Ethanol induces embryonic malformations by competing for retinaldehyde dehydrogenase activity during vertebrate gastrulation

Human embryos exposed to alcohol (ethanol) develop a complex developmental phenotype known as fetal alcohol spectrum disorder (FASD). In Xenopus embryos, ethanol reduces the levels of retinoic acid (RA) signaling during gastrulation. RA, a metabolite of vitamin A (retinol), is required for vertebrate embryogenesis, and deviation from its normal levels results in developmental malformations. Retinaldehyde dehydrogenase 2 (RALDH2) is required to activate RA signaling at the onset of gastrulation. We studied the effect of alcohol on embryogenesis by manipulating retinaldehyde dehydrogenase activity in ethanol-treated embryos. In alcohol-treated embryos, we analyzed RA signaling levels, phenotypes induced and changes in gene expression. Developmental defects that were characteristic of high ethanol concentrations were phenocopied by a low ethanol concentration combined with partial RALDH inhibition, whereas Raldh2 overexpression rescued the developmental malformations induced by high ethanol. RALDH2 knockdown resulted in similar RA signaling levels when carried out alone or in combination with ethanol treatment, suggesting that RALDH2 is the main target of ethanol. The biochemical evidence that we present shows that, at the onset of RA signaling during early gastrulation, the ethanol effect centers on the competition for the available retinaldehyde dehydrogenase activity. In light of the multiple regulatory roles of RA, continued embryogenesis in the presence of abnormally low RA levels provides an etiological explanation for the malformations observed in individuals with FASD.

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title: Ethanol exposure affects gene expression in the embryonic organizer and reduces retinoic acid levels.
abstract: Fetal Alcohol Spectrum Disorder (FASD) is a set of developmental malformations caused by alcohol consumption during pregnancy. Fetal Alcohol Syndrome (FAS), the strongest manifestation of FASD, results in short stature, microcephally and facial dysmorphogenesis including microphthalmia. Using Xenopus embryos as a model developmental system, we show that ethanol exposure recapitulates many aspects of FAS, including a shortened rostro-caudal axis, microcephally and microphthalmia. Temporal analysis revealed that Xenopus embryos are most sensitive to ethanol exposure between late blastula and early/mid gastrula stages. This window of sensitivity overlaps with the formation and early function of the embryonic organizer, Spemann's organizer. Molecular analysis revealed that ethanol exposure of embryos induces changes in the domains and levels of organizer-specific gene expression, identifying Spemann's organizer as an early target of ethanol. Ethanol also induces a defect in convergent extension movements that delays gastrulation movements and may affect the overall length. We show that mechanistically, ethanol is antagonistic to retinol (Vitamin A) and retinal conversion to retinoic acid, and that the organizer is active in retinoic acid signaling during early gastrulation. The model suggests that FASD is induced in part by an ethanol-dependent reduction in retinoic acid levels that are necessary for the normal function of Spemann's organizer.

title: Retinoic acid metabolism and mechanism of action: a review.
abstract: Retinoids are vitamin A (retinol) derivatives essential for normal embryo development and epithelial differentiation. These compounds are also involved in chemoprevention and differentiation therapy of some cancers, with particularly impressive results in the management of acute promyelocytic leukemia (APL). Although highly effective in APL therapy, resistance to retinoic acid (RA) develops rapidly. The causes of this resistance are not completely understood and the following factors have been involved: increased metabolism, increased expression of RA binding proteins, P-glycoprotein expression, and mutations in the ligand binding domain of RARalpha. RA exerts its molecular actions mainly through RAR and RXR nuclear receptors. In addition to the nuclear receptor based mechanism of RA action, covalent binding of RA to cell macromolecules has been reported. RA derives from retinol by oxidation through retinol and retinal dehydrogenases, and several cytochrome p450s (CYPs). RA is thereafter oxidized to several metabolites by a panel of CYPs that differs for the different RA isomers. Phase II metabolism, mainly glucuronidation, is also observed. The role RA metabolism plays in the expression of its biological actions is not completely understood: in several systems, metabolism decreases RA activity, whereas in other systems metabolism appears involved in its action. In addition, several RA metabolites have shown activity and cannot be classified as only catabolites. Therapy of cancer with retinoids is still in its infancy, but the use of new analogues with improved pharmacological properties, along with combination with other drugs, could undoubtedly improve the management of several cancers in the future.

title: Expression of a retinoic acid response element-hsplacZ transgene defines specific domains of transcriptional activity during mouse embryogenesis.
abstract: Treatment with retinoic acid (RA) is known to produce complex teratogenic effects in vertebrates, and its presence in the developing embryo as an endogenous substance has led to the suggestion that RA might be a natural morphogenetic agent. Although our understanding of the molecular mechanism of RA action has improved considerably with the identification of nuclear receptors for RA (RARs) and RA-responsive genes, the exact relationship between the proposed morphogenetic activity of RA and its teratogenic effects remains to be characterized. Here, we show that a RA response element (RARE) present in the RAR beta gene can direct specific spatial and temporal expression of an hsplacZ transgene during mouse embryogenesis. In the early embryo, the transgene is expressed in a specific anterior-posterior domain that is completely obliterated by treatment of pregnant mice with teratogenic doses of RA. The expression of the transgene becomes more restricted as organogenesis progresses and mimics closely the reported expression of the RAR beta gene. These results suggest that, in vivo, some of the morphogenetic effects of RA could be mediated through localized transcriptional activity controlled by the various RARs. The specific pattern of expression of the RAREhsplacZ transgene does not correlate with the proposed sites of action of RA as defined by its teratogenic effects but does support a role for RA in early anterior-posterior patterning along the body axis.

