Research School of Biology, Australian National University, ACT 2601, Canberra, Australia.
Dev Genes Evol. 2012 Nov;222(6):361-8. doi: 10.1007/s00427-012-0414-8. Epub 2012 Sep 4.
The Rho GTP exchange factor, Pebble (Pbl), long recognised as an essential activator of Rho during cytokinesis, also regulates mesoderm migration at gastrulation. Like other cell cycle components, pbl expression patterns broadly correlate with proliferative tissue. Surprisingly, in spite of its role in the early mesoderm, pbl is downregulated in the presumptive mesoderm before ventral furrow formation. Here, we show that this mesoderm-specific repression of pbl is dependent on the transcriptional repressor Snail (Sna). pbl repression was lost in sna mutants but was unaffected when Sna was ectopically expressed, showing that Sna is necessary, but not sufficient, for pbl repression. Using DamID, the first intron of pbl was identified as a Sna-binding region. Nine sites with the Sna-binding consensus motif CAGGT[GA] were identified in this intron. Mutating these to TAGGC[GA] abolished the ventral repression of pbl. Surprisingly, Sna-dependent repression of pbl was not essential for viability or fertility. Loss of repression did, however, increase the frequency of low-penetrance gastrulation defects. Consistent with this, expression of a pbl-GFP transgene in the presumptive mesoderm generated similar gastrulation defects. Finally, we show that a cluster of Snail-binding sites in the middle of the first intron of pbl orthologues is a conserved feature in the other 11 sequenced Drosophila species. We conclude that pbl levels are precisely regulated to ensure that there is enough protein available for its role in early mesoderm development but not so much as to inhibit the orderly progression of gastrulation.
Rho GTP 交换因子 Pebble（Pbl）长期以来被认为是胞质分裂过程中 Rho 的必需激活因子，它还调节原肠胚形成时的中胚层迁移。与其他细胞周期成分一样，pbl 的表达模式与增殖组织广泛相关。令人惊讶的是，尽管 pbl 在早期中胚层中起作用，但在腹侧沟形成之前，其在假定的中胚层中被下调。在这里，我们表明，这种 pbl 的中胚层特异性抑制依赖于转录抑制因子 Snail（Sna）。在 sna 突变体中，pbl 的抑制作用丧失，但当 Sna 异位表达时，pbl 的抑制作用不受影响，表明 Sna 是必需的，但不是充分的，pbl 的抑制作用。使用 DamID，鉴定了 pbl 的第一个内含子是 Sna 结合区域。在这个内含子中，发现了九个具有 Sna 结合基序 CAGGT[GA]的位点。将这些突变为 TAGGC[GA]，则会破坏 pbl 的腹侧抑制。令人惊讶的是，pbl 的 Sna 依赖性抑制对于存活力或繁殖力不是必需的。然而，抑制作用的丧失确实增加了低 penetrance 原肠胚形成缺陷的频率。这与表达 GFP 标记的 pbl 转基因在假定的中胚层中产生类似的原肠胚形成缺陷一致。最后，我们表明，pbl 同源物第一个内含子中间的一组 Snail 结合位点是在其他 11 个已测序的果蝇物种中保守的特征。我们得出结论，pbl 水平受到精确调节，以确保有足够的蛋白质用于其在早期中胚层发育中的作用，但不会抑制原肠胚形成的有序进展。
