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Vitamin A in Reproduction and Development


Abstract and Figures

The requirement for vitamin A in reproduction was first recognized in the early 1900's, and its importance in the eyes of developing embryos was realized shortly after. A greater understanding of the large number of developmental processes that require vitamin A emerged first from nutritional deficiency studies in rat embryos, and later from genetic studies in mice. It is now generally believed that all-trans retinoic acid (RA) is the form of vitamin A that supports both male and female reproduction as well as embryonic development. This conclusion is based on the ability to reverse most reproductive and developmental blocks found in vitamin A deficiency induced either by nutritional or genetic means with RA, and the ability to recapitulate the majority of embryonic defects in retinoic acid receptor compound null mutants. The activity of the catabolic CYP26 enzymes in determining what tissues have access to RA has emerged as a key regulatory mechanism, and helps to explain why exogenous RA can rescue many vitamin A deficiency defects. In severely vitamin A-deficient (VAD) female rats, reproduction fails prior to implantation, whereas in VAD pregnant rats given small amounts of carotene or supported on limiting quantities of RA early in organogenesis, embryos form but show a collection of defects called the vitamin A deficiency syndrome or late vitamin A deficiency. Vitamin A is also essential for the maintenance of the male genital tract and spermatogenesis. Recent studies show that vitamin A participates in a signaling mechanism to initiate meiosis in the female gonad during embryogenesis, and in the male gonad postnatally. Both nutritional and genetic approaches are being used to elucidate the vitamin A-dependent pathways upon which these processes depend.
Metabolism of vitamin A (retinol) to all- trans retinoic acid (RA), and the mechanism of RA action. ( A ) Metabolic scheme proposed by Dowling and Wald in 1960 [11]; ( B ) Mechanism ca. 2011. Vitamin A (retinol, ROL) circulates bound to the plasma retinol-binding protein (RBP4) and transthyretin (not shown). RBP4 binds to the membrane receptor STRA6 to facilitate the cellular uptake of retinol in some cells. Vitamin A circulating as part of a chylomicron remnant (CMRE) can also serve as a source of vitamin A for the cell. Note that cellular retinol and RA binding proteins have been omitted for simplicity. Retinol is either esterified by lecithin:retinol acyltransferase (LRAT) and stored, or is oxidized reversibly to retinaldehyde (RAL) by retinol dehydrogenases (RDH/ADH), and further oxidized in irreversible fashion to RA by retinaldehyde dehydrogenase (RALDH 1, 2, or 3). In the nucleus, the RAR/RXR complex is bound to a specific sequence of DNA called the retinoic acid response element (RARE). Binding of RA to the RAR leads to release of the corepressor complex (CoRep) and association with coactivator proteins (CoAct), followed by altered transcription of downstream target genes and ultimately changes in cellular function. RA also undergoes further oxidation by the cytochrome P450 (CYP) 26 family to more polar metabolites. The lipophilic molecule, RA, can act within the same cell in which it is synthesized (autocrine) or can diffuse through the cell membrane to act in nearby cells (paracrine). Abbreviations: ADH, alcohol dehydrogenase; RDH, retinol dehydrogenase; REH, retinyl ester hydrolase; RE, retinyl ester.
( A ) Germ cell development and gametogenesis. Primordial germ cells colonize the gonad in both male and female embryos. The first morphological marker of sex-specific germ cell development is seen in the female embryo when the oogonia enter meiosis. Primary oocytes proceed through the leptotene, zygotene and pachytene stages of meiotic prophase before birth, when they arrest in diplotene of meiosis I. At ovulation, meiosis I is completed, and the secondary oocytes enter meiosis II and arrest again in metaphase. Meiosis II is completed after fertilization. In the male embryo, germ cells are committed to the spermatogenic program but arrest in G0/G1, and do not complete mitosis and enter meiosis until after they are born. Primary spermatocytes entering meiosis I are seen during the first week of life; secondary spermatocytes complete meiosis II forming spermatids and functional gametes called spermatozoa or sperm. In the male, waves of meiosis continue throughout life. ( B ) In the female embryo access to RA or alternatively, another factor indicated as (?), promotes entry into meiosis whereas embryonic male germ cells are maintained in a pluripotent state. Either RA or another factor acts in the embryonic female germ cell to increase Stra8 , essential for entry into meiosis. In the ovary, Fgf9 levels are low. In the male embryo, entry into meiosis is prevented by the action of CYP26B1; high levels of Fgf9 in the testes antagonize Stra8 expression and maintain germ cells in a pluripotent state. (Adapted from [77,78]).
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Nutrients 2011, 3, 385-428; doi:10.3390/nu3040385
ISSN 2072-6643
Vitamin A in Reproduction and Development
Margaret Clagett-Dame
* and Danielle Knutson
Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison,
WI 53706, USA; E-Mail:
School of Pharmacy, Pharmaceutical Sciences Division, University of Wisconsin-Madison,
777 Highland Ave., Madison, WI 53705, USA
* Author to whom correspondence should be addressed; E-Mail:;
Tel.: +1-608-262-3450; Fax: +1-608-262-7122.
Received: 24 November 2010; in revised form: 28 February 2011 / Accepted: 22 March 2011 /
Published: 29 March 2011
Abstract: The requirement for vitamin A in reproduction was first recognized in the early
1900’s, and its importance in the eyes of developing embryos was realized shortly after.
A greater understanding of the large number of developmental processes that require
vitamin A emerged first from nutritional deficiency studies in rat embryos, and later from
genetic studies in mice. It is now generally believed that all-trans retinoic acid (RA) is the
form of vitamin A that supports both male and female reproduction as well as embryonic
development. This conclusion is based on the ability to reverse most reproductive and
developmental blocks found in vitamin A deficiency induced either by nutritional or
genetic means with RA, and the ability to recapitulate the majority of embryonic defects in
retinoic acid receptor compound null mutants. The activity of the catabolic CYP26
enzymes in determining what tissues have access to RA has emerged as a key regulatory
mechanism, and helps to explain why exogenous RA can rescue many vitamin A
deficiency defects. In severely vitamin A-deficient (VAD) female rats, reproduction fails
prior to implantation, whereas in VAD pregnant rats given small amounts of carotene or
supported on limiting quantities of RA early in organogenesis, embryos form but show a
collection of defects called the vitamin A deficiency syndrome or late vitamin A
deficiency. Vitamin A is also essential for the maintenance of the male genital tract and
spermatogenesis. Recent studies show that vitamin A participates in a signaling mechanism
to initiate meiosis in the female gonad during embryogenesis, and in the male gonad
postnatally. Both nutritional and genetic approaches are being used to elucidate the vitamin
A-dependent pathways upon which these processes depend.
Nutrients 2011, 3
Keywords: retinoic acid; vitamin A deficiency; embryonic
1. Background
It has been nearly 100 years since the essential micronutrient, vitamin A, was first described.
In 1913 McCollum and Davis reported that the addition of an ether extract from egg yolk or butter, but
not lard or olive oil, could reinstate growth in rats maintained for several months on a purified ration of
casein, carbohydrates and salt mixtures [1]. We now know that this essential accessory article in
foodstuffs is vitamin A. Using dietary manipulation to induce deficiency in rats, the importance of
vitamin A in both male and female reproduction was soon discovered [2,3]. For a time, there was
confusion over how both yellow-colored foods and those that were colorless, for example extract from
pork liver or cod liver oil, could both yield vitamin A activity [4]. This problem was solved when
Moore fed carotenoids to vitamin A-deficient (VAD) rats in amounts that enabled the animals to
resume normal growth, and showed that only the ―colorless‖ form of vitamin A was found in the livers
collected from these animals [5]. Thus, it was realized that carotenoids (at least a subset) could be
converted to vitamin A, a fact that was fully appreciated when Karrer et al. published the chemical
structures of both carotene and vitamin A [6,7]. In 1946, Arens and van Dorp synthesized vitamin A
acid (retinoic acid), and reported it was as potent as vitamin A in supporting the growth of VAD rats
but could not be converted back to vitamin A [810]. The metabolic scheme in which vitamin A
(retinol) generates the vitamin A aldehyde (retinaldehyde) to support synthesis of the visual pigments,
and its further irreversible oxidation to the vitamin A acid (all-trans retinoic acid, RA) that supports
growth and tissue maintenance was first reported in the landmark paper by Dowling and Wald and this
metabolic scheme stands essentially unchanged today (Figure 1A) [11].
All-trans retinol (retinol, vitamin A) is obtained in the diet from plant sources (carotenoids with
vitamin A activity) or as retinyl esters from animal sources. Retinol has two major fates:
(1) esterification and tissue storage, and (2) oxidative metabolism to all-trans retinaldehyde and further
oxidation to RA. The enzyme lecithin:retinol acyltransferase (LRAT) is responsible for esterifying the
majority of retinol into retinyl esters [12]. The first and rate-limiting step in the production of RA from
retinol results from the action of cytosolic alcohol dehydrogenases (ADH) and microsomal retinol
dehydrogenases (RDH) yielding all-trans retinaldehyde [13]. The irreversible oxidation of all-trans
retinaldehyde to all-trans retinoic acid is catalyzed by several aldehyde dehydrogenases (RALDH),
of the ALDH1A class (ALDH 1A1, 1A2, and 1A3 also known as RALDH 1, 2 and 3) [14,15].
RALDH-independent generation of RA from retinol by CYP1B1 has also been reported [16].
The metabolism of RA at the C4 and C18 positions to oxidative metabolites including 4-hydroxy-RA,
18-hydroxy-RA, and 4-oxo-RA occurs by the action of cytochrome P450 enzymes of the CYP26
family (A1, B1 and C1) [1723]. Vitamin A and metabolites are lipophilic compounds that are
generally found in association with serum and cellular binding proteins [24]. Retinol-binding protein
(RBP or RBP4) carries the majority of retinol in the circulation [25], and a membrane receptor, STRA6,
binds to RBP to enable efficient retinol uptake by a number of cells [26]. In addition, cellular proteins
that bind to retinol (CRBP I, II, and III), and RA (CRABP I and II) have been studied in null mutant
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mice; some have been found to be dispensable, while roles for others have been revealed when animals
are fed a vitamin A-restricted diet [12,27,28].
Figure 1. Metabolism of vitamin A (retinol) to all-trans retinoic acid (RA), and the
mechanism of RA action. (A) Metabolic scheme proposed by Dowling and Wald in
1960 [11]; (B) Mechanism ca. 2011. Vitamin A (retinol, ROL) circulates bound to the
plasma retinol-binding protein (RBP4) and transthyretin (not shown). RBP4 binds to the
membrane receptor STRA6 to facilitate the cellular uptake of retinol in some cells.
Vitamin A circulating as part of a chylomicron remnant (CMRE) can also serve as a source
of vitamin A for the cell. Note that cellular retinol and RA binding proteins have been
omitted for simplicity. Retinol is either esterified by lecithin:retinol acyltransferase (LRAT)
and stored, or is oxidized reversibly to retinaldehyde (RAL) by retinol dehydrogenases
(RDH/ADH), and further oxidized in irreversible fashion to RA by retinaldehyde
dehydrogenase (RALDH 1, 2, or 3). In the nucleus, the RAR/RXR complex is bound to a
specific sequence of DNA called the retinoic acid response element (RARE). Binding of
RA to the RAR leads to release of the corepressor complex (CoRep) and association with
coactivator proteins (CoAct), followed by altered transcription of downstream target genes
and ultimately changes in cellular function. RA also undergoes further oxidation by the
cytochrome P450 (CYP) 26 family to more polar metabolites. The lipophilic molecule,
RA, can act within the same cell in which it is synthesized (autocrine) or can diffuse
through the cell membrane to act in nearby cells (paracrine). Abbreviations: ADH, alcohol
dehydrogenase; RDH, retinol dehydrogenase; REH, retinyl ester hydrolase; RE, retinyl ester.
Nutrients 2011, 3
RA is a ligand for the nuclear retinoic acid receptor (RAR) proteins. There are three major subtypes
of RAR protein (α, β and γ) with additional isoforms resulting from alternate promoter usage and
splicing [29]. The nuclear RARs act as ligand-activated transcription factors to regulate gene
transcription in a cell-type and tissue-specific manner [30]. The all-trans isomer of RA is the highest
affinity endogenous ligand for the RAR [31]. Members of a second protein family, the retinoid
(rexinoid)-X receptors (RXR) heterodimerize with RAR to confer high-affinity binding to DNA. The
DNA to which the RAR/RXR heterodimer binds is called a retinoic acid response element (RARE).
The consensus RARE is composed of two direct repeats of PuG(G/T)TCA separated most often by
5 bases [32]. However, more complex elements that do not adhere to this rule have been described [33].
RAREs serve as enhancer elements and when occupied by the RA/RAR/RXR complex, facilitate
chromatin opening and changes in RA target gene transcriptional activity [34,35]. A large number of
genes are altered when cells or tissues are exposed to RA, however, only a small subset are primary
(direct) targets via RARE-mediated transcription, with the remainder representing downstream
targets [36,37]. A schematic summarizing the metabolism of vitamin A and the cellular mechanism of
RA action is shown in Figure 1B. Note in this review retinoid is a term that refers to compounds
structurally-related to retinol, and in this review, is used to refer to vitamin A and its metabolites.
2. Vitamin A and Reproduction
2.1. Male Reproduction
Vitamin A is required for male reproduction. Early work in the laboratories of Wolbach and Howe
as well as Mason showed that in vitamin A deficiency, the epithelia of the epididymis, prostate, and
seminal vesicle is replaced with stratified squamous keratinizing epithelium, and spermatogenesis
ceases [2,38]. Later work showed that in the VAD rat testes, undifferentiated spermatogonia, Sertoli
cells and a small number of preleptotene spermatocytes remain [3941], whereas in the mouse,
spermatogenesis is arrested at the spermatogonia stage [42]. Upon addition of vitamin A,
spermatogenesis can be reinstituted by stimulating A to A1 spermatogonial differentiation in a
synchronized manner [4244]. The block in adult spermatogenesis resulting from vitamin A deficiency
is shown in Figure 2. Recent work supports the conclusion that the vitamin A metabolite, RA, is
needed both for adult male spermatogonial differentiation (transition to A1) and the entrance into
meiosis [4547].
In 1991, Van Pelt and de Rooij determined that a large dose of RA (5 mg) given by injection twice
a week, when combined with a RA-containing diet, supported the development of spermatocytes, and
their subsequent development into spermatids in VAD rats supporting that RA is the active form of
vitamin A in male reproduction [48]. A CYP26-mediated catabolic barrier comprised of peritubular
myoid cells surrounds the seminiferous tubule, and may prevent RA in the general circulation from
reaching cells in the interior of the tubule, thus explaining why such high doses of exogenous RA were
required [49,50]. Within the normal tubule, the Sertoli cell is believed to generate RA by the action of
Raldh1 [49,51,52], and possibly Raldh2 [50]. Raldh2 is also found in late pachytene and diplotene
spermatocytes, and early stage spermatids.
Nutrients 2011, 3
Figure 2. Spermatogenesis in the adult. Spermatogenesis takes place in the seminiferous
epithelium of testis tubules from puberty through adulthood. Undifferentiated (A-type)
spermatogonia at the base of the seminiferous epithelium divide mitotically until they enter
the differentiation pathway to become A1 spermatogonia. A1 spermatogonia undergo
division to A1A4 and finally B spermatogonia. B spermatogonia divide to produce
preleptotene (primary) spermatocytes that migrate away from the base of the seminiferous
tubule to undergo meiosis. The first meiotic division produces secondary spermatocytes,
and after the second meiotic division, spermatids (haploid cells) begin the differentiation
process (spermiogenesis) to spermatozoa. In vitamin A deficiency, the transition from A to
A1 spermatogonia is blocked [4244].
RARγ null males are sterile and exhibit squamous metaplasia of the seminal vesicles and prostate
glands [53]. RARα null mutants are sterile and show a reduction in spermatozoa, indicating that
nuclear RAR is needed for spermiation [54]. RARα is expressed primarily in the Sertoli cell, Rarβ in
spermatids and RARγ in A spermatogonia [49]. Expression of RARα is needed for differentiation of
the spermatogonia during prepuberty [55]. However, RAR signaling in the Sertoli cell cannot account
for the VAD-induced arrest in spermatogonia differentiation, as deletion of all RARs in the Sertoli
cell does not cause arrest of spermatogonia differentiation in the adult mouse. The cell types
in which RA and its receptors act to support spermatogenesis continues to be a subject of active
investigation [45,49,5557].
2.2. Female Reproduction
In the female, the effect of vitamin A deficiency on reproductive outcome is dependent upon the
time when deficiency is imposed, as well as its severity [58]. When severe vitamin A deficiency is
imposed prior to mating, cornified cells are continuously present in vaginal smears [59,60] and
reproduction fails prior to implantation [3]. VAD female rats continue to ovulate and form corpora
Nutrients 2011, 3
lutea irregularly or at normal intervals, however, degenerated eggs are found in the last portion of the
tube, and there is no evidence that blastogenesis has occurred.
Warkany and Schraffenberger showed that when limited amounts of provitamin A carotenoid are
provided to VAD female rats prior to mating, a less severe maternal vitamin A deficiency is produced
which enables fertilization and implantation to occur, but embryonic death at midgestation often
results [61]. If provided in adequate amounts, retinol will support reproduction and embryonic
development in full [62]. RA in amounts ranging from 2 to 12 mcg/g diet or 40 to 230 mcg/rat/day
given to a VAD female rat is sufficient to maintain normal fertilization, implantation and early
embryogenesis. However, pregnant animals maintained on this level of RA will invariably resorb all
the fetuses. Higher amounts of RA (250 mcg/g diet or approximately 4.5 mg/rat/day) or retinol is
needed by embryonic day 8.5 (E8.5) (late gastrula/early neurula) to support normal embryonic
development and overcome midgestational resorption [6367].
Maternal vitamin A also plays a role in placental development and/or maintenance, as the
chorioallantoic placenta undergoes widespread necrosis by E15.5 in VAD rats supported on
insufficient amounts of RA [68]. Microscopic analysis of the placenta from E14 to E17 reveals
changes in the central region of the junctional zone and the labyrinthine zone of VAD rats supported
on insufficient vitamin A acid, whereas addition of retinyl acetate to the diet prevents these
changes [69]. Microscopic findings suggest the differentiation of the parenchymal cells of the
junctional zone to glycogen cells, and of the chorionic trophoblast cells to the inner trophoblastic
lamina of the trichorial placenta is affected. In summary, the relative vitamin A status of the female,
both at the time of conception and throughout pregnancy, is a critical determinant in reproductive
outcome, and deficiency can lead to either a complete failure of reproduction prior to implantation or
fetal resorption or malformation.
2.3. Germ Cell Development
The generation of sperm and oocytes requires germ cells to undergo meiosis, the process in which
diploid cells give rise to haploid cells. In the female, germ cells enter meiosis I during embryogenesis,
whereas in the male, this process occurs postnatally (Figure 3A). It has been proposed that access of
primordial germ cells (gonocytes) to RA plays an important role in determining when they will enter
meiosis, with female germ cells entering into meiosis after exposure to embryonic RA, whereas, in the
male embryo, this pathway is blocked by the action of CYP26B1, but is enabled after birth [7073].
