PHYSIOLOGICAL REVIEWS
Vol. 80, No. 3, July 2000
Printed in U.S.A.
Retinoids in Embryonal Development
SHARON A. ROSS, PETER J. MCCAFFERY, URSULA C. DRAGER, AND LUIGI M. DE LUCA
Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Nutritional Products,
Labeling, and Dietary Supplements, Washington, DC; E. Kennedy Shriver Center, Waltham; Department
of Psychiatry, Harvard Medical School, Boston, Massachusetts; and National Cancer Institute,
National Institutes of Health, Bethesda, Maryland
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I. Introduction
II. Retinoid Structure
III. Retinoid Requirement During Embryogenesis
A. Introduction
B. Retinoid ligand knockout models
IV. Metabolism
A. Introduction
B. Retinol dehydrogenase
C. Retinaldehyde dehydrogenases
D. Synthesis of other bioactive retinoids in the embryo
E. Influence of substrate concentration and distribution
F. RA catabolism
G. Retinoid binding proteins
H. Hypothesis: CRABP captures RA for cells that lack RA synthesis
V. Retinoid Receptors/Function
A. Introduction
B. Speculations on orders of receptor interactions
C. Receptors and embryogenesis
VI. Retinoid Receptor Knockout Mutants
A. Isoform-specific knockouts
B. Single subtype knockouts
C. Double/compound mutants
D. Retinoid receptor mutants and limb malformations
E. Retinoid receptor mutants and malformations of the vertebral column
F. Response of mutants to excess RA
G. Conclusions of mutant mice studies
VII. Hox Genes and Retinoids in Development
A. Hox genes in development
B. Retinoids and Hox genes
C. Conclusions on the induction of Hox genes by retinoids
VIII. Teratology/Excess Retinoids
A. Introduction
B. Retinoids and limb morphogenesis
C. Animal teratogenic models
D. Molecular mechanism of excess retinoids in teratogenesis
E. Prescription oral retinoids as teratogens in humans
F. Human epidemiological evidence: intake of vitamin A and risk of birth defects
IX. Conclusions and Future Aspects
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Ross, Sharon A., Peter J. McCaffery, Ursula C. Drager, and Luigi M. De Luca. Retinoids in Embryonal
Development. Physiol Rev 80: 1021–1054, 2000.—The key role of vitamin A in embryonal development is reviewed.
Special emphasis is given to the physiological action of retinoids, as evident from the retinoid ligand knockout
models. Retinoid metabolism in embryonic tissues and teratogenic consequences of retinoid administration at high
doses are presented. Physiological and pharmacological actions of retinoids are outlined and explained on the basis
of their interactions as ligands of the nuclear retinoid receptors. Immediate target genes and the retinoid response
www.physrev.physiology.org
0031-9333/00 $15.00 Copyright © 2000 the American Physiological Society
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Volume 80
elements of their promoters are summarized. The fundamental role of homeobox genes in embryonal development
and the actions of retinoids on their expression are discussed. The similarity of the effects of retinoid ligand
knockouts to effects of compound retinoid receptor knockouts on embryogenesis is presented. Although much
remains to be clarified, the emerging landscape offers exciting views for future research.
I. INTRODUCTION
II. RETINOID STRUCTURE
Figure 1 shows the chemical structure of the salient,
physiologically important retinoids so far identified. The
parent compound retinol and its oxidation product, retinoic acid (RA), are shown in the stretched-out all-transconfiguration at the top of the figure. Derivatives of retinol include 4-hydroxy-retinol, 4-oxo-retinol, and 14hydroxy-4,14-retro-retinol. Of the RA derivatives,
4-hydroxy-RA, 4-oxo-RA, 3,4-didehydro-RA (ddRA), and
III. RETINOID REQUIREMENT
DURING EMBRYOGENESIS
A. Introduction
Retinol (also referred to as ROL and/or vitamin A) is
the only retinoid known to be capable of sustaining all
vitamin A functions, including development, growth, vision, and reproduction (Fig. 2). On the other hand, RA
maintains differentiation and growth of the adult organism; it, however, is insufficient to support gestation of the
embryo. Because RA cannot fulfill all vitamin A functions,
it is obvious that several retinoids, in addition to RA and
which are not RA metabolites, act in concert to maintain
the health and well being of the entire organism. Also
possible are limitations of cellular metabolism and transport, where cells require RA but must transport retinol
across a tissue barrier. For a more detailed discussion of
the different roles of vitamin A, the reader is referred to
the following reviews (11, 263, 307).
As early as the 1930s it was realized that maternal
insufficiency of vitamin A results in death of the fetus as
well as congenital malformations (79, 172). Later, Wilson
and co-workers (279, 305) defined congenital abnormalities resulting from vitamin A deficiency during gestation.
Teratogenic targets of vitamin A deficiency were the
heart, ocular tissues, and respiratory, urogenital and circulatory systems (321).These abnormalities were prevented by inclusion of vitamin A in the diet.
Excess dietary vitamin A, on the other hand, has been
shown to also cause teratogenesis (26). Several other
studies followed this original observation and are discussed in section VIII. Suffice it to say here that major
excess vitamin A targets include the heart, the skull,
skeleton, limbs, brain, eyes, CNS, as well as craniofacial
structures (15, 110, 111, 189, 191, 203, 234, 252, 322).
Similarity of teratogenic responses between vitamin
A deficiency and excess supports the concept that the
same molecules are involved. It also suggests a fundamental role for this nutrient in embryonal development.
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Vitamin A and its derivatives (the retinoids) are essential for both normal embryonic development (87) and
maintenance of differentiation in the adult organism (35,
36, 78), accounting for the intense interest in these compounds in biology and medicine. Embryo segmentation
and growth fail, vascularization stops, and the embryo is
eventually resorbed in the absence of sufficient vitamin A
(151, 279, 297). Excess vitamin A, on the other hand,
results in terata during embryogenesis and is membranolytic and hepatotoxic in the adult organism (204). In the
adult, epithelial differentiation requires vitamin A. Its deficiency causes squamous metaplasia, a preneoplastic lesion, which eventually also alters functional epithelial
characteristics and leads to infection and death (36, 247,
306). These considerations make it obvious that maintenance of retinoid homeostasis is tantamount to maintenance of normal physiology of the intact organism.
The use of the retinoid ligand knockout models to
study embryonic development has unequivocally linked
the physiological function of vitamin A to development of
the heart, the embryonal circulation, the central nervous
system (CNS), and the normal left-to-right cardiac symmetry (321). Recent work has also emphasized that the
developmentally regulated generation of bioactive retinoids is fundamental to the control of embryonic development (47, 321).
Remarkable progress has occurred in the past 10
years in our understanding of the mode of action of
vitamin A and its derivatives, the retinoids (37). The discovery of the nuclear receptors for retinoic acid and other
retinoids has provided a conceptual basis to explain how
these compounds preside over a large network of gene
activation processes (35).
It is the purpose of this review to present a balanced
synopsis of the actions of retinoids in embryonal development and to provide suggestions for future studies.
the rexinoid receptor (RXR) ligand 9-cis-RA are shown.
All-trans RA is the natural ligand for the retinoic acid
receptors (RAR) (71, 221), and 9-cis-RA for the RXR (137),
although the latter compound binds to both receptor families. The specificity of interactions would also suggest the
possibility that various retinoids, both natural and synthetic, may specifically be useful as drugs to combat
diverse diseases.
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RETINOIDS IN EMBRYONAL DEVELOPMENT
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The work of Wellick and De Luca (297) has demonstrated that retinol is essential and RA alone appears
insufficient to allow gestation in the rat to reach completion. In fact, vitamin A-deficient pregnant rats resorb their
fetuses at about day 15 of gestation , even if given retinoic
acid daily. Retinol, on the other hand, prevents this resorption when administered no later than day 10 of gestation. In fact, the administration of as little as 2 ␮g on
day 10 is sufficient to allow continuation of gestation
through parturition (298). Claggett-Dame recently proposed that extremely high RA doses may, in fact, rescue
the dpc 14.5 gestational barrier identified by Wellick and
De Luca (298). Whether this reflects rescue by traces of
alternative retinoids in not known at this time. Countering
this is Niederreither’s RALDH2 knockout, which is incompletely rescued by exogenous RA (206, 207).
The recommended dietary allowance (RDA) for vitamin A for nonpregnant and pregnant women is 800 ␮g RA,
which is equivalent to ⬃2,700 IU (210). No increment of
vitamin A is recommended during pregnancy (210, 214).
For a discussion of the data which justify this recom-
mended intake, the reader is referred to the 10th edition
of the RDA (210).
Different retinoid requirements between the avian and
mammalian systems have been highlighted and are summarized as follows: 1) ddRA and its precursor 3,4-didehydroretinol (ddROH) were undetectable in mouse limb buds,
although prominent in chick limbs; 2) a relatively high concentration of retinyl esters (1.5 ␮M) is evident in chick limb
buds but not in mouse; and 3) there is a higher concentration of cellular retinoic acid binding proteins (CRABP), especially CRABPII, than their ligands RA and ddRA in both
species. Retinol and ddROH, on the other hand, were
present in much higher concentration than cellular retinol
binding proteins (CRBP) (245). The concentration of “free”
RA was found to be near the range for the dissociation
constant (Kd) value of the murine RAR.
Principal approaches to study the essential function
of vitamin A during embryonal development are exposure
to excess retinoid, retinoid receptor knockout studies,
and finally retinoid-ligand knockout models. This last approach is discussed in section IIIB.
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FIG. 1. Structures of the most common natural retinoids.
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2. Highlights of the various functions of vitamin A in males and females.
B. Retinoid Ligand Knockouts Models
The studies of Zile (321) have demonstrated the usefulness of the vitamin A-deficient (retinoid ligand knockout) avian embryo. These embryos display a strict dependence on vitamin A for their development of early
vasculature. These avian embryos die at 3 days of embryonic life in the absence of vitamin A (43, 279). After the
initial discovery by Heine et al. (83) that vitamin A is
necessary for the establishment of the early vasculature,
Zile (321) extended these studies to early development.
Dong and Zile (53) and Chen et al. (25) had established
that RA given to the hen is not transported to the egg. In
this way a completely vitamin A-deficient embryo can be
obtained from hens that are RA sufficient (53). This model
was used by Zile and collaborators to define primary
target tissues of vitamin A action during embryogenesis.
The vitamin A-deficient quail embryo presents with
gross abnormalities in the cardiovascular system, head,
CNS, hematopoietic organs, and trunk (43, 151, 152, 283,
321, 322). Bioactive retinoids can “rescue” the vitamin
A-deficient embryo by preventing abnormal development
when available at early development (43, 83, 115, 117, 279,
321). Because both retinoic excess as well as deficiency
had similar teratogenic effects on the heart, Zile’s group
has launched an in depth investigation of the function of
vitamin A in early heart development, using the vitamin
A-deficient quail model (115–117). These studies are of
particular interest because of the obvious relevance to
human cardiovascular malformations, which may well be
diet-related during pregnancy. Availability of vitamin A
may be a relevant contributory element to the incidence
of heart malformation in developing countries, where vitamin A deficiency is a significant problem (261). Very
little is understood of its etiology in the industrialized
world, hence, the importance of better definition of etiological factors, especially dietary ones during pregnancy,
since diet is likely to contribute the majority of xenobiotic
as well as homobiotic factors to the physiology and pathology of the body. Vitamin A is also required for normal
specification of heart left-right asymmetry. In a large percentage of vitamin A-deficient quail embryos, the heart
appears on the wrong side (randomization) (281, 283, 284,
323). Retinoids, although not directly involved in assigning cardiac asymmetry genes to their asymmetry-specific
sites, are absolutely essential for normal heart sidedness
to occur (M. H. Zile, personal communication). Importantly, administration of vitamin A to deficient embryos as
late as stage 8 (neurulation) prevents the anticipated vitamin A-deficient phenotype, including situs inversus (43,
115, 117, 323). They propose that the critical retinoidrequiring developmental window is at the four/five-somite
stage during neurulation, when retinoid presence is absolutely essential for normal embryonic development to
proceed. Excess retinoid resulting from implantation of
retinoid impregnated pellets also causes cardiac abnor-
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FIG.
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RETINOIDS IN EMBRYONAL DEVELOPMENT
heart which eventually progresses to develop abnormally
in the absence of vitamin A, the lateral mesoderm posterior to the heart. These authors also report that administration of retinol to the vitamin A-deficient embryos is
able to restore GATA-4 expression and completely rescues the vitamin A-deficient phenotype. The authors conclude that their results indicate GATA-4 is a component of
the retinoid-mediated cardiogenic pathway. They also
conclude that this pathway appears unlinked to the differentiation of cardiomyocytes, and it is involved in the
morphogenesis of the posterior heart tube as well as the
development of the cardiac inflow tract, which does not
form in the vitamin A-deficient embryo.
IV. METABOLISM
A. Introduction
RA regulates development by activating gene transcription in many different locations within the embryo. A
particular cell will only respond to RA if 1) it expresses
RA receptors and 2) the RA concentration lies within a
range that is appropriate for its response. Because different genes are known to be activated by different RA levels
(13), the effectiveness of RA as a developmental regulator
involves the precise control of its distribution and concentration. Several mouse strains transgenic for a RAactivated reporter gene show that RA is not homogeneously distributed throughout the vertebrate embryo,
but is instead localized to specific territories within a
restricted number of developing organs in the embryo (8,
236). Studies on RA metabolism in the embryo indicate
that the RA-synthesizing and catabolic enzymes are localized to subregions of developing tissues (175, 194, 206,
319), which resemble the activation patterns in the RAreporter mice (8, 236). The pathways of synthesis and
catabolism thus establish the local levels of RA and determine where and when RA regulation occurs. The most
detailed description of this localization is available for the
embryonic retina (54) and spinal cord (175). In addition,
several other regions exhibit localized RA synthesis, both
in the CNS, such as the substantia nigra and corpus
striatum (176), and in mesoderm-derived tissues, such as
the meninges (206), heart (194), and somites (206). Several regions in the developing CNS, e.g., the limb motor
neurons in the spinal cord (319), express very high levels
of RA-synthesizing enzymes, resulting in remarkably high
RA concentrations locally, up to micromolar levels. The
RA-rich regions can be located next to areas of high RA
catabolism that contain little detectable RA, as is the case
for the embryonic retina described in more detail below
(181). The analysis of the distribution of RA synthetic and
catabolic enzymes shows that several developing tissues
contain compartments of high and low RA levels, a mor-
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malities, including duplicate heart, cardia bifida, abnormal looping, and situs inversus (46, 215). Genes coding for
extracellular matrix proteins appear important during
heart embryogenesis and are regulated by retinoid status
(267). Kostetskii and collaborators (116, 117) have found
that RAR␤2 expression in the retinoid ligand knockout
quail model is reduced, as is RAR␣. They also reported
that several retinoids, including all-trans-, 9-cis-, 4-oxo-,
and didehydro-RA could rescue embryonic development,
when provided to the developing embryo at up to the
five-somite stage of development, but not later. These
retinoids induced RAR␤ expression; 4-oxo-RA is least
active.
Metabolic routes of retinoid activation and disposal
are highly relevant to the occurrence of orderly developmental events and are covered separately in this review.
Rat embryos made deficient in retinoids during embryogenesis (gestational days 11.5–13.5) show specific
cardiac, limb, ocular, and nervous system deficits (258).
