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.