Zebrafish as a Model to Investigate Dynamin 2-Related Diseases.

Cinzia Bragato,Marina Mora,Flavia Blasevich,Giulio Pavesi,Germano Gaudenzi,Lorenzo Maggi,Michele Giunta,Franco Cotelli

doi:10.1038/srep20466

Abstract

Mutations in the dynamin-2 gene (DNM2) cause autosomal dominant centronuclear myopathy (CNM) and dominant intermediate Charcot-Marie-Tooth (CMT) neuropathy type B (CMTDIB). As the relation between these DNM2-related diseases is poorly understood, we used zebrafish to investigate the effects of two different DNM2 mutations. First we identified a new alternatively spliced zebrafish dynamin-2a mRNA (dnm2a-v2) with greater similarity to human DNM2 than the deposited sequence. Then we knocked-down the zebrafish dnm2a, producing defects in muscle morphology. Finally, we expressed two mutated DNM2 mRNA by injecting zebrafish embryos with human mRNAs carrying the R522H mutation, causing CNM, or the G537C mutation, causing CMT. Defects arose especially in secondary motor neuron formation, with incorrect branching in embryos injected with CNM-mutated mRNA, and total absence of branching in those injected with CMT-mutated mRNA. Muscle morphology in embryos injected with CMT-mutated mRNA appeared less regularly organized than in those injected with CNM-mutated mRNA. Our results showing, a continuum between CNM and CMTDIB phenotypes in zebrafish, similarly to the human conditions, confirm this animal model to be a powerful tool to investigate mutations of DNM2 in vivo.

Highlights

Dynamin-2 (DNM2) related diseases are a heterogeneous group of conditions that affect muscular and nervous systems
Dynamins 1, 2 and 3 have a 5-domain structure: an N-terminal GTPase domain, a middle domain (MD), a pleckstrin-homology (PH) domain that binds phosphoinositides, a GTPase effector domain (GED) that is involved in oligomerization and regulation of GTPase activity, and a C-terminal proline-rich domain (PRD) that interacts with SH3 domains[6]
We investigated the spatial localization of dnm2a using whole mount in situ hybridization (WISH), using a specific probe recognizing a 456 bp region shared by the original dnm[2] and the dnm2a-v2 transcript

Summary

Introduction

Dynamin-2 (DNM2) related diseases are a heterogeneous group of conditions that affect muscular and nervous systems. Mutations in DNM2 cause centronuclear myopathy (CNM), a rare hereditary disease characterized by centrally located nuclei in muscle fibres. Other mutations in DNM2 cause the dominant intermediate axonal form of Charcot-Marie-Tooth neuropathy type B (hereafter CMT), a motor and sensory neuropathy that primarily affects peripheral nerves[1,2,3,4]. Mutations in the PH domain of dynamin-2, which binds phosphatidylinositol-4, 5-bisphosphate to mediate localization on the membrane, are responsible for severe forms of both CNM and CMT7,8. Several pathogenic mechanisms related to DNM2 function have been suggested[9]; despite the knowledge gained so far, the pathogenic mechanisms that cause CNM versus CMT are still unknown

Methods

Results

Conclusion