Alcohol induces neural tube defects by reducing retinoic acid signaling and promoting neural plate expansion.

Introduction: Neural tube defects (NTDs) are among the most debilitating and common developmental defects in humans. The induction of NTDs has been attributed to abnormal folic acid (vitamin B9) metabolism, Wnt and BMP signaling, excess retinoic acid (RA), dietary components, environmental factors, and many others. In the present study we show that reduced RA signaling, including alcohol exposure, induces NTDs. Methods: Xenopus embryos were exposed to pharmacological RA biosynthesis inhibitors to study the induction of NTDs. Embryos were treated with DEAB, citral, or ethanol, all of which inhibit the biosynthesis of RA, or injected to overexpress Cyp26a1 to reduce RA. NTD induction was studied using neural plate and notochord markers together with morphological analysis. Expression of the neuroectodermal regulatory network and cell proliferation were analyzed to understand the morphological malformations of the neural plate. Results: Reducing RA signaling levels using retinaldehyde dehydrogenase inhibitors (ethanol, DEAB, and citral) or Cyp26a1-driven degradation efficiently induce NTDs. These NTDs can be rescued by providing precursors of RA. We mapped this RA requirement to early gastrula stages during the induction of neural plate precursors. This reduced RA signaling results in abnormal expression of neural network genes, including the neural plate stem cell maintenance genes, geminin, and foxd4l1.1. This abnormal expression of neural network genes results in increased proliferation of neural precursors giving rise to an expanded neural plate. Conclusion: We show that RA signaling is required for neural tube closure during embryogenesis. RA signaling plays a very early role in the regulation of proliferation and differentiation of the neural plate soon after the induction of neural progenitors during gastrulation. RA signaling disruption leads to the induction of NTDs through the mis regulation of the early neuroectodermal network, leading to increased proliferation resulting in the expansion of the neural plate. Ethanol exposure induces NTDs through this mechanism involving reduced RA levels.

Reduced Retinoic Acid Signaling During Gastrulation Induces Developmental Microcephaly.

Retinoic acid (RA) is a central signaling molecule regulating multiple developmental decisions during embryogenesis. Excess RA induces head malformations, primarily by expansion of posterior brain structures at the expense of anterior head regions, i.e., hindbrain expansion. Despite this extensively studied RA teratogenic effect, a number of syndromes exhibiting microcephaly, such as DiGeorge, Vitamin A Deficiency, Fetal Alcohol Syndrome, and others, have been attributed to reduced RA signaling. This causative link suggests a requirement for RA signaling during normal head development in all these syndromes. To characterize this novel RA function, we studied the involvement of RA in the early events leading to head formation in Xenopus embryos. This effect was mapped to the earliest RA biosynthesis in the embryo within the gastrula Spemann-Mangold organizer. Head malformations were observed when reduced RA signaling was induced in the endogenous Spemann-Mangold organizer and in the ectopic organizer of twinned embryos. Two embryonic retinaldehyde dehydrogenases, ALDH1A2 (RALDH2) and ALDH1A3 (RALDH3) are initially expressed in the organizer and subsequently mark the trunk and the migrating leading edge mesendoderm, respectively. Gene-specific knockdowns and CRISPR/Cas9 targeting show that RALDH3 is a key enzyme involved in RA production required for head formation. These observations indicate that in addition to the teratogenic effect of excess RA on head development, RA signaling also has a positive and required regulatory role in the early formation of the head during gastrula stages. These results identify a novel RA activity that concurs with its proposed reduction in syndromes exhibiting microcephaly.

A coregulator shift, rather than the canonical switch, underlies thyroid hormone action in the liver.

Thyroid hormones (THs) are powerful regulators of metabolism with major effects on body weight, cholesterol, and liver fat that have been exploited pharmacologically for many years. Activation of gene expression by TH action is canonically ascribed to a hormone-dependent "switch" from corepressor to activator binding to thyroid hormone receptors (TRs), while the mechanism of TH-dependent repression is controversial. To address this, we generated a mouse line in which endogenous TRß1 was epitope-tagged to allow precise chromatin immunoprecipitation at the low physiological levels of TR and defined high-confidence binding sites where TRs functioned at enhancers regulated in the same direction as the nearest gene in a TRß-dependent manner. Remarkably, although positive and negative regulation by THs have been ascribed to different mechanisms, TR binding was highly enriched at canonical DR4 motifs irrespective of the transcriptional direction of the enhancer. The canonical NCoR1/HDAC3 corepressor complex was reduced but not completely dismissed by TH and, surprisingly, similar effects were seen at enhancers associated with negatively as well as positively regulated genes. Conversely, coactivator CBP was found at all TH-regulated enhancers, with transcriptional activity correlating with the ratio of CBP to NCoR rather than their presence or absence. These results demonstrate that, in contrast to the canonical "all or none" coregulator switch model, THs regulate gene expression by orchestrating a shift in the relative binding of corepressors and coactivators.

