Role of retinoic acid during forebrain development begins late when Raldh3 generates retinoic acid in the ventral subventricular zone

Retinoic acid (RA) synthesized by Raldh3 in the frontonasal surface ectoderm of chick embryos has been suggested to function in early forebrain patterning by regulating Fgf8, Shh, and Meis2 expression. Similar expression of Raldh3 exists in E8.75 mouse embryos, but Raldh2 is also expressed in the optic vesicle at this stage suggesting that both genes may play a role in early forebrain patterning. Furthermore, Raldh3 is expressed later in the forebrain itself (lateral ganglionic eminence; LGE) starting at E12.5, suggesting a later role in forebrain neurogenesis. Here we have analyzed mouse embryos carrying single or double null mutations in Raldh2 and Raldh3 for defects in forebrain development. Raldh2(-/-);Raldh3(-/-) embryos completely lacked RA signaling activity in the early forebrain, but exhibited relatively normal expression of Fgf8, Shh, and Meis2 in the forebrain. Thus, we find no clear requirement for RA in controlling expression of these important forebrain patterning genes, but Raldh3 expression in the frontonasal surface ectoderm was found to be needed for normal Fgf8 expression in the olfactory pit. Our studies revealed that later expression of Raldh3 in the subventricular zone of the LGE is required for RA signaling activity in the ventral forebrain. Importantly, expression of dopamine receptor D2 in E18.5 Raldh3(-/-) embryos was essentially eliminated in the developing nucleus accumbens, a tissue lying close to the source of RA provided by Raldh3. Our results suggest that the role of RA during forebrain development begins late when Raldh3 expression initiates in the ventral subventricular zone.     

Shifting boundaries of retinoic acid activity control hindbrain segmental gene expression

Retinoic acid (RA) generated by Raldh2 in paraxial mesoderm is required for specification of the posterior hindbrain, including restriction of Hoxb1 expression to presumptive rhombomere 4 (r4). Hoxb1 expression requires 3' and 5' RA response elements for widespread induction up to r4 and for r3/r5 repression, but RA has previously been detected only from r5-r8, and vHnf1 is required for repression of Hoxb1 posterior to r4 in zebrafish. We demonstrate in mouse embryos that an RA signal initially travels from the paraxial mesoderm to r3, forming a boundary next to the r2 expression domain of Cyp26a1 (which encodes an RA-degrading enzyme). After Hoxb1 induction, the RA boundary quickly shifts to r4/r5, coincident with induction of Cyp26c1 in r4. A functional role for Cyp26c1 in RA degradation was established through examination of RA-treated embryos. Analysis of Raldh2-/- and vHnf1-/- embryos supports a direct role for RA in Hoxb1 induction up to r4 and repression in r3/r5, as well as an indirect role for RA in Hoxb1 repression posterior to r4 via RA induction of vHnf1 up to the r4/r5 boundary. Our findings suggest that Raldh2 and Cyp26 generate shifting boundaries of RA activity, such that r3-r4 receives a short pulse of RA and r5-r8 receives a long pulse of RA. These two pulses of RA activity function to establish expression of Hoxb1 and vHnf1 on opposite sides of the r4/r5 boundary.     

Retinoic acid regulates endothelial cell proliferation during vasculogenesis

A dietary deficiency of vitamin A is associated with cardiovascular abnormalities in avian and murine systems. Retinoic acid (RA) is the active metabolite of vitamin A and whether it directly regulates mammalian blood vessel formation has not been determined and is investigated herein. We used mice rendered RA-deficient via targeted deletion of retinaldehyde dehydrogenase 2 (Raldh2(-/-)), the enzyme required to produce active RA in the embryo. Histological examination at E8.0-8.5, prior to cardiac function and systemic blood circulation, revealed that capillary plexi formed in Raldh2(-/-) yolk sacs and embryos, but were dilated, and not appropriately remodeled or patterned. Raldh2(-/-) endothelial cells exhibited significantly increased expression of phosphohistone 3 and decreased expression of p21 and p27, suggesting that RA is required to control endothelial cell cycle progression during early vascular development. Uncontrolled endothelial cell growth, in Raldh2(-/-) mutants, was associated with decreased endothelial cell maturation, disrupted vascular plexus remodeling and lack of later stages of vessel assembly, including mural cell differentiation. Maternally administrated RA restored endothelial cell cycle control and vascular patterning. Thus, these data indicate that RA plays a crucial role in mammalian vascular development; it is required to control endothelial cell proliferation and vascular remodeling during vasculogenesis.     

