The role of the zebrafish scube gene family in Hedgehog signalling and slow muscle development.

Abstract

Hedgehog (Hh) signalling from the notochord induces the slow muscle cell fate in the adaxial cells of the developing zebrafish embryo. Slow muscle formation is disrupted in zebrafish you-type mutants resulting in U-shaped somites. In many you-type mutants, genes encoding components of the Hh signalling pathway are mutated. scube2, a gene not previously known to be involved in Hh signalling, is disrupted in the you-type mutant you . you mutants are deficient in several Hh dependent cell types and show decreased expression of Hh target genes. The Scube (signal peptide-CUB domain-EGF-related) family of proteins act as secreted glycoproteins or cell-surface proteins and are thought to be involved in protein-protein interactions and ligand binding. At the protein level, the Scube family resembles the endocytic receptor Cubilin. Cubilin is known to interact with another endocytic receptor Megalin, which can function as an endocytic receptor for Sonic Hedgehog (SHH) in vitro. Megalin endocytosis of Shh may be an important part of the Hh signal transduction pathway. An anti-Scube2 antibody was developed during this work to investigate the intracellular localization pattern of Scube2 and facilitate the identification of potential Scube2 binding partner(s). In addition, this work identified and characterized two homologs of scube2 in zebrafish, scube 1 and scube 3. The high level of similarity amongst the Scube family of proteins and the weak phenotype of the you mutant suggested scube1 and scube3 might also be involved in slow muscle development. Loss of function experiments performed by antisense morpholino knockdown of scube1 and scube3 in the you mutant decreases the expression of Hh target genes to levels seen in embryos lacking Hh signalling and dramatically enhances the loss of slow muscle fibres compared to you mutants alone. Thus, injecting both scube1 and scube3 morpholinos into you blocks Hh signalling and these embryos fail to develop slow muscle. Inhibition of the three partially redundant scube genes inhibits Hh signalling in zebrafish embryos, thereby demonstrating the essential requirement for scube gene function in the Hh signalling pathway.