Effects of Excess Vitamin a on the Cranial Neural Crest in the Chick Embryo

Ainnals of the Royal College of Surgeons of England (I977) vol 59

Effects of excess vitamin A on the cranial neural crest in the chick embryo

John Keith BSC Teratology Laboratories, Royal College of Surgeons of England, Downe, Kent Summary resembles a breaking wave in section, the two Experiments on fertile hens' eggs have shown waves being symmetrically opposed so that that an excess of vitamin A has a highly when the 'crests' of the 'waves' meet they specific effect on the migration of cranial will enclose the neuroectoderm of the neural neural crest cells in the early stages of chick tube (Fig. i). The cells which migrate, the embryogenesis. Migration is disrupted and neural crest cells, are, as is implied by the retarded. This is consistent with the causal name, situated at the crest of the neural folds. mechanism proposed by Poswillo for thu' Their origin is the ectoderm at the point where Treacher Collins syndrome. the neuroectoderm meets the skin ectoderm. In the cranial region cells start to migrate away from the neural crests before the two neural Introduction folds come in contact, the process continuing Poswillo' has emphasized the value of until the superficial ectoderm is continuous appreciating the causal mechanisms of and the margins of the neural tube have malformation to the clinician proposing to fused. At first the cells are migrating between carry out surgical correction. It provides a two layers of ectoderm, but these become rational basis for the timing of the attempted separated and mesoderm cells intervene so that reconstruction. To this end an animal model the neural crest cells are migrating in a cell- has been worked out for the causal mechanism free space between the skin ectoderm and of the symmetrical first arch syndrome of the the mesoderm'5. type known as the Treacher Collins syndrome2. There are a few reports of studies on the The animal model of the human syndrome effects of excess vitamin A in the chick6-9. Of results from a dose of IOO 000 IU of vitamin these only the last, by Hansborough, used a A administered to a pregnant rat at Day 8.5 comparable dosing regimen, but the results in gestation. It has been proposed that the were presented in general terms and there defects in the syndrome are the result of a is no indication whether or not the neural deficiency of the neural-crest-derived compon- crest was affected by vitamin A. To test ent of the first and second branchial arches whether excess vitamin A could produce effects and are thus manifest in facial structures which in the chick embryo consistent with the develop from these embryonic precursors. Early mechanism proposed by Poswillo2 the follow- stages of the effect cannot be demonstrated ing study was carried out. in the rat as the neural crest cells are difficult to identify. The early migration of neural Materials and methods crest cells is easier to demonstrate in the chick Fertile White Leghorn eggs were used. Doses embryo', '. were administered by injection. For this The process is as described by Johnston4. purpose the shell was first punctured with a Briefly, the early stages of migration in the vial saw. The injections were made from below cranial region are as follows. The neural folds usin,g a o.63-,gauge 38-mm needle. The aim are rising and arching over the neural groove of this was to ensure that the dose was to meet in the midline. Each of these folds administered within the yolk of the egg. Dosing 480 John Keith

