Upstream signals of left-bide specific nodal expression in the lateral plate mesoderm

为了了解侧板中胚层的左右决定途径，我重点研究了结节的转录调控。ASE是侧板中胚层结节和Lefty2的左侧增强子。Node基因还受另一种增强子--左侧特异性增强子LSE的调控。利用转基因技术，我在节点基因上游一个300b的5kb区域鉴定了这个增强子的活性。对该区域的详细缺失分析揭示了LSE活性所必需的三个独立区域。这些区域不包含FoxH1结合序列，FoxH1结合序列调节节点和左侧2的酶。为了研究LSE在左侧结节特异表达中的作用，利用Cre-loxP系统建立了LSE基因敲除小鼠模型。用插入在loxP序列之间的pGK-neo基因替换LSE区，然后用Cre重组酶切割pGK-neo基因。与野生型小鼠相比，突变小鼠的出生后发育略有延迟。由于一些突变纯合子表现出房间隔缺陷，LSE基因的缺失影响了左右信息。因此，在交配后8天检测左侧表达基因的表达模式，如Nodeal、Lefties和Pitx2。这些基因在突变的胚胎中得到了表达。但是，突变胚节的表达开始时间推迟了约1个体节时期。

I focused on transcriptional regulation of nodal in order to understand the left-right determination pathway in the lateral plate mesoderm. ASE is a left side enhancer of nodal and lefty2 in the lateral plate mesoderm. Nodal gene is also regulated by another enhancer, the left side specific enhancer LSE. Using transgenic technique, I identified this enhancer activity in a 300b region 5 kb upstream of nodal gene. Deletion analysis of this region in detail revealed three independent regions essential for LSE activity. These regions did not contain FoxH1 binding sequences which regulate ASE of nodal and lefty2. Therefore LSE was regulated by at last three different signals other than FoxH1.In order to study the function of LSE in the left-side specific expression of nodal, LSE deleted knockout mice were generated by using the Cre-loxP system. The LSE region was replaced by a PGK-neo gene inserted between loxP sequences, and subsequently PGK-neo gene was excised by Cre recombinase. Postnatal development of the mutants was slightly delayed compared with wild type mice.Since some mutant homozygotes showed atrial septum defects left-right information was affected by the deletion of LSE. Therefore, expression patterns of left-side expressed genes such as nodal, lefties and Pitx2 were examined at 8 days post coitum. These genes were expressed in the mutant embryos. However, the beginning of the expression of nodal was delayed for about 1 somite period in mutant embryos.