Effect of retinoic acid signaling on Wnt/β-catenin and FGF signaling during body axis extension

Cell–cell signaling regulated by retinoic acid (RA), Wnt/β-catenin, and fibroblast growth factor (FGF) is important during body axis extension, and interactions between these pathways have been suggested. At early somite stages, Wnt/β-catenin and FGF signaling domains exist both anterior and posterior to the developing trunk, whereas RA signaling occurs in between in the trunk under the control of the RA-synthesizing enzyme retinaldehyde dehydrogenase-2 (Raldh2). Previous studies demonstrated that vitamin A deficient quail embryos and Raldh2^−/− mouse embryos lacking RA synthesis exhibit ectopic expression of Fgf8 and Wnt8a in the developing trunk. Here, we demonstrate that Raldh2^−/− mouse embryos display an expansion of FGF signaling into the trunk monitored by Sprouty2 and Pea3 expression, and an expansion of Wnt/β-catenin signaling detected by expression of Axin2, Tbx6, Cdx2, and Cdx4. Following loss of RA signaling, the caudal expression domains of Fgf8, Wnt8a, and Wnt3a expand anteriorly into the trunk, but no change is observed in caudal expression of Fgf4 or Fgf17 plus caudal expression of Fgf18 and Cdx1 is reduced. These findings suggest that RA repression of Fgf8, Wnt8a, and Wnt3a in the developing trunk functions to down-regulate FGF signaling and Wnt/β-catenin signaling as the body axis extends.     

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.     

Retinoic-acid signalling in node ectoderm and posterior neural plate directs left-right patterning of somitic mesoderm

Somitogenesis requires bilateral rhythmic segmentation of paraxial mesoderm along the antero-posterior axis. The location of somite segmentation depends on opposing signalling gradients of retinoic acid (generated by retinaldehyde dehydrogenase-2; Raldh2) anteriorly and fibroblast growth factor (FGF; generated by Fgf8) posteriorly. Retinoic-acid-deficient embryos exhibit somite left-right asymmetry, but it remains unclear how retinoic acid mediates left-right patterning. Here, we demonstrate that retinoic-acid signalling is uniform across the left-right axis and occurs in node ectoderm but not node mesoderm. In Raldh2(-/-) mouse embryos, ectodermal Fgf8 expression encroaches anteriorly into node ectoderm and neural plate, but its expression in presomitic mesoderm is initially unchanged. The late stages of somitogenesis were rescued in Raldh2(-/-) mouse embryos when the maternal diet was supplemented with retinoic acid until only the 6-somite stage, demonstrating that retinoic acid is only needed during node stages. A retinoic-acid-reporter transgene marking the action of maternal retinoic acid in rescued Raldh2(-/-) embryos revealed that the targets of retinoic-acid signalling during somitogenesis are the node ectoderm and the posterior neural plate, not the presomitic mesoderm. Our findings suggest that antagonism of Fgf8 expression by retinoic acid occurs in the ectoderm and that failure of this mechanism generates excessive FGF8 signalling to adjacent mesoderm, resulting initially in smaller somites and then left-right asymmetry.     

Requirement of mesodermal retinoic acid generated by Raldh2 for posterior neural transformation

Studies in amphibian embryos have suggested that retinoic acid (RA) may function as a signal that stimulates posterior differentiation of the nervous system as postulated by the activation-transformation model for anteroposterior patterning of the nervous system. We have tested this hypothesis in retinaldehyde dehydrogenase-2 (Raldh2) null mutant mice lacking RA synthesis in the somitic mesoderm. Raldh2^−/− embryos exhibited neural induction (activation) as evidenced by expression of Sox1 and Sox2 along the neural plate, but differentiation of spinal cord neuroectodermal progenitor cells (posterior transformation) did not occur as demonstrated by a loss of Pax6 and Olig2 expression along the posterior neural plate. Spinal cord differentiation in Raldh2^−/− embryos was rescued by maternal RA administration, and during the rescue RA was found to act directly in the neuroectoderm but not the somitic mesoderm. RA generated by Raldh2 in the somitic mesoderm was found to normally travel as a signal throughout the mesoderm and neuroectoderm of the trunk and into tailbud neuroectoderm, but not into tailbud mesoderm. Raldh2^−/− embryos also exhibited increased Fgf8 expression in the tailbud, and decreased cell proliferation in tailbud neuroectoderm. Our findings demonstrate that RA synthesized in the somitic mesoderm is necessary for posterior neural transformation in the mouse and that Raldh2 provides the only source of RA for posterior development. An important concept to emerge from our studies is that the somitic mesodermal RA signal acts in the neuroectoderm but not mesoderm to generate a spinal cord fate.     

