Genetic requirement of <i>dact1/2</i> to regulate noncanonical Wnt signaling and <i>calpain 8</i> during embryonic convergent extension and craniofacial morphogenesis.

Abstract

Wnt signaling plays crucial roles in embryonic patterning including the regulation of convergent extension (CE) during gastrulation, the establishment of the dorsal axis, and later, craniofacial morphogenesis. Further, Wnt signaling is a crucial regulator of craniofacial morphogenesis. The adapter proteins Dact1 and Dact2 modulate the Wnt signaling pathway through binding to Disheveled. However, the distinct relative functions of Dact1 and Dact2 during embryogenesis remain unclear. We found that <i>dact1</i> and <i>dact2</i> genes have dynamic spatiotemporal expression domains that are reciprocal to one another suggesting distinct functions during zebrafish embryogenesis. Both <i>dact1</i> and <i>dact2</i> contribute to axis extension, with compound mutants exhibiting a similar CE defect and craniofacial phenotype to the <i>wnt11f2</i> mutant. Utilizing single-cell RNAseq and an established noncanonical Wnt pathway mutant with a shortened axis (<i>gpc4</i>), we identified <i>dact1/2</i>-specific roles during early development. Comparative whole transcriptome analysis between wildtype and <i>gpc4</i> and wildtype and <i>dact1/2</i> compound mutants revealed a novel role for <i>dact1/2</i> in regulating the mRNA expression of the classical calpain <i>capn8</i>. Overexpression of <i>capn8</i> phenocopies <i>dact1/2</i> craniofacial dysmorphology. These results identify a previously unappreciated role of <i>capn8</i> and calcium-dependent proteolysis during embryogenesis. Taken together, our findings highlight the distinct and overlapping roles of <i>dact1</i> and <i>dact2</i> in embryonic craniofacial development, providing new insights into the multifaceted regulation of Wnt signaling.