However, a report appearing when this manuscript was under review raises questions concerning a role
for RA in this model [74].
The culture of embryonic rat ovaries with RA promotes meiotic entry [75], whereas murine
embryonic ovaries (E11.5) cultured in the presence of the RAR panantagonist BMS-204493 for 2 days
do not express the RA-responsive gene Stra8 [70], a gene that is required for meiotic initiation in
female gonocytes [76]. However, a recent report shows that female RALDH2 and RALDH2/3
knockout gonads express Stra8 and undergo meiosis in the absence of detectable RA activity as
assessed using a β2-RARE-lacZ (RARE-lacZ) reporter [74]. If the reporter accurately detects all RA
activity, this new work indicates that meiotic initiation can occur independently of RA signaling.
Nutrients 2011, 3
Figure 3. (A) Germ cell development and gametogenesis. Primordial germ cells colonize
the gonad in both male and female embryos. The first morphological marker of sex-specific
germ cell development is seen in the female embryo when the oogonia enter meiosis.
Primary oocytes proceed through the leptotene, zygotene and pachytene stages of meiotic
prophase before birth, when they arrest in diplotene of meiosis I. At ovulation, meiosis I is
completed, and the secondary oocytes enter meiosis II and arrest again in metaphase.
Meiosis II is completed after fertilization. In the male embryo, germ cells are committed to
the spermatogenic program but arrest in G0/G1, and do not complete mitosis and enter
meiosis until after they are born. Primary spermatocytes entering meiosis I are seen during
the first week of life; secondary spermatocytes complete meiosis II forming spermatids and
functional gametes called spermatozoa or sperm. In the male, waves of meiosis continue
throughout life. (B) In the female embryo access to RA or alternatively, another factor
indicated as (?), promotes entry into meiosis whereas embryonic male germ cells are
maintained in a pluripotent state. Either RA or another factor acts in the embryonic female
germ cell to increase Stra8, essential for entry into meiosis. In the ovary, Fgf9 levels are
low. In the male embryo, entry into meiosis is prevented by the action of CYP26B1; high
levels of Fgf9 in the testes antagonize Stra8 expression and maintain germ cells in a
pluripotent state. (Adapted from [77,78]).
There is clear in vivo evidence that vitamin A is required for the normal onset of meiotic prophase
in ovarian germ cells [79]. While fetal ovarian germ cell number is unaffected by vitamin A
deficiency, the germ cells in embryos with the most severe vitamin A deficiency fail to enter meiosis
as evidenced by a lack of immunostaining for SYCP3 (a gene that encodes a component of the
Nutrients 2011, 3
synaptonemal complex), and the critical RA-responsive gene, Stra8 is nearly undetectable.
When RA is included in the maternal diet at a slightly higher level, but one insufficient to support most
other vitamin A-dependent embryonic processes, a small number of cells undergo meiosis (30%)
compared to 75% of cells in the vitamin A-sufficient group. Thus, it is possible that only a very low
level of RA is needed to initiate meiosis, or alternatively, vitamin A may support meiotic entry by an
alternative mechanism.
The mesonephros has been proposed as the source of RA that initiates the entry of primordial
gonocytes into meiosis [71]. Raldh2 is largely responsible for RA synthesis in the mesonephros, with
some contribution from Raldh3 in the region of the mesonephric duct [71,80]. Expression of Raldh1
mRNA is reported in the adjacent male gonad from E11.5 to 13.5, and to a lesser extent in the female
gonad by E13.5 [81]. The gene encoding the RA-metabolizing cytochrome P450 enzyme, Cyp26b1, is
expressed early in the genital ridge, but is decreased after E12.5 in the female mouse embryo, thus
enabling access of the gonocyte to RA [71]. In contrast, the expression of Cyp26b1 persists in the male
embryonic gonad, and has been proposed to prevent RA from stimulating the gonocytes to undergo
meiosis [70,71]. Embryonic male germ cells undergo G
mitotic cell cycle arrest and do not enter
meiosis until a week after birth. In CYP26B1 null mutant male embryos, RA levels in the testes are
increased and germ cells prematurely enter meiosis at embryonic day 13.5, and this is followed by
apoptosis [73]. CYP26B1 is also required later in embryogenesis to maintain cells in an
undifferentiated state [82]. However, a new report shows that inhibition of CYP26 activity with
ketoconazole results in a similar increase in Stra8 mRNA in cultured wild-type and RALDH2 null
mutant testis/mesonephros complexes, whereas no expression is seen in testes cultured in isolation [74].
This suggests that some factor other than RA from the mesonephros may be affected by CYP26, and
thus control entry into meiosis. Thus, either a very low level of RA that is undetectable by the
RARE-lacZ reporter and that is generated by an enzyme other than RALDH2 is responsible for meiotic
entry, or alternatively, CYP26B1 functions by degrading an unknown inducer of Stra8 or participates
in the synthesis of a factor that inhibits Stra8 expression.
Recently, Li et al. showed that vitamin A is required in the male neonate for germ cells to enter the
first round of spermatogenesis [83]. Vitamin A deficiency was produced in prepubertal life by
maintaining LRAT
female mice on a diet devoid of all vitamin A during pregnancy and lactation.
It was possible to generate deficiency at this time because LRAT
mice cannot store retinol in the
form of retinyl esters in most tissues, and thus the mothers and pups can be more rapidly depleted of
their vitamin A stores than wild-type mice. In the wild-type mouse, Stra8 begins to increase in the
testis by postnatal day 6 [84] and spermatogenesis begins shortly thereafter [85]. However, in the VAD
gonad the expected increase in Stra8 did not occur. Germ cells, although present in normal
numbers, did not undergo meiosis as was observed in the vitamin A-sufficient controls and instead
remained undifferentiated [83]. The addition of retinol to VAD LRAT
pups at postnatal day 5
prevented meiotic failure. Thus, it appears that an environment depleted of vitamin A maintains germ
cells in an undifferentiated state, and vitamin A sufficiency enables entry into meiosis I in both the
developing female and male gonad.
Nutrients 2011, 3
3. Vitamin A and Embryonic Development
3.1. Embryonic Vitamin A Deficiency Studies
Very early evidence that vitamin A is required for embryonic development came from reports of
abnormalities in pigs born to gilts on a VAD diet [86,87]. A range of reproductive outcomes were
reported in gilts fed a vitamin A-free ration from 160 days before breeding and for 30 days thereafter.
Some gilts on the deficient diet failed to show the symptoms of estrous, whereas others became
pregnant, but resorbed all the fetuses. The remaining offspring from litters that survived to term
showed a lack of eye development or no eyes. Cleft palate, accessory ears and arrested ascension of
the kidneys were also observed, but at a lower frequency. In the 1940’s, Warkany and colleagues
published their landmark studies describing a syndrome of defects in rat embryos from VAD mothers
given limited amounts of carotene [61,8895]. Approximately 70% of the VAD female rats
supplemented in this fashion conceived, however, the majority of pregnancies did not continue beyond
midgestation [61]. Ocular defects were most frequently observed in offspring from the few
pregnancies that progressed to term. In order to understand how maternal vitamin A deficiency
affected development, fetuses ranging from embryonic days 12.5 to 20.5 (near term) were taken by
cesarean section whenever evidence of maternal bleeding was observed, and histology was performed.
Of the fetuses recovered in this fashion, defects in eye development were most frequent (49% with one
or more abnormality) and included coloboma, retinal eversion, penetration of the retina by mesodermal
tissue, low insertion of the optic stalk and the cup, and defects in the iris. Defects at lower penetrance
were noted in other systems including the genitourinary tract (42%), kidney (38%), diaphragm (31%),
lung (4%), aortic arch (9%) and heart (4%) [95]. The low penetrance of many of these defects likely
resulted from variation both in the timing and severity of maternal vitamin A deficiency.
Taking advantage of the fact that RA will support early fetal development in VAD rats [65],
See et al. was able to generate the previously described vitamin A deficiency syndrome in 100% of
fetuses (with the exception of cardiovascular defects) from mothers supported on a sufficient amount
of RA up to E10.5, and fed a suboptimal amount of RA thereafter (late VAD rat model, Table 1) [67].
Rapid initiation of VAD is possible using this approach because RA, unlike retinol, is not stored and
has a very short biological half-life. The low penetrance of cardiovascular defects was due to
institution of deficiency after aortic arch and septal development had largely been completed. The
addition of retinol after E10.5 prevented all fetal anomalies from appearing, and the addition of a
higher level of RA led to either a complete or partial rescue of fetal anomalies, supporting the idea that
RA is the functional form of vitamin A in embryonic development. Using nutritional models in which
RA deficiency is imposed either early, or after the 1215 somite stage, a number of defects have been
added to the original fetal vitamin A deficiency syndrome. Nervous system, cardiovascular, and axial
patterning defects result from early deficiency [6366,96], whereas a less well-developed nasal region,
salivary gland hypoplasia, agenesis of the Harderian glands, hypoplasia of the intestinal villi and a
number of skeletal abnormalities arise if RA is limiting at later times [67].
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Table 1. Summary of abnormalities in VAD rat embryos given insufficient RA (late
VAD) * [67], insufficient carotene (VAD syndrome) [95], or observed in RAR null
mutants [9799].
Late VAD
Observed in RAR
null mutants
Eversion of retina
Fibrous retrolenticular membrane
Heartinterventricular septal defect
Diaphragmatic hernia
Intestinal villi hypoplastic
Not reported
Kidneys too close or fused
Not reported
Ureterectopic termination
Undescended testes
Not reported
Skeletal defects
Glandular defects
Nasal region less developed
* Dietary RA restricted after E10.5 (1215 somite stage) of development.
3.2. Role of the Retinoic Acid Receptors
The Rars are widely expressed in development [100]. A series of genetic experiments revealed the
importance of the RAR in mediating the actions of RA in developing embryos [99,101,102]. These key
experiments showed that compound RAR mutants die either in utero or shortly after birth, and
recapitulate most of the defects described as part of the vitamin A deficiency syndrome [61,8895,103]
and the late VAD rat embryo model [67,104]. These genetic studies provide clear evidence that
the vitamin A metabolite, RA, acts through a RAR signaling mechanism in support of normal
embryonic development.
RAR/RXR heterodimers form the functional unit that transduces the RA signal, with one genomic
copy of the Rxrα sufficient to support RXR function in embryogenesis [105]. Ocular defects are
present in some RXRα mutants lacking only the AF-2 domain (activation function) in the
ligand-binding domain [99,106,107]. However, work from the Duester group indicates that ligand
binding to the RAR is sufficient to transduce the signal, as a RAR- but not a RXR- selective ligand is
able to rescue the developmental defects in RALDH2 null mutant embryos [108]. Thus, the need for a
RXR ligand in RAR-driven events remains to be established.
Nutrients 2011, 3
3.3. Transport of Retinoid from Maternal to Fetal Compartment
There are at least two ways in which retinoid is transferred from the maternal blood to the fetus.
The major form of transport is the binding of retinol to a specific binding protein (RBP or
RBP4) [109,110]. Retinyl esters can also be transported in the form of chylomicron remnants or as a part
of very low-density and low-density lipoproteins. During early placentation, RBP is localized in the
endoderm of the yolk sac visceral wall surrounding the embryo, and after definitive placentation, in the
yolk sac membranes as well as in the uterus (decidua basalis) [111,112]. RBP does not cross the placenta
into the fetal circulation [113]. Maternal retinol must be transferred to fetal RBP [114], or alternatively,
retinol may be esterified in the placenta, and delivered to tissues in lipoproteins [115,116]. Although
RBP null mutant embryos from null mutant mothers develop normally on a vitamin A-sufficient diet,
embryogenesis is perturbed when maternal vitamin A intake is restricted [110]. Thus, when RBP is not
present, circulating retinyl ester appears to represent the main pathway for the provision of retinoid
from mother to embryo, provided maternal vitamin A intake is adequate [113].
3.4. Embryonic RA Synthesis and Catabolism
It is clear that RA is essential for embryonic development. However, too much RA at critical stages
can result in embryo lethality or malformation [117120]. Thus, regulation of the amount of RA that is
available to the embryo at specific times and to a given tissue site is of critical importance. Both the
distribution and function of enzymes involved in RA synthesis and degradation have been studied
intensively in developing embryos. Interestingly, both the levels and domains of Cyp26 gene expression
in the developing embryo are affected by retinoid status, whereas the Raldhs are unaffected [121].
Of the retinol dehydrogenase enzymes, RDH10 plays a key role, as loss results in embryonic
lethality by approximately E13.0 [122]. These embryos (trex mutants) show a spectrum of defects
consistent with vitamin A deficiency, although they do not die as early in gestation as severely
RA-depleted embryos [65,123], indicating that one or more additional enzymes must also be active in
generating retinaldehyde for RA synthesis. Supplementation with RA rescues the abnormalities,
supporting a role for RDH10 in RA biosynthesis. A recent detailed examination of Rdh10 in the avian
embryo shows that the expression domain is often smaller than that of the corresponding Raldh,
leading the authors to suggest that retinaldehyde may be transferred between cells [124]. Recent work
in Xenopus shows that RA down regulates XRDH10 transcripts, and this may serve as an additional
mechanism to regulate endogenous retinoid levels [125]. Adh 1, 3 and 4 enzyme family members are
also expressed in developing embryos [126,127], however, mutation does not result in embryo
lethality [128,129]. It should be noted, that ADH3 and ADH4 null mutant mice do undergo early
postnatal lethality if maintained for two generations on a VAD diet [129]. Thus, to date, RDH10 is the
only retinol dehydrogenase shown to be indispensable for embryonic development under normal
dietary conditions.
Numerous retinaldehyde dehydrogenase single and compound deletion mutants have been
generated and studied [123,130135]. Deletion of RALDH2 is lethal early in development, whereas
RALDH3 null mutants die at birth [131], and RALDH1 mutants are viable [133]. Thus, RALDH2 is
the earliest expressed family member to produce RA in the embryo, and is believed responsible for all
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RA signaling activity from E7.5E8.5 in the mouse [136]. Shortly thereafter, RALDH1 and RALDH3
contribute to RA synthesis in the eye and olfactory pit. Cyp1B1 can also generate RA and is expressed
at known sites of RALDH-independent retinoid signaling [16], however, null mutant mice for this
gene are viable [137]. Recent work in zebrafish indicates that the availability of retinaldehyde for RA
synthesis may represent another point of control, with a short-chain dehydrogenase/reductase family
member, DHRS3a, which catalyzes reduction of retinaldehyde to retinol serving as a RA-induced
feedback inhibitor of RA biosynthesis [138].
The importance of CYP26 enzymes in restricting RA distribution and availability is illustrated by
studies in which one or more is genetically ablated. Mutation of Cyp26A1 is embryonic lethal and
mutants die during mid- to late gestation with symptoms mirroring those observed in RA toxicity
including caudal defects and occasionally exencephaly [139141]. CYP26B1 null mutants die
immediately after birth and exhibit limb, male germ cell, and craniofacial abnormalities [73,142,143];
whereas loss of CYP26C1 alone does not produce an overt phenotype [144]. Deletion of both mouse
CYP26 A1 and C1 is lethal at E9.5 to E10.5 [144], and deletion of all three CYP26 family members in
the mouse is also lethal early in embryonic life, and produces a duplication of the body axis [145].
Thus, CYP26 enzymes play a very early and important role in regulating access of the embryo to RA.
3.5. RA in the Early Embryo
Using direct identification by HPLC, all-trans retinaldehyde is detected in embryos as early as the
egg cylinder or pre-primitive streak stage, whereas RA is detected between the mid-primitive streak
stage and the late allantoic bud stage [146]. Expression of a β2-RARE-lacZ (RARE-lacZ) transgene that
is activated in reporter mice when retinoid interacts with the RAR, has been used extensively as a
marker of retinoid-signaling activity in vivo [147]. In the post-implantation embryo, definitive staining
is noted throughout the length of the primitive streak and the appearance of staining is coincident with
formation of the neural plate. The time of initiation of RA synthesis within the embryo proper has also
been deduced from studies of retinaldehyde dehydrogenase mRNA (Raldh2). It is first detected at the
mid-primitive streak stage adjacent to the node and primitive streak [148,149]. The early activity of
Raldh2 in the embryo, along with expression in the visceral endoderm (extraembryonic) is required for
vascular development in both the embryo proper, as well as the yolk sac [150152]. Prior to this time
RA in the embryo is believed to be of maternal origin [145]. HPLC studies of embryonic tissues at
later times confirm that all-trans retinol and RA are the primary retinoids detected, with retinol clearly
the most abundant [153155].
The importance of protecting the embryo from RA at times when it is not needed is highlighted by
recent work on embryos null for all three CYP26 family members. RA is available from the maternal
circulation, and Raldh2 is expressed in the endometrial stroma and decidua [156]. Raldh2 is highly
expressed in the endometrial stroma by E2.5, remains high after implantation, and expression is
reduced as stromal cells undergo decidualization. Uehara et al. report that the developing embryo is
protected from maternal sources of RA by Cyp26 family members expressed in extraembryonic cell
types including extraembryonic endoderm and ectoderm, visceral endoderm and ectoplacental cone
that surround the murine embryo proper at E5.5 and E6.25 [145]. When all three CYP26 family
members are deleted, embryos show a duplication of the body axis resulting from expanded Nodal
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expression driven by RA throughout the epiblast. LacZ staining that results from RA-driven expression
of the RARE-lacZ reporter is not normally detected in wild-type embryos at E6.25, but is abundantly
expressed in CYP26A1/B1/C1 triple knockout embryos. Thus, the CYP26 catabolic enzymes play a
key role in regulating the exposure of the early embryo to maternal RA.
3.6. Early Nervous System Development
Vitamin A is required for many aspects of nervous system development including patterning and
neural differentiation [157,158]. One of the best-studied functions of RA is in the developing hindbrain
where it contributes to the anteroposterior patterning of the neural plate [159]. When induced, the
neural plate is initially anterior or forebrain-like. Induction of more posterior domains occurs by the
actions of RA, Wnts and FGFs, with RA specifying the posterior hindbrain and cervical spinal
cord [160]. Patterning in the vertebrate hindbrain involves segmentation, a strategy that is used to direct
the diverse range of nerves and craniofacial structures essential to hindbrain function [161]. Hindbrain
segments called rhombomeres (r), are transient structures that express a distinct set of cellular and
molecular properties, including an ordered pattern of Hox gene expression. Using the VAD quail
model, the Maden and Zile laboratories first observed that the posterior region of the hindbrain
(rhombomeres 47) does not develop in VAD embryos [162]. The effect of depleting vitamin A by a
number of means in a variety of organisms was then studied in great detail. Loss of function mutation
of RALDH2 in both mice and zebrafish results in anteriorization of the hindbrain [163165]. VAD rat
embryos generated by severely restricting the amount of RA fed to deficient mothers at the beginning
of pregnancy results in a loss of posterior rhombomere segmentation, with a significant shortening of
the hindbrain [65]. In addition, ectopic otic-like vesicles appear posterior to the orthotopic vesicle. By
varying the amount of RA added back to the maternal diet, this study provided evidence that an
increasing amount of retinoid is needed for the correct specification of more posterior rhombomeres.
