Special Article

Vitamin A and clefting: putative biological mechanisms nure_425 613..624

Mignon MG Ackermans, Huiqing Zhou, Carine EL Carels, Frank ADTG Wagener, and
Johannes W Von den Hoff

Nutritional factors such as vitamin intake contribute to the etiology of cleft palate.
Vitamin A is a regulator of embryonic development. Excess vitamin A can cause

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congenital malformations such as spina bifida and cleft palate. Therefore,
preventive nutritional strategies are required. This review identifies putative
biological mechanisms underlying the association between maternal vitamin A
intake and cleft palate. Excessive vitamin A may disturb all three stages of
palatogenesis: 1) during shelf outgrowth, it may decrease cell proliferation and thus
prevent tissue development; 2) it may prevent shelf elevation by affecting
extracellular matrix composition and hydration; and 3) during shelf fusion, it may
affect epithelial differentiation and apoptosis, which precludes the formation of a
continuous palate. In general, high doses of vitamin A affect palatogenesis through
interference with cell proliferation and growth factors such as transforming growth
factor b and platelet-derived growth factor. The effects of lower doses of vitamin A
need to be investigated in greater depth in order to improve public health
recommendations.
© 2011 International Life Sciences Institute

INTRODUCTION lism. The maternal diet during the first trimester is

Orofacial clefts are the most common craniofacial birth
defects in humans. These disorders are caused by
impaired fusion of one or more orofacial structures in the
embryo.1 In the etiology of orofacial clefts, both genetic
and environmental factors are involved. The genetic
background of orofacial clefting is starting to become
unraveled because of advances in epidemiological
research. However, the environmental factors that are
involved, as well as their interaction with genetic factors,
are still incompletely understood.2
One of the most extensively studied environmental
factors in congenital disorders is maternal nutrition.
During pregnancy, the nutritional status of the embryo
is fully dependent on maternal food intake and metabo-
crucial for the development of various organs such as
the brain and the heart. A nutritional excess or defi-
ciency can lead to birth defects of the neural tube, other
neurological abnormalities, congenital heart disease, and
intestinal malformations.3 The maternal diet is also
associated with orofacial clefting.4 For example deficien-
cies in maternal nutrient intake, such as zinc and myo-
inositol, can increase the risk of cleft palate (CP) in
offspring.5 Vitamins are of particular interest because
they cannot be synthesized by the body. Maternal folate
deficiency may increase the risk of neural tube defects,
but it is also reported to increase the risk of CP in off-
spring.6,7 Evidence for this association, however, remains
inconclusive.8 Vitamins are obtained through the diet,
and their sources within the diet can be divided into

Affiliations: MMG Ackermans, CEL Carels, FADTG Wagener, and JW Von den Hoff are with the Department of Orthodontics and Craniofacial
Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. H Zhou is
with the Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre,
Nijmegen, The Netherlands.
Correspondence: JW Von den Hoff, Department of Orthodontics and Craniofacial Biology, Radboud University Nijmegen Medical Centre,
Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands. E-mail: h.vondenhoff@dent.umcn.nl, Phone: +31-243614084,
Fax: +31-243540631.
Key words: cleft palate, embryonic development, growth factors, nutrition, vitamin A

doi:10.1111/j.1753-4887.2011.00425.x
Nutrition Reviews® Vol. 69(10):613–624 613
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Figure 1 The retinoic acid signaling pathway. Retinol from the diet is converted into retinoic acid through retinal. Binding of
retinoic acid to its receptor on the DNA regulates gene transcription. Abbreviations: RAR, retinoic acid receptor; RXR, retinoid X
receptor; RARE, retinoic acid response element; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; CRBP, cellular
retinol-binding protein; CRABP, cellular retinoic-acid-binding protein; SDR, short-chain dehydrogenase/reductase.

