Raldh2 Gene Research Articles

The ubiquitin ligase HECTD1 promotes retinoic acid signaling required for development of the aortic arch.

ABSTRACTThe development of the aortic arch is a complex process that involves remodeling of the bilaterally symmetrical pharyngeal arch arteries (PAAs) into the mature asymmetric aortic arch. Retinoic acid signaling is a key regulator of this process by directing patterning of the second heart field (SHF), formation of the caudal PAAs and subsequent remodeling of the PAAs to form the aortic arch. Here, we identify the HECTD1 ubiquitin ligase as a novel modulator of retinoic acid signaling during this process. Hectd1opm/opm homozygous mutant embryos show a spectrum of aortic arch abnormalities that occur following loss of 4th PAAs and increased SHF marker expression. This sequence of defects is similar to phenotypes observed in mutant mouse models with reduced retinoic acid signaling. Importantly, HECTD1 binds to and influences ubiquitination of the retinoic acid receptor, alpha (RARA). Furthermore, reduced activation of a retinoic acid response element (RARE) reporter is detected in Hectd1 mutant cells and embryos. Interestingly, Hectd1opm/+ heterozygous embryos exhibit reduced retinoic acid signaling, along with intermediate increased expression of SHF markers; however, heterozygotes show normal development of the aortic arch. Decreasing retinoic acid synthesis by reducing Raldh2 (also known as Aldh1a2) gene dosage in Hectd1opm/+ heterozygous embryos reveals a genetic interaction. Double heterozygous embryos show hypoplasia of the 4th PAA and increased incidence of a benign aortic arch variant, in which the transverse arch between the brachiocephalic and left common carotid arteries is shortened. Together, our data establish that HECTD1 is a novel regulator of retinoic acid signaling required for proper aortic arch development.

Kinetic characterization and regulation of the human retinaldehyde dehydrogenase 2 enzyme during production of retinoic acid.

Retinoic acid (RA) is an important regulator of embryogenesis and tissue homoeostasis. Perturbation of RA signalling causes developmental disorders, osteoarthritis, schizophrenia and several typesof tumours. RA is produced by oxidation of retinaldehyde from vitamin A. The main enzyme producing RA in the early embryo is retinaldehyde dehydrogenase 2 (RALDH2, ALDH1A2). In the present study we describe in depth the kinetic properties and regulation of the human RALDH2 (hRALDH2) enzyme. We show that this enzyme produces RA using invivo and invitro assays. We studied the naturally occurring all-trans-, 9-cis- and 13-cis-retinaldehyde isomers as substrates of hRALDH2. Based on the values measured for the Michaelis-Menten constant Km and the maximal rate Vmax, invitro hRALDH2 displays the same catalytic efficiency for their oxidation. We characterized two known inhibitors of the vertebrate RALDH2 and determined their kinetic parameters on hRALDH2. In addition, RA was studied as a possible inhibitor of hRALDH2 and a regulator of its activity. We show that hRALDH2 is not inhibited by its oxidation product, all-trans-RA, suggesting the absence of a negative feedback regulatory loop. Expression of the Raldh2 gene is known to be regulated by RA itself, suggesting that the main regulation of the hRALDH2 activity level is transcriptional.

Wt1 controls retinoic acid signalling in embryonic epicardium through transcriptional activation of Raldh2.

Epicardial-derived signals are key regulators of cardiac embryonic development. An important part of these signals is known to relate to a retinoic acid (RA) receptor-dependent mechanism. RA is a potent morphogen synthesised by Raldh enzymes, Raldh2 being the predominant one in mesodermal tissues. Despite the importance of epicardial retinoid signalling in the heart, the molecular mechanisms controlling cardiac Raldh2 transcription remain unknown. In the current study, we show that Wt1-null epicardial cells display decreased expression of Raldh2 both in vivo and in vitro. Using a RA-responsive reporter, we have confirmed that Wt1-null epicardial cells actually show reduced synthesis of RA. We also demonstrate that Raldh2 is a direct transcriptional target of Wt1 in epicardial cells. A secondary objective of this study was to identify the status of RA-related receptors previously reported to be critical to epicardial biology (PDGFRα,β; RXRα). PDGFRα and PDGFRβ mRNA and protein levels are downregulated in the absence of Wt1, but only Pdgfra expression is rescued by the addition of RA to Wt1-null epicardial cells. RXRα mRNA levels are not affected in Wt1-null epicardial cells. Taken together, our results indicate that Wt1 critically regulates epicardial RA signalling via direct activation of the Raldh2 gene, and identify a role for Wt1 in the regulation of morphogen receptors involved in the proliferation, migration, and differentiation of epicardial and epicardially-derived cells (EPDC).

