Expression of zebrafish aldh1a3 (raldh3) and absence of aldh1a1 in teleosts

The vitamin A-derived morphogen retinoic acid (RA) plays important roles during the development of chordate animals. The Aldh1a-family of RA-synthesizing enzymes consists of three members, Aldh1a1-3 (Raldh1-3), that are dynamically expressed throughout development. We have searched the known teleost genomes for the presence of Raldh family members and have found that teleost fish possess orthologs of Aldh1a2 and Aldh1a3 only. Here we describe the expression of aldh1a3 in the zebrafish, Danio rerio. Whole mount in situ hybridization shows that aldh1a3 is expressed during eye development in the retina flanking the optic stalks and later is expressed ventrally, opposite the expression domain of aldh1a2. During inner ear morphogenesis, aldh1a3 is expressed in developing sensory epithelia of the cristae and utricular macula and is specifically up-regulated in epithelial projections throughout the formation of the walls of the semicircular canals and endolymphatic duct. In contrast to the mouse inner ear, which expresses all three Raldhs, we find that only aldh1a3 is expressed in the zebrafish otocyst, while aldh1a2 is present in the periotic mesenchyme. During larval stages, additional expression domains of aldh1a3 appear in the anterior pituitary and the swim bladder. Our analyses provide a starting point for genetic studies to examine the role of RA in these organs and emphasize the suitability of the zebrafish inner ear in dissecting the contribution of RA signaling to the development of the vestibular system.     

Cyp26C1 encodes a novel retinoic acid-metabolizing enzyme expressed in the hindbrain,inner ear,first branchial arch and tooth buds during murine development

Retinoic acid (RA), an active metabolite of vitamin A, is a crucial signaling molecule involved in tissue morphogenesis during embryonic development. RA distribution and concentration is precisely regulated during embryogenesis by balanced complementary activities of RA synthesizing (RALDH) and metabolizing (CYP26) enzymes. Here, we describe the identification of a novel murine p450 cytochrome belonging to the CYP26 family, mCYP26C1. Sequence alignment show that mCYP26C1 is more closely related to mCYP26B1 than mCYP26A1. At early developmental stages (E8.0-E8.5), mCyp26C1 is expressed in prospective rhombomeres 2 and 4, in the first branchial arch and along the lateral surface mesenchyme adjacent to the rostral hindbrain. At E9.5, mCyp26C1 expression persists in rhombomere 2 and in the maxillary and mandibular components of the first branchial arch, and is strongly induced in the lateral cervical mesenchyme. By mid-gestation, mCyp26C1 is weakly expressed in the cervical mesenchyme and in the maxillary component of the first branchial arch. At E11.5, mCyp26C1 can only be seen in a narrow band in the lateral cervical mesenchyme. During late gestation, mCyp26C1 exhibits region-specific expression in the inner ear epithelium and a persistent expression in the inner dental epithelium of the developing teeth. This pattern of expression suggests that mCYP26C1 may play an important role in protecting the hindbrain, first branchial arch, otocyst and tooth buds against RA exposure during embryonic development.     

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.     

Complementary expression patterns of retinoid acid-synthesizing and -metabolizing enzymes in pre-natal mouse inner ear structures

Retinoic acid (RA) plays a pivotal role in patterning and differentiation of the embryonic inner ear. Despite its documented effects during embryonic development, the cellular sites that synthesize or metabolize RA in the inner ear have yet to be determined. Here we describe the distribution of three synthesizing enzymes, retinaldehyde dehydrogenases 1, 2 and 3 (RALDH1, RALDH2 and RALDH3) and two catabolizing enzymes (CYP26A1 and CYP26B1) in the mouse inner ear at embryonic day 18.5 when active cell differentiation is underway. Two detection methods, radioactive and non-radioactive in situ hybridization, were employed to elucidate the tissue distribution and cellular localization of these enzymes, respectively. All of the five enzymes examined, with the exception of CYP26A1, were expressed in both vestibular and cochlear end organs. While expression of the three RALDHs was observed in various cell types, CYP26B1 expression was found only in supporting cells of the vestibular and cochlear end organs. In the cochlea, expression domains of RALDH1-3 and CYP26B1 were complementary to one another. These results reveal specific tissue- and cellular expression patterns of RA synthesizing and catabolizing enzymes in the pre-natal inner ear, and suggest that a precise control of RA concentrations in various cell types of the inner ear is achieved by the balance between RALDHs and CYP26B1 activities.     