Many of these abnormalities also occur in retinoid receptor null mutants as discussed in section VI. Retinoid-ligand
knockout models reveal additional defects, however,
clearly supporting redundancy of the retinoid receptors
(258).
Stratford et al. (267) have recently identified target
genes in the development of the early limb bud of the
quail, in the retinoid ligand knockout model. The retinoiddeficient quail embryos were found to develop to about
stage 20/21. In a study of genes involved in anteroposterior axis, these authors found that Hoxb-8 was upregulated and its border was shifted anteriorly. In sharp contrast, shh and mesodermal bmp-2 were downregulated.
These authors also studied the apical ectodermal genes
and found that fgf-8 and ectodermal bmp-2 were not
affected (267). A strong effect of retinoid deficiency was
observed for genes involved in dorsoventral polarity.
Wnt-7a was expressed in ventral ectoderm, although normally it is expressed exclusively in dorsal ectoderm. On
the other hand, the corresponding dorsal mesodermal
gene Lmx-1 was found to extend its expression into the
ventral mesoderm. En-1 expression was absent from its
normal expression site, the ventral ectoderm (267). This
study exemplifies the usefulness of this model to study
the role of retinoids in the regulation of gene expression
during development.
Kostetskii et al. (115) have investigated the expression of cardiomyocyte differentiation genes. They have
reported that vitamin A deficiency in the quail embryo
fails to alter these genes including atrial-specific myosin
heavy chain, ventricular-specific myosin, and sarcomeric
myosin as well as the putative cardiomyocyte specification gene Nkx-2.5. However, the expression of the transcription factor GATA-4 was greatly reduced in the heartforming region of stage 7–10 embryo in vitamin A
deficiency. This downregulation occurs in the areas of the
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Volume 80
phogenetic RA pattern in addition to, and different from,
the earlier model of patterning through RA gradients
(280).
B. Retinol Dehydrogenase
The first step in RA synthesis is the reversible oxidation of retinol to retinaldehyde (22) (see Fig. 3). The full
complement of enzymes that catalyze this reaction in the
embryo remains to be determined. It is likely, though, that
most of the retinaldehyde in the embryo is generated by
alcohol dehydrogenases. A point of contention is, however, whether the medium-chain cytoplasmic alcohol
dehydrogenases or the short-chain membrane-bound
alcohol dehydrogenases are more important. Several
members of the medium-chain, cytoplasmic class are
competitively inhibited by ethanol, whereas the shortchain membrane-bound class is resistant to ethanol inhibition. The answer is important in reference to the hypothesis that the abnormalities evident in fetal alcohol
syndrome result from the inhibition of RA synthesis by
ethanol (58, 227, 315). This hypothesis has been proposed,
because of the similarities between the embryonic abnormalities evident in RA teratogenicity and in fetal alcohol
syndrome.
1. Medium-chain alcohol dehydrogenase family
This family of enzymes consists of at least four
classes of zinc-dependent cytosolic enzymes: classes I, II,
III, and IV (Adh1, -2, -3, and -4). Both Adh1 and Adh4
dehydrogenases can oxidize retinol to retinaldehyde, but
only the Adh1 enzymes are inhibited by ethanol. In the
adult, ethanol has been shown to inhibit RA synthesis in
several tissues, including testes (289), liver (186), and
cornea (94). Moreover, the alcohol dehydrogenase specific inhibitor 4-methylpyrazole has been shown to inhibit
retinaldehyde synthesis in the testes (289). It is clear,
however, that several retinol dehydrogenases are neither
competitively inhibited by ethanol nor inhibited by
4-methylpyrazole. The alcohol dehydrogenase negative
deermouse lacks all ethanol dehydrogenase activity but
can oxidize retinol normally (226). Because this strain of
deermouse is apparently normal, the ethanol-sensitive
form of retinol dehydrogenase does not seem to be essential for normal development. The house mouse expresses only a single gene of the Adh1 group, and although its human homolog contains a RA response
element, this is not the case in the mouse (292). In the
mouse embryo, Adh1 is distributed in only a few of the
regions known to synthesize RA, such as the developing
kidneys (236, 292), and it is entirely absent from the CNS
(292). It seems thus unlikely that Adh1, the only enzyme in
the mouse competitively inhibited by ethanol, is important for RA synthesis during most of embryogenesis. Another candidate for embryonic retinol dehydrogenase is
Adh4, since it oxidizes retinol to retinaldehyde and is
expressed in several regions of embryonic RA synthesis
(4). In particular, its expression commences at embryonic
day 7.5, the time when RA synthesis begins (236). It is
present in the early CNS and also in the tissue around the
developing eye, both regions of high RA synthesis, as well
as the otic vesicles and migrating neural crest (81). Adh4
is, however, absent from the embryonic retina at all times
and from the entire embryo after embryonic day 10.5 (4);
hence, retinaldehyde for embryonic development from
this stage onward must be synthesized by another enzyme. In addition, null mutations for Adh4 , as well as for
Adh1, develop normally (34), indicating that, even at early
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FIG. 3. The metabolic flow from retinol to retinoic acid and its oxidation products is shown. Note that the oxidation
of retinal to retinoic acid and further oxidation reactions are irreversible.
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RETINOIDS IN EMBRYONAL DEVELOPMENT
developmental stages, redundancy in the retinol dehydrogenases exists.
2. Short-chain dehydrogenase/reductases and
9-cis-RA synthesis
the teratogenic actions of ethanol in the brain are due to
the abnormal induction of RA synthesis (P. J. McCaffery
and D. Ullman, unpublished observations).
C. Retinaldehyde Dehydrogenases
The embryonic enzymes that catalyze the irreversible
oxidation of retinaldehyde to RA are predominantly of the
cytosolic class I aldehyde dehydrogenase family (22, 177).
Although retinaldehyde oxidases exist (163, 232) and a
proportion of rat embryonic RA synthesis is believed to
occur via oxidases (22), our work (175, 194, 206) demonstrates that the retinaldehyde dehydrogenases are almost
totally responsible for the RA distribution observable in
the mouse embryo.
In our search for retinaldehyde dehydrogenases we
initially employed a zymographic technique (178). For this
assay, native tissue extracts are separated by isoelectric
focusing (IEF), and retinaldehyde dehydrogenases are
identified with RA-reporter cells by their capacity to oxidize retinaldehyde to RA in a NAD-dependent reaction.
This bioassay is highly sensitive, allowing the use of very
low substrate concentrations and detection of synthesized RA levels similar to those present in vivo. This
feature enabled the detection of two previously unknown
retinaldehyde dehydrogenases in the embryo, which we
initially termed V1 and V2. The V2 gene is now cloned and
has been renamed RALDH2 (319), while V1 remains to be
characterized at the molecular level. A third enzyme
present in the eye had already been described as the
predominant enzyme for RA synthesis in the adult mouse
liver (133); this third enzyme is referred to as AHD2 in the
mouse, following its gene locus name.
1. AHD2 and V1
The two enzymes AHD2 and V1 create the very high
and spatiotemporally regulated RA levels in the embryonic retina. Apart from the embryonic retina, they are
only expressed in a few restricted locations within the
embryo, i.e., within the CNS. AHD2 is found only in a
subpopulation of dopaminergic cells in the substantia
nigra and adjoining ventral tegmentum, and within the
head, V1 is only expressed in the maxillar regions of the
developing face and the lateral ganglionic eminence of the
brain (unpublished observations). In the retina (see Fig.
4), AHD2 is restricted to a dorsal (D) region, whereas V1
is expressed in the ventral (V) portion (179). AHD2 is
relatively inefficient in synthesizing RA from the low concentrations of retinaldehyde present in the retina. In contrast, V1 shows a much greater efficiency at these retinaldehyde concentrations, and the net result is greater RA
synthesis in the ventral than dorsal retina (179). It would
be expected that the boundary region between the two
enzymes contains an intermediate concentration of RA
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At least five microsomal retinol dehydrogenases of
the short-chain dehydrogenase/reductase are known to be
expressed in the adult (270). These enzymes are typically
resistant to inhibition by 4-methylpyrazole and ethanol.
The type II microsomal dehydrogenase was reported to be
associated with the cytochrome P-450/CYP2D1 oxidase
which, combined with NADPH-P-450 reductase, promotes
retinaldehyde oxidase activity (92). It is possible, then,
that the short-chain dehydrogenase/reductases may be
particularly important for retinaldehyde reduction to retinol, competing with the oxidation of retinaldehyde to
bioactive RA. The embryonic distribution of the five dehydrogenase/reductases is largely unknown. Two recently
identified short-chain dehydrogenase/reductases, however, have interesting properties; they are specific for
cis-retinol isomers and do not catalyze all-trans-retinol
oxidation (185, 233); they are named 9-cis-retinol dehydrogenases. One of the two was found to be broadly
expressed in many regions of the embryo, including the
CNS, eye, ear, and somites (233). The other one is identical to the 11-cis-retinol dehydrogenase required for regeneration of the visual chromophore in the retinal pigment epithelium (56, 256). In addition to the eye, it is
expressed in several other tissues in the adult organism,
including the testes, but its localization in the embryo is
not yet known. The identification of a 9-cis-retinol dehydrogenases implies that 9-cis-RA, the only RA isomer that
activates the RXR receptors, is generated from 9-cis-retinoid precursors, rather than by a RA isomerase from
all-trans-RA. Such a route for 9-cis-RA synthesis is attractive, because a pool of 9-cis-retinol is known to exist, at
least in the adult (124), and because a RA isomerase has
remained elusive (285, 286). If RA isomerization does
occur during normal development, then it may not be
enzymatically catalyzed (285, 286), although tissues are
known to differ in their ability to isomerize all-trans-RA to
9-cis-RA (131). It is also relevant that, so far, Xenopus is
the only species in which 9-cis-RA has been identified in
the embryo (118), although this may reflect lower levels of
the isomer in embryos of other species.
The 9-cis/11-cis-retinol dehydrogenase is similar to
the other short-chain microsomal enzyme in that it is
insensitive to ethanol inhibition (185). An intriguing characteristic of type I, and perhaps other short-chain retinol
dehydrogenases, is that its activity is stimulated by ethanol. This is consistent with the features of the mouse fetal
alcohol syndrome (275, 295), which are more similar to
the effects of a RA excess (252), than to the effects of
vitamin A deficiency (305). We have evidence that some of
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and that a ventral-to-dorsal gradient extends across the
entire retina. Instead, between the two RA-synthesizing
enzymes lies a horizontal stripe of the RA catabolic enzyme CYP26 (181). This arrangement creates three regions along the dorsoventral axis of the embryonic retina,
a dorsal zone of intermediate RA concentration, an intervening region of very low RA and a ventral zone of high
RA concentration (Fig. 4). Gradients of RA concentration
may exist within each region. This is the first description
of a mechanism by which zones of RA concentration can
be created. Inhibition of RA synthesis by citral in zebrafish blocks the development of the ventral half of the
retina in similar fashion to the effects of null mutations of
the RA receptors (98, 140, 167). Thus the ventral retina is
a zone distinguished by its expression of the V1 RALDH,
by its high RA synthesis, and by its dependence on RA for
development. The dorsal, medial, and ventral territories
of differing RA levels are likely to regulate transcription of
some of the genes with differential expression patterns
along the dorsoventral axis of the retina. Such genes
include the tyrosine receptor kinase EphB2 and its ligand
ephrin B2, which are believed to influence the target
selectivity of outgrowing retinal axons (164), and the
green and blue cone photoreceptors in the mouse (276).
The ligand ephrin B2 and the green cones are restricted to
the dorsal retina, and the receptor EphB2 and the blue
cones are located predominantly in the ventral retina. It is
likely that other regions of the embryo contain a similar
zonal patterning of RA, created by the juxtapositioning of
RA synthetic and catabolic enzymes.
Another intriguing property of the class I aldehyde
dehydrogenases, in addition to their enzymatic function,
is their affinity for several ligands of the nuclear receptor
family. The promoter of the human ALDH1 gene contains
a putative response elements for androgen (313), and the
enzyme has been demonstrated to bind androgen (218).
The corresponding cytosolic aldehyde dehydrogenase in
Xenopus has been identified as the major thyroid-hormone binding protein in this species (312). We found that
AHD2 in the embryonic dorsal retina and substantia nigra
is present in amounts far exceeding the concentrations of
the other retinaldehyde dehydrogenases (180). This suggests this enzyme may have a dual function, both synthesizing RA and also modulating the local concentration of
other nuclear receptor ligands that can influence RA signaling in the embryonic retina.
2. RALDH2
The retinaldehyde dehydrogenase RALDH2 exhibits
the greatest substrate specificity of the three dehydrogenases, and its distribution provides the most accurate
guide to the localization of all-trans-RA in the embryo.
RALDH2 is essential for normal development, and its
knockout results in a complete failure of embryo survival
and early morphogenesis (207). The trunk of the embryo
is severely shortened, and the neural tube remains open,
whereas the anterior region is less affected. Unfortunately, the embryos die before the effect on more differentiated structures can be observed. Studies on these
structures await the creation of conditional knockouts.
A guide to the pattern of all-trans-RA signaling in the
embryo is provided by the RA-reporter mice, a strain
transgenic for ␤-galactosidase controlled by a sensitive
RA response element (RARE-lacZ) (27, 236). The expression of ␤-galactosidase in a particular cell is the result of
the balance of RA synthesis and catabolism in that region,
as well as the capacity of the cell to respond to RA
depending on its expression of RA receptors, coactivators, and corepressors. Given the number of factors on
which ␤-galactosidase expression depends, it is surprising
that the distribution of RALDH2 (206) matches quite well
with the distribution of ␤-galactosidase in the RA reporter
mice (236), including expression in the spinal cord, motoneurons, and developing eye. When examined in greater
detail, there was a very good correlation, for instance, in
the embryonic heart (194). This suggests that a significant
proportion of the patterning of RA signaling in the embryo
may rely on localized RA synthesis. Regions do exist,
however, in which ␤-galactosidase distribution does not
correlate with RALDH2 expression, and this includes the
RARE-lacZ induction in the telencephalic vesicles and
nasolachrimal groove. This is likely to be the result of RA
synthesis by other RALDH. Analysis of RA distribution by
HPLC also indicates a pattern similar to that of RALDH2
(90). With the exceptions of the restricted regions of V1
and AHD2 expression, which include the retina, face, and
the striatum (176), RALDH2 appears to be responsible for
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FIG. 4. The distribution of retinoic acid (RA) in the embryonic
mouse retina. The AHD-2 retinaldehyde dehydrogenase is specific to the
dorsal (top) region and is distributed as a gradient from dorsal tip to the
boundary of the catabolic enzyme CYP26, creating a gradient of RA in
this orientation. The boundary stripe of CYP26 degrades RA creating a
zone of very low RA concentration. Below CYP26 the “V1” retinaldehyde
dehydrogenase is expressed, possibly in a gradient from ventral (bottom) tip to the CYP26 boundary creating a second RA gradient in this
orientation. The overall amount of RA is greater in the ventral half
compared with the dorsal half.