RARα/γ compound mutants show a hindbrain phenotype similar to that seen in vitamin A
deficiency [166], whereas the RARα/β compound null mutants present a less severe phenotype [167],
indicating that RARα and RARγ play an important role at early stages of hindbrain patterning.
Elimination of all RARs in zebrafish severely disrupts hindbrain patterning [168]. Using cultured chick
embryos treated with RAR antagonist at various stages of development, distinct developmental time
windows were identified when RA specifies specific rhombomeres in a rostrocaudal sequence [169].
The RA needed for antero-posterior patterning is produced in the anterior paraxial mesoderm by
RALDH2 and diffuses into the adjacent central nervous system (Figure 4A). After this, Raldh2 mRNA
is found in somitic as well as presomitic mesoderm, but not in the node [170]. The generation and
diffusion of RA has been proposed to form a gradient (higher caudal/lower rostral) that patterns the
hindbrain. However, the ability of exogenous RA to rescue development implies that mechanisms
other than simple diffusion of RA from a localized posterior source must be involved in generating
differential responsiveness along the hindbrain anterior to posterior axis.
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Figure 4. Schematic showing the location of RA and Cyp26 expression in a presomitic
mouse embryo undergoing (A) patterning of the neural plate and (B) later during the
course of hindbrain patterning. (A) RA generated by RALDH2 in the posterior mesoderm
forms an early anterior boundary of activity in the neural plate at presumptive rhombomere
(pr) 2/pr3, whereas Cyp26A1 and Cyp26C1 are expressed rostral to the pr2 border;
(B) By E8.0 to E8.5, RA is being expressed by the somites and anterior presomitic
mesoderm, and acts on the overlying hindbrain and spinal cord. The activity of the CYP26
enzymes regulate access of the neuroepithelium to RA (Adapted from [139]).
The importance of CYP26 family members in restricting access of the developing hindbrain to RA
was first exemplified by genetic ablation of CYP26A1, resulting in hindbrain posteriorization. The
rhombomere-specific expression pattern of the Cyp26 mRNAs led to the proposal that boundaries of
RA activity are created by their expression [23,141,171] (Figure 4B). For example, Cyp26A1 is
initially expressed in the anterior epiblast and neural plate [18] and forms a boundary at presumptive
r2/r3 [141]. This is followed by the later expression of Cyp26A1 in r2 and Cyp26C1 in r2 and
r4 [18,21,139,144]. Accordingly, CYP26A1 mutants show hindbrain abnormalities just rostral
to the region affected by retinoid deficiency, including posterior transformation of r2/3 to a r4-like
character [139,140], however, individual CYP26B1 and CYP26C1 mutants develop normally [143,144].
A series of detailed studies using mouse embryos carrying the RARE-lacZ transgene led to a further
posit that the initial gradient of RA entering the posterior hindbrain is converted into RA boundaries
that shift over time [172]. It was proposed that CYP26C1 could play a role in restricting RA activity to
the rhombomere 4/5 border, although CYP26C1 null mutants reportedly do not show abnormalities at
this boundary as assessed by HoxB1 and RARE-lacZ staining [144]. However, CYP26A1/C1 double
knockouts do exhibit even more severe hindbrain abnormalities than CYP26A1 mutants alone,
including a grossly enlarged posterior hindbrain at the expense of anterior structures [144].
Interestingly, in Cyp26 morpholino studies in zebrafish, restriction of the RA-responsive gene, Vhnff1,
is determined by the posterior limit of Cyp26C1 at the r4/r5 boundary, a function that can be
compensated for by CYP26B1 [173]. Thus, the combined action of these enzymes along with the
dynamic changes in where they are expressed during development must prevent RA from reaching
specific anterior structures. Although not identical, recent models explaining how RA patterns the
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developing hindbrain all include an important role for CYP26 family members in regulating the
formation of waves or pulses of RA in this dynamic process [23,144,172,173].
When hindbrain patterning is underway, exposure of the anterior brain to RA is prevented by the
action of CYP26A1 and CYP26C1, and loss of CYP26A1 renders embryos more sensitive to the
teratogenic effects of excess exogenous RA on head truncation [174]. By regulating levels of RA in
the anterior brain, the CYPs may enable a ligand independent function of the RARs. Based on studies
in Xenopus embryos, unliganded RARs in the head region at this early time are proposed to function as
transcriptional repressors, that if activated inappropriately by RA, lead to activation of target genes that
should remain off [175]. Thus, teratogenesis may result from over activation of genes that are normally
upregulated by RA and/or a loss of gene repression by unliganded RAR.
A role for RA in forebrain patterning was initially proposed based on studies in chick embryos
exposed to RAR and RXR antagonists [176] and from the VAD quail model in which the size of the
telencephalic vessel is reduced and the diencephalon is abnormally patterned [177]. Additional studies
in VAD quail indicated that the vitamin was needed to correctly position anterior and dorsal
boundaries in the forebrain via modulation of FGF8 and Wnt signaling [178]. RA was also reported in
chick to contribute to regionalization of the telencephalon along the dorsoventral axis, and RALDH3
produced by the head ectoderm was proposed as the retinoid source [179]. However, RALDH2/3
compound null mutant mice reportedly do not show defects in the early molecular determinants of
forebrain patterning [178,180]. Instead, Molotkova and colleagues propose that RA functions at later
times in forebrain development, and provide evidence that RA generated by RALDH3 in the lateral
ganglionic eminence is required for normal expression of the dopamine D2 receptor in the nucleus
accumbens and medial striatum [180].
Additional roles for RA in later development of the central nervous system (CNS) are beginning to
emerge [181]. McCaffery’s group found that RA is produced by RALDH2 in the meninges overlying
the hindbrain at mouse embryonic day 13, and that RA activity as assessed by RARE-lacZ is present in
precerebellar neurons migrating around the hindbrain circumference to form the inferior olive and
pontine nuclei [182]. This group went on to show that RA is required for the generation of posterior
neurons in the inferior olive, as the posterior inferior olive is significantly smaller in late VAD rat
embryos compared to vitamin A-sufficient controls [183]. In the meninges covering the cortex, levels
of Raldh2 increase in the mouse embryo from E13 to E14 onward, to a maximal level in the newborn
brain [184]. Recently, RA from the meninges was reported to function in cortical development by
regulating the switch of radial glia cells in the ventricular zone from symmetric to asymmetric
division, resulting in the formation of neurons or intermediate progenitor cells [185]. It will be
important to determine whether RA regulates stem cell division/differentiation in any other areas of
the CNS.
3.7. Spinal Cord and Other Neuronal Development
RA plays a role in the development of caudal structures, including the neural tube that forms the
spinal cord and in somite development. These tissues arise from the node-streak border, a region that
comprises the caudal end of the node and the rostral end of the primitive streak [186]. Although primary
neurulation occurs in both RALDH2 mutant mice and VAD chick and rat embryos [65,123,187], the
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RALDH2 mutant is reported to have a thinner neuroepithelium [149] and VAD quail embryos have
fewer spinal cord neurons and smaller somites [162,188]. Raldh2 is first expressed during gastrulation
in the mesenchyme adjacent to the node and the primitive streak [148]. In the mouse and chick
embryonic axis, RA from the anterior presomitic mesoderm and somites promotes neural
differentiation by inhibiting the expression of Fgf8 [189]. In RALDH2 mutants, primitive streak and
mesodermal markers are expanded at the expense of those representative of the prospective
neuroepithelium [149].
RA from the adjacent somites is needed to generate certain interneurons and ventral spinal cord
motor neurons [190,191]. RA generated from within the spinal cord is required for the differentiation
of a subset of lateral motor column (LMC) neurons that extend axons into the limb [192,193].
Interactions between HOXD10, which defines rostral boundaries of the lumbosacral LMC, and
HOXD11, which opposes the effects of HOXD10, influence the distribution of motor neuron subtypes
along the rostrocaudal axis. HOXD11 may also serve to restrict RALDH expression to regions where it
is needed to support LMC neuron survival and maturation [194].
There is evidence that RA promotes the survival of sympathetic neurons by inducing
responsiveness to neurotrophins [195197]. RA is also linked to the promotion of neurite
outgrowth [157,162]. Recently, a number of genes including neuron navigator 2 (Nav2) and Nedd9
were discovered in a screen for RA-responsive genes in a neuroblastoma cell line that elaborates
neurites in response to RA [198200]. Nedd9 is a direct downstream target of RA, whereas a
functional RARE has not yet been identified in the Nav2 gene [33]. When NAV2 is knocked down, the
neuroblastoma cell is no longer able to extend neurites in response to RA [201]. The human Nav2 gene
can rescue defects in axonal elongation in the C. elegans mutant, UNC-53 (the homolog of Nav2 in the
worm), and a mouse mutant hypomorphic for NAV2 also shows defects in cranial nerve development
and general neurite density [202]. NAV2 may function as a link between actin remodeling and
microtubule dynamics during neurite outgrowth.
3.8. Eye Development
Recognition of the importance of vitamin A in eye development first came from the experiments of
Hale in which piglets from deficient mothers were born blind [86,87]. The morphologic
consequences of vitamin A deficiency in rat embryos were examined in great detail by Warkany and
Scharffenberger [61,88] and Wilson et al. [95]. They noted dysmorphogenesis of the anterior eye
segment, retina and optic disc, although the penetrance of the defects was extremely variable. Using a
model in which normal development is supported in VAD rat embryos up to embryonic day 10.5
(1215 somite stage) with RA and deficiency is induced thereafter, See et al. was able to produce eye
defects in 100% of embryos which included; coloboma, absence of the anterior chamber of the eye,
rudimentary iris or loss, fusion of the lens and cornea, thickening of the eyelid tissues, reduced size of
the conjunctival sac, folding of the retina, absence of the vitreous body and the presence of a fibrous
retrolenticular membrane [67,104]. The addition of retinol at E10.5 provided a full rescue of all
defects, showing that all the defects were attributable to retinoid deficiency imposed after this time.
The addition of a high level of RA after E10.5 dramatically improved eye development, supporting
that vitamin A acid is the active moiety.
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The RA synthesizing enzymes, RALDHs, show a very dynamic pattern of expression in the
developing eye. In the mouse, Raldh2 (also called V2 activity) is transiently expressed in the optic
vesicle neuroepithelium at E8.5 [132,148,203], after which Raldh3 (also called V1 activity) is
expressed in the ventral neural retina and pigmented neuroepithelium [134,204206] and Raldh1 (also
called ADH-2) is expressed in the dorsal retina from E9.5 onward [134,207]. RARα is expressed in all
layers of the developing murine neural retina, whereas RARβ is expressed in the inner nuclear layer
from embryonic day 14 to postnatal day 7 [208,209]. Genetic ablation of two or more retinoid
receptors in mice results in eye defects that are largely similar to those seen in VAD rat
embryos [97,209211]. A number of eye defects in RALDH3 and RALDH1/3 compound
mutants [134,135,178] are also similar to those observed in late VAD rat embryos.
Using the late VAD rat embryo model, it is possible to identify the times when vitamin A must be
present to support eye development by adding back retinol at successively later times ranging from
E11.5 to E15.5, and evaluating eye development at E18.5 [104]. A full rescue of all structures is
achieved if retinol is added by E11.5, whereas addition on or after E14.5 is ineffective. Retinol added
as late as E13.5 completely prevents retinal folding, formation of a fibrous retrolenticular membrane,
and loss of the vitreous body but is ineffective in preventing anterior eye segment defects, whereas
addition by E12.5 improves or rescues the majority of defects in anterior eye segment development
(absence of anterior chamber, rudimentary iris, corneal-lenticular stalk fusion, and small conjunctival
sac) and prevents coloboma of the retina and optic disc in the majority of fetuses. This study also
revealed that the cells within the retina of late VAD embryos lose their characteristic shape and
orientation along the apical-to-basal axis of the retina, with the appearance of gaps or holes in the
neural retina that worsen as retinol is added at successively later times. Interestingly, the increase in
cell adhesion proteins, N-cadherin and β-catenin, that normally occurs with development is not seen in
VAD retinas. Additionally, a reduction in cyclin D1 labeling is observed in the retina of late VAD
fetuses, suggesting that cell proliferation is also disrupted. These effects may contribute to retinal
collapse seen in VAD rat embryos receiving no supplemental retinol, or retinol on or after E14.5.
Using this temporal model, See et al. also clearly showed that a lack of optic fissure closure does not
lead to retinal folding/collapse, as a well formed retina is produced by adding retinol at a time that is
too late to prevent coloboma [104]. Retinal thinning and disruption of cellular organization and
adhesion is a more likely reason for collapse of the retina seen at late stages of severe deficiency.
RARα has been reported to be the sole receptor transducing the RA signal in the retina beyond
E10.5 based on the inability to detect RARE-lacZ expression in the RARα null mutant mouse
retina [212]. In the absence of this receptor, however, the mouse retina appears to develop normally
leading to the conclusion that RARα is unnecessary for the developing mouse retina [213].
If RARE-lacZ expression is an accurate readout of all RAR-mediated signaling in the neural retina of
RARα null mutants, then the neural retina would appear not to be a direct target of RA and its
receptors. If so, then it is possible that adverse effects of vitamin A deficiency on neural retina
development could occur by a loss of paracrine action of RA in a secondary tissue, as is proposed for
retinoid support of anterior eye segment development (Figure 5). Thus, it is unknown whether RA
generated in the retina acts locally to regulate retina development.
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Figure 5. Schematic showing the proposed sites of RA function during eye morphogenesis
(left) and differentiation (right). At early stages of eye development, RA generated by
RALDH1 and RALDH3 acts as a paracrine signal binding to RARs located in the perioptic
mesenchyme to support anterior eye segment development and closure of the optic fissure.
Pitx2 is a RA/RAR-regulated transcription factor that is required both for anterior eye
segment morphogenesis, as well as closure of the optic fissure. At later stages of
development, RA promotes differentiation of the neural retina. The mechanism is unclear,
but could involve either a paracrine effect of RA outside of the neural retina, or a direct
effect on the cells within the retina itself.
The molecular basis for the vitamin A deficiency-induced defects in anterior eye segment
development and in the appearance of coloboma has been the subject of several recent studies. It does
not involve abnormalities in dorsoventral patterning genes, but rather, disruption of paracrine signaling
from the retina to the perioptic mesenchyme. Compound deletion of both RARβ and RARγ in the
neural crest cell-derived periocular mesenchyme using Wnt1-cre to direct excision results in eye
defects including abnormalities of the cornea and anterior chamber [134,212]. Similarly, RALDH1/3
compound null mutants show malformations of the anterior segment of the eye, including agenesis of
the corneal and iris stroma, agenesis of the anterior chamber and less frequently, coloboma of the
retina [134,135]. Thus, RA from retina is believed to signal in paracrine fashion to the perioptic
mesenchyme to support the development of anterior eye structures. Defects result from the
loss of control of programmed cell death in this region, and by loss of RA-mediated Pitx2,
Foxc1, Eya2 and Dkk2 expression, and subsequent loss of repression of Wnt/β-catenin
signaling [134,135,212,214]. Pitx2 encodes a homeodomain transcription factor that is essential for
anterior eye segment development, and is also required for optic fissure closure [215]. Pitx2 is down
regulated in the periocular mesenchyme of late VAD embryos that not only show defects in anterior
eye segment development, but also retinal/optic disc coloboma at 100% penetrance [67,104]. Very
recently it was shown that the Pitx2 gene contains a RARE located 4.3 kb upstream of the promoter,
and thus is a direct target of RA and its receptors [214]. Duester and colleagues propose that RA
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signaling activates Pitx2 in the perioptic mesenchyme, which in turn, induces Dkk2 and suppresses
local Wnt signaling in the regulation of normal anterior segment development. Zacharias et al. has
extended this model to include a role for canonical Wnt signaling in the maintenance of Pitx2
expression after its initiation by RA, and prior to the time that Wnt signaling is suppressed [216].
3.9. Somites and Skeleton
RA plays a role in the initiation of differentiation of the anterior region of the presomitic mesoderm
from which the new somites originate [217]. VAD quail embryos have smaller somites than their
vitamin A-sufficient counterparts, and also have an expanded FGF8 domain in the presomitic
mesoderm [188]. In RALDH2 null mutants, the expression of Fgf8 mRNA in the primitive ectoderm
(epiblast) is shifted anteriorly such that it enters the node ectoderm and neuroectoderm, and somite
development is abnormal [170]. FGF signaling is important in the process of somitogenesis, and the
opposition of FGF and RA signaling is also important in regulating somite size. A reduction in FGF in
the presomitic mesoderm below a threshold value is needed to position the future somite
boundary [218,219]. Higher levels of FGF8 and Wnt3a signaling maintain cells in an undifferentiated
state, whereas exposure to RA along with lower FGF8 and Wnt3a initiates differentiation, and these
factors are believed to control the developmental switch at this boundary [217]. Recent work in chick
indicates that RA may also be involved in the termination of the process of segmentation [220].
RA signaling is involved both in the specification of the axial identity of future somites as well as in
later stages of skeletal development. Somites, although similar by appearance, develop into distinct
structures dependent upon their axial position. Determination of initial axial identity is believed to
occur in mesodermal cells prior to somite formation [221]. VAD rat embryos show anterior vertebral
transformations throughout the axial skeleton that can be rescued only if vitamin A is provided on or
before embryonic day 8.75, a time that precedes appearance of the first somite [66]. Genetic deletion
of several RARs produces defects in axial development with anterior cervical transformations similar
to VAD embryos [53,97,209]. However, in these mutants, either posteriorization at the cervical/thoracic
junction is observed, or no vertebral changes caudal to this region are reported [97,209]. Alteration of
Hox gene expression appears to be major way in which retinoids affect positional information along
the anteroposterior body axis [222225].
Vitamin A is also required for skeletal development beyond its role in presomitic embryos. Rat
embryos given sufficient RA up to embryonic day 10.5 (approximately the 1215 somite stage), but
made deficient thereafter exhibit hypoplastic cranial bones, defects of the thyroid, cricoid and tracheal
cartilages as well as agenesis of the neural arch of cervical vertebrae 1 (C1) and ectopic bone in the
dorsal regions of C1 [67]; defects in this region bear many similarities to those observed in RARα/γ
compound null mutants [97,98]. Late VAD rat embryos also exhibit gross malformation of the sternal
and pelvic regions [67]. Sternal malformations have been reported in RBP null mutant embryos from
RBP null mothers fed inadequate vitamin A [110]. Surprisingly, late VAD rat embryos have anterior
vertebral transformations in the cervical axial skeleton up to and including the thoracic juncture, along
with posteriorization events at the thoracic and sacral levels of the skeleton [67]. As discussed above, a
number of RAR mutants show both cervical anteriorizations and posteriorization at the cervical
thoracic junction [97,209]. It is also interesting that administration of excess RA to the mouse at E7
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(presomitic; equivalent to E8.59 rat) produces posteriorizing transformations throughout the skeleton;
however, excess RA at E8.5 (equivalent to E1010.5 rat), produces anterior transformations starting at
vertebrae 15 (thoracic vertebra 8, T8) whereas excess RA at even later times, yields both rostral
posteriorizing and caudal anteriorizations [223,224]. Thus, vertebral identities, initially specified at the
late primitive streak phase, can also be respecified at later times in mouse development when the
vertebrae precursors (the somites), differentiate, and the sclerotome cells begin to form vertebrae.