whole food, foods fortified with synthetic vitamins, and
dietary supplements. Vitamins play key roles in growth
and development and can, therefore, be considered vital
ingredients of embryonic nutrition.
The essential vitamin A, particularly its derivate ret-
inoic acid (RA), is an important regulator of embryo-
genesis. RA regulates proliferation, differentiation, and
apoptosis during the morphogenesis of embryonic
structures.9 In addition, RA regulates the specification of
cell identity and gene expression through activation of
nuclear receptors of the retinoid X receptor and retinoic
acid receptor families, which bind to specific DNA ele-
ments in the regulatory regions of target genes called
retinoic acid response elements.10–12 Precursor forms of
vitamin A are present in vegetables and animal tissues.
Vegetables contain carotenoids, while meat contains
retinyl esters. After ingestion, both are transformed
into retinol and stored in the liver. In the liver, the
stellate cells control blood plasma concentration by
retinol storage and mobilization. Retinol in the blood
enters the target cell and is oxidized to RA in a two-
step process (Figure 1). The first step of RA synthesis
is the reversible oxidation of retinol to retinal, which
is mainly mediated by alcohol dehydrogenases and
short-chain dehydrogenases/reductases. The second
step is the irreversible oxidation of retinal to RA by
aldehyde dehydrogenase. Alternatively, RA can be
directly formed from b-carotene.13 The availability of
RA is controlled by proteins such as cellular retinol-
binding proteins and cellular retinoic-acid-binding
proteins.
RA receptor knockout studies have illustrated the
importance of RA-signaling in palatogenesis. Among
other malformations, these knockouts result in orofacial
clefting.14 In general, over- or under-exposure to RA
during embryonic and fetal development disturbs the
closure of the neural tube and the development of many
other organs and limbs.15,16 According to the 2001 recom-
mendations of the US Food and Nutrition Board, the
recommended dietary allowance (RDA) for vitamin A is
900 mg for adult males, 700 mg for adult females, and
770 mg during pregnancy.17 Epidemiological studies in
humans performed to date have not demonstrated a clear
association between vitamin A and CP.18–20 However, evi-
dence from animal studies shows a clear increase in the
risk of CP related to excess vitamin A.21–23
The putative effects of low doses of RA in the human
diet are also discussed in this review. The developmental
stage at the time of exposure determines the sensitivity
to CP induction. In the first weeks of pregnancy,
RA-induced apoptosis in cell populations that derive
from the first branchial arch leads to CP.24,25 However, in
humans, RA exposure seems most critical between devel-
opmental weeks 4 and12.26 This review discusses the role
of RA in the regulation of palatogenesis during this
particular period and identifies putative biological
mechanisms to explain the association between RA and
orofacial clefting. The majority of studies in this field are
based on mouse models. First, a brief overview of normal
facial and palatal development is given. Subsequently, the
effects of RA in the different stages of palatogenesis are
discussed in detail.

614 Nutrition Reviews® Vol. 69(10):613–624


Figure 2 Facial processes of a human embryo. Scanning electron micrograph of a human embryo at 7 weeks. The processes
that form the face are indicated, except for the frontal process that lies above the area shown. Reproduced from Nanci (2003)27
with permission.
DEVELOPMENT OF THE CRANIOFACIAL COMPLEX
The development of the face starts in week 4 of human
embryonic development, when cells migrating from the
neural crest combine with the core mesoderm and the
overlying epithelium to establish the facial primor-
dia.27,28 The topographical center of the developing facial
structures is the stomodeum, or primitive mouth. It is
delimited by five distinct processes (Figure 2). The
process rostral to the stomodeum is called the frontal
process. Laterally, the stomodeum is delimited by a pair
of maxillary processes and caudally by the mandibular
processes.
In week 5 of human embryonic development
(embryonic day E10 in mice), the epithelium on both
sides of the ventrolateral part of the frontal process thick-
ens and gives rise to the nasal placodes. Directed growth
and expansion around the nasal placodes results in for-
mation of the nasal pits and the lateral and medial nasal
processes (Figure 2).27,29,30 After fusion of the lateral and
medial nasal processes, the maxillary and medial nasal
processes fuse to form the upper lip. The fusion of the
upper lip takes place between developmental weeks 5 and
7 (E10.5–12.5 in mice) and is based on mechanisms
similar to those involved in the fusion of the secondary
palate. A failure of lip fusion may secondarily affect the
later fusion of the secondary palate, which leads to a com-
bined cleft lip and palate (CLP). The secondary palate is
formed between weeks 6 and 12 of human development
(E12–16 in mice). The inner parts of the maxillary pro-
cesses develop bilateral shelf-like outgrowths that grow
downward on either side of the developing tongue
(Figure 3A–D). As palatogenesis progresses, the palatal
shelves move upward and grow towards the midline.
After contact, the medial edge epithelia (MEE) of the
opposing processes adhere and form a midline epithelial
Nutrition Reviews® Vol. 69(10):613–624
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seam (MES).31 Subsequently, the MES disappears, but the
exact fate of these cells remains controversial. The pro-
posed mechanisms of MES disappearance are discussed
later in this review. Palatal confluence is achieved when
the MES has completely disappeared and thus mesenchy-
mal continuity across the secondary palate is established.
Fusion spreads from the middle third of the palate in
anterior and posterior directions and is completed by
week 12 of development (E16 in mice).28,30 Subsequently,
the ossified hard palate forms out of the anterior two-
thirds of the palate, while the posterior one-third of the
palate forms the soft palate.32 Interestingly, palatal fusion
can also be reproduced in cultured mouse embryonic
palatal shelves (Figure 3E–G).
In summary, the development of the human cranio-
facial complex takes place between weeks 4 and 12 of
gestation. The rapid proliferative expansion and complex
morphogenetic events that coordinate facial development
make it highly sensitive to the effects of gene mutations
and to environmental influences. This may explain the
relatively high incidence of craniofacial birth defects like
CP compared with other craniofacial disorders. In the
next sections, palatogenesis is reviewed in detail, and
putative biological effects of RA leading to a cleft are
identified.
SHELF OUTGROWTH: PUTATIVE EFFECTS OF
RETINOIC ACID
The secondary palate develops from bilateral out-
growths of the maxillary processes, generally referred to
as the palatal shelves. The shelves increase in size by
mesenchymal cell proliferation and by the production
and hydration of extracellular matrix (ECM) compo-
nents.28 In pregnant mice, RA overexposure reduces the
growth of the palatal shelves in the embryo.33 This
615

