Dok-7 promotes slow muscle integrity as well as neuromuscular junction formation in a zebrafish model of congenital myasthenic syndromes

Abstract

The small signalling adaptor protein Dok-7 has recently been reported as an essential protein of the neuromuscular junction (NMJ). Mutations resulting in partial loss of Dok-7 activity cause a distinct limb-girdle subtype of the inherited NMJ disorder congenital myasthenic syndromes (CMSs), whereas complete loss of Dok-7 results in a lethal phenotype in both mice and humans. Here we describe the zebrafish orthologue of Dok-7 and study its in vivo function. Dok-7 deficiency leads to motility defects in zebrafish embryos and larvae. The relative importance of Dok-7 at different stages of NMJ development varies; it is crucial for the earliest step, the formation of acetylcholine receptor (AChR) clusters in the middle of the muscle fibre prior to motor neuron contact. At later stages, presence of Dok-7 is not absolutely essential, as focal and non-focal synapses do form when Dok-7 expression is downregulated. These contacts however are smaller than in the wild-type zebrafish, reminiscent of the neuromuscular endplate pathology seen in patients with DOK7 mutations. Intriguingly, we also observed changes in slow muscle fibre arrangement; previously, Dok-7 has not been linked to functions other than postsynaptic AChR clustering. Our results suggest an additional role of Dok-7 in muscle. This role seems to be independent of the muscle-specific tyrosine kinase MuSK, the known binding partner of Dok-7 at the NMJ. Our findings in the zebrafish model contribute to a better understanding of the signalling pathways at the NMJ and the pathomechanisms of DOK7 CMSs.

The cellular and molecular progression of mitochondrial dysfunction induced by 2,4-dinitrophenol in developing zebrafish embryos

“…8). The developing neuromuscular system of zebrafish has revealed mechanistic insights into many neuromuscular diseases (McGown et al, 2013; Muller et al, 2010; Steele et al, 2014) and is likely to provide important information linking our observations with neuromuscular pathology observed in patients with mitochondrial diseases.…”

Genetic heterogeneity and pathophysiological mechanisms in congenital myasthenic syndromes

“…14-55-58 More recent animal studies using a zebrafish model suggest that Dok-7 deficiency may also impair slow muscle organisation, independent of Musk. 59 9…”

Interação funcional entre o sistema colinérgico e adrenérgico na manutenção da massa muscular e da placa motora

“…Anaesthesia and preperation of mice for microscopy and electrophysiology was done as described previously [34][35][36] . Zebrafish embryos were raised and staged in hours postfertilization (hpf) according to standard protocols as described previously 27 . Given that all treated animals, unless excluded for having reached predetermined exclusion criteria,…”

“…Since corresponding mouse models are lethal in the early postnatal period 26 limiting the use of these animals in pharmacological assays, and to address the general importance of our findings across phylogenetic groups, we tested the effect of SM on a previously established DOK-7 knowdown Zebrafish model, which exhibits strongly reduced motility 27 . Incubation with SM led to increased size, increased presynaptic ramifications and more postsynaptic AChR plaques (Fig.…”

“…Touch-evoked swimming response was observed by touching the head or tail of the Zebrafish with a fine pipette tip. Alternating tail movements were observed under Leica stereomicroscope equipped with a digital camera (model LEICA DFC 420C) and were analyzed as described previously 27 .…”