Differentiation and functioning of the lateral line organ in zebrafish require Smpx activity.

The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in the inner ear hair cells (HCs), possibly regulating auditory function. In the last decade, several mutations in SMPX have been associated with X-chromosomal progressive non syndromic hearing loss in humans and, in line with this, Smpx-deficient animal models, namely zebrafish and mouse, showed significant impairment of inner ear HCs development, maintenance, and functioning. In this work, we uncovered smpx expression in the neuromast mechanosensory HCs of both Anterior and Posterior Lateral Line (ALL and PLL, respectively) of zebrafish larvae and focused our attention on the PLL. Smpx was subcellularly localized throughout the cytoplasm of the HCs, as well as in their primary cilium. Loss-of-function experiments, via both morpholino-mediated gene knockdown and CRISPR/Cas9 F0 gene knockout, revealed that the lack of Smpx led to fewer properly differentiated and functional neuromasts, as well as to a smaller PLL primordium (PLLp), the latter also Smpx-positive. In addition, the kinocilia of Smpx-deficient neuromast HCs appeared structurally and numerically altered. Such phenotypes were associated with a significant reduction in the mechanotransduction activity of the neuromast HCs, in line with their positivity for Smpx. In summary, this work highlights the importance of Smpx in lateral line development and, specifically, in proper HCs differentiation and/or maintenance, and in the mechanotransduction process carried out by the neuromast HCs. Because lateral line HCs are both functionally and structurally analogous to the cochlear HCs, the neuromasts might represent an invaluable-and easily accessible-tool to dissect the role of Smpx in HCs development/functioning and shed light on the underlying mechanisms involved in hearing loss.

Inner Ear and Muscle Developmental Defects in Smpx-Deficient Zebrafish Embryos.

The last decade has witnessed the identification of several families affected by hereditary non-syndromic hearing loss (NSHL) caused by mutations in the SMPX gene and the loss of function has been suggested as the underlying mechanism. In the attempt to confirm this hypothesis we generated an Smpx-deficient zebrafish model, pointing out its crucial role in proper inner ear development. Indeed, a marked decrease in the number of kinocilia together with structural alterations of the stereocilia and the kinocilium itself in the hair cells of the inner ear were observed. We also report the impairment of the mechanotransduction by the hair cells, making SMPX a potential key player in the construction of the machinery necessary for sound detection. This wealth of evidence provides the first possible explanation for hearing loss in SMPX-mutated patients. Additionally, we observed a clear muscular phenotype consisting of the defective organization and functioning of muscle fibers, strongly suggesting a potential role for the protein in the development of muscle fibers. This piece of evidence highlights the need for more in-depth analyses in search for possible correlations between SMPX mutations and muscular disorders in humans, thus potentially turning this non-syndromic hearing loss-associated gene into the genetic cause of dysfunctions characterized by more than one symptom, making SMPX a novel syndromic gene.

Expression pattern of the small muscle protein, X-linked (smpx) gene during zebrafish embryonic and larval developmental stages.

The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in both cardiac and skeletal muscles, as well as in fetal inner ears, with suggested roles as mechanotransductor. Recently, several mutations in the SMPX gene have been associated with X-chromosomal progressive deafness in human. However, very little information is known concerning the roles of SMPX, and no in-vivo models are currently available. Therefore, we characterized the zebrafish ortholog of SMPX to pave the way towards the establishment of a biotool for future functional studies. Despite the genome duplication occurred in the ancestry of teleosts, zebrafish retain only one copy of smpx which shares a high degree of similarity with the mammalian counterpart in terms of genomic organization, syntenic map, and encoded protein. RT-PCR, as well as whole-mount in-situ hybridization and immunofluorescence analyses, revealed that smpx is expressed in several embryonic areas starting from the 4-somite stage. Specifically, smpx mRNA marked the Kupffer's vesicle (KV), the somites, the myocardium, the hair cells of the anterior and the posterior macula of the inner ear, the pronephric ducts, and the muscles of the branchial arches, eyes and pectoral fins. According to our data, zebrafish smpx expression pattern closely resembles that observed in mouse and human, supporting the notion that zebrafish might represent a suitable in-vivo model to disclose the cellular and molecular mechanisms underlying the involvement of SMPX in development and disease.