Figure 3 Palatogenesis in vivo and in vitro in mice. In
vivo (A–D): Frontal sections through the developing mouse
head, showing the palatal shelves (A) E13: The bilateral
palatal shelves grow down vertically along the two sides of
the tongue. (B) E14: The palatal shelves are elevated above
the dorsum of the tongue and come into contact to form the
MES. (C) E15: The MES disappears, and palatal fusion is com-
pleted. (D) E16: Ossification of the palate has started. Abbre-
viations: MES, midline epithelial seam; N, nasal septum; T,
tongue; P, palatal shelf. In vitro (E–G): Palatal shelves were
dissected from E13 mouse embryos, cultured for 1, 2, or 3
days, and stained with HandE stain. (E) Day 1: The palatal
shelves come into contact with each other. (F) Day 2: The
MES has partly disintegrated. (G) Day 3: The MES has totally
disappeared, and palatal fusion is completed. Osteoid-like
tissue (O) is present in the lateral palatal mesenchyme. The
bar represents 100 mm. Abbreviations: C, connective tissue;
E, epithelium; MES, midline epithelial seam. Reproduced
from Meng et al. (2009)32 with permission.
seems to be a consequence of reduced mesenchymal cell
proliferation as well as inhibition of ECM produc-
tion.34,35 Some authors also found evidence of mesen-
chymal apoptosis in response to RA,36 but these findings
are controversial.37 The effects of RA on mesenchymal
616
proliferation and ECM production will be discussed in
detail in the next section of this review. Proliferation is
the main process in shelf outgrowth, while ECM pro-
duction is crucial in palatal shelf elevation.
The general effects of RA are summarized in
Tables 1 and 2. RA inhibits the proliferation of mouse
palatal mesenchyme by arresting the cells in the G1
phase.34 This occurs through upregulation of cyclin-
dependent kinase inhibitor p21(Cip1) and subsequent
hypophosphorylation of the tumor suppressor Rb.37,38
RA also interferes with specific signaling pathways in
cell proliferation by modulating platelet-derived growth
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factors (PDGFs).39,40 PDGF-C is downregulated by RA
in mouse embryonic mesenchymal palatal cells. During
palatogenesis, downregulation of PDGF signaling by RA
results in hypoplastic palatal shelves that are not able to
fuse,39 probably due to inhibition of nuclear regulator
genes that control cell proliferation, such as members of
the signal transducer and activator or transcription
family and nuclear factor kB.41 Moreover, RA stimulates
expression of the small proteoglycan decorin, which
might inhibit mesenchymal proliferation.33 In several
cell types, decorin activates the epidermal growth factor
receptor (EGFR) that induces activation of mitogen-
activated protein kinases, mobilization of intracellular
calcium, upregulation of p21, and, finally, growth
suppression.42
In addition, RA interacts with members of the trans-
forming growth factor b (TGF-b) superfamily, which
contains, among other items, the bone morphogenetic
proteins and the TGF-bs. These are multifunctional
growth factors that mediate a variety of biological pro-
cesses such as the proliferation of mesenchymal cells, the
synthesis of ECM proteins, and apoptosis.43 During
palatal shelf outgrowth, bone morphogenetic protein 2
stimulates mesenchymal proliferation,44 but its expres-
sion is decreased by RA.45 The effect of TGF-bs depends
highly on the cell type and the developmental stage at the
time of exposure. For that reason, the interactions
between RA and the TGF-b signaling pathway are often
contradictory. Some studies show an increase in TGF-b1
and TGF-b2 signaling in response to RA, while others
show a decrease (Table 1). Decreased expression of the
TGF-b1 and -b2 isoforms reduces mesenchymal prolif-
eration. The TGF-b3 isoform is mainly involved in a later
stage and will be discussed in another section. TGF-b
signaling is downregulated by RA through suppression of
Smad2/3 phosphorylation, probably by binding of the
Smad transcriptional corepressor TGIF (TG-interacting
factor).32,46,47
In conclusion, the major mechanisms through which
excess RA may disturb palatal shelf outgrowth include
inhibition of mesenchymal proliferation and signaling of
growth factors.
Nutrition Reviews® Vol. 69(10):613–624
 Table 1 In vitro effects of retinoic acid.
Cell type RA form and
concentration
Mesenchymal MC-3T3-El ATRA 1 ¥ 10-6 mol/L
cells
MEMP ATRA 5 ¥ 10-6 and
10 ¥ 10-6 mol/L
ATRA 0.33 ¥ 10-6 and
3.3 ¥ 10-6 mol/L
ATRA 0.03 ¥ 10-6,
0.33 ¥ 10-6, and
3.3 ¥ 10-6 mol/L
13-cis RA (isotretinoin)
1 ¥ 10-5, 1 ¥ 10-4, and
4 ¥ 10-4 mol/L
ATRA 1 ¥ 10-6 to
1 ¥ 10-8 mol/L
ATRA 1 ¥ 10-6 to
1 ¥ 10-10 mol/L
ATRA 1 ¥ 10-6 mol/L
MMF ATRA 1 ¥ 10-6 mol/L
PMS ATRA 1 ¥ 10-6 mol/L
PNHOF ATRA 10 ¥ 10-6 mol/L
REMP Retinyl palmitate