Volume 80
July 2000
RETINOIDS IN EMBRYONAL DEVELOPMENT
D. Synthesis of Other Bioactive Retinoids
in the Embryo
1. Didehydro-RA
Although RA is the predominant active retinoid in the
embryonic mouse, this is not the case in several other
vertebrate classes. In the chick, didehydro-RA was found
to be the principal active retinoid in the developing limb
bud (278), as well as in the developing spinal cord and the
somites (156). Moreover, the likely precursor didehydroretinol has been detected in the embryonic chick. The
distribution of didehydro-RA in the chick embryo is similar to the distribution of all-trans-RA in the mouse. Furthermore, the patterns of RALDH2 expression in both
mouse (206) and chick (10) are similar. It is thus likely
that RALDH2 will oxidize didehydro-retinaldehyde to didehydro-RA, although this has still to be demonstrated
directly. Didehydro-RA is also present in both Xenopus
and zebrafish (29, 30) but has yet to be detected in mouse
(90).
2. 4-Oxo-RA, 4-oxo-retinaldehyde, and 4-oxo-retinol
In the embryonic Xenopus (223), all-trans-4-oxo-RA
influences the development of the anteroposterior body
axis in a fashion that is unresponsive to all-trans-RA. Both
retinoids act through RA receptors, and it is assumed that
they activate differing configurations of receptors. Similarly, the 9-cis-isomer of 4-oxo-RA differs from 9-cis-RA in
receptor selectivity, since it is able to specifically activate
RAR-RXR heterodimers and not RXR-RXR homodimers
(222). Presumably, both isomers of 4-oxo-RA can be synthesized from their respective RA isomers via a cytochrome P-450-linked oxidase, such as CYP26. Another
potential source of 4-oxo-RA is 4-oxo-retinaldehyde, and
this retinoid has been identified in early Xenopus embryos
(12). Surprisingly, 4-oxo-retinaldehyde itself can activate
the RAR, and it is the major bioactive retinoid in Xenopus
(12). Another transactivator of RAR is 4-oxo-retinol, a
compound present in both Xenopus and the mouse F9
teratocarcinoma cell line (12). The existence of such active ketone derivatives of the retinoids suggests the potential importance of cytochrome P-450-linked oxidases
in activating retinoids, in addition to their role in retinoid
catabolism (see sect. IVF).
E. Influence of Substrate Concentration
and Distribution
In embryonic mouse (90) and human (119), retinol is
present at micromolar concentrations, whereas retinaldehyde levels are barely detectable. This contrasts with
several cold-blooded organisms including zebrafish (29)
and Xenopus (7), in which high levels of retinaldehyde are
present, bound via the labile Schiff-base linkage to vitellogenin. High retinaldehyde concentrations in the zebrafish embryo place even greater emphasis on the retinaldehyde dehydrogenases as the determinant of RA
synthesis (29). The substrates all-trans-retinaldehyde, alltrans-retinol, and didehydro-retinol all appear to be localized to specific regions of the early Xenopus embryo
(118), a distribution that is likely to influence the distribution of RA and didehydro-RA. We have found this to be
the case for the retinaldehyde distribution in the zebrafish
trunk (170). The distribution of the retinyl esters has not
been investigated in the developing embryo, but both
retinyl acetate and retinyl palmitate have been identified
in cultured cells from the chick retina (266).
F. RA Catabolism
A number of pathways of RA catabolism have been
described, which may be tissue or species specific (6, 70,
113, 118, 254). These routes include oxidation, isomerization, and formation of glucuronides and taurine conjugates. Recently, a new member of the cytochrome P-450
family, CYP26 (or P-450RAI), has been identified as an
enzyme in the embryo that specifically mediates RA oxidation (68), and it is presumed to mediate RA catabolism.
CYP26 was first identified in the adult zebrafish as a
RA-inducible catabolic enzyme (301) and was later cloned
in mouse and human cells by several laboratories (230,
300). Its predominant metabolites have been described as
either 4-hydroxy-RA, 18-oxo-RA, and 4-oxo-RA (230, 300),
or as 5,8-epoxy-RA (68). The CYP26 promoter contains a
RA response element; however, RA exposure of the
mouse embryo induces expression only in a limited region
(68), indicating the existence of other transcriptional regulators for this gene. Its normal expression pattern in the
embryo is suggestive of a function of regulating RA distribution. In the mouse (68), at embryonic day 7.25, it is
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retinaldehyde oxidation in most other regions of early
embryonic all-trans-RA synthesis. Within the embryonic
CNS, the limb motor neurons in the spinal cord (175, 260,
319) represent major sites of RALDH2 expression,
whereas most of the remaining CNS shows lower RA
synthesis. It is of note, however, that the meninges surrounding the brain contain extremely high levels of
RALDH2, and this potent source of RA may be important
in brain development and plasticity (unpublished observations). For the developing cerebellar region, the meninges are likely to represent a major source of RA required
for the differentiation of granule cells in the adjacent
external granular layer. RALDH2 does not discriminate
between all-trans- and 9-cis-retinaldehyde (M. Warren,
personal communication); hence, this enzyme does not
appear to influence the all-trans- and 9-cis-RA ratios.
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ROSS, McCAFFERY, DRAGER, AND DE LUCA
G. Retinoid Binding Proteins
The retinoid binding proteins are likely to play a role
in the regulation of retinoid metabolism, and they are
widely expressed in the developing embryo (40, 51, 148,
219, 241, 242). Generally, they act as cytoplasmic carriers
for the lipophilic retinoids, which they shuttle between
both subcellular compartments and metabolic enzymes.
They are not, however, absolutely essential for the embryo, because the loss of function of either CRBP-I or
CRABP-I and -II has little effect on normal development
(127). It has been suggested that their importance may
become more evident under circumstances of low dietary
vitamin A supply. It is also likely that alternative retinoid
binding proteins exist (310).
CRBP-I is believed to promote RA synthesis by presenting retinol to the retinol dehydrogenase (200). After
oxidation to retinaldehyde, this substrate can again bind
to CRBP and is available to the retinaldehyde dehydrogenase (216). Apo-CRBP-I is also known to promote retinyl
ester hydrolysis, leading to the release of retinol (200),
which is then accessible to the retinaldehyde dehydrogenases.
CRABP-I is thought to promote the breakdown of RA
(14) because the catabolic enzyme prefers holo-CRABP-I
as a substrate (64). The embryonic distributions of
CRABP-I is generally complementary to that of CRBP-I,
possibly reflecting its contrasting function. Embryonic
regions sensitive to the teratogenic effects of retinoids,
including the developing neural crest and hindbrain, express high levels of CRABP-I, which led to the suggestion
that CRABP-I serves a protective role in RA vulnerable
tissues (40, 242, 287), consistent with a role in RA buffering and catabolism. In contrast to CRABP-I, CRABP-II has
been shown to be associated with cells that synthesize RA
in the adult uterus and testes, and it may play a role in RA
synthesis or secretion (16, 320).
H. Hypothesis: CRABP Captures RA for Cells That
Lack RA Synthesis
RA can function as an embryonic morphogen when
synthesized by discrete organizer regions to create gradients of transcriptional activation. This is likely to occur in
several regions of the embryo including the early eye
anlage (55). The observations reviewed here demonstrate
another mode of patterning in the form of zones of differing RA concentration. These territories are created by
the ordered patterning of RA synthetic and catabolic enzymes in the embryo, and they have been observed in
several regions during development, including the early
heart (194) and the retina at later stages of embryonic
development (181). A sharp drop off in RA levels occurs
also between the spinal cord and the hindbrain in the
embryonic mouse (175) and chick (156), and it was noted
that gradients are not evident in the RA reporter mice
(236). It appears that the zonal patterning of RA levels
may be an important form of organization in addition to
RA gradients. Several regions of retinaldehyde dehydrogenase expression show exceptionally high levels of enzyme activity, including the spinal cord and the retina (54,
175). The resulting RA concentration in the ventral retina
probably exceeds 1 ␮M, far beyond the concentrations
necessary for the activation of the RA receptors. Because
the ventral retina is bounded by the catabolic enzyme, this
high concentration cannot serve to create a continuous
RA gradient across the entire retina. One possible explanation for the high retinaldehyde dehydrogenase levels in
neurons may relate to the observations that high enzyme
levels are not restricted to the cell bodies but extend also
into the axons. Very high AHD2 concentrations are
present in the retinal axons (180) and in the dopaminergic
axons innervating the corpus striatum (176), and RALDH2
is found in axons of the motoneurons projecting from the
embryonic spinal cord (10) Axons from the retina, the
ventral tegmentum, and the spinal cord innervate regions
that do not contain synthetic enzyme but do express RA
receptors. If the axon terminals represent sources of RA,
then high enzyme activities would be required in the soma
region, to provide amounts for the transport through the
thin axons which are sufficient for target activation. Territories of high RA may also represent RA sources for
neurons whose processes make contact or pass through
these regions, because RA diffuses readily through cell
membranes. Fibers innervating the spinal cord motoneurons, or passing by in their vicinity, are exposed to the
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present in a posterior to anterior gradient in the mesoderm. High expression at the posterior end continues
through embryonic day 8.5 to be present in the posterior
neural plate and mesoderm of the hindgut and tailbud at
embryonic days 9.5–10.5. Localized areas of anterior expression also exist, with high levels in the neural crest
cells of the hindbrain which migrate to the cranial ganglia.
In the CNS, expression is also present in the otic vesicle
and eye (68). As described above, the presence of CYP26
in the retina is particularly revealing since it sits as a
midline stripe subdividing the two RA synthetic enzymes
in the dorsal and ventral (181). This suggests a possible
role for CYP26 in boundary formation, acting as a perimeter between regions of high RA synthesis. A function, for
CYP26, of boundary delineation in the Xenopus hindbrain
is also suggested by Hollemann et al. (88). The existence
of catabolic enzymes in the mouse hindbrain in addition
to CYP26 has also been reported (309). It is likely that the
patterning of the catabolic enzymes will be just as important as that of the synthetic enzymes for determining the
sites of RA’s action.
Volume 80
July 2000
RETINOIDS IN EMBRYONAL DEVELOPMENT
V. RETINOID RECEPTORS/FUNCTION
A. Introduction
The cloning and characterization of the retinoid receptors represent a landmark in our understanding of the
physiology of the retinoids. The discovery was a direct
consequence of basic work in the field of steroid and
thyroid hormone receptors. A characteristic of these receptors is their modular structure with six different domains (A to F) that fulfill different functions (21, 160). The
DNA-binding domain or DBD, also known as the C domain, is very highly conserved between these different
receptors. Retinoid receptor cloning was possible because of the common 24-nucleotide probe within this C
region used to identify new orphan receptors (71, 221).
These approaches permitted the characterization of three
genes (␣, ␤, and ␥) for RAR and three genes for RXR
(159). The latter class of receptors specifically bind 9-cisRA, whereas RAR bind both RA and 9-cis-RA (35, 137,
159).
Genes containing retinoic acid response elements
(RARE) in their promoters are known to be involved in
diverse and yet interconnected biological processes, such as
embryogenesis, growth, and differentiation (35, 160, 161).
The complexity of interactions becomes greater if one considers that RXR, in addition to forming homodimers (159),
also form heterodimers with other receptors (135, 159, 166,
314), including RAR, the thyroid hormone receptor TR, the
1,25-dihydroxy-vitamin D3 receptor VDR (314), the peroxisomal proliferator activated receptor PPAR (105, 108, 282),
NGFI-B, NURR1 (220), and COUP-TF (106). A distinctive
characteristic of the RXR is their ability to interact with
other nuclear receptors of the superfamily resulting in their
binding to their respective DNA response elements in the
promoters of different target genes (107). Gene transcription
responses are RXR partner receptor dependent as well as
dependent on the presence of the ligand of their heterodimeric partner, e.g., vitamin D3 for the VDR/RXR combination, thyroid hormone for the TR/RXR combination, and
so on. It is therefore easy to conceptualize how RXR may
control a complex network of hormone-dependent pathways. Complexity increases if one also considers that there
are some 18 isoforms of the RAR and probably just as many
RXR formed through the use of different promoters or alternative splicing (134).
In addition to the activation function AF-1 in the
NH2-terminal A/B regions of the receptors, the use of
various reporter gene assays has made it possible to
identify a transcriptional activation function (AF-2),
which overlaps the ligand binding domain (LBD) in the E
region of the RAR and RXR. RAR AF-2 are activated
similarly by all-trans- and 9-cis-RA and their 3,4-didehydroderivatives (Fig. 1), whereas RXR AF-2 are only efficiently activated by 9-cis-RA and by 9-cis-3,4-ddRA (see
Ref. 134 and references therein).
A third level of complexity accrues from interactive
elements on different promoters. For instance, response
elements containing direct repeats (DR) of the canonical
sequence AGGTCA with a spacer of two nucleotides
(DR2) or five (DR5) are known to occur in the homeobox
b1 (169, 269) and RAR␤2 genes. In addition, the actual
sequence of the direct repeats and the types of flanking
bases appear to be important determinants for RAR and
RXR binding efficiencies (157). Moreover, interactions
with other proteins, which may either function as transcriptional repressors (73, 89, 122, 316) or ligand-dependent activators (20, 23, 97), such as the cAMP response
element binding protein (CBP), have also been reported
and may add another level of complexity as well as permit
a variety of interactive pathways to regulate specific gene
expression.
B. Speculations on Orders
of Receptor Interactions
These considerations suggest different orders of interactions that may lead to diverse biological outcomes.
We suggest that effects of interactions between RXR-RAR
heterodimers and target gene promoters are the most
immediate and would be the first to respond to retinoid
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very high RA levels present here. Cells of the neural crest,
which require RA (151) but do not express RALDH2 (10),
migrate through mesenchyme which expresses high levels of RALDH2 (206). Such exposure may be a necessary
part of their maturation. Cell types that do not express
retinaldehyde dehydrogenases but that respond to external sources of RA may have to be efficient at RA capture.
This may be a function of CRABP-I, since it is almost
exclusively expressed in cells that respond to RA but that
do not themselves synthesize RA. Examples include the
neural crest (104), cerebellum (311), and interneurons of
the spinal cord (253). The normal response of these tissues to low concentrations of RA might explain why these
regions are so sensitive to RA teratogenicity (9, 287); the
exposure to abnormally high RA levels will disturb their
normal sequence of gene expression. The axons of several
neurons are known to express CRABP-I (147, 154, 253),
and the binding proteins may assist in the capture of RA,
allowing for its transport back to the cell body. The
polarization of neurons into soma and far-reaching axonal
projections makes it likely that axonal transport plays a
role in the localized capture and release of RA. There can
be little doubt that the developing CNS will be an interesting area of investigation for the interactions between
retinoid metabolism, signaling, and patterning of gene
expression.