Identification of a conserved roof plate enhancer in the RALDH2 gene

Identification of a conserved roof plate enhancer in the RALDH2 gene

Rescue of morphogenetic defects and of retinoic acid signaling in retinaldehyde dehydrogenase 2 ( Raldh2) mouse mutants by chimerism with wild-type cells

Retinoic acid (RA), the active vitamin A derivative, is an important developmental signaling molecule in vertebrates. In this study, we have assessed whether minimal numbers and/or specific distributions of RA-producing cells can support normal mouse embryonic development. Retinaldehyde dehydrogenase 2 (RALDH2) is the main RA-synthesizing enzyme acting during development. We have generated an embryonic stem (ES) cell line homozygous for an Raldh2 gene disruption, and have analyzed chimeric embryos with various contributions of wild-type cells. Whereas embryos almost completely derived from Raldh2 −/− cells phenocopy the corresponding germline null mutants, the presence of even small numbers (<10%) of wild-type cells can rescue most of the morphogenetic defects, including embryonic turning and axial elongation, and left–right looping of the heart tube. No consistent bias in the distribution of wild-type cells was observed in the phenotypically rescued Raldh2 −/− chimeras. Analysis of an RA-sensitive transgene indicates that RA can diffuse from wild-type cells and elicit a widespread transcriptional response in Raldh2-deficient cells. Our results show that few wild-type RA-producing cells, even when present in apparent random distributions, can support early morphogenesis of the mouse embryo. However, the Raldh2 −/− chimeric fetuses display lung abnormalities, persistent truncus arteriosus, and abnormal myocardial differentiation, showing that subsequent RA-dependent events cannot be fully rescued by the mosaic presence of wild-type cells.

Retinoids control anterior and dorsal properties in the developing forebrain

We have previously shown that retinoic acid (RA) synthesized by the retinaldehyde dehydrogenase 2 (RALDH2) is required in forebrain development. Deficiency in RA due to inactivation of the mouse Raldh2 gene or to complete absence of retinoids in vitamin-A-deficient (VAD) quails, leads to abnormal morphogenesis of various forebrain derivatives. In this study we show that double Raldh2/Raldh3 mouse mutants have a more severe phenotype in the craniofacial region than single null mutants. In particular, the nasal processes are truncated and the eye abnormalities are exacerbated. It has been previously shown that retinoids act mainly on cell proliferation and survival in the ventral forebrain by regulating SHH and FGF8 signaling. Using the VAD quail model, which survives longer than the Raldh-deficient mouse embryos, we found that retinoids act in maintaining the correct position of anterior and dorsal boundaries in the forebrain by modulating FGF8 anteriorly and WNT signaling dorsally. Furthermore, BMP4 and FGF8 signaling are affected in the nasal region and BMP4 is ventrally expanded in the optic vesicle. At the optic cup stage, Pax6, Tbx5 and Bmp4 are ectopically expressed in the presumptive retinal pigmented epithelium (RPE), while Otx2 and Mitf are not induced, leading to a dorsal transdifferentiation of RPE to neural retina. Therefore, besides being required for survival of ventral structures, retinoids are involved in restricting anterior identity in the telencephalon and dorsal identity in the diencephalon and the retina.

A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field.

Establishment of anteroposterior (AP) polarity is one of the earliest decisions in cardiogenesis and plays an important role in the coupling between heart and blood vessels. Recent research implicated retinoic acid (RA) in the communication of AP polarity to the heart. We utilized embryo culture, in situ hybridization, morphometry, fate mapping and treatment with the RA pan-antagonist BMS493 to investigate the relationship between cardiac precursors and RA signalling. We describe two phases of AP signalling by RA, reflected in RALDH2 expression. The first phase (HH4-7) is characterized by increasing proximity between sino-atrial precursors and the lateral mesoderm expressing RALDH2. In this phase, RA signalling is consistent with diffusion of the morphogen from a large field rather than a single hot spot. The second phase (HH7-8) is characterized by progressive encircling of cardiac precursors by a field of RALDH2 originating from a dynamic and evolutionary-conserved caudorostral wave pattern in the lateral mesoderm. At this phase, cardiac AP patterning by RA is consistent with localized action of RA by regulated activation of the Raldh2 gene within an embryonic domain. Systemic treatment with BMS493 altered the cardiac fate map such that ventricular precursors were found in areas normally devoid of them. Topical application of BMS493 inhibited atrial differentiation in left anterior lateral mesoderm. Identification of the caudorostral wave of RALDH2 as the endogenous source of RA establishing cardiac AP fates provides a useful model to approach the mechanisms whereby the vertebrate embryo confers axial information on its organs.

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.