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.     

Regulation of retinoic acid distribution is required for proximodistal patterning and outgrowth of the developing mouse limb

Exogenous retinoic acid (RA) induces marked effects on limb patterning, but the precise role of endogenous RA in this process has remained unknown. We have studied the role of RA in mouse limb development by focusing on CYP26B1, a cytochrome P450 enzyme that inactivates RA. Cyp26b1 was shown to be expressed in the distal region of the developing limb bud, and mice that lack CYP26B1 exhibited severe limb malformation (meromelia). The lack of CYP26B1 resulted in spreading of the RA signal toward the distal end of the developing limb and induced proximodistal patterning defects characterized by expansion of proximal identity and restriction of distal identity. CYP26B1 deficiency also induced pronounced apoptosis in the developing limb and delayed chondrocyte maturation. Wild-type embryos exposed to excess RA phenocopied the limb defects of Cyp26b1(-/-) mice. These observations suggest that RA acts as a morphogen to determine proximodistal identity, and that CYP26B1 prevents apoptosis and promotes chondrocyte maturation, in the developing limb.     

The regional pattern of retinoic acid synthesis by RALDH2 is essential for the development of posterior pharyngeal arches and the enteric nervous system

Targeted inactivation of the mouse retinaldehyde dehydrogenase 2 (RALDH2/ALDH1a2), the enzyme responsible for early embryonic retinoic acid synthesis, is embryonic lethal because of defects in early heart morphogenesis. Transient maternal RA supplementation from E7.5 to (at least) E8.5 rescues most of these defects, but the supplemented Raldh2(-/-) mutants die prenatally, from a lack of septation of the heart outflow tract (Niederreither, K., Vermot, J., Messaddeq, N., Schuhbaur, B., Chambon, P. and Dollé, P. (2001). Development 128, 1019-1031). We have investigated the developmental basis for this defect, and found that the RA-supplemented Raldh2(-/-) embryos exhibit impaired development of their posterior (3rd-6th) branchial arch region. While the development of the first and second arches and their derivatives, as well as the formation of the first branchial pouch, appear to proceed normally, more posterior pharyngeal pouches fail to form and the pharyngeal endoderm develops a rudimentary, pouch-like structure. All derivatives of the posterior branchial arches are affected. These include the aortic arches, pouch-derived organs (thymus, parathyroid gland) and post-otic neural crest cells, which fail to establish segmental migratory pathways and are misrouted caudally. Patterning and axonal outgrowth of the posterior (9th-12th) cranial nerves is also altered. Vagal crest deficiency in Raldh2(-/-) mutants leads to agenesis of the enteric ganglia, a condition reminiscent of human Hirschprung's disease. In addition, we provide evidence that: (i) wildtype Raldh2 expression is restricted to the posteriormost pharyngeal mesoderm; (ii) endogenous RA response occurs in both the pharyngeal endoderm and mesoderm, and extends more rostrally than Raldh2 expression up to the 2nd arch; (iii) RA target genes (Hoxa1, Hoxb1) are downregulated in both the pharyngeal endoderm and mesoderm of mutant embryos. Thus, RALDH2 plays a crucial role in producing RA required for pharyngeal development, and RA is one of the diffusible mesodermal signals that pattern the pharyngeal endoderm.     

