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.

发育生物学的基本问题是了解有限数量的信号通路 指导许多细胞类型的规范。这样的信号通路之一是规范的Wnt信号通路， 在整个动物之间是高度保守的，在无数的发展过程中起作用，及其 失调在疾病中很常见。要指导不同的发展结果，Wnt信号必须激活 在不同情况下，不同的基因调节网络（GRN）。揭示如何配体的一般原则 可以激活独特的grn，我将使用Hydra ufgaris发现两种不同的细胞类型如何独特地响应 Wnt信号通路。 Hydra提供了研究Wnt定向GRN的几个优点：1）Hydra是一个 相关的简单生物体，我们具有分子和经常定义的所有细胞类型，2）成年九头蛇是 在恒定的发展状态下，所有发展途径，包括WNT指导的途径， 是连续活动的。 Wnt信号在Hydra的口腔端（即头部）高，并指导分化 多个不同的口服细胞命运。规范Wnt信号传导的主要作用是β-稳定 Catenin（BCAT）蛋白质及其结合伴侣TCF激活靶基因的转录。到 在特定的发育环境中激活靶基因表达，BCAT/TCF必须在组合中起作用 与其他TF的时尚。但是，在很大程度上未知TF支持Wnt目标的激活和 这些相互作用是否在物种和疾病期间保存。根据我的初步数据 我假设外胚层同型TFS和内胚层BHLH TFS以组合方式起作用 用BCAT/TCF指导Hydra中的细胞类型特异性GRN模块。要检验这一假设，我将使用 chip-seq以识别在两种口腔上皮细胞类型的hydra中BCAT/TCF的细胞类型的特定直接靶标 （外胚层和内胚层）（AIM 1）。然后，我将使用Hydra单细胞图集来确定表达模式 直接目标。为了确定直接目标是否由同源ox或BHLH TF共同调节，我将敲门 这些TF在上皮细胞中测试是否需要规格。然后，我将确定WNT目标 还需要这些TF通过在敲低hydra上进行RNA-seq来适当表达的基因（AIM 2）。最后，我将采取公正的方法来识别用于功能测试的其他共同调节的TF（AIM 3），如果需要，这也将提供替代假设。该项目完成后，我将生成 BCAT/TCF在Hydra和Hydra及 同型和BHLH TF在不同的调节这些主要目标的情况下。在小鼠中进行的芯片实验， 已经表明，BCAT可以结合特定的同型TF，以控制不同的靶基因表达 发展环境。因此，我的结果可能表明在 在不同的发育环境中，从CNIDARIAN到双侧的不同发育环境中调节不同的WNT目标。