Solving cell-type specific differences of the Wnt-directed gene regulatory network in Hydra vulgaris.

发育生物学的一个基本问题是了解有限数量的信号通路是如何 指导多种细胞类型的规范。其中一个这样的信号通路是典型的Wnt信号通路， 它在动物中高度保守，在无数的发育过程中发挥着作用，它的 失调在疾病中很常见。为了引导不同的发育结果，Wnt信号必须被激活 在不同的背景下，不同的基因调控网络(GRN)。揭示WNT配体的一般原理 可以激活独特的GRN，我将使用普通九头蛇来发现两种不同的细胞类型如何独特地响应 Wnt信号通路。九头蛇为研究WNT定向GRN提供了几个优势：1)九头蛇是一种 相对简单的生物体，我们已经在分子和空间上定义了所有细胞类型，2)成体九头蛇是 在不断发展的状态下，所有的发育途径，包括WNT导向的途径， 是持续活跃的。WNT信号在九头蛇的口腔末端(即头部)很高，并指导分化 多种截然不同的口腔细胞命运。规范的Wnt信号的主要作用是稳定β- 连环蛋白(BCAT)，它与其结合伙伴TCF一起激活靶基因的转录。至 在特定的发育环境中激活靶基因表达，BCAT/TCF必须在组合中发挥作用 与其他女性一起时尚。然而，在很大程度上还不清楚是什么因子促进了WNT靶标的激活和 这些相互作用是否在物种间和疾病期间是保守的。根据我的初步数据， 我假设外胚层同源盒因子和内胚层bHLHTF以一种组合方式起作用。 通过BCAT/TCF将特定单元类型的GRN模块定向到九头蛇中。为了验证这一假设，我将使用 CHIP-SEQ在两种口腔上皮细胞类型中鉴定BCAT/TCF的细胞类型特异性直接靶点 (外胚层和内胚层)(目标1)。然后我将使用我们的Hydra单细胞图谱来确定表达模式 直接目标的数量。为了确定直接靶标是否受到Homeobox或bHLHTF的共同调控，我将敲除 这些转录因子在上皮细胞中测试它们是否需要规格。然后我会确定WNT的目标 也需要这些转录因子通过在击倒的Hydra(AIM)上进行RNA-SEQ来正确表达的基因 2)。最后，我将执行不偏不倚的方法来确定用于功能测试(AIM)的其他共同调节的TF 3)，如果需要，它还将提供替代假设。在这个项目完成后，我将生成 BCAT/TCF在九头蛇中的主要目标的综合清单，并可能已发现 Homeobox和bHLHTF在差异调节这些主要靶点中的作用。在老鼠身上进行的芯片实验， 已经证明BCAT可以结合特定的同源盒转录因子来控制不同基因在不同组织中的表达 发展背景。因此，我的结果可能表明，Homeobox TF在 在不同的发育环境中调节不同的WNT靶标，从线虫到双边动物。

A fundamental question of developmental biology is to understand how a limited number of signaling pathways direct the specification of many cell types. One such signaling pathway is the canonical Wnt signaling pathway, which is highly conserved across animals, plays a role in a myriad of developmental processes, and its dysregulation is common in disease. To direct different developmental outcomes, Wnt signaling must activate different gene regulatory networks (GRNs) in different contexts. To reveal general principles of how Wnt ligands can activate unique GRNs, I will use Hydra vulgaris to discover how two distinct cell types uniquely respond to the Wnt signaling pathway. Hydra offer several advantages for studying Wnt directed-GRNs: 1) Hydra is a relatively simple organism and we have molecularly and spatially defined all cell types and 2) the adult Hydra is in a constant state of development such that all developmental pathways, including the Wnt-directed pathways, are continuously active. Wnt signaling is high at Hydra’s oral end (i.e., the head) and directs the differentiation of multiple distinct oral cell fates. The principal effect of canonical Wnt signaling is the stabilization of the beta- catenin (Bcat) protein, which together with its binding partner TCF activates transcription of target genes. To activate target gene expression in specific developmental contexts, Bcat/TCF must work in a combinatorial fashion with other TFs. However, it is largely unknown what TFs are facilitating the activation of Wnt targets and whether these interactions are conserved across species and during disease. Based on my preliminary data, I hypothesize that ectodermal Homeobox TFs and endodermal bHLH TFs work in a combinatorial manner with Bcat/TCF to direct cell-type specific GRN modules in Hydra. Towards testing this hypothesis, I will use ChIP-seq to identify the cell-type specific direct targets of Bcat/TCF in the two oral epithelial cell types of Hydra (ectoderm and endoderm) (Aim 1). I will then use our Hydra single cell Atlas to determine the expression pattern of the direct targets. To determine if direct targets are co-regulated by Homeobox or bHLH TFs, I will knockdown these TFs in the epithelial cells to test if they are required for specification. I will then identify the Wnt target genes that also require these TFs for proper expression by conducting RNA-seq on the knockdown Hydra (Aim 2). Finally, I will perform unbiased approaches to identify additional co-regulating TFs for functional testing (Aim 3), which will also provide alternative hypotheses if needed. Upon completion of this project, I will have generated a comprehensive list of the primary targets of Bcat/TCF in Hydra and potentially have discovered a role for Homeobox and bHLH TFs in differentially regulating these primary targets. ChIP experiments conducted in mice, have shown that Bcat can bind specific Homeobox TFs to control target gene expression in different developmental contexts. Therefore, my results could suggest a deeply conserved role for Homeobox TFs in regulating different Wnt targets in different developmental contexts from cnidarians to bilaterians.