1031
1032
TABLE
ROSS, McCAFFERY, DRAGER, AND DE LUCA
Volume 80
1. Natural retinoic acid response elements
1) mRAR␣2
2) hRAR␣2
3) mRAR␤2
4) hRAR␤2
5) hRAR␥2
6) hADH3
7) mCRBPI
8) mLamBI
9) hapoAI
10) mCP-H
11) rCRBPII
12) hMCAD
5⬘-(-59)GGCGAGTTCAGCAAGAGTTCAGCCGA(-34)-3⬘ (136)
5⬘-(-58)GGCGAGTTCAGCGAGAGTTCAGCCGC(-33)-3⬘ (174)
5⬘-(-57)GAAGGGTTCACCGAAAGTTCACTCGC(-32)-3⬘ (273)
5⬘-(-57)GTAGGGTTCACCGAAAGTTCACTCGC(-32)-3⬘ (45, 86)
5⬘-(401)GGCCGGGTCAGGAGGAGGTGAGCGCGC(-375)-3⬘ (174)
5⬘-(-280)ACAGGGGTCATTCAGAGTTCAGTTTT(-305)-3⬘ (59)
5⬘-(-1015)TAGTAGGTCAAAAGGTCAGACAC(-993)-3⬘ (259)
5⬘-(-432)GAGGTGAGCTAGGTTAAGCCCTTAGAA AAAGGGTCAA(-468)-3⬘ (290, 291)
5⬘-(-192)AGGGCAGGGGTCAAGGGTTCAGTGGG(-217)-3⬘ (238)
5⬘-(-147)CAGCAGGTCACTGACAGGGCATAGTA(-122)-3⬘ (196)
5⬘-(-639)GCTGTCACAGGTCACAGGTCAC AGGTCACAGTTCA(-605)-3⬘ (162)
5⬘-(-341)GGGTTTGACCTTTCTCTCC GGGTAAAGGTGAAGG(-308)-3⬘ 3⬘CCCAAACTGGAAAGAGAGGCCCATTTCCACTTCC-5⬘ (228)
33333
33333
5⬘-共-351兲AAGTGTCACAGGTCAAGGTAACCCAC共-326兲 共132兲
TTCACAGTGTCCAGTTCCATTGGGTG
ERE
4444444444
14) mCRABPII
RARE1
RARE2
15) rPEPCK
RARE1
RARE2
16) mTGase (mTGRRE1)
17) mHox1.6
18) mHoxb-1
19) mHoxb-1
20) mSTAT1
21) rDopamine D2 receptor
5⬘-(-1162)CCCCAGTTCACCAGGTCAGGGCT(-1140)-3⬘ (61)
5⬘-(-657)CTGTGACCTCTGCCCTTCT(-639)-3⬘ (61)
5⬘-(-451)TGACCTTTGGCCGTGGGA(-434)-3⬘ (143)
5⬘-(-351)GATCCGTCCCGGCCAGCCCTGTCCT TGACCCCCACCTGACAATTAAGGCAAGAGCCT(-295)-3 (244, 274)
5⬘-(-1703)CATGGGGGTCACTGTGAGAGGTCCCAG TGGGGTCAGGATTA(-1751)-3⬘ antisense (199)
3⬘-enhancer 5⬘-(Sac-74)CAGGTTCACCGAAAGTTCAAG(Sac-55)-3⬘ (128)
3⬘-enhancer 5⬘-CTTAGAGGTAAAAAGGTCAGCCCAG-3⬘ (169, 269)
5⬘-repressor 5⬘-AGGGCAAGAGTTCA-3⬘ (169, 269)
5⬘-(-274)TCGAGATGGGTCAGGGTGATAAC(-252)-3⬘ (114)
5⬘-(-71)CCTCG TGGCCAGGGTGACCCC(-51)-3⬘ (288)
Consensus
G
5⬘-
T
G
A
1
2
A
T
G
3
4
C
5
A
6
共X兲
T
G
G
1
2
G
3
T
C
A-3⬘
4
5
6
Shown are the natural retinoic acid response elements (RARE) from the promoters of the following genes: 1) mouse (m) RAR␣2 (136); 2)
human (h) RAR␣2 (174); 3) mRAR␤2 (273); 4) hRAR␤2 (45, 86); 5) hRAR␥2 (174); 6) human alcohol dehydrogenase 3 (ADH3) (59); 7) mouse cellular
retinol binding protein I (mCRBPI) (259); 8) mouse laminin BI (mLB1) (290, 291); 9) human apolipoprotein AI (238); 10) complement factor H
(mCP-H) (196); 11) rat cellular retinol binding protein II (rCRBPII) (162); 12) putative RARE of the human medium chain acyl-CoA dehydrogenase
(hMCAD) (228); 13) composite element containing an RARE (RXRE) and ERE from the human lactoferrin promoter (132); 14) two RARE from the
mouse cellular retinoic acid binding protein II (mCRABPII) (61); 15) two RARE from the rat phosphoenolpyruvate carboxykinase (rPEPCK) (143,
274); 16) mouse transglutaminase type II (mTGRRE1) (199), which contains three hexad repeats (DR5 and DR7 motif) capable to bind both RXR
homodimers as well as RXR-RAR heterodimers; 17) repeated hexad motifs in the 3⬘-enhancer of the mouse Hox1.6 (mHox1.6) (128); 18) mouse
Hoxb1 (169, 269); 19) mouse Hoxb1 5⬘-repressor (169, 269); 20) mouse STAT1 (114); and 21) rat dopamine D2 receptor (288). Key motifs (157) are
highlighted. Reference numbers are given in parentheses.
depletion or excess. These reactions would acquire the
characteristic immediate response, if the RARE is located
in the promoter of the target gene itself. This is the case
for the RAR␤2 gene, which is therefore a “first-order
dependence gene.” Other first-order dependence genes
would be the RAR␥2 and ␣2 and all the genes that contain
a RARE in their promoters. Some of these are listed in
Table 1.
First-order dependence would also be observed with
ligands activating RAR-RAR or RXR-RXR homodimers,
such as for 9-cis-RA in the activation of the CRBPII gene
(Table 1). RXR cognate receptors other than RAR, such as
VDR, TR (17, 107, 166, 318), COUP-TF (106), and PPAR
(282), would mediate second-order types of retinoid responses, in that their responses, although possibly depen-
dent on 9-cis-RA (188), are also dependent on other hormonal ligands such as vitamin D3, thyroxine (314), as well
as xenobiotic agents.
We suggest that genes belonging to the “second-order
retinoid dependence” are probably much more numerous
than those of first-order dependence. These genes control
most gene activation processes dependent on thyroid hormone, vitamin D, and other important hormonal and xenobiotic ligands, which also depend on heterodimer formation with RXR. We call “third-order retinoid-dependent
genes” all those genes that would be activated as the
result of secondary transcriptional events. An example of
this type of dependence is genes that depend on AP-1
complexes. It is well known that transcriptional transrepression occurs at the AP-1 site concomitant with RA-
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13) hLactoferrin composite
element
RARE
July 2000
RETINOIDS IN EMBRYONAL DEVELOPMENT
C. Receptors and Embryogenesis
Data from in situ hybridization studies show that
different RAR and RXR subtypes are widely expressed
during embryogenesis and that each subtype has its own
individual expression patterns that may or may not overlap with the other subtypes. Each retinoid receptor subtype can give rise to different isoforms, each with their
own unique expression patterns during embryogenesis
(62). Retinoid nuclear receptors of different types appear
to be expressed along the entire anterior/posterior (A/P)
axis in the CNS. The data suggest that individual receptor
subtypes have specific and probably multiple functions.
The RAR␣ subtype is expressed in a general fashion during murine embryogenesis, whereas RAR␤ and RAR␥ are
more restricted (52, 183, 239 –241). In the hindbrain,
RAR␤ has a rostral limit of expression at the rhombomere
6/7 boundary (149), and RAR␥ is expressed in the open
neural tube before the fusion of neural folds (239). Among
the RXR, murine RXR␤ is expressed in a general fashion,
but RXR␣ and RXR␥ have more restricted patterns during
embryogenesis (48, 158).
The patterned expression or suppression of developmental genes (e.g., the homeobox b1 gene) has been
demonstrated to be strictly retinoid responsive, in specific
rhombomeres of the mouse hindbrain (168). In fact,
RARE have been demonstrated both 5⬘ as well as 3⬘ of the
gene with the 5⬘-RARE functioning as a suppressor and
the 3⬘-RARE as an inducer of transcriptional activity for
the homeobox b1 gene (38, 168, 269). The developmental
time dependency of the function of the two RARE permits
and controls the switching on and off of the homeobox
␤1-gene expression and termination of expression at the
border of different rhombomeres, possibly controlling
segment identity. It is also evident that a highly ordered
retinoid delivery system must be operative to ensure the
regulated series of developmental events. Availability of
excess retinoids or their deficiency may lead to teratogenesis and/or resorption of the embryo.
VI. RETINOID RECEPTOR KNOCKOUT
MUTANTS
A. Isoform-Specific Knockouts
Much effort has been applied over the past few years
to the identification of the specific functions of the different retinoid receptors during embryogenesis. One strategy has been to evaluate the phenotype of mice lacking
the gene for a retinoid receptor or receptor subtype.
Although the different receptor isoforms have unique distributions during development, knocking out one specific
isoform revealed a surprising redundancy between members of each receptor subtype. Mice homozygous for either RAR␣1, RAR␤2, or RAR␥2 mutation were viable and
did not display phenotypic abnormalities (99). Since these
initial studies, a stepwise progression of mutant mice
from single isoform retinoid receptor mutants to RAR
subtype mutants to RAR/RXR compound mutant mice
have been created to examine the in vivo function of these
receptors (Table 2).
B. Single Subtype Knockouts
Developmental malformations can arise as a result of
knocking out the entire gene of a single retinoid receptor.
For example, homozygous RAR␣ mutant mice, which express no RAR␣ isoform transcripts, exhibited early postnatal lethality and testis degeneration (145). Of the RAR
single-type null mutations, RAR␥ knockout mice showed
malformations (138). RAR␥ mutant mice exhibited several abnormalities previously associated with vitamin A
deficiency, including homeotic transformations of the cervical vertebra and occipital region of the skull, fusion of
the first and second ribs, and irregularities of the tracheal
rings, among others (138).
Because of their interaction with several members of
the steroid/thyroid hormone receptor superfamily, the ab-
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mediated gene activation on various RARE, especially
when RA is in excess. Genes containing 12-O-tetradecanoylphorbol-13-acetate (TPA) responsive elements
(TRE), such as the stromelysin or the collagenase genes,
are negatively regulated by RA. This negative regulation
was thought to be due to binding of the AP-1 Jun-Fos
protein to the RAR, thus removing them from interactions
with the TRE. More recent results indicate that transrepression is the result of limiting concentrations of the
cAMP response element binding protein (CREB) and the
protein that binds to CREB or CBP. This CBP apparently
binds with high affinity to CREB as well as to RAR,
estrogen receptor (ER), and possibly a variety of other
receptors (97). Whereas the binding and transcriptional
activation by the CREB homodimer is independent of
ligands, CBP binding to RAR and other hormone receptors and consequent transcriptional activation is ligand
dependent (97). Because CBP is present in limiting
amounts, AP-1 trans-repression normally occurs when
RAR or the ER concentrations are increased. When CBP
is nonlimiting, trans-repression is not observed. Therefore, the concentration of CBP and of other liganddependent coactivators, as well as that of corepressors,
add another level of transcriptional control. Recent reviews on the important aspects of structural configuration
and space-filling models (159) of the retinoid receptors
and on the genetic knockout (99) of their genes have
recently been published.
1033
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TABLE
ROSS, McCAFFERY, DRAGER, AND DE LUCA
Volume 80
2. Comparison of RAD and retinoid receptor null mutant embryos
RAR␣/␥
RAR␣/␤2
RAR␤2/␥(2)
RXR␣
RAR␣/RXR␣
RAR␥/RXR␣
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹a
⫹b
NR
NR
⫹c
⫺
⫹
⫹
⫹
⫺
⫺
⫺
⫺
NR
NR
⫺
⫺
⫺
⫹
⫺
⫹
NR
⫺
⫺
NR
⫹
⫹c
⫺
⫹d
⫺
⫺
⫺
NR
⫹a
⫺
NR
NR
⫺
⫺
⫹
⫹
⫺
⫺
NR
NRa
⫺
NR
NR
⫾c
⫺
⫺
⫹
⫺
⫺
NR
⫹a
⫺
NR
NR
⫾c
⫺
⫹
⫹
⫺
⫺
NR
⫹
⫹
⫾
⫹
⫹g
⫹e
⫹
⫺
⫹f
⫹h
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
NR
⫺
⫺
NR
⫺
NR
⫺
⫺
NR
⫺
NR
⫺
⫺
NR
⫺
a
b
c
NR, not reported or assessed.
Also smaller ventral retina.
Not detailed; one instance of absent lens.
⫾ indicates retinal
d
e
f
g
eversion, no obvious gap.
Poorly differentiated.
Open rhombencephalon.
Motor nuclei of CNVI only.
Thinning of marginal
h
layer.
Small cerebral hemispheres, collapsed ventricles. [Data from Smith and co-workers (47, 258), Grondona et al. (75a), Kastner et al. (98),
Lohnes and co-workers (139, 140), Mendelsohn et al. (183), and Sucov et al. (271).]
sence of a RXR would be expected to have more serious
consequences during embryogenesis. One such gene
knockout, RXR␤, resulted in morphologically normal
mice, with the exception that males were sterile (101).
Homozygous RXR␣ mutant mice died between gestation
days 13.5 and 16.5 (271). The embryonic lethality in
RXR␣-deficient mice was due to hypoplastic development
of the ventricular chambers of the heart, which resulted in
a very thin ventricular wall and defects in ventricular
septation. RXR␣ null mutants showed abnormalities of
the eye (e.g., shortening of the ventral retina) as also
observed in vitamin A deficiency (Table 2).
None of the phenotypes resulting from single knockouts, however, was expected on the basis of expression
patterns of retinoid nuclear receptors during embryogenesis. Although some specific aberrations have been found
to be associated with the lack of certain nuclear receptor
subtypes, these studies seem to suggest a high degree of
functional redundancy among these receptors. The results of single RAR and RXR null mutants suggest that
embryogenesis is well protected by a system that apparently allows most or all functions of an absent receptor to
be substituted by another.
C. Double/Compound Mutants
1. RAR
Unlike RAR single mutants, RAR double or compound mutants died either in utero or shortly after birth
(139, 140, 146, 184). Histological and anatomical analyses
of these transgenic mice revealed many defects charac-
teristic of fetal vitamin A deficiency (Table 2). Multiple
eye abnormalities were found in various RAR double
mutant fetuses that were similar to those previously seen
in vitamin A-deficient fetuses. A majority of these abnormalities recapitulated those observed in the fetal vitamin
A-deficient syndrome, initially described more than 40
years ago (Table 2) (305). A number of additional malformations not described in vitamin A deficiency studies,
however, were also observed (140, 184). These findings
probably reflect the difficulty of achieving severe vitamin
A deficiency by dietary deprivation (99). Taken collectively, these results demonstrate that RAR are essential
for vertebrate development and that, most likely, retinoic
acid is the active retinoid, required at several stages of the
development of numerous tissues and organs (Table 2). In
contrast to the apparent necessary role for RAR in transducing the RA signal, CRABPI and CRABPII are probably
not critical to these processes, since CRABPI/CRABPII
double null mutant mice were found viable and morphologically normal (127).
2. RXR
Double or triple mutations of RXR would be expected to reproduce the defects associated with inactivations of RAR/RXR heterodimers or, at least, produce defects similar to the RAR double mutant defects. Strikingly,
RXR␤ ⫺/⫺ RXR␥ ⫺/⫺ double and RXR␣ ⫹/⫺ RXR␤ ⫺/⫺
RXR␥ ⫺/⫺ triple mutant phenotypes were viable, displaying no obvious congenital or even postnatal abnormalities, except a marked growth deficiency and male sterility
due to loss of function of RXR␤ (121). Therefore, it ap-
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
Eye
Microphthalmia
Lens elongation/fiber anomaly
Lens cell apoptosis
Neural retina apoptosis
Intraretinal gap
Absent choroid fissure
Thickened precorneal stroma
Heart defects
Abnormal limb shape
Absent tracheal-esophageal septum
Absent cranial flexure
Head and nervous system
Hindbrain defects
Frontonasal hypoplasia, cell death
Mandibular hypoplasia, cell death
Cranial nerve hypoplasia, absence
Brain hypoplasia
RAD
July 2000
RETINOIDS IN EMBRYONAL DEVELOPMENT
pears that one copy of RXR␣ is sufficient to perform most
of the functions of the RXR.