title: Involvement of alcohol dehydrogenase, short-chain dehydrogenase/reductase, aldehyde dehydrogenase, and cytochrome P450 in the control of retinoid signaling by activation of retinoic acid synthesis.
abstract: The effects of vitamin A (retinol) on growth and development are mediated by the active metabolite retinoic acid which controls a nuclear receptor signaling pathway. While elegant work on the retinoic acid receptor family has focused attention upon how the receptor controls this pathway, there now exists a relatively large gap in our understanding of how retinol is activated to form the ligand. During vertebrate embryogenesis and in adult organs retinoic acid is detected in a distinct spatiotemporal pattern, suggesting that it is produced from retinol in a regulated fashion. Enzymes involved in retinol and retinal metabolism are likely candidates for regulators of tissue retinoic acid levels. Members of the alcohol dehydrogenase and short-chain dehydrogenase/reductase enzyme families catalyze the reversible interconversion of retinol and retinal, the rate-limiting step, whereas members of the aldehyde dehydrogenase and cytochrome P450 enzyme families catalyze the irreversible oxidation of retinal to retinoic acid. The identification of enzymes likely to catalyze retinol oxidation in vivo has been particularly controversial, and this is made even more difficult by the reversible nature of this reaction. Taking into account enzymatic properties and coenzyme preferences, a case can be made that class IV alcohol dehydrogenase catalyzes retinol oxidation to provide retinal for retinoic acid synthesis, whereas microsomal retinol dehydrogenase (a short-chain dehydrogenase/reductase) catalyzes the reduction of retinal to retinol to promote retinoid storage. Further studies on these enzyme families will allow this layer of control in the retinoid signaling pathway to be understood.

title: Respecification of vertebral identities by retinoic acid.
abstract: In higher vertebrates, the formation of the body axis proceeds in a craniocaudal direction during gastrulation. Cell biological evidence suggests that mesoderm formation and specification of axial positions occur simultaneously. Exposure of gastrulating embryos to retinoic acid induces changes in axial patterns, e.g. anterior and posterior homeotic transformations of vertebrae. These morphological changes are accompanied by changes in the nonidentical, overlapping expression domains of Hox genes. In this report the influence of retinoic acid, administered at the end of and after gastrulation, on vertebral patterns is described. Anterior transformations and truncations affecting the caudal part of the vertebral column characterize animals exposed on day 8 and 9. 4 hours after retinoic acid administration on day 8 + 5 hours, Hox-1.8, Hox-1.9, and Hox-4.5 transcripts were not detected in their usual posterior expression domains, whereas transcripts of the anterior Hox-1.5 gene remained unaffected. 4 days after RA exposure on day 8 + 5 hours, Hox-1.8 expression was shifted posteriorly by an effectively low dose of RA, which induced the formation of supernumerary ribs. Hox-1.8 expression was limited to posterior, disorganized mesenchyme, bulging out neural tube, some intestinal loops and the hindlimb in truncated embryos exposed to a high dose of RA. A causal relation between the delayed activation of posterior Hox genes and anterior transformations or agenesis of vertebrae is discussed. On day 10.5 posterior transformations begin to occur in the cervical region, while later exposures again affect more caudal structures. The distribution of the transformations along the vertebral column indicates an influence of RA on migrating sclerotome cells before they are finally fixed in the cartilagenous vertebrae. The findings show that the mesodermal segments originally specified during gastrulation can be respecified in their second migratory phase, with effects spreading for a second time in a craniocaudal direction. The transformations are discussed with regard to a molecular specification of axial levels by Hox codes, defined as combinations of expressed Hox genes.

title: Vitamin A-deficient quail embryos have half a hindbrain and other neural defects
abstract: Retinoic acid (RA) is a morphogenetically active signalling molecule thought to be involved in the development of severely embryonic systems (based on its effect when applied in excess and the fact that it can be detected endogenously in embryos). Here, we adopt a novel approach and use the vitamin A-deficient (A-) quail embryo to ask what defects these embryos show when they develop in the absence of RA, with particular reference to the nervous system.



We have examined the anatomy, the expression domains of a variety of genes and the immunoreactivity to several antibodies in these A- embryos. In addition to the previously documented cardiovascular abnormalities, we find that the somites are smaller in A- embryos, otic vesicle development is abnormal and the somites continue up to and underneath the otic vesicle. In the central nervous system, we find that neural crest cells need RA for normal development and survival, and the neural tube fails to extend any neurites into the periphery. Using general hindbrain morphology and the expression patterns of Hoxa-2, Hoxb-1, Hoxb-4, Krox-20 and FGF-3 as markers, we conclude that segmentation in the myelencephalon (rhombomeres 4-8) is disrupted. In contrast, the dorsoventral axis of the neural tube using Shh, islet-1 and Pax-3 as markers is normal.



These results demonstrate at least three roles for RA in central nervous system development: neural crest survival, neurite outgrowth and hindbrain patterning.

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

title: 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.
abstract: 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.