Retinoic acid activates myogenesis in vivo through Fgf8 signalling

Retinoic acid (RA) has been shown to regulate muscle differentiation in vitro. Here, we have investigated the role of RA signalling during embryonic myogenesis in zebrafish. We have altered RA signalling from gastrulation stages onwards by either inhibiting endogenous RA synthesis using an inhibitor of retinaldehyde dehydrogenases (DEAB) or by addition of exogenous RA. DEAB reduces expression of the myogenic markers myoD and myogenin in somites, whereas RA induces increased expression of these genes and strongly induces premature myoD expression in the presomitic mesoderm (psm). The expression dynamics of myf5 in presomitic and somitic mesoderm suggest that RA promotes muscle differentiation, a role supported by the fact that RA activates expression of fast myosin, while DEAB represses it. We identify Fgf8 as a major relay factor in RA-mediated activation of myogenesis. We show that fgf8 expression in somites and anterior psm is regulated by RA, and find that in the absence of Fgf8 signalling in the acerebellar mutant RA fails to promote myoD expression. We propose that, in the developing embryo, localised synthesis of RA by Raldh2 in the anterior psm and in somites activates fgf8 expression which in turn induces the expression of myogenic genes and fast muscle differentiation.     

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.     

Retinoic acid synthesis controlled by Raldh2 is required early for limb bud initiation and then later as a proximodistal signal during apical ectodermal ridge formation

We present evidence for the existence of two phases of retinoic acid (RA) signaling required for vertebrate limb development. Limb RA synthesis is under the control of retinaldehyde dehydrogenase-2 (Raldh2) expressed in the lateral plate mesoderm, which generates a proximodistal RA signal during limb outgrowth. We report that Raldh2(-/-) embryos lack trunk mesodermal RA activity and fail to initiate forelimb development. This is associated with deficient expression of important limb determinants Tbx5, Meis2, and dHand needed to establish forelimb bud initiation, proximal identity, and the zone of polarizing activity (ZPA), respectively. Limb expression of these genes can be rescued by maternal RA treatment limited to embryonic day 8 (E8) during limb field establishment, but the mutant forelimbs obtained at E10 display a significant growth defect associated with a smaller apical ectodermal ridge (AER), referred to here as an apical ectodermal mound (AEM). In these RA-deficient forelimbs, a ZPA expressing Shh forms, but it is located distally adjacent to the Fgf8 expression domain in the AEM rather than posteriorly as is normal. AER formation in Raldh2(-/-) forelimbs is rescued by continuous RA treatment through E10, which restores RA to distal ectoderm fated to become the AER. Our findings indicate the existence of an early phase of RA signaling acting upstream of Tbx5, Meis2, and dHand, followed by a late phase of RA signaling needed to expand AER structure fully along the distal ectoderm. During ZPA formation, RA acts early to activate expression of dHand, but it is not required later for Shh activation.     

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.     

Retinoic acid functions as a key GABAergic differentiation signal in the basal ganglia

Although retinoic acid (RA) has been implicated as an extrinsic signal regulating forebrain neurogenesis, the processes regulated by RA signaling remain unclear. Here, analysis of retinaldehyde dehydrogenase mutant mouse embryos lacking RA synthesis demonstrates that RA generated by Raldh3 in the subventricular zone of the basal ganglia is required for GABAergic differentiation, whereas RA generated by Raldh2 in the meninges is unnecessary for development of the adjacent cortex. Neurospheres generated from the lateral ganglionic eminence (LGE), where Raldh3 is highly expressed, produce endogenous RA, which is required for differentiation to GABAergic neurons. In Raldh3?/? embryos, LGE progenitors fail to differentiate into either GABAergic striatal projection neurons or GABAergic interneurons migrating to the olfactory bulb and cortex. We describe conditions for RA treatment of human embryonic stem cells that result in efficient differentiation to a heterogeneous population of GABAergic interneurons without the appearance of GABAergic striatal projection neurons, thus providing an in vitro method for generation of GABAergic interneurons for further study. Our observation that endogenous RA is required for generation of LGE-derived GABAergic neurons in the basal ganglia establishes a key role for RA signaling in development of the forebrain.     

