homozygous and heterozygous shorttailed Brachyury embryos 9 days after fertilization. The abnormalities de- scribed by Chesley for the Brachyury condition manifest themselves in highly irregular twisting and folding of the neural tube, presence of blebs and occasional doubling of the neural tube resembling very much the situation found in our own trypan blue induced malformations. But whereas Chesley ( '35 ) described absence of notochord or presence of notochordal diverticula as the main abnormal feature of Brachyury embryos, notochordal abnormalities were observed in only a small number of cases in our material.

Gluecksohn-Schoenheimer ( '45) on the other hand studied the embryology of the Sd tail mutation in homozygous and heterozygous condition. She reported that tail formation pro- ceeds normally up to the age of 10 days after fertilization in these mutants Sd. Subsequently small hematomata are found in the tail tip, which eventually increase in size and are ac- companied by a breakdown of all tail structures, such as neural tube, notochord, somites and mesenchyme. This de- generation of the tail resembles the histological picture found to occur in the development of the majority of our own trypan blue induced tail abnormalities. The similarities of develop- ment of several tail mutations to each other and to the trypan blue induced tail malformations lead one to assume that the number of modes by which tail abnormalities may occur is rather limited. Two of the possibilities are exemplified by the picture represented (1) by the developwent of Dunforth's short tail wawtatiom as described by Gluecksohn-Schoenheimer ('45) and (2 ) by the developmental h k t o r y of the Brachyury nzutution as analyzed by Chesley ' 35 ) . Our own analysis

420 MAX HAMBURGH

has shown that elements of both types of abnormal develop- ment represented by the two mutations may be found in the trypan blue induced abnormalities.

One of the most interesting questions in connection with the effect of trypan blue on embryonic development concerns the physiological disturbances which trypan blue must create within the developing organism in order to bring about the kind of developmental deviations which were described in the preceding pages.

Among the first problems to be attacked in this connection is that of the ability of trypan blue to penetrate the placental barrier. Microscopic examination of the yolk sac and chorion of litters from trypan blue injected animals revealed that the dye penetrated the yolk sac epithelium, but no trypan blue granules were ever found in the embryos proper.

For a more direct test 9 embryos from 4 different litters were removed from the maternal uterus 7 hours after in- jection and suspended in 5 em3 of 70% trichloroacetic acid. Approximately equivalent amounts of uterine tissue and chorion, which unlike the embryos showed the blue coloration of the dye, were suspended in the same amounts of trichloro- acetic acid. The dye was extracted from the latter tissue until the acid achieved an ink-like deep blue color. No effects of extraction were observed in TCA suspended embryos. Unless the dye penetrates the embryonic tissue in minute amounts, too small to be detected by any color change or granular accumulation, and measurable only by colorimetric procedure, it must be assumed that penetration of trypan blue is barred through the placenta as well as through the yolk sac. Similar conclusions were reached by Goldmann ('09), Wislocki ('20) and Everett ( '35).

Essentially two alternatives present themselves for an ex- planation of the possible physiological mechanism of the dye. (1) The trypan blue at the dosage used might produce a subthreshold poison in the injected organism, which, unlike the dye itself, might be capable of passing through either the placenta or the yolk sac, or through both. In the embryo

IK’DCCED RlALFORMATIONS IN MICE 421

such a poison might exert physiological effects which in the growing and differentiating embryo might lead to the mor- phological consequences described in this paper. (2) The trypan blue molecules accumulating in the placental or yolk- sac tissue might form an obstruction blocking the passage of vital substances to the embryo. Such a block might be of general nature or might be specific for particular substance.

SUMMARY

Pregnant females of the Bagg albino mouse strain were injected with 3 em:+ of 0 5 1 % trypan blue solution on the 8th day after fertilization.. The effects of such injections on diff went developmental systems of embryos were studied.

The gross analysis of embryos from 84-14 days after fer- tilization revealed two regularly occurring types of abnor- malities: (1) those of the anterior axis, such as brain malformations resembling the condition known as pseuden- cephaly or exencephaly, and (2 ) those of the posterior axis, such as tail malformations resembling in some respects the phenotypes of certain tail mutations in mice.