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

title: Increased XRALDH2 activity has a posteriorizing effect on the central nervous system of Xenopus embryos
abstract: Retinoic acid (RA) metabolizing enzymes play important roles in RA signaling during vertebrate embryogenesis. We have previously reported on a RA degrading enzyme, XCYP26, which appears to be critical for the anteroposterior patterning of the central nervous system (EMBO J. 17 (1998) 7361). Here, we report on the sequence, expression and function of its counterpart, XRALDH2, a RA generating enzyme in Xenopus. During gastrulation and neurulation, XRALDH2 and XCYP26 show non-overlapping, complementary expression domains. Upon misexpression, XRALDH2 is found to reduce the forebrain territory and to posteriorize the molecular identity of midbrain and individual hindbrain rhombomeres in Xenopus embryos. Furthermore, ectopic XRALDH2, in combination with its substrate, all-trans-retinal (ATR), can mimic the RA phenotype to result in microcephalic embryos. Taken together, our data support the notion that XRALDH2 plays an important role in RA homeostasis by the creation of a critical RA concentration gradient along the anteroposterior axis of early embryos, which is essential for proper patterning of the central nervous system in Xenopus.

title: Retinoic acid is enriched in Hensen's node and is developmentally regulated in the early chicken embryo.
abstract: Retinoic acid (RA) has been considered as a potential morphogen in the chicken limb and has also been suggested to be involved in early embryonic development. On the basis of biological activity, previous reports suggest that Hensen's node, the anatomical equivalent in the chicken of the Spemann's organizer, may contain RA. Here, by using a molecular assay system, we demonstrate that Hensen's node contains retinoids in a concentration approximately 20 times more than that in the neighboring tissues. Furthermore, stage 6 Hensen's node contains approximately 3 times more retinoid than that of stage 4 embryos. These endogenous retinoids may establish a concentration gradient from Hensen's node to adjacent tissues and play a role in establishing the primary embryonic axis in the vertebrate. The results also suggest that the retinoid concentration in Hensen's node is developmentally regulated.

title: A concentration gradient of retinoids in the early Xenopus laevis embryo.
abstract: Previous studies have postulated that Xenopus embryos contain an endogenous retinoic acid (RA) concentration gradient from posterior to anterior during the process of primary axis formation, since RA is able to alter profoundly primary axis formation in Xenopus embryos, to increase the expression of some posterior markers, and to inhibit the expression of some anterior markers, including homeobox-containing genes. Here, we provide direct evidence for this hypothesis. By using a reporter cell system, we demonstrate that the endogenous biologically active retinoid concentration in whole Xenopus embryos increases 3-fold from the two-cell stage to the neurula stage, and that the active retinoid concentration in the dorsal marginal zone, a region wherein the Spemann's organizer is located, increases about 5-fold from the early gastrula to late gastrula stages, suggesting the developmental regulation of the retinoid levels. In the early neurula stage (stage 13-14), endogenous active retinoids are present in a concentration gradient with the highest level at the posterior end, about 10-fold higher than that at the anterior end, of the embryo. This concentration gradient may be established during gastrulation and may provide positional cues for primary axis formation.

title: Temporal distribution, localization and metabolism of all-trans-retinol, didehydroretinol and all-trans-retinal during Xenopus development.
abstract: Recently, the temporal and spatial distribution patterns of the retinoid receptor ligands 9-cis-retinoic acid and all-trans-retinoic acid were described in Xenopus embryos during early development [Creech Kraft, Schuh, Juchau and Kimelman (1994) ]. The present study demonstrates the presence and distribution of their likely precursors, all-trans-retinol, didehydroretinol, didehydroretinal and all-trans-retinal, as well as the occurrence of 4-oxo metabolites, in Xenopus embryos. The temporal and spatial distribution patterns of all-trans-retinol, didehydroretinol and all-trans-retinal did not coincide with that observed for 9-cis-retinoic acid but, in certain regards, were similar to the patterns delineated for all-trans-retinoic acid and all-trans-retinoyl beta-glucuronide. Evidence is presented that 9-cis-retinoic acid can be synthesized from both all-trans-retinoic acid and all-trans-retinol in Xenopus embryos, suggesting that the difference between the distributions of 9-cis-retinoic acid and the other retinoids may be caused by selective synthesis and/or protein binding of the 9-cis isomer.

title: Alcohol dehydrogenases in Xenopus development: Conserved expression of ADH1 and ADH4 in epithelial retinoid target tissues
abstract: Mammalian alcohol dehydrogenases ADH1 (class I ADH) and ADH4 (class IV ADH) function as retinol dehydrogenases contributing to the synthesis of retinoic acid, the active form of vitamin A involved in growth and development. Xenopus laevis ADH1 and ADH4 genes were isolated using polymerase chain reaction primers corresponding to conserved motifs of vertebrate ADHs. The predicted amino acid sequence of Xenopus ADH1 was clearly found to be an ortholog of ADH1 from the related amphibian Rana perezi. Phylogenetic tree analysis of the Xenopus ADH4 sequence suggested this enzyme is likely to be an ADH4 ortholog, and this classification was more confidently made when based also on the unique expression patterns of Xenopus ADH1 and ADH4 in several retinoid‐responsive epithelial tissues. Northern blot analysis of Xenopus adult tissues indicated nonoverlapping patterns of ADH expression, with ADH1 mRNA found in small intestine, large intestine, liver, and mesonephros and ADH4 mRNA found in esophagus, stomach, and skin. These nonoverlapping tissue‐specific patterns are identical to those previously observed for mouse ADH1 and ADH4, thus providing further evidence that Xenopus ADH1 and ADH4 are orthologs of mouse ADH1 and ADH4, respectively. During Xenopus embryonic development ADH1 mRNA was first detectable by Northern blot analysis at stage 35, whereas ADH4 mRNA was undetectable through stage 47. Whole‐mount in situ hybridization indicated that ADH1 expression was first localized in the pronephros during Xenopus embryogenesis, thus conserved with mouse embryonic ADH1 which is first expressed in the mesonephros. ADH4 expression was not detected in Xenopus embryos by whole‐mount in situ hybridization but was localized to the gastric mucosa of the adult stomach, a property shared by mouse ADH4. Conserved expression of ADH1 and ADH4 in retinoid‐responsive epithelial tissues of amphibians and mammals argue that these enzymes may perform essential retinoid signaling functions during development of the pronephros, mesonephros, liver, and lower digestive tract in the case of ADH1 and in the skin and upper digestive tract in the case of ADH4.

