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.

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Environmental concentrations of cocaine and its main metabolites modulated antioxidant response and caused cyto-genotoxic effects in zebrafish embryo cells.

Illicit drugs have been recently identified as a serious environmental problem because of the growing evidence regarding their occurrence in aquatic environment and potential toxicity towards non-target organisms. Among them, cocaine (COC) and its main metabolites, namely benzoylecgonine (BE) and ecgonine methyl ester (EME), are commonly measured in freshwaters worldwide at levels that might cause diverse sub-lethal effects to aquatic organisms. Thus, the present study was aimed at investigating the potential adverse effects induced by the exposure to environmental concentrations (0.04, 0.4, 4 and 40 nM) of COC, BE, and EME on zebrafish (Danio rerio) embryos at 96 h post fertilization. Cytotoxicity was assessed by the Trypan Blue exclusion method, while primary and fixed genetic damages were evaluated by the Single Cell Gel Electrophoresis (SCGE) assay, and the DNA diffusion assay together with the Micronucleus test, respectively. The involvement of oxidative stress in the mechanism of action (MoA) of all tested drugs was assessed by measuring the activity of defense enzymes (SOD, CAT, GPx, and GST) and the expression of their encoding genes. Exposure to COC and both metabolites significantly reduced cell viability, increased DNA fragmentation and promoted the onset of apoptotic cells and micronuclei in zebrafish embryos. Results from oxidative stress-related endpoints and gene expression suggested that the observed genotoxicity may be caused by an overproduction of free radicals that imbalanced the oxidative status of embryos. The integration of biomarker responses into a synthetic index showed that at each tested concentration, BE and EME had a similar toxicity and were both more toxic than COC. Our data confirmed the potential toxicity of environmental concentrations of COC, BE, and EME, suggesting the need of further in-depth studies to shed light on their MoA and long-term toxicity towards non-target aquatic species.

Intronless WNT10B-short variant underlies new recurrent allele-specific rearrangement in acute myeloid leukaemia.

Defects in the control of Wnt signaling have emerged as a recurrent mechanism involved in cancer pathogenesis and acute myeloid leukaemia (AML), including the hematopoietic regeneration-associated WNT10B in AC133bright leukaemia cells, although the existence of a specific mechanism remains unproven. We have obtained evidences for a recurrent rearrangement, which involved the WNT10B locus (WNT10BR) within intron 1 (IVS1) and flanked at the 5' by non-human sequences whose origin remains to be elucidated; it also expressed a transcript variant (WNT10BIVS1) which was mainly detected in a cohort of patients with intermediate/unfavorable risk AML. We also identified in two separate cases, affected by AML and breast cancer respectively, a genomic transposable short form of human WNT10B (ht-WNT10B). The intronless ht-WNT10B resembles a long non-coding RNA (lncRNA), which suggests its involvement in a non-random microhomology-mediated recombination generating the rearranged WNT10BR. Furthermore, our studies supports an autocrine activation primed by the formation of WNT10B-FZD4/5 complexes in the breast cancer MCF7 cells that express the WNT10BIVS1. Chemical interference of WNT-ligands production by the porcupine inhibitor IWP-2 achieved a dose-dependent suppression of the WNT10B-FZD4/5 interactions. These results present the first evidence for a recurrent rearrangement promoted by a mobile ht-WNT10B oncogene, as a relevant mechanism for Wnt involvement in human cancer.

INaP selective inhibition reverts precocious inter- and motorneurons hyperexcitability in the Sod1-G93R zebrafish ALS model.

The pathogenic role of SOD1 mutations in amyotrophic lateral sclerosis (ALS) was investigated using a zebrafish disease model stably expressing the ALS-linked G93R mutation. In addition to the main pathological features of ALS shown by adult fish, we found remarkably precocious alterations in the development of motor nerve circuitry and embryo behavior, and suggest that these alterations are prompted by interneuron and motor neuron hyperexcitability triggered by anomalies in the persistent pacemaker sodium current INaP. The riluzole-induced modulation of INaP reduced spinal neuron excitability, reverted the behavioral phenotypes and improved the deficits in motor nerve circuitry development, thus shedding new light on the use of riluzole in the management of ALS. Our findings provide a valid phenotype-based tool for unbiased in vivo drug screening that can be used to develop new therapies.