LncRNA-SULT1C2A regulates Foxo4 in congenital scoliosis by targeting rno-miR-466c-5p through PI3K-ATK signalling.

Congenital scoliosis (CS) is the result of anomalous vertebrae development, but the pathogenesis of CS remains unclear. Long non-coding RNAs (lncRNAs) have been implicated in embryo development, but their role in CS remains unknown. In this study, we investigated the role and mechanisms of a specific lncRNA, SULT1C2A, in somitogenesis in a rat model of vitamin A deficiency (VAD)-induced CS. Bioinformatics analysis and quantitative real-time PCR (qRT-PCR) indicated that SULT1C2A expression was down-regulated in VAD group, accompanied by increased expression of rno-miR-466c-5p but decreased expression of Foxo4 and somitogenesis-related genes such as Pax1, Nkx3-2 and Sox9 on gestational day (GD) 9. Luciferase reporter and small interfering RNA (siRNA) assays showed that SULT1C2A functioned as a competing endogenous RNA to inhibit rno-miR-466c-5p expression by direct binding, and rno-miR-466c-5p inhibited Foxo4 expression by binding to its 3' untranslated region (UTR). The spatiotemporal expression of SULT1C2A, rno-miR-466c-5p and Foxo4 axis was dynamically altered on GDs 3, 8, 11, 15 and 21 as detected by qRT-PCR and northern blot analyses, with parallel changes in Protein kinase B (AKT) phosphorylation and PI3K expression. Taken together, our findings indicate that SULT1C2A enhanced Foxo4 expression by negatively modulating rno-miR-466c-5p expression via the PI3K-ATK signalling pathway in the rat model of VAD-CS. Thus, SULT1C2A may be a potential target for treating CS.

Dietary vitamin A intake below the recommended daily intake during pregnancy and the risk of congenital diaphragmatic hernia in the offspring.

BACKGROUND: Vitamin A has been related to the etiology of congenital diaphragmatic hernia (CDH). We performed a case-control study to investigate whether maternal dietary vitamin A intake is related to CDH in the offspring. METHODS: Thirty-one pregnancies diagnosed with CDH and 46 control pregnancies were included during the study. After CDH diagnosis and inclusion of controls by risk set sampling, maternal vitamin A intake was investigated with a food frequency questionnaire. Serum retinol and retinol-binding protein were determined. Univariable and multivariable logistic regression models were used to calculate risk estimates with adjustment for potential confounders. RESULTS: We found no significant differences in the overall nutrient and vitamin A intake between case and control mothers. After stratification in body mass index (BMI) categories, case mothers with normal weight showed a lower energy adjusted vitamin A intake (685 vs. 843 µg retinol activity equivalents [RAEs] / day; p = 0.04) and a slightly lower serum retinol (1.58 vs. 1.67 µmol/L; p = 0.08) than control mothers. Vitamin A intake <800 µg retinol activity equivalents (recommended daily intake) in normal weight mothers was associated with a significantly increased CDH risk (odds ratio [OR], 7.2; 95% confidence interval [CI], 1.5-34.4; p = 0.01). Associations were not significantly different in underweight and overweight mothers. CONCLUSIONS: In normal-weight mothers, dietary vitamin A intake during pregnancy below the recommended daily intake is significantly associated with an increased risk of a child with CDH. This finding supports the retinoid hypothesis in human CDH, but warrants further investigation in larger study populations. Birth Defects Research (Part A), 2013. © 2013 Wiley Periodicals, Inc.

Ectopic expression of retinoic acid receptors and change of myocardial structure in the offspring heart with vitamin A deficiency.

Vitamin A is a key micronutrient required during crucial stages of embryonic development and vitamin A deficiency (VAD) results in embryonic heart malformation. The pleiotropic functions of vitamin A are mediated by specific nuclear receptors: the retinoic acid receptors (RARα, -ß, and -γ) and the retinoic X receptors (RXRα, -ß, and -γ). The action of nuclear receptors has been implicated in controlling of cell proliferation, differentiation and apoptosis, and the expressions of these receptor genes are regulated by retinoic acid levels during the early stages of embryonic development. GATA-4 is one of the earliest transcription factors expressed in developing cardiac cells. However, the functional links of specific nuclear receptors to heart development in VAD embryos are not clearly understood. In our study, weaning female Sprague-Dawley rats were fed a modified diet containing different concentrations of vitamin A according to the American Institute of Nutrition 93 Growth Purified Diet. After 10-wk feeding, the female rats were mated with normal male rats, and a portion of them were transferred to a diet with enough added vitamin A for the pregnancy cycle. The embryo hearts were dissected out at embryonic day 13.5 (E13.5) to study the expression of RARs, RXRs and GATA-4. The embryo hearts from E18.5 were for observation of ultrastructural changes. In comparison to vitamin A supplemented groups, the embryo hearts from vitamin A insufficient groups exhibited ultrastructural changes and significantly lower expression of GATA-4, RARα, and -γ, and higher expression of RXRα and -ß. Our findings suggest that the down-regulation of RARs and the up-regulation of RXRs resulted from VAD affected GATA-4 gene expression, which resulted in ultrastructural changes in embryo hearts due to maternal insufficiency of vitamin A during pregnancy.

