VegT activation of Sox17 at the midblastula transition alters the response to nodal signals in the vegetal endoderm domain

In Xenopus, the prospective endoderm and mesoderm are localized to discrete, adjacent domains at the beginning of gastrulation, and this is made evident by the expression of Sox17 in vegetal blastomeres and Brachyury (Xbra) in marginal blastomeres. Here, we examine the regulation of Sox17alpha expression and the role of Sox17alpha in establishing the vegetal endodermal gene expression domain. Injection of specific inhibitors of VegT or Nodal resulted in a loss of Sox17alpha expression in the gastrula. However, the onset of Sox17alpha expression at the midblastula transition was dependent on VegT, but not on Nodal function, indicating that Sox17alpha expression is initiated by VegT and then maintained by Nodal signals. Consistent with these results, VegT, but not Xenopus Nodal-related-1 (Xnr1), can activate Sox17alpha expression at the midblastula stage in animal explants. In addition, VegT activates Sox17alpha in the presence of cycloheximide or a Nodal antagonist, suggesting that Sox17alpha is an immediate-early target of VegT in vegetal blastomeres. Given that Nodal signals are necessary and sufficient for both mesodermal and endodermal gene expression, we propose that VegT activation of Sox17alpha at the midblastula transition prevents mesodermal gene expression in response to Nodal signals, thus establishing the vegetal endodermal gene expression domain. Supporting this idea, Sox17alpha misexpression in the marginal zone inhibits the expression of multiple mesodermal genes. Furthermore, in animal explants, Sox17alpha prevents the induction of Xbra and MyoD, but not Sox17beta or Mixer, in response to Xnr1. Therefore, VegT activation of Sox17alpha plays an important role in establishing a region of endoderm-specific gene expression in vegetal blastomeres.     

Effects of ethanol on the primitive streak stage mouse embryo

Recent studies of mouse models have suggested that malformations associated with the fetal alcohol syndrome (FAS) are caused by the effects of ethanol on early embryos during gastrulation and neurulation. A study of Xenopus laevis embryos showed that exposure of gastrula stage amphibian embryos to ethanol inhibits migration of the mesodermal cells, causes formation of small neural plates, and subsequently causes hypoplastic craniofacial malformations in tadpoles. We now report effects of ethanol on the primitive streak stage mouse embryos. An ethanol solution (25%) was injected intraperitonealy twice into mice of 6.5-7.0 days of pregnancy at a dose of 0.015 ml/gm of body weight. Histological and morphometric examinations of 7.5-day embryos, 20 hr after the second injection, showed that the epiblast layer was disorganized and shrunk with formation of many blebs. In addition, formation of the mesodermal cell layer was retarded in the ethanol-treated embryos, suggesting that exposure of gastrula stage embryos to ethanol causes similar abnormalities in mouse and Xenopus embryos. These results suggest that the inhibition of the morphogenetic movements during gastrulation may be the primary effect of ethanol in causing major craniofacial malformations of FAS.     

The mRNA coding for Xenopus glutamate receptor interacting protein 2 (XGRIP2) is maternally transcribed, transported through the late pathway and localized to the germ plasm

Using a large-scale in situ hybridization screening, we found that the mRNA coding for Xenopus glutamate receptor interacting protein 2 (XGRIP2) was localized to the germ plasm of Xenopus laevis. The mRNA is maternally transcribed in oocytes and, during maturation, transported to the vegetal germ plasm through the late pathway where VegT and Vg1 mRNAs are transported. In the 3'-untranslated region (UTR) of the mRNA, there are clusters of E2 and VM1 localization motifs that were reported to exist in the mRNAs classified as the late pathway group. With in situ hybridization to the sections of embryos, the signal could be detected in the cytoplasm of migrating presumptive primordial germ cells (pPGCs) until stage 35. At stage 40, when the cells cease to migrate and reach the dorsal mesentery, the signal disappeared. A possible role of XGRIP2 in pPGCs of Xenopus will be discussed.     