Fetal Alcohol Spectrum Disorder: Embryogenesis Under Reduced Retinoic Acid Signaling Conditions.

Fetal Alcohol Spectrum Disorder (FASD) is a complex set of developmental malformations, neurobehavioral anomalies and mental disabilities induced by exposing human embryos to alcohol during fetal development. Several experimental models and a series of developmental and biochemical approaches have established a strong link between FASD and reduced retinoic acid (RA) signaling. RA signaling is involved in the regulation of numerous developmental decisions from patterning of the anterior-posterior axis, starting at gastrulation, to the differentiation of specific cell types within developing organs, to adult tissue homeostasis. Being such an important regulatory signal during embryonic development, mutations or environmental perturbations that affect the level, timing or location of the RA signal can induce multiple and severe developmental malformations. The evidence connecting human syndromes to reduced RA signaling is presented here and the resulting phenotypes are compared to FASD. Available data suggest that competition between ethanol clearance and RA biosynthesis is a major etiological component in FASD.

Retinoic acid signaling reduction recapitulates the effects of alcohol on embryo size.

Intrauterine growth restriction (IUGR) is commonly observed in human pregnancies and can result in severe clinical outcomes. IUGR is observed in Fetal Alcohol Syndrome (FAS) fetuses as a result of alcohol (ethanol) exposure during pregnancy. To further understand FAS, the severe form of Fetal Alcohol Spectrum Disorder, we performed an extensive quantitative analysis of the effects of ethanol on embryo size utilizing our Xenopus model. Ethanol-treated embryos exhibited size reduction along the anterior-posterior axis. This effect was evident primarily from the hindbrain caudally, while rostral regions appeared refractive to ethanol-induced size changes, also known as asymmetric IUGR. Interestingly, some embryo batches in addition to shortening from the hindbrain caudally also exhibited an alcohol-dependent reduction of the anterior head domain, known as symmetric IUGR. To study the connection between ethanol exposure and reduced retinoic acid levels we treated embryos with the retinaldehyde dehydrogenase inhibitors, DEAB and citral. Inhibition of retinoic acid biosynthesis recapitulated the growth defects induced by ethanol affecting mainly axial elongation from the hindbrain caudally. To study the competition between ethanol clearance and retinoic acid biosynthesis we demonstrated that, co-exposure to alcohol reduces the teratogenic effects of treatment with retinol (vitamin A), the retinoic acid precursor. These results further support the role of retinoic acid in the regulation of axial elongation.

Acetaldehyde inhibits retinoic acid biosynthesis to mediate alcohol teratogenicity.

Alcohol consumption during pregnancy induces Fetal Alcohol Spectrum Disorder (FASD), which has been proposed to arise from competitive inhibition of retinoic acid (RA) biosynthesis. We provide biochemical and developmental evidence identifying acetaldehyde as responsible for this inhibition. In the embryo, RA production by RALDH2 (ALDH1A2), the main retinaldehyde dehydrogenase expressed at that stage, is inhibited by ethanol exposure. Pharmacological inhibition of the embryonic alcohol dehydrogenase activity, prevents the oxidation of ethanol to acetaldehyde that in turn functions as a RALDH2 inhibitor. Acetaldehyde-mediated reduction of RA can be rescued by RALDH2 or retinaldehyde supplementation. Enzymatic kinetic analysis of human RALDH2 shows a preference for acetaldehyde as a substrate over retinaldehyde. RA production by hRALDH2 is efficiently inhibited by acetaldehyde but not by ethanol itself. We conclude that acetaldehyde is the teratogenic derivative of ethanol responsible for the reduction in RA signaling and induction of the developmental malformations characteristic of FASD. This competitive mechanism will affect tissues requiring RA signaling when exposed to ethanol throughout life.

Competition between ethanol clearance and retinoic acid biosynthesis in the induction of fetal alcohol syndrome.

