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

基本信息

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
项目类别：
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

项目摘要

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) 对于细胞内 F-肌动蛋白动力学至关重要。细胞核转录和表观遗传调控中的基本但尚未充分表征的作用 WASL 的这些多方面作用之间的平衡是肌动蛋白依赖性和肌动蛋白非依赖性的关键。了解其将外部环境信号与细胞行为和分化联系起来的调节功能。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中，了解 WASL 调节的动态至关重要。 WASL 在调节发育模式中的令人惊讶的作用：我们发现 WASL 对于骨骼模式的形成和 WASL 信号传导的增加导致新骨骼元素的形成。重要的是，我们发现 WASL 的这种模式作用在脊椎动物中是保守的。 WASL 以前是未知的，并且由于其在单元中的核心功能而出人意料。 WASL 活性调节不当会导致细胞质 F-肌动蛋白调节的破坏以及转录，目前尚不清楚 WASL 如何协调这些发育事件背后的特定信号。在这里，我们利用斑马鱼和小鼠的新配对功能获得和丧失模型来解决 WASL 通过转录调节来调节骨骼发育的中心假设并且与其在细胞质 F-肌动蛋白动力学中的作用无关，我们概述了三种独立的方法。在目标 1 中，我们将利用 WASL 蛋白的模块化性质来直接解决这一假设。去除建立 F-肌动蛋白成核所需的蛋白质的特定区域。这些 ΔVCA 突变等位基因。 WASL 的基因将与我们已经确定的特定功能获得性 WASL 等位基因进行比较，两者都是分开的以及顺式，通过分析细胞内的 WASL 定位、细胞质 F-肌动蛋白形成、Hox 基因然后在目标 2 中，我们将使用 WASL 的损失和增益模型。定义与 WASL 调节相关的特定转录和表观遗传变化的活性这使我们能够识别发育中 WASL 的明确转录特征。以及它们对 Wasl F-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节及其如何影响转录调节最后，在目标 3 中，我们利用 WASL 氨基酸序列的自然变异来改进。使用我们的新方法，将特定的磷酸化残基作为骨骼多样化的潜在关键调节剂。实验工具，我们将解析WASL活性和功能在骨骼生长和模式形成中的调节通过完成这些方法，我们将拓宽我们对开发过程的理解。 WASL 在细胞行为和分化中的复杂且具有指导意义的作用，以及这种整合的转变如何影响导致新颖结构的产生和形式的变化。