Molecular analysis of the CNS dorsalizing factor Tiarin

为了寻找在发育中的中枢神经系统中提供位置信息的新因子，我们对神经特异性分泌因子进行了系统的信号序列陷阱筛选和差异杂交。我们已经分离出一种新的中枢神经系统前部的背化因子，Xenopus Tiarin，它属于olfactomedin相关家族。表达开始于原肠期晚期，位于靠近前神经板的非神经外胚层。Tiarin在胚胎中的过度表达导致背神经标记物的扩张和腹侧标记物的抑制。在眼形成领域，Tiarin过表达诱导视网膜标记物Rx和Pax6，抑制视柄标记物。Tiarin在缺乏中胚层组织的情况下直接激活神经组织，并拮抗Shh的腹化活性。共注射试验表明，Tiarin不直接干扰或协同BMP、Wnt或Shh信号。这表明Tiarin是一种使用新型信号通路的模式因子。在本项目中，我们过量生产了Tiarin及其相关蛋白，用于分析该家族蛋白的信号传导。通过拉下实验，我们确定了几个候选的与tiarin家族蛋白结合的膜蛋白。我们还在鸡和小鼠胚胎中分离到了几个新的Tiarin家族成员。我们培育了3个月的lacz敲入小鼠，并观察了其在发育中的中枢神经系统和中胚层组织中的表达。我们还发现，鸡胚中cONT1的过度表达会导致神经嵴细胞的过量产生。

To search for new factors that provide positional information in the developing CNS, we have performed a systematic signal-sequence-trap screen combined with differential hybridization for neural-specific secreted factors. We have isolated a novel dorsalizing factor of the anterior CNS, Xenopus Tiarin, which belongs to the Olfactomedin-related family. Expression starts at the late gastrula stage in the non-neural ectoderm adjacent to the anterior neural plate. Overexpression of Tiarin in the embryo causes expansion of dorsal neural markers and suppression of ventral markers. In the eye-forming field, Tiarin overexpression induces the retinal markers Rx and Pax6 and represses optic stalk markers. Tiarin directly dorsalizes neural tissues in the absence of mesodermal tissues, and antagonizes the ventralizing activity of Shh. Co-injection assays have shown that Tiarin does not directly interfere or cooperate with BMP, Wnt or Shh signaling. This suggests that Tiarin is a patterning factor using a novel signaling pathway. In the present project, we have overproduced Tiarin and its related proteins for analyzing signal traduction of this family proteins. By using pulldown assay, we have identified a few candidate membrane proteins that bind to Tiarin-family proteins. We have also isolated several new Tiarin family members in chick and mouse embryos. We have generated LacZ-knock-in mice for mONT3,and observed its expression in the developing CNS and mesodermal tissues. We have also found that overexpression of cONT1 in the chick embryo causes overproduction of neural crest cells.