3. RAR/RXR double mutants
D. Retinoid Receptor Mutants
and Limb Malformations
Mouse embryos lacking RXR␣ have normal limbs and
display resistance to limb malformations normally induced by teratogenic RA exposure (272). RA treatments
that cause limb defects in 100% of wild-type embryos
failed to elicit malformations in RXR␣ null homozygotes,
implicating RXR␣ as a component in the teratogenic process in the limbs. Furthermore, heterozygous embryos are
intermediate in their sensitivity to RA, suggesting the
importance of RXR␣ gene dosage in limb teratogenesis.
These investigators, however, found that expression of
the RA-inducible gene RAR␤2 was equivalent between
wild-type and homozygous RXR␣ embryos after RA treatment. The spatial expression of sonic hedgehog (Shh) and
Hoxd12 was also similar for both wild-type and RXR␣
embryos, following RA treatment. Hoxd12 expression,
however, was elevated in RXR␣ embryos. These observations indicate that transcriptional processes, which are
inappropriately influenced in the mouse limb by exogenous RA, require RXR␣ for their execution.
Results from double mutant mice provide additional
information for the role of RA in limb patterning (100).
Because both RAR␣ and RAR␥ transcripts are uniformly
expressed in the early stage mouse limb bud (51), it was
surprising that neither RAR␣ nor RAR␥ single knockout
mice produced limb malformations (138, 145). These observations, however, suggested a functional redundancy
between these two receptors. This may well be the case
since limbs from RAR␣/RAR␥ double mutants consistently exhibited malformations, including size reduction
of the scapula, perforated scapula, radius agenesis, and
abnormal digit number (140). Many of these limb defects
appear to be fairly restricted to the forelimb skeleton and
may reflect a requirement of RA to generate the proper
amount of limb mesenchyme, since a deficit of mesenchyme leads to preferential loss of anterior skeletal elements in frog hindlimbs and preferential loss of posterior
skeletal elements in salamander hindlimbs (3). The defects do not appear to result from an early zone of polarizing activity (ZPA) defect because limbs displayed a clear
A/P asymmetry. The investigators suggest this does not
exclude a role for RA in normal A/P limb patterning, since
RAR␤ transcripts are expressed in a region that overlaps
with the ZPA and appear to be unaffected in the limbs of
RAR␣/RAR␥ double mutants (140). The observations
from the limb defects in RAR␣/RAR␥ double mutants
suggest that either RA plays different roles in forelimb
and hindlimb development, or the observation is related
to events occurring at different developmental time periods for the two limbs. It has also been suggested that
inactivation of all three RAR might result in more dramatic effects on limb patterning (99).
E. Retinoid Receptor Mutants and Malformations
of the Vertebral Column
RAR␥ and occasionally RAR␣ single mutants show
homeotic transformations and malformations of vertebrae (138, 140). These studies establish that RA plays an
important role in patterning of the main body A/P axis.
Penetrance and expressivity of cervical anterior transformations observed in RAR␥ null mice increased in a graded
manner, with subsequent loss of RAR␣1 and RAR␣2 isoforms from the RAR␥ background (140). Furthermore,
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Kastner and co-workers (99, 100) performed a massive undertaking to determine the functional significance
of RXR/RAR heterodimeric signaling in embryogenesis.
These investigators created compound mutant fetuses
bearing null alleles in one RXR (␣, ␤, or ␥) and one RAR
(␣, ␤, or ␥) subtype or isoform gene (100). The data
presented show synergy between the effects of RXR␣ and
RAR mutations, but no such synergy was observed when
RXR␤ or RXR␥ mutations were combined with RAR mutations. It is also noteworthy that all defects of fetal
vitamin A deficiency are recapitulated by the various
RXR␣/RAR compound mutations (Table 2). Some of the
abnormalities were specific to one type of RXR␣/RAR
mutant combination, whereas others were seen in several
types of RXR␣/RAR double mutants. As an example of
specificity, the study of RAR double mutants has suggested that RAR␣ and RAR␤2, as well as RAR␣ and RAR␥,
are functionally redundant for the formation of the aorticopulmonary septum (184). In all RXR␣/RAR␣ double
mutants, a complete absence of this septum was observed. This suggests little functional redundancy between the RAR subtypes, since RAR␣ can never be functionally replaced by either RAR␥ or RAR␤2 in a RXR␣
mutant background. The case for multiplicity of function
may indicate that more than one RXR␣/RAR pair is normally involved in the underlying developmental process,
which may require the expression of several RA target
genes. These data confirm the role of RXR/RAR heterodimers as the functional units that transduce the retinoid signal for a large number of RA-dependent processes. These results further implicate RXR␣ as the main
RXR in the developmental functions of RAR. However, it
is noted that a tight functional specificity of RXR␣ for
heterodimerization with RAR is unlikely, because many
RAR-dependent developmental processes proceed normally in mutants that express RXR␤ as the only RXR
(121).
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ROSS, McCAFFERY, DRAGER, AND DE LUCA
F. Response of Mutants to Excess RA
Both RAR␣ and RAR␤ null mutants show the same
teratogenic responses to RA excess as wild-type mice
(193). Furthermore, RAR␣ ⫺/⫺ RAR␤ double mutant
mice were also susceptible to the teratogenic effects of
RA, demonstrating that RAR␣1 and RAR␤ isoforms, singly
or in combination, do not play a major role in RA-induced
craniofacial malformation and limb deformities (146).
The response of RAR␥ null mice to RA excess, however, is significantly different from wild-type mice (138).
RA treatment at 8.5–9.0 dpc results in craniofacial abnormalities and axial defects (truncations of the lumbosacral
spine and fusions of the lower ribs) in wild-type mice. The
RAR␥ null mutants show the same RA-induced pattern of
craniofacial abnormalities as the wild-type mice. Interestingly, RA administration to RAR␥ null heterozygotes
shows a mild version of the axial abnormality. Therefore,
RAR␥ may be required for the teratogenic RA signal in the
generation of the trunk, but not for craniofacial skeletal
abnormalities. The toxic effects of repeated doses of RA
and two synthetic retinoids, Ro-15–1570 and Ro-40 – 6055,
on RAR␥ null mice have also been examined (141). Surprisingly, homozygous mutant mice were resistant to
fourfold higher doses of RA than wild-type mice as well as
to elevated doses of the synthetic retinoids. The results
indicated that RAR␥ may have a major role in mediating
retinoid toxicity. Furthermore, these findings may have
practical implications for reducing the toxicity of synthetic retinoids in clinical use. Mouse embryos lacking
RXR␣ have been described as resistant to RA-induced
teratogenicity, in particular for limb defects (272). RA
treatments, which caused limb defects in 100% of wildtype embryos, failed to elicit malformations in RXR␣
homozygotes, implicating RXR␣ as a component in the
teratogenic process in the limbs.
G. Conclusions of Mutant Mice Studies
Much knowledge has been obtained on the possible
function of retinoid receptors during embryogenesis by
analyzing their expression patterns and the effects of
“knocking out” receptor classes. It is not, however, within
the scope of this review to describe all the various phenotypes arising from the several retinoid receptor mutant
mice; instead, we provide a few of the highlights. The
defects are diverse, as might be expected from the complex expression patterns of the different RAR and RXR,
and some of the effects are common in the offspring of
vitamin A-deficient mice. Simple knockouts of a given
receptor do not appear to result in the unambiguous
characterization of the receptor’s role in vivo, most likely
because it involves a family of receptors and because it
functions as a heterodimer with other receptors. Additionally, many of the mutations may not reveal the functions of a given receptor, because they result in either a
lethal phenotype or agenesis of that organ. Expression
patterns of RAR/RXR during embryogenesis suggest conserved specialized functions for particular receptor subtypes. Genes that appear to be functionally redundant
from knockout studies, however, may not be redundant in
normal development. The studies of single and double
RAR mutations reveal that a given RAR subtype can exert
some of the functions of another RAR subtype, only when
the latter, which performs these functions and hinders the
activity of the former under physiological conditions, has
been knocked out. Such artifactual redundancies generated by knockouts of genes that belong to a multigene
family may limit our ability to deduce the in vivo functions
of a given gene product. The RXR␣/RAR double mutants,
however, provide some information on functional specificity among retinoid receptors. Specific phenotypes do
arise in these double mutants, indicating that the different
receptor types probably possess different functions during development. Furthermore, the observation that developmental malformations arising from RXR/RAR double mutants are similar to malformations in vitamin A
deficiency suggests that the RXR/RAR heterodimer most
likely functions to express the retinoid signal in vivo.
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concomitant inactivation of all RAR␣ and -␥ isoforms
resulted in severe degeneration of the cervical vertebrae
(140). RAR␤2 may also be involved in axial patterning,
because RAR␣␤2 (but not RAR␣) double mutants displayed a high frequency of anterior transformations of the
sixth and seventh vertebrae (140). Cervical region patterning has also been examined after inactivation of one
RXR␣ allele within several RAR mutant backgrounds
(100). Of these, the RXR␣ ⫹/⫺, RAR␥ ⫹/⫺, as well as
RXR␣ ⫹/⫺ RAR␣ ⫹/⫺ newborns exhibited a high frequency of defects in the cervical region. The nature of the
vertebral abnormalities in RAR single and double mutants
as well as RXR␣/RAR mutant mice is of significance in
view of the interactions between RA and Hox gene signaling. In the somitic mesoderm of the future vertebral
column, each vertebral level has a specific Hox gene
specification (18, 193). The Hox code in this region can be
modulated by either altered expression of Hox genes via
overexpression or mutational analyses or by excess RA
(193). Furthermore, several of the homeotic transformations observed in RAR mutants (single and double) or
RXR␣/RAR mutants appear similar to those described for
Hox null mutants (229). The homeotic transformations,
observed in RAR and RXR/RAR null mutants, suggest that
retinoids may play a role in Hox gene specification in the
early vertebral column. The results from the RXR/RAR
mutant studies suggest a synergism between RXR␣ and
RAR␣ or RAR␥ mutations for generating defects during
cervical vertebrae morphogenesis (100).
Volume 80
July 2000
RETINOIDS IN EMBRYONAL DEVELOPMENT
VII. HOX GENES AND RETINOIDS
IN DEVELOPMENT
A. Hox Genes in Development
Homeobox genes have fundamental roles in the organization of developmental pattern in both invertebrate
and vertebrate embryos (182). Vertebrate homeobox
genes of the Hox family are expressed during segmental
patterning of the mammalian hindbrain as well as in axial
patterning of the limb (77). Hox genes contain homeoboxes, which encode a helix-turn-helix motif that
functions to bind DNA (76). These DNA-binding proteins
are thought to control pattern formation by providing
positional cues that determine organization within the
developing embryo (75, 76, 255). The mammalian genome
contains up to 38 Hox genes subdivided in four clusters
(Hox A, B, C, and D). These Hox gene clusters have been
identified in mouse and human genomes, each cluster on
a different chromosome. A Hox gene cluster contains at
least nine genes organized in a homologous linear arrangement of ⬃100 –150 kb. Furthermore, all Hox genes
share the same transcriptional orientation. During early
development, Hox genes display colinearity with respect
to their temporal order of activation. Generally, the 3⬘genes are activated earlier in development than the 5⬘genes (2). In addition to temporal order of expression, the
Hox genes are expressed in sequential zones along the
A/P axis in the hindbrain and trunk regions. Hox genes
show A/P colinearity of expression, such that progressively more 5⬘-genes are expressed in progressively more
posterior zones (74, 120, 182). Therefore, Hox genes at the
3⬘-ends of the clusters are expressed earlier in embryonic
development and in more anterior regions, whereas Hox
genes closer to the 5⬘-ends of the clusters are expressed at
later times in development and in posterior regions of the
embryo.
B. Retinoids and Hox Genes
Retinoids have been shown to affect the expression
of Hox genes in vitro, in teratocarcinoma cells, and in
early embryos (62). Recent investigations have identified
RARE in the enhancer regions of certain Hox genes. In F9
cells, Langston and Gudas (128) have shown that at ⬃5 kb
3⬘ of the Hoxa1 gene transcriptional start site there is an
RA-inducible enhancer that contains an RARE of the DR5
type (RAIDR5) that specifically binds RAR. A similar enhancer was also found in the murine and chick Hoxb1
gene and was shown to be required for expression in the
developing foregut, which gives rise to various organs
including the esophagus, lung, stomach, liver, and pancreas. This enhancer is necessary and sufficient for RA
activation of the Hoxa1 gene, the 3⬘-most gene in the A
cluster. Retinoid response elements have also been identified for the human and murine Hoxb1 (169, 213, 269, and
described below) and murine and human Hoxd4 genes
(190, 225). From expression analysis of Hoxb1-lacZ transgenic mice, Gudas and collaborators (91) have shown that
a wild-type (wt) transgene with 15 kb of Hoxb1 genomic
DNA, which includes the Hoxb1 3⬘-RAIDR5, displays a
pattern of localization and developmental expression similar to that of the endogenous Hoxb1 gene. Introduction of
a point mutation in the DR5 RARE caused it not to be
expressed in the developing foregut (91). Exogenous RA
also resulted in enhanced expression of the wt transgene
in the gut, with an anterior expansion of expression in this
tissue. Therefore, these studies conclude that Hoxb1 is
expressed in the developing gut and that this expression
is regulated through the DR5 RARE. The authors suggest
a role for Hoxb1 in the A/P axis patterning of the gut and
a critical role for endogenous retinoids in early gut development.
The Hox B cluster of genes is expressed in the developing hindbrain and spinal cord in head to tail order
with a 3⬘ to 5⬘ sequence of expression. Some of the genes
in the Hox B cluster may help establish pattern and
positional identity in neural development. However, the
Hoxa1 is expressed before Hoxb1 and is thought to be
involved in Hoxb1 initiation (268). Interest in Hoxb1 is
based on the observation that this gene is among the early
genes to be expressed in the mesoderm and neuroectoderm of primitive streak and presomite embryo, before its
expression in hindbrain rhombomeres (67, 197, 198, 304).
In mice, the expression of the Hoxb1 gene varies at different times of development. The initial expression takes
place at 7 dpc in a diffuse manner starting at the r3/r4
boundary and reaching caudally to the end of the developing spinal cord. Hoxb1 continues to be expressed in the
posterior hindbrain and trunk as well as r4 until very late
in development. Excess exogenous RA can cause craniofacial malformations and malformations in the hindbrain
region. The ability of RA to alter the segmental organiza-
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In the past it has been widely accepted that the
teratogenic effects of exogenous RA reflect a physiological role for endogenous RA in embryogenesis. The findings from RAR and RXR mutant mice, however, cast some
doubt on the normal physiological relevance of RA excess
studies. In the two cases for which the involvement of a
given RAR or RXR in the mediation of a teratogenic event
was demonstrated, the same receptor was not required
for development of the same structure during normal
embryogenesis [e.g., RAR␥ in the vertebrae (138) and
RXR␣ in the limbs (272)]. Therefore, it appears that specific teratogenic processes, as well as specific normal
developmental processes under vitamin A control, occur
through individual members of the RXR and RAR families
and that these receptor-specific processes appear to be
different in the excess RA situation, compared with the
normal condition.
