Recent Advances in Nutritional Sciences
Function of Vitamin A in Vertebrate
Embryonic Development1,2
Maija H. Zile
Department of Food Science and Human Nutrition, Michigan
State University, East Lansing, MI 48824
ABSTRACT Advances in molecular biology and retinoic
acid receptor research have significantly contributed to the
understanding of the role of vitamin A during vertebrate
development. Examination of the function of this vitamin
during very early developmental stages using the com-
pletely vitamin A– depleted avian embryo has revealed that
the vitamin A requirement begins at the time of formation of
the primitive heart, circulation and specification of hind-
brain. The lack of vitamin A at this critical time results in
gross abnormalities and early embryonic death. In rodent
models, vitamin A deficiency can be targeted to later ges-
tational windows and documents the need for vitamin A for
more advanced stages of development. Major target tis-
sues of vitamin A deficiency include the heart, central ner-
vous system and structures derived from it, the circulatory,
urogenital and respiratory systems, and the development of
skull, skeleton and limbs. These abnormalities are also
evident in mice mutants from retinoid receptor knockouts;
they have revealed both morphological and molecular as-
pects of vitamin A function during development. Retinoic
acid receptors (RAR) in partnership with retinoid X receptor
(RXR)␣ appear to be the important retinoid receptor tran-
scription factors regulating vitamin A function at the gene
level during development via the physiologic ligand all-
trans-retinoic acid. Homeostasis of retinoic acid is main-
tained by developmentally regulated vitamin A metabolism
enzyme systems. Inadequate vitamin A nutrition during
early pregnancy may account for some pediatric congenital
abnormalities. J. Nutr. 131: 705–708, 2001.
KEY WORDS: ● vitamin A ● avian development ● retinoic acid
The essentiality of vitamin A nutrition throughout the life
cycle has been well established. Most importantly, the require-
ment for vitamin A begins with embryonic life as noted by
early nutritionists who reported that maternal insufficiency of
vitamin A during pregnancy results in fetal death or congen-
ital abnormalities in the offspring (1– 4). During the last
decade, there has been great interest in the area of vitamin A
function in development as reflected in a number of recent
detailed reviews on the subject (4 – 8).
1
Supported by the U.S. Department of Agriculture, the National Institutes of
Health and Michigan AES.
2
Manuscript received 8 December 2000.
0022-3166/01 $3.00 © 2001 American Society for Nutritional Sciences.
705
Retinoic Acid, a Critical Signaling Molecule in the
Vertebrate Embryo. The elucidation of how vitamin A
exerts its pleiotropic effects has become a molecular biology–
driven endeavor for many nutritionists since the discovery and
realization that the biological effects of vitamin A, except for
its role in vision, are mediated by its active form, all-trans-
retinoic acid via specific nuclear receptors that are members of
the steroid superfamily of ligand-activated transcription factors
(4,8 –10), i.e., the retinoic acid receptors (RAR)3 ␣, ␤ and
␦ that are activated by retinoic acid (RA), and the retinoid X
receptors (RXR) ␣, ␤ and ␦ that are obligatory partners for the
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RAR. The RAR-RXR heterodimers are the functional units in
transducing the retinoid signal at the gene level. The pleio-
tropic effects of vitamin A are explained by the discovery that
many of the RA target genes are genes involved in diverse
biological processes (4,8 –10). Redundancy of retinoid recep-
tors, evidenced in studies with receptor gene knockouts (4),
attests to the paramount importance of vitamin A in devel-
opment. During the course of normal embryonic development,
distinct developmental events are regulated by the presence of
the vitamin A active form, RA. The function of vitamin A is
inseparable from its metabolism because the biogeneration of
RA in the embryo is the first developmental step in the
initiation of RA-regulated signaling pathways and must be
linked to regulated metabolic systems for the inactivation of
unused RA. All of the physiologically important vitamin A
metabolites and enzyme systems that regulate vitamin A me-
tabolism have been demonstrated in embryos (3– 8,11–15).
Model Systems for the Study of Vitamin A Function
in Embryonic Development. Vitamin A deficiency and
excess have profound effects on the development of the ver-
tebrate embryo (3– 8). Almost every organ or tissue can be
affected by RA if the embryo is treated with it at a critical time
in development (16). A vast amount of information has been
gathered on vitamin A function at the molecular level from
cell culture (4,10,17) and from in vivo studies by perturbing
normal embryonic development with exogenous retinoids (4 –
8,10). The abnormalities observed in these studies, when
viewed together with similar abnormalities reported for the
vitamin A– deficient (VAD) embryo and fetus, identify com-
mon development pathways that are severely disrupted by an
imbalance in vitamin A status. The function of vitamin A in
development has been addressed recently by the use of trans-
genic mice with changes in retinoid receptor gene structure
(4,9,18 –20). Many of the abnormalities in these mutant mice
resemble those observed in fetuses from the VAD animals
reported earlier. These studies have provided valuable insights
into the pleiotropic effects of vitamin A and have demon-
strated the critical role of retinoid receptors in vertebrate
ontogenesis. The interpretation of the data has been compli-
cated by retinoid receptor redundancy and by the broad role of
the RXR as coreceptors for other nuclear receptors (4,9).