Histological analysis showed the nervous system and the circulatory system of the embryos to be most severely af- fected by the trypan blue treatment. Minor effects on noto- chord and somites were observed, too.

A comparison is made between the disturbances of develop- mental pathways leading to the same type of abnormalities in mutants and in trypan blue treated animals. Similarities and differences of developmental steps in the two types of abnormalities are discussed in connection with the concept of phcnocopies.

LITERATURE CITED

RONNEVIE, K. 1936 Pseudencephalie als spontane recessive mutation. Norske

CHESLEY, P. Development of the short tailed mutant in the house mouse.

EVERETT, J. W. Morphological and physiological studies of the placental

Videnskaps Akad. Oslo., Mat.-Natur. Klasse, 9 : 217-251.

J. Exp. Zool., 70: 429455. 1935

in the albino rat. J. Exp. Zool., 70: 243-280.

1935

422 MAX HAMBURGH

GILLNAN, J., CH. GILBERT, TH. GILLMANN AND I. SPENCE 1948 Preliminary report on hyrrocephaly, spina bifida and other congenital malformations in rats produced by trypan blue. South African J . Med. Sci., 13:

GLUECKSOHN-SCHOENEEIMER, S. 1945 The embryonic development of mutants of the Sd strain in mice. Genetics, 30: 29-38.

GLUECKSOHN-WAELSCH, S. 1951 Physiological genetics of the mouse. Advances in Genetics, 4 : 2-25.

GOLDMANN, E. E. 1909 DiGussere and Sekretion des gesunden und kranken Organismus im Lichte der Vitalen Farbung. Beitr. z. klin. Chirurgie,

GOLDSCHMIDT, R. 1945 Additional data on phenocopies and genic action. J.

HALL, TEOS. 1942 The mode of action of lithium salts in amphibian derelop- ment. J. Exp. Zool., 89: 1-30.

HAMBURGH, M. 1952 Malformations in mouse embryos induced by trypan blue. Nature, 169: 27.

HEFGBST, C. 1892 Experimentelle Untersuchungen uber den Einfluss der veriin- derten chemisehen Zusammensetzung des nmgebeiiden Mediums auf die Entwicklung der Tiere. I. Zeits. f . Wiss. Zool., 55: 446.

1952 An experimental analysis of the development of the spinal column. J. Exp. Zool., 121: 121-147.

1950 Anoxia as a cause of fetal death and congenital defect i n the mouse. A. J. Diseases of Children, 80 : 34-45.

KRAVEN, A. 1938 Rontgenmodifikation bei Mausen. Zeits. mensch. Vererbungs u. Konstitutionslehre, 22: 238-246.

LANDAUER, W. 1948 Hereditary abnormalities and their chemically induced phenocopies. Growth Symposium, 12: 171-200.

Genetic factors in chicken embryos. Ann. N. Y. Acad. Sci.,

MOSELEY, H. R. 1947 Insulin induced rumplessness of chickens. J. Exp. Zool.,

RUSSELL, L. B. X-ray induced developinental abnormalities in the mouse and their use in the analysis of embryological patterns. J . Exp. Zool.,

1934 Abnormal development in tho mouse caused by chromosome imbalance. J. Exp. Zool., 67:

The artificial production of a single median cyelopimi eye in the fish embryo by means of seawater salt solution of RlgCI. Arch. f . Entwick. d. Organismen, 23: 250-258.

WADDINGTON, C. H., AND T. c. CARTER Malformations in mouse embryos induced by trypan blue. Nature, 169: 27-28.

WILSON, J. G., H. C. JORDAN AND R. L. BRENT 1953 Effects of irradiation on development. Am. J . Anat., 92: 153-176.

WISLOCKI, G. B. 1920 Experimental studies on fetal absorption. Carnegie Con- trib. to Embry., 2 1 : 47-55.

47-90.

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EX^. ZOO^., 100: 193-201.

HOLTZER, H.

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1952

PLATES

PLATE 1

EXPLANATION O F FIGURES

1 Young mouse approximately 5 weeks old from mother illjected with trypan blue showing an abnormally formed (helical) tail. Life size.