1037
1038
ROSS, McCAFFERY, DRAGER, AND DE LUCA
genes, tissue formed in the region of r4 lacks the earliest
r4 marker, Eph tyrosine kinase receptor EphA2. This
suggests a failure to initiate rather than maintain the
specification of the r4 identity by both Hoxb1 and Hoxa1.
This genetic analysis shows that individual members of
the vertebrate labial-related gene family have multiple
roles in different steps governing segmental processes in
the developing hindbrain (268). However, studies on mice
lacking expression of Hoxb1 showed a very limited phenotype, which is not consistent with a quantitatively major role of this gene in embryogenesis (69, 268). Even the
double a-1/b-1 mutants show little change in phenotype
from the single a-1 mutant (69, 268). Even though this
change may be crucial for the development of certain
organs like the ear, it does not appear to support a role for
b-1 in hindbrain segmentation or overall hindbrain neural
arrangement. It appears, therefore, that the role of b-1 is
mainly at the level of a single segment identity. Importantly, the normality of trunk development in the a-1/b-1
double mutants argues against a requirement for these
genes for initiation of more posterior Hox genes (69, 268).
A comparison of Hox misexpression experiments
with the results of RA teratogenesis suggests that retinoids influence development via Hox gene regulation.
Exposure of embryos to exogenous RA, which effectively
increases the levels of retinoids along the body axis,
extends the anterior boundaries of some of the Hox genes
(129) and may cause transformation of anterior rhombomeres into morphologically posterior rhombomeres
(168, 192). Interestingly, transgenic overexpression of
Hoxa1 transformed anterior rhombomeres 2 and 3 (normally not expressing Hoxa1) into rhombomeres that resemble rhombomere 4 (the most anterior boundary of
Hoxa1 in normal embryogenesis) (317). Expression of
Hoxb1 occurred in the neuroepithelial cells in r2 following overexpression of Hoxa1, which suggests possible
interactions between Hox genes. With the exception of
Hoxb1, however, the expression patterns of other Hox
genes were unaffected by the overexpression of Hoxa1.
Furthermore, abolishing Hoxa1 expression by introduction of a null mutation reduces some of the segmentation
of the hindbrain leading to the loss of rhombomeres 4 and
5 (50, 144, 165). Interestingly, the r4-restricted pattern of
Hoxb1 expression was maintained in Hoxa1 mutant mice
(165). The similarities in phenotypes between retinoid
teratogenicity and models generating Hox gene mutations
suggest that endogenous retinoids may establish the A/P
graded expression of the Hox genes and thus influence
A/P identity along the axis.
In an attempt to describe some of the physiological
requirements for vitamin A in development, Maden and
co-workers (151, 152) characterized neural defects using
the vitamin A-deficient quail embryo as a model. Among
the defects observed in the vitamin A-deficient quail system is the absence of posterior rhombomeres. In addition
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tion of the developing hindbrain may result in part from
its ability to alter the pattern of expression of Hox genes
(28). In fact, both the early broad pattern and the later
restricted pattern of the Hoxb1 expression are regulated
by RA through RARE (169, 269).
Regulatory elements required to generate the early
neuroectodermal and mesodermal phase of Hoxb1 expression were localized to the 3⬘-flanking region of the
gene (169). Two separate enhancers were revealed in this
3⬘-flanking region, each regulating different aspects of the
early phase of Hoxb1 expression. The enhancer regulating
the expression of Hoxb1 in neuroectoderm was found to
contain a RARE. Point mutations within this 3⬘-RARE
prevented the establishment of neural expression of
Hoxb1 in transgenic mice. The position of this Hoxb1
3⬘-RARE (DR2 type) is similar to that seen with the Hoxa1
RAIDR5 (91, 128). The demonstration of 3⬘-RARE enhancer regions in both Hoxa1 and Hoxb1, the first genes
of the Hox A and Hox B clusters, respectively, suggests
that RA has important molecular functions during morphogenesis. The restriction of Hoxb1 expression to r4
during later stages of hindbrain development is due to two
DNA sequences located upstream, i.e., 5⬘ of the Hoxb1
gene (269). A combination of activation and repression
determines the limitation of Hoxb1 to r4. An enhancer
sequence at the 5⬘-end of the Hoxb1 gene is necessary for
expression in r4 as well as in r3 and r5 (265). However,
next to this 5⬘-enhancer sequence is another DNA sequence that sharply limits Hoxb1 expression to r4 (269).
This sequence was found to turn off Hoxb1 expression in
r3 and r5. Furthermore, the sequence of this repressor
element contains a DR2-RARE. Mutations in this sequence abolish its repressive activity. Therefore, the
Hoxb1 5⬘-RARE is an essential component of the repressor region and cooperates with the r4 enhancer to refine
Hoxb1 expression in the hindbrain. Gudas and collaborators (130) have discovered an enhancer containing a DR5
RARE (RAIDR5) located at a DNase I-hypersensitive site
3⬘ of the murine Hoxb1 gene. This enhancer regulates the
RA responsiveness of the Hoxb1 gene and is different in
location and sequence from the RA-regulated 3⬘ Hoxb1
enhancers described by Marshall et al. (169) but is similar
in sequence to the Hoxa1 RARE enhancer discovered
previously by the same group (128). These 3⬘-enhancers
may be important in the activation of the entire Hox loci
as well as in the regulation of Hoxa1 and Hoxb1. A
targeted mutation in the Hoxb1 3⬘-RARE showed that its
expression is required to attain early high levels of Hoxb1
in neural ectoderm. Double mutant analysis with this
(3⬘-RARE) allele and other targeted loss of function alleles from both Hoxa1 and Hoxb1 revealed synergy between these genes. Consistent with these studies is the
recent report that shows a synergy between Hoxa1 and
Hoxb1 in patterning the hindbrain, cranial nerves, and the
second pharyngeal arch (69). In the absence of both
Volume 80
July 2000
RETINOIDS IN EMBRYONAL DEVELOPMENT
C. Conclusions on the Induction of Hox Genes
by Retinoids
A direct molecular link between RA, RAR, and the
activation of specific homeobox genes has been observed
during embryogenesis. The availability of retinoid response elements in Hoxa1, Hoxb1, and Hoxd4 genes
suggests that retinoids act as an early developmental
signal, possibly conditioning the posterior-to-anterior gradient in the gastrula and providing positional specification
of the A/P axis in the developing vertebrate embryo. How
other Hox genes located in more 5⬘-positions of the cluster are regulated by RA is not known. The Hoxa1 and
Hoxb1 genes appear to be modulated directly via the
RARE, and the protein products of these genes may activate the proximal 5⬘-genes in the clusters, that is Hoxa2
and Hoxb2, respectively. This scenario would allow for a
sequential activation of Hox genes. It may also be that
downstream targets of Hox genes, as well as other developmental genes regulated by RA, are involved in the
regulation of the Hox genes located in more 5⬘-positions.
Specific combinations of Hox proteins, expressed for
example at different axial levels in the hindbrain, appear
to result in expression patterns of target genes and unique
tissue identity.
Mechanisms in normal development and RA-induced
malformations may further be elucidated by determining
downstream targets of the homeobox proteins. The target
genes for both the homeobox proteins and retinoid receptors are a current area of investigation in embryogenesis.
For example, evidence suggests that genes involved in
cell shape determination are targets of the Hoxa1 homeobox protein (75).
In conclusion, Hox gene regulation is mediated partly
by positional information supplied by retinoids, and the
effects of excess RA on axial patterning are partly mediated through the disruption of Hox gene expression in the
hindbrain and spinal cord. Studies suggest, however, that
RA, along with its nuclear receptors, is an important
signaling molecule in this developmental process.
VIII. TERATOLOGY/EXCESS RETINOIDS
A. Introduction
Embryonic exposure to either an excess of retinoid
or a deficiency of vitamin A leads to abnormal development (149, 204). From these results, it was understood
very early that the embryo requires a precisely regulated
supply of retinoids. Although the spectrum of malformations that have been observed due to either vitamin A
deficiency or excess appears to overlap, there are examples of selective responses to only deficiency or excess.
No single mechanism is likely to explain the action of
retinoids in normal or teratogenic development. It is more
likely that each tissue utilizes RA in a unique process
during development. Much work has been performed to
relate the mechanism of malformations from excess exogenous retinoids to the role that retinoids play in normal
developmental processes. Here we summarize some of
the important work that has been performed utilizing
excess retinoids, as well as a summary of epidemiologic
evidence for human teratology from exposure to retinoids. This provides an historical context for our current
thinking on retinoid action in embryogenesis and also
reflects our interest in teratogenesis resulting from retinoid toxicity.
B. Retinoids and Limb Morphogenesis
The developing chick limb bud has been extensively
used to study morphogenesis (87); cells in the limb bud
differentiate into muscle, cartilage, and bone cells of the
mature wing (66). The digits of the wing are oriented
along the A/P axis of the wing bud, and they are ordered
by the ZPA in the posterior region of the bud. A mirror
image duplication of digits (296) can result from either
grafting the ZPA to the anterior region of the bud or by the
local application of RA to this area. This implied that RA
could mimic the behavior of the cells of the ZPA. It is
possible that high concentrations of RA may give rise to a
new ZPA region adjacent to an RA implant (inert carrier
material impregnated with RA). In support of the hypoth-
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to observing hindbrain morphology, these investigators
confirmed the loss of posterior hindbrain by utilizing expression patterns of Hoxa2, Hoxb1, Hoxb4, Krox20, and
FGF-3 as markers and found that segmentation of the
myelencephalon (rhombomeres 4 – 8) was disrupted
(151). Expression patterns of other early developmental
genes revealed the presence of rhombomeres r1, r2, and
r3. Maden et al. (153) later observed that the absence of
the posterior hindbrain was caused by a highly localized
region of cell death occurring as the hindbrain becomes
respecified at the seven-somite stage. The authors suggest
that a posterior regression of Hox gene expression may
have resulted in the absence of the posterior hindbrain
(152). The results imply that Hox genes, or other developmental genes, play a role in the A/P patterning of the
CNS and are under the control of RA in the early embryo.
Using the vitamin A-deficient quail model, Maden and
co-workers (151, 152) also observed that neural crest cells
need RA for normal development and survival and that
the neural tube failed to extend neurites into the periphery. The influence of RA on the expression of HoxD
(Hox4) genes during limb and vertebrae morphogenesis
has been reviewed (87).
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ROSS, McCAFFERY, DRAGER, AND DE LUCA
bud stages results in digital fusions and truncations,
bowed radius, and retarded growth and skeletogenesis
(308). These teratogenic effects of exogenous retinoids
may indicate that endogenous retinoids play a role in limb
bud patterning in the mouse. In support of this theory, it
has been shown that mouse limb buds contain RA (90,
243). Higher levels of RA, however, on the posterior side
versus the anterior side have not been observed in mouse
limb buds. In addition to RA, both CRABP (123, 155) and
RAR (51) are found in the mouse limb bud at the right
place and time, suggesting a role for retinoids in limb bud
morphogenesis. Furthermore, homeobox genes, such as
the Hox D complex (Hox4, according to the older nomenclature), are expressed in the mouse limb bud in a spatial
and temporal pattern (49). Because homeobox genes have
been shown to be regulated by RA in certain tissues,
current work continues to explore RA’s regulation of Hox
genes during mouse limb bud morphogenesis as well as
the teratogenic effect of RA on Hox gene expression.
Exogenous retinoids are also known to influence patterning of other structures, such as the anteroposterior body
axis which is reviewed elsewhere (87).
C. Animal Teratogenic Models
The teratogenic effects of high doses of vitamin A in
pregnant rats were first described by Cohlan in 1953 (26;
see Ref. 231; as reviewed in Ref. 5). Cohlan demonstrated
that feeding pregnant female rats 35,000 IU/day of a “natural vitamin A” preparation (most likely a retinyl ester
preparation) on gestation days 2–16 resulted in a large
number of fetal anomalies. These anomalies included exencephaly, cleft lip, cleft palate, brachygnathia, and eye
defects. RA was later shown to be a more potent teratogen than retinol in several animal models (109, 252).
Similar anomalies to those described above have been
observed in embryos of many species of experimental
animals including monkeys, rabbits, rats, mice, and hamsters after excess retinoid administration (63, 95, 96, 103,
109, 112, 252). In addition to malformations in the offspring, the treatment of pregnant female experimental
animals with excessive amounts of vitamin A (retinyl
acetate or palmitate) or one of its metabolites, e.g., RA or
13-cis-RA, has resulted in dramatically increased rates of
fetal resorptions and stillbirths. Generalizations concerning retinoids in teratogenesis have emerged from these
and other studies. First, the anomalies observed in animals in response to retinoids are stage dependent. Early
postimplantation exposure [gestational days (GD) 8 –10]
typically leads to craniofacial and overt CNS defects,
whereas exposure on GD 12–14 is often associated with
limb and genitourinary defects (103). Second, the teratogenic response for each retinoid is dose dependent (1,
246). Higher doses increase the frequency and severity of
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esis that RA may induce a ZPA region is the observation
that retinoid receptor antagonists blocked the formation
of the ZPA when applied to the chick limb bud (85, 111).
The intercalary interaction model of pattern formation has also been postulated to explain the action of RA
in limb bud duplication (87). In this model, RA or the ZPA
influences short-range cell-to-cell interactions, and each
cell has a positional characteristic that represents its
spatial coordinate within an embryonic tissue. In the RA
implant-induced digit duplication experiments, it is
thought that RA “converts” cells next to the implant into
posterior cells. These posterior cells interact with existing
adjacent anterior cells, giving rise to cells with progressively greater anterior positional values, eventually resulting in digit duplications.
Molecular evidence suggests that the Hox4 genes
may encode positional information (93, 212). The Hox4
homeobox genes have been found to be coordinately
expressed in partially overlapping domains during chick
wing development. Local application of RA has been
shown to induce transcription of Hox4 genes in a mirrorimage pattern of expression, which correlated with the
subsequent development of mirror-image patterns of digits. Additional molecular evidence suggests that the product of the sonic hedgehog (Shh) gene may be the morphogen in limb bud duplication and that RA may function
indirectly in limb patterning by inducing Shh (209, 231,
257). Chen et al. (25), however, have demonstrated that
the expression of Shh in Hensen’s node is not affected by
the endogenous vitamin A status in chick embryos, further complicating the role that RA plays in limb morphogenesis (25). Stratford et al. (267) find diminished Shh
expression in vitamin A-deficient quail limb. Additional
evidence suggests that other factors, e.g., fibroblast
growth factors (FGF), play a role in generating and maintaining ZPA activity and limb patterning (209). It was
found that Shh gene expression was activated in limb
mesenchyme by RA plus FGF-4, but, once induced, Shh
expression was maintained by FGF-4 alone (209). It can
be hypothesized from these developments that retinoids
are most likely part of a cascade of signals providing
positional information in the limb, which may also include
signals from Hox genes, Shh, and FGF. The role of retinoids in limb bud duplication and in normal morphogenesis of the limb is unclear; the effects from high exogenous as well as endogenous levels of retinoids both need
to be further evaluated in this system. One hypothesis is
that, during normal development, high local concentrations of RA at the future posterior side of the developing
limb may provide a signal(s) to promote posterior but
suppress anterior limb development. In this regard, endogenous levels of RA have been shown to be in higher
concentration on the posterior side than on the anterior
side of the chick limb bud (150).