Patterning of forelimb bud myogenic precursor cells requires retinoic acid signaling initiated by Raldh2

Limb skeletal muscle is derived from cells of the dermomyotome that detach and migrate into the limb buds to form separate dorsal and ventral myogenic precursor domains. Myogenic precursor cell migration is dependent on limb bud mesenchymal expression of hepatocyte growth factor/scatter factor (Hgf), which encodes a secreted ligand that signals to dermomyotome through the membrane receptor tyrosine kinase Met. Here, we find that correct patterning of Hgf expression in forelimb buds is dependent on retinoic acid (RA) synthesized by retinaldehyde dehydrogenase 2 (Raldh2) expressed proximally. Raldh2(-/-) forelimb buds lack RA and display an anteroproximal shift in expression of Hgf such that its normally separate dorsal and ventral expression domains are joined into a single anterior-proximal domain. Met and MyoD are expressed in this abnormal domain, indicating that myogenic cell migration and differentiation are occurring in the absence of RA, but in an abnormal location. An RA-reporter transgene revealed that RA signaling in the forelimb bud normally exists in a gradient across the proximodistal axis, but uniformly across the anteroposterior axis, with all proximal limb bud cells exhibiting activity. Expression of Bmp4, an inhibitor of Hgf expression, is increased and shifted anteroproximally in Raldh2(-/-) limb buds, thus encroaching into the normal expression domain of Hgf. Our studies suggest that RA signaling provides proximodistal information for limb buds that counterbalances Bmp signaling, which in turn helps mediate proximodistal and anteroposterior patterning of Hgf expression to correctly direct migration of Met-expressing myogenic precursor 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.     

Inhibition of Tgf beta signaling by endogenous retinoic acid is essential for primary lung bud induction

Disruption of retinoic acid (RA) signaling during early development results in severe respiratory tract abnormalities, including lung agenesis. Previous studies suggest that this might result from failure to selectively induce fibroblast growth factor 10 (Fgf10) in the prospective lung region of the foregut. Little is known about the RA-dependent pathways present in the foregut that may be crucial for lung formation. By performing global gene expression analysis of RA-deficient foreguts from a genetic [retinaldehyde dehydrogenase 2 (Raldh2)-null] and a pharmacological (BMS493-treated) mouse model, we found upregulation of a large number of Tgfbeta targets. Increased Smad2 phosphorylation further suggested that Tgfbeta signaling was hyperactive in these foreguts when lung agenesis was observed. RA rescue of the lung phenotype was associated with low levels of Smad2 phosphorylation and downregulation of Tgfbeta targets in Raldh2-null foreguts. Interestingly, the lung defect that resulted from RA-deficiency could be reproduced in RA-sufficient foreguts by hyperactivating Tgfbeta signaling with exogenous TGF beta 1. Preventing activation of endogenous Tgfbeta signaling with a pan-specific TGFbeta-blocking antibody allowed bud formation and gene expression in the lung field of both Raldh2-null and BMS493-treated foreguts. Our data support a novel mechanism of RA-Tgfbeta-Fgf10 interactions in the developing foregut, in which endogenous RA controls Tgfbeta activity in the prospective lung field to allow local expression of Fgf10 and induction of lung buds.     

Genetic evidence that retinaldehyde dehydrogenase Raldh1 (Aldh1a1) functions downstream of alcohol dehydrogenase Adh1 in metabolism of retinol to retinoic acid

Vitamin A (retinol) is a nutrient that is essential for developmental regulation but toxic in large amounts. Previous genetic studies have revealed that alcohol dehydrogenase Adh1 is required for efficient clearance of excess retinol to prevent toxicity, thus demonstrating that the mechanism involves oxidation of excess retinol to retinoic acid (RA). Whereas Adh1 plays a dominant role in the first step of the clearance pathway (oxidation of retinol to retinaldehyde), it is unknown what controls the second step (oxidation of retinaldehyde to RA). We now present genetic evidence that aldehyde dehydrogenase Aldh1a1, also known as retinaldehyde dehydrogenase Raldh1, plays a dominant role in the second step of retinol clearance in adult mice. Serum RA levels following a 50 mg/kg dose of retinol were reduced 72% in Raldh1-/- mice and 82% in Adh1-/- mice. This represented reductions in RA synthesis of 77-78% for each mutant after corrections for altered RA degradation in each. After retinol dosing, serum retinaldehyde was increased 2.5-fold in Raldh1-/- mice (indicating defective retinaldehyde clearance) and decreased 3-fold in Adh1-/- mice (indicating defective retinaldehyde synthesis). Serum retinol clearance following retinol administration was decreased 7% in Raldh1-/- mice and 69% in Adh1-/- mice. LD50 studies indicated a small increase in retinol toxicity in Raldh1-/- mice and a large increase in Adh1-/- mice. These observations demonstrate that Raldh1 functions downstream of Adh1 in the oxidative metabolism of excess retinol and that toxicity correlates primarily with accumulating retinol rather than retinaldehyde.     

