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 指导许多细胞类型的规格。一种这样的信号传导途径是经典Wnt信号传导途径， 在动物中高度保守，在无数的发育过程中发挥作用， 失调在疾病中是常见的。为了指导不同的发育结果，Wnt信号必须激活 不同的基因调控网络（GRNs）在不同的背景下。为了揭示Wnt配体 可以激活独特的GRNs，我将使用Hydra vulgaris来发现两种不同的细胞类型如何独特地响应 Wnt信号通路。Hydra为研究Wnt定向GRNs提供了几个优势：1）Hydra是一个 相对简单的有机体，我们已经在分子和空间上定义了所有的细胞类型，2）成年水螅是 处于恒定的发育状态，使得所有的发育途径，包括Wnt导向的途径， 持续活跃。Wnt信号在Hydra的口端是高的（即，头部），并指导分化 多种不同的口腔细胞命运经典Wnt信号传导的主要作用是稳定β- 连环蛋白（Bcat）蛋白，其与其结合伴侣TCF一起激活靶基因的转录。到 在特定的发育环境中激活靶基因的表达，Bcat/TCF必须在一个组合中起作用。 与其他TF一起时尚。然而，在很大程度上不知道什么样的TF促进Wnt靶点的激活， 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， 我假设外胚层同源异型盒转录因子和内胚层bHLH转录因子以组合方式起作用 与Bcat/TCF一起指导Hydra中的细胞类型特定GRN模块。为了验证这个假设，我将使用 ChIP-seq鉴定两种口腔上皮细胞类型Hydra中Bcat/TCF的细胞类型特异性直接靶点 （外胚层和内胚层）（目的1）。然后我会用我们的九头蛇单细胞图谱来确定 的直接目标。为了确定直接靶点是否受同源异型框或bHLH TF的共同调节，我将敲低 上皮细胞中的这些TF，以测试它们是否是规格所需的。然后我会确认Wnt目标 通过对敲低的Hydra进行RNA-seq，也需要这些TF来正确表达的基因（Aim 2）。最后，我将执行无偏的方法，以确定额外的共同调节TF的功能测试（目的 3），如果需要，还将提供替代假设。在这个项目完成后，我将产生 Bcat/TCF在Hydra中的主要目标的全面列表，并可能发现了 同源框和bHLH转录因子在差异调节这些主要目标。在小鼠中进行的ChIP实验， 已经表明Bcat可以结合特异性同源框TF以控制不同细胞中靶基因的表达， 发展背景。因此，我的研究结果可能表明同源异型框转录因子在人类免疫系统中的作用非常保守。 从刺胞动物到两侧体动物，在不同的发育环境中调节不同的Wnt靶点。

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.