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

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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.