Retinoic acid-metabolizing enzyme Cyp26a1 is essential for determining territories of hindbrain and spinal cord in zebrafish

Retinoic acid (RA) plays a critical role in neural patterning and organogenesis in the vertebrate embryo. Here we characterize a mutant of the zebrafish named giraffe (gir) in which the gene for the RA-degrading enzyme Cyp26a1 is mutated. The gir mutant displayed patterning defects in multiple organs including the common cardinal vein, pectoral fin, tail, hindbrain, and spinal cord. Analyses of molecular markers suggested that the lateral plate mesoderm is posteriorized in the gir mutant, which is likely to cause the defects of the common cardinal vein and pectoral fin. The cyp26a1 expression in the rostral spinal cord was strongly upregulated in the gir mutant, suggesting a strong feedback control of its expression by RA signaling. We also found that the rostral spinal cord territory was expanded at the expense of the hindbrain territory in the gir mutant. Such a phenotype is the opposite of that of the mutant for Raldh2, an enzyme that synthesizes RA. We propose a model in which Cyp26a1 attenuates RA signaling in the prospective rostral spinal cord to limit the expression of hox genes and to determine the hindbrain-spinal cord boundary.     

Uncoupling of retinoic acid signaling from tailbud development before termination of body axis extension

During the early stages of body axis extension, retinoic acid (RA) synthesized in somites by Raldh2 represses caudal fibroblast growth factor (FGF) signaling to limit the tailbud progenitor zone. Excessive RA down-regulates Fgf8 and triggers premature termination of body axis extension, suggesting that endogenous RA may function in normal termination of body axis extension. Here, we demonstrate that Raldh2-/- mouse embryos undergo normal down-regulation of tailbud Fgf8 expression and termination of body axis extension in the absence of RA. Interestingly, Raldh2 expression in wild-type tail somites and tailbud from E10.5 onwards does not result in RA activity monitored by retinoic acid response element (RARE)-lacZ. Treatment of wild-type tailbuds with physiological levels of RA or retinaldehyde induces RARE-lacZ activity, validating the sensitivity of RARE-lacZ and demonstrating that deficient RA synthesis in wild-type tail somites and tailbud is due to a lack of retinaldehyde synthesis. These studies demonstrate an early uncoupling of RA signaling from mouse tailbud development and show that termination of body axis extension occurs in the absence of RA signaling.     

Targeted disruption of Aldh1a1 (Raldh1) provides evidence for a complex mechanism of retinoic acid synthesis in the developing retina

Genetic studies have shown that retinoic acid (RA) signaling is required for mouse retina development, controlled in part by an RA-generating aldehyde dehydrogenase encoded by Aldh1a2 (Raldh2) expressed transiently in the optic vesicles. We examined the function of a related gene, Aldh1a1 (Raldh1), expressed throughout development in the dorsal retina. Raldh1(-/-) mice are viable and exhibit apparently normal retinal morphology despite a complete absence of Raldh1 protein in the dorsal neural retina. RA signaling in the optic cup, detected by using a RARE-lacZ transgene, is not significantly altered in Raldh1(-/-) embryos at embryonic day 10.5, possibly due to normal expression of Aldh1a3 (Raldh3) in dorsal retinal pigment epithelium and ventral neural retina. However, at E16.5 when Raldh3 is expressed ventrally but not dorsally, Raldh1(-/-) embryos lack RARE-lacZ expression in the dorsal retina and its retinocollicular axonal projections, whereas normal RARE-lacZ expression is detected in the ventral retina and its axonal projections. Retrograde labeling of adult Raldh1(-/-) retinal ganglion cells indicated that dorsal retinal axons project to the superior colliculus, and electroretinography revealed no defect of adult visual function, suggesting that dorsal RA signaling is unnecessary for retinal ganglion cell axonal outgrowth. We observed that RA synthesis in liver of Raldh1(-/-) mice was greatly reduced, thus showing that Raldh1 indeed participates in RA synthesis in vivo. Our findings suggest that RA signaling may be necessary only during early stages of retina development and that if RA synthesis is needed in dorsal retina, it is catalyzed by multiple enzymes, including Raldh1.     

