Genetic requirement ofdact1/2to regulate noncanonical Wnt signaling andcalpain 8during embryonic convergent extension and craniofacial morphogenesis

Wnt signaling plays crucial roles in embryonic patterning including the regulation of convergent extension 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 thatdact1anddact2genes have dynamic spatiotemporal expression domains that are reciprocal to one another suggesting distinct functions during zebrafish embryogenesis. Bothdact1anddact2contribute to axis extension, with compound mutants exhibiting a similar convergent extension defect and craniofacial phenotype to thewnt11f2mutant. Utilizing single-cell RNAseq and an established noncanonical Wnt pathway mutant with a shortened axis (gpc4), we identifieddact1/2specific roles during early development. Comparative whole transcriptome analysis between wildtype andgpc4and wildtype anddact1/2compound mutants revealed a novel role fordact1/2in regulating the mRNA expression of the classical calpaincapn8. Over-expression ofcapn8phenocopiesdact1/2craniofacial dysmorphology. These results identify a previously unappreciated role ofcapn8and calcium-dependent proteolysis during embryogenesis. Taken together, our findings highlight the distinct and overlapping roles ofdact1anddact2in embryonic craniofacial development, providing new insights into the multifaceted regulation of Wnt signaling.