Molecular analysis of the CNS dorsalizing factor Tiarin

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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.