The role of CYP26 enzymes in defining appropriate retinoic acid exposure during embryogenesis.

Retinoic acid (RA) is a pleiotropic derivative of vitamin A, or retinol, which is responsible for all of the bioactivity associated with this vitamin. The teratogenic influences of vitamin A deficiency and excess RA in rodents were first observed more than 50 years ago. Efforts over the last 15-20 years have refined these observations by defining the molecular mechanisms that control RA availability and signaling during murine embryonic development. This review will discuss our current understanding of the role of RA in teratogenesis, with specific emphasis on the essential function of the RA catabolic CYP26 enzymes in preventing teratogenic consequences caused by uncontrolled distribution of RA. Particular focus will be paid to the RA-sensitive tissues of the caudal and cranial regions, the limb, and the testis, and how genetic mutation of factors controlling RA distribution have revealed important roles for RA during embryogenesis.

Vitamin A facilitates enteric nervous system precursor migration by reducing Pten accumulation.

Hirschsprung disease is a serious disorder of enteric nervous system (ENS) development caused by the failure of ENS precursor migration into the distal bowel. We now demonstrate that retinoic acid (RA) is crucial for GDNF-induced ENS precursor migration, cell polarization and lamellipodia formation, and that vitamin A depletion causes distal bowel aganglionosis in serum retinol-binding-protein-deficient (Rbp4(-/-)) mice. Ret heterozygosity increases the incidence and severity of distal bowel aganglionosis induced by vitamin A deficiency in Rbp4(-/-) animals. Furthermore, RA reduces phosphatase and tensin homolog (Pten) accumulation in migrating cells, whereas Pten overexpression slows ENS precursor migration. Collectively, these data support the hypothesis that vitamin A deficiency is a non-genetic risk factor that increases Hirschsprung disease penetrance and expressivity, suggesting that some cases of Hirschsprung disease might be preventable by optimizing maternal nutrition.

Vitamin A-not for your eyes only: requirement for heart formation begins early in embryogenesis.

Vitamin A insufficiency has profound adverse effects on embryonic development. Major advances in understanding the role of vitamin A in vertebrate heart formation have been made since the discovery that the vitamin A active form, all-trans-retinoic acid, regulates many genes, including developmental genes. Among the experimental models used, the vitamin A-deficient avian embryo has been an important tool to study the function of vitamin A during early heart formation. A cluster of retinoic acid-regulated developmental genes have been identified that participate in building the heart. In the absence of retinoic acid the embryonic heart develops abnormally leading to embryolethality.

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.

Mammalian eye development requires vitamin A (retinol, ROL). The role of vitamin A at specific times during eye development was studied in rat fetuses made vitamin A deficient (VAD) after embryonic day (E) 10.5 (late VAD). The optic fissure does not close in late VAD embryos, and severe folding and collapse of the retina is observed at E18.5. Pitx2, a gene required for normal optic fissure closure, is dramatically downregulated in the periocular mesenchyme in late VAD embryos, and dissolution of the basal lamina does not occur at the optic fissure margin. The addition of ROL to late VAD embryos by E12.5 restores Pitx2 expression, supports dissolution of the basal lamina, and prevents coloboma, whereas supplementation at E13.5 does not. Surprisingly, ROL given as late as E13.5 completely prevents folding of the retina despite the presence of an open fetal fissure, showing that coloboma and retinal folding represent distinct VAD-dependent defects. Retinal folding due to VAD is preceded by an overall reduction in the percentage of cyclin D1 positive cells in the developing retina, (initially resulting in retinal thinning), as well as a dramatic reduction in the cell adhesion-related molecules, N-cadherin and beta-catenin. Reduction of retinal cell number combined with a loss of the normal cell-cell adhesion proteins may contribute to the collapse and folding of the retina that occurs in late VAD fetuses.

Role of vitamin A in determining nephron mass and possible relationship to hypertension.

Vitamin A (retinol) and its analogs (retinoids) are important regulators of cell proliferation, differentiation, immune function, and apoptosis. The kidneys are target organs for vitamin A action. Retinoic acid (RA), a vitamin A metabolite, is involved in embryonic kidney patterning through the control of receptor tyrosine kinase expression, which modulates ureteric bud branching morphogenesis. Vitamin A status of the mother profoundly affects kidney organogenesis of the newborn. In rodents, mild vitamin A deficiency results in a 20% reduction of nephron number. In adult humans, nephron number varies between 0.3 and 1.3 million per kidney, which is accepted as normal. However, recent studies indicate that humans at the low end of nephron number are predisposed to primary hypertension. Because RA regulates nephron mass, its optimal availability during nephrogenesis is critical. RA levels in the embryo are affected by several factors, such as maternal vitamin A nutrition and disturbances in retinol metabolism. Maternal vitamin A deficiency during pregnancy is widespread in developing countries and segments of these populations may be exposed to low vitamin A during fetal life when nephron number is determined. Infants are likely to be born with suboptimal nephrons and may develop primary hypertension later in life. Although maternal vitamin A deficiency is not common in developed countries, congenital nephron number nevertheless varies widely, indicating low fetal RA levels due to common variants of the enzymes that convert retinol to RA. These infants might require heightened surveillance for hypertension later in life.

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.

Vitamin A plays an essential role in vertebrate embryogenesis. In the present study, pregnant vitamin A-deficient (VAD) rats were maintained during early pregnancy on the short half-life vitamin A metabolite, all-trans retinoic acid (atRA), in an amount sufficient to support normal development to E10.5, with a higher level of atRA (250 microg atRA/g diet) provided from embryonic day (E) 8.5-10.5 to prevent mid-gestational resorption. When limiting amounts of atRA (1.5 or 12 microg/g diet) were provided after E10.5, a highly reproducible and penetrant state of late fetal vitamin A deficiency (late VAD) was induced in the organs of developing fetuses. In addition, late VAD fetuses displayed both anteriorization of cervical regions and novel posteriorization events at the thoracic and sacral levels of the skeleton, and showed sternal and pelvic malformations not previously observed in early VAD or genetic models. The expression of several Hox genes (Hoxd3 and Hoxb4) was altered in late VAD embryos, with a reduction in Hoxd3 noted as early as 1 day after instituting deficiency. All late VAD-induced malformations were prevented by the addition of retinol starting at E10.5, whereas provision of a high level of atRA throughout pregnancy improved but could not completely rescue the development of all organ systems. This work defines a nutritional model in which vitamin A deficiency can be induced during fetal development, and reveals new functions for the vitamin in the development of the axial and appendicular skeleton.

Prenatal vitamin A deficiency causes laryngeal malformation in rats.

OBJECTIVES: Our previous research demonstrated that vitamin A might be related to vocal fold development. The purpose of this study was to determine whether vitamin A deficiency affects prenatal laryngeal development in rats. METHODS: Two considerations were necessary in designing a study using a rat model: for embryonic survival, vitamin A is necessary through day 10 of gestation, and laryngeal formation occurs primarily after day 11. Thus, we created a rat model that developed vitamin A deficiency after embryonic day 11. Ten pregnant rats (5 vitamin A-deficient rats and 5 control rats) were studied. Embryos were collected at embryonic day 18.5 and analyzed histologically. RESULTS: Eighteen percent of the vitamin A-deficient embryos were alive and demonstrated laryngotracheal cartilage malformation, incomplete separation of the glottis, and/or laryngoesophageal clefts. CONCLUSIONS: These results document the important role played by vitamin A in laryngeal development.

Retinoic acid is required for specification of the ventral eye field and for Rathke's pouch in the avian embryo.

We have investigated the role of retinoic acid (RA) in eye development using the vitamin A deficient quail model system, which overcomes problems of retinoic acid synthesising enzyme redundancy in the embryo. In the absence of retinoic acid, the ventral optic stalk and ventral retina are missing, whereas the dorsal optic stalk and dorsal retina develop appropriately. Other ocular abnormalities observed were a thinner retina and the lack of differentiation of the lens. In an attempt to explain this, we studied the expression of various dorsally and ventrally expressed genes such as Pax2, Pax6, Tbx6, Vax2, Raldh1 and Raldh3 and noted that they were unchanged in their expression patterns. In contrast, the RA catabolising enzymes Cyp26A1 and Cyp26B1 which are known to be RA-responsive were not expressed at all in the developing eye. At much earlier stages, the expression domain of Shh in the prechordal plate was reduced, as was Nkx2.1 and we suggest a model whereby the eye field is specified according to the concentration of SHH protein that is present. We also describe another organ, Rathke's pouch which fails to develop in the absence of retinoic acid. We attribute this to the down-regulation of Bmp2, Shh and Fgf8 which are known to be involved in the induction of this structure.

Metabolism of retinol during mammalian placental and embryonic development.

Retinol (vitamin A) is a fat-soluble nutrient indispensable for a harmonious mammalian gestation. The absence or excess of retinol and its active derivatives [i.e., the retinoic acids (RAs)] can lead to abnormal development of embryonic and extraembryonic (placental) structures. The embryo is unable to synthesize the retinol and is strongly dependent on the maternal delivery of retinol itself or precursors: retinyl esters or carotenoids. Before reaching the embryonic tissue, the retinol or the precursors have to pass through the placental structures. During this placental step, a simple diffusion of retinol can occur between maternal and fetal compartments; but retinol can also be used in situ after its activation into RA(1) or stored as retinyl esters. Using retinol-binding protein knockout model, an alternative way of embryonic retinol supply was described using retinyl esters incorporated into maternal chylomicrons. In the embryo, the principal metabolic event occurring for retinol is its conversion into RAs, the active molecules implicated on the molecular control of embryonic morphogenesis and organogenesis. All these placental and embryonic events of retinol transport and metabolism are highly regulated. Nevertheless, some genetic and/or environmental abnormalities in the transport and/or metabolism of retinol can be related to developmental pathologies during mammalian development.

Bmp2 and Gata4 function additively to rescue heart tube development in the absence of retinoids.

We used the vitamin A-deficient (VAD) quail model to investigate the retinoid-dependent mechanism that regulates heart tube development. We showed previously that decreased levels of Gata4 in cardiogenic mesoderm and endoderm correlate with the cardiomyopathy caused by VAD, but that this could be rescued by transplanting normal anterior endoderm. Bmp2 is a known cardiogenic factor that is expressed normally in lateral plate mesoderm and cardiac-associated pharyngeal endoderm. Here we show that (like Gata4) transcripts encoding Bmp2 and BMP-dependent signaling activity are decreased throughout the heart-forming region of the VAD embryo. Addition of Bmp2 protein or forced expression of Gata4 in cultured VAD embryos leads to a partial rescue of the cardiomyopathy, and addition of both Bmp2 and Gata4 has an additive positive effect. Our data are consistent with a requirement for retinoid signaling to maintain expression of Bmp2, which regulates Gata4, and in addition acts with Gata4 to regulate genes important for normal morphogenesis of the primitive heart tube.

Retinaldehyde dehydrogenase 2 (RALDH2)-mediated retinoic acid synthesis regulates early mouse embryonic forebrain development by controlling FGF and sonic hedgehog signaling.

Although retinoic acid (RA) has been implicated as one of the diffusible signals regulating forebrain development, patterning of the forebrain has not been analyzed in detail in knockout mouse mutants deficient in embryonic RA synthesis. We show that the retinaldehyde dehydrogenase 2 (RALDH2) enzyme is responsible for RA synthesis in the mouse craniofacial region and forebrain between the 8- and 15-somite stages. Raldh2-/- knockout embryos exhibit defective morphogenesis of various forebrain derivatives, including the ventral diencephalon, the optic and telencephalic vesicles. These defects are preceded by regionally decreased cell proliferation in the neuroepithelium, correlating with abnormally low D-cyclin gene expression. Increases in cell death also contribute to the morphological deficiencies at later stages. Molecular analyses reveal abnormally low levels of FGF signaling in the craniofacial region, and impaired sonic hedgehog signaling in the ventral diencephalon. Expression levels of several regulators of diencephalic, telencephalic and optic development therefore cannot be maintained. These results unveil crucial roles of RA during early mouse forebrain development, which may involve the regulation of the expansion of neural progenitor cells through a crosstalk with FGF and sonic hedgehog signaling pathways.

The control of morphogen signalling: regulation of the synthesis and catabolism of retinoic acid in the developing embryo.

We consider here how morphogenetic signals involving retinoic acid (RA) are switched on and off in the light of positive and negative feedback controls which operate in other embryonic signalling systems. Switching on the RA signal involves the synthetic retinaldehyde dehydrogenase (RALDH) enzymes and it is currently thought that switching off the RA signal involves the CYP26 enzymes which catabolise RA. We have tested whether these enzymes are regulated by the presence or absence of all-trans-RA using the vitamin A-deficient quail model system and the application of excess retinoids on beads to various locations within the embryo. The Raldhs are unaffected either by the absence or presence of excess RA, whereas the Cyps are strongly affected. In the absence of RA some, but not all domains of Cyp26A1, Cyp26B1 and Cyp26C1 are down-regulated, in particular the spinal cord (Cyp26A1), the heart and developing vasculature (Cyp26B1) and the rhombomeres (Cyp26C1). In the presence of excess RA, the Cyps show a differential regulation-Cyp26A1 and Cyp26B1 are up-regulated whereas Cyp26C1 is down-regulated. We tested whether the Cyp products have a similar influence on these genes and indeed 4-oxo-RA, 4-OH-RA and 5,6-epoxy-RA do. Furthermore, these 3 metabolites are biologically active in that they fully rescue the vitamin A-deficient quail embryo. Finally, by using retinoic acid receptor selective agonists we show that these compounds regulate the Cyps through the RARalpha receptor. These results are discussed with regard to positive and negative feedback controls in developing systems.

Distinct roles for hindbrain and paraxial mesoderm in the induction and patterning of the inner ear revealed by a study of vitamin-A-deficient quail.

The hindbrain and cranial paraxial mesoderm have been implicated in the induction and patterning of the inner ear, but the precise role of the two tissues in these processes is still not clear. We have addressed these questions using the vitamin-A-deficient (VAD) quail model, in which VAD embryos lack the posterior half of the hindbrain that normally lies next to the inner ear. Using a battery of molecular markers, we show that the anlagen of the inner ear, the otic placode, is induced in VAD embryos in the absence of the posterior hindbrain. By performing grafting and ablation experiments in chick embryos, we also show that cranial paraxial mesoderm which normally lies beneath the presumptive otic placode is necessary for otic placode induction and that paraxial mesoderm from other locations cannot induce the otic placode. Two members of the fibroblast growth factor family, FGF3 and FGF19, continue to be expressed in this mesodermal population in VAD embryos, and these may be responsible for otic placode induction in the absence of the posterior hindbrain. Although the posterior hindbrain is not required for otic placode induction in VAD embryos, the subsequent patterning of the inner ear is severely disrupted. Several regional markers of the inner ear, such as Pax2, EphA4, SOHo1 and Wnt3a, are incorrectly expressed in VAD otocysts, and the sensory patches and vestibulo-acoustic ganglia are either greatly reduced or absent. Exogenous application of retinoic acid prior to 30 h of development is able rescue the VAD phenotype. By performing such rescue experiments before and after 30 h of development, we show that the inner ear defects of VAD embryos correlate with the absence of the posterior hindbrain. These results show that induction and patterning of the inner ear are governed by separate developmental processes that can be experimentally uncoupled from each other.

Pathways of vitamin A delivery to the embryo: insights from a new tunable model of embryonic vitamin A deficiency.

Circulating retinoids (vitamin A and its derivatives) are found predominantly as retinol bound to retinol-binding protein (RBP), which transports retinol from liver stores to target tissues, or as retinyl ester incorporated in lipoproteins of dietary origin. The transport of retinoids from maternal to fetal circulation is poorly understood, especially under conditions of inadequate dietary vitamin A intake. Here we present RBP-/- mice as a tunable model of embryonic vitamin A deficiency. This model has enabled us to analyze metabolic links between maternal nutrition and retinoid delivery to the fetus. Our data show that retinol-RBP is the primary contributor to fetal development, whereas retinyl ester are largely responsible for accumulation of fetal retinoid stores. Furthermore, these studies indicate the importance of embryonic RBP in distributing vitamin A to certain developing tissues under restrictive diets. We also show differences among developing tissues in their dependency on the embryonic retinol-RBP pathway. Finally, we demonstrate that accumulation of embryonic vitamin A stores does not depend on the expression of RBP in the fetal liver.

Retinoic acid selectively regulates Fgf10 expression and maintains cell identity in the prospective lung field of the developing foregut.

Although respiratory tract defects that result from disruption of retinoic acid (RA) signaling have been widely reported, the mechanism by which endogenous RA regulates early lung morphogenesis is unknown. Here, we provide novel evidence that a major role for RA is to selectively maintain mesodermal proliferation and induce fibroblast growth factor 10 (Fgf10) expression in the foregut region where the lung forms. By using a pan-RAR antagonist (BMS493) in foregut explant cultures, we show that bud initiation is selectively blocked in the prospective respiratory region by failure to induce Fgf10 in the corresponding mesoderm. The RA regulation of Fgf10 expression occurs only in this region, within a defined developmental window, and is not seen in other foregut derivatives such as thyroid and pancreas where Fgf10 is also required for normal development. Furthermore, we show that RA activity is essential in the lung field to maintain lung cell identity in the endoderm; RAR antagonism disrupts expression of thyroid transcription factor 1 (Ttf1), an early marker of the respiratory region in the endoderm, and surfactant protein C (Sp-C) mRNAs. Our observations in mouse foregut cultures are corroborated by data from an in vivo model of vitamin A deficiency in rats. Our study supports RA as an essential regulator of gene expression and cellular activities during primary bud formation.

The role of retinoic acid in the morphogenesis of the neural tube.

We have examined the role of the signalling molecule, retinoic acid, in the process of neurulation and the subsequent growth and differentiation of the central nervous system using quail embryos that have developed in the absence of retinoic acid. Such retinoic acid-free embryos undergo abnormal neural tube formation in terms of its shape and structure, but the embryos do not display spina bifida or exencephaly. The neural tubes have a wider floor plate, a thicker roof plate and a different dorsoventral shape. Phalloidin staining and electron microscopy revealed alterations in the actin filaments and the junctional complexes of the cell layer lining the lumen. Initially the neural tubes proliferated at the same rate as normal, but later the proliferation rate declined drastically and neuronal differentiation was highly deficient. There were very few motoneurons extending neurites into the periphery, and within the neural tube axon trajectories were chaotic. These results reveal several functions for retinoic acid in the morphogenesis and growth of the neural tube, many of which can be explained by defective notochord signalling, but they do not suggest that this molecule plays a role in neural tube closure.

Retinoid receptors and vitamin A deficiency: differential patterns of transcription during early avian development and the rapid induction of RARs by retinoic acid.

The functional links of specific retinoid receptors to early developmental events in the avian embryo are not known. Before such studies are undertaken, knowledge is required of the spatiotemporal expression patterns of the receptor genes and their regulation by endogenous retinoic acid levels during the early stages of development. Here, we report the expression patterns of mRNAs for RARalpha, RARalpha2, RARbeta2, RARgamma, RARgamma2, RXRalpha, and RARgamma from neurulation to HH10 in the normal and vitamin A-deficient (VAD) quail embryo. The transcripts for all retinoid receptors are detectable at HH5, except for RXRgamma, which is detected at the beginning of HH6. At the 4/5 somite stage of HH8, when retinoid signaling is initiated in the avian embryo, mRNAs of all receptors are present, with very strong and ubiquitous expression patterns for RARalpha, RARalpha2, RARgamma, RARgamma2, and RXRalpha, a persistent expression of RARgamma in the neural tissues, a strong expression of RARbeta2 in lateral plate mesoderm and somites, and an anterior expression of RXRgamma. All retinoid receptors are expressed in the heart primordia. In the VAD quail embryo, the general pattern of retinoid receptor transcript localization is similar to that of the normal, except that the expression of RARalpha2 and RARbeta2 is severely diminished. Administration of retinol or retinoic acid to VAD embryos at or before the 4/5 somite stage rescues the expression of RARalpha2 and RARbeta2 within approximately 45 min and restores normal development. RARbeta2 expression requires the expression of RARalpha2. After neurulation, the expression of all retinoid receptors in the VAD quail embryo becomes independent of vitamin A status and is similar to that of the normal. The mRNA levels and sites of expression of the key enzyme for retinoic acid biosynthesis, Raldh-2, are not affected by vitamin A status; the expression pattern is restricted and does not correspond to that of retinoid receptors at all sites. The general patterns and intensity of retinoid receptor gene expression during early quail development are comparable to those of the mammalian and thus validate the application of results from retinoid-regulated avian development studies to those of the mammalian.