In summary, vitamin A plays a normal role in the maintenance of vertebral identity as well as in the
development of skeletal elements.
Much of the craniofacial skeleton originates from neural crest cells [226]. Frontonasal agenesis has
been reported in compound RAR null mutant mice [97] and RBP null mutant mice on a
vitamin A-deficiency diet [110]. Dupe and Pellerin recently showed that selective ablation of RARα
and RARγ subtypes using the Wnt1-Cre promoter leads to agenesis of the frontonasal skeletal
elements, but does not appear to adversely affect the early survival and migration of neural crest
cells [227]. Deletion of all three RAR subtypes using the selective promoter produces a similar
phenotype, indicating that RA and RA act cell-autonomously in neural crest cells to direct
morphogenesis of these skeletal elements.
3.10. Heart Development
Cardiac and aortic arch defects were observed as a part of the early vitamin A deficiency syndrome
in rat embryos [93,94] and in RAR compound null mutant mouse embryos [98,102]. When severe
VAD is imposed in quail, the initiation of heart morphogenesis is disrupted [88,228,229].
In VAD quail embryos, vascular networks are absent and the heart appears ballooned and
non-compartmentalized, and is randomly-positioned without an inflow tract at the posterior site [228].
Inability of the heart tube to undergo looping is also observed in RALDH2 mouse mutants [230]. The
sinuatrial (venous) valve, a transient structure that flanks the orifice between the sinus venosus and
right atrium, is not formed properly in rat embryos deprived of RA for one day (E9.510.5), and
results in improper channeling of venous blood and anterior cardinal vein distension [64]. If RA
deficiency is imposed after E10.5 in the rat embryo, a time when the cardiac primordium has
completed looping and the primitive vasculature is established, early cardiac and aortic arch defects
are not observed [67]. In summary, roles for vitamin A in mammalian heart development include:
heart tube patterning and looping, chamber and outflow tract septation, ventricular trabeculation,
cardiomyocyte differentiation and coronary vessel development [231233]. A more in depth
discussion of the molecular pathways involved in heart morphogenesis can be found in several recent
reviews [229,232,234].
3.11. Kidney and Urinary Tract Development
The requirement for RA in the developing kidney and urogenital tract has been illustrated in several
animal models. In rodents, maternal vitamin A deficiency results in embryonic renal hypoplasia, the
severity of which depends on the extent of vitamin A deprivation [67,91,110,235]. Ectopic kidneys,
renal fusion and failure of the renal pelvis and calyx to undergo dilatation, in addition to ectopic
ureteric openings and other genitourinary tract defects have been reported in VAD rat fetuses [67,91].
Nutrients 2011, 3
RALDH2 null mutant mice lack nephric ducts [123] and in RARα/β2 double receptor mutants,
nephron progenitors, stromal cells and ureteric bud tips are all greatly reduced or completely absent at
birth [98,236]. These compound RAR mutants also have incorrectly positioned distal ureters,
hydronephrosis and megaureter.
Signaling between the ureteric bud epithelium that forms the collecting duct system, the
metanephric mesenchyme that differentiates into nephron, and the stromal mesenchyme that
differentiates into the renal interstitium is important during early kidney development. In the
embryonic kidney, Raldh2 is localized in stromal mesenchyme of the outer cortex and Raldh3 is
expressed in the ureteric bud [130,237,238]. RA and its receptors are needed to maintain Ret
expression [237], a gene critically required for formation of the ureteric bud and its branching in the
kidney [239241]. The forced expression of Ret in ureteric bud cells in RARα/β2 double receptor
mutants rescues renal development, restoring ureteric bud growth and stromal cell patterning [237].
Recent work shows that RA generated by RALDH2 in stromal mesenchyme acts in paracrine fashion
to activate RA-receptor signaling and Ret expression in ureteric bud cells [242].
In kidney, the number of embryonic branching events determines the final number of nephrons an
individual will have for life, and it has been proposed that suboptimal nephron number at birth
increases susceptibility to acquired renal disease and essential hypertension later in life [243246].
Studies in rodents suggest that even mild vitamin A deficiency (a 50% decrease in circulating vitamin
A concentrations) can lead to impaired branching and a 20% reduction in the number of
nephrons [235]. Quite remarkably, a single injection of RA to a control group of pregnant rats at
midgestation (E11) led to supernumerary nephron endowment in the kidneys of their offspring. RA
given intraperitoneally at E11.5 is also able to increase nephron endowment in offspring exposed to
maternal protein restriction without affecting body weight or kidney size, such that the number of
nephrons per volume of kidney tissue is increased in these rat pups above that seen in the kidneys of
control offspring [247]. Hence mild vitamin A deficiency in pregnancy may correlate to sub-clinical
deficiencies in nephron number and slight nephron deficits that are not recognized at birth, but could
possibly contribute in the long-term to renal failure and hypertension.
3.12. Diaphragm
The diaphragm functions as the primary muscle of respiration and forms a physical barrier between
the thoracic and abdominal cavities. Congenital diaphragmatic hernia (CDH) occurs in approximately
one in 3000 births, and is associated with high neonatal mortality [248]. Vitamin A is essential for
normal diaphragm development, and it has been hypothesized that disruption of retinoid signaling may
contribute to the etiology of the human disorder. A recent report shows that there is a relationship
between low cord retinol and RBP levels and CDH in newborn infants [249].
A herniated diaphragm was observed as a part of the early vitamin A deficiency syndrome [89], and
appears at a 100% penetrance in RA-supported VAD embryos given insufficient RA after E10.5 of
development, whereas a normal diaphragm results if the mother is supplemented with retinol after this
time [67]. Diaphragmatic hernia is also observed at low penetrance in RARα/β compound null mutant
mice [98]. Congenital diaphragmatic hernia in humans is linked to mutations in Stra6, which encodes
Nutrients 2011, 3
for a membrane RBP receptor [26,250], as well as to CDH-affected chromosome loci encoding for
several other retinoid-related genes [251].
The pleuroperitoneal fold (PPF) is a transient structure formed at the union of the pleuro-pericardial
folds and the septum transversum and represents the component of the primordial diaphragm through
which muscle precursor cells and pioneer axons of the phrenic nerve migrate to form the mature
diaphragm. The PPF is fully formed by E13.5 in vitamin A-sufficient rat embryos, but it is abnormal in
late VAD embryos [252]. Similar defects in PPF development are seen in rat embryos treated with
nitrofen and several other CDH-inducing teratogens that interfere with the synthesis of RA and RA
signaling in vivo [253,254]. Raldh2 mRNA is expressed in the PPF, and RARs α, γ and RXRα are
most strongly expressed in the nonmuscular mesenchymal cells of the PPF. Thus, vitamin A signaling
in the developing PPF appears to play a key role in the developing diaphragm.
3.13. Lung and Upper Respiratory Tract and Airways
Respiratory defects including left lung agenesis, bilateral lung hypoplasia, and agenesis of the
esophagotracheal septum were described in early VAD syndrome embryos but were characterized as
rare anomalies [90,95]. Lung hypoplasia is observed with 100% penetrance in rat embryos in which
RA deficiency is imposed after E10.5, and the severity is increased as RA in the maternal diet is
reduced [67]. Rars are expressed throughout lung development and RAR compound null mutant mice
(RARα/β) show left lung agenesis and hypoplasia [209]. Additionally, a wide array of RA
synthesizing, metabolizing and binding proteins are found in developing lung [255257]. Mice null for
RALDH2 or RDH10, also have lung agenesis or hypoplastic phenotypes [122,258,259]. The lung is
second only to the liver as the major retinoid storage organ [260].
The lung arises from foregut endoderm during early development of the embryo. RA from the
splanchnic mesoderm surrounding the foregut endoderm has been found to be essential for primordial
lung bud formation at E9.5 in the mouse [256,258,259,261]. It was recently shown that in the foregut
mesoderm, RA controls the Fgf10 expression required for bud formation by balancing the activation of
canonical Wnt signaling through direct transcriptional repression of its antagonist Dkk1, and repression
of Tgfβ signaling [262]. This conclusion is reinforced by work showing that simultaneous activation of
Wnt and repression of Tgfβ in RA-deficient foregut rescues lung bud formation.
While RA signaling is required for initial budding, by E10.5E11.5, as secondary buds form, levels
are down regulated by the appearance of the RA-degrading enzyme CYP26A1 to enable more distal
branching and distal airway formation to proceed to completion [256,263]. A role for RA in alveoli
formation is supported by the finding that RARs are required for correct lung alveoli septation, and
reports that exogenous RA can stimulate alveoli formation in immature rat and mouse lung [264269].
Figure 6 summarizes the proposed functions of RA in lung development.
A recent report in the New England Journal of Medicine showed that, in a region with endemic
vitamin A (retinol) deficiency, children whose mothers had received vitamin A supplementation
before, during, and for 6 months after pregnancy had better lung function when they were tested at 9 to
11 years of age than children whose mothers had received beta carotene supplementation or placebo.
Additionally, they found that the period during which supplementation with vitamin A was most
important was from gestation through a postnatal age of 6 months [270].
Nutrients 2011, 3
Figure 6. Schematic showing the proposed sites of RA action in lung development. In the
developing embryo, RA is needed for primary bud formation (grey oval), but signaling is
down-regulated during the time that lung differentiation occurs. A later role for RA in
alveolar formation (grey oval) is also proposed. During induction of the lung buds, RA
regulates mesodermal Fgf10 levels by negatively regulating Tgfβ and enabling induction of
the Wnt pathway by repression of the Dickkopf homolog 1 (Dkk1) known to antagonize
Wnt ligand-receptor binding. RA may also influence the response of the foregut endoderm
(origin of lung progenitors) to Fgf10. (Adapted from [262]).
RA acting through the RAR is also necessary for the morphogenesis of other components of the
respiratory tract including partitioning of the primitive foregut into oesophagus and trachea, and for the
opening between the nasal and oropharyngeal cavities [67,131,209,256,258,261].
3.14. Pancreas
Although not observed either as a part of the vitamin A deficiency syndrome or in compound RAR
null mutant mice, a requirement for RA in pancreatic development has been proposed based on studies
in RALDH null mutant mice [271,272] and in several other organisms in which retinoid signaling is
deficient or inhibited, including Xenopus, zebrafish, chick, and quail [273276]. RDH10 knockout
mice do not have a pancreas [122]. RA produced by RALDH2 is required for early development of the
dorsal pancreas in the mouse [271,272]. Specification of dorsal pancreatic tissue can be rescued in
RALDH2-deficient embryos by low-dose maternal administration of RA.
The effects of RA deficiency on pancreatic lineages appear to be due to the loss of pancreatic field
specification within the endoderm [274]. Specification of the pancreas occurs between E8.08.5 of
mouse development, and is followed by the development of the dorsal and ventral pancreatic buds at
E9.09.5 [271]. RALDH2 null mutant embryos do not develop a dorsal pancreatic bud. It has been
suggested that the differential expression of retinoic acid receptors (RARs) in gastrula stage endoderm
is at least partially responsible for the distinct responsiveness of dorsal versus ventral pancreas [277].
Both defects in RA signaling and RA treatment have been shown to affect the expression of
PDX-1 [271,272,275,278] an essential regulator of early pancreas development required for the
Nutrients 2011, 3
pancreatic buds to grow and differentiate [279281]. RALDH2 mutant embryos lack PDX1 expression
in dorsal but not ventral endoderm [271,272]. Conversely, exogenous RA has been shown to expand
the pancreatic field [273275], and CYP26A1 has recently been shown to play a critical role in setting
the anterior limit of the pancreas field endodermal Cyp26 expression [282]. Additional information
regarding the role of RA in pancreas development can be found in several recent reviews [283,284].
3.15. Limb Development and Interdigital Cell Death
Functions for RA in limb development have been forwarded and debated for many years. Inhibitors
of RA synthesis and vitamin A deficiency inhibit limb outgrowth in the quail [285,286]. In the absence
of RALDH2, murine forelimb buds do not develop and embryonic growth ceases prior to the stage
when hindlimb buds are initiated [287,288]. Zebrafish RALDH2 mutants lack pectoral fins and fin bud
induction does not occur [164,165,276,289]. Limb defects in mice also result from treatment with
excess exogenous RA, or inappropriate exposure to endogenous levels of RA due to the absence of
CYP26B1. These studies show that controlled exclusion of RA from the limb bud is essential for
proper limb morphology [142].
RA has been proposed to play a role in patterning the developing limb by regulating the pattern of
expression of genes such as Hand2 (activates SHH), Shh (patterns the anterior-to-posterior axis), and
Meis1/2 (transcription factors that mark the proximal limb bud mesenchyme) [287,290292].
However, a new report from Zhao and colleagues using a RALDH2/3 double mouse knockout
suggests that RA acts as a permissive signal at an earlier stage to allow limb bud initiation rather than
acting in an instructive manner, and that the role of RA is to antagonize early axial FGF signals which
otherwise inhibit the limb field [80,141,293]. According to this new model, RA signaling within the
forelimb bud proper is not required for normal patterning to occur. A similar idea was proposed by
Gilbert et al., who found that axial retinoic acid signals played a permissive role in the induction
of zebrafish pectoral fins [289]. RA appears to be dispensable for hindlimb budding and
patterning [80,287]. It is notable that trex embryos carrying a mutation in Rdh10, have small, abnormal
forelimbs but have hindlimbs that are relatively unaffected [122].
At later stages of development, RA is also essential for interdigital cell death, the mechanism by
which digit separation occurs. Webbed digits have been described in compound RAR mutant mice due
to a loss of apoptosis [209,294]. Around E12.5 (mouse) RA signaling becomes confined to the
interdigital zones by a combination of interdigital Raldh2 expression and Cyp26B1 expression in the
developing digits [295]. The mechanism by which RA activates cell death is currently a subject of
active investigation [294297].
4. Conclusions
In summary, the vitamin A metabolite, RA, is essential for reproduction in both the male and
female, as well as for many events in the developing embryo. Nutritional as well as genetic approaches
are being used to identify the cell types and pathways that are dependent upon RA signaling in support
of these processes. Paracrine signaling appears to play a prominent role in RA action. Regulation of
RA synthesis as well as its catabolism is important in determining when and where RA signaling will
be activated. Future studies are needed to develop a more detailed understanding of when in
Nutrients 2011, 3
development, and in what specific cell types RA and its receptors are acting. Elucidation of the
pathways that are involved in support of vitamin A functions in stem/germ cell division/differentiation,
patterning and tissue/organ development remain major tasks for future work.
The work from the author’s laboratory discussed in this review was supported in part by funds from
NIH grant DK-14881, a fellowship to EMM NIH T32 DK07665, and the USDA CSREES WIS04305,
WIS04768 and CSRSG 9400543 and 9900802. Support to the author was also provided by NIH
CA49837. We also thank Laura Vanderploeg in the Biochemistry Media Lab for the artwork.
1. McCollum, E.V.; Davis, M. The necessity of certain lipins in the diet during growth. J. Biol.
Chem. 1913, 15, 167175.
2. Wolbach, S.B.; Howe, P.R. Tissue changes following deprivation of fat-soluble A vitamin.
J. Exp. Med. 1925, 42, 753777.
3. Evans, H.M. The effects of inadequate vitamin A on the sexual physiology of the female.
J. Biol. Chem. 1928, 77, 651654.
4. Carpenter, K.J.; Harper, A.E.; Olson, R.E. Experiments That Changed Nutritional Thinking.
J. Nutr. 1997, 127, 1017S1053S.
5. Moore, T. Vitamin A and carotene: The absence of the liver oil vitamin A from carotene. VI. The
conversion of carotene to vitamin A in vivo. Biochem. J. 1930, 24, 692702.
6. Karrer, P.; Morf, R.; Schoepp, K. Zur Kenntnis des Vitamins A in Gischtranen. Helv. Chim. Acta
1931, 14, 14311436.
7. Karrer, P. Carotenoids, flavins and vitamin A and B
: Nobel lecture, December 11, 1937.
In Nobel Lectures, Chemistry (19221941); Elsevier: Amsterdam, The Netherlands, 1966;
pp. 433448.
8. Arens, J.F.; van Dorp, D.A. Synthesis of some compounds possessing vitamin A activity. Nature
1946, 157, 190191.
9. Arens, J.F.; van Dorp, D.A. Activity of vitamin A-acid in the rat. Nature 1946, 158, 622.
10. van Dorp, D.A.; Arens, J.F. Biological activity of vitamin A acid. Nature 1946, 158, 60.
11. Dowling, J.E.; Wald, G. The biological function of vitamin A acid. Proc. Natl. Acad. Sci. USA
1960, 46, 587608.
12. Moise, A.R.; Noy, N.; Palczewski, K.; Blaner, W.S. Delivery of retinoid-based therapies to target
tissues. Biochemistry 2007, 46, 44494458.
13. Pares, X.; Farres, J.; Kedishvili, N.; Duester, G. Medium- and short-chain dehydrogenase/reductase
gene and protein families: Medium-chain and short-chain dehydrogenases/reductases in retinoid
metabolism. Cell. Mol. Life Sci. 2008, 65, 39363949.
14. Duester, G. Families of retinoid dehydrogenases regulating vitamin A function: Production of
visual pigment and retinoic acid. Eur. J. Biochem. 2000, 267, 43154324.
Nutrients 2011, 3
15. Sima, A.; Parisotto, M.; Mader, S.; Bhat, P.V. Kinetic characterization of recombinant mouse
retinal dehydrogenase types 3 and 4 for retinal substrates. Biochim. Biophys. Acta 2009, 1790,
16. Chambers, D.; Wilson, L.; Maden, M.; Lumsden, A. RALDH-independent generation of retinoic
acid during vertebrate embryogenesis by CYP1B1. Development 2007, 134, 13691383.
17. White, J.A.; Beckett-Jones, B.; Guo, Y.D.; Dilworth, F.J.; Bonasoro, J.; Jones, G.; Petkovich, M.
cDNA cloning of human retinoic acid-metabolizing enzyme (hP450RAI) identifies a novel
family of cytochromes P450. J. Biol. Chem. 1997, 272, 1853818541.
18. Fujii, H.; Sato, T.; Kaneko, S.; Gotoh, O.; Fujii-Kuriyama, Y.; Osawa, K.; Kato, S.; Hamada, H.
Metabolic inactivation of retinoic acid by a novel P450 differentially expressed in developing
mouse embryos. EMBO J. 1997, 16, 41634173.