3 ¥ 10-2 mol/L
Epithelial MEE ATRA 1 ¥ 10-5, 1 ¥ 10-7,
cells and 1 ¥ 10-9 mol/L
ATRA 1 ¥ 10-5 mol/L
PNGE ATRA 1 ¥ 10-6 mol/L
PNK 1 ¥ 10-6 mol/L, form
unknown, most
probably ATRA
NT2 ATRA 10 ¥ 10-6 mol/L
Abbreviations and symbols: ↑, Stimulation; ↓, inhibition; –, no effect; ATRA, all-trans retinoic acid; BMP, bone morphogenetic protein;
EGF, epidermal growth factor; GAGs, glycosaminoglycans; HA, hyaluronan; MC-3T3-El, fibroblast-like mouse cell line; MEE, medial edge
epithelial cell; MEMP, mouse embryonic mesenchymal palatal cell; MMF, multipotent mesenchymal fibroblast; NT2, human
teratocarcinoma cell line; PDGF, platelet-derived growth factor; PMS, pluripotent mesenchymal stem cell; PNGE, postnatal gingival
epithelial cell; PNHOF, postnatal human oral fibroblast; PNK, postnatal keratinocytes; REMP, rat embryonic mesenchymal palatal cell;
TGF, transforming growth factor.
SHELF ELEVATION: PUTATIVE EFFECTS OF
RETINOIC ACID
The shelves elevate to a horizontal position above the
dorsum of the tongue, while the mandible elongates and
the tongue moves downward (Figures 3A,B). Excess RA
was found to increase apoptosis in the base of the fetal
tongue, which might physically impair the horizontal
elevation of the palatal shelves.37 Furthermore, excess RA
prevents tongue withdrawal by disturbance of tongue
muscle development.48 As shown in several models,
tongue withdrawal is a prerequisite for proper palatoge-
nesis.49 RA prevents tongue withdrawal through down-
Nutrition Reviews® Vol. 69(10):613–624
Effect Reference
↑ Apoptosis Guo et al. (2008)36
↓ Proliferation, BMP-7 expression
↓ PDGF-C Han et al. (2006)39
– TGF-b1
↑TGF-b2 and TGF-b3 Nugent et al. (1998)105
↓ Mesenchymal proliferation Nugent et al. (1998)105
Watanabe et al. (1988)35
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↓ Mesenchymal proliferation, and Yoshikawa et al. (1987)34
HA synthesis
↓ Sulfated GAG synthesis
↓ TGF-b3 through stimulation of the
expression of TG-interacting factor Zhang et al. (2009)46
↓ TGF-b receptors
↓ TGF-b3, BMP-2 and BMP-4 Makhijani et al. (2005)106
↓ GAGs, TGF-b receptors Gazit et al. (1993)107
↑TGF-b3 Baroni et al. (2006)62
↓ GAGs, collagen, and collagen
crosslinks Sauer and Evans (1980)55
↑ EGF and MEE proliferation, altered
differentiation of MEE cells Abbott et al. (1988)108
Altered differentiation of MEE cells
↓ Desmosomes and Abbott and Pratt (1987)68
hemidesmosomes Hatakeyama et al. (2004)72
↓ Desmosomes
Wanner et al. (1999)73
↓ WNT3A, WNT8A, WNT8B, WNT10B, Katoh (2002)109
and WNT11
↑ WNT2, WNT7B, and WNT14B
regulation of Tbx1, a candidate gene for DiGeorge
syndrome, which includes CP.37,48 Furthermore, a recent
study showed that Wnt1Cre;Tgfbr2 conditional knockout
mice develop microglossia and CP by downregulation of
fibroblast growth factor 10 (FGF10).50 Interestingly, RA is
a physiological regulator of FGF10 expression during the
development of various organs.51 In general, FGFs coor-
dinate epithelial-mesenchymal interactions. Impaired
FGF signaling also contributes to about 3% of the non-
syndromic CLP cases.52 In FGF10 knockout mice, the
elevation of the palatal shelves is physically prevented by
adhesion and fusion of the shelves to the tongue and
mandible.53 The unusual fusion pattern probably occurs
617
as a result of ectopic expression of TGF-b3, a downstream
target of FGF10 that is essential for the adhesion and
fusion of the palatal shelves. Disturbance of the intrinsic
mechanisms that cause shelf elevation may cause CP as
well. ECM production and hydration are the key factors
in the elevation of the palatal shelves. The main compo-
nent of the palatal ECM is collagen. Collagen fibers form
a network to provide tensile strength to the palatal
shelves. In addition, the stiffness of the collagen network
is determined by the number of intermolecular cross-
links. Glycosaminoglycans (GAGs) and proteoglycans are
also essential components of the ECM. They form aggre-
gates that bind large amounts of water, and the resulting
swelling pressure is the driving force behind shelf
elevation.30
Prior to elevation, excess RA may directly suppress
collagen synthesis through binding to retinoic acid
response element sites in the collagen promoter region.54
In addition, the formation of collagen crosslinks may
be reduced.55 Hypothetically, these weakened palatal
shelves are not able to elevate to a horizontal position.
The mesenchymal cells that secrete the palatal ECM also
produce matrix metalloproteinases (MMPs) that are
responsible for ECM remodeling.56 The level of active
MMPs increases during palatal shelf elevation, while the
expression of tissue inhibitors of matrix metalloprotein-
ases (TIMPs) is constant.57 RA inhibits MMP synthesis
and stimulates the expression of TIMPs.58 The latter
