Redundant and novel functions of scube genes during zebrafish development

The N-glycoprotein SCUBE family (Scube1, Scube2, and Scube3) plays diverse roles in vertebrate development and disease, yet many specific functions of the three family members remain unclear. These proteins exhibit broad tissue expression patterns, exist as soluble or membrane-tethered forms, and can form homo- or heteromeric complexes with each other, exerting both short- and long-range effects. Individual functional characterisation has proved challenging because of overlapping expression patterns and compensatory mechanisms that likely obscure specific roles. To elucidate Scube function, we generated single, double, and triple mutant combinations of the three scube genes in zebrafish and assessed their roles in Hedgehog signalling. Our findings indicate that all three Scube proteins contribute to muscle development at distinct levels, with triple knockout zebrafish larvae displaying near-complete Hedgehog loss-of-function phenotypes. RNA sequencing analysis revealed potential roles for all three scube genes, and specific functions for Scube1 and Scube3 in apoptosis regulation and macrophage biology were identified. Furthermore, the Scube family contains multiple functional domains, and the role of EGF domains in Hedgehog signalling remains contentious. Comparative rescue analysis revealed that full-length scube2 mRNA restored slow muscle fibre numbers more efficiently than EGF domain-truncated constructs in scube2 knockout zebrafish, demonstrating the importance of EGF domains in Scube2 function. Our results illustrate both redundant and non-redundant functions among Scube family members and provide new insights into the roles of this enigmatic class of developmental regulators.