FIG. I Diagrams illustrat- b ing early stages of cranial neural crest cell migration in the chick. (a) Transverse section of neural folds as they approach each other. NC = neural crests- arrows indicate that the crests are folding and meeting; C = neural crest cells (stippled)- arrows indicate that the cells are moving away from the neural c crest; SE = skin ectoderm; NE = neural ectoderm; N = notochord. (b) Detail of neural crest region showing typical cellular relationships. (c) Transverse section of chick cranial region after fusion of neural folds, showing relations of germ layers and neural crest. Stipple = area occupied by neural crest cells; M = mesoderm; NT = neural tube. was at either o h or after 24 h of incubation. Results Vitamin A was in the form of the ester One hundred and fifty eggs were used, of (vitamin A palmitate, water-soluble, IOO 000 which two-thirds were experimental and the IU/ml, Roche Products Ltd) or crystalline remainder controls. Of the experimentally retinol (Roche Products Ltd). The former was diluted with sterile saline, the latter dissolved dosed eggs, 27 were examined at stages during in ethyl alcohol and the solution then diluted incubation when cranial neural crest migra- some with sterile saline or ethylene glycol to give tion was taking place. Of the remainder, were too young, some were just too old, and a final concentration of i 7o ethyl alcohol in were gross water or ethylene glycol. In each case the others examined for malformations at stages. a were concentration of vitamin A was determined later Finally small proportion or or gross mal- so that the desired dose could be given in infertile, lost, damaged, had formations not relevant to this study. A back- 0.05 ml of solution. Doses were 0.03 and 0.3 mg of vitamin A palmitate and a range from ground malformation rate of about 5'o is 0.012 to 0.7 mg of retinol per egg. Controls concomitant with any experiment on eggs"0 were dosed with the identical canrier but and with artificial incubation conditions"' 12 without the vitamin A component. The The specimens were classified on examin- majority of specimens were collected after a ation according to Hamburger and Hamilton'3 total of between 36 and 72 h of incubation; into developmental horizons. As mentioned, some were examined at later stages up to Day 27 were collected at stages when cranial neural I5 of incubation. The blastodisc was cut free crest migration was taking place (Hamburger from the yolk in saline and placed flat on and Hamilton (H & H) IX-XI). Fifteen of a Petri dish to be washed and fixed with I o7o these embryos (56%) had abnormalities in- forrmol saline. The blastodiscs were photo,. corporating the following features in the cranial graphed as whole specimens using a Zeiss region: (a) ectomesenchymal deficiency; (b) Tessovar Macrophot system and embedded aggregation or 'clumping' of cells in the neural in 2% agar to facilitate orientation. The crests; (c) retarded migration of cells from specimens were then embedded in Paraplast the neural crests; (d) neural crest cell damage; wax, serially sectioned at 4-7 ym, and stained and (e) neural tube distortion related to these by a standard haematoxylin and eosin method. effects. These features are illustrated and Effects of excess vitamin A on the cranial neural crest in the chick embryo contrasted with a normal embryo in Figures 2-6. Figure 2 illustrates the normal features of a transverse section through the cranial region of a Stage XI (H & H) chick embryo. The neural tube is ring-shaped and the ectoderm is continuous over it. In earlier stages the rais- ing and fusion of the neural folds in this region occupies embryonic Stages VII-XI, taking about I2 h. The initial fusion occurs in this region. It occurs at Stage IX, about 7 h before Stage XI. Neural crest cell migration from FIG. 3 Comparable section of experimental the apices of the neural folds takes place in embryo. H & H Stage XI (X 40). Neural those 7 hours. The ectomesenchyme cells crest cells are identifiable (arrowed). There are lateral to the netural tube, lying between the lacunae in the ectomesenchyme, with an over- neural tube and ectoderm, are a mixture of all deficiency in the cell population. Although mesenchyme cells, which are migrating cranial- the rest of the tissues look normal, the ecto- ly from the primitive streak, and the neural mesenchyme looks 'moth-eaten'. crest cells migrating ventrolaterally from over the neural tube. The cells lying between the neural tube and the ectoderm can be identified, pared with 270 in the control. This reduced by virtue of their position and orientation, as ectomesenchyme cell population is a consistent neural crest cells. The ectomesenchyme cell finding in the experimental embryos; in this population is relatively dense. Figure 3 shows case it is the only abnormality seen. Figures an experimental specimen which has achieved 4-6 show the range of abnormal features and the same developmental stage as the specimen indicate how they develop. Figure 4 shows in Figure 2; the neural tube is a discrete an embryo of Stage X, although the effects of cylinder separate from the ectoderm, which the vitamin A make it hard to assign. The has fused. There is, however, a big difference mass of cells between the neural tube and the in the ectomesenchyme population, I 50 cells ectodermn are neural crest cells which have between the neural tube and ectoderm com- been unable to migrate normally laterally away from the neural crest. This results in retarded fusion of the ectoderm, distortion of the dorsal region of the neural tube, and failure of fusion of its dorsal edges. In addition there is a reduced ectomesenchyme population, the cells of which are scattered and sparse. Figure 5 shows a slightly earlier stage. In sections of normal embryos at this stage, with the neural folds in apposition, the migrating neural crest cells are a striking feature4. In this experimental embryo the cells at the apices of the neural fold are very closely packed and FIG. 2 Normal chick embryo, transverse are preventing its normal fusion. Migration section cranial region, H & H Stage XI of the neural crest cells is retarded and the (X 40). E = ectoderm; N = neural tube; mesenchyme is sparse. In Figure 6, again an C = notochord; E-M = ectomesenchyme. experimental embryo, the neural crest cells The ectomesenchyme includes both mesen- are in the process of migrating but they appear chymal and neural-crest-derived cells. The rela- to have been retarded. The alignment of the tionships between neural crest and mesen- cells in the dorsal region of the neural tube chymal cells will be indicated in the has been disrupted, there is a clump of cells experimental specimens. in between neural tube and ectoderm, and 482 John Keith