Litter mate of animal in figure 1 showing a kinky tail. Life size. 2

3 Embryo 1000-2R. 9f days after fertilization. Microcephalic head folds. X 56.

4 Embryo 1003-6R. 8-84 days after fertilization. Anterior neural folds open and bulging out and blister present laterally. X 42.

5 Embryo 74-5B. 113-12 days after fertilization. Lateral view showing ex- tensire growth reduction. X 12.

6 Embryo 1001-1L. 9-94 days after fertilization. Headfolds remain unclosed as f a r as level of the heart. A large blister is present lateral.

Embryo 18-1. 16 days after fertilization. The head shows the typical pseud- eneephalic condition with the everted brain sitting like a cap on top of the skull. The tail is bent and curly. Life size.

X 35.

7

8 Embryo 4-2L. 124-13 days after fertilization. The brain shows advanced degree of psendeneephaly. The left forelimb is rotated dorsad. X 24.

9 Embryo 18-2. 16 days after fertilization. Littermate of animal in figure 7, showing advanced degree of pseudencephalp. Life size.

10 Embryo 46-3R. 113-12 days after fertilization. Lateral view showing “kinky” tail condition. X 24.

11 Embryo 10-2R. 123-13 days af ter fertilization. Lateral view showing “short ’’ tail condition. X 24.

12 Embryo 67-31,. 116-12 days after fertilization. Lateral view showing eon- stricted tail. Distally the diameter of the tail is reduced t o a thin filament. X 24.

424

INDUCED MALFORMATIONS I N MICE MA): HAMBURGH

PLATE 1

425

PLATE 2

EXPLANATION 05’ FIGURES

13

14

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17

18

19

20

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24

Embryo 838-2L. 9 days after fertilization. Cross section t.hrougll lieart regioli. The neural folds are open as far down as tlie lcrcl of the heart. A large blister is located dorso-lateral. X 55 .

Eiiibryo 838-GR. 9 days after fertilization. Section through t runk region showing irregular path of the neural tube and presence of blisters hterallp. x 55.

Eiiil)ryo 14-2R. 114-12 days after fertilizntion. Sagittal section of pseud- eiicephalic embryo. The brain is everted and telenccjtllaloii : i d dienceplialoii are not closed. x 55.

Embryo 181-4R. 114-12 days a f te r fertilization. Cross section tlirougli region of the optic cup of pseudenccplialic embryo. Tlie wills of the dieiiceplialoii form deep folds and diverticula. X 55.

Embryo 100-2L. 103-11 days after fertilization. Cross section t l i ro~rg l~ t!lc region of the oiic, vesicle showing h t e ra l I~yprrtropliy of tlie walls of the rlioinbeneeplialon. x‘ 55.

Embryo 101-2L. 12;-13 days a f t e r fertilixxtion. Section tlirougli posterior trunk region shoi~ing RII irregulnr and folded ncur:iI tube. T h e notochord is iiorrnal. x 55.

Embryo 3-2L. 131-14 days after fertilization. Cross swtion tlirougli posterior trunk region showing a secoiidary neur:il tube. Tlie notorhord :I ppe:irs “\mvy.’ x 55 .

S:inie embryo as 19. Sagittal section of tail regioii sl~owiiig two 1iciir:il tubes riiiiiiiiig parallel t o each other. The tip of the tail ~mdcrgoes degener:itire changes. X 55.

Embryo 11-2. 124-13 days a f te r fertilization. Cross wet ion through pnsterior trunk rcgion showing ‘ [ n.:iry ’ ’ notochord.

Eiiibryo 145-3L. 113-12 days after fertilization. S:igitt:il section of the tail showing liematomata in the t ip of tlir tail. Tlie neiiral tnlie is irregular in outline. x 82.

Eni1)ryo 106-lR. 11$-12 days after fertilization. Cross section tlirougll t;lil region showing invasion of I)lootl cell rluii!ps iuto somite tissiw.

1Snibryo 160-GL. 10 -11 clays after fertiiiz:ition. Sagittal sectioii tlirougli tail region showing the somites arrnnged along crooked path following the irregclar outline of the iieurnl t ube .

X 250.

x 250.

x 82.

426

PLATE 2

427