Administration of RA to mouse embryos at early limb
Volume 80
July 2000
RETINOIDS IN EMBRYONAL DEVELOPMENT
D. Molecular Mechanism of Excess Retinoids
in Teratogenesis
One approach to unravel the molecular events in
RA-induced teratogenesis is to examine the spatial distribution of active retinoids during embryogenesis. For example, radiolabeled RA administered to pregnant mice at
postimplantation stages (approximately GD 8 –11) was
shown to accumulate in restricted areas along the neural
tube (39, 41). These areas were identified as rhombomeres of the hindbrain, neural crest cells, and neural
crest-derived structures, putative targets for the teratogenic action of retinoids (126, 294, 303). Other work suggested the distribution of RA was found to be correlated
with expression patterns of CRABPI (40, 42), suggesting
that CRABPI may be an integral part of the teratogenic
effect of RA. For example, CRABPI may become increasingly saturated upon “dosing” the embryo with exogenous
RA, resulting in an enhanced availability of unbound RA
for nuclear translocation and consequent gene modulation. Horton and Maden (90) had an interesting result,
where the regional accumulation of teratogenic RA had
no relationship to the distribution of CRABPI or -II.
Other approaches have been utilized to study the
molecular events in RA-induced teratogenesis.
Determining the pattern of expression of retinoid
nuclear receptors in embryos exposed to teratogenic
doses of RA is one such approach. Studies using GD 11
mouse embryos demonstrated that mRNA levels of the
RAR␤2 isoform were elevated after a teratogenic dose of
RA and distributed in regions of the mouse embryo that
are most susceptible to the effects of RA (80). For example, RAR␤2 mRNA levels were elevated 12-fold in limb
buds (highly sensitive), 8-fold in the head (moderately
sensitive), and only 3-fold in the remainder of the body
(relatively insensitive) after treatment of the pregnant
dams with teratogenic doses of RA. Furthermore, on GD
14, when the mouse embryo is no longer sensitive to the
teratogenic effects of retinoids, the elevation in RAR␤2
mRNA levels in all embryonic tissues was only two- to
threefold (80). These observations are consistent with the
possibility that elevation in the mRNA level of specific
RAR (or RXR) isoforms may mediate abnormal development in RA-treated embryos (262). Furthermore, an alteration in the relative amounts of one receptor could have
a cascadelike effect and perhaps result in wide-ranging
effects on the expression of a variety of genes.
Regionalized synthesis of RA creates regions of high
and low RA (see Fig. 4). Teratogenic RA may ablate these
high/low differences and disrupt the patterning created by
these. Regions of low RA content (which generally express CRABPI) would be most sensitive (54, 55).
Altered expression of specific homeobox genes has
been associated with RA-induced teratogenesis. For example, alterations in Hoxb1 expression have been linked
to defects in the hindbrain in embryos treated with excess
RA (28, 169). These studies suggest a link between RA,
CRABP, RAR/RXR, and transcriptional modulation of homeobox genes during teratogenesis.
E. Prescription Oral Retinoids as Teratogens
in Humans
A relationship between high levels of retinoids and
human birth defects has been known for some time (82
and reviewed in Ref. 217). The teratogenic effects of
retinoids become manifest during the first trimester of
embryonic development, and many defects likely arise
from the abnormal migration of cranial-neural crest cells
or a defect in early axial patterning. The threshold dosage
of retinoids required to cause these malformations is
unknown (204). Maternal ingestion of 13-cis-RA (isotretinoin or Accutane), prescribed for the treatment of severe
cystic acne, has resulted in spontaneous abortions and
the manifestation of severe malformations in the offspring
(126). Abnormalities, such as microtia/anotia, micrognathia, cleft palate, conotruncal heart defects and aorticarch abnormalities, thymic defects, retinal or optic-nerve
abnormalities, and CNS malformations (e.g., hydrocephalus), were observed in the fetuses of women ingesting
therapeutic doses of 13-cis-RA (0.5–1.5 mg/kg) during the
first trimester of pregnancy, and these retinoids are, thus,
contraindicated for use during pregnancy (126). The pattern of malformations arising from exposure to isotretinoin in humans is thought to closely resemble those ab-
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malformations and are more likely to be lethal to the
embryo (109). When both stage and dose dependency are
considered, it is noted that relatively lower doses ⱕ10
mg/kg) are toxic early in the critical phase of development, whereas higher doses (ⱖ100 mg/kg) are required
later in the critical phase of organogenesis (1). Third,
some retinoids are more potent than others. In mice, for
example, 13-cis-RA and retinol are ⬃20 and 4 times less
potent teratogens than RA (262), respectively. Furthermore, the potency of the retinoid can vary with different
animal models. Humans, for example, are more sensitive
to 13-cis-RA than monkeys and rabbits, whereas mice and
rats are relatively insensitive to this retinoid (201, 202).
Species differences in response to the teratogenic potency of 13-cis-RA can be explained by variation in pharmacokinetics (204). In many of the animal studies described above, the dosages tested were generally far in
excess of likely human exposures. However, the spectrum of malformations observed in animal models are
similar to those observed with human exposure to pharmacological doses of 13-cis-RA and, although seen less
frequently, retinol (251).
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TABLE
ROSS, McCAFFERY, DRAGER, AND DE LUCA
Volume 80
3. Epidemiologic studies of the association of vitamin A supplements and birth defects
Retinoid and Source
Reference
Study type
Exposure window
Control or comparison
group (n)
Cases or exposed
group (n)
Martinez-Frias and
Salvador (171)
Case-control
All months of
pregnancy
n ⫽ 11,193
Strengths
Limits
ⱖ10,000 IU of retinol
or retinol palmitate
alone or in
combination with
other vitamins
ⱖ10,000 IU not associated with
risk (OR: 1.1; CI: 0.5–2.5);
ⱖ40,000 IU increase but not
significant (OR: 2.7; CI: 0.8–
11.7)
Controls matched to
cases on sex,
hospital; considered
the effects of dose
and time of
exposure during
pregnancy;
interviewed within
the first 3 days after
delivery about
exposure
Low frequency of exposure
to vitamin A; imputed
dose from brand name;
included both major and
minor birth defects;
diagnostic period for
birth defects limited to
1st 3 days of life
Vitamin A supplements:
1) retinol alone
group ⫽
retinol/esters alone
or with vitamin D or
fish oils; 2)
multivitamin group
Suggestive, but not significant
association for exposure to
retinol alone during the 1st
month (OR: 2.4; CI: 0.9–6.2);
no association between
multivitamin use in month 1
(OR: 0.9; CI: 0.7–1.1)
Well-defined group of
cranial neural crest
defects; reduced
reporting bias (cases
and controls both
malformed);
structured in-home
interview;
confounding effects
considered
Data on dose not available;
inappropriate control
group (uncertain of full
spectrum of birth
defects associated with
vitamin A use); low
frequency of exposure to
vitamin A
Vitamin A dose changed
during study;
inappropriate control
group
n ⫽ 11,293
Reference
Study type
Exposure window
Control or comparison
group (n)
Werler et al. (299)
Case-control
⫹1 to ⫹3 mo gestation
n ⫽ 2,609 infants with
other malformations
Cases or exposed
group (n)
n ⫽ 2,658 infants with
craniofacial and
cardiac
malformations
Reference
Study type
Czeizel and Dudas (31)
Randomized, controlled,
clinical trial
Peri-conception use
n ⫽ 2,052 women
receiving traceelement supplements
n ⫽ 2,104 women
receiving
multivitamins
Multivitamins with
4,000–6,000 IU
vitamin A
(preformed)
Lower rate of birth defects in
multivitamin users
Randomized; blinded
Reference
Study type
Exposure window
Control or comparison
group (n)
Cases or exposed
group (n)
Rothman et al. (237)
Prospective
⫺3 to ⫹3 mo gestation
ⱕ5,000 IU
(supplements)
n ⫽ 11,083
⬎10,000 IU
(supplements)
n ⫽ 317
Retinol in supplements
and combination of
retinol in
supplements and
food
Mothers consuming ⬎10,000 IU
from supplements
(compared with ⱕ5,000 IU)
increased prevalence of
cranial-neural crest tissue
birth defects (PR: 4.8; CI:
2.2–10.5)
Confounding effects
Potential misclassification
considered; collected
of cranial neural crest
information on
defects; estimated
exposure variable
supplemental dose from
before knowledge of
brand name information;
birth outcome
selected foods only for
dietary exposure
Reference
Study type
Exposure window
Control or comparison
group (n)
Cases or exposed
group (n)
Khoury et al. (102)
Case-control
⫺1 to ⫹3 mo gestation
n ⫽ 3,029
Vitamin A (preformed)
supplement plus
multivitamin
supplement
No increased risk among
women who regularly took
both vitamin A supplements
plus multivitamins (OR: 0.54;
CI: 0.22–1.33)
Similar case
classification as
Rothman et al.;
controls frequency
matched to cases by
period of birth, race,
and hospital of birth
No data on dose available;
insufficient information
on methods to interpret
study adequately
Reference
Study type
Exposure window
Control or comparison
group (n)
Cases or exposed
group (n)
Shaw et al. (250)
Case-control
⫺1 to ⫹3 mo gestation
n ⫽ 734
Single supplements of
vitamin A
(preformed)
No increased risk for orofacial
clefts with vitamin A
supplement use (OR: 0.55;
CI: 0.21–1.5); no increased
risk for conotruncal heart
defects
Malformations
associated with
neural crest cellsvitamin A target
tissue
No data on dose available;
interviewed 3.5 yr after
pregnancy for exposure
information
Reference
Study type
Exposure window
Control or comparison
group (n)
Cases or exposed
group (n)
Mills et al. (187)
Case-control
Peri-conception use
Normal pregnancy
outcome (n ⫽ 573)
1) Neural tube defects
(n ⫽ 548)
2) Malformations other
than neural tube
defects (n ⫽ 387)
Preformed vitamin A in
fortified cereals and
dietary supplements;
also asked yes/no for
consumption of
organ meat
No association at doses ⬎8,000
IU (OR: 0.79; CI: 0.4–1.53) or
⬎10,000 IU (OR: 0.73; CI:
0.27–1.96) compared with
doses ⬍5,000 IU
56.8% supplied dose
information from the
vitamin bottle;
detailed info on
types and doses of
vitamin supplements
and cereals; short
time interval
between
organogenesis and
interview;
geographically based
nature of sample;
blinded interviews
Possible recall bias of
vitamin A intake; low
frequency of exposure to
high-dose vitamin A;
compared ⱖ8,000 IU
with ⬍5,000 IU and no
comparison of ⱖ8,000
with the 5,000–8,000 IU
range; lack of complete
dietary information
Exposure window
Control or comparison
group (n)
Cases or exposed
group (n)
n ⫽ 4,918
n ⫽ 731
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Findings
TABLE
1043
RETINOIDS IN EMBRYONAL DEVELOPMENT
July 2000
3 —Continued
Retinoid and Source
Shaw et al. (249)
Case-control
⫺3 to ⫹3 mo gestation
n ⫽ 611
Reference
Czeizel and
Rockenbauer (32)
Case-control
First trimester
n ⫽ 35,727
Study type
Exposure window
Control or comparison
group (n)
Cases or exposed
group (n)
Reference
Study type
Exposure window
Control or comparison
group (n)
Cases or exposed
group (n)
Limits
Retinol from prenatal,
multivitamin, and
single source
supplements; retinol
in food
ⱖ10,000 IU (food and
supplement sources) not
associated with risk (OR:
1.2; CI: 0.6–2.2); ⱖ10,000 IU
(supplement sources only)
not associated with risk
(OR: 0.7; CI: 0.2–2.3)
Interviewer
administered food
frequency
questionnaire
obtained; amount of
vitamin A intake
from supplements
obtained directly
from brand/product
information or
imputed by
standardized method
Exposure information
collected between 4th
and 5th mo after actual
or projected date of
term delivery; potential
overestimation of dose
from single sources if
dose was estimated
Vitamin A (retinol)
alone and
multivitamins with
vitamin A
Decreased risk of birth defects
with vitamin A use (OR:
0.79; CI: 0.7–0.8)
Large population-based
cohort; controls
matched to cases
according to sex,
birth week, and
residence; 2 or 3
matched controls
selected for each
case
No dietary information
collected; response rate
of control was lower
than of cases; small
subgroup exposed to
⬎10,000 IU vitamin A
supplements
ⱖ10,000 IU/day
preformed vitamin A
for at least 1 wk
during the first 9 wk
ⱖ10,000 IU not associated with
risk when compared with
either control group (study
group vs. control group
exposed to high vitamin A
after 9 wk of gestation PR:
0.28; CI: 0.06–1.23)
(Study group vs. control
group exposed to
nonteratogenic agents PR:
0.50; CI: 0.14–1.76)
Prospective
information about
timing and dose of
vitamin A; selection
bias minimized
because exposed
and nonexposed
subjects selected
from same
population of
women who
contacted a
European Teratology
Information Services
Definition of use of vitamin
A in first trimester was
liberal; no dietary
information collected;
ascertainment of major
malformations not
obtained directly by
investigators
n ⫽ 613
n ⫽ 20,830
Mastroiacovo et al.
(173)
Prospective
First 9 wk of gestation
1) n ⫽ 116 exposed to
vitamin A after 9 wk
of gestation
2) n ⫽ 679 exposed to
nonteratogenic agents
n ⫽ 311
Strengths
OR, odds ratio; CI, 95% confidence interval; PR, prevalence ratio; periconceptional use, around the period of conception; ⫺ months, month before pregnancy; ⫹
months, month of pregnancy.
normalities produced in animal studies of retinoid
teratogenesis. Because 13-cis-RA is a metabolite of vitamin A, the possibility exists that retinol, in sufficiently
large doses, is also teratogenic. Furthermore, 13-cis-RA
occurs physiologically in very small amounts, and it
shares many metabolic and binding properties with alltrans-RA, and therefore 13-cis-RA may be involved in the
teratogenicity of preformed vitamin A. This is further
strengthened by the hypothesis that the teratogenic potency of vitamin A is thought to be mediated by its metabolism to active retinoids (204).
Synthetic retinoids have also been implicated in teratogenesis. For example, human exposure to etretinate, a
synthetic, aromatic retinoid prescribed for the treatment
of psoriasis, has resulted in spontaneous abortions or the
manifestation of severe malformations (e.g., craniofacial
and skeletal) in offspring (235). One case of etretinate
embryopathy occurred in an infant conceived 1 yr after
termination of etretinate, which unlike isotretinoin is
stored in maternal adipose tissue (125). Although there
appears to be a characteristic set of birth defects with
ingestion of these retinoids, the pattern of malformations
is too complex to reconcile, at present, with the known
effects of retinoids on morphogenesis or with the molecular mechanisms elucidated to date (110).