A novel cytochrome P450, zebrafish Cyp26D1, is involved in metabolism of all-trans retinoic acid

Retinoid signaling is essential for development of vertebrate embryos, and its action is mainly through retinoic acid (RA) binding to its RA receptors and retinoid-X receptors, while the critical concentration and localization of RA in embryos are determined by the presence and activity of retinal dehydrogenases (for RA synthesis) and cytochrome P450 RAs (Cyp26s) (for degradation of RA). Previously, we identified a novel cyp26 gene (cyp26d1) in zebrafish that is expressed in hindbrain during early development. Using reverse-phase HPLC analyses, we show here that zebrafish Cyp26D1 expressed in 293T cells could metabolize all-trans RA, 9-cis RA, and 13-cis RA, but could not metabolize retinol or retinal. The metabolites of all-trans RA produced by Cyp26D1 were the same as that produced by Cyp26A1, which are mainly 4-hydroxy-all-trans-RA and 4-oxo-all-trans-RA. Performing mRNA microinjection into zebrafish embryos, we demonstrated that overexpression of Cyp26D1 in embryos not only caused the distance between rhombomere 5 and the first somite of the injected embryos to be shorter than control embryos but also resulted in left-right asymmetry of somitogenesis in the injected embryos. These alterations were similar to those caused by the overexpression of cyp26a1 in zebrafish embryos and to that which resulted from treating embryos with 1 microm 4-diethylamino-benzaldehyde (retinal dehydrogenase inhibitor), implying that cyp26d1 can antagonize RA activity in vivo. Together, our in vitro and in vivo results provided direct evidence that zebrafish Cyp26D1 is involved in RA metabolism.     

Differential expression of espin isoforms during epithelial morphogenesis, stereociliogenesis and postnatal maturation in the developing inner ear

The espins are a family of multifunctional actin cytoskeletal proteins. They are present in hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction. Here, we demonstrate that the different espin isoforms are expressed in complex spatiotemporal patterns during inner ear development. Espin 3 isoforms were prevalent in the epithelium of the otic pit, otocyst and membranous labyrinth as they underwent morphogenesis. This espin was down-regulated ahead of hair cell differentiation and during neuroblast delamination. Espin also accumulated in the epithelium of branchial clefts and pharyngeal pouches and during branching morphogenesis in other embryonic epithelial tissues, suggesting general roles for espins in epithelial morphogenesis. Espin reappeared later in inner ear development in differentiating hair cells. Its levels and compartmentalization to stereocilia increased during the formation and maturation of stereociliary bundles. Late in embryonic development, espin was also present in a tail-like process that emanated from the hair cell base. Increases in the levels of espin 1 and espin 4 isoforms correlated with stereocilium elongation and maturation in the vestibular system and cochlea, respectively. Our results suggest that the different espin isoforms play specific roles in actin cytoskeletal regulation during epithelial morphogenesis and hair cell differentiation.     

Distinct roles for Fgf,Wnt and retinoic acid in posteriorizing the neural ectoderm

Early neural patterning in vertebrates involves signals that inhibit anterior (A) and promote posterior (P) positional values within the nascent neural plate. In this study, we have investigated the contributions of, and interactions between, retinoic acid (RA), Fgf and Wnt signals in the promotion of posterior fates in the ectoderm. We analyze expression and function of cyp26/P450RAI, a gene that encodes retinoic acid 4-hydroxylase, as a tool for investigating these events. Cyp26 is first expressed in the presumptive anterior neural ectoderm and the blastoderm margin at the late blastula. When the posterior neural gene hoxb1b is expressed during gastrulation, it shows a strikingly complementary pattern to cyp26. Using these two genes, as well as otx2 and meis3 as anterior and posterior markers, we show that Fgf and Wnt signals suppress expression of anterior genes, including cyp26. Overexpression of cyp26 suppresses posterior genes, suggesting that the anterior expression of cyp26 is important for restricting the expression of posterior genes. Consistent with this, knock-down of cyp26 by morpholino oligonucleotides leads to the anterior expansion of posterior genes. We further show that Fgf- and Wnt-dependent activation of posterior genes is mediated by RA, whereas suppression of anterior genes does not depend on RA signaling. Fgf and Wnt signals suppress cyp26 expression, while Cyp26 suppresses the RA signal. Thus, cyp26 has an important role in linking the Fgf, Wnt and RA signals to regulate AP patterning of the neural ectoderm in the late blastula to gastrula embryo in zebrafish.     