19. Chithalen, J.V.; Luu, L.; Petkovich, M.; Jones, G. HPLC-MS/MS analysis of the products
generated from all-trans-retinoic acid using recombinant human CYP26A. J. Lipid Res. 2002, 43,
20. White, J.A.; Ramshaw, H.; Taimi, M.; Stangle, W.; Zhang, A.; Everingham, S.; Creighton, S.;
Tam, S.P.; Jones, G.; Petkovich, M. Identification of the human cytochrome P450, P450RAI-2,
which is predominantly expressed in the adult cerebellum and is responsible for all-trans-retinoic
acid metabolism. Proc. Natl. Acad. Sci. USA 2000, 97, 64036408.
21. Tahayato, A.; Dolle, P.; Petkovich, M. Cyp26C1 encodes a novel retinoic acid-metabolizing
enzyme expressed in the hindbrain, inner ear, first branchial arch and tooth buds during murine
development. Gene Expr. Patterns 2003, 3, 449454.
22. Taimi, M.; Helvig, C.; Wisniewski, J.; Ramshaw, H.; White, J.; Amad, M.; Korczak, B.;
Petkovich, M. A novel human cytochrome P450, CYP26C1, involved in metabolism of 9-cis and
all-trans isomers of retinoic acid. J. Biol. Chem. 2004, 279, 7785.
23. White, R.J.; Schilling, T.F. How degrading: Cyp26s in hindbrain development. Dev. Dyn. 2008,
237, 27752790.
24. D’Ambrosio, D.N.; Clugston, R.D.; Blaner, W.S. Vitamin A Metabolism: An Update. Nutrients
2011, 3, 63103.
25. Soprano, D.R.; Blaner, W.S. Plasma Retinol-Binding Protein. In The Retinoids: Biology,
Chemistry, and Medicine, 2nd ed.; Sporn, M.B., Roberts, A.B., Goodman, D.S., Eds.; Raven
Press: New York, NY, USA, 1994; pp. 257282.
26. Kawaguchi, R.; Yu, J.; Honda, J.; Hu, J.; Whitelegge, J.; Ping, P.; Wiita, P.; Bok, D.; Sun, H.
A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science
2007, 315, 820825.
27. Xueping, E.; Zhang, L.; Lu, J.; Tso, P.; Blaner, W.S.; Levin, M.S.; Li, E. Increased neonatal
mortality in mice lacking cellular retinol-binding protein II. J. Biol. Chem. 2002, 277,
28. Theodosiou, M.; Laudet, V.; Schubert, M. From carrot to clinic: an overview of the retinoic acid
signaling pathway. Cell. Mol. Life Sci. 2010, 67, 14231445.
29. Chambon, P. A decade of molecular biology of retinoic acid receptors. FASEB J. 1996, 10,
Nutrients 2011, 3
30. Rochette-Egly, C.; Germain, P. Dynamic and combinatorial control of gene expression by
nuclear retinoic acid receptors (RARs). Nucl. Recept. Signal. 2009, 7, e005.
31. Repa, J.J.; Hanson, K.K.; Clagett-Dame, M. All-trans-retinol is a ligand for the retinoic acid
receptors. Proc. Natl. Acad. Sci. USA 1993, 90, 72937297.
32. Mangelsdorf, D.; Umesono, K.; Evans, R.M. The Retinoid Receptors. In The Retinoids: Biology,
Chemistry and Medicine, 2nd ed.; Sporn, M.B., Roberts, A.B., Goodman, D.S., Eds.; Raven
Press: New York, NY, USA, 1994; pp. 319350.
33. Knutson, D.C.; Clagett-Dame, M. atRA Regulation of NEDD9, a gene involved in neurite
outgrowth and cell adhesion. Arch. Biochem. Biophys. 2008, 477, 163174.
34. Glass, C.K.; Rosenfeld, M.G. The coregulator exchange in transcriptional functions of nuclear
receptors. Genes Dev. 2000, 14, 121141.
35. Bastien, J.; Rochette-Egly, C. Nuclear retinoid receptors and the transcription of retinoid-target
genes. Gene 2004, 328, 116.
36. Balmer, J.E.; Blomhoff, R. Gene expression regulation by retinoic acid. J. Lipid Res. 2002,
43, 17731808.
37. Blomhoff, R.; Blomhoff, H.K. Overview of retinoid metabolism and function. J. Neurobiol.
2006, 66, 606630.
38. Mason, K.E. Differences in testis injury and repair after vitamin A-deficiency,
vitamin E-deficiency, and inanition. Am. J. Anat. 1933, 52, 153239.
39. Mitranond, V.; Sobhon, P.; Tosukhowong, P.; Chindaduangrat, W. Cytological changes in the
testes of vitamin-A-deficient rats. I. Quantitation of germinal cells in the seminiferous tubules.
Acta Anat. (Basel) 1979, 103, 159168.
40. Huang, H.F.; Hembree, W.C. Spermatogenic response to vitamin A in vitamin A deficient rats.
Biol. Reprod. 1979, 21, 891904.
41. Unni, E.; Rao, M.R.; Ganguly, J. Histological & ultrastructural studies on the effect of vitamin A
depletion & subsequent repletion with vitamin A on germ cells & Sertoli cells in rat testis.
Indian J. Exp. Biol. 1983, 21, 180192.
42. van Pelt, A.M.; de Rooij, D.G. Synchronization of the seminiferous epithelium after vitamin A
replacement in vitamin A-deficient mice. Biol. Reprod. 1990, 43, 363367.
43. Morales, C.; Griswold, M.D. Retinol-induced stage synchronization in seminiferous tubules of
the rat. Endocrinology 1987, 121, 432434.
44. van Pelt, A.M.; de Rooij, D.G. The origin of the synchronization of the seminiferous epithelium
in vitamin A-deficient rats after vitamin A replacement. Biol. Reprod. 1990, 42, 677682.
45. Hogarth, C.A.; Griswold, M.D. The key role of vitamin A in spermatogenesis. J. Clin. Invest.
2010, 120, 956962.
46. Matson, C.K.; Murphy, M.W.; Griswold, M.D.; Yoshida, S.; Bardwell, V.J.; Zarkower, D. The
mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis
versus meiosis decision in male germ cells. Dev. Cell 2010, 19, 612624.
47. Snyder, E.M.; Small, C.; Griswold, M.D. Retinoic acid availability drives the asynchronous
initiation of spermatogonial differentiation in the mouse. Biol. Reprod. 2010, 83, 783790.
Nutrients 2011, 3
48. van Pelt, A.M.; de Rooij, D.G. Retinoic acid is able to reinitiate spermatogenesis in
vitamin A-deficient rats and high replicate doses support the full development of spermatogenic
cells. Endocrinology 1991, 128, 697704.
49. Vernet, N.; Dennefeld, C.; Rochette-Egly, C.; Oulad-Abdelghani, M.; Chambon, P.;
Ghyselinck, N.B.; Mark, M. Retinoic acid metabolism and signaling pathways in the adult and
developing mouse testis. Endocrinology 2006, 147, 96110.
50. Ghyselinck, N.B.; Vernet, N.; Dennefeld, C.; Giese, N.; Nau, H.; Chambon, P.; Viville, S.;
Mark, M. Retinoids and spermatogenesis: lessons from mutant mice lacking the plasma retinol
binding protein. Dev. Dyn. 2006, 235, 16081622.
51. Zhai, Y.; Sperkova, Z.; Napoli, J.L. Cellular expression of retinal dehydrogenase types 1 and 2:
effects of vitamin A status on testis mRNA. J. Cell. Physiol. 2001, 186, 220232.
52. Livera, G.; Rouiller-Fabre, V.; Pairault, C.; Levacher, C.; Habert, R. Regulation and perturbation
of testicular functions by vitamin A. Reproduction 2002, 124, 173180.
53. Lohnes, D.; Kastner, P.; Dierich, A.; Mark, M.; LeMeur, M.; Chambon, P. Function of retinoic
acid receptor gamma in the mouse. Cell 1993, 73, 643658.
54. Lufkin, T.; Lohnes, D.; Mark, M.; Dierich, A.; Gorry, P.; Gaub, M.P.; LeMeur, M.; Chambon, P.
High postnatal lethality and testis degeneration in retinoic acid receptor alpha mutant mice.
Proc. Natl. Acad. Sci. USA 1993, 90, 72257229.
55. Vernet, N.; Dennefeld, C.; Guillou, F.; Chambon, P.; Ghyselinck, N.B.; Mark, M. Prepubertal
testis development relies on retinoic acid but not rexinoid receptors in Sertoli cells. EMBO J.
2006, 25, 58165825.
56. Doyle, T.J.; Braun, K.W.; McLean, D.J.; Wright, R.W.; Griswold, M.D.; Kim, K.H. Potential
functions of retinoic acid receptor A in Sertoli cells and germ cells during spermatogenesis.
Ann. N. Y. Acad. Sci. 2007, 1120, 114130.
57. Chung, S.S.; Choi, C.; Wang, X.; Hallock, L.; Wolgemuth, D.J. Aberrant distribution of
junctional complex components in retinoic acid receptor alpha-deficient mice. Microsc. Res.
Tech. 2010, 73, 583596.
58. Clagett-Dame, M.; DeLuca, H.F. The role of vitamin A in mammalian reproduction and
embryonic development. Annu. Rev. Nutr. 2002, 22, 347381.
59. Evans, H.M.; Bishop, K.S. On an invariable and characteristic disturbance of reproductive
function in animals reared on a diet poor in fat soluble vitamine A. Anat. Rec. 1922, 23, 1718.
60. Mason, K.E.; Ellison, E.T. Changes in the vaginal epithelium of the rat after
vitamin A-deficiency. J. Nutr. 1935, 9, 735755.
61. Warkany, J.; Schraffenberger, E. Congenital malformations induced in rats by maternal vitamin
A deficiency. I. Defects of the eye. Arch. Ophthalmol. 1946, 35, 150169.
62. Thompson, J.N.; Howell, J.M.; Pitt, G.A. Vitamin a and Reproduction in Rats. Proc. R. Soc.
Lond. B Biol. Sci. 1964, 159, 510535.
63. White, J.C.; Shankar, V.N.; Highland, M.; Epstein, M.L.; DeLuca, H.F.; Clagett-Dame, M.
Defects in embryonic hindbrain development and fetal resorption resulting from vitamin A
deficiency in the rat are prevented by feeding pharmacological levels of all-trans-retinoic acid.
Proc. Natl. Acad. Sci. USA 1998, 95, 1345913464.
Nutrients 2011, 3
64. White, J.C.; Highland, M.; Clagett-Dame, M. Abnormal development of the sinuatrial venous
valve and posterior hindbrain may contribute to late fetal resorption of vitamin A-deficient rat
embryos. Teratology 2000, 62, 374384.
65. White, J.C.; Highland, M.; Kaiser, M.; Clagett-Dame, M. Vitamin A deficiency results in the
dose-dependent acquisition of anterior character and shortening of the caudal hindbrain of the rat
embryo. Dev. Biol. 2000, 220, 263284.
66. Kaiser, M.E.; Merrill, R.A.; Stein, A.C.; Breburda, E.; Clagett-Dame, M. Vitamin A deficiency
in the late gastrula stage rat embryo results in a one to two vertebral anteriorization that extends
throughout the axial skeleton. Dev. Biol. 2003, 257, 1429.
67. See, A.W.; Kaiser, M.E.; White, J.C.; Clagett-Dame, M. A nutritional model of late embryonic
vitamin A deficiency produces defects in organogenesis at a high penetrance and reveals new
roles for the vitamin in skeletal development. Dev. Biol. 2008, 316, 171190.
68. Howell, J.M.; Thompson, J.N.; Pitt, G.A. Histology of the Lesions Produced in the Reproductive
Tract of Animals Fed a Diet Deficient in Vitamin A Alcohol but Containing Vitamin A Acid. II.
The Female Rat. J. Reprod. Fertil. 1964, 7, 251258.
69. Noback, C.R.; Takahashi, Y.I. Micromorphology of the placenta of rats reared on marginal
vitamin-A-deficient diet. Acta Anat. (Basel) 1978, 102, 195202.
70. Koubova, J.; Menke, D.B.; Zhou, Q.; Capel, B.; Griswold, M.D.; Page, D.C. Retinoic acid
regulates sex-specific timing of meiotic initiation in mice. Proc. Natl. Acad. Sci. USA 2006, 103,
71. Bowles, J.; Knight, D.; Smith, C.; Wilhelm, D.; Richman, J.; Mamiya, S.; Yashiro, K.;
Chawengsaksophak, K.; Wilson, M.J.; Rossant, J.; Hamada, H.; Koopman, P. Retinoid signaling
determines germ cell fate in mice. Science 2006, 312, 596600.
72. Bowles, J.; Koopman, P. Retinoic acid, meiosis and germ cell fate in mammals. Development
2007, 134, 34013411.
73. MacLean, G.; Li, H.; Metzger, D.; Chambon, P.; Petkovich, M. Apoptotic extinction of germ
cells in testes of Cyp26b1 knockout mice. Endocrinology 2007, 148, 45604567.
74. Kumar, S.; Chatzi, C.; Brade, T.; Cunningham, T.J.; Zhao, X.; Duester, G. Sex-specific timing of
meiotic initiation is regulated by Cyp26b1 independent of retinoic acid signalling. Nat. Commun.
2011, 2, 151.
75. Livera, G.; Rouiller-Fabre, V.; Valla, J.; Habert, R. Effects of retinoids on the meiosis in the fetal
rat ovary in culture. Mol. Cell. Endocrinol. 2000, 165, 225231.
76. Baltus, A.E.; Menke, D.B.; Hu, Y.C.; Goodheart, M.L.; Carpenter, A.E.; de Rooij, D.G.;
Page, D.C. In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes
premeiotic DNA replication. Nat. Genet. 2006, 38, 14301434.
77. Barrios, F.; Filipponi, D.; Pellegrini, M.; Paronetto, M.P.; Di Siena, S.; Geremia, R.; Rossi, P.;
De Felici, M.; Jannini, E.A.; Dolci, S. Opposing effects of retinoic acid and FGF9 on Nanos2
expression and meiotic entry of mouse germ cells. J. Cell Sci. 2010, 123, 871880.
78. Bowles, J.; Feng, C.W.; Spiller, C.; Davidson, T.L.; Jackson, A.; Koopman, P. FGF9 suppresses
meiosis and promotes male germ cell fate in mice. Dev. Cell 2010, 19, 440449.
79. Li, H.; Clagett-Dame, M. Vitamin A deficiency blocks the initiation of meiosis of germ cells in
the developing rat ovary in vivo. Biol. Reprod. 2009, 81, 9961001.
Nutrients 2011, 3
80. Zhao, X.; Sirbu, I.O.; Mic, F.A.; Molotkova, N.; Molotkov, A.; Kumar, S.; Duester, G. Retinoic
acid promotes limb induction through effects on body axis extension but is unnecessary for limb
patterning. Curr. Biol. 2009, 19, 10501057.
81. Bowles, J.; Feng, C.W.; Knight, D.; Smith, C.A.; Roeszler, K.N.; Bagheri-Fam, S.; Harley, V.R.;
Sinclair, A.H.; Koopman, P. Male-specific expression of Aldh1a1 in mouse and chicken fetal
testes: implications for retinoid balance in gonad development. Dev. Dyn. 2009, 238, 20732080.
82. Li, H.; MacLean, G.; Cameron, D.; Clagett-Dame, M.; Petkovich, M. Cyp26b1 expression in
murine Sertoli cells is required to maintain male germ cells in an undifferentiated state during
embryogenesis. PLoS ONE 2009, 4, e7501.
83. Li, H.; Palczewski, K.; Baehr, W.; Clagett-Dame, M. Vitamin A deficiency results in meiotic
failure and accumulation of undifferentiated spermatogonia in prepubertal mouse testis. Biol.
Reprod. 2011, 84, 336341.
84. Zhou, Q.; Nie, R.; Li, Y.; Friel, P.; Mitchell, D.; Hess, R.A.; Small, C.; Griswold, M.D.
Expression of stimulated by retinoic acid gene 8 (Stra8) in spermatogenic cells induced by
retinoic acid: an in vivo study in vitamin A-sufficient postnatal murine testes. Biol. Reprod. 2008,
79, 3542.
85. Bellve, A.R.; Millette, C.F.; Bhatnagar, Y.M.; O’Brien, D.A. Dissociation of the mouse testis and
characterization of isolated spermatogenic cells. J. Histochem. Cytochem. 1977, 25, 480494.
86. Hale, F. Pigs born without eye balls. J. Hered. 1933, 24, 105106.
87. Hale, F. The relation of vitamin A to anophthalmos in pigs. Am. J. Ophthalmol. 1935, 18,
88. Warkany, J.; Schraffenberger, E. Congenital malformations of the eyes induced in rats by
maternal vitamin A deficiency. Proc. Soc. Exp. Biol. Med. 1944, 57, 4952.
89. Warkany, J.; Roth, C.B. Congenital malformations induced in rats by maternal vitamin A
deficiency. II. Effect of varying the preparatory diet upon the yield of abnormal young. J. Nutr.
1948, 35, 111.
90. Warkany, J.; Roth, C.B.; Wilson, J.G. Multiple congenital malformations: a consideration of
etiologic factors. Pediatrics 1948, 1, 462471.
91. Wilson, J.G.; Warkany, J. Malformations in the genito-urinary tract induced by maternal vitamin
A deficiency in the rat. Am. J. Anat. 1948, 83, 357407.
92. Wilson, J.G.; Barch, S. Fetal death and maldevelopment resulting from maternal vitamin A
deficiency in the rat. Proc. Soc. Exp. Biol. Med. 1949, 72, 687693, illust.
93. Wilson, J.G.; Warkany, J. Aortic-arch and cardiac anomalies in the offspring of vitamin A
deficient rats. Am. J. Anat. 1949, 85, 113155.
94. Wilson, J.G.; Warkany, J. Cardiac and aortic arch anomalies in the offspring of vitamin A
deficient rats correlated with similar human anomalies. Pediatrics 1950, 5, 708725.
95. Wilson, J.G.; Roth, C.B.; Warkany, J. An analysis of the syndrome of malformations induced by
maternal vitamin A deficiency. Effects of restoration of vitamin A at various times during
gestation. Am. J. Anat. 1953, 92, 189217.
96. Dickman, E.D.; Thaller, C.; Smith, S.M. Temporally-regulated retinoic acid depletion produces
specific neural crest, ocular and nervous system defects. Development 1997, 124, 31113121.
Nutrients 2011, 3
97. Lohnes, D.; Mark, M.; Mendelsohn, C.; Dolle, P.; Dierich, A.; Gorry, P.; Gansmuller, A.;
Chambon, P. Function of the retinoic acid receptors (RARs) during development (I). Craniofacial
and skeletal abnormalities in RAR double mutants. Development 1994, 120, 27232748.
98. Mendelsohn, C.; Lohnes, D.; Decimo, D.; Lufkin, T.; LeMeur, M.; Chambon, P.; Mark, M.
Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities
at various stages of organogenesis in RAR double mutants. Development 1994, 120, 27492771.