F. Human Epidemiological Evidence: Intake
of Vitamin A and Risk of Birth Defects
The embryo can be exposed to teratogenic doses of
vitamin A when a pregnant woman consumes a chronic
dietary intake of foods that contain high concentrations of
vitamin A, either an acute large dose or chronic high
doses of vitamin A dietary supplement products, or prescription drugs that contain retinoids. One case report of
a single dose of supplemental vitamin A in early pregnancy (500,000 IU in the second month of pregnancy)
provided evidence for the human teratogenicity of vitamin A (195, 235). In this case report, the fetus presented
with preauricular appendices and eye defects. Less massive doses taken daily for several weeks or longer have
been shown to produce defects such as facial dysmorphism, absence of external genitalia and of anal and ure-
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Reference
Study type
Exposure window
Control or comparison
group (n)
Cases or exposed
group (n)
Findings
1044
ROSS, McCAFFERY, DRAGER, AND DE LUCA
of the earlier case-control studies did not question participants about vitamin A consumption until a few years
after the diagnosis of a birth defect (250). In addition to
supplemental vitamin A, this study also examined the
consumption of breakfast cereals with added vitamin A
and queried as to whether the women consumed organ
meat. Neither breakfast cereal nor organ meat consumption changed the conclusions of the study. It is important
to note that in studies evaluating dietary intake, provitamin A sources (e.g., ␤-carotene) have not been associated
with an increased risk of birth defects. This may be due to
the inefficient absorption and conversion of ␤-carotene to
retinol.
Two prospective studies have been conducted (173,
237) (Table 3). These studies are important in that investigators collect information on vitamin A exposure before
knowledge about birth outcome, which minimizes potential recall bias. In one prospective study, history of vitamin A supplementation use was obtained before pregnancy outcome on more than 22,000 pregnant women
(237). One of the most significant findings of this study
was an association of the consumption of ⬎10,000 IU
vitamin A/day from supplements and an increased risk of
birth defects of all types. The consumption of more than
10,000 IU of vitamin A from supplements was also associated with an increased risk of birth defects originating
from cranial-neural crest tissue, defects thought to be
strongly correlated with high intakes of vitamin A. The
results of this study raise concerns about the safety of
high doses of vitamin A during early pregnancy. However,
there are a number of methodological questions concerning the study that prevent reaching the conclusion that the
dosages of vitamin A (⬎10,000 IU) examined in the study
cause certain types of birth defects. One of the limitations
of this study was the broad definition used for cranialneural crest defects. Thus some birth defects arose in
sites unlikely to be of true cranial-neural crest origin,
which may have led to a birth defect being attributed to
vitamin A, when in reality it was the result of some other
factor. This misclassification problem, however, does not
influence the finding that consumption of ⬎10,000 IU of
vitamin A daily from supplements was associated with
increased risk of birth defects of all types.
In a more recent study, pregnant women exposed to
10,000 IU/day or more of vitamin A during the first 9 wk of
pregnancy were identified prospectively and their pregnancy outcomes were compared with two control groups:
1) pregnant women exposed to high vitamin A exposure
later in pregnancy or 2) pregnant women exposed to
nonteratogenic agent exposures (173). The birth prevalence rate of major malformations in the study group
compared with either of the control groups did not provide evidence for an increased risk of major malformations from 10,000 IU/day or more of vitamin A during
organogenesis. Although timing and dose of vitamin A use
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thral openings, among others (235, 264, 293). Case reports
of adverse pregnancy outcomes associated with supplemental vitamin A intakes of 25,000 IU or more per day
have also been documented (235). These reports suggest
that vitamin A may be teratogenic in humans when consumed in amounts greatly in excess of the RDA during
pregnancy [the RDA for pregnant women is 800 ␮g RE,
which is equivalent to about 2,700 IU (210); the reference
daily intake (RDI) for vitamin A established by the Food
and Drug Administration for food labeling purposes in
Title 21 of the Code of Federal Regulations Part 101.9 is
5,000 IU], but confirmation of human teratogenicity requires comparisons with control populations. Therefore,
similar results from either case-control studies (comparisons of dose exposures in controls with those in selected
birth defect categories) or prospective studies (comparisons of defect outcomes in high-dose exposed to low-dose
exposed women) would provide stronger scientific evidence for a relationship between high-dose vitamin A and
birth defects.
The results of the small number of case-control studies performed have shown that children born to women
who consumed supplemental vitamin A at levels found in
multivitamin preparations were not at increased risk for
birth defects (32, 102, 171, 187, 249, 250, 299) (Table 3).
Two of these case-control studies found a relationship
between higher-dose vitamin A exposure during organogenesis and malformations, but the results were not statistically significant (171, 299). For example, one of these
studies suggested an increased risk of birth defects associated with the consumption of ⬎40,000 IU of vitamin A
daily (171). Two additional case-control studies also
found no association between vitamin A supplement use
and birth defects, but in these studies, only estimated data
on retinol dose were available (102, 250). Another more
recent population-based case-control study of California
pregnancies (1989 –1991) found no relationship between
maternal daily intake of vitamin A (ⱖ10,000 IU, from both
food and supplement sources) and risk of neural tube
defect affected pregnancies (249). Interestingly, when
women who consumed ⱖ10,000 IU vitamin A from supplements alone were compared with those women who
consumed less, an odds ratio of 0.7 (95% confidence interval: 0.2–2.3) was found. Furthermore, comparing
women who consumed ⱖ10,000 IU from supplements
alone with women who did not use any vitamin supplements during the periconceptional period revealed an
odds ratio of 0.5 (95% confidence interval ⫽ 0.2–1.7).
In a more recent case-control study, Mills et al. (187)
found that doses greater than 8,000 or 10,000 IU of supplemental vitamin A during pregnancy did not appear to
be associated with birth defects. The strengths of this
study included more detailed information on the types
and doses of vitamin A used and the fact that the women
were questioned shortly after the time of diagnosis. Some
Volume 80
July 2000
RETINOIDS IN EMBRYONAL DEVELOPMENT
trations reported in this study include the following: alltrans-RA (0.68 –2.18 ng/ml), 4-oxo-RA (0.26 –2.04 ng/ml),
13-cis-RA (0.72– 4.72 ng/ml), and 4-oxo-13-cis-RA (0.84 –
7.72 ng/ml). The authors suggested that retinoid plasma
levels in this range may be nonteratogenic because these
pregnancies did not result in fetal malformations. Vitamin
A intake from dietary sources was not reported in this
population of pregnant women. The same investigators
report plasma levels of vitamin A metabolites following
daily vitamin A supplements of 4,000, 10,000, and 30,000
IU for 3 wk to nonpregnant women (302). Plasma levels
were found to be in the range or slightly above the range
observed in early pregnancy. Furthermore, after a 3-wk
treatment with 30,000 IU of vitamin A/day, peak plasma
levels of RA and isotretinoin were within or just slightly
above the range of their physiological levels. These findings lend insight into possible safe upper intake levels of
vitamin A.
Marginal vitamin A status of pregnant women in underdeveloped countries has been documented, and although the association of marginal deficiency status and
risk of birth defects has been hypothesized, it has not
been confirmed because of concurrent nutrient deficiencies in these populations. The vitamin A status of specific
populations groups in the United States may also be at
risk for marginal deficiency. The vitamin A status of lowincome women during the third trimester of pregnancy in
a population in Iowa was examined using the modified
relative dose response test (MRDR) (60). Twenty-six percent of the study population was found to be in a marginal
vitamin A status with MRDR values of ⱖ0.03, whereas 9%
had values of ⱖ0.06. As vitamin A is depleted in an
individual, the MRDR value increases. These cutoff values
correspond to tentative cutoff points that have been set at
0.03 in the United States and at 0.06 in Indonesia. These
findings, therefore, suggest that additional research is
needed to examine the relationship between low vitamin
A intake and risk of birth defects.
Because of the teratogenic potential of retinoids,
women treated with large doses of oral retinoids for
various skin disorders and some types of cancer should
avoid pregnancy during this period. Furthermore,
women using vitamin A supplements need to be aware
of the teratogenic risk during early pregnancy. Multivitamins, in addition to their beneficial effects, remain a
potential hazard because women may take large doses
in the belief that more is better. Additionally, capsules
containing 25,000 IU of preformed vitamin A are available on store shelves in the United States. Multivitamins targeted for adult consumption, and potentially
used by women of childbearing age, may contain high
doses of preformed vitamin A. Many multivitamins,
however, also contain folic acid, which reduces the risk
of neural tube defects and possibly other birth defects
(211). Several randomized, controlled intervention tri-
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
was collected prospectively, limitations of this study include the representativeness of the study population to
healthy women of childbearing age and the liberal definition of vitamin A use in the study group, i.e., ⱖ10,000
IU/day for at least 1 wk during the first trimester. Furthermore, confounding factors such as diet and other maternal factors were not controlled for in this study.
The results from randomized, placebo-controlled
studies can potentially provide important information in
assessing a relationship because the design minimizes the
effects of confounding and bias. A randomized controlled
trial in Hungary compared a multivitamin containing 4,000
or 6,000 IU of vitamin A (in addition to 0.8 mg folic acid)
with a placebo. The study found a lower incidence of
spina bifida, anencephaly, and other birth defects among
the infants of multivitamin users (31, 57) (Table 3). This
study, however, was undertaken to determine whether
folic acid in a multivitamin supplement reduced the occurrence of neural tube defects rather than as an examination of the association between vitamin A supplement
use and birth defects. Following this trial these same
authors utilized a large population-based, case-control
data set, the Hungarian Case-Control Surveillance of Congenital Abnormalities (1980 –1994), to explore whether
cases of congenital abnormalities were more likely to be
exposed to high vitamin A intake than healthy controls
(32). These authors report that maternal vitamin A use
was significantly associated with pregnancies resulting in
healthy newborn infants. Smoking, alcohol, and dietary
intake were not assessed in this case-control study.
Preformed vitamin A may be teratogenic; however,
the teratogenic threshold in humans has not yet been
determined. The teratogenicity in humans of doses above
10,000 IU/day of preformed vitamin A has been reported,
but not yet confirmed. Before the Rothman study, the
lowest maternal dose reported to be teratogenic in humans was ⬃25,000 IU (235). It is clear, however, that
exposure to therapeutic doses of certain analogs of vitamin A, e.g., 13-cis-RA (0.5–1.5 mg/kg), results in substantially increased risk of birth defects. As the dose decreases, however, the strength of the apparent
relationship between excess vitamin A ingestion and birth
defects becomes more tenuous. In many of the studies
cited above, it was rare for women to take more than
10,000 IU of supplemental vitamin A per day. This fact has
impacted on the ability to detect an association between
high potency vitamin A supplements and birth defects. It
is encouraging that the frequency of exposure to highpotency vitamin A supplements is apparently rare.
To approximate safe levels of vitamin A intake during
pregnancy, an assessment of vitamin A metabolites in
plasma during pregnancy has been performed. In this
study, endogenous plasma concentrations of vitamin A
metabolites during pregnancy ranged form 0.26 to 7.72
ng/ml (302). The vitamin A metabolites and their concen-
1045
1046
ROSS, McCAFFERY, DRAGER, AND DE LUCA
als have been performed to investigate the use of multivitamin supplements containing folic acid, with or
without vitamin A, during the periconceptual period for
the prevention of neural tube defects. In many trials,
the rate of malformations was significantly lower in the
supplemented compared with placebo groups, or there
was no effect. Because of the wide range of vitamin A
intake in the human population, additional research,
i.e., epidemiologic as well as mechanistic, is needed to
determine whether excess consumption of vitamin A
from supplements or foods, as well as diets deficient in
vitamin A, or the therapeutic use of retinoids constitutes a public health problem.
The discovery, over a decade ago, of the retinoid
receptor families has led to the present view of the retinoids as transcriptional activators, which regulate gene
expression during embryonal development and differentiation. In addition to the important insights into mechanisms responsible for the control of morphogenesis at the
molecular level, these discoveries have also offered suggestions as to how vitamin A deficiency or excess may
disrupt the finely tuned developmental processes, perhaps through a disregulation of RAR and the consequent
effects on homeobox genes, which might be either not
expressed or overexpressed at the wrong place and time,
finally resulting in teratogenesis.
An interesting area for future investigation is the
characterization of the ligands and functions of the retinoid-related orphan nuclear receptors. Among these are
the ROR/RZR (19, 72), RVR/Rev-erb (65), COUP-TF (106),
and NGFI-B (220), and NURR1 (220). Some of these orphan receptors have demonstrated transactivation activity via the same responsive elements as the retinoid receptors. Future work may show that retinoid-like effects
may be evoked by the activation of one or more of these
orphan receptors (224).
This review has highlighted the retinoid ligand and
retinoid receptor knockout models to understand their
essentiality during embryogenesis. New models are being
developed to study retinoid-dependent developmental
events. The use of retinoid receptor antagonists is one
example. A recent study reported that a single oral dose
of an RAR antagonist, AGN-193109, given on 8 dpc produced severe craniofacial anomalies, including eye malformation (111).
An important focus of current research is the metabolism of retinoids during embryogenesis. This area of
research is likely to lend additional insights into the normal process of embryonal development as well as of
teratogenesis. It is known that many of the functions of
vitamin A in embryogenesis are carried out through the
active metabolite retinoic acid, formed by the oxidation of
retinol in a two-step process. Human embryos undergo
normal morphogenesis when converting a small amount
of maternally derived vitamin A (retinol) into RA in a
tissue-specific fashion, but may undergo teratogenesis,
when responding to events (e.g., ethanol intoxication),
which increase or decrease retinoic acid levels above or
below the normal range. Antiepileptic agents (e.g., valproic acid) are other exogenous factors implicated as
human teratogens. Recent evidence suggests that these
compounds may disrupt RA metabolism (205). Additional
work is necessary to clarify if, as well as how, disruption
of retinoid metabolism affects fetal alcohol syndrome,
and perhaps malformations in general.
More research is needed on the enzymes involved in
the conversion of retinol to retinal and the consequent
effects of increases or decreases of the retinol metabolites on gene expression during critical periods of morphogenesis. Mechanisms regulating expression as well as
enzyme activity for both alcohol dehydrogenase and aldehyde dehydrogenase enzymes need to be better understood.
It also reasonable to suggest that vitamin A deficiency pauses a clinical risk similar to excess retinoids. In
support of this concept, Olson and collaborators have
identified a surprisingly profound deficiency in a Iowa
woman enrolled in the Women Infants and Children Program (60).
Finally, it should be considered that research in embryogenesis and teratogenesis may have an important
impact on our understanding of the molecular events
taking place during carcinogenesis. This is because this
process may reestablish, in adult tissues, the high growth
potential characteristic of embryonic tissues. Therefore,
genes that are involved in ontogenesis may also play
fundamental roles during carcinogenesis. Particularly, homeobox genes may represent an interesting area of investigation. The findings that RAR are downregulated (33,
142) or mutated (44) during carcinogenesis and that retinoic acid is an effective differentiation agent in acute
promyelocytic leukemia (248) indicates the involvement
of the retinoid receptors in this process as well and offers
clues for preventive approaches (24, 277).
We are very grateful to the reviewers, whose suggestions
have resulted in a much improved manuscript. We thank Dr.
Lorraine J. Gudas and Dr. Maija H. Zile for help with specific
sections of the review. We thank Matteo De Cosmo for the art
work on Figure 2. Finally, we thank Deborah Smith for help with
generating Figure 4.
Address for reprint requests and other correspondence:
L. M. De Luca, Bldg. 37, Rm. 3A-17, National Institutes of Health,
National Cancer Institute, 37 Convent Dr., Bethesda, MD 20892–
4255 (E-mail: [email protected]).
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IX. CONCLUSIONS AND FUTURE ASPECTS
Volume 80
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RETINOIDS IN EMBRYONAL DEVELOPMENT
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