3
Abbreviations used: CNS, central nervous system; RA, retinoic acid; RAR,
retinoic acid receptor; RXR, retinoid X receptor; VAD, vitamin A-deficient.
706
An important approach to the examination of molecular
mechanisms of retinoid action in developmental regulation is
the use of in vivo embryo model systems in which the function
of vitamin A has been diminished or completely eliminated by
removing the vitamin. The absolute essentiality of vitamin A
for embryogenesis is most clearly demonstrated in the VAD
avian embryo, i.e., the quail embryo retinoid ligand knockouts.
The completely VAD embryos develop gross abnormalities in
the cardiovascular and central nervous systems and trunk and
die by d 4 of embryonic life (3,4,6,7,21–23). Importantly, the
VAD embryo can be “rescued” and normal development re-
stored by administration of the physiologic ligand for RAR,
all-trans-retinoic acid, or its precursor, retinol. Bioactive reti-
noids must be administered to these embryos during early
development so as to be present during the critical window of
time in which important developmental events are specified,
i.e., the formation of heart, cardiovascular system, hindbrain,
foregut and probably other events (21,23). With this model, it
is possible to examine morphological, anatomical and molec-
ular biology aspects during development that are solely attrib-
utable to vitamin A. The ability to rescue the VAD embryo at
a precise time during development makes the avian retinoid
ligand knockout model a powerful tool for the elucidation of
vitamin A function during early development.
Using rats as a mammalian model, it is possible to obtain
near vitamin A deficiency in the dams and to target embryonal
vitamin A insufficiency to distinct gestational windows
(3,4,6,7,24 –26). These rat embryos exhibit specific cardiac,
limb, ocular and central nervous system (CNS) abnormalities,
some of which have certain features similar to those reported
in retinoid receptor knockout mice (25). Abnormalities in
hindbrain development of the rat embryos (26) are similar to
those reported in VAD quail embryos (7,27) and suggest that
even partial vitamin A deficiency affects the sensitive devel-
oping CNS. These studies have also revealed the importance
of vitamin A in fetal lung and kidney development (24,28,29).
Another in vivo approach to eliminate vitamin A–active
forms is to block RA with an anti-RA antibody (30) or to
inactivate the RA generation pathways (4,7). Knockout mice
embryos of the RA synthesizing enzyme RALDH2 (11) have
many abnormalities similar to those of the VAD quail embryo,
but complete vitamin A deficiency was not obtained, probably
due to the presence of other RA-generating systems. Partial
depletion of RA may also be achieved by an overexpression of
CYP26, an enzyme that degrades RA (4,13).
Heart and Blood Vessel Development. A great deal of
information on the effects of retinoids on heart development has
been gathered from ectopic application of excess retinoids to
embryos (6,31,32) but physiologically more relevant are the ex-
perimental approaches that diminish or eliminate vitamin A
function. Maternal insufficiency of vitamin A during pregnancy
results in fetal death or severe abnormalities in the offspring,
including abnormal heart development; many of the heart defects
have been recapitulated in fetuses generated from various com-
binations of retinoid receptor knockouts. Heart abnormalities
obtained include a thin-walled dilated heart cavity, abnormalities
in ventricular chambers and defects in the outflow tract
(3,4,6,18 –20,22,25,31). Important information has also been ob-
tained by molecular analysis (9,18 –20), but the primary retinoid
targets have not been identified. The phenotypes obtained may
reflect secondary lesions and abnormalities due to RXR functions
with other nuclear receptors (4,9). In contrast, the quail embryo
retinoid ligand knockout model allows an analysis of develop-
mental regulation solely attributable to vitamin A and reveals the
full function of this vitamin. The model has an advantage for
ZILE
studies of vitamin A function during early precardiac and subse-
quent heart-forming stages by “rescue” manipulations to restore
normal gene expression and cardiogenesis. The VAD cardiac
phenotype in this avian model is highly reproducible and, to-
gether with molecular analysis, allows us to draw certain conclu-
sions about the initial cardiogenic events regulated by vitamin A
(3,6,21–23). The developing heart is a grossly abnormal, thin-
walled, dilated and distended structure, without chambers, but it
contracts until the embryo dies. This is not surprising because the
expression of early cardiogenic genes that regulate heart precursor
cell differentiation into cardiomyocytes is not affected by the lack
of vitamin A (22). In contrast, exogenous treatments with RA
induce differentiation of precursor cells into cardiomyocytes (31).