99. Mark, M.; Ghyselinck, N.B.; Chambon, P. Function of retinoic acid receptors during embryonic
development. Nucl. Recept. Signal. 2009, 7, e002.
100. Dolle, P. Developmental expression of retinoic acid receptors (RARs). Nucl. Recept. Signal.
2009, 7, e006.
101. Lohnes, D.; Mark, M.; Mendelsohn, C.; Dolle, P.; Decimo, D.; LeMeur, M.; Dierich, A.; Gorry, P.;
Chambon, P. Developmental roles of the retinoic acid receptors. J. Steroid Biochem. Mol. Biol.
1995, 53, 475486.
102. Mark, M.; Ghyselinck, N.B.; Chambon, P. Function of retinoid nuclear receptors: lessons from
genetic and pharmacological dissections of the retinoic acid signaling pathway during mouse
embryogenesis. Annu. Rev. Pharmacol. Toxicol. 2006, 46, 451480.
103. Warkany, J. Disturbance of embryonic development by maternal vitamin deficiencies.
J. Cell. Physiol. 1954, 43, 207236.
104. See, A.W.; Clagett-Dame, M. The temporal requirement for vitamin A in the developing eye:
mechanism of action in optic fissure closure and new roles for the vitamin in regulating cell
proliferation and adhesion in the embryonic retina. Dev. Biol. 2009, 325, 94105.
105. Krezel, W.; Dupe, V.; Mark, M.; Dierich, A.; Kastner, P.; Chambon, P. RXR gamma null mice
are apparently normal and compound RXR alpha +/−/RXR beta −/−/RXR gamma −/− mutant
mice are viable. Proc. Natl. Acad. Sci. USA 1996, 93, 90109014.
106. Mascrez, B.; Ghyselinck, N.B.; Chambon, P.; Mark, M. A transcriptionally silent RXRalpha
supports early embryonic morphogenesis and heart development. Proc. Natl. Acad. Sci. USA
2009, 106, 42724277.
107. Mascrez, B.; Mark, M.; Dierich, A.; Ghyselinck, N.B.; Kastner, P.; Chambon, P. The RXRalpha
ligand-dependent activation function 2 (AF-2) is important for mouse development. Development
1998, 125, 46914707.
108. Mic, F.A.; Molotkov, A.; Benbrook, D.M.; Duester, G. Retinoid activation of retinoic acid
receptor but not retinoid X receptor is sufficient to rescue lethal defect in retinoic acid synthesis.
Proc. Natl. Acad. Sci. USA 2003, 100, 71357140.
109. Morriss-Kay, G.M.; Ward, S.J. Retinoids and mammalian development. Int. Rev. Cytol. 1999,
188, 73131.
110. Quadro, L.; Hamberger, L.; Gottesman, M.E.; Wang, F.; Colantuoni, V.; Blaner, W.S.;
Mendelsohn, C.L. Pathways of vitamin A delivery to the embryo: insights from a new tunable
model of embryonic vitamin A deficiency. Endocrinology 2005, 146, 44794490.
111. Soprano, D.R.; Soprano, K.J.; Goodman, D.S. Retinol-binding protein and transthyretin mRNA
levels in visceral yolk sac and liver during fetal development in the rat. Proc. Natl. Acad. Sci.
USA 1986, 83, 73307334.
Nutrients 2011, 3
112. Sapin, V.; Ward, S.J.; Bronner, S.; Chambon, P.; Dolle, P. Differential expression of transcripts
encoding retinoid binding proteins and retinoic acid receptors during placentation of the mouse.
Dev. Dyn. 1997, 208, 199210.
113. Quadro, L.; Hamberger, L.; Gottesman, M.E.; Colantuoni, V.; Ramakrishnan, R.; Blaner, W.S.
Transplacental delivery of retinoid: the role of retinol-binding protein and lipoprotein retinyl
ester. Am. J. Physiol. Endocrinol. Metab 2004, 286, E844E851.
114. Bavik, C.; Ward, S.J.; Chambon, P. Developmental abnormalities in cultured mouse embryos
deprived of retinoic by inhibition of yolk-sac retinol binding protein synthesis. Proc. Natl. Acad.
Sci. USA 1996, 93, 31103114.
115. Farese, R.V., Jr.; Cases, S.; Ruland, S.L.; Kayden, H.J.; Wong, J.S.; Young, S.G.; Hamilton, R.L.
A novel function for apolipoprotein B: lipoprotein synthesis in the yolk sac is critical for
maternal-fetal lipid transport in mice. J. Lipid Res. 1996, 37, 347360.
116. Sapin, V.; Chaib, S.; Blanchon, L.; Alexandre-Gouabau, M.C.; Lemery, D.; Charbonne, F.;
Gallot, D.; Jacquetin, B.; Dastugue, B.; Azais-Braesco, V. Esterification of vitamin A by the
human placenta involves villous mesenchymal fibroblasts. Pediatr. Res. 2000, 48, 565572.
117. Kochhar, D.M. Teratogenic activity of retinoic acid. Acta Pathol. Microbiol. Scand. 1967, 70,
118. Conlon, R.A. Retinoic acid and pattern formation in vertebrates. Trends Genet. 1995, 11,
119. Shenefelt, R.E. Morphogenesis of malformations in hamsters caused by retinoic acid: relation to
dose and stage at treatment. Teratology 1972, 5, 103118.
120. Collins, M.D.; Mao, G.E. Teratology of retinoids. Annu. Rev. Pharmacol. Toxicol. 1999, 39,
121. Reijntjes, S.; Blentic, A.; Gale, E.; Maden, M. The control of morphogen signalling: regulation
of the synthesis and catabolism of retinoic acid in the developing embryo. Dev. Biol. 2005, 285,
122. Sandell, L.L.; Sanderson, B.W.; Moiseyev, G.; Johnson, T.; Mushegian, A.; Young, K.; Rey, J.P.;
Ma, J.X.; Staehling-Hampton, K.; Trainor, P.A. RDH10 is essential for synthesis of embryonic
retinoic acid and is required for limb, craniofacial, and organ development. Genes Dev. 2007, 21,
123. Niederreither, K.; Subbarayan, V.; Dolle, P.; Chambon, P. Embryonic retinoic acid synthesis is
essential for early mouse post-implantation development. Nat. Genet. 1999, 21, 444448.
124. Reijntjes, S.; Zile, M.H.; Maden, M. The expression of Stra6 and Rdh10 in the avian embryo and
their contribution to the generation of retinoid signatures. Int. J. Dev. Biol. 2010, 54, 12671275.
125. Strate, I.; Min, T.H.; Iliev, D.; Pera, E.M. Retinol dehydrogenase 10 is a feedback regulator of
retinoic acid signalling during axis formation and patterning of the central nervous system.
Development 2009, 136, 461472.
126. Ang, H.L.; Deltour, L.; Hayamizu, T.F.; Zgombic-Knight, M.; Duester, G. Retinoic acid
synthesis in mouse embryos during gastrulation and craniofacial development linked to class IV
alcohol dehydrogenase gene expression. J. Biol. Chem. 1996, 271, 95269534.
Nutrients 2011, 3
127. Ang, H.L.; Deltour, L.; Zgombic-Knight, M.; Wagner, M.A.; Duester, G. Expression patterns of
class I and class IV alcohol dehydrogenase genes in developing epithelia suggest a role for alcohol
dehydrogenase in local retinoic acid synthesis. Alcohol. Clin. Exp. Res. 1996, 20, 10501064.
128. Molotkov, A.; Deltour, L.; Foglio, M.H.; Cuenca, A.E.; Duester, G. Distinct retinoid metabolic
functions for alcohol dehydrogenase genes Adh1 and Adh4 in protection against vitamin A
toxicity or deficiency revealed in double null mutant mice. J. Biol. Chem. 2002, 277,
129. Molotkov, A.; Fan, X.; Deltour, L.; Foglio, M.H.; Martras, S.; Farres, J.; Pares, X.; Duester, G.
Stimulation of retinoic acid production and growth by ubiquitously expressed alcohol
dehydrogenase Adh3. Proc. Natl. Acad. Sci. USA 2002, 99, 53375342.
130. Mic, F.A.; Haselbeck, R.J.; Cuenca, A.E.; Duester, G. Novel retinoic acid generating activities in
the neural tube and heart identified by conditional rescue of Raldh2 null mutant mice.
Development 2002, 129, 22712282.
131. Dupe, V.; Matt, N.; Garnier, J.M.; Chambon, P.; Mark, M.; Ghyselinck, N.B. A newborn lethal
defect due to inactivation of retinaldehyde dehydrogenase type 3 is prevented by maternal
retinoic acid treatment. Proc. Natl. Acad. Sci. USA 2003, 100, 1403614041.
132. Mic, F.A.; Molotkov, A.; Molotkova, N.; Duester, G. Raldh2 expression in optic vesicle
generates a retinoic acid signal needed for invagination of retina during optic cup formation. Dev.
Dyn. 2004, 231, 270277.
133. Fan, X.; Molotkov, A.; Manabe, S.; Donmoyer, C.M.; Deltour, L.; Foglio, M.H.; Cuenca, A.E.;
Blaner, W.S.; Lipton, S.A.; Duester, G. Targeted disruption of Aldh1a1 (Raldh1) provides
evidence for a complex mechanism of retinoic acid synthesis in the developing retina. Mol. Cell.
Biochem. 2003, 23, 46374648.
134. Matt, N.; Dupe, V.; Garnier, J.M.; Dennefeld, C.; Chambon, P.; Mark, M.; Ghyselinck, N.B.
Retinoic acid-dependent eye morphogenesis is orchestrated by neural crest cells. Development
2005, 132, 47894800.
135. Molotkov, A.; Molotkova, N.; Duester, G. Retinoic acid guides eye morphogenetic movements
via paracrine signaling but is unnecessary for retinal dorsoventral patterning. Development 2006,
133, 19011910.
136. Duester, G. Retinoic acid synthesis and signaling during early organogenesis. Cell 2008, 134,
137. Buters, J.T.; Sakai, S.; Richter, T.; Pineau, T.; Alexander, D.L.; Savas, U.; Doehmer, J.;
Ward, J.M.; Jefcoate, C.R.; Gonzalez, F.J. Cytochrome P450 CYP1B1 determines susceptibility
to 7,12-dimethylbenz[a]anthracene-induced lymphomas. Proc. Natl. Acad. Sci. USA 1999, 96,
138. Feng, L.; Hernandez, R.E.; Waxman, J.S.; Yelon, D.; Moens, C.B. Dhrs3a regulates retinoic acid
biosynthesis through a feedback inhibition mechanism. Dev. Biol. 2010, 338, 114.
139. Abu-Abed, S.; Dolle, P.; Metzger, D.; Beckett, B.; Chambon, P.; Petkovich, M. The retinoic
acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral
identity, and development of posterior structures. Genes Dev. 2001, 15, 226240.
Nutrients 2011, 3
140. Sakai, Y.; Meno, C.; Fujii, H.; Nishino, J.; Shiratori, H.; Saijoh, Y.; Rossant, J.; Hamada, H. The
retinoic acid-inactivating enzyme CYP26 is essential for establishing an uneven distribution of
retinoic acid along the anterio-posterior axis within the mouse embryo. Genes Dev. 2001, 15,
141. Pennimpede, T.; Cameron, D.A.; Maclean, G.A.; Li, H.; Abu-Abed, S.; Petkovich, M. The
role of CYP26 enzymes in defining appropriate retinoic acid exposure during embryogenesis.
Birth Defects Res. A Clin. Mol. Teratol. 2010, 88, 883894.
142. Yashiro, K.; Zhao, X.; Uehara, M.; Yamashita, K.; Nishijima, M.; Nishino, J.; Saijoh, Y.;
Sakai, Y.; Hamada, H. Regulation of retinoic acid distribution is required for proximodistal
patterning and outgrowth of the developing mouse limb. Dev. Cell 2004, 6, 411422.
143. MacLean, G.; Dolle, P.; Petkovich, M. Genetic disruption of CYP26B1 severely affects
development of neural crest derived head structures, but does not compromise hindbrain
patterning. Dev. Dyn. 2009, 238, 732745.
144. Uehara, M.; Yashiro, K.; Mamiya, S.; Nishino, J.; Chambon, P.; Dolle, P.; Sakai, Y. CYP26A1
and CYP26C1 cooperatively regulate anterior-posterior patterning of the developing brain and
the production of migratory cranial neural crest cells in the mouse. Dev. Biol. 2007, 302, 399411.
145. Uehara, M.; Yashiro, K.; Takaoka, K.; Yamamoto, M.; Hamada, H. Removal of maternal
retinoic acid by embryonic CYP26 is required for correct Nodal expression during early
embryonic patterning. Genes Dev. 2009, 23, 16891698.
146. Ulven, S.M.; Gundersen, T.E.; Weedon, M.S.; Landaas, V.O.; Sakhi, A.K.; Fromm, S.H.;
Geronimo, B.A.; Moskaug, J.O.; Blomhoff, R. Identification of endogenous retinoids, enzymes,
binding proteins, and receptors during early postimplantation development in mouse: important
role of retinal dehydrogenase type 2 in synthesis of all-trans-retinoic acid. Dev. Biol. 2000, 220,
147. Rossant, J.; Zirngibl, R.; Cado, D.; Shago, M.; Giguere, V. Expression of a retinoic acid response
element-hsplacZ transgene defines specific domains of transcriptional activity during mouse
embryogenesis. Genes Dev. 1991, 5, 13331344.
148. Niederreither, K.; McCaffery, P.; Drager, U.C.; Chambon, P.; Dolle, P. Restricted expression and
retinoic acid-induced downregulation of the retinaldehyde dehydrogenase type 2 (RALDH-2)
gene during mouse development. Mech. Dev. 1997, 62, 6778.
149. Ribes, V.; Le Roux, I.; Rhinn, M.; Schuhbaur, B.; Dolle, P. Early mouse caudal development
relies on crosstalk between retinoic acid, Shh and Fgf signalling pathways. Development 2009,
136, 665676.
150. Lai, L.; Bohnsack, B.L.; Niederreither, K.; Hirschi, K.K. Retinoic acid regulates endothelial cell
proliferation during vasculogenesis. Development 2003, 130, 64656474.
151. Bohnsack, B.L.; Hirschi, K.K. Red light, green light: signals that control endothelial cell
proliferation during embryonic vascular development. Cell Cycle 2004, 3, 15061511.
152. Bohnsack, B.L.; Lai, L.; Dolle, P.; Hirschi, K.K. Signaling hierarchy downstream of retinoic acid
that independently regulates vascular remodeling and endothelial cell proliferation. Genes Dev.
2004, 18, 13451358.
Nutrients 2011, 3
153. Satre, M.A.; Kochhar, D.M. Elevations in the endogenous levels of the putative morphogen
retinoic acid in embryonic mouse limb-buds associated with limb dysmorphogenesis. Dev. Biol.
1989, 133, 529536.
154. Horton, C.; Maden, M. Endogenous distribution of retinoids during normal development and
teratogenesis in the mouse embryo. Dev. Dyn. 1995, 202, 312323.
155. Scott, W.J., Jr.; Walter, R.; Tzimas, G.; Sass, J.O.; Nau, H.; Collins, M.D. Endogenous status of
retinoids and their cytosolic binding proteins in limb buds of chick vs. mouse embryos. Dev.
Biol. 1994, 165, 397409.
156. Vermot, J.; Fraulob, V.; Dolle, P.; Niederreither, K. Expression of enzymes synthesizing
(aldehyde dehydrogenase 1 and reinaldehyde dehydrogenase 2) and metabolizaing (Cyp26)
retinoic acid in the mouse female reproductive system. Endocrinology 2000, 141, 36383645.
157. Clagett-Dame, M.; McNeill, E.M.; Muley, P.D. Role of all-trans retinoic acid in neurite
outgrowth and axonal elongation. J. Neurobiol. 2006, 66, 739756.
158. Maden, M. Retinoic acid in the development, regeneration and maintenance of the nervous
system. Nat. Rev. Neurosci. 2007, 8, 755765.
159. Glover, J.C.; Renaud, J.S.; Rijli, F.M. Retinoic acid and hindbrain patterning. J. Neurobiol. 2006,
66, 705725.
160. Maden, M. Retinoids and spinal cord development. J. Neurobiol. 2006, 66, 726738.
161. Gavalas, A.; Krumlauf, R. Retinoid signalling and hindbrain patterning. Curr. Opin. Genet. Dev.
2000, 10, 380386.
162. Maden, M.; Gale, E.; Kostetskii, I.; Zile, M. Vitamin A-deficient quail embryos have half a
hindbrain and other neural defects. Curr. Biol. 1996, 6, 417426.
163. Niederreither, K.; Vermot, J.; Schuhbaur, B.; Chambon, P.; Dolle, P. Retinoic acid synthesis and
hindbrain patterning in the mouse embryo. Development 2000, 127, 7585.
164. Begemann, G.; Schilling, T.F.; Rauch, G.J.; Geisler, R.; Ingham, P.W. The zebrafish neckless
mutation reveals a requirement for raldh2 in mesodermal signals that pattern the hindbrain.
Development 2001, 128, 30813094.
165. Grandel, H.; Lun, K.; Rauch, G.J.; Rhinn, M.; Piotrowski, T.; Houart, C.; Sordino, P.;
Kuchler, A.M.; Schulte-Merker, S.; Geisler, R.; Holder, N.; Wilson, S.W.; Brand, M. Retinoic
acid signalling in the zebrafish embryo is necessary during pre-segmentation stages to pattern the
anterior-posterior axis of the CNS and to induce a pectoral fin bud. Development 2002, 129,
166. Wendling, O.; Ghyselinck, N.B.; Chambon, P.; Mark, M. Roles of retinoic acid receptors in early
embryonic morphogenesis and hindbrain patterning. Development 2001, 128, 20312038.
167. Dupe, V.; Ghyselinck, N.B.; Wendling, O.; Chambon, P.; Mark, M. Key roles of retinoic acid
receptors alpha and beta in the patterning of the caudal hindbrain, pharyngeal arches and otocyst
in the mouse. Development 1999, 126, 50515059.
168. Linville, A.; Radtke, K.; Waxman, J.S.; Yelon, D.; Schilling, T.F. Combinatorial roles for
zebrafish retinoic acid receptors in the hindbrain, limbs and pharyngeal arches. Dev. Biol. 2009,
325, 6070.
169. Dupe, V.; Lumsden, A. Hindbrain patterning involves graded responses to retinoic acid
signalling. Development 2001, 128, 21992208.
Nutrients 2011, 3
170. Sirbu, I.O.; Duester, G. Retinoic-acid signalling in node ectoderm and posterior neural plate
directs left-right patterning of somitic mesoderm. Nat. Cell Biol. 2006, 8, 271277.
171. MacLean, G.; Abu-Abed, S.; Dolle, P.; Tahayato, A.; Chambon, P.; Petkovich, M. Cloning of a
novel retinoic-acid metabolizing cytochrome P450, Cyp26B1, and comparative expression
analysis with Cyp26A1 during early murine development. Mech. Dev. 2001, 107, 195201.
