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

Wiskott-Aldrich综合征样，WASL是细胞内F-肌动蛋白动力学所必需的。Wasl还拥有 在核转录和表观遗传调控中的基础作用，但尚未得到很好的表征 肌动蛋白依赖和不依赖肌动蛋白。WASL的这些多方面角色之间的平衡是 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。 因为这些细胞过程的失调与癌症侵袭性和肿瘤的增加有关 在细胞转化过程中，了解WASL调控的动力学是至关重要的。我们最近发现了一种 Wasl在调节发育模式中的令人惊讶的作用：我们发现Wasl对于 骨架模式的形成和WASL信号的增加导致了新的骨架元件的形成。 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。一种发育功能 对于Wasl来说，考虑到它在细胞中的核心功能，它以前是未知的和意想不到的。然而，由于 失控的WasL活性导致细胞质F-肌动蛋白调节中断以及 转录，目前尚不清楚Was1如何协调这些发育事件背后的特定信号。 在这里，我们利用斑马鱼和老鼠的新的成对功能获得和丧失模型来解决 WASL通过转录调控调节骨骼发育的中心假说 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 来直接解决这一假设。在目标1中，我们将利用Was1蛋白质和 去除建立F-肌动蛋白成核所需的特定蛋白质区域。这些∆vca突变等位基因 将与我们鉴定的特定功能增益等位基因Was1进行比较，两者分别 以及顺式基因，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，以及骨骼图案。然后在目标2中，我们将使用我们的WASS损失和收益模型 确定与WASL调控相关的特定转录和表观遗传变化的活性 肢体和鳍的发育。这使我们能够确定发育中的Wasl的最终转录签名 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对肢芽细胞成软骨分化的调控及其对转录调控的影响 在开发过程中。最后，在目标3中，我们利用Was1氨基酸序列的自然变异来提炼 特定的磷酸化残基是骨骼多样化的潜在关键调节因素。使用我们的新产品 实验工具，我们将解析WasL活性和功能在骨骼生长和图案化过程中的调节 在开发过程中。通过这些方法的完成，我们将拓宽我们对 Wasl在细胞行为和分化中复杂而有指导意义的作用，以及这种整合中的变化如何 导致了新结构的产生和形式的变化。

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.