title: Retinoic acid biosynthetic enzyme ALDH1 localizes in a subset of retinoid‐dependent tissues during Xenopus development
abstract: Control of retinoic acid synthesis in vertebrate organisms is undoubtedly important for regulating the numerous retinoid signaling events which occur during development. The mechanisms which accomplish this task involve enzymes such as class I aldehyde dehydrogenase (ALDH1), which has recently been found to be conserved from amphibians to mammals and which functions as a retinoic acid biosynthetic enzyme in vivo. Here we have found that Xenopus ALDH1 mRNA and protein is expressed in a subset of retinoid‐dependent tissues which develop shortly after neurulation during the tail bud stages. ALDH1 mRNA was first clearly detectable by in situ hybridization in stage 28 tail bud embryos localized in the olfactory placode and pronephros, and at stage 35 mRNA was also detected in the pronephric duct. Antibodies were generated against Xenopus ALDH1, and immunohistochemistry was used to demonstrate that ALDH1 protein accumulates in the olfactory placode, pronephros, and dorsal retina at stage 28, and additionally in the lens placode and pronephric duct at stage 35. Neither ALDH1 mRNA nor protein was detected in the posterior region of Xenopus embryos during the tail bud stages. In contrast to neurula stage embryos in which retinoic acid is distributed in an anteroposterior gradient with the high end posteriorly, we found that tail bud stage embryos have retinoic acid present in significant levels in both the head and trunk regions, but with no detection in the posterior region. These findings are consistent with ALDH1 contributing to retinoic acid synthesis needed for development of certain head structures (olfactory placodes, dorsal retina, lens placode) and certain trunk structures (pronephros and pronephric duct).

title: 4-Methylpyrazole partially ameliorated the teratogenicity of retinol and reduced the metabolic formation of all-trans-retinoic acid in the mouse
abstract: Oral administration of retinol (50 mg/kg) to NMRI mice on day 11 of gestation (vaginal plug = day 0) led to the metabolic formation of high quantities of all-trans retinoic acid and all-trans-4-oxoretinoic acid, both known as potent teratogenic agents in the mouse. A 96% reduction of the area under the concentration-versus-timecurve (AUC) of metabolically generated alltrans retinoic acid in maternal plasma, and an 84% decrease in the embryonic AUC were observed when mice had been pretreated with the alcohol dehydrogenase inhibitor 4-methylpyrazole. A similar reduction was observed for the major metabolite of all-trans retinoic acid in the mouse, all-trans-4-oxoretinoic acid. However, 4-methylpyrazole pretreatment decreased the AUC of retinol by 10% in maternal plasma and 15% in embryo. Treatment with retinol alone resulted in 55.6%, 43.9% and 56.0% skeletal anomalies of the forelimbs, hindlimbs and craniofacial structures, respectively. Pretreatment with 4-methylpyrazole lowered the retinol induced skeletal defects to 31.3%, 24.0% and 31.3%, respectively, in the forelimb, hindlimb and craniofacial region. Typical retinoid-induced malformations for gestational day 11, e.g. bent or reduced zeugopod or stylopod elements, or cleft palate, were significantly reduced by 4-methylpyrazole pretreatment but were still detected in significantly higher prevalence than in control mice. These data suggest that the teratogenic activity of a single high dose of vitamin A in mouse is partially but not exclusively dependent on the metabolic activation of retinol to all -trans retinoic acid. Thus it could be hypothesized that retinol is either a proximate teratogen or a coteratogen with all -trans retinoic acid.

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

title: Early molecular effects of ethanol during vertebrate embryogenesis.
abstract: Fetal alcohol spectrum disorder (FASD) is the combination of developmental, morphological, and neurological defects that result from exposing human embryos to ethanol (EtOH). Numerous embryonic structures are affected, leading to a complex viable phenotype affecting among others, the anterior/posterior axis, head, and eye formation. Recent studies have provided evidence suggesting that EtOH teratogenesis is mediated in part through a reduction in retinoic acid (RA) levels, targeting mainly the embryonic organizer (Spemann's organizer) and its subsequent functions. EtOH-treated Xenopus embryos were subjected to an analysis of gene expression patterns. Analysis of organizer-specific genes revealed a transient delay in the invagination of gsc- and chordin-positive cells that eventually reach their normal rostro-caudal position. Dorsal midline genes show defects along the rostro-caudal axis, lacking either their rostral (Xbra and Xnot2) or caudal (FoxA4b and Shh) expression domains. Head-specific markers like Otx2, en2, and Shh show abnormal expression patterns. Otx2 exhibits a reduction in expression levels, while en2 becomes restricted along the dorsal/ventral axis. During neurula stages, Shh becomes up-regulated in the rostral region and it is expressed in an abnormal pattern. These results and histological analysis suggest the existence of malformations in the brain region including a lack of the normal fore brain ventricle. An increase in the size of both the prechordal plate and the notochord was observed, while the spinal cord is narrower. The reduction in head and eye size was accompanied by changes in the eye markers, Pax6 and Tbx3. Our results provide evidence for the early molecular changes induced by EtOH exposure during embryogenesis, and may explain some of the structural changes that are part of the EtOH teratogenic phenotype also in FASD individuals.