172. Sirbu, I.O.; Gresh, L.; Barra, J.; Duester, G. Shifting boundaries of retinoic acid activity control
hindbrain segmental gene expression. Development 2005, 132, 26112622.
173. Hernandez, R.E.; Putzke, A.P.; Myers, J.P.; Margaretha, L.; Moens, C.B. Cyp26 enzymes
generate the retinoic acid response pattern necessary for hindbrain development. Development
2007, 134, 177187.
174. Ribes, V.; Fraulob, V.; Petkovich, M.; Dolle, P. The oxidizing enzyme CYP26a1 tightly
regulates the availability of retinoic acid in the gastrulating mouse embryo to ensure proper head
development and vasculogenesis. Dev. Dyn. 2007, 236, 644653.
175. Koide, T.; Downes, M.; Chandraratna, R.A.; Blumberg, B.; Umesono, K. Active repression of
RAR signaling is required for head formation. Genes Dev. 2001, 15, 21112121.
176. Schneider, R.A.; Hu, D.; Rubenstein, J.L.; Maden, M.; Helms, J.A. Local retinoid signaling
coordinates forebrain and facial morphogenesis by maintaining FGF8 and SHH. Development
2001, 128, 27552767.
177. Halilagic, A.; Zile, M.H.; Studer, M. A novel role for retinoids in patterning the avian forebrain
during presomite stages. Development 2003, 130, 20392050.
178. Halilagic, A.; Ribes, V.; Ghyselinck, N.B.; Zile, M.H.; Dolle, P.; Studer, M. Retinoids control
anterior and dorsal properties in the developing forebrain. Dev. Biol. 2007, 303, 362375.
179. Marklund, M.; Sjodal, M.; Beehler, B.C.; Jessell, T.M.; Edlund, T.; Gunhaga, L. Retinoic acid
signalling specifies intermediate character in the developing telencephalon. Development 2004,
131, 43234332.
180. Molotkova, N.; Molotkov, A.; Duester, G. Role of retinoic acid during forebrain development
begins late when Raldh3 generates retinoic acid in the ventral subventricular zone. Dev. Biol.
2007, 303, 601610.
181. Luo, T.; Wagner, E.; Drager, U.C. Integrating retinoic acid signaling with brain function. Dev.
Psychol. 2009, 45, 139150.
182. Zhang, J.; Smith, D.; Yamamoto, M.; Ma, L.; McCaffery, P. The meninges is a source of retinoic
acid for the late-developing hindbrain. J. Neurosci. 2003, 23, 76107620.
183. Yamamoto, M.; Fujinuma, M.; Hirano, S.; Hayakawa, Y.; Clagett-Dame, M.; Zhang, J.;
McCaffery, P. Retinoic acid influences the development of the inferior olivary nucleus in the
rodent. Dev. Biol. 2005, 280, 421433.
184. Smith, D.; Wagner, E.; Koul, O.; McCaffery, P.; Drager, U.C. Retinoic acid synthesis for the
developing telencephalon. Cereb. Cortex 2001, 11, 894905.
185. Siegenthaler, J.A.; Ashique, A.M.; Zarbalis, K.; Patterson, K.P.; Hecht, J.H.; Kane, M.A.;
Folias, A.E.; Choe, Y.; May, S.R.; Kume, T.; Napoli, J.L.; Peterson, A.S.; Pleasure, S.J. Retinoic
acid from the meninges regulates cortical neuron generation. Cell 2009, 139, 597609.
186. Wilson, V.; Olivera-Martinez, I.; Storey, K.G. Stem cells, signals and vertebrate body axis
extension. Development 2009, 136, 15911604.
Nutrients 2011, 3
187. Molotkova, N.; Molotkov, A.; Sirbu, I.O.; Duester, G. Requirement of mesodermal retinoic acid
generated by Raldh2 for posterior neural transformation. Mech. Dev. 2005, 122, 145155.
188. Diez del Corral, R.; Olivera-Martinez, I.; Goriely, A.; Gale, E.; Maden, M.; Storey, K. Opposing
FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and
segmentation during body axis extension. Neuron 2003, 40, 6579.
189. Diez del Corral, R.; Storey, K.G. Opposing FGF and retinoid pathways: a signalling switch that
controls differentiation and patterning onset in the extending vertebrate body axis. Bioessays
2004, 26, 857869.
190. Pierani, A.; Brenner-Morton, S.; Chiang, C.; Jessell, T.M. A sonic hedgehog-independent,
retinoid-activated pathway of neurogenesis in the ventral spinal cord. Cell 1999, 97, 903915.
191. Novitch, B.G.; Wichterle, H.; Jessell, T.M.; Sockanathan, S. A requirement for retinoic
acid-mediated transcriptional activation in ventral neural patterning and motor neuron
specification. Neuron 2003, 40, 8195.
192. Sockanathan, S.; Perlmann, T.; Jessell, T.M. Retinoid receptor signaling in postmitotic motor
neurons regulates rostrocaudal positional identity and axonal projection pattern. Neuron 2003,
40, 97111.
193. Vermot, J.; Schuhbaur, B.; Le Mouellic, H.; McCaffery, P.; Garnier, J.M.; Hentsch, D.; Brulet, P.;
Niederreither, K.; Chambon, P.; Dolle, P.; Le Roux, I. Retinaldehyde dehydrogenase 2 and
Hoxc8 are required in the murine brachial spinal cord for the specification of Lim1+
motoneurons and the correct distribution of Islet1+ motoneurons. Development 2005, 132,
194. Misra, M.; Shah, V.; Carpenter, E.; McCaffery, P.; Lance-Jones, C. Restricted patterns of
Hoxd10 and Hoxd11 set segmental differences in motoneuron subtype complement in the
lumbosacral spinal cord. Dev. Biol. 2009, 330, 5472.
195. Rodriguez-Tebar, A.; Rohrer, H. Retinoic acid induces NGF-dependent survival response and
high-affinity NGF receptors in immature chick sympathetic neurons. Development 1991, 112,
196. Plum, L.A.; Clagett-Dame, M. All-trans retinoic acid stimulates and maintains neurite outgrowth
in nerve growth factor-supported developing chick embryonic sympathetic neurons. Dev. Biol.
1996, 205, 5263.
197. Plum, L.A.; Parada, L.F.; Tsoulfas, P.; Clagett-Dame, M. Retinoic acid combined with
neurotrophin-3 enhances the survival and neurite outgrowth of embryonic sympathetic neurons.
Exp. Biol. Med. 2001, 226, 766775.
198. Merrill, R.A.; Plum, L.A.; Kaiser, M.E.; Clagett-Dame, M. A mammalian homolog of unc-53 is
regulated by all-trans retinoic acid in neuroblastoma cells and embryos. Proc. Natl. Acad. Sci.
USA 2002, 99, 34223427.
199. Merrill, R.A.; Ahrens, J.M.; Kaiser, M.E.; Federhart, K.S.; Poon, V.Y.; Clagett-Dame, M.
All-trans retinoic acid-responsive genes identified in the human SH-SY5Y neuroblastoma cell
line and their regulated expression in the nervous system of early embryos. Biol. Chem. 2004,
385, 605614.
Nutrients 2011, 3
200. Merrill, R.A.; See, A.W.; Wertheim, M.L.; Clagett-Dame, M. Crk-associated substrate (Cas)
family member, NEDD9, is regulated in human neuroblastoma cells and in the embryonic
hindbrain by all-trans retinoic acid. Dev. Dyn. 2004, 231, 564575.
201. Muley, P.D.; McNeill, E.M.; Marzinke, M.A.; Knobel, K.M.; Barr, M.M.; Clagett-Dame, M. The
atRA-responsive gene neuron navigator 2 functions in neurite outgrowth and axonal elongation.
Dev. Neurobiol. 2008, 68, 14411453.
202. McNeill, E.M.; Roos, K.P.; Moechars, D.; Clagett-Dame, M. Nav2 is necessary for cranial nerve
development and blood pressure regulation. Neural Dev. 2010, 5, 6.
203. Wagner, E.; McCaffery, P.; Drager, U.C. Retinoic acid in the formation of the dorsoventral retina
and its central projections. Dev. Biol. 2000, 222, 460470.
204. Drager, U.C.; Li, H.; Wagner, E.; McCaffery, P. Retinoic acid synthesis and breakdown in the
developing mouse retina. Prog. Brain Res. 2001, 131, 579587.
205. Mic, F.A.; Molotkov, A.; Fan, X.; Cuenca, A.E.; Duester, G. RALDH3, a retinaldehyde
dehydrogenase that generates retinoic acid, is expressed in the ventral retina, otic vesicle and
olfactory pit during mouse development. Mech. Dev. 2000, 97, 227230.
206. Li, H.; Wagner, E.; McCaffery, P.; Smith, D.; Andreadis, A.; Drager, U.C. A retinoic acid
synthesizing enzyme in ventral retina and telencephalon of the embryonic mouse. Mech. Dev.
2000, 95, 283289.
207. McCaffery, P.; Tempst, P.; Lara, G.; Drager, U.C. Aldehyde dehydrogenase is a positional
marker in the retina. Development 1991, 112, 693702.
208. Mori, M.; Ghyselinck, N.B.; Chambon, P.; Mark, M. Systematic immunolocalization of retinoid
receptors in developing and adult mouse eyes. Invest. Ophthalmol. Vis. Sci. 2001, 42, 13121318.
209. Ghyselinck, N.B.; Dupe, V.; Dierich, A.; Messaddeq, N.; Garnier, J.M.; Rochette-Egly, C.;
Chambon, P.; Mark, M. Role of the retinoic acid receptor beta (RARbeta) during mouse
development. Int. J. Dev. Biol. 1997, 41, 425447.
210. Kastner, P.; Grondona, J.M.; Mark, M.; Gansmuller, A.; LeMeur, M.; Decimo, D.; Vonesch, J.L.;
Dolle, P.; Chambon, P. Genetic analysis of RXR alpha developmental function: convergence of
RXR and RAR signaling pathways in heart and eye morphogenesis. Cell 1994, 78, 9871003.
211. Kastner, P.; Mark, M.; Ghyselinck, N.; Krezel, W.; Dupe, V.; Grondona, J.M.; Chambon, P.
Genetic evidence that the retinoid signal is transduced by heterodimeric RXR/RAR functional
units during mouse development. Development 1997, 124, 313326.
212. Matt, N.; Ghyselinck, N.B.; Pellerin, I.; Dupe, V. Impairing retinoic acid signalling in the neural
crest cells is sufficient to alter entire eye morphogenesis. Dev. Biol. 2008, 320, 140148.
213. Cammas, L.; Trensz, F.; Jellali, A.; Ghyselinck, N.B.; Roux, M.J.; Dolle, P. Retinoic acid
receptor (RAR)-alpha is not critically required for mediating retinoic acid effects in the
developing mouse retina. Invest. Ophthalmol. Vis. Sci. 2010, 51, 32813290.
214. Kumar, S.; Duester, G. Retinoic acid signaling in perioptic mesenchyme represses Wnt signaling
via induction of Pitx2 and Dkk2. Dev. Biol. 2010, 340, 6774.
215. Gage, P.J.; Suh, H.; Camper, S.A. Dosage requirement of Pitx2 for development of multiple
organs. Development 1999, 126, 46434651.
Nutrients 2011, 3
216. Zacharias, A.L.; Gage, P.J. Canonical Wnt/beta-catenin signaling is required for maintenance but
not activation of Pitx2 expression in neural crest during eye development. Dev. Dyn. 2010, 239,
217. Aulehla, A.; Pourquie, O. Signaling gradients during paraxial mesoderm development. Cold
Spring Harb. Perspect. Biol. 2010, 2, a000869.
218. Dubrulle, J.; McGrew, M.J.; Pourquie, O. FGF signaling controls somite boundary position and
regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 2001, 106,
219. Sawada, A.; Shinya, M.; Jiang, Y.J.; Kawakami, A.; Kuroiwa, A.; Takeda, H. Fgf/MAPK
signalling is a crucial positional cue in somite boundary formation. Development 2001, 128,
220. Tenin, G.; Wright, D.; Ferjentsik, Z.; Bone, R.; McGrew, M.J.; Maroto, M. The chick
somitogenesis oscillator is arrested before all paraxial mesoderm is segmented into somites.
BMC Dev. Biol. 2010, 10, 24.
221. Kieny, M.; Mauger, A.; Sengel, P. Early regionalization of somitic mesoderm as studied by the
development of axial skeleton of the chick embryo. Dev. Biol. 1972, 28, 142161.
222. Gruss, P.; Kessel, M. Axial specification in higher vertebrates. Curr. Opin. Genet. Dev. 1991, 1,
223. Kessel, M. Respecification of vertebral identities by retinoic acid. Development 1992, 115,
224. Kessel, M.; Gruss, P. Homeotic transformations of murine vertebrae and concomitant alteration
of Hox codes induced by retinoic acid. Cell 1991, 67, 89104.
225. Marshall, H.; Nonchev, S.; Sham, M.H.; Muchamore, I.; Lumsden, A.; Krumlauf, R. Retinoic
acid alters hindbrain Hox code and induces transformation of rhombomeres 2/3 into a 4/5
identity. Nature 1992, 360, 737741.
226. Hall, B.K.; Horstadius, S. The Neural Crest; Oxford University Press: New York, NY, USA,
1988; pp. 1303.
227. Dupe, V.; Pellerin, I. Retinoic acid receptors exhibit cell-autonomous functions in cranial neural
crest cells. Dev. Dyn. 2009, 238, 27012711.
228. Dersch, H.; Zile, M.H. Induction of normal cardiovascular development in the vitamin A-deprived
quail embryo by natural retinoids. Dev. Biol. 1993, 160, 424433.
229. Zile, M.H. Vitamin ANot for Your Eyes Only: Requirement for Heart Formation Begins Early
in Embryogenesis. Nutrients 2010, 2, 532550.
230. Niederreither, K.; Vermot, J.; Messaddeq, N.; Schuhbaur, B.; Chambon, P.; Dolle, P. Embryonic
retinoic acid synthesis is essential for heart morphogenesis in the mouse. Development 2001,
128, 10191031.
231. Wagner, M.; Siddiqui, M.A. Signal transduction in early heart development (II): ventricular
chamber specification, trabeculation, and heart valve formation. Exp. Biol. Med. (Maywood)
2007, 232, 866880.
232. Hoover, L.L.; Burton, E.G.; Brooks, B.A.; Kubalak, S.W. The expanding role for retinoid
signaling in heart development. Sci. World J. 2008, 8, 194211.
Nutrients 2011, 3
233. Lin, S.C.; Dolle, P.; Ryckebusch, L.; Noseda, M.; Zaffran, S.; Schneider, M.D.; Niederreither, K.
Endogenous retinoic acid regulates cardiac progenitor differentiation. Proc. Natl. Acad. Sci. USA
2010, 107, 92349239.
234. Rochais, F.; Mesbah, K.; Kelly, R.G. Signaling pathways controlling second heart field
development. Circ. Res. 2009, 104, 933942.
235. Lelievre-Pegorier, M.; Vilar, J.; Ferrier, M.L.; Moreau, E.; Freund, N.; Gilbert, T.;
Merlet-Benichou, C. Mild vitamin A deficiency leads to inborn nephron deficit in the rat. Kidney
Int. 1998, 54, 14551462.
236. Mendelsohn, C.; Batourina, E.; Fung, S.; Gilbert, T.; Dodd, J. Stromal cells mediate
retinoid-dependent functions essential for renal development. Development 1999, 126,
237. Batourina, E.; Gim, S.; Bello, N.; Shy, M.; Clagett-Dame, M.; Srinivas, S.; Costantini, F.;
Mendelsohn, C. Vitamin A controls epithelial/mesenchymal interactions through Ret expression.
Nat. Genet. 2001, 27, 7478.
238. Niederreither, K.; Fraulob, V.; Garnier, J.M.; Chambon, P.; Dolle, P. Differential expression of
retinoic acid-synthesizing (RALDH) enzymes during fetal development and organ differentiation
in the mouse. Mech. Dev. 2002, 110, 165171.
239. Schuchardt, A.; D’Agati, V.; Larsson-Blomberg, L.; Costantini, F.; Pachnis, V. Defects in the
kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 1994,
367, 380383.
240. Schuchardt, A.; D’Agati, V.; Pachnis, V.; Costantini, F. Renal agenesis and hypodysplasia in
ret-k- mutant mice result from defects in ureteric bud development. Development 1996, 122,
241. Batourina, E.; Choi, C.; Paragas, N.; Bello, N.; Hensle, T.; Costantini, F.D.; Schuchardt, A.;
Bacallao, R.L.; Mendelsohn, C.L. Distal ureter morphogenesis depends on epithelial cell
remodeling mediated by vitamin A and Ret. Nat. Genet. 2002, 32, 109115.
242. Rosselot, C.; Spraggon, L.; Chia, I.; Batourina, E.; Riccio, P.; Lu, B.; Niederreither, K.;
Dolle, P.; Duester, G.; Chambon, P.; Costantini, F.; Gilbert, T.; Molotkov, A.; Mendelsohn, C.
Non-cell-autonomous retinoid signaling is crucial for renal development. Development 2010,
137, 283292.
243. Hoy, W.E.; Hughson, M.D.; Bertram, J.F.; Douglas-Denton, R.; Amann, K. Nephron number,
hypertension, renal disease, and renal failure. J. Am. Soc. Nephrol. 2005, 16, 25572564.
244. Brenner, B.M.; Mackenzie, H.S. Nephron mass as a risk factor for progression of renal disease.
Kidney Int. Suppl. 1997, 63, S124S127.
245. Poladia, D.P.; Kish, K.; Kutay, B.; Bauer, J.; Baum, M.; Bates, C.M. Link between reduced
nephron number and hypertension: studies in a mutant mouse model. Pediatr. Res. 2006, 59,
246. Keller, G.; Zimmer, G.; Mall, G.; Ritz, E.; Amann, K. Nephron number in patients with primary
hypertension. New Engl. J. Med. 2003, 348, 101108.
247. Makrakis, J.; Zimanyi, M.A.; Black, M.J. Retinoic acid enhances nephron endowment in rats
exposed to maternal protein restriction. Pediatr. Nephrol. 2007, 22, 18611867.
Nutrients 2011, 3
248. Torfs, C.P.; Curry, C.J.; Bateson, T.F.; Honore, L.H. A population-based study of congenital
diaphragmatic hernia. Teratology 1992, 46, 555565.
249. Beurskens, L.W.; Tibboel, D.; Lindemans, J.; Duvekot, J.J.; Cohen-Overbeek, T.E.; Veenma, D.C.;
de Klein, A.; Greer, J.J.; Steegers-Theunissen, R.P. Retinol status of newborn infants is
associated with congenital diaphragmatic hernia. Pediatrics 2010, 126, 712720.