The concept of a specific retinoid function in axial speci-
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fication has a long history, originating with the identification
of RAR responsive elements in some of the evolutionarily
conserved axial patterning genes (33). It has been perpetuated
by results from numerous studies in which exogenous RA
applied to embryos of various species at various stages of
development affects the specification of body axes, heart asym-
metry and limb patterning (5,8,31–33). These studies sug-
gested a specific role for RA in heart asymmetry determination
(23,31,32). However, recent evidence points to vitamin A
having a general rather than a specific role, i.e., it appears that
vitamin A is required to provide a proper environment for the
expression of adequate levels of heart asymmetry genes (23).
This concept is supported by evidence from the literature that
the generation and distribution of RA in the embryo as well as
the expression patterns of vitamin A metabolism enzymes and
retinoid receptors are symmetric (7,11–14,21).
A major developmental defect that may be linked directly
to the early embryolethality of the VAD quail embryo is the
absence of a cardiac inflow tract (3,6,22), i.e., the VAD heart
has no opening at its caudal end where the extraembryonal
blood vessels converge into vitelline veins to deliver blood to
the embryonic heart for distribution to the embryo. This
defect has not been reported in any of the mammalian in vivo
models addressing vitamin A function during embryogenesis.
The formation of the cardiac inflow tract may be linked to the
expression of the retinoid-regulated cardiac transcription fac-
tor GATA-4 in the posterior heart–forming area where this
gene is involved in a BMP2 pathway that specifies the
endodermal structures of the heart and the underlying foregut
primordia, and where apoptosis is observed in the VAD em-
bryo (34). In these embryos, the extraembryonal vascular
networks are sparse and fail to converge at the level of the
cardiac inflow tract (35; Fig. 1). Defects in vitelline vessel
formation have also been observed in cultured mouse embryos
when the transfer of retinol from the yolk-sac to the embryo is
prevented (36). Anomalies in vasculogenesis have not been
reported in retinoid receptor knockout mice, but may have
contributed to embryolethality.
The Central Nervous System. Vitamin A plays an impor-
tant role in development of the central nervous system (CNS)
(4,7,10,14,26,27,37). Rescue studies and functional analysis of
VAD quail embryos have revealed that vitamin A is required
at the time of specification of the posterior hindbrain, for the
subsequent specification of segments in that region and for
neurite outgrowth and neural crest survival (7,27). Studies
with in vivo vitamin A– deprived rodent embryos (14,26,38)
revealed less severe CNS defects, suggesting that the defi-
ciency was not complete. When such embryos survive to more
advanced developmental stages, abnormalities are seen in the
developing visual system and the retina, in inner ear primordia
and in craniofacial and spinal cord development (4). Retinoid
 VITAMIN A IN VERTEBRATE EMBRYONIC DEVELOPMENT
FIGURE 1 Vascular development in the 36 –38 h normal and vita-
min A– deficient (VAD) quail embryo. In the normal embryo, well-formed
vascular networks (arrowhead) converge into vitelline veins at the car-
diac inflow tract (black arrows); the VAD embryo has sparse, disorga-
nized vascular networks and poorly developed vitelline veins. Heart
(large arrows) has begun to loop in the normal embryo but is abnormal
in the VAD embryo. Note the distorted head and short body in the VAD
embryo. All views are ventral.
receptor knockouts have also provided important information
on the role of retinoids in CNS (4,14,18 –20,39) and confirm
the vitamin A requirement for the development of hindbrain,
the visual and auditory systems, for various structures and
organs arising from neural crest, for craniofacial structures,
pharyngeal and branchial arches, for heart outflow tract and
the thymus, thyroid and parathyroid glands (4,7,10,19,37).
Increased apoptosis of the migrating neural crest cells due to
lack of vitamin A may be responsible for the abnormalities (7).
Development of Limbs, Respiratory System and Other
Systems. Retinoids have long been linked to vertebrate limb
morphogenesis, mainly as the result of studies involving in-
duction of genes and morphologic anomalies with exogenous
retinoids. Results from these numerous studies have been
summarized in several recent publications which detail the
morphology and the genetic pathways and propose mecha-
nisms for retinoid involvement (4,5,8,40). Recent examina-
tion of limb patterning in a physiologically relevant model
suggests that studies with ectopic retinoids may not accurately
reflect the function of vitamin A in normal limb development
(40,41). The completely VAD quail embryo does not live long
enough to develop limbs, but in contrast to earlier notions, the
initiation of limb bud development occurs in the absence of
vitamin A. Analysis of the expression of developmental genes
in these limb buds has revealed an important role for vitamin
A in patterning the dorsoventral axis (40). It is not clear how
the abnormal limb patterning pathways in VAD avian limb
buds relate to the limb malformations observed in certain
retinoid receptor mutants (4); the malformed limbs may be the
result of marginal retinoid activity during a later period in
development. Interestingly, mice knockouts of the RALDH2
707
gene, an enzyme involved in the generation of RA, do not
appear to have limb buds (11), suggesting species differences.