title: Stimulation of premature retinoic acid synthesis in Xenopus embryos following premature expression of aldehyde dehydrogenase ALDH1.
abstract: In order for nuclear retinoic acid receptors to mediate retinoid signaling, the ligand retinoic acid must first be produced from its vitamin A precursor retinal. Biochemical studies have shown that retinal can be metabolized in vitro to retinoic acid by members of the aldehyde dehydrogenase enzyme family, including ALDH1. Here we describe the first direct evidence that ALDH1 plays a physiological role in retinoic acid synthesis by analysis of retinoid signaling in Xenopus embryos, which have plentiful stores of maternally derived retinal. The Xenopus ALDH1 gene was cloned and shown to be highly conserved with chick and mammalian homologs. Xenopus ALDH1 was not expressed at blastula and gastrula stages, but was expressed at the neurula stage. We used a retinoic acid bioassay to demonstrate that retinoic acid is normally undetectable in embryos from fertilization to the initial gastrula stage, but that a tremendous increase in retinoic acid occurs during neurulation when ALDH1 is first expressed. Overexpression of ALDH1 by injection of Xenopus embryos with mRNAs encoding the mouse, chick or Xenopus ALDH1 homologs induced high levels of retinoic acid detection during the blastula stage. Thus, premature expression of ALDH1 stimulates premature synthesis of retinoic acid. These findings reveal an important conserved role for ALDH1 in retinoic acid synthesis in vivo, and demonstrate that conversion of retinoids from the aldehyde form to the carboxylic acid form is a crucial regulatory step in retinoid signaling.

title: Regionalized metabolic activity establishes boundaries of retinoic acid signalling
abstract: The competence of a cell to respond to the signalling molecule retinoic acid (RA) is thought to depend largely on its repertoire of cognate zinc finger nuclear receptors. XCYP26 is an RA hydroxylase that is expressed differentially during early Xenopus development. In Xenopus embryos, XCYP26 can rescue developmental defects induced by application of exogenous RA, suggesting that the enzymatic modifications introduced inhibit RA signalling activities in vivo. Alterations in the expression pattern of a number of different molecular markers for neural development induced upon ectopic expression of XCYP26 reflect a primary function of RA signalling in hindbrain development. Progressive inactivation of RA signalling results in a stepwise anteriorization of the molecular identity of individual rhombomeres. The expression pattern of XCYP26 during gastrulation appears to define areas within the prospective neural plate that develop in response to different concentrations of RA. Taken together, these observations appear to reflect an important regulatory function of XCYP26 for RA signalling; XCYP26‐mediated modification of RA modulates its signalling activity and helps to establish boundaries of differentially responsive and non‐responsive territories.

title: Feedback mechanisms regulate retinoic acid production and degradation in the zebrafish embryo
abstract: Retinoic acid (RA) signaling in vertebrate embryos occurs in a distinct physical and temporal pattern. Regulating this spatial distribution is crucial to the development of the embryo, as RA in excess or in inappropriate tissues is teratogenic. In order to understand how RA availability is determined in zebrafish we have investigated the expression of cyp26a1, an enzyme that inactivates RA, and its relationship to raldh2, one of the enzymes that produce RA from retinal. cyp26a1 expression follows three phases: in presumptive anterior neurectoderm and in a circumblastoporal ring during gastrulation, in the tailbud throughout somitogenesis, and in multiple specific tissue types beginning at mid-somitogenesis and continuing through 48 h postfertilization (hpf). This expression was either adjacent or opposite to those tissues expressing raldh2. We then investigated how RA production might regulate these relationships. Endogenous RA produced by raldhs did not play a role in setting cyp26a1 expression in most tissues. However, exogenous RA regulates expression of both enzymes. cyp26a1 is up regulated in the embryo in a time, concentration, and tissue-dependent manner. Conversely, raldh2 expression is reduced with RA treatment. Tests of the raldh2 promoter in cell transfections proved that RA directly represses its activity. These data demonstrate that the feedback mechanisms regulating production and degradation of RA must be considered in any experiments altering levels of RA in the developing vertebrate embryo.

title: Morpholino oligos: making sense of antisense?
abstract: Since morpholino oligos were first introduced as a means to inhibit gene function in embryos, in the Spring of 2000, they have been tested in a range of model organisms, including sea urchin, ascidian, zebrafish, frog, chick, and mouse. This review surveys the results of these studies and examines the successes and limitations of the approach for targeting maternal and zygotic gene function. The evidence so far suggests that, with careful controls, morpholinos provide a relatively simple and rapid method to study gene function.

title: Function of vitamin A in vertebrate embryonic development.
abstract: Advances in molecular biology and retinoic acid receptor research have significantly contributed to the understanding of the role of vitamin A during vertebrate development. Examination of the function of this vitamin during very early developmental stages using the completely vitamin A-depleted avian embryo has revealed that the vitamin A requirement begins at the time of formation of the primitive heart, circulation and specification of hindbrain. The lack of vitamin A at this critical time results in gross abnormalities and early embryonic death. In rodent models, vitamin A deficiency can be targeted to later gestational windows and documents the need for vitamin A for more advanced stages of development. Major target tissues of vitamin A deficiency include the heart, central nervous system and structures derived from it, the circulatory, urogenital and respiratory systems, and the development of skull, skeleton and limbs. These abnormalities are also evident in mice mutants from retinoid receptor knockouts; they have revealed both morphological and molecular aspects of vitamin A function during development. Retinoic acid receptors (RAR) in partnership with retinoid X receptor (RXR)alpha appear to be the important retinoid receptor transcription factors regulating vitamin A function at the gene level during development via the physiologic ligand all-trans-retinoic acid. Homeostasis of retinoic acid is maintained by developmentally regulated vitamin A metabolism enzyme systems. Inadequate vitamin A nutrition during early pregnancy may account for some pediatric congenital abnormalities.