might be mediated by nuclear receptors similar to the
action of glucocorticoids and other hormones.58–60 This
might lead to reduced remodeling of the ECM and
impaired elevation of the palatal shelves. Another
mechanism involves RA-induced decorin, which binds
to collagen and thereby stimulates collagen fiber assem-
bly.61 This might enhance the stiffness of collagen fibers
and prevent elevation.33 In human as well as murine
mesenchymal cells, RA inhibits the synthesis of
GAGs,34,55,62 possibly by interference with the p38
mitogen-activated protein kinase pathway in TGF-b1
and -2 signaling.63,64 This may result in a reduced swell-
ing pressure and impaired shelf elevation.
In conclusion, RA may prevent palatal shelf elevation
by impeding tongue withdrawal and by disrupting the
collagen network. Furthermore, RA inhibits the synthesis
of GAGs, which decreases the hydration of the ECM and
might thus reduce the driving force for shelf elevation.
SHELF FUSION: PUTATIVE EFFECTS OF RETINOIC ACID
After the palatal shelves have reached a horizontal posi-
tion, the MEE approach and adhere to each other in
order to form the MES. Prior to the contact of the bilat-
eral palatal shelves, the MEE consists of two cell layers;
the basal MEE cells attached to the basement mem-
618
brane, and the superficial MEE cells, also called the peri-
derm. The first contact takes place in the middle third of
the palate and proceeds in anterior and posterior direc-
tions.28 In order to make contact, the peridermal cells
develop protrusions, mainly filopodia.31 These filopodia
not only increase the surface area of the MEE available
for fusion but also possess large numbers of cell
adhesion molecules.65 In this section, the effects of
RA on the expression of adhesion molecules and the
formation of filopodia will be discussed. Subsequently,
the focus will be on the actual fusion of the palatal
shelves.
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If the shelves are able to make contact, excess RA
may also impair subsequent differentiation of MEE cells
into MES cells. RA exposure of palatal shelves prolongs
EGFR expression in the MEE.66,67 This induces an aber-
rant ciliated secretory cell phenotype in the MEE, which
prevents normal cell-to-cell adherence.26,68 The normal
expression of filopodia and chondroitin sulfate pro-
teoglycans that mediate adhesion is regulated by growth
factors like PDGF and TGF-b3. Altered growth factor
signaling by RA may thus interfere with palatal shelf
adherence by reducing the number of filopodia and the
expression of chondroitin sulfate proteoglycans in peri-
dermal cells (Table 1).31,65,69,70 In contrast, RA does not
affect the expression of E-cadherin, which is needed for
epithelial adhesion.71 In oral and dermal postnatal kera-
tinocytes, excess RA also leads to a loss of desmosomes
and hemidesmosomes by blocking the synthesis of des-
mosomal proteins,72,73 but the effect of RA on desmo-
somes in the MEE is not clear. Upon adhesion of the
palatal shelves, the peridermal cells migrate to the oral
and nasal sides and contribute to the formation of the
epithelial triangles. Subsequently, most peridermal cells
undergo apoptosis. Then, the basal MEE cells of both
shelves intercalate, resulting in a single epithelial layer
that is delimited on both sides by a basement mem-
brane.74 With the formation of this MES, the actual palatal
fusion starts (Figures 3F–G).
A critical step in palatal fusion is the disappearance
of the MES, by which a continuous mesenchymal tissue
is formed. Recent findings from animal studies indicate
that persistence of the MES can induce a secondary
separation of the adhered palatal shelves at later fetal
stages or in newborns.69,75 The adhering but unfused
shelves are pulled apart by lateral growth of the head.
This might correspond with submucous palatal clefts in
humans. The exact fate of the MES cells is still contro-
versial. Currently, most authors believe that the MES
cells disappear mainly by apoptosis,74,76 but migration77
and epithelial-to-mesenchymal transition (EMT)75 may
also occur. In the migration theory, it is hypothesized
that MES cells migrate out of the seam and incorporate
into the nasal and oral epithelia.77 EMT refers to the
Nutrition Reviews® Vol. 69(10):613–624
 Table 2 In vivo effects of retinoic acid.
Animal Exposure day RA form and Effect Reference
concentration
Rat E9 Retinyl acetate ↓ Mesenchymal proliferation Kochhar and Johnson
118 mg/kg for 3 (1965)21
consecutive days
Mouse 13-cis RA (isotretinoin) ↑ Apoptosis in cell Sulik et al. (1987)24
400 mg/kg populations that derived
from the first branchial arch
Mouse E10 ATRA 100 mg/kg ↓ TGF-a, mesenchymal Abbott and Birnbaum
proliferation (1990)110
– TGF-b1 in mesenchymal cells
↑ TGF-b2 in nasal epithelial