Several models have been proposed to explain the neurodevelopmental syndrome induced by exposure of human embryos to alcohol, which is known as fetal alcohol spectrum disorder (FASD). One of the proposed models suggests a competition for the enzymes required for the biosynthesis of retinoic acid. The outcome of such competition is development under conditions of reduced retinoic acid signaling. Retinoic acid is one of the biologically active metabolites of vitamin A (retinol), and regulates numerous embryonic and differentiation processes. The developmental malformations characteristic of FASD resemble those observed in vitamin A deficiency syndrome as well as from inhibition of retinoic acid biosynthesis or signaling in experimental models. There is extensive biochemical and enzymatic overlap between ethanol clearance and retinoic acid biosynthesis. Several lines of evidence suggest that in the embryo, the competition takes place between acetaldehyde and retinaldehyde for the aldehyde dehydrogenase activity available. In adults, this competition also extends to the alcohol dehydrogenase activity. Ethanol-induced developmental defects can be ameliorated by increasing the levels of retinol, retinaldehyde, or retinaldehyde dehydrogenase. Acetaldehyde inhibits the production of retinoic acid by retinaldehyde dehydrogenase, further supporting the competition model. All of the evidence supports the reduction of retinoic acid signaling as the etiological trigger in the induction of FASD.

ADHFe1: a novel enzyme involved in retinoic acid-dependent Hox activation.

Retinoic acid (RA) signaling is a central pathway regulating anterior-posterior patterning of the embryo through its targets, the Hox genes. RA is produced by two sequential oxidations from vitamin A (retinol) and this biosynthesis has to be regulated temporally, spatially and quantitatively. Mining Xenopus embryonic expression databases identified a novel component of the RA metabolic network, ADHFe1. Using Xenopus laevis embryos as our experimental system we determined the temporal and spatial pattern of AdhFe1 expression. Gain- and loss-of-function of ADHFe1 were induced to study its function and the regulation of the AdhFe1 gene by RA was studied. Expression analysis localized the ADHFe1 protein to the late Spemann's organizer, the trunk organizer. Subsequently, ADHFe1 can be detected in the prechordal mesoderm, the notochord and the lateral plate mesoderm. Manipulation of ADHFe1 levels affects the normal Hox gene expression. The effects of ADHFe1 manipulation can by rescued by increasing the levels of RA or its biosynthesis. Expression of the AdhFe1 gene is regulated by RA itself. ADHFe1 is an enzyme active already during gastrula stages and the protein is still present during neurula stages. ADHFe1 regulates the expression of the Hox genes during the early patterning of the trunk. The effect of ADHFe1 on Hox expression is mediated through regulation of RA levels. ADHFe1 probably reduces retinaldehyde to retinol thereby restricting the availability of retinaldehyde, the substrate needed by retinaldehyde dehydrogenases to produce RA making it a novel regulator of RA concentrations in the embryo and RA homeostasis.

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

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

Cdx1 is essential for the initiation of HoxC8 expression during early embryogenesis.

The Hox genes pattern the anterior-posterior axis in developing embryos through tightly regulated, partially overlapping, temporal and spatial expression domains. Initial regulation of Hox expression is important to establish these overlapping transcription domains. The Cdx homeodomain factors have been proposed as Hox regulators, but their precise role and mechanism during this regulatory interaction remain unclear. In Xenopus embryos, HoxC8 transcripts begin to accumulate during mid/late gastrula. Cdx1 overexpression and knockdown lead to precocious or slower HoxC8 expression, respectively. The mouse HoxC8 early enhancer when introduced into Xenopus embryos recapitulates the endogenous XHoxC8 temporal expression pattern and shows the same responsiveness to Cdx1 regulation. Three pairs of conserved Cdx binding sites were identified within the HoxC8 early enhancer. We demonstrate that Cdx1 binds directly these responsive elements during embryogenesis, as part of the mechanism for the timely activation of HoxC8 expression. We define the function and mechanism of Cdx1 regulation on HoxC8 expression and suggest the possibility that the temporal changes in Cdx activity levels during gastrulation, combined with differential DNA binding affinity, might play a role in the establishment of Hox sequential activation.

Negative autoregulation of Oct3/4 through Cdx1 promotes the onset of gastrulation.

Gastrulation marks the onset of germ layer formation from undifferentiated precursor cells maintained by a network including the Pou5f1 gene, Oct3/4. Negative regulation of the undifferentiated state is a prerequisite for germ layer formation and subsequent development. A novel cross-regulatory network was characterized including the Pou5f1 and Cdx1 genes as part of the signals controlling the onset of gastrulation. Of particular interest was the observation that, preceding gastrulation, the Xenopus Oct3/4 factors, Oct60, Oct25, and Oct91, positively regulate Cdx1 expression through FGF signaling, and during gastrulation the Oct3/4 factors become repressors of Cdx1. Cdx1 negatively regulates the Pou5f1 genes during gastrulation, thus contributing to the repression of the network maintaining the undifferentiated state and promoting the onset of gastrulation. These regulatory interactions suggest that Oct3/4 initiates its own negative autoregulation through Cdx1 up-regulation to begin the repression of pluripotency in preparation for the onset of gastrulation and germ layer differentiation.