Retinoic Acid Signaling Regulates Sonic Hedgehog and Bone Morphogenetic Protein Signalings During Genital Tubercle Development

Retinoic acid (RA) plays pivotal roles in organogenesis, and both excessive and reduced amounts of RA cause developmental abnormalities. Reproductive organs are susceptible to teratogen toxigenicity, and the genital tubercle (GT) is one such representative organ. The physiological function of endogenous RA signaling and the mechanisms of RA‐induced teratogenicity are poorly understood during the GT development. The objective of this study is to understand the developmental and teratogenic roles of RA during GT development by analyzing genetically modified mouse models. We found dynamic patterns of gene expression for the RA‐synthesizing enzyme, Raldh2, and for the RA‐catabolizing enzyme, Cyp26b1, during GT development. Rarb, an indicator gene for RA signaling, starts its expression in the prospective corpus cavernosum penis and in the urethral plate epithelium (UE), which plays central roles during GT development. Excessive RA signaling in Cyp26b1^?/? mutants leads to abnormal extents of cell proliferation and differentiation during GT development, and also upregulates expression of growth factor signalings. They include Sonic hedgehog (Shh) signaling and Bone morphogenetic protein (Bmp) signaling, which are expressed in the UE and its bilateral mesenchyme. RA signaling positively regulatesShh and Bmp4 expression during GT development as testified also by the experiment of RA administration and analyses of loss–of‐function of RA signaling mutants. Thus, RA signaling is involved in the developmental cascade necessary for UE formation and GT development.     

Restriction of retinoic acid activity by Cyp26b1 is required for proper timing and patterning of osteogenesis during zebrafish development

Skeletal syndromes are among the most common birth defects. Vertebrate skeletogenesis involves two major cell types: cartilage-forming chondrocytes and bone-forming osteoblasts. In vitro, both are under the control of retinoic acid (RA), but its exact in vivo effects remained elusive. Here, based on the positional cloning of the dolphin mutation, we have studied the role of the RA-oxidizing enzyme Cyp26b1 during cartilage and bone development in zebrafish. cyp26b1 is expressed in condensing chondrocytes as well as in osteoblasts and their precursors. cyp26b1 mutants and RA-treated wild-type fish display a reduction in midline cartilage and the hyperossification of facial and axial bones, leading to fusions of vertebral primordia, a defect not previously described in the context of RA signaling. Fusions of cervical vertebrae were also obtained by treating mouse fetuses with the specific Cyp26 inhibitor R115866. Together with data on the expression of osteoblast markers, our results indicate that temporal and spatial restriction of RA signaling by Cyp26 enzymes is required to attenuate osteoblast maturation and/or activity in vivo. cyp26b1 mutants may serve as a model to study the etiology of human vertebral disorders such as Klippel-Feil anomaly.     

Restriction of Wnt signaling in the dorsal otocyst determines semicircular canal formation in the mouse embryo

The mouse inner ear develops from a simple epithelial pouch, the otocyst, with the dorsal and ventral portions giving rise to the vestibule and cochlea, respectively. The otocyst undergoes a morphological change to generate flattened saclike structures, known as outpocketings, in the dorsal and lateral regions. The semicircular canals of the vestibule form from the periphery of the outpocketings, with the central region (the fusion plate) undergoing de-epithelialization and disappearing. However, little is known of the mechanism that orchestrates formation of the semicircular canals. We now show that the area of canonical Wnt signaling changes dynamically in the dorsal otocyst during its morphogenesis. The genes for several Wnt ligands were found to be expressed in the dorsal otocyst according to specific patterns, whereas those for secreted inhibitors of Wnt ligands were expressed exclusively in the ventral otocyst. With the use of whole-embryo culture in combination with potent modulators of canonical Wnt signaling, we found that forced persistence of such signaling resulted in impaired formation both of the lateral outpocketing and of the fusion plates of the dorsal outpocketing. Canonical Wnt signaling was found to suppress Netrin1 expression and to preserve the integrity of the outpocketing epithelium. In addition, inhibition of canonical Wnt signaling reduced the size of the otocyst, likely through suppression of cell proliferation and promotion of apoptosis. Our stage-specific functional analysis suggests that strict regulation of canonical Wnt signaling in the dorsal otocyst orchestrates the process of semicircular canal formation.     