and absence of a positive relationship between size of dose and degree of effect). This has to be borne in mind when considering the relevance of the findings reported. It is felt, however, that the variability of the results was mainly caused by the dosing technique used and that an improved technique would give a reproducible experiment. The surest way to remove this variability FIG. 4 Experimental embryo, H & H Stage would be to use a window in the eggshell X (X 40). See text for description. and inject under direct vision. This has the disadvantage of increasing the trauma inherent there is an abnormal stream of cells and frag- mented debris in the process of migration ventrolaterally. This may be a less severely affected embryo of the same stage as in Figure ...... 4 or it may be an embryo of a later stage in which damaged cells are attempting to follow the normal pattern. In younger embryos of the series neural crest cell migration has not started and the neural folds appear normal. In later specimens there seems to be little if any disparity in mesenchyme in the branchial arches or in development of cranial ganglia related to vitamin A dosage. Discussion There is an undesirable degree of variability FIG. 6 Experimental embryo, H & H Stage in the results presented (only 56% of speci- X (X 200). See text for description. mens of the relevant stages showing any effects in the experiment and it is time-consuming. These disadvantages are even more significant -~~~~~~~~~~~. when considering the use of in-vitro techniques. The results, as illustrated, show that the neural crest cells in the cranial region of the * 7 t0~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... chick are specifically affected by excess vitamin A. It is possible that mesenchymal cell migration anteriorly in the cranial regioin has been disrupted and that this results in interference with neural crest migration, but while this might explain the picture seen in Figures 4 and 5, it does not fit with the appearance of Figures 3 and 6. Furthermore, the sequence of events of neural crest migration5 does not ~~~~~~~4T 'it4 i seem to depend upon the presence of normal developing mesoderm in this region. FIG. 5 Experimental embryo, H & H Stage It is very tempting toi suggest that the vita- X (X i6o). (Plastic embedded, sectioned at min A is affecting the neural crest cells directly. 2 plm, stained with toluidine blue). A similar There are two reasons why they might have specimen to Fig. 4 in a higher magnification. a peculiar sensitivity to, vitamin A, one of See text for description. whose properties is the disruption of biological Effects of excess vitamin A on the cranial neural crest in the chick embryo 483 membranes"4 15 Firstly, the distinguishing References characteristics of neural crest cells are that they I Poswillo, D E (I 974) Journal of Maxillofacial move out of the ectoderm in the neural folds Surgery, 2, 64. and migrate through the embryo. These ac- 2 Poswillo, D E (1975) British Journal of Oral tivities are functions of the cell membrane. Surgery, 13, I. Secondly, these activities imply that neural crest cells will be particularly sensitive to any 3 Di Virgilio, G, Lavenda, N, and Worden, J L damage to the mitochondrial membrane where (I967) Acta Anatomica, 68, I27. adenosine triphosphate-producing enzyme sys- 4 Johnston, M C (I966) Anatomical Record, I56, tems are located. '43. The relevance of the present study is that 5 Pratt, R M, Larsen, M A, and Johnston, M C it showvs that vitamin A has a highly specific (1975) Developmental Biology, 44, 298. effect on the migration of cranial neural crest 6 Abramovici, A (i97i) Comptes rendus hebdoma- cells in the early stages of chick embryogenesis. daires des se'ances de l'Acade'mie des Sciences, This agrees with the proposed mechanism of 272, Series D, 2726. the animal model of hypervitaminosis A in the 7 Thompson, J N, Howell, J McC, Pitt, G A J, rat and is additional evidence for the role of and Houghton, C I (I965) Nature, 205, ioo6. the neural crest in the causation of some human 8 Pelagalli, G V (I963) Bolletino della Societa craniofacial syndromes. italiana di biologia sperimentale, 39, I626. 9 Hansborough, L A (I947) Growth, II, 77. Conclusion io Romanoff, A L (1972) Pathogenesis of the Avian The evidence provided by this study supports Embryo, p. 363. London, Wiley. A the suggestion that vitamin does affect the ii Romanoff, A L (1972) Pathogenesis of the Avian cells of the neural crest. In a more extreme Embryo, p. 57. London, Wiley. form this effect can result in malformation of 12 Landauer, W (I967) The Hatchability of Chicken the type of the Treacher Collins syndrome. Eggs as Influenced by Environment and Heredity, Hence the answer to the question posed in p. 2 I 1. Connecticut, Storrs. the introduction is a qualified affirmative. 13 Hamburger, V, and Hamilton, H (I95I) Journal I would like to thank Dr Pollitt, of Roche Products of Morphology, 88, 45. Ltd, for his gift of crystalline retinol and Dr J Mc- Kenzie for valuable discussion. I4 Dingle, J T, and Lucy, J A (i965) Biological Reviews, 40, 422. This investigation was supported by a grant from the National Fund for Research into Crippling I5 Morriss, G M (I975) Journal of Embryology and Diseases to Professor D E Poswillo. Experimental Morphology, 30, 2I9.