Mmp25ß facilitates elongation of sensory neurons during zebrafish development.

Matrix metalloproteinases (MMPs) are a large and complex family of zinc-dependent endoproteinases widely recognized for their roles in remodeling the extracellular matrix (ECM) during embryonic development, wound healing, and tissue homeostasis. Their misregulation is central to many pathologies, and they have therefore been the focus of biomedical research for decades. These proteases have also recently emerged as mediators of neural development and synaptic plasticity in vertebrates, however, understanding of the mechanistic basis of these roles and the molecular identities of the MMPs involved remains far from complete. We have identified a zebrafish orthologue of mmp25 (a.k.a. leukolysin; MT6-MMP), a membrane-type, furin-activated MMP associated with leukocytes and invasive carcinomas, but which we find is expressed by a subset of the sensory neurons during normal embryonic development. We detect high levels of Mmp25ß expression in the trigeminal, craniofacial, and posterior lateral line ganglia in the hindbrain, and in Rohon-Beard cells in the dorsal neural tube during the first 48 h of embryonic development. Knockdown of Mmp25ß expression with morpholino oligonucleotides results in larvae that are uncoordinated and insensitive to touch, and which exhibit defects in the development of sensory neural structures. Using in vivo zymography, we observe that Mmp25ß morphant embryos show reduced Type IV collagen degradation in regions of the head traversed by elongating axons emanating from the trigeminal ganglion, suggesting that Mmp25ß may play a pivotal role in mediating ECM remodeling in the vicinity of these elongating axons.

Unc45b is essential for early myofibrillogenesis and costamere formation in zebrafish.

Despite the prevalence of developmental myopathies resulting from muscle fiber defects, the earliest stages of myogenesis remain poorly understood. Unc45b is a molecular chaperone that mediates the folding of thick-filament myosin during sarcomere formation; however, Unc45b may also mediate specific functions of non-muscle myosins (NMMs). unc45b Mutants have specific defects in striated muscle development, which include myocyte detachment indicative of dysfunctional adhesion complex formation. Given the necessity for non-muscle myosin function in the formation of adhesion complexes and premyofibril templates, we tested the hypothesis that the unc45b mutant phenotype is not mediated solely by interaction with muscle myosin heavy chain (mMHC). We used the advantages of a transparent zebrafish embryo to determine the temporal and spatial patterns of expression for unc45b, non-muscle myosins and mMHC in developing somites. We also examined the formation of myocyte attachment complexes (costameres) in wild-type and unc45b mutant embryos. Our results demonstrate co-expression and co-regulation of Unc45b and NMM in myogenic tissue several hours before any muscle myosin heavy chain is expressed. We also note deficiencies in the localization of costamere components and NMM in unc45b mutants that is consistent with an NMM-mediated role for Unc45b during early myogenesis. This represents a novel role for Unc45b in the earliest stages of muscle development that is independent of muscle mMHC folding.

UNC-45 is required for NMY-2 contractile function in early embryonic polarity establishment and germline cellularization in C. elegans.

The Caenorhabditis elegans UNC-45 protein is required for proper body wall muscle assembly and acts as a molecular co-chaperone for type II myosins. In contrast to other body wall muscle components, UNC-45 is also abundant in the germline and embryo. We show that maternally provided UNC-45 acts with non-muscle myosin II (NMY-2) during embryonic polarity establishment, cytokinesis and germline cellularization. In embryos depleted for UNC-45, myosin contractility is eliminated resulting in embryonic defects in polar body extrusion, cytokinesis and establishment of polarity. Despite a lack of contractility in an unc-45(RNAi) embryo, NMY-2::GFP localizes to the cortex and accumulates at the presumptive cytokinetic furrow indicating that UNC-45 is not required for cortical localization. UNC-45 and NMY-2 are also required for fertility since the lack of either component results in complete sterility due to failed initiation of the cellularization furrows that separate syncytial nuclei into germ cells. In the absence of UNC-45, the actomyosin cytoskeleton does not contract despite non-functional myosin still directly binding actin. UNC-45 has been previously suggested to be required for the folding of the myosin head, and our results refine this hypothesis suggesting that UNC-45 is not required to fold or maintain the actin binding domain but is still required for myosin function.