Redundant functions of Rac GTPases in inner ear morphogenesis

Development of the mammalian inner ear requires coordination of cell proliferation, cell fate determination and morphogenetic movements. While significant progress has been made in identifying developmental signals required for inner ear formation, less is known about how distinct signals are coordinated by their downstream mediators. Members of the Rac family of small GTPases are known regulators of cytoskeletal remodeling and numerous other cellular processes. However, the function of Rac GTPases in otic development is largely unexplored. Here, we show that Rac1 and Rac3 redundantly regulate many aspects of inner ear morphogenesis. While no morphological defects were observed in Rac3(-/-) mice, Rac1(CKO); Rac3(-/-) double mutants displayed enhanced vestibular and cochlear malformations compared to Rac1(CKO) single mutants. Moreover, in Rac1(CKO); Rac3(-/-) mutants, we observed compromised E-cadherin-mediated cell adhesion, reduced cell proliferation and increased cell death in the early developing otocyst, leading to a decreased size and malformation of the membranous labyrinth. Finally, cochlear extension was severely disrupted in Rac1(CKO); Rac3(-/-) mutants, accompanied by a loss of epithelial cohesion and formation of ectopic sensory patches underneath the cochlear duct. The compartmentalized expression of otic patterning genes within the Rac1(CKO); Rac3(-/-) mutant otocyst was largely normal, however, indicating that Rac proteins regulate inner ear morphogenesis without affecting cell fate specification. Taken together, our results reveal an essential role for Rac GTPases in coordinating cell adhesion, cell proliferation, cell death and cell movements during otic development.     

Rescue of cytochrome P450 oxidoreductase (Por) mouse mutants reveals functions in vasculogenesis, brain and limb patterning linked to retinoic acid homeostasis

Cytochrome P450 oxidoreductase (POR) acts as an electron donor for all cytochrome P450 enzymes. Knockout mouse Por(-/-) mutants, which are early embryonic (E9.5) lethal, have been found to have overall elevated retinoic acid (RA) levels, leading to the idea that POR early developmental function is mainly linked to the activity of the CYP26 RA-metabolizing enzymes (Otto et al., Mol. Cell. Biol. 23, 6103-6116). By crossing Por mutants with a RA-reporter lacZ transgene, we show that Por(-/-) embryos exhibit both elevated and ectopic RA signaling activity e.g. in cephalic and caudal tissues. Two strategies were used to functionally demonstrate that decreasing retinoid levels can reverse Por(-/-) phenotypic defects, (i) by culturing Por(-/-) embryos in defined serum-free medium, and (ii) by generating compound mutants defective in RA synthesis due to haploinsufficiency of the retinaldehyde dehydrogenase 2 (Raldh2) gene. Both approaches clearly improved the Por(-/-) early phenotype, the latter allowing mutants to be recovered up until E13.5. Abnormal brain patterning, with posteriorization of hindbrain cell fates and defective mid- and forebrain development and vascular defects were rescued in E9.5 Por(-/-) embryos. E13.5 Por(-/-); Raldh2(+/-) embryos exhibited abdominal/caudal and limb defects that strikingly phenocopy those of Cyp26a1(-/-) and Cyp26b1(-/-) mutants, respectively. Por(-/-); Raldh2(+/-) limb buds were truncated and proximalized and the anterior-posterior patterning system was not established. Thus, POR function is indispensable for the proper regulation of RA levels and tissue distribution not only during early embryonic development but also in later morphogenesis and molecular patterning of the brain, abdominal/caudal region and limbs.     

Regulation of CYP26 (cytochrome P450RAI) mRNA expression and retinoic acid metabolism by retinoids and dietary vitamin A in liver of mice and rats.

Retinoic acid (RA), through nuclear retinoid receptors, regulates the expression of numerous genes. However, little is known of the biochemical mechanisms that regulate RA concentration in vivo. CYP26 (P450RAI), a novel cytochrome P450, is expressed during embryonic development, induced by all-trans RA, and capable of catalyzing the oxidation of [3H]RA to polar retinoids including 4-oxo-RA. Here we report that CYP26 expression in adult liver is regulated by all-trans RA and dietary vitamin A, and is correlated with the metabolism of all-trans RA to polar metabolites. In normal mouse and rat liver, CYP26 mRNA was barely detectable; however, after acute treatment with all-trans RA CYP26 mRNA and RA metabolism by liver microsomes were significantly induced. Aqueous-soluble RA metabolites were detected, but their formation was not induced. The expression of retinoid receptors, RAR-gamma and RXR-alpha, was not changed after RA treatment in vivo. In a model of chronic vitamin A ingestion during aging, CYP26 mRNA expression, determined by Northern blot and RT-PCR analysis, increased progressively with dietary vitamin A (P<0.0001; marginal < control < supplemented) and age (P<0.003). The relative expression of CYP26 mRNA was positively correlated with liver total retinol (log10), ranging from undetectable CYP26 expression at liver retinol concentrations below approximately 20 nmol/g to a three- to fourfold elevation at concentrations >10,000 nmol/g (r=0.90, P<0.0001). We conclude that CYP26 expression and RA metabolism are regulated in adult liver not only acutely by RA administration, as may be relevant to retinoid therapy, but under chronic dietary conditions relevant to vitamin A nutrition in humans.     