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cells
Rat ATRA 100 mg/kg ↓ BMP-7 Guo et al. (2008)36
Rat Retinyl acetate ↓ Mesenchymal proliferation Kochhar and Johnson
118 mg/kg for 3 (1965)21
consecutive days
Mouse E11,5 10 mg/kg, form – E-cadherin in the MEE cells Luning et al. (1994)71
unknown
Mouse ATRA 100 mg/kg ↑ Apoptosis in the tongue Okano et al. (2007)37
primordium
↓ Tongue withdrawal,
elevation of palatal shelves,
mesenchymal proliferation,
filopodia on the MEE cells,
altered differentiation of
MEE cells
Mouse E12 ATRA 100 mg/kg ↑ TGF-a, TGF-b1 in Okano et al. (2007)37
mesenchymal cells, TGF-b2
in nasal epithelial cells,
altered differentiation of
MEE cells
Mouse ATRA 70 mg/kg ↓ ECM components, hydration, Degitz et al. (1998)94
and elevation of palatal
shelves
Mouse ATRA 70 mg/kg ↓ TGF-b1 in mesenchymal Degitz et al. (1998)94
cells, hydration
Mouse 100 mg/kg, form ↓ BMP-2,4,5 Lu et al. (2000)45
unknown
Mouse ATRA 75 mg/kg ↑ EGF signaling Abbott et al. (1988)108
Mouse E12,5 ATRA 70 mg/kg ↓ Decorin expression, Zhang et al. (2003)33
mesenchymal proliferation,
and elevation of palatal
shelves
Abbreviations and symbols: ↑, Stimulation; ↓, inhibition; –, no effect; ATRA, all-trans retinoic acid; BMP, bone morphogenetic protein;
ECM, extracellular matrix; EGF, epidermal growth factor; MEE, medial edge epithelial; TGF, transforming growth factor.