title: Retinoid receptors and vitamin A deficiency: differential patterns of transcription during early avian development and the rapid induction of RARs by retinoic acid.
abstract: The functional links of specific retinoid receptors to early developmental events in the avian embryo are not known. Before such studies are undertaken, knowledge is required of the spatiotemporal expression patterns of the receptor genes and their regulation by endogenous retinoic acid levels during the early stages of development. Here, we report the expression patterns of mRNAs for RARalpha, RARalpha2, RARbeta2, RARgamma, RARgamma2, RXRalpha, and RARgamma from neurulation to HH10 in the normal and vitamin A-deficient (VAD) quail embryo. The transcripts for all retinoid receptors are detectable at HH5, except for RXRgamma, which is detected at the beginning of HH6. At the 4/5 somite stage of HH8, when retinoid signaling is initiated in the avian embryo, mRNAs of all receptors are present, with very strong and ubiquitous expression patterns for RARalpha, RARalpha2, RARgamma, RARgamma2, and RXRalpha, a persistent expression of RARgamma in the neural tissues, a strong expression of RARbeta2 in lateral plate mesoderm and somites, and an anterior expression of RXRgamma. All retinoid receptors are expressed in the heart primordia. In the VAD quail embryo, the general pattern of retinoid receptor transcript localization is similar to that of the normal, except that the expression of RARalpha2 and RARbeta2 is severely diminished. Administration of retinol or retinoic acid to VAD embryos at or before the 4/5 somite stage rescues the expression of RARalpha2 and RARbeta2 within approximately 45 min and restores normal development. RARbeta2 expression requires the expression of RARalpha2. After neurulation, the expression of all retinoid receptors in the VAD quail embryo becomes independent of vitamin A status and is similar to that of the normal. The mRNA levels and sites of expression of the key enzyme for retinoic acid biosynthesis, Raldh-2, are not affected by vitamin A status; the expression pattern is restricted and does not correspond to that of retinoid receptors at all sites. The general patterns and intensity of retinoid receptor gene expression during early quail development are comparable to those of the mammalian and thus validate the application of results from retinoid-regulated avian development studies to those of the mammalian.

title: The zebrafish neckless mutation reveals a requirement for raldh2 in mesodermal signals that pattern the hindbrain.
abstract: We describe a new zebrafish mutation, neckless, and present evidence that it inactivates retinaldehyde dehydrogenase type 2, an enzyme involved in retinoic acid biosynthesis. neckless embryos are characterised by a truncation of the anteroposterior axis anterior to the somites, defects in midline mesendodermal tissues and absence of pectoral fins. At a similar anteroposterior level within the nervous system, expression of the retinoic acid receptor a and hoxb4 genes is delayed and significantly reduced. Consistent with a primary defect in retinoic acid signalling, some of these defects in neckless mutants can be rescued by application of exogenous retinoic acid. We use mosaic analysis to show that the reduction in hoxb4 expression in the nervous system is a non-cell autonomous effect, reflecting a requirement for retinoic acid signalling from adjacent paraxial mesoderm. Together, our results demonstrate a conserved role for retinaldehyde dehydrogenase type 2 in patterning the posterior cranial mesoderm of the vertebrate embryo and provide definitive evidence for an involvement of endogenous retinoic acid in signalling between the paraxial mesoderm and neural tube.

title: Decreased embryonic retinoic acid synthesis results in a DiGeorge syndrome phenotype in newborn mice
abstract: Retinoic acid (RA), the active derivative of vitamin A, is involved in various developmental and homeostatic processes. To define whether certain developmental events are particularly sensitive to a decrease in embryonic RA levels, we generated mice bearing a hypomorphic allele of the RA-synthesizing enzyme Raldh2. The resulting mutant mice, which die perinatally, exhibit the features of the human DiGeorge syndrome (DGS) with heart outflow tract septation defects and anomalies of the aortic arch-derived head and neck arteries, laryngeal-tracheal cartilage defects, and thymus/parathyroid aplasia or hypoplasia. Analysis of Raldh2 hypomorph embryos reveal selective defects of the posterior (third to sixth) branchial arches, including absence or hypoplasia of the corresponding aortic arches and pharyngeal pouches, and local down-regulation of RA-target genes. Thus, a decreased level of embryonic RA (through genetic and/or nutritional causes) could represent a major modifier of the expressivity of human 22q11del-associated DiGeorge/velocardiofacial syndromes and, if severe enough, could on its own lead to the clinical features of the DiGeorge syndrome.

title: Endogenous retinoids in the zebrafish embryo and adult
abstract: Retinoic acid and its isoforms are considered to be endogenous compounds which regulate embryonic development. In the work reported here we have determined which retinoids are present in zebrafish embryos and how their levels change throughout development and into adulthood. All‐trans‐RA is present and its level does not change significantly during embryogenesis. We failed to detect other retinoic acid isomers such as 9‐cis‐RA and 4‐oxo‐RA, but we did observe a rapid rise in the level of didehydroretinol after gastrulation. The most striking result is that the zebrafish embryo, like Xenopus and tunicates, contains a vast excess of t‐retinal whereas the embryos of higher vertebrates have an excess of t‐retinol. However, as the zebrafish grows, the levels of t‐retinol rise so that by adulthood t‐retinol and t‐retinal concentrations are more equivalent, indicating a changing pattern of retinoid metabolism with growth. To examine the significance of the use of t‐retinal as a precursor of t‐RA we treated embryos with disulphiram, an inhibitor of retinaldehyde dehydrogenase. This resulted in embryos with an undulating notochord and correspondingly abnormal somites and ventral floor plate. In contrast to this effect, 4‐methylpyrazole, which inhibits alcohol dehydrogenases, had no effect on development. This effect of disulphiram suggests that t‐RA may be involved in the establishment of the anteroposterior axis of the embryo.