Retinoids, eye development, and maturation of visual function

Vitamin A is known to be critical for the beginning of eye development as well as for photoreception in the functional retina. Hardly anything, however, is known about whether retinoic acid (RA)-regulated gene expression also plays a role in the long intervening period, during which the neurobiological retinal structure takes shape. The eye contains a highly intricate architecture of RA-synthesizing (RALDH) and degrading (CYP26) enzymes. Whereas the RALDHs are integrated in the early molecular mechanisms through which the dorso-ventral retina organization is established, the CYP26 enzymes are not necessary for this process and no molecular targets that match their retinal expression pattern have yet been identified. In this article we describe that CYP26 expression in the mouse is most distinctive during later stages of retina formation. Throughout development CYP26A1 degrades RA in a horizontal region that extends across the retina, but during later embryonic and postnatal retina maturation this function is reinforced by another enzyme, CYP26C1. RA applications at this stage do not affect the RALDHs but cause differential changes in CYP26 expression: Cyp26a1 is up-regulated, but more rapidly by 9-cis than all-trans RA, Cyp26c1 is down-regulated, and Cyp26b1, which is undetectable in the normal mouse retina, is strongly activated in retinal ganglion cells. The dynamic regulation in RA-difference patterns by the CYP26 enzymes may set up spatial constellations for expression of genes involved in formation of retinal specializations for higher acuity vision, which are known to form over a prolonged period late in retina development.     

Retinoid signaling can repress blastula Wnt signaling and impair dorsal development in Xenopus embryo

Vitamin A derivatives (retinoids) are actively involved during vertebrate embryogenesis. However, exogenous retinoids have also long been known as potent teratogens. The defects caused by retinoid treatment are complex. Here, we provided evidence that RAR-mediated retinoid signaling can repress Xenopus blastula Wnt signaling and impair dorsal development. Exogenous retinoic acid (RA) could antagonize the dorsalizing effects of lithium chloride-mediated Wnt activation in blastula embryos. The Wnt-responsive reporter gene transgenesis and luciferase assay showed that excess RA can repress the Wnt signaling in blastula embryos. In addition, the downstream target genes of the Wnt signaling that direct embryonic dorsal development, were also down-regulated in the RA-treated embryos. Mechanically, RA did not interfere with the stability of beta-catenin, but promoted its nuclear accumulation. The inverse agonist of retinoic acid receptors (RAR) rescued the Wnt signaling repression by RA and relieved the RA-induced nuclear accumulation of beta-catenin. Our results explain one of the reasons for the complicated teratogenic effects of retinoids and shed light on the endogenous way of interactions between two developmentally important signaling pathways.     

Ectopic noggin blocks sensory and nonsensory organ morphogenesis in the chicken inner ear

Bone morphogenetic protein 4 (Bmp4) is expressed during multiple stages of development of the chicken inner ear. At the otocyst stage, Bmp4 is expressed in each presumptive sensory organ, as well as in the mesenchymal cells surrounding the region of the otocyst that is destined to form the semicircular canals. After the formation of the gross anatomy of the inner ear, Bmp4 expression persists in some sensory organs and restricted domains of the semicircular canals. To address the role of this gene in inner ear development, we blocked BMP4 function(s) by delivering one of its antagonists, Noggin, to the developing inner ear in ovo. Exogenous Noggin was delivered to the developing otocyst by using a replication-competent avian retrovirus encoding the Noggin cDNA (RCAS-N) or implanting beads coated with Noggin protein. Noggin treatment resulted in a variety of phenotypes involving both sensory and nonsensory components of the inner ear. Among the nonsensory structures, the semicircular canals were the most sensitive and the endolymphatic duct and sac most resistant to exogenous Noggin. Noggin affected the proliferation of the primordial canal outpouch, as well as the continual outgrowth of the canal after its formation. In addition, Noggin affected the structural patterning of the cristae, possibly via a decrease of Msx1 and p75NGFR expression. These results suggest that BMP4 and possibly other BMPs are required for multiple phases of inner ear development.