250. Pasutto, F.; Sticht, H.; Hammersen, G.; Gillessen-Kaesbach, G.; Fitzpatrick, D.R.; Nurnberg, G.;
Brasch, F.; Schirmer-Zimmermann, H.; Tolmie, J.L.; Chitayat, D.; et al. Mutations in STRA6
cause a broad spectrum of malformations including anophthalmia, congenital heart defects,
diaphragmatic hernia, alveolar capillary dysplasia, lung hypoplasia, and mental retardation. Am.
J. Hum. Genet. 2007, 80, 550560.
251. Goumy, C.; Gouas, L.; Marceau, G.; Coste, K.; Veronese, L.; Gallot, D.; Sapin, V.; Vago, P.;
Tchirkov, A. Retinoid pathway and congenital diaphragmatic hernia: hypothesis from the
analysis of chromosomal abnormalities. Fetal Diagn. Ther. 2010, 28, 129139.
252. Clugston, R.D.; Klattig, J.; Englert, C.; Clagett-Dame, M.; Martinovic, J.; Benachi, A.;
Greer, J.J. Teratogen-induced, dietary and genetic models of congenital diaphragmatic hernia
share a common mechanism of pathogenesis. Am. J. Pathol. 2006, 169, 15411549.
253. Mey, J.; Babiuk, R.P.; Clugston, R.; Zhang, W.; Greer, J.J. Retinal dehydrogenase-2 is inhibited
by compounds that induce congenital diaphragmatic hernias in rodents. Am. J. Pathol. 2003, 162,
254. Clugston, R.D.; Zhang, W.; Alvarez, S.; de Lera, A.R.; Greer, J.J. Understanding abnormal
retinoid signaling as a causative mechanism in congenital diaphragmatic hernia. Am. J. Respir.
Cell Mol. Biol. 2010, 42, 276285.
255. Hind, M.; Corcoran, J.; Maden, M. Temporal/spatial expression of retinoid binding proteins and
RAR isoforms in the postnatal lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 2002, 282,
256. Malpel, S.; Mendelsohn, C.; Cardoso, W.V. Regulation of retinoic acid signaling during lung
morphogenesis. Development 2000, 127, 30573067.
257. Mollard, R.; Viville, S.; Ward, S.J.; Decimo, D.; Chambon, P.; Dolle, P. Tissue-specific
expression of retinoic acid receptor isoform transcripts in the mouse embryo. Mech. Dev. 2000,
94, 223232.
258. Wang, Z.; Dolle, P.; Cardoso, W.V.; Niederreither, K. Retinoic acid regulates morphogenesis
and patterning of posterior foregut derivatives. Dev. Biol. 2006, 297, 433445.
259. Desai, T.J.; Chen, F.; Lu, J.; Qian, J.; Niederreither, K.; Dolle, P.; Chambon, P.; Cardoso, W.V.
Distinct roles for retinoic acid receptors alpha and beta in early lung morphogenesis. Dev. Biol.
2006, 291, 1224.
260. Hind, M.; Gilthorpe, A.; Stinchcombe, S.; Maden, M. Retinoid induction of alveolar
regeneration: from mice to man? Thorax 2009, 64, 451457.
261. Mollard, R.; Ghyselinck, N.B.; Wendling, O.; Chambon, P.; Mark, M. Stage-dependent
responses of the developing lung to retinoic acid signaling. Int. J. Dev. Biol. 2000, 44, 457462.
262. Chen, F.; Cao, Y.; Qian, J.; Shao, F.; Niederreither, K.; Cardoso, W.V. A retinoic acid-dependent
network in the foregut controls formation of the mouse lung primordium. J. Clin. Invest. 2010,
120, 20402048.
Nutrients 2011, 3
263. Wongtrakool, C.; Malpel, S.; Gorenstein, J.; Sedita, J.; Ramirez, M.I.; Underhill, T.M.;
Cardoso, W.V. Down-regulation of retinoic acid receptor alpha signaling is required for
sacculation and type I cell formation in the developing lung. J. Biol. Chem. 2003, 278,
264. Massaro, D.; Massaro, G.D. Retinoids, alveolus formation, and alveolar deficiency: clinical
implications. Am. J. Respir. Cell Mol. Biol. 2003, 28, 271274.
265. Massaro, G.D.; Massaro, D. Retinoic acid treatment partially rescues failed septation in rats and
in mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 2000, 278, L955L960.
266. Massaro, G.D.; Massaro, D. Postnatal treatment with retinoic acid increases the number of
pulmonary alveoli in rats. Am. J. Physiol. 1996, 270, L305L310.
267. Massaro, G.D.; Massaro, D.; Chambon, P. Retinoic acid receptor-alpha regulates pulmonary
alveolus formation in mice after, but not during, perinatal period. Am. J. Physiol. Lung Cell. Mol.
Physiol. 2003, 284, L431L433.
268. Massaro, G.D.; Massaro, D.; Chan, W.Y.; Clerch, L.B.; Ghyselinck, N.; Chambon, P.;
Chandraratna, R.A. Retinoic acid receptor-beta: an endogenous inhibitor of the perinatal
formation of pulmonary alveoli. Physiol. Genomics 2000, 4, 5157.
269. McGowan, S.; Jackson, S.K.; Jenkins-Moore, M.; Dai, H.H.; Chambon, P.; Snyder, J.M. Mice
bearing deletions of retinoic acid receptors demonstrate reduced lung elastin and alveolar
numbers. Am. J. Respir. Cell Mol. Biol. 2000, 23, 162167.
270. Checkley, W.; West, K.P., Jr.; Wise, R.A.; Baldwin, M.R.; Wu, L.; LeClerq, S.C.; Christian, P.;
Katz, J.; Tielsch, J.M.; Khatry, S.; Sommer, A. Maternal vitamin A supplementation and lung
function in offspring. New Engl. J. Med. 2010, 362, 17841794.
271. Martin, M.; Gallego-Llamas, J.; Ribes, V.; Kedinger, M.; Niederreither, K.; Chambon, P.;
Dolle, P.; Gradwohl, G. Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice.
Dev. Biol. 2005, 284, 399411.
272. Molotkov, A.; Molotkova, N.; Duester, G. Retinoic acid generated by Raldh2 in mesoderm is
required for mouse dorsal endodermal pancreas development. Dev. Dyn. 2005, 232, 950957.
273. Stafford, D.; Hornbruch, A.; Mueller, P.R.; Prince, V.E. A conserved role for retinoid signaling
in vertebrate pancreas development. Dev. Genes Evol. 2004, 214, 432441.
274. Stafford, D.; Prince, V.E. Retinoic acid signaling is required for a critical early step in zebrafish
pancreatic development. Curr. Biol. 2002, 12, 12151220.
275. Chen, Y.; Pan, F.C.; Brandes, N.; Afelik, S.; Solter, M.; Pieler, T. Retinoic acid signaling is
essential for pancreas development and promotes endocrine at the expense of exocrine cell
differentiation in Xenopus. Dev. Biol. 2004, 271, 144160.
276. Alexa, K.; Choe, S.K.; Hirsch, N.; Etheridge, L.; Laver, E.; Sagerstrom, C.G. Maternal and
zygotic aldh1a2 activity is required for pancreas development in zebrafish. PLoS ONE 2009,
4, e8261.
277. Pan, F.C.; Chen, Y.; Bayha, E.; Pieler, T. Retinoic acid-mediated patterning of the pre-pancreatic
endoderm in Xenopus operates via direct and indirect mechanisms. Mech. Dev. 2007, 124,
Nutrients 2011, 3
278. Tulachan, S.S.; Doi, R.; Kawaguchi, Y.; Tsuji, S.; Nakajima, S.; Masui, T.; Koizumi, M.;
Toyoda, E.; Mori, T.; Ito, D.; Kami, K.; Fujimoto, K.; Imamura, M. All-trans retinoic acid
induces differentiation of ducts and endocrine cells by mesenchymal/epithelial interactions in
embryonic pancreas. Diabetes 2003, 52, 7684.
279. Ahlgren, U.; Jonsson, J.; Edlund, H. The morphogenesis of the pancreatic mesenchyme is
uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice. Development
1996, 122, 14091416.
280. Jonsson, J.; Carlsson, L.; Edlund, T.; Edlund, H. Insulin-promoter-factor 1 is required for
pancreas development in mice. Nature 1994, 371, 606609.
281. Offield, M.F.; Jetton, T.L.; Labosky, P.A.; Ray, M.; Stein, R.W.; Magnuson, M.A.; Hogan, B.L.;
Wright, C.V. PDX-1 is required for pancreatic outgrowth and differentiation of the rostral
duodenum. Development 1996, 122, 983995.
282. Kinkel, M.D.; Sefton, E.M.; Kikuchi, Y.; Mizoguchi, T.; Ward, A.B.; Prince, V.E. Cyp26
enzymes function in endoderm to regulate pancreatic field size. Proc. Natl. Acad. Sci. USA 2009,
106, 78647869.
283. Gittes, G.K. Developmental biology of the pancreas: a comprehensive review. Dev. Biol. 2009,
326, 435.
284. Pearl, E.J.; Bilogan, C.K.; Mukhi, S.; Brown, D.D.; Horb, M.E. Xenopus pancreas development.
Dev. Dyn. 2009, 238, 12711286.
285. Stratford, T.; Horton, C.; Maden, M. Retinoic acid is required for the initiation of outgrowth in
the chick limb bud. Curr. Biol. 1996, 6, 11241133.
286. Stratford, T.; Logan, C.; Zile, M.; Maden, M. Abnormal anteroposterior and dorsoventral
patterning of the limb bud in the absence of retinoids. Mech. Dev. 1999, 81, 115125.
287. Niederreither, K.; Vermot, J.; Schuhbaur, B.; Chambon, P.; Dolle, P. Embryonic retinoic acid
synthesis is required for forelimb growth and anteroposterior patterning in the mouse.
Development 2002, 129, 35633574.
288. Mic, F.A.; Sirbu, I.O.; Duester, G. Retinoic acid synthesis controlled by Raldh2 is required early
for limb bud initiation and then later as a proximodistal signal during apical ectodermal ridge
formation. J. Biol. Chem. 2004, 279, 2669826706.
289. Gibert, Y.; Gajewski, A.; Meyer, A.; Begemann, G. Induction and prepatterning of the zebrafish
pectoral fin bud requires axial retinoic acid signaling. Development 2006, 133, 26492659.
290. Mercader, N.; Leonardo, E.; Piedra, M.E.; Martinez, A.C.; Ros, M.A.; Torres, M. Opposing RA
and FGF signals control proximodistal vertebrate limb development through regulation of Meis
genes. Development 2000, 127, 39613970.
291. Benazet, J.D.; Zeller, R. Vertebrate limb development: moving from classical morphogen
gradients to an integrated 4-dimensional patterning system. Cold Spring Harb. Perspect. Biol.
2009, 1, a001339.
292. Zeller, R.; Lopez-Rios, J.; Zuniga, A. Vertebrate limb bud development: moving towards
integrative analysis of organogenesis. Nat. Rev. Genet. 2009, 10, 845858.
293. Lewandoski, M.; Mackem, S. Limb development: the rise and fall of retinoic acid. Curr. Biol.
2009, 19, R558R561.
Nutrients 2011, 3
294. Dupe, V.; Ghyselinck, N.B.; Thomazy, V.; Nagy, L.; Davies, P.J.; Chambon, P.; Mark, M.
Essential roles of retinoic acid signaling in interdigital apoptosis and control of BMP-7
expression in mouse autopods. Dev. Biol. 1999, 208, 3043.
295. Zhao, X.; Brade, T.; Cunningham, T.J.; Duester, G. Retinoic acid controls expression of tissue
remodeling genes Hmgn1 and Fgf18 at the digit-interdigit junction. Dev. Dyn. 2010, 239, 665671.
296. Rodriguez-Leon, J.; Merino, R.; Macias, D.; Ganan, Y.; Santesteban, E.; Hurle, J.M. Retinoic
acid regulates programmed cell death through BMP signalling. Nat. Cell Biol. 1999, 1, 125126.
297. Hernandez-Martinez, R.; Castro-Obregon, S.; Covarrubias, L. Progressive interdigital cell death:
regulation by the antagonistic interaction between fibroblast growth factor 8 and retinoic acid.
Development 2009, 136, 36693678.
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... Vitamin A is an essential nutrient involved in multiple biological processes including vision, reproduction, growth, development, and differentiation [1]. Retinoids derivatives) are derived from the diet as retinyl esters or β-carotene (Fig 1). ...
... 4-Oxo-atRA and 4-oxo-13cisRA have been detected previously in human blood although the concentrations of 4-oxo-atRA were below the detection limit in most samples [6]. During pregnancy, retinoids are essential for the maintenance of the placenta as well as the development of the embryo [1]. However, current data suggests that as long as sufficient vitamin A is provided to the fetus from maternal stores the embryo/fetus can self-regulate retinoic acid concentrations and gradients necessary for appropriate morphogenesis and fetal development. ...
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Retinoids and vitamin A are essential for multiple biological functions, including vision and immune responses, as well as the development of an embryo during pregnancy. Despite its importance, alterations in retinoid homeostasis during normal human pregnancy are incompletely understood. We aimed to characterize the temporal changes in the systemic retinoid concentrations across pregnancy and postpartum period. Monthly blood samples were collected from twenty healthy pregnant women, and plasma concentrations of retinol, all-trans-retinoic acid (atRA), 13-cis-retinoic acid (13cisRA), and 4-oxo-retinoic acids were measured using liquid chromatography-tandem mass spectrometry. Significant decreases in 13cisRA concentrations over the pregnancy were observed, with rebound increases in retinol and 13cisRA levels after delivery. Of note, atRA concentrations exhibited a unique temporal pattern with levels peaking at mid-pregnancy. While the 4-oxo-atRA concentration was below the limit of quantification, 4-oxo-13cisRA was readily detectable, and its temporal change mimicked that of 13cisRA. The time profiles of atRA and 13cisRA remained similar after correction by albumin levels for plasma volume expansion adjustment. Together, the comprehensive profiling of systemic retinoid concentrations over the course of pregnancy provides insights into pregnancy-mediated changes in retinoid disposition to maintain its homeostasis.
... Retinoids play an important role in the morphogenesis of various organs in mammals. In particular, maternal retinoids can cause syndromic malformations of the urogenital tract like those seen in humans (17). They also affect mesenchymal/epithelial interactions in the developing kidneys, and other organs (18). ...
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Background: Vitamin A (retinol) and its derivatives are essential for maintaining cell differentiation in adult organisms as well as for normal embryonic development in fetuses. On the other hand, high amounts of vitamin A are known to be teratogenic. The formation of urogenital structures depends heavily on retinoic acid receptors. The effects of subteratogenic dosages of retinol on the urinary system have not been adequately studied. The aim of the current study was to investigate the effects of moderate and low doses of vitamin A on the fetal kidney. Materials and Methods: Pregnant rats were divided into 6 groups. On day 10 to 12 of pregnancy (P10-P12) the first group was administered 10000 IU/kg, the second group 20000 IU/kg, the third group 30000 IU/kg, the fourth group 40000 IU/kg and the fifth group 50000 IU/kg oral vitamin A. The control group only received 1 ml of corn oil on the same days. The fetuses were delivered via cesarean section at P19. The kidneys of the fetuses were removed after cardiac perfusion was used to fixate them. After histological preparation of the kidneys, the slides were stained with hematoxylin and eosin. By using stereological methods, the kidneys' volume (V), glomeruli per unit area (NAg), and mean glomeruli diameter (D) were all estimated. The results were statistically analyzed. Results: The renal volumes of the 20000, 30000 and 40000 IU/kg groups were higher than those of the other groups. It was also found that the NAg levels of the group receiving 50000 IU/kg Vitamin A were lower than those of all other groups. Moreover, the NAg levels of the groups receiving 20000, 30000 and 40000 IU/kg vitamin A were higher than those of the control group and the group receiving 10000 IU/kg. While the glomeruli diameters of the experimental groups were not different from those of the control group, the glomeruli diameters of the group receiving 20000 and 50000 IU/kg retinol were larger than those of the groups receiving 10000 and 40000 IU/kg vitamin A. Conclusions: Given the estimated higher V, Na, and D values of the group receiving 20000 IU/kg vitamin A, we can assume that this particular dose has a significant effect on renal morphology and development.
... Vitamin A is one of the key components involved with visual function, but also in development and cellular differentiation, primary epithelial cells and bone tissue [94]. It also sustains fetal development and embryonic growth [95]. Furthermore, it keeps the immune system active and strengthens its humoral and cellular components [96]. ...
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The aim of this review is to provide an overview of different compositions, in terms of main minerals and vitamins, of milk from animal species that represent the most common source of this food for humans, highlighting the uniqueness of nutritional qualities linked to animal species. It is known that milk is an important and valuable food for human nutrition, representing an excellent source of nutrients. Indeed, it contains both macronutrients (proteins, carbohydrates, and fat) that contribute to its nutritive and biological value and micronutrients represented by minerals and vitamins, which play a relevant role in the body's various vital functions. Although their supply is represented by small quantities, vitamins and minerals are important components for a healthy diet. Milk composition in terms of minerals and vitamins differs between various animal species. Micronutrients are important components for human health as their deficiency is causes of malnutrition. Furthermore, we report on the most significant metabolic and beneficial effects of certain micronutrients in the milk, emphasizing the importance of this food for human health and the need for some milk enrichment procedures with the most relevant micronutrients to human health.
... Retinol is a common fat-soluble vitamin essential for animal visual function, embryonic development, immune regulation, reproduction, cell growth, and differentiation, etc. (Saari et al., 2004;Yadu and Kumar, 2019;Clagett-Dame and Knutson, 2011;Huang et al., 2018;Polcz and Barbul, 2019;Ayuso et al., 2015;Yang et al., 2018). However, retinol must produce active retinoic acid to perform its function (Kedishvili et al., 2013). ...
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As one of the most successful domesticated animals in the Neolithic age, sheep gradually migrated all over the world with human activities. During the domestication process, remarkable changes have taken place in morphology, physiology, and behavior, resulting in different breeds with different characters via artificial and natural selection. However, the genetic background responsible for these phenotypic variations remains largely unclear. Here, we used whole genome resequencing technology to compare and analyze the genome differences between Asiatic mouflon wild sheep (Ovis orientalis) and Hu sheep (Ovis aries). A total of 755 genes were positively selected in the process of domestication and selection, and the genes related to sensory perception had directional evolution in the autosomal region, such as OPRL1, LEF1, TAS1R3, ATF6, VSX2, MYO1A, RDH5, and some novel genes. A missense mutation of c.T722C/p.M241T in exon 4 of RDH5 existing in sheep were found, and the T allele was completely fixed in Hu sheep. In addition, the mutation with the C allele reduced the retinol dehydrogenase activity encoding by RDH5, which can impair retinoic acid metabolism and further influenced the visual cycle. Overall, our results showed significant enrichment for positively selected genes involved in sensory perception development during sheep domestication; RDH5 and its variants may be related to the retinal degeneration in sheep. We infer that the wild sheep ancestors with weaker visual sensitivity were weeded out by humans, and the mutation was selective, swept by the dual pressures of natural and artificial selection.