Novel retinoic acid generating activities in the neural tube and heart identified by conditional rescue of Raldh2 null mutant mice

Retinoid control of vertebrate development depends upon tissue-specific metabolism of retinol to retinoic acid (RA). The RA biosynthetic enzyme RALDH2 catalyzes much, but not all, RA production in mouse embryos, as revealed here with Raldh2 null mutants carrying an RA-responsive transgene. Targeted disruption of Raldh2 arrests development at midgestation and eliminates all RA synthesis except that associated with Raldh3 expression in the surface ectoderm of the eye field. Conditional rescue of Raldh2(-/-) embryos by limited maternal RA administration allows development to proceed and results in the establishment of additional sites of RA synthesis linked to Raldh1 expression in the dorsal retina and to Raldh3 expression in the ventral retina, olfactory pit and urinary tract. Unexpectedly, conditionally rescued Raldh2(-/-) embryos also possess novel sites of RA synthesis in the neural tube and heart that do not correspond to expression of Raldh1-3. RA synthesis in the mutant neural tube was localized in the spinal cord, posterior hindbrain and portions of the midbrain and forebrain, whereas activity in the mutant heart was localized in the conotruncus and sinus venosa. In the posterior hindbrain, this novel RA-generating activity was expressed during establishment of rhombomeric boundaries. In the spinal cord, the novel activity was localized in the floorplate plus in the intermediate region where retinoid-dependent interneurons develop. These novel RA-generating activities in the neural tube and heart fill gaps in our knowledge of how RA is generated spatiotemporally and may, along with Raldh1 and Raldh3, contribute to rescue of Raldh2(-/-) embryos by producing RA locally.     

Cyp26 enzymes generate the retinoic acid response pattern necessary for hindbrain development

Retinoic acid (RA) is essential for normal vertebrate development, including the patterning of the central nervous system. During early embryogenesis, RA is produced in the trunk mesoderm through the metabolism of vitamin A derived from the maternal diet and behaves as a morphogen in the developing hindbrain where it specifies nested domains of Hox gene expression. The loss of endogenous sources of RA can be rescued by treatment with a uniform concentration of exogenous RA, indicating that domains of RA responsiveness can be shaped by mechanisms other than the simple diffusion of RA from a localized posterior source. Here, we show that the cytochrome p450 enzymes of the Cyp26 class, which metabolize RA into polar derivatives, function redundantly to shape RA-dependent gene-expression domains during hindbrain development. In zebrafish embryos depleted of the orthologs of the three mammalian CYP26 genes CYP26A1, CYP26B1 and CYP26C1, the entire hindbrain expresses RA-responsive genes that are normally restricted to nested domains in the posterior hindbrain. Furthermore, we show that Cyp26 enzymes are essential for exogenous RA to rescue hindbrain patterning in RA-depleted embryos. We present a ;gradient-free' model for hindbrain patterning in which differential RA responsiveness along the hindbrain anterior-posterior axis is shaped primarily by the dynamic expression of RA-degrading enzymes.     

Retinoic acid metabolizing factor xCyp26c is specifically expressed in neuroectoderm and regulates anterior neural patterning in Xenopus laevis

Anterior-posterior neural patterning is determined during gastrulation when head structure is induced. Induction of anterior neural structures requires inhibition of Wnt signaling by several Wnt antagonists. We performed microarray analysis to isolate genes regulated by canonical Wnt signaling and abundantly expressed in the anterior neuroectoderm at the early neurula stage. We identified xCyp26c, a Cyp26 (RA-metabolizing protein)-family gene. In situ hybridization showed xCyp26c expression restricted to the anterior region of neurula, while xCyp26a was expressed in both anterior and posterior regions. At the tadpole stage, xCyp26c was also expressed in restricted sets of cranial nerves. Microarray, RT-PCR and in situ hybridization analyses revealed decreased xCyp26c expression with overexpression of beta-catenin, suggesting regulation by Wnt/beta-catenin signaling. We also assessed the effects of retinoic acid (RA) on xCyp26c expression. Embryos treated with 10(-7) M RA showed an anterior shift in the spatial expression of xCyp26c, reflecting a posteriorization effect. Conversely, expression patterns in embryos treated with more than 10(-6) M RA were less affected and remained restricted to the most anterior region. Moreover, injection of xCyp26c mRNA into animal poles caused head defects, and exogenous expression of xCyp26c rescued the posteriorizing effect of RA treatment. Taken together, these results implicated a role for xCyp26c in anterior patterning via RA signaling.     

