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

解决水螅中 Wnt 导向基因调控网络的细胞类型特异性差异。

• 批准号: 10751675
• 负责人: Hannah Morris Little
• 金额: $ 4.03万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-03 至 2025-07-02
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751675
• 关键词: ATAC-seq; Address; Adult; Animals; Atlases; Binding; Cells; ChIP-seq; Cnidaria; Complex; Data; Development; Developmental Biology; Developmental Gene; Developmental Process; Disease; Ectoderm; Endoderm; Epithelial Cells; Gene Expression; Genes; Genetic Transcription; Goals; Head; Homeobox; Ligands; Malignant Neoplasms; Mediating; Modeling; Molecular; Morphology; Mus; Oral; Organism; Outcome; Output; Pathway interactions; Pattern; Proteins; RNA Interference; Regulation; Resolution; Role; Signal Pathway; Signal Transduction; Sorting; Specific qualifier value; System; Testing; Transcriptional Activation; WNT Signaling Pathway; Work; beta catenin; cell type; combinatorial; developmental disease; directed differentiation; experimental study; gene regulatory network; knock-down; oral cavity epithelium; response; single-cell RNA sequencing; transcription factor; transcriptome sequencing

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.

发育生物学的一个基本问题是了解有限数量的信号通路如何 指导许多细胞类型的规范的一种信号传导途径是典型的 Wnt 信号传导途径， 它在动物中高度保守，在无数的发育过程中发挥着作用，其 失调在疾病中很常见。为了指导不同的发育结果，Wnt 信号传导必须激活。 不同背景下的不同基因调控网络 (GRN) 揭示 Wnt 配体如何发挥作用的一般原理。 可以激活独特的 GRN，我将使用普通水螅来发现两种不同的细胞类型如何独特地响应 Wnt 信号通路为研究 Wnt 定向 GRN 提供了几个优势：1) Hydra 是一种 相对简单的生物体，我们在分子和空间上定义了所有细胞类型，2）成年水螅是 处于持续的发育状态，使得所有发育途径，包括 Wnt 定向途径， Wnt 信号在水螅的口端（即头部）持续活跃并指导分化。 经典 Wnt 信号传导的主要作用是 β- 的稳定。 连环蛋白 (Bcat) 蛋白，与其结合伙伴 TCF 一起激活靶基因的转录。 在特定的发育环境中激活靶基因表达，Bcat/TCF 必须以组合方式发挥作用 然而，目前尚不清楚哪些 TF 能够促进 Wnt 靶标的激活和 根据我的初步数据，这些相互作用在物种间和疾病期间是否保守。 我发现外胚层同源框 TF 和内胚层 bHLH TF 以组合方式发挥作用 为了测试这个假设，我将使用 Bcat/TCF 来指导 Hydra 中的细胞类型特定 GRN 模块。 ChIP-seq 用于识别 Hydra 两种口腔上皮细胞类型中 Bcat/TCF 的细胞类型特异性直接靶标 （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。 为了确定直接目标是否受到同源盒或 bHLH TF 的共同调控，我将击倒。 上皮细胞中的这些 TF 来测试它们是否是规范所必需的，然后我将确定 Wnt 靶点。 通过对敲低 Hydra 进行 RNA 测序，也需要这些 TF 才能正确表达的基因（目标 2).最后，我将采用公正的方法来确定用于功能测试的其他共同调节 TF（目标 3），如果需要的话，我还将提供替代假设。 Bcat/TCF 在 Hydra 中的主要目标的完整列表，并可能发现了其作用 在小鼠中进行的同源框和 bHLH TF 差异调节这些主要靶标的实验。 研究表明，Bcat 可以结合特定的同源盒转录因子 (Homeobox TF) 来控制不同细胞中靶基因的表达。 因此，我的结果可能表明同源框转录因子在发育中具有高度保守的作用。 从刺胞动物到两侧对称动物，在不同的发育背景下调节不同的 Wnt 靶点。