/ . Embryol. exp. Morph. Vol. 39, pp. 267-27J, 1977
Printed in Great Britain
267
Inhibition of cranial neural crest cell development
by vitamin A in the cultured chick embryo
JOHN R. HASSELL,1 JUDITH H. GREENBERG 2 AND MALCOLM
C. JOHNSTON 2
From the Laboratory of Developmental Biology and Anomalies, National Institute
of Dental Research, National Institutes of Health, Bethesda, Maryland
SUMMARY
Chick embryos at stage 8, prior to neural crest cell migration, were explanted on whole
egg medium with or without vitamin A and cultured for 3 days. Sections through the head
regions showed that the cranial neural crest cells had migrated into the first visceral arch in
the controls but were absent from this structure in the treated embryos. These observations
suggest that vitamin A inhibits neural crest cell development or migration, an effect which
may in part account for the facial malformations produced by excess vitamin A.
INTRODUCTION
Vitamin A causes a variety of facial and limb malformations in rodents,
depending upon the gestational age at which it is administered. Morriss (1973)
has shown that a single dose of retinol palmitate given to the rat on day 8, 9,
or 10 produces mainly facial malformations while administration on day 11 or
12 results predominantly in defects of the limbs.
[3H]Thymidine cell marking procedures used for the study of chick embryos
have shown that most of the facial mesenchyme is derived from the neural crest
cells (Johnston, 1966). In the rat, where early facial development is basically
similar to that of the chick, the neural crest cells migrate from the neural plate
and into the facial region during days 9 and 10 (Johnston & Hassell, unpublished
observations). This period of greatest suspectibility to vitamin A-induced
teratogenicity with regard to facial malformations appears to coincide with the
migration of the neural crest cells. These observations suggest that vitamin A
may alter neural crest cell development. Similar hypotheses has been put forward by Morriss (1975) and Poswillo (1975).
The explanted chick embryo system (Spratt, 1947; Klein, McConnell &
Requier, 1964) permits the use of precisely staged embryos which can be
1
Authors' address: Building 30, Room 414, National Institute of Dental Research, Bethesda,
Maryland 20014, U.S.A.
2
Present address: University of North Carolina, School of Dentistry, Chapel Hill, North
Carolina 27514, U.S.A.
268
J. R. HASSELL, J. H. GREENBERG AND M. C. JOHNSTON
exposed to a variety of agents during a 3-day culture period. Klein et aJ. (1964)
has shown that compared to in ovo development, in vitro organ development
proceeds normally but growth is slower. This system was employed in this
study to examine the effect of vitamin A on cranial neural crest cell development.
MATERIALS AND METHODS
Fertile eggs from White Leghorn hens were obtained from Truslow Farms
(Chestertown, Md.) and incubated for approximately 30 h at 37-5 °C prior to
explantation. The embryos were then dissected and cultured according to the
technique of Spratt (1947) as modified by Klein et al. (1964). Embryos with
extra-embryonic membranes were cut free of the yolk, and those embryos of
stage 8 (Hamburger & Hamilton, 1951; 5 or 6 pairs of somites) were used for
culture. After removal of the overlying vitelline membrane, the extra-embryonic
membrane peripheral to the sinus terminalis was trimmed away. The explants
were placed ventral surface down on a semi-solid nutrient medium and cultured
at 37-5 °C in an atmosphere 25 % O2 + 75 % air for the first 24 h and in 100 % Oa
for the next 48 h. At the end of the 72 h culture period the embryos were fixed
in 2-5 % glutaraldehyde (LADD) in 0-1 M cacodylate buffer, pH 7-4. The embryos
were embedded in paraffin, cut at 8 /an and stained with hematoxylin and eosin.
The culture medium used was the whole egg medium of Britt & Herrmann
(1959) in which the white and yolk of a fresh egg were combined with an equal
volume of a 1-5 % agar solution in chick Ringer's at 50 °C. Retinol (Sigma, final
concentration 10, 50, 100 /*g/ml of medium) was dissolved in 95 % ethanol and
mixed with the culture medium before it was dispensed in 1 ml aliquots and
allowed to solidify.
