biosensing-motor-neuron-membrane-potential-in-live-zebrafish-embryos

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 ...

Università degli Studi di Milano

The Grb10-Interacting GYF Protein-2 (GIGYF2) gene has been proposed as the Parkinson-disease (PD) gene underlying the PARK11 locus. However, association of GIGYF2 with PD has been challenged and a functional validation of GIGYF2 mutations is lacking. In this frame, we performed a mutational screening of GIGYF2 in an Italian PD cohort. Exons containing known mutations were analyzed in 552 cases and 552 controls. Thereafter, a subset of 184 familial PD cases and controls were subjected to a full coding-exon screening. These analyses identified 8 missense variations in 9 individuals (4 cases, 5 controls). Furthermore, we developed a zebrafish model of gigyf2 deficiency. Abrogation of gigyf2 function in zebrafish embryos did not lead to a drastic cell loss in diencephalic dopaminergic (DA) neuron clusters, suggesting that gigyf2 is not required for DA neuron differentiation. Notably, gigyf2 functional abrogation did not increase diencephalic DA neurons susceptibility to the PD-inducing drug MPP+. These data, together with those recently reported by other groups, suggest that GIGYF2 is unlikely to be the PARK11 gene.

Mutational screening and zebrafish functional analysis of GIGYF2 as a Parkinson-disease gene.

The lymphatic vascular system, draining interstitial fluids from most tissues and organs, exerts crucial functions in several physiological and pathological processes. Lymphatic system development depends on Prox1, the first marker to be expressed in the endothelial cells of the cardinal vein from where lymph vessels originate. Prox1 ortholog in the optically clear, easily manipulated zebrafish model has been previously isolated and its contribution to lymphangiogenesis has been clarified. Because of a round of genome duplication occurred at the base of teleosts radiation, several zebrafish genes have been retained in duplicate through evolution. We investigated for the presence of additional prox1 genes and determined their role in zebrafish lymphangiogenesis.

prox1b Activity is essential in zebrafish lymphangiogenesis.

Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC) populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/β-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs) in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10) and leukemic (n = 33) human long-term reconstituting AC133(+) cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133(bright) AML cells and shows a diffuse expression and release of WNT10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133(+) AML cell culture (A46) demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated β-catenin in vivo. We tested the LSC functional activity in AC133(+) cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2(-/-)γc(-/-) mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133(bright) LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration.

Regeneration-associated WNT signaling is activated in long-term reconstituting AC133bright acute myeloid leukemia cells.

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.

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

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.

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

Anna Ghilardi

Department of Biosciences

Università degli Studi di Milano

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos