RDH10 oxidation of Vitamin A is a critical control step in synthesis of retinoic acid during mouse embryogenesis

Retinoic Acid (RA) is a small lipophilic signaling molecule essential for embryonic development and adult tissue maintenance. Both an excess of RA and a deficiency of RA can cause pathogenic anomalies, hence it is critical to understand the mechanisms controlling the spatial and temporal distribution of RA. However, our current understanding of these processes remains incomplete. Vitamin A is metabolized to RA via two sequential enzymatic reactions. The first requires retinol dehydrogenase (RDH) activity to oxidize Vitamin A (retinol) to retinal, and the second requires retinaldehyde activity (RALDH) to oxidize retinal into RA. The first reaction has previously been attributed to the alcohol dehydrogenase (ADH) family, whose genes are ubiquitously or redundantly expressed. Consequently, the specificity of RA synthesis was thought to reside exclusively at the level of the second reaction. To better understand the metabolism of Vitamin A into RA during embryogenesis, we generated new mouse models that disrupt this process. Here we describe a new targeted knockout of Rdh10 in which RA synthesis is severely impaired, particularly at critical early embryonic stages. We also introduce a new mutant allele of Aldh1a2. Both mutations produce similar developmental defects resulting in lethality around embryonic day 10.5 (E10.5). The severity of the Rdh10 null phenotype demonstrates that embryonic oxidation of retinol is carried out primarily by RDH10 and that neither ADHs nor other enzymes contribute significantly to this reaction. We also show that reduced RA production results in upregulation of Rdh10. These data demonstrate that RDH10 plays a critical role in mediating the rate limiting RDH step of Vitamin A metabolism and functions as a nodal point in feedback regulation of RA synthesis. Moreover, RDH10-mediated oxidation of retinol plays as important a role in the control and regulation of RA production during embryogenesis as does the subsequent RALDH-mediated reaction.     

Morphological defects in a novel Rdh10 mutant that has reduced retinoic acid biosynthesis and signaling

Retinoic acid (RA) signaling is necessary for proper patterning and morphogenesis during embryonic development. Tissue-specific RA signaling requires precise spatial and temporal synthesis of RA from retinal by retinaldehyde dehydrogenases (Raldh) and the conversion of retinol to retinal by retinol dehydrogenases (Rdh) of the short-chain dehydrogenase/reducatase gene family (SDR). The SDR, retinol dehydrogenase 10 (RDH10), is a major contributor to retinal biosynthesis during mid-gestation. We have identified a missense mutation in the Rdh10 gene (Rdh10(m366Asp) ) using an N-ethyl-N-nitrosourea-induced forward genetic screen that result in reduced RA levels and signaling during embryonic development. Rdh10(m366Asp) mutant embryos have unique phenotypes, such as edema, a massive midline facial cleft, and neurogenesis defects in the forebrain, that will allow the identification of novel RA functions.     

Retinoic acid signaling in regulation of meiosis during embryonic development in mice

In the embryonic gonads of mice, the genetic and epigenetic regulatory programs for germ cell sex specification and meiosis induction or suppression are intertwined. The quest for garnering comprehensive understanding of these programs has led to the emergence of retinoic acid (RA) as an important extrinsic factor, which regulates initiation of meiosis in female fetal germ cells that have attained a permissive epigenetic ground state. In contrast, germ cells in fetal testis are protected from the exposure to RA due to the activity of CYP26B1, an RA metabolizing enzyme, which is highly expressed in fetal testis. In this review, we provide an overview of the molecular mechanisms operating in fetal gonads of mice, which enable regulation of meiosis via RA signaling.     

Retinoic acid synthesis and functions in early embryonic development

Retinoic acid (RA) is a morphogen derived from retinol (vitamin A) that plays important roles in cell growth, differentiation, and organogenesis. The production of RA from retinol requires two consecutive enzymatic reactions catalyzed by different sets of dehydrogenases. The retinol is first oxidized into retinal, which is then oxidized into RA. The RA interacts with retinoic acid receptor (RAR) and retinoic acid X receptor (RXR) which then regulate the target gene expression. In this review, we have discussed the metabolism of RA and the important components of RA signaling pathway, and highlighted current understanding of the functions of RA during early embryonic development.     

Wnt signaling pathway in rheumatoid arthritis,with special emphasis on the different roles in synovial inflammation and bone remodeling

Rheumatoid arthritis (RA) is a chronic symmetrical autoimmune disease of unknown etiology that affects primarily the diarthrodial joints. Characteristic features of RA pathogenesis are synovial inflammation and proliferation accompanied by cartilage erosion and bone loss. Fibroblast-like synoviocytes (FLS) display an important role in the pathogenesis of RA. Several lines of evidence show that the Wnt signaling pathway significantly participates in the RA pathogenesis. The Wnt proteins are glycoproteins that bind to the Fz receptors on the cell surface, which leads to several important biological functions, such as cell differentiation, embryonic development, limb development and joint formation. Accumulated evidence has suggested that this signaling pathway plays a key role in the FLS activation, bone resorption and joint destruction during RA development. Greater knowledge of the role of the Wnt signaling pathway in RA could improve understanding of the RA pathogenesis and the differences in RA clinical presentation and prognosis. In this review, new advances of the Wnt signaling pathway in RA pathogenesis are discussed, with special emphasis on its different roles in synovial inflammation and bone remodeling. Further studies are needed to reveal the important role of the members of the Wnt signaling pathway in the RA pathogenesis and treatment.     

Opposing effects of retinoid signaling on astrogliogenesis in embryonic day 13 and 17 cortical progenitor cells

All-trans retinoic acid (RA) is a differentiation factor in many tissues. However, its role in astrogliogenesis has not been extensively studied. Here, we investigated the effect of RA on the regulation of astrogliogenesis at different cortical developmental stages. We prepared rat cortical progenitor cells from embryonic day (E) 13 and E17, which correspond to the beginning of neurogenic and astrogliogenic periods, respectively. Surprisingly, RA promoted astrogliogenesis at E17 but inhibited astrogliogenesis induced by ciliary neurotrophic factor (CNTF) at E13. The inhibitory effect of RA on astrogliogenesis at E13 was not due to premature commitment of progenitors to a neuronal or oligodendroglial lineage. Rather, RA retained more progenitors in a proliferative state. Furthermore, RA inhibition of astrogliogenesis at E13 was independent of STAT3 signaling and required the function of the α and β isoforms of the RA receptors (RAR). Moreover, the differential response of E13 and E17 progenitors to RA was due to differences in the intrinsic properties of these cells that are preserved in vitro . The inhibitory effect of RA on cytokine-induced astrogliogenesis at E13 may contribute to silencing of any potential precocious astrogliogenesis during the neurogenic period.     

Depletion of Retinoic Acid Receptors Initiates a Novel Positive Feedback Mechanism that Promotes Teratogenic Increases in Retinoic Acid

Normal embryonic development and tissue homeostasis require precise levels of retinoic acid (RA) signaling. Despite the importance of appropriate embryonic RA signaling levels, the mechanisms underlying congenital defects due to perturbations of RA signaling are not completely understood. Here, we report that zebrafish embryos deficient for RA receptor αb1 (RARαb1), a conserved RAR splice variant, have enlarged hearts with increased cardiomyocyte (CM) specification, which are surprisingly the consequence of increased RA signaling. Importantly, depletion of RARαb2 or concurrent depletion of RARαb1 and RARαb2 also results in increased RA signaling, suggesting this effect is a broader consequence of RAR depletion. Concurrent depletion of RARαb1 and Cyp26a1, an enzyme that facilitates degradation of RA, and employment of a novel transgenic RA sensor line support the hypothesis that the increases in RA signaling in RAR deficient embryos are the result of increased embryonic RA coupled with compensatory RAR expression. Our results support an intriguing novel mechanism by which depletion of RARs elicits a previously unrecognized positive feedback loop that can result in developmental defects due to teratogenic increases in embryonic RA.     

Targeted disruption of retinoic acid receptor alpha (RAR alpha) and RAR gamma results in receptor-specific alterations in retinoic acid-mediated differentiation and retinoic acid metabolism.

F9 embryonic teratocarcinoma stem cells differentiate into an epithelial cell type called extraembryonic endoderm when treated with retinoic acid (RA), a derivative of retinol (vitamin A). This differentiation is presumably mediated through the actions of retinoid receptors, the RARs and RXRs. To delineate the functions of each of the different retinoid receptors in this model system, we have generated F9 cell lines in which both copies of either the RAR alpha gene or the RAR gamma gene are disrupted by homologous recombination. The absence of RAR alpha is associated with a reduction in the RA-induced expression of both the CRABP-II and Hoxb-1 (formerly 2.9) genes. The absence of RAR gamma is associated with a loss of the RA-inducible expression of the Hoxa-1 (formerly Hox-1.6), Hoxa-3 (formerly Hox-1.5), laminin B1, collagen IV (alpha 1), GATA-4, and BMP-2 genes. Furthermore, the loss of RAR gamma is associated with a reduction in the metabolism of all-trans-RA to more polar derivatives, while the loss of RAR alpha is associated with an increase in metabolism of RA relative to wild-type F9 cells. Thus, each of these RARs exhibits some specificity with respect to the regulation of differentiation-specific gene expression. These results provide an explanation for the expression of multiple RAR types within one cell type and suggest that each RAR has specific functions.     

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

Retinoic acid (RA) activity plays sequential roles during the development of the ventral spinal cord. Here, we have investigated the functions of local RA synthesis in the process of motoneuron specification and early differentiation using a conditional knockout strategy that ablates the function of the retinaldehyde dehydrogenase 2 (Raldh2) synthesizing enzyme essentially in brachial motoneurons, and later in mesenchymal cells at the base of the forelimb. Mutant (Raldh2L-/-) embryos display an early embryonic loss of a subset of Lim1+ brachial motoneurons, a mispositioning of Islet1+ neurons and inappropriate axonal projections of one of the nerves innervating extensor limb muscles, which lead to an adult forepaw neuromuscular defect. The molecular basis of the Raldh2L-/- phenotype relies in part on the deregulation of Hoxc8, which in turn regulates the RA receptor RARbeta. We further show that Hoxc8 mutant mice, which exhibit a similar congenital forepaw defect, display at embryonic stages molecular defects that phenocopy the Raldh2L-/- motoneuron abnormalities. Thus, interdependent RA signaling and Hox gene functions are required for the specification of brachial motoneurons in the mouse.     

The cellular retinoic-acid-binding protein is expressed in tissues associated with retinoic-acid-induced malformations

Retinoic acid (RA) is thought to play a role in embryonic pattern formation in vertebrates. A naturally occurring gradient of endogenous RA has been demonstrated in the developing chick limb bud, while local application of RA leads to the formation of additional digits. In mammals, a well-defined spectrum of birth defects has been reported as a result of fetal exposure to excess RA. In analogy to the chick limb bud, it may be speculated that these malformations are the result of disturbance of morphogenetic RA concentration gradients. A candidate gene involved in the regulation of endogenous RA concentrations is the gene encoding cellular RA binding protein (CRABP). We have isolated a partial cDNA clone corresponding to the chicken homolog of CRABP, and performed in situ hybridization experiments on sections of embryos at various stages of development. CRABP expression was detected in the CNS, the craniofacial mesenchyme, ganglia of the peripheral nervous system, the limb bud, and the visceral arch area. Our results indicate that the spatiotemporally specified expression pattern displayed by the CRABP gene exhibits a striking correspondence to the tissues that are affected by exposure of avian or mammalian embryos to RA. We hypothesize that CRABP plays an important role in normal embryogenesis and that embryonic tissues showing high CRABP expression are susceptible to the adverse effects of excess RA.     

Retinoic acid signaling is essential for pancreas development and promotes endocrine at the expense of exocrine cell differentiation in Xenopus

How and when the vertebrate endoderm is first subdivided into discrete progenitor cell populations that will give rise to the different major organs, including pancreas and liver, are only poorly understood. We have used Xenopus laevis as a model system to characterize these events, since it is particularly suited to study the early embryonic patterning in vertebrates. Our experimental results support the notion that retinoic acid (RA) functions as an essential endodermal patterning signal in Xenopus and that it acts as early as during gastrulation. As a result of RA treatment, the expression of Sonic Hedgehog (Shh), a known inhibitor of pancreas development in other vertebrate systems, is negatively regulated in the dorsal prepancreatic endoderm. Furthermore, RA is found to promote endocrine at the expense of exocrine differentiation in the dorsal pancreas, correlating with a specific inhibition of Notch signaling activities in this territory. Conversely, RA enhances exocrine marker gene expression in the ventral pancreas.     

The functional basis of tissue-specific retinoic acid signalling in embryos

Retinoic acid (RA) is essential for normal embryonic development. In mammals it is sequestered from the maternal circulation in the form of retinol. In rodents, embryonic uptake relies upon the presence of retinol binding protein (RBP) in the yolk sac, probably involving an RBP receptor. The molecular activity of RA in the nucleus is well established, but less is known about cytoplasmic events including tissue-specific intraembryonic RA synthesis and intracellular transport of both retinol and RA. The cellular binding proteins for retinol and RA may play important roles in these processes.     

Retinoic acid-dependent signaling pathways and lineage events in the developing mouse spinal cord

Studies in avian models have demonstrated an involvement of retinoid signaling in early neural tube patterning. The roles of this signaling pathway at later stages of spinal cord development are only partly characterized. Here we use Raldh2-null mouse mutants rescued from early embryonic lethality to study the consequences of lack of endogenous retinoic acid (RA) in the differentiating spinal cord. Mid-gestation RA deficiency produces prominent structural and molecular deficiencies in dorsal regions of the spinal cord. While targets of Wnt signaling in the dorsal neuronal lineage are unaltered, reductions in Fibroblast Growth Factor (FGF) and Notch signaling are clearly observed. We further provide evidence that endogenous RA is capable of driving stem cell differentiation. Raldh2 deficiency results in a decreased number of spinal cord derived neurospheres, which exhibit a reduced differentiation potential. Raldh2-null neurospheres have a decreased number of cells expressing the neuronal marker β-III-tubulin, while the nestin-positive cell population is increased. Hence, in vivo retinoid deficiency impaired neural stem cell growth. We propose that RA has separable functions in the developing spinal cord to (i) maintain high levels of FGF and Notch signaling and (ii) drive stem cell differentiation, thus restricting both the numbers and the pluripotent character of neural stem cells.