Using partially vitamin A– depleted rodent models in
which the fetuses survive longer and later gestational windows
can be examined, it was demonstrated that vitamin A is
specifically required during midgestation for fetal lung devel-
opment and neonatal survival (24,42). Abnormal cell differ-
entiation and diminished expression of elastin gene and a
growth-arrest gene were associated with abnormal lung mor-
phology (42), also seen in mice with deletions in RAR genes
(29). Compound mutants of RAR also exhibit congenital
respiratory tract abnormalities (4,19). The importance of ad-
equate maternal/fetal vitamin A nutrition in prevention of
neonatal lung injury has been demonstrated clinically and in
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animal models (28). Similarly, renal development is affected
by retinoids (43), also noted in the RALDH2 knockout mice
(11); congenital malformations in the urogenital system are
seen in fetuses of RAR compound mutant mice (4).
Summary and Perspectives. Early animal studies on vita-
min A requirement during pregnancy as well as more recent
rodent depletion models have provided important information
regarding the role of this vitamin during specific gestational
windows. However, complete vitamin A deficiency is neces-
sary to reveal the full function of vitamin A during early
development as best illustrated in the avian embryo vitamin A
knockout model. Results from these experiments suggest that
the requirement for vitamin A activity in the embryo begins at
the time of first organ system initiation, not earlier. This
hypothesis is in agreement with the conclusions reached by
Durston et al (33) who noted that the gain and loss of function
experiments with retinoid receptors do not demonstrate sig-
nificant changes during early embryogenesis and thus chal-
lenge the existing hypothesis that RA signaling is essential at
the time of embryonal cell organization and initial axial pat-
terning. Results from the avian studies provide strong evidence
that the absolute vitamin A requirement begins at the time of
formation of the primordial heart tube, the linking of the
extraembryonal vascular system to the developing heart and
the specification of hindbrain. These critical events in the
avian embryo take place at the same developmental time and
correlate to first 2–3 wk of human pregnancy, emphasizing the
importance of adequate vitamin A nutrition during initial
stages of pregnancy. If vitamin A is completely lacking during
this critical time, development takes place along a default
pathway, resulting in gross abnormalities and early embryole-
thality. In marginally vitamin A– deprived rat embryos or with
some retinoid receptor knockouts, the requirement for vitamin
A can be demonstrated for later, more advanced developmen-
tal events. Certain retinoid receptor knockouts recapitulate
many of the vitamin A deficiency symptoms, but do not
provide a clear vitamin A deficiency phenotype because of
receptor redundancy and the pleiotropic effects from RXR
acting as partners with multiple nuclear receptors (4,9,18,31).
Altogether, all observations support the requirement for vita-
min A during multiple stages of development for numerous
tissues and organs. Consistent with the many roles of vitamin
A in development, retinoid receptors are widely expressed in
the vertebrate embryo (4,12,31). Retinoid receptor knockout
studies have revealed that for developmental function, there is
very little redundancy among the RAR subtypes, and that
RXR␣ is the major partner for the RAR in RA signaling (4).
The RAR-RXR heterodimer is most likely activated by the
RAR ligand, all-trans-retinoic acid, with RXR acting as en-
hancers in RA signal transduction (9). The present data
suggest that during embryogenesis, all-trans-retinoic acid is the
708
sole active form for all retinoid functions involving retinoid
receptors. Vitamin A function during embryogenesis is locally
regulated by a spatiotemporal developmental expression of RA-
generating and -inactivating enzymes and further fine-tuned
through a diversity of receptors and their isoform combinations.
A number of genes are known to have RAR responsive elements
(4,8,10), but it is not known which genes are causally and directly
linked to the VAD phenotype. At this time, no immediate RA
target genes have been identified for any developmental event,
and no single protein, other than the RAR and RXR proteins, is
known to be linked directly to vitamin A function. RA effector
genes may be important genes involved in mainstream functions
such as cell division, differentiation and energy metabolism, bi-
ological processes that have long been recognized as hallmarks of
vitamin A action.
Understanding the function of vitamin A nutrition during
development is of great clinical importance because popula-
tion studies estimate that ⬃3% of all children born in the
United States have major malformations at birth (44) with
70% of these of unknown etiology, including congenital heart
disease, the most prevalent human birth defect (45). Some of
the heart abnormalities obtained with retinoid receptor
knockouts or resulting from insufficient vitamin A resemble
the most common heart defects in humans (45).
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