Abstract：
Using a transgenic zebrafish line harboring a heat-inducible dominant-interference pou5f3 gene (en-pou5f3), we reported that this PouV gene is involved in isthmus development at the midbrain-hindbrain boundary (MHB), which patterns the midbrain and cerebellum. Importantly, the functions of pou5f3 reportedly differ before and after the end of gastrulation. In the present study, we examined in detail the effects of en-pou5f3 induction on isthmus development during embryogenesis. When en-pou5f3 was induced around the end of gastrulation (bud stage), the isthmus was abrogated or deformed by the end of somitogenesis (24 hours post-fertilization). At this stage, the expression of MHB markers -- such as pax2a, fgf8a, wnt1, and gbx2 -- was absent in embryos lacking the isthmus structure, whereas it was present, although severely distorted, in embryos with a deformed isthmus. We further found that, after en-pou5f3 induction at late gastrulation, pax2a, fgf8a, and wnt1 were immediately and irreversibly downregulated, whereas the expression of en2a and gbx2 was reduced only weakly and slowly. Induction of en-pou5f3 at early somite stages also immediately downregulated MHB genes, particularly pax2a, but their expression was restored later. Overall, the data suggested that pou5f3 directly upregulates at least pax2a and possibly fgf8a and wnt1, which function in parallel in establishing the MHB, and that the role of pou5f3 dynamically changes around the end of gastrulation. We next examined the transcriptional regulation of pax2a using both in vitro and in vivo reporter analyses; the results showed that two upstream 1.0-kb regions with sequences conserved among vertebrates specifically drove transcription at the MHB. These reporter analyses confirmed that development of the isthmic organizer is regulated by PouV through direct regulation of pax2/pax2a in vertebrate embryos.
摘要：
使用带有热诱导显性干扰pou5f3基因（en-pou5f3）的转基因斑马鱼品系，我们报道了这个PouV基因参与中脑-后脑边界（MHB）的峡部发育，形成中脑和小脑的图案。重要的是,据报道，pou5f3的功能在原肠胚形成之前和之后有所不同。在本研究中,我们详细研究了en-pou5f3诱导对胚胎发生过程中峡部发育的影响。当在原肠胚形成结束时(芽阶段)诱导en-pou5f3，在体发生结束时（受精后24小时），峡部被废除或变形。在这个阶段,MHB标记的表达——如pax2a，fgf8a,wnt1和gbx2-在缺乏峡部结构的胚胎中不存在，虽然它存在，虽然严重扭曲，地峡变形的胚胎。我们进一步发现,在晚期原肠胚形成的en-pou5f3诱导后，pax2a,fgf8a,wnt1被立即和不可逆转地下调，而en2a和gbx2的表达仅微弱且缓慢地降低。在早期体节阶段诱导en-pou5f3也立即下调MHB基因，尤其是pax2a，但是他们的表情后来恢复了。总的来说,数据表明，pou5f3直接上调至少pax2a和可能的fgf8a和wnt1，它们在建立MHB时并行起作用，并且pou5f3的作用在原肠胚形成结束时动态变化。接下来，我们使用体外和体内报告分子分析检查了pax2a的转录调节；结果表明，在脊椎动物中具有保守序列的两个上游1.0kb区域特异性地驱动了MHB的转录。这些报告分子分析证实，PouV通过直接调节脊椎动物胚胎中的pax2/pax2a来调节峡部组织者的发育。