Distinct functions for Aldh1 and Raldh2 in the control of ligand production for embryonic retinoid signaling pathways

During vertebrate embryogenesis retinoic acid (RA) synthesis must be spatiotemporally regulated in order to appropriately stimulate various retinoid signaling pathways. Various forms of mammalian aldehyde dehydrogenase (ALDH) have been shown to oxidize the vitamin A precursor retinal to RA in vitro. Here we show that injection of Xenopus embryos with mRNAs for either mouse Aldh1 or mouse Raldh2 stimulates RA synthesis at low and high levels, respectively, while injection of human ALDH3 mRNA is unable to stimulate any detectable level of RA synthesis. This provides evidence that some members of the ALDH gene family can indeed perform RA synthesis in vivo. Whole-mount immunohistochemical analyses of mouse embryos indicate that ALDH1 and RALDH2 proteins are localized in distinct tissues. RALDH2 is detected at E7.5-E10.5 primarily in trunk tissue (paraxial mesoderm, somites, pericardium, midgut, mesonephros) plus transiently from E8.5-E9.5 in the ventral optic vesicle and surrounding frontonasal region. ALDH1 is first detected at E9.0-E10. 5 primarily in cranial tissues (ventral mesencephalon, dorsal retina, thymic primordia, otic vesicles) and in the mesonephros. As previous findings indicate that embryonic RA is more abundant in trunk rather than cranial tissues, our findings suggest that Raldh2 and Aldh1 control distinct retinoid signaling pathways by stimulating high and low RA biosynthetic activities, respectively, in various trunk and cranial tissues.     

An FGF3-BMP Signaling Axis Regulates Caudal Neural Tube Closure,Neural Crest Specification and Anterior-Posterior Axis Extension

During vertebrate axis extension, adjacent tissue layers undergo profound morphological changes: within the neuroepithelium, neural tube closure and neural crest formation are occurring, while within the paraxial mesoderm somites are segmenting from the presomitic mesoderm (PSM). Little is known about the signals between these tissues that regulate their coordinated morphogenesis. Here, we analyze the posterior axis truncation of mouse Fgf3 null homozygotes and demonstrate that the earliest role of PSM-derived FGF3 is to regulate BMP signals in the adjacent neuroepithelium. FGF3 loss causes elevated BMP signals leading to increased neuroepithelium proliferation, delay in neural tube closure and premature neural crest specification. We demonstrate that elevated BMP4 depletes PSM progenitors in vitro, phenocopying the Fgf3 mutant, suggesting that excessive BMP signals cause the Fgf3 axis defect. To test this in vivo we increased BMP signaling in Fgf3 mutants by removing one copy of Noggin, which encodes a BMP antagonist. In such mutants, all parameters of the Fgf3 phenotype were exacerbated: neural tube closure delay, premature neural crest specification, and premature axis termination. Conversely, genetically decreasing BMP signaling in Fgf3 mutants, via loss of BMP receptor activity, alleviates morphological defects. Aberrant apoptosis is observed in the Fgf3 mutant tailbud. However, we demonstrate that cell death does not cause the Fgf3 phenotype: blocking apoptosis via deletion of pro-apoptotic genes surprisingly increases all Fgf3 defects including causing spina bifida. We demonstrate that this counterintuitive consequence of blocking apoptosis is caused by the increased survival of BMP-producing cells in the neuroepithelium. Thus, we show that FGF3 in the caudal vertebrate embryo regulates BMP signaling in the neuroepithelium, which in turn regulates neural tube closure, neural crest specification and axis termination. Uncovering this FGF3-BMP signaling axis is a major advance toward understanding how these tissue layers interact during axis extension with important implications in human disease.     

