Ethanol induced acetylation of histone at G9a exon1 and G9a-mediated histone H3 dimethylation leads to neurodegeneration in neonatal mice.

The transient exposure of immature rodents to ethanol during postnatal day 7 (P7), comparable to a time point within the third trimester of human pregnancy, induces neurodegeneration. However, the molecular mechanisms underlying the deleterious effects of ethanol on the developing brain are poorly understood. In our previous study, we showed that a high dose administration of ethanol at P7 enhances G9a and leads to caspase-3-mediated degradation of dimethylated H3 on lysine 9 (H3K9me2). In this study, we investigated the potential role of epigenetic changes at G9a exon1, G9a-mediated H3 dimethylation on neurodegeneration and G9a-associated proteins in the P7 brain following exposure to a low dose of ethanol. We found that a low dose of ethanol induces mild neurodegeneration in P7 mice, enhances specific acetylation of H3 on lysine 14 (H3K14ace) at G9a exon1, G9a protein levels, augments the dimethylation of H3K9 and H3 lysine 27 (H3K27me2). However, neither dimethylated H3K9 nor K27 underwent degradation. Pharmacological inhibition of G9a activity prior to ethanol treatment prevented H3 dimethylation and neurodegeneration. Further, our immunoprecipitation data suggest that G9a directly associates with DNA methyltransferase (DNMT3A) and methyl-CpG-binding protein 2 (MeCP2). In addition, DNMT3A and MeCP2 protein levels were enhanced by a low dose of ethanol that was shown to induce mild neurodegeneration. Collectively, these epigenetic alterations lead to association of G9a, DNMT3A and MeCP2 to form a larger repressive complex and have a significant role in low-dose ethanol-induced neurodegeneration in the developing brain.

Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo.

The region of the amniote embryo corresponding to Spemann's organizer in amphibians is Hensen's node, which lies at the tip of the primitive streak during gastrulation. It is a special site in the embryo that can be defined by the presence of progenitors of several axial tissues (notochord, prechordal mesoderm, somites, gut endoderm), by characteristic cell movements, by specific patterns of gene expression (e.g. goosecoid, HNF-3beta, Sonic hedgehog) and, most importantly, by its ability to induce a complete axis, including host-derived neural tissue, when transplanted to an ectopic site. Here, we show that complete removal not only of the node but also of the anterior 40% of the primitive streak leads to the development of normal embryos containing cells with all the fates normally produced by the node. Cell movement pathways through the regenerated node are identical to those seen in the normal embryo. The patterns of expression of HNF-3beta and Sonic hedgehog are also restored, as is their left/right asymmetry, but goosecoid expression is not. When the regenerated node is transplanted to an ectopic site, it induces a complete embryonic axis that includes a fully patterned, host-derived central nervous system. Analysis of the properties of cells surrounding the site of ablation shows that they acquire these properties gradually. We suggest that the organizer is a region of the embryo that is defined by cell interactions and that the node normally inhibits the organizer state in neighbouring cells.

Fates and migratory routes of primitive streak cells in the chick embryo.

We have used carbocyanine dyes to fate map the primitive streak in the early chick embryo, from stages 3+ (mid-primitive streak) to 9 (8 somites). We show that presumptive notochord, foregut and medial somite do not originate solely from Hensen's node, but also from the anterior primitive streak. At early stages (4- and 4), there is no correlation between specific anteroposterior levels of the primitive streak and the final position of their descendants in the notochord. We describe in detail the contribution of specific levels of the primitive streak to the medial and lateral halves of the somites. To understand how the descendants of labelled cells reach their destinations in different tissues, we have followed the movement of labelled cells during their emigration from the primitive streak in living embryos, and find that cells destined to different structures follow defined pathways of movement, even if they arise from similar positions in the streak. Somite and notochord precursors migrate anteriorly within the streak and pass through different portions of the node; this provides an explanation for the segregation of notochord and somite territories in the node.