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（WASL）是细胞内F-肌动蛋白动力学所必需的。WASL也有 基本的，但还没有很好地表征，在转录和细胞核中的表观遗传调控的作用， 肌动蛋白依赖型和肌动蛋白非依赖型。WASL的这些多方面作用之间的平衡是关键， 了解其调节功能，将外部环境信号与细胞行为和分化联系起来。 由于这些细胞过程的失调与癌症侵袭性和肿瘤性增加有关， 细胞转化，理解WASL调节的动力学是至关重要的。我们最近发现了 WASL在调节发育模式中的惊人作用：我们发现WASL对发育模式的形成是必要的。 骨骼模式的形成和增加的WASL信号传导导致新的骨骼元件的形成。 重要的是，我们发现WASL的这种模式化作用在脊椎动物中是保守的。发展功能 对于WASL来说，考虑到其在细胞中的核心功能，这是以前未知和意外的。但随着 WASL活性的失调导致细胞质F-肌动蛋白调节的破坏以及细胞内 转录，WASL如何编排这些发育事件背后的特定信号仍不清楚。 在这里，我们利用斑马鱼和小鼠中新配对的功能获得和丧失模型来解决 WASL通过调节转录调节骨骼发育的中心假设 并且独立于其在细胞质F-肌动蛋白动力学中的作用。我们概述了三种独立的方法 来直接解决这个假设。在目标1中，我们将利用WASL蛋白的模块化性质， 去除建立F-肌动蛋白成核所需的蛋白质的特定区域。这些突变的等位基因 WASL的基因将与我们已经鉴定的特异性功能获得性WASL等位基因进行比较， 以及顺式，通过分析WASL在细胞内的定位，细胞质F-肌动蛋白的形成，Hox基因 转录，以及骨骼模式。然后在目标2中，我们将使用我们的WASL损失和收益模型 活性，以确定与WASL调节相关的特定转录和表观遗传变化， 肢和鳍的发育。这使我们能够确定明确的转录签名的WASL在发展中 以及它们对Was 1F-肌动蛋白结合的依赖性。我们将进一步评估F-肌动蛋白形成的依赖性， WASL对肢芽细胞软骨分化的调控及其对转录调控的影响 在发展过程中。最后，在目标3中，我们利用WASL氨基酸序列的自然变异来改进 特定的磷酸化残基作为骨骼多样化的潜在关键调节因子。使用我们的新 实验工具，我们将解析WASL的活动和功能，在骨骼生长和模式的调节 在发展过程中。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指导性作用，以及这种整合中的变化如何 导致产生新颖结构和形式上的变化。