phenotypical change of epithelial cells into mesenchy-
mal cells. EMT occurs in embryonic development78 but
is also thought to be involved in tumor progression and
fibrotic disorders.79,80 The EMT theory suggests that
MES cells undergo EMT and become part of the palatal
mesenchyme. Since the evidence for MES migration and
EMT is relatively weak, only the effect of RA on apop-
tosis will be discussed.
RA mediates apoptosis during normal palatogenesis
and is synthesized by MES cells soon after shelf contact.
There is no evidence that excess RA directly affects apo-
ptosis in the MES,81 but it may interfere with signaling
pathways that regulate apoptosis, such as the WNT and
TGF-b pathways. Twelve of 19 members of the WNT
family are expressed in the developing palate.82 WNT11
is expressed in the MES and induces apoptosis by inhi-
bition of fibroblast growth factor receptor 1b.83 A single
nucleotide polymorphism in the WNT11 gene is also
associated with clinical nonsyndromic CP and CLP.84
Since RA was shown to repress the expression of several

Nutrition Reviews® Vol. 69(10):613–624 619

WNT genes during early body axis extension in the
embryo,85 it might also affect WNT signaling during
palatogenesis. RA-induced downregulation of WNT11
signaling may inhibit MES apoptosis, but the exact
interaction between these factors during palatal fusion
remains to be elucidated. Another interesting regulatory
protein in the RA-mediated apoptosis during palatoge-
nesis is p63. The p63 protein is a master regulator of
ectodermal development. Mutations in p63 give rise to
at least seven ectoderm-related developmental disor-
ders,86 of which CP is one of the cardinal features. RA
treatment of differentiated human primary kerati-
nocytes results in an elevated level of DNp63a,87 the
most abundant isoform expressed in skin keratinocytes
as well as in oral and nasal palatal epithelia. Normally,
expression of p63 is reduced in MEE during palatal
shelve fusion, and downregulation of p63 is suggested to
be a prerequisite for proper periderm development
and palatal fusion.88 Recently, an enzyme involved in RA
metabolism, the retinal dehydrogenase/reductase
retSDR1/DHRS3, has been identified as a direct tran-
scriptional target of p63.89,90 This finding raises the pos-
sibility that p63 regulates RA levels and that the
interplay between RA and p63 affects palatogenesis.
Apoptosis of the MES cells subsequently activates the
proteolytic breakdown of the basement membrane by
MMPs.56,74,91,92 RA reduces synthesis of MMPs and stimu-
lates the transcription of their inhibitors, the TIMPs,58
possibly through interference with EGFR and TGF-b3
signaling pathways.91,92 This may lead to persistence of the
basement membrane and impaired fusion of the palatal
shelves.
In summary, RA may prevent initial shelf contact by
inducing apoptosis within the MEE. In addition, it may
prevent shelf adherence by disrupting normal epithelial
differentiation. After adhesion, RA may impair the disap-
pearance of the MES, which precludes the formation of a
continuous palate.
HIGH VERSUS LOW DOSES OF VITAMIN A
The previously discussed research primarily involved
high doses of vitamin A in animal models. These experi-
ments showed clear effects on the different stages of
palatogenesis that might lead to clefting. According to the
2001 report of the US Food and Nutrition Board, the
recommended RDA for vitamin A is 770 mg during preg-
nancy.17 For an average person, the RDA means a dosage
of 10.5 mg/kg. The dosages tested in animal experiments
lie in the range of 10.5·103 mg/kg to 21·104 mg/kg, which is
1,000 to 20,000 times the RDA.36,37,81,93,94
Epidemiological studies on nutritional exposure to
vitamin A are scarce. Only a few studies clearly describe
the dosage used and have an acceptable statistical power.
620
Extreme vitamin A deficiency is lethal for the embryo,
while minor deficiencies, below half the RDA, mainly