Conditional mutation of Rb causes cell cycle defects without apoptosis in the central nervous system

Targeted disruption of the retinoblastoma gene in mice leads to embryonic lethality in midgestation accompanied by defective erythropoiesis. Rb(-/-) embryos also exhibit inappropriate cell cycle activity and apoptosis in the central nervous system (CNS), peripheral nervous system (PNS), and ocular lens. Loss of p53 can prevent the apoptosis in the CNS and lens; however, the specific signals leading to p53 activation have not been determined. Here we test the hypothesis that hypoxia caused by defective erythropoiesis in Rb-null embryos contributes to p53-dependent apoptosis. We show evidence of hypoxia in CNS tissue from Rb(-/-) embryos. The Cre-loxP system was then used to generate embryos in which Rb was deleted in the CNS, PNS and lens, in the presence of normal erythropoiesis. In contrast to the massive CNS apoptosis in Rb-null embryos at embryonic day 13.5 (E13.5), conditional mutants did not have elevated apoptosis in this tissue. There was still significant apoptosis in the PNS and lens, however. Rb(-/-) cells in the CNS, PNS, and lens underwent inappropriate S-phase entry in the conditional mutants at E13.5. By E18.5, conditional mutants had increased brain size and weight as well as defects in skeletal muscle development. These data support a model in which hypoxia is a necessary cofactor in the death of CNS neurons in the developing Rb mutant embryo.     

视黄酸挽救的视黄酸合成酶Raldh2基因敲除鼠E10.75胚胎后肢发育正常

视黄酸合成酶Raldh2基因敲除鼠胚胎没有肢体的发育在胚胎E6.75-E 8.25期间,喂给怀孕母鼠含视黄酸（0.1 mg/g食物）食物后,Raldh2基因敲除鼠E10.75胚胎后肢形态正常,前肢发育较小.原位杂交结果表明,决定肢体近 远端轴发育的标志基因(marker gene)Fgf8,决定前-后轴发育的标志基因Shh以及后肢发育特异性基因Tbx4 和Pitx1在视黄酸挽救的Raldh2基因敲除鼠E10.75胚胎的后肢表达正常.上述结果提示,视黄酸可以挽救Raldh2基因敲除鼠E10.75胚胎后肢的正常发育.     

Retinoic acid and Cyp26b1 are critical regulators of osteogenesis in the axial skeleton

Retinoic acid (RA) plays important roles in diverse biological processes ranging from germ cell specification to limb patterning. RA ultimately exerts its effect in the nucleus, but how RA levels are being generated and maintained locally is less clear. Here, we have analyzed the zebrafish stocksteif mutant, which exhibits severe over-ossification of the entire vertebral column. stocksteif encodes cyp26b1, a cytochrome P450 member that metabolizes RA. The mutant is completely phenocopied by treating 4 dpf wild-type embryos with either RA or the pharmacological Cyp26 blocker R115866, thus identifying a previously unappreciated role for RA and cyp26b1 in osteogenesis of the vertebral column. Cyp26b1 is expressed within osteoblast cells, demonstrating that RA levels within these cells need to be tightly controlled. Furthermore, we have examined the effect of RA on osteoblasts in vivo. As numbers of osteoblasts do not change upon RA treatment, we suggest that RA causes increased activity of axial osteoblasts, ultimately resulting in defective skeletogenesis.