Clarifying The Molecular Events Of Early Myofibrillogenesis In Zebrafish

Sarcomeres are highly-structured protein arrays, consisting of precisely-aligned thick and thin filaments. The contractile mechanisms of sarcomeres are generally well understood, but how their patterning is initiated during early striated muscle development remains uncertain. Two of the most widely-accepted hypotheses for this process include the "molecular ruler" model, in which the massive protein titin provides a scaffold along which the myosin thick filament is assembled, and t he "pre-myofibril" model, which proposes that thick filament formation rather involves "pre-myofibril" templates, modified stress fibers consisting of non-muscle myosin (NMM) and cytoskeletal actin. These models have proven difficult to test in vivo, but zebrafish motility mutants with developmental defects in sarcomere patterning are useful for the elucidation of such mechanisms. One such mutant, steif, lacks Unc45b, a molecular chaperone that mediates the folding of thick-filament myosin during sarcomere formation. However, Unc45b may also mediate specific functions of NMMs, and unc45b mutants display myocyte detachment, indicative of dysfunctional adhesion complex formation. Given the necessity for non-muscle myosin function in the formation of adhesion complexes and pre-myofibril templates, we tested the hypothesis that the unc45b mutant phenotype is not mediated solely by interaction with muscle myosin. Our results demonstrate co-expression and co-localization of Unc45b and NMM in myogenic tissue several hours before any muscle myosin is expressed. We also noted deficiencies in the localization of adhesion complex components and NMM in unc45b mutants, that areconsistent with a NMM-mediated role for Unc45b during early myogenesis. Further, we report the analysis of the previously uncharacterized herzschlag mutant, which has similar striated muscle deficits. The herzschlag mutant produces a truncated titin protein, lacking the C-terminal rod domain that is proposed to act as a thick filament scaffold, yet muscle patterning was still initiated, with grossly normal thick and thin filament assembly. Only after embryonic muscle contraction begins is breakdown of sarcomeric myosin patterning observed, consistent with a role for titin in maintaining the contractile integrity of mature sarcomeres. These results support the pre-myofibril model, and conflict with the molecular ruler model of early sarcomere patterning, while demonstrating a novel role for Unc45b in early myogenesis.