A novel role for Wasl signaling in the regulation of skeletal patterning

Wasl 信号在骨骼模式调节中的新作用

• 批准号: 10718448
• 负责人: Matthew P Harris
• 金额: $ 53.42万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10718448
• 关键词: ANGPTL2 gene; ATAC-seq; Actins; Address; Adopted; Affect; Alleles; Amino Acid Sequence; Articulation; Behavior; Binding; C-terminal; Cell Differentiation process; Cell Nucleus; Cell physiology; Cells; Chromatin; Complement; Cytoplasm; Cytoskeleton; DNA-Directed RNA Polymerase; Data; Dependence; Development; Developmental Gene; Elements; Environment; Epigenetic Process; Equilibrium; Event; Experimental Models; Extracellular Matrix; F-Actin; Family member; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Expression Regulation; Generations; Genes; Genetic Epistasis; Genetic Transcription; Growth; Homeobox Genes; Investigation; Limb Bud; Limb Development; Limb structure; Link; Mediating; Microfilaments; Modeling; Morphology; Mus; Mutation; Nature; Neoplastic Cell Transformation; Nuclear; Pattern; Phenotype; Phosphorylation; Protein Region; Proteins; Regulation; Regulator Genes; Reporting; Role; Shapes; Signal Transduction; Site; Skeletal Development; Skeleton; Specificity; Stains; Structure; Testing; Tetrapoda; Time; Transcriptional Regulation; Variant; Vertebrates; Wasps; Wiskott-Aldrich Syndrome; Zebrafish; appendage; cancer invasiveness; cell behavior; density; epigenetic regulation; gain of function; gene discovery; gene function; in vivo; long bone; loss of function; mRNA sequencing; migration; mouse development; mutant; novel; response; skeletal; stem; tomography; tool; transcriptome sequencing

Wiskott-Aldrich Syndrome-Like, WASL, is essential for F-actin dynamics within cells. WASL also has fundamental, yet not well characterized, roles in transcription and epigenetic regulation in the nucleus that are actin-dependent and actin-independent. The balance between these multifaceted roles of WASL is key in understanding its regulatory function tying external environmental signals to cellular behavior and differentiation. As dysregulation of these cellular processes have been tied to increased cancer invasiveness and neoplastic cell transformation, it is essential to understand the dynamics of WASL regulation. We recently uncovered a surprising role for WASL in regulating developmental patterning: We find that WASL is necessary for the formation of skeletal pattern and increased WASL signaling leads to the formation of novel skeletal elements. Importantly, we find that this patterning role for WASL is conserved across vertebrates. A developmental function for WASL was previously unknown and unexpected given its core functionality in the cell. However, as misregulated WASL activity results in disruption of both cytoplasmic F-actin regulation as well as changes in transcription, it remains unclear how WASL orchestrates specific signals underlying these developmental events. Here, we capitalize on new paired gain- and loss-of-function models in the zebrafish and the mouse to address the central hypothesis that WASL regulates skeletal development through modulation of transcription and is independent of its role in cytoplasmic F-actin dynamics. We outline three independent approaches to directly address this hypothesis. In Aim 1, we will take advantage of the modular nature of WASL protein and remove specific regions of the protein required for establishing F-actin nucleation. These ∆VCA mutant alleles of WASL will be compared against the specific gain-of-function WASL allele we have identified, both separately as well as in cis, through analysis of WASL localization within the cell, cytoplasmic F-actin formation, Hox gene transcription, as well as skeletal patterning. Then in Aim 2, we will use our models of loss and gain of WASL activity to define the specific transcriptional and epigenetic changes associated with WASL regulation during limb and fin development. This allows us to identify definitive transcriptional signatures of WASL in development and their dependence on Wasl F-actin binding. We will further assess the dependence of F-actin formation in WASL regulation of chondrogenic differentiation of limb bud cells and how this affects transcriptional modulation during development. Lastly, in Aim 3, we capitalize on natural variation in WASL amino acid sequence to refine specific phosphorylated residues as potential key regulators of skeletal diversification. Using our new experimental tools, we will parse the regulation of WASL activity and function in skeletal growth and patterning during development. Through the completion of these approaches we will broaden our understanding of the intricate, and instructive roles of WASL in cell behavior and differentiation and how shifts in this integration can lead to generation of novel structures and variation in form.

Wiskott-Aldrich综合征样的Wasl对于细胞内的F-肌动蛋白动力学至关重要。 Wasl也有 基本的，但没有很好地表征在转录和表观遗传调节中的作用 肌动蛋白依赖性和肌动蛋白独立。 WASL的这些多面角色之间的平衡是关键 了解其调节功能将外部环境信号与细胞行为和分化联系在一起。 由于这些细胞过程的失调与癌症的侵入性和肿瘤相关 细胞转化，必须了解WASL调节的动力学。我们最近发现了 WASL在监管发展模式中的惊人作用：我们发现WASL对于 骨骼模式的形成和增加的WASL信号传导导致新型骨骼元素的形成。 重要的是，我们发现WASL的这种图案作用是在脊椎动物之间配置的。发展功能 因为WASL以前是未知的，并且鉴于其在细胞中的核心功能鉴于其核心功能。但是，如 误导的WASL活性导致细胞质F-肌动蛋白调节的破坏以及变化 转录，目前尚不清楚Wasl如何协调这些发展事件的特定信号。 在这里，我们利用斑马鱼和鼠标的新配对增益和功能丧失模型来解决 WASL通过调节转录来调节骨骼发育的中心假设 并且独立于其在细胞质F-肌动力学中的作用。我们概述了三种独立的方法 直接解决这一假设。在AIM 1中，我们将利用Wasl蛋白的模块化性质和 去除建立F-肌动蛋白成核所需的蛋白质的特定区域。这些∆VCA突变等位基因 将WASL与我们已经识别的特定功能获得的废水等位基因进行比较，两者单独 以及在顺式中，通过分析细胞中的WASL定位，细胞质F-肌动蛋白形成，HOX基因 转录以及骨骼图案。然后在AIM 2中，我们将使用损失模式和瓦斯尔的收益 活性以定义与WASL调节相关的特定转录和表观遗传变化 肢体和鳍开发。这使我们能够确定WASL开发的确定转录特征 以及它们对WASL F-肌动蛋白结合的依赖。我们将进一步评估F-肌动蛋白在 WASL调节肢体芽细胞的软骨分化以及这如何影响转录调节 在开发过程中。最后，在AIM 3中，我们利用了Wasl氨基酸序列的自然变化以完善 特定的磷酸化残留物作为骨骼多样化的潜在关键调节剂。使用我们的新 实验工具，我们将解析WASL活性和骨骼生长中功能的调节 在开发过程中。通过完成这些方法，我们将扩大我们对 WASL在细胞行为和分化中的复杂和有指导的作用以及这种整合中的变化如何可以 导致新颖结构的产生和形式变化。