A number of studies have suggested that retinoic acid (RA) is an important signal for patterning the hindbrain, the branchial arches and the limb bud. Retinoic acid is thought to act on the posterior hindbrain and the limb buds at somitogenesis stages in chick and mouse embryos. Here we report a much earlier requirement for RA signalling during pre-segmentation stages for proper development of these structures in zebrafish. We present evidence that a RA signal is necessary during pre-segmentation stages for proper expression of the spinal cord markers hoxb5a and hoxb6b, suggesting an influence of RA on anteroposterior patterning of the neural plate posterior to the hindbrain. We report the identification and expression pattern of the zebrafish retinaldehyde dehydrogenase2 (raldh2/aldh1a2) gene. Raldh2 synthesises retinoic acid (RA) from its immediate precursor retinal. It is expressed in a highly ordered spatial and temporal fashion during gastrulation in the involuting mesoderm and during later embryogenesis in paraxial mesoderm, branchial arches, eyes and fin buds, suggesting the involvement of RA at different times of development in different functional contexts. Mapping of the raldh2 gene reveals close linkage to no-fin (nof), a newly discovered mutant lacking pectoral fins and cartilaginous gill arches. Cloning and functional tests of the wild-type and nof alleles of raldh2 reveal that nof is a raldh2 mutant. By treating nof mutants with RA during different time windows and by making use of a retinoic acid receptor antagonist, we show that RA signalling during pre-segmentation stages is necessary for anteroposterior patterning in the CNS and for fin induction to occur.

Specific expression of the retinoic acid-synthesizing enzyme RALDH2 during mouse inner ear development.

Retinoid binding proteins and nuclear receptors are expressed in the developing mouse inner ear. Here, we report that the retinaldehyde dehydrogenase 2 (Raldh2) gene, whose product is involved in the enzymatic generation of retinoic acid (RA), exhibits a restricted expression pattern during mouse inner ear ontogenesis. The Raldh2 gene is first expressed at embryonic day (E) 10.5 in a V-shaped medio-dorsal region of the otocyst outer epithelium, which evolves as two separate domains upon otocyst morphogenesis. At E14.5, Raldh2 is expressed in two areas of the utricle epithelium and specific regions of the saccule and cochlear mesenchyme. Later, Raldh2 transcripts are restricted to two cochlear areas, the stria vascularis and Reissner membrane. Raldh2 mesenchymal expression did not correlate with migrating neural crest-derived melanoblasts. These restricted expression domains may correspond to specific sites of RA synthesis during inner ear morphogenesis.

Analysis of Mouse Retinal Dehydrogenase Type 2 Promoter and Expression

The mouse RALDH2 gene spans >50 kb, has a structure similar to that of human class 1 aldehyde dehydrogenase genes, and localizes to the central region of chromosome 9 by single-strand polymorphism analysis. Expression of mouse RALDH2 was detected in testis, lung, brain, and heart (Northern blot) and in liver and kidney (RNase protection assays). Expression was not detected by RNase protection assay in testis of vitamin A-deficient rats, and all-trans-retinoic acid dosing did not increase expression in vitamin A-deficient rat testis. A 2.3-kb section of the gene 5′ to the transcription start site included neither retinoic acid nor retinoid X response elements, but included TATA and CCAAT motifs and AP, AHR, CREB, ER, Ets, and SREBP sites. The promoter initiated transcription of a luciferase reporter in human embryonic kidney cells (EBNA) and mouse Leydig- (TM3) and Sertoli-derived (TM4) cell lines, but neither all-trans-retinoic acid nor 9-cis-retinoic acid affected reporter transcription. These data suggest that relatively weak RALDH2 expression in vitamin A-deficient testis reflects vastly decreased numbers of germ cells, the major site of expression.

Embryonic retinoic acid synthesis is essential for early mouse post-implantation development.

A number of studies have suggested that the active derivative of vitamin A, retinoic acid (RA), may be important for early development of mammalian embryos. Severe vitamin A deprivation in rodents results in maternal infertility, precluding a thorough investigation of the role of RA during embryogenesis. Here we show that production of RA by the retinaldehyde dehydrogenase-2 (Raldh2) enzyme is required for mouse embryo survival and early morphogenesis. Raldh2 is an NAD-dependent aldehyde dehydrogenase with high substrate specificity for retinaldehyde. Its pattern of expression during mouse development has suggested that it may be responsible for embryonic RA synthesis. We generated a targeted disruption of the mouse Raldh2 gene and found that Raldh2-/- embryos, which die at midgestation without undergoing axial rotation (body turning), exhibit shortening along the anterioposterior axis and do not form limb buds. Their heart consists of a single, medial, dilated cavity. Their frontonasal region is truncated and their otocysts are severely reduced. These defects result from a block in embryonic RA synthesis, as shown by the lack of activity of RA-responsive transgenes, the altered expression of an RA-target homeobox gene and the near full rescue of the mutant phenotype by maternal RA administration. Our data establish that RA synthesized by the post-implantation mammalian embryo is an essential developmental hormone whose lack leads to early embryo death.