Zebrafish deficient for Muscleblind-like 2 exhibit features of myotonic dystrophy

Abstract

SUMMARYMyotonic dystrophy (DM; also known as dystrophia myotonica) is an autosomal dominant disorder that affects the heart, eyes, brain and endocrine system, but the predominant symptoms are neuromuscular, with progressive muscle weakness and wasting. DM presents in two forms, DM1 and DM2, both of which are caused by nucleotide repeat expansions: CTG in the DMPK gene for DM1 and CCTG in ZNF9 (CNBP) for DM2. Previous studies have shown that the mutant mRNAs containing the transcribed CUG or CCUG repeats are retained within the nuclei of cells from individuals with DM, where they bind and sequester the muscleblind-like proteins MBNL1, MBNL2 and MBNL3. It has been proposed that the sequestration of these proteins plays a key role in determining the classic features of DM. However, the functions of each of the three MBNL genes are not completely understood. We have generated a zebrafish knockdown model in which we demonstrate that a lack of mbnl2 function causes morphological abnormalities at the eye, heart, brain and muscle levels, supporting an essential role for mbnl2 during embryonic development. Major features of DM are replicated in our model, including muscle defects and splicing abnormalities. We found that the absence of mbnl2 causes disruption to the organization of myofibrils in skeletal and heart muscle of zebrafish embryos, and a reduction in the amount of both slow and fast muscle fibres. Notably, our findings included altered splicing patterns of two transcripts whose expression is also altered in DM patients: clcn1 and tnnt2. The studies described herein provide broader insight into the functions of MBNL2. They also lend support to the hypothesis that the sequestration of this protein is an important determinant in DM pathophysiology, and imply a direct role of MBNL2 in splicing regulation of specific transcripts, which, when altered, contributes to the DM phenotype.

Deterministic splicing ofDscam2is regulated by Muscleblind

“…Consistent with what has been reported in nematodes (Norris et al, 2017), zebrafish (Machuca-Tzili et al, 2011) and chickens (Huang et al, 2008), we demonstrated cell-typespecific expression of mbl. Mbl was present in all cells tested that express Dscam2.10B and absent from Dscam2.10A cells.…”

Myotonic Dystrophy Type 1: Focus on the RNA Pathology and Therapy

“…Importantly, this DM1 mouse model exhibited severe muscle wasting, which has not been reported previously in models in which MBNL1 depletion was the main feature (Orengo et al, 2008). More recently, Machuca-Tzili et al generated an mbnl2 knockdown zebrafish model, which exhibits features of DM (Machuca-Tzili et al, 2011). They showed that loss of zebrafish mbnl2 function causes muscle defects and splicing abnormalities of clcn1 and tnnt2 transcripts, similar to those observed in DM1 patients (Machuca-Tzili et al, 2011).…”

Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions

“…Evolutionary conservation of muscle-enriched alternative splicing events has also been reported by others(Bland et al, 2010), with Rbfox proteins implicated as critical regulators along with members of the MBNL and CELF families of splicing factors. Disruption of splicing networks mediated by the latter factors is a hallmark of myotonic dystrophy(Cooper et al, 2009; Du et al, 2010) and zebrafish MBNL2 morphants also exhibit splicing defects and features of myotonic dystrophy (Machuca-Tzili et al, 2011). Mouse knockout experiments have implicated additional splicing factors such as SF2/ASF (SFRS1) and SC35 (SFRS2) as important contributors to alternative splicing in muscle (Ding et al, 2004; Xu et al, 2005).…”