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.

Specific signaling molecules play a pivotal role in the induction and specification of tissues during early vertebrate embryogenesis. BMP-4 specifies ventral mesoderm differentiation and inhibits neural induction in Xenopus, whereas three molecules secreted from the organizer, noggin, follistatin and chordin dorsalize mesoderm and promote neural induction. Here we report that follistatin antagonizes the activities of BMP-4 in frog embryos and mouse teratocarcinoma cells. In Xenopus embryos follistatin blocks the ventralizing effect of BMP-4. In mouse P19 cells follistatin promotes neural differentiation. BMP-4 antagonizes the action of follistatin and prevents neural differentiation. In addition we show that the follistatin and BMP-4 proteins can interact directly in vitro. These data provide evidence that follistatin might play a role in modulating BMP-4 activity in vivo.

The in vitro proliferation of the spontaneous lymphoid T-cell leukemia designated LB was enhanced by physiological, intermediate and supraphysiological concentrations of insulin. The enhancing effect was observed in both serum-free medium (SFM) and medium containing low concentrations of serum. Guinea-pig anti-insulin serum, but not guinea-pig normal serum, inhibited the proliferation of LB cells incubated either in medium containing serum alone or in medium containing serum and supplemented with insulin. This finding suggests that LB cells use serum insulin as a growth factor. Insulin-like growth factors I (IGF-I) and II (IGF-II) failed to stimulate an appreciable proliferation in LB cells, whereas in the same experiment insulin markedly enhanced the proliferation of this lymphoid leukemia. Furthermore, the concentration of unlabelled insulin required to displace 50% of 125I-insulin bound to LB cells was 3 orders of magnitude lower than the concentration of IGF-I required to achieve the same displacement. Our findings indicate that interaction of insulin with its own receptor, and not with IGF-I receptor, triggers the proliferation of LB cells. Radio-receptor assays revealed that LB cells express approximately 3,200 molecules of high affinity (Kd = 10(-9) M) insulin receptor per cell. None of 7 other tumor cell lines tested responded to insulin. The proliferation of insulin-stimulated LB cells was also inhibited with tyrphostin, a tyrosine kinase blocker analogous to tyrosine, which perhaps blocks the tyrosine kinase activity of the insulin receptor beta-chain.

Expression of the Xenopus Xcad-1 and Xcad-2 genes initiates during early gastrulation exhibiting a dorsoventral asymmetry in their domains of transcription. At mid-gastrulation the ventral preference becomes stronger and the caudal genes take up a posterior localization in their expression, which they will maintain until their downregulation along the dorsal midline. Comparison of the three Xenopus caudal genes revealed a temporal and spatial nested set of expression patterns. The transcription of the caudal genes is sequentially downregulated with the one expressed most caudally (Xcad-2) being shut down first, this sequence is most evident along the dorsal midline. This pattern of expression suggests a role for the caudal genes as posterior determinants along the anteroposterior axis. In chicken, mouse, man and Xenopus three members of the caudal family have been identified in the genome. Even though in Xenopus the Xcad-3 gene has been previously described, in order to obtain a better insight on the role of the caudal genes a comparative study of all three frog genes was performed.

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.