RESULTS
Figure 1 shows a section through the head of a control embryo cultured for
72 h and which has reached stage 20. By this stage, the neural crest cells (NC)
have migrated from the neural folds and appear in the first visceral arch as loosely
packed cells surrounding a densely packed core of mesodermal cells (M). The
arrows indicate the path and direction of neural crest cell migration. Fig. 2
shows a cross-section through the head of an embryo cultured in the presence of
50 fig of retinol/ml medium for 72 h. Although the densely packed mesodermal
core of cells is present in the first aortic arch, the number of neural crest cells
surrounding the mesodermal core is greatly reduced. Furthermore, there are a
number of other differences: the epithelial lining of the foregut is thickened,
the cavity of the foregut is distorted and enlarged, the dorsal aortas are enlarged and there appears to be an increased cell density in the region (circled
areas) near the base of the visceral arch. Similar results were obtained at
100 jug retinol/ml, but embryos cultured on medium containing 10/tg/ml were
unaffected.
Vitamin A and neural crest cell development in chick embryos
269
Fig. 1. Cross-section through the head region of a control embryo stage 20 cultured
for 72 h. Neural crest cells (NC) have migrated into the first visceral arch. The
arrows indicate the path and direction of neural crest cell migration. NT, neural
tube; DA, dorsal aorta; FG, foregut; NC, neural crest cells; M, mesodermal cells;
N, notochord. x 130.
Fig. 2. Cross-section through the head region of an embryo cultured for 72 h on
medium containing 50 /tg retinol/ml. The number of neural crest cells in the first
visceral arch is greatly reduced and an increased cell density (circled areas) is
observed near the base of the visceral arch. NT, neural tube; DA, dorsal aorta;
FG, foregut; M, mesodermal cells; N, notochord. x 130.
DISCUSSION
The results of this study show that vitamin A prevents the appearance of the
cranial neural crest cells in the first aortic arch of cultured chick embryos. This
particular arch gives rise to the lower jaw, a structure which was shown to
develop abnormally in rats treated with vitamin A at a comparable developmental age (Morriss, 1973). It may be that altered neural crest cell development
accounts for the facial malformations produced by vitamin A.
Normally, the neural crest cells migrate out from the neural tube towards the
aortic arches in a hyaluronate-rich cell-free space between the mesoderm and the
ectoderm (Johnston, 1966; Pratt, Larson & Johnston, 1975). Although the fate
of the neural crest cells in the treated embryos cannot be conclusively determined
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270
J. R. HASSELL, J. H. GREENBERG AND M. C. JOHNSTON
from the data presented, the increased cell density observed near the base of the
visceral arch may actually represent some of the crest cells or their remnants that
failed to reach the first visceral arch. In contrast, the core of condensed mesodermal cells in the first arch does not appear to be affected. These observations
suggest that vitamin A may, in part, alter the migration of the crest cells. In
support of this suggestion, Kwasigroch & Kochhar (1975) have also reported
that vitamin A impairs mesenchymal cell movement in cell culture. However,
one cannot rule out the possibility that vitamin A inhibits neural crest cell
proliferation or causes cell death, which could also account for the reduced
numbers of neural crest cells seen in the first aortic arch.
Vitamin A has been shown to alter the biochemical and morphological properties of cultured epidermal cells (Yuspa & Harris, 1974; DeLuca & Yuspa, 1974),
cartilage cells (Fell & Dingle, 1963), and 3T3 cells (Kochhar, Dingle & Lucy,
1968) at concentrations of 3-12 /*g/ml, which were lower than the effective concentrations (50-100/Ag/ml) required in the present study. However, in the experiments reported here, the explants were cultured on top of the solidified
medium rather than submerged and so must rely on the extra-embryonic membrane for metabolism and transfer of components of the medium to the embryo (Hassell & Klein, 1971).
Although the biochemical mechanisms by which vitamin A causes teratological effects has not yet been determined, there are several possibilities. First,
Fell & Dingle (1963) have shown a reduction of matrix staining in cartilage
cells treated with vitamin A and have suggested that vitamin A causes a destabilization of lysosomal membranes, leading to the release of enzymes that degrade
the matrix. In the case of the neural crest cells, released lysosomal enzymes may
alter the hyaluronate-rich matrix through which they migrate. However, more
recent evidence suggests that vitamin A may also alter synthesis of glycosaminoglycans or glycoproteins. Yuspa & Harris (1974) and DeLuca & Yuspa (1974)
have shown that vitamin A causes an increase in glycoprotein synthesis or glycosylation and PAS staining in epidermal cells. Kochhar et ah (1968), using 3T3
cells, showed that vitamin A increased the secretion of a glucosamine-labeled
product into the medium. Since it has been shown the neural crest cells synthesize glycoproteins (Greenberg & Pratt, 1977) and migrate through a hyaluronaterich matrix (Pratt et al 1975) synthesized at the time of migration, it may be
that vitamin A alters cell surface glycoproteins or the secretion and distribution
of the matrix in which the neural crest cells migrate.
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GREENBERG,
{Received 25 August 1976)
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