FGF signaling directs myotube guidance by regulating Rac activity.

Nascent myotubes undergo a dramatic morphological transformation during myogenesis, in which the myotubes elongate over several cell diameters and are directed to the correct muscle attachment sites. Although this process of myotube guidance is essential to pattern the musculoskeletal system, the mechanisms that control myotube guidance remain poorly understood. Using transcriptomics, we found that components of the Fibroblast Growth Factor (FGF) signaling pathway were enriched in nascent myotubes in Drosophila embryos. Null mutations in the FGF receptor heartless (htl), or its ligands, caused significant myotube guidance defects. The FGF ligand Pyramus is expressed broadly in the ectoderm, and ectopic Pyramus expression disrupted muscle patterning. Mechanistically, Htl regulates the activity of Rho/Rac GTPases in nascent myotubes and effects changes in the actin cytoskeleton. FGF signals are thus essential regulators of myotube guidance that act through cytoskeletal regulatory proteins to pattern the musculoskeletal system.

A diagnostic ceiling for exome sequencing in cerebellar ataxia and related neurological disorders.

Genetic ataxias are associated with mutations in hundreds of genes with high phenotypic overlap complicating the clinical diagnosis. Whole-exome sequencing (WES) has increased the overall diagnostic rate considerably. However, the upper limit of this method remains ill-defined, hindering efforts to address the remaining diagnostic gap. To further assess the role of rare coding variation in ataxic disorders, we reanalyzed our previously published exome cohort of 76 predominantly adult and sporadic-onset patients, expanded the total number of cases to 260, and introduced analyses for copy number variation and repeat expansion in a representative subset. For new cases (n = 184), our resulting clinically relevant detection rate remained stable at 47% with 24% classified as pathogenic. Reanalysis of the previously sequenced 76 patients modestly improved the pathogenic rate by 7%. For the combined cohort (n = 260), the total observed clinical detection rate was 52% with 25% classified as pathogenic. Published studies of similar neurological phenotypes report comparable rates. This consistency across multiple cohorts suggests that, despite continued technical and analytical advancements, an approximately 50% diagnostic rate marks a relative ceiling for current WES-based methods and a more comprehensive genome-wide assessment is needed to identify the missing causative genetic etiologies for cerebellar ataxia and related neurodegenerative diseases.

Prevalence of spinocerebellar ataxia 36 in a US population.

OBJECTIVE: To assess the prevalence and clinical features of individuals affected by spinocerebellar ataxia 36 (SCA36) at a large tertiary referral center in the United States. METHODS: A total of 577 patients with undiagnosed sporadic or familial cerebellar ataxia comprehensively evaluated at a tertiary referral ataxia center were molecularly evaluated for SCA36. Repeat primed PCR and fragment analysis were used to screen for the presence of a repeat expansion in the NOP56 gene. RESULTS: Fragment analysis of triplet repeat primed PCR products identified a GGCCTG hexanucleotide repeat expansion in intron 1 of NOP56 in 4 index cases. These 4 SCA36-positive families comprised 2 distinct ethnic groups: white (European) (2) and Asian (Japanese [1] and Vietnamese [1]). Individuals affected by SCA36 exhibited typical clinical features with gait ataxia and age at onset ranging between 35 and 50 years. Patients also suffered from ataxic or spastic limbs, altered reflexes, abnormal ocular movement, and cognitive impairment. CONCLUSIONS: In a US population, SCA36 was observed to be a rare disorder, accounting for 0.7% (4/577 index cases) of disease in a large undiagnosed ataxia cohort.

Noncanonical roles for Tropomyosin during myogenesis.

For skeletal muscle to produce movement, individual myofibers must form stable contacts with tendon cells and then assemble sarcomeres. The myofiber precursor is the nascent myotube, and during myogenesis the myotube completes guided elongation to reach its target tendons. Unlike the well-studied events of myogenesis, such as myoblast specification and myoblast fusion, the molecules that regulate myotube elongation are largely unknown. In Drosophila, hoi polloi (hoip) encodes a highly conserved RNA-binding protein and hoip mutant embryos are largely paralytic due to defects in myotube elongation and sarcomeric protein expression. We used the hoip mutant background as a platform to identify novel regulators of myogenesis, and uncovered surprising developmental functions for the sarcomeric protein Tropomyosin 2 (Tm2). We have identified Hoip-responsive sequences in the coding region of the Tm2 mRNA that are essential for Tm2 protein expression in developing myotubes. Tm2 overexpression rescued the hoip myogenic phenotype by promoting F-actin assembly at the myotube leading edge, by restoring the expression of additional sarcomeric RNAs, and by promoting myoblast fusion. Embryos that lack Tm2 also showed reduced sarcomeric protein expression, and embryos that expressed a gain-of-function Tm2 allele showed both fusion and elongation defects. Tropomyosin therefore dictates fundamental steps of myogenesis prior to regulating contraction in the sarcomere.

Post-transcriptional regulation of myotube elongation and myogenesis by Hoi Polloi.

Striated muscle development requires the coordinated expression of genes involved in sarcomere formation and contractility, as well as genes that determine muscle morphology. However, relatively little is known about the molecular mechanisms that control the early stages of muscle morphogenesis. To explore this facet of myogenesis, we performed a genetic screen for regulators of somatic muscle morphology in Drosophila, and identified the putative RNA-binding protein (RBP) Hoi Polloi (Hoip). Hoip is expressed in striated muscle precursors within the muscle lineage and controls two genetically separable events: myotube elongation and sarcomeric protein expression. Myotubes fail to elongate in hoip mutant embryos, even though the known regulators of somatic muscle elongation, target recognition and muscle attachment are expressed normally. In addition, a majority of sarcomeric proteins, including Myosin Heavy Chain (MHC) and Tropomyosin, require Hoip for their expression. A transgenic MHC construct that contains the endogenous MHC promoter and a spliced open reading frame rescues MHC protein expression in hoip embryos, demonstrating the involvement of Hoip in pre-mRNA splicing, but not in transcription, of muscle structural genes. In addition, the human Hoip ortholog NHP2L1 rescues muscle defects in hoip embryos, and knockdown of endogenous nhp2l1 in zebrafish disrupts skeletal muscle development. We conclude that Hoip is a conserved, post-transcriptional regulator of muscle morphogenesis and structural gene expression.