induce ocular abnormalities.95,96 Doses slightly above the
RDA (1,500 mg) were found to protect against CLP97 and
CP19 in the offspring. On the other hand, other studies
did not find such an effect.98 A study on high vitamin A
intake (4,500 mg) shows that defects related to cranial
neural-crest-derived structures, such as craniofacial,
cardiac, and thymic defects, are 3.5 times more prevalent
than after low intake (ⱕ1,500 mg).99 In summary, these
studies suggest harmful effects for vitamin A deficiency
(ⱕ375 mg),95,96 possible protective effects in the range of
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the RDA,19,97 and harmful effects at high doses
(ⱖ4,500 mg),99 consistent with a U-shaped dose-effect
curve. Thus far, no safe upper limit for vitamin A intake
has been established, but the evidence suggests an intake
of up to 4 times the RDA (3,000 mg) is harmless.20,99–101
The same amount is suggested by the US Food and
Nutrition Board as the tolerable upper level of intake for
vitamin A.17 In the light of the limited number of avail-
able studies, the question remains whether slight nutri-
tional overexposure to vitamin A contributes to clefting
in humans. Theoretically, this might increase the risk of
clefting in genetically predisposed individuals with spe-
cific polymorphisms of enzymes or other proteins
involved in vitamin A metabolism.102 However, as for
folate, such interactions have not yet been definitely
established.103,104
CONCLUSION
Nutritional factors such as vitamin intake are important
in the etiology of CP. Therefore, the development of pre-
ventive nutritional strategies is of great interest. Overex-
posure to vitamin A can cause congenital malformations
such as CP. The putative biological mechanisms underly-
ing RA-induced CP are summarized in Figure 4. Depend-
ing on the time of exposure, excess RA might disturb all
three stages of palatogenesis: 1) during shelf outgrowth, it
may decrease mesenchymal proliferation and thus
prevent tissue expansion; 2) in the next stage, RA may
prevent shelf elevation by affecting the ECM composition
and hydration; and 3) during the actual fusion of the
shelves, it may affect epithelial differentiation and apop-
tosis, which precludes the formation of a continuous
palate.
In general, RA seems to act through interference with
growth factor signaling in all stages of palatogenesis. The
exact interactions of RA with relevant signaling pathways
in palatogenesis need to be investigated in more depth.
Future research should focus specifically on the effects of
slight nutritional overexposure of vitamin A to improve
the dietary recommendations to pregnant women or
Nutrition Reviews® Vol. 69(10):613–624
 Downloaded from https://academic.oup.com/nutritionreviews/article/69/10/613/1867002 by guest on 08 February 2024
Figure 4 Biological effects of retinoic acid on palatogenesis. Retinoic acid (RA) binds to its receptors RAR and RXR, which
activate the RA response element (RARE) on the DNA and thereby regulate the transcription of target genes. The affected
signaling pathways are shown below each of the three dotted frames. The dotted frames represent frontal histological sections
of the main stages of palatogenesis: 1 outgrowth; 2 elevation; 3 fusion. The main biological targets of RA are indicated.
Abbreviations: CLP, cleft lip and palate; ECM, extracellular matrix; EGF, epidermal growth factor; FGF, fibroblast growth factor;
MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; NS, nasal septum; PS, palatal shelf; RAR, retinoic acid
receptor; RARE, retinoic acid response element; RXR, retinoid X receptor; T, tongue; TGF, transforming growth factor.

those who want to become pregnant, thereby reducing
the risk of CP in offspring.
Acknowledgments
Funding. No special funding was received for this
project.
Declaration of interest. The authors have no relevant
interests to declare.
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