dact1/2 modifies noncanonical Wnt signaling and calpain 8 expression to regulate convergent extension and craniofacial development.

Wnt signaling plays a crucial role in the early embryonic patterning and development, to regulate convergent extension during gastrulation and the establishment of the dorsal axis. 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 dact1 and dact2 genes have dynamic spatiotemporal expression domains that are reciprocal to one another and to wnt11f2l, that suggest distinct functions during zebrafish embryogenesis. We found that both dact1 and dact2 contribute to axis extension, with compound mutants exhibiting a similar convergent extension defect and craniofacial phenotype to the wnt11f2 mutant. Utilizing single-cell RNAseq and gpc4 mutant that disrupts noncanonical Wnt signaling, we identified dact1/2 specific roles during early development. Comparative whole transcriptome analysis between wildtype, gpc4 and dact1/2 mutants revealed a novel role for dact1/2 in regulating the mRNA expression of the classical calpain capn8. Over-expression of capn8 phenocopies dact1/2 craniofacial dysmorphology. These results identify a previously unappreciated role of capn8 and calcium-dependent proteolysis during embryogenesis. Taken together, our findings highlight the distinct and overlapping roles of dact1 and dact2 in embryonic craniofacial development, providing new insights into the multifaceted regulation of Wnt signaling.