title: Expression of the murine retinol dehydrogenase 10 (Rdh10) gene correlates with many sites of retinoid signalling during embryogenesis and organ differentiation
abstract: Retinoic acid acts as a signalling molecule regulating many developmental events in vertebrates. As this molecule directly influences gene expression by activating nuclear receptors, its patterns of synthesis have to be tightly regulated, and it is well established that at least three retinaldehyde dehydrogenases (RALDHs) are involved in such tissue‐specific synthesis. Whereas embryos from oviparous species can obtain retinaldehyde by metabolizing carotenoids stored in the yolk, placental embryos rely on retinol transferred from the maternal circulation. Here, we show that the gene encoding one of the murine retinol dehydrogenases, Rdh10, is expressed according to complex profiles both during early embryogenesis and organ differentiation. Many of its expression sites correlate with regions of active retinoid signalling and Raldh gene expression, especially with Raldh2 in the early presomitic and somitic mesoderm, retrocardiac and posterior branchial arch region, or later in the pleural mesothelium and kidney cortical region. Rdh10 also shows cell‐type and/or regional specificity during development of the palate, teeth, and olfactory system. During limb bud development, it may participate in retinoic acid production in proximal/posterior cells, and eventually in interdigital mesenchyme. These data implicate the retinol to retinaldehyde conversion as the first step in the tissue‐specific regulation of retinoic acid synthesis, at least in mammalian embryos. Developmental Dynamics 236:2899–2908, 2007.

title: RDH10 is essential for synthesis of embryonic retinoic acid and is required for limb, craniofacial, and organ development.
abstract: Regulation of patterning and morphogenesis during embryonic development depends on tissue-specific signaling by retinoic acid (RA), the active form of Vitamin A (retinol). The first enzymatic step in RA synthesis, the oxidation of retinol to retinal, is thought to be carried out by the ubiquitous or overlapping activities of redundant alcohol dehydrogenases. The second oxidation step, the conversion of retinal to RA, is performed by retinaldehyde dehydrogenases. Thus, the specific spatiotemporal distribution of retinoid synthesis is believed to be controlled exclusively at the level of the second oxidation reaction. In an N-ethyl-N-nitrosourea (ENU)-induced forward genetic screen we discovered a new midgestation lethal mouse mutant, called trex, which displays craniofacial, limb, and organ abnormalities. The trex phenotype is caused by a mutation in the short-chain dehydrogenase/reductase, RDH10. Using protein modeling, enzymatic assays, and mutant embryos, we determined that RDH10(trex) mutant protein lacks the ability to oxidize retinol to retinal, resulting in insufficient RA signaling. Thus, we show that the first oxidative step of Vitamin A metabolism, which is catalyzed in large part by the retinol dehydrogenase RDH10, is critical for the spatiotemporal synthesis of RA. Furthermore, these results identify a new nodal point in RA metabolism during embryogenesis.

title: Craniofacial malformation in Xenopus laevis tadpoles caused by the exposure of early embryos to ethanol.
abstract: The principal features of fetal alcohol syndrome (FAS) include microcephaly, reduced body size, and characteristic facial malformation, most of which are associated with hypoplasticity of structures around the mouth. A recent study using pregnant mice suggested that the malformations in FAS are caused by the effects of ethanol on early embryos during gastrulation and neurulation. Exposure of Xenopus laevis early embryos to 1-2% ethanol until they developed to the late neurula stage produced craniofacial malformations in tadpoles which have many similarities with the described features of FAS. They include reduced length of brain, reduced body size, and hypoplasticity of the anterior end of the body around the mouth. Size reduction from the control group was about 20% in the mouth region, and 10% in other head regions. Ethanol inhibited migration of mesodermal cells toward the animal pole during gastrulation, thus inducing a smaller neural plate with reduced anterior-posterior length. These very early effects of ethanol in development seem to produce various craniofacial malformations in tadpoles, and possibly also in human infants with FAS. In contrast to mammalian embryos, one can conveniently study in detail the effects of ethanol on morphogenetic movements in various in vitro experiments by using a large number of amphibian embryos. Thus, amphibian embryos represent a valuable animal model for the study of mechanisms of the teratogenic effects of ethanol.

title: Ethanol impairs migration of the prechordal plate in the zebrafish embryo.
abstract: Exposure of vertebrate embryos to ethanol causes cyclopia, but little is known about the underlying mechanisms of this effect. Here we show that cyclopia can be induced in the zebrafish by a short ethanol treatment during early gastrula stages and is accompanied by loss of gene expression characteristic of the ventral aspects of the fore- and midbrain. Interestingly, defects in the expression of ventral brain markers are linked to impaired migration of the prechordal plate mesoderm indicating that the correct position of the prechordal plate mesoderm under the anterior neural plate in the zebrafish embryo is required for specification of the anterior neural midline. Ethanol-induced cyclopia does not, however, impair the induction of anterior neuroectodermal structures in general. Finally, as genes like goosecoid and islet-1 are expressed in prechordal plate cells in a temporal pattern similar to control embryos despite the ectopic position of expressing cells, it appears that regulation of prechordal plate-specific gene expression is largely independent of the final position of the prechordal plate.