Beyond the neckless phenotype: influence of reduced retinoic acid signaling on motor neuron development in the zebrafish hindbrain

Retinoic acid (RA) has been identified as a key signal involved in the posteriorization of vertebrate neural ectoderm. The main biosynthetic enzyme responsible for RA signaling in the hindbrain and spinal cord is Raldh2. However, neckless/raldh2-mutant (nls) zebrafish exhibit only mild degrees of anteriorization in the neural ectoderm, compared to full vitamin A deficiency in amniotes and the Raldh2-/- mouse. Here we investigated the role of RA during neuronal development in the zebrafish hindbrain and anterior spinal cord using DEAB, an inhibitor of retinaldehyde dehydrogenases. We show that the nls hindbrain and spinal cord are not fully devoid of RA, since blocking Raldh-mediated RA signaling leads to a more severe hindbrain phenotype than in nls. The anteroposterior distribution of branchiomotor neurons in the facial and more posterior nuclei depends on full RA signaling throughout early and late gastrula stages. In contrast, inhibition of RA synthesis after gastrulation reduces the number of branchiomotor neurons in the vagal nucleus, but has no effect on anteroposterior cell fates. In addition, blockage of RA-mediated signaling not only interferes with the differentiation of branchiomotor neurons and their axons in the hindbrain, but also affects the development of the posterior lateral line nerve.     

Expression of Raldh2, Cyp26 and Hox-1 in normal and retinoic acid-treated Ciona intestinalis embryos

Spatially regulated synthesis and degradation of retinoic acid (RA) organize embryonic pattern formation in vertebrate embryos. Here, we show expression pattern of genes encoding Ciona intestinalis homologs of the retinaldehyde dehydrogenase, RALDH2, and the cytochrome P450 RA-degrading enzyme, CYP26, in normal and RA-treated embryos. The Ciona homolog of Raldh2, Ci-Raldh2, was expressed in a few muscle-lineage blastomeres in the middle gastrula. Strong expression was then restricted to the anterior-most three muscle cells on each side of the tailbud embryo. The Ciona homolog of Cyp26, Ci-Cyp26, was expressed in the presumptive brain cells in the middle gastrula. The expression was then upregulated in the neck region. The posterior end of the tail was also weakly stained. Non-overlapping expression domains of Ci-Raldh2 and Ci-Cyp26 look similar to those in vertebrates, although the expression of both genes was restricted to a small number of cells in Ciona embryos. RA upregulated Ci-Cyp26 expression and slightly downregulated Ci-Raldh2 expression in the tailbud embryo. We also show expression pattern of a Hox-1 ortholog (CiHox-1) in the Ciona embryo. CiHox-1 was expressed in two separated regions of the nerve cord and neck epidermis at the neurula stage. Expression pattern of these three genes are essentially similar to that in vertebrates.     

Retinoic Acid Activity in Undifferentiated Neural Progenitors Is Sufficient to Fulfill Its Role in Restricting Fgf8 Expression for Somitogenesis

Bipotent axial stem cells residing in the caudal epiblast during late gastrulation generate neuroectodermal and presomitic mesodermal progeny that coordinate somitogenesis with neural tube formation, but the mechanism that controls these two fates is not fully understood. Retinoic acid (RA) restricts the anterior extent of caudal fibroblast growth factor 8 (Fgf8) expression in both mesoderm and neural plate to control somitogenesis and neurogenesis, however it remains unclear where RA acts to control the spatial expression of caudal Fgf8. Here, we found that mouse Raldh2-/- embryos, lacking RA synthesis and displaying a consistent small somite defect, exhibited abnormal expression of key markers of axial stem cell progeny, with decreased Sox2+ and Sox1+ neuroectodermal progeny and increased Tbx6+ presomitic mesodermal progeny. The Raldh2-/- small somite defect was rescued by treatment with an FGF receptor antagonist. Rdh10 mutants, with a less severe RA synthesis defect, were found to exhibit a small somite defect and anterior expansion of caudal Fgf8 expression only for somites 1–6, with normal somite size and Fgf8 expression thereafter. Rdh10 mutants were found to lack RA activity during the early phase when somites are small, but at the 6-somite stage RA activity was detected in neural plate although not in presomitic mesoderm. Expression of a dominant-negative RA receptor in mesoderm eliminated RA activity in presomitic mesoderm but did not affect somitogenesis. Thus, RA activity in the neural plate is sufficient to prevent anterior expansion of caudal Fgf8 expression associated with a small somite defect. Our studies provide evidence that RA restriction of Fgf8 expression in undifferentiated neural progenitors stimulates neurogenesis while also restricting the anterior extent of the mesodermal Fgf8 mRNA gradient that controls somite size, providing new insight into the mechanism that coordinates somitogenesis with neurogenesis.     

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.