Integration of Lef/Tcf isoforms in regulatory networks involved in brain pattering

LEF/TCF家族序列特异性HMG-box转录因子作为致癌Wnt/b-catenin信号通路的核介质，在胚胎发生和恶性肿瘤的发生发展中起着重要的决定作用。在之前的资助期间，我们通过鉴定这些分子中的几个抑制和激活结构域，表征了爪蟾中单个LEF/TCF家族成员的不同特性。XTCF3、xtcf - 4a、B、C和XLEF-1的不同表达模式和特性暗示了这些转录因子在典型wnt信号传导中作为细胞应答调节剂的功能。为了验证这一假设，我们将重点关注单个LEF/TCF转录因子在大脑模式中的作用，它们在大脑中表现出不同的非重叠表达谱。为了确定单个转录因子的功能，我们将使用反义morpholino注射与其他LEF/TCF家族成员的异位表达联合使用。大脑模式的改变将伴随着标记基因的表达。这些实验将在整个胚胎以及神经化动物帽外植体上进行。在第二部分中，我们将进一步澄清转录因子的不同性质是如何在其结构中奠定的。为了寻找特定的相互作用伙伴，我们将使用重要的LEF/TCF结构域的双标记融合蛋白进行串联亲和纯化（TAP），然后对共沉淀进行MALDI-TOF分析。有趣的交互伙伴将被隔离并进一步表征。它们在典型wnt信号传导中的调节功能将通过双轴形成和报告基因检测进行研究，如果它们在某些脑区与LEF/TCF转录因子共表达，则主要在脑模式中进行研究。

As nuclear mediators of the oncogenic Wnt/b-catenin signaling pathway, the sequence specific HMG-box transcription factors of LEF/TCF family graduate important decisions in embryogenesis and in the development of malignat carcinoma. In the previous funding period we characterized the different properties of the individual LEF/TCF family members in Xenopus by identifying several repressive and activating domains within these molecules. The different expression patterns and properties of XTCF3, XTCF-4 A, B, C and XLEF-1 implicate a function of these transcription factors as modifiers of the cellular response in canonical Wnt-signaling. To test this hypothesis we will focus on the role of individual LEF/TCF transcription factors in patterning the brain where they exhibit distinct non-overlapping expression profiles. To identify the function of single transcription factors we will apply antisense morpholino injections in combination with ectopic expression of the other LEF/TCF family members. Altered patterning of the brain will be followed by marker gene expression. These experiments will be performed with whole embryos as well as with neuralized animal cap explants. In a second part we will further clear up how the different properties of the transcription factors are layed down in their structure. In search for specific interaction partners we will perform Tandem-Affinity Purification (TAP) using double tagged fusion proteins of important LEF/TCF domains followed by MALDI-TOF analyses of the coprecipitates. Interesting interaction partners will be isolated and further caracterized. Their regulative function in canonical Wnt-signaling will be studied by double axis formation and reporter gene assays, and predominantly in brain patterning if they are co-expressed with LEF/TCF transcription factors in certain brain compartiments.