title: Formation and function of Spemann's organizer.
abstract: The organizer is formed in an equatorial sector of the blastula stage amphibian embryo by cells that have responded to two maternal agents: a general mesoendoderm inducer (involving the TFG-beta signaling pathway) and a dorsal modifier (probably involving the Wnt signaling pathway). The meso-endoderm inducer is secreted by most vegetal cells, those containing maternal materials that had been localized in the vegetal hemisphere of the oocyte during oogenesis. As a consequence of the inducer's distribution and action, the competence domains of prospective ectoderm, mesoderm, and endoderm are established in an animal-to-vegetal order in the blastula. The dorsal modifier signal is secreted by a sector of cells of the animal and vegetal hemispheres on one side of the blastula. These cells contain maternal materials transported there in the first cell cycle from the vegetal pole of the egg along microtubules aligned by cortical rotation. The Nieuwkoop center is the region of blastula cells secreting both maternal signals, and hence specifying the organizer in an equatorial sector. Final steps of organizer formation at the late blastula or early gastrula stage may involve locally secreted zygotic signals as well. At the gastrula stage, the organizer secretes a variety of zygotic proteins that act as antagonists to various members of the BMP and Wnt families of ligands, which are secreted by cells of the competence domains surrounding the organizer. BMPs and Wnts favor ventral development, and cells near the organizer are protected from these agents by the organizer's inducers. The nearby cells are derepressed in their inherent capacity for dorsal development, which is apparent in the neural induction of the ectoderm, dorsalization of the mesoderm, and anteriorization of the endoderm. The organizer also engages in extensive specialized morphogenesis, which brings it within range of responsive cell groups. It also self-differentiates to a variety of axial tissues of the body.

title: Patterning of the embryo along the anterior-posterior axis: the role of the caudal genes.
abstract: Patterning along the anterior-posterior axis takes place during gastrulation and early neurulation. Homeobox genes like Otx-2 and members of the Hox family have been implicated in this process. The caudal genes in Drosophila and C. elegans have been shown to determine posterior fates. In vertebrates, the caudal genes begin their expression during gastrulation and they take up a posterior position. By injecting sense and antisense RNA of the Xenopus caudal gene Xcad-2, we have studied a number of regulatory interactions among homeobox genes along the anterior-posterior axis. Initially, the Xcad-2 and Otx-2 genes are mutually repressed and, by late gastrulation, they mark the posterior- or anterior-most domains of the embryo, respectively. During late gastrulation and neurulation, Xcad-2 plays an additional regulatory function in relation to the Hox genes. Hox genes normally expressed anteriorly are repressed by Xcad-2 overexpression while those normally expressed posteriorly exhibit more anterior expression. The results show that the caudal genes are part of a posterior determining network which during early gastrulation functions in the subdivision of the embryo into anterior head and trunk domains. Later in gastrulation and neurulation these genes play a role in the patterning of the trunk region.

title: Molecular nature of Spemann's organizer: the role of the Xenopus homeobox gene goosecoid

abstract: This study analyzes the function of the homeobox gene goosecoid in Xenopus development. First, we find that goosecoid mRNA distribution closely mimics the expected localization of organizer tissue in normal embryos as well as in those treated with LiCl and UV light. Second, goosecoid mRNA accumulation is induced by activin, even in the absence of protein synthesis. It is not affected by bFGF and is repressed by retinoic acid. Lastly, microinjection of goosecoid mRNA into the ventral side of Xenopus embryos, where goosecoid is normally absent, leads to the formation of an additional complete body axis, including head structures and abundant notochordal tissue. The results suggest that the goosecoid homeodomain protein plays a central role in executing Spemann's organizer phenomenon.

title: Xenopus chordin: A novel dorsalizing factor activated by organizer-specific homeobox genes
abstract: A Xenopus gene whose expression can be activated by the organizer-specific homeobox genes goosecoid and Xnot2 was isolated by differential screening. The chordin gene encodes a novel protein of 941 amino acids that has a signal sequence and four Cys-rich domains. The expression of chordin starts in Spemann's organizer subsequent to that of goosecoid, and its induction by activin requires de novo protein synthesis. Microinjection of chordin mRNA induces twinned axes and can completely rescue axial development in ventralized embryos. This molecule is a potent dorsalizing factor that is expressed at the right time and in the right place to regulate cell-cell interactions in the organizing centers of head, trunk, and tail development.

title: Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization.
abstract: We have adapted a non-radioactive technique to detect localized mRNAs in whole-mount Xenopus embryos. Synthetic antisense RNA transcribed in the presence of digoxygenin-UTP is used as a probe and is detected via an anti-digoxygenin antibody. We show that localized mRNAs can be detected from late gastrula to tadpole stages and that high as well as low abundance RNAs can be detected. The method was tested on muscle actin and alpha-globin RNAs, whose localization has previously been characterized. In addition, we used the method to determine the distribution of XA-1 RNA, an anterior ectoderm-specific RNA, which we show is expressed in the periphery of the cement gland as well as in the region of the hatching gland. The sequence of an XA-1 cDNA predicts a protein rich in proline and histidine.

title: A single morphogenetic field gives rise to two retina primordia under the influence of the prechordal plate.
abstract: Two bilaterally symmetric eyes arise from the anterior neural plate in vertebrate embryos. An interesting question is whether both eyes share a common developmental origin or they originate separately. We report here that the expression pattern of a new gene ET reveals that there is a single retina field which resolves into two separate primordia, a suggestion supported by the expression pattern of the Xenopus Pax-6 gene. Lineage tracing experiments demonstrate that retina field resolution is not due to migration of cells in the median region to the lateral parts of the field. Removal of the prechordal mesoderm led to formation of a single retina both in chick embryos and in Xenopus explants. Transplantation experiments in chick embryos indicate that the prechordal plate is able to suppress Pax-6 expression. Our results provide direct evidence for the existence of a single retina field, indicate that the retina field is resolved by suppression of retina formation in the median region of the field, and demonstrate that the prechordal plate plays a primary signaling role in retina field resolution.