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.

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.

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.

Temporal analysis of the early BMP functions identifies distinct anti-organizer and mesoderm patterning phases.

BMP signaling performs multiple important roles during early embryogenesis. Signaling through the BMP pathway is mediated by different BMP ligands expressed in partially overlapping temporal and spatial patterns. Assignment of different BMP-dependent activities to the individual ligands has relied on the patterns of expression of the various BMP genes. Temporal analysis of BMP signaling prior to and during gastrulation was performed using glucocorticoid-controlled Smad proteins. Overexpression of the BMP-specific Smad1 and Smad5 revealed that suppression of Spemann's organizer formation in Xenopus embryos can only take place by activating the BMP pathway prior to the onset of gastrulation. Blocking BMP signaling with the inhibitory Smad, Smad6, results in dorsalized embryos or secondary axis induction, only when activated up to early gastrula stages. BMP2 efficiently represses organizer-specific transcription from the midblastula transition onwards while BMP4 is unable to prevent the early activation of organizer-specific genes. Manipulation of the BMP pathway during mid/late gastrula affects mesodermal patterning with no external phenotypic effects. These observations suggest that the malformations resulting from inhibition or promotion of organizer formation, ventralized or dorsalized, respectively, are the result of a very early BMP function, through its antagonism of organizer formation. This function is apparently fulfilled by BMP2 and only at its latest phase by BMP4. Subsequently, BMP functions in the patterning of the mesoderm with no apparent phenotypic effects.

Ethanol exposure affects gene expression in the embryonic organizer and reduces retinoic acid levels.

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.

Gbx2 interacts with Otx2 and patterns the anterior-posterior axis during gastrulation in Xenopus.

Anterior-posterior patterning of the embryo requires the activity of multiple homeobox genes among them Hox, caudal (Cdx, Xcad) and Otx2. During early gastrulation, Otx2 and Xcad2 establish a cross-regulatory network, which is an early event in the anterior-posterior patterning of the embryo. As gastrulation proceeds and the embryo elongates, a new domain forms, which expresses neither, Otx2 nor Xcad2 genes. Early transcription of the Xenopus Gbx2 homologue, Xgbx2a, is spatially restricted between Otx2 and Xcad2. When overexpressed, Otx2 and Xcad2 repress Xgbx2a transcription, suggesting their role in setting the early Xgbx2a expression domain. Homeobox genes have been shown to play crucial roles in the specification of the vertebrate brain. The border between the transcription domains of Otx2 and Gbx2 is the earliest known marker of the region where the midbrain/hindbrain boundary (MHB) organizer will develop. Xgbx2a is a negative regulator of Otx2 and a weak positive regulator of Xcad2. Using obligatory activator and repressor versions of Xgbx2a, we demonstrate that, during early embryogenesis, Xgbx2a acts as a transcriptional repressor. In addition, taking advantage of hormone-inducible versions of Xgbx2a and its antimorph, we show that the ability of Xgbx2a to induce head malformations is restricted to gastrula stages and correlates with its ability to repress Otx2 during the same developmental stages. We therefore suggest that the earliest known step of the MHB formation, the establishment of Otx2/Gbx2 boundary, takes place via mutual inhibitory interactions between these two genes and this process begins as early as at midgastrulation.

Otx2 can activate the isthmic organizer genetic network in the Xenopus embryo.

Development and differentiation of the vertebrate caudal midbrain and anterior hindbrain are dependent on the isthmic organizer signals at the midbrain/hindbrain boundary (MHB). The future MHB forms at the boundary between the Otx2 and Gbx2 expression domains. Recent studies in mice and chick suggested that the apposition of Otx2- and Gbx2-expressing cells is instrumental for the positioning and early induction of the MHB genetic cascade. We show that Otx2 and Gbx2 perform different roles in this process. We find that ectopically expressed Otx2 on its own can induce a substantial part of the MHB genetic network, namely En2, Wnt1, Pax-2, Fgf8 and Gbx2, in a concentration-dependent manner. This induction does not require protein synthesis and ends during neurulation. In contrast, Gbx2 is a negative regulator of Otx2 and the MHB genes. Based on the temporal patterns of expression of the genes involved, we propose that Otx2 might be the early inducer of the isthmic organizer genetic network while Gbx2 restricts Otx2 expression along the anterior-posterior axis and establishes an Otx2 gradient.