Expression of Sox1 during Xenopus early embryogenesis

Sox B1 group genes, Sox1, Sox2, and Sox3 (Sox1-3), are involved in neurogenesis in various species. Here, we identified the Xenopus homolog of Sox1, and investigated its expression patterns and neural inducing activity. Sox1 was initially expressed in the anterior neural plate of Xenopus embryos, with expression restricted to the brain and optic vesicle by the tailbud stage. Expression subsequently decreased in the eye region by the tadpole stage. Sox1 expression in animal cap explants was induced by inhibition of BMP signaling in the same manner as Sox2, Sox3, and SoxD. In addition, overexpression of Sox1 induced neural markers in ventral ectoderm and in animal caps. These results implicate Xenopus Sox1 in neurogenesis, especially brain and eye development.     

GATA4, 5 and 6 mediate TGFbeta maintenance of endodermal gene expression in Xenopus embryos

The individual contributions of the three vertebrate GATA factors to endoderm formation have been unclear. Here we detail the early expression of GATA4, 5 and 6 in presumptive endoderm in Xenopus embryos and their induction of endodermal markers in presumptive ectoderm. Induction of HNF3beta by all three GATA factors was abolished when protein synthesis was inhibited, showing that these inductions are indirect. In contrast, whereas induction of Sox17alpha and HNF1beta by GATA4 and 5 was substantially reduced when protein synthesis was inhibited, induction by GATA6 was minimally affected, suggesting that GATA6 is a direct activator of these early endodermal genes. GATA4 induced GATA6 expression in the same assay and antisense morpholino oligonucleotides (MOs), designed to knock down translation of GATA6, blocked induction of Sox17alpha and HNF1beta by GATA4, suggesting that GATA4 induces these genes via GATA6 in this assay. All three GATA factors were induced by activin, although GATA4 and 6 required lower concentrations. GATA MOs inhibited Sox17alpha and HNF1beta induction by activin at low and high concentrations in the order: GATA6>GATA4>GATA5. Together with the timing of their expression and the effects of GATA MOs in vivo, these observations identify GATA6 as the predominant GATA factor in the maintenance of endodermal gene expression by TGFbeta signaling in gastrulating embryos. In addition, examination of gene expression and morphology in later embryos, revealed GATA5 and 6 as the most critical for the development of the gut and the liver.     

Six3 activation of Pax6 expression is essential for mammalian lens induction and specification

The homeobox gene Six3 regulates forebrain development. Here we show that Six3 is also crucial for lens formation. Conditional deletion of mouse Six3 in the presumptive lens ectoderm (PLE) disrupted lens formation. In the most severe cases, lens induction and specification were defective, and the lens placode and lens were absent. In Six3-mutant embryos, Pax6 was downregulated, and Sox2 was absent in the lens preplacodal ectoderm. Using ChIP, electrophoretic mobility shift assay, and luciferase reporter assays, we determined that Six3 activates Pax6 and Sox2 expression. Misexpression of mouse Six3 into chick embryos promoted the ectopic expansion of the ectodermal Pax6 expression domain. Our results position Six3 at the top of the regulatory pathway leading to lens formation. We conclude that Six3 directly activates Pax6 and probably also Sox2 in the PLE and regulates cell autonomously the earliest stages of mammalian lens induction.     

TAT-mediated PRDX6 protein transduction protects against eye lens epithelial cell death and delays lens opacity

A diminished level of endogenous antioxidant in cells/tissues is associated with reduced resistance to oxidative stress. Peroxiredoxin 6 (PRDX6), a protective molecule, regulates gene expression/function by controlling reactive oxygen species (ROS) levels. Using PRDX6 protein linked to TAT, the transduction domain from human immunodeficiency virus type 1 TAT protein, we demonstrated that PRDX6 was transduced into lens epithelial cells derived from rat or mouse lenses. The protein was biologically active, negatively regulating apoptosis and delaying progression of cataractogenesis by attenuating deleterious signaling. Lens epithelial cells from cataractous lenses bore elevated levels of ROS and were susceptible to oxidative stress. These cells harbored increased levels of active transforming growth factor (TGF)-beta 1 and of alpha-smooth muscle actin and beta ig-h3, markers for cataractogenesis. Importantly, cataractous lenses showed a 10-fold reduction in PRDX6 expression, whereas TGF-beta1 mRNA and protein levels were elevated. The changes were reversed, and cataractogenesis was delayed when PRDX6 was supplied. Results suggest that delivery of PRDX6 can postpone cataractogenesis, and this should be an effective approach to delaying cataracts and other degenerative diseases that are associated with increased ROS.