Generation of glial cell-derived neurotrophic factor (gdnf) morphants in zebrafish larvae by cerebroventricular microinjection of vivo morpholino.

Dopaminergic neurons in the brain are an important source of dopamine, which is a crucial neurotransmitter for wellbeing, memory, reward, and motor control. Deficiency of dopamine due to advanced age and accumulative dopaminergic neuron defects can lead to movement disorders such as Parkinson's disease. Glial cell-derived neurotrophic factor (GDNF) is one of many factors involved in dopaminergic neuron development and/or survival. However, other endogenous GDNF functions in the brain await further investigation. Zebrafish is a well-established genetic model for neurodevelopment and neurodegeneration studies. Importantly, zebrafish shares approximately 70% functional orthologs with human genes including GDNF. To gain a better understanding on the precise functional role of gdnf in dopaminergic neurons, our laboratory devised a targeted knockdown of gdnf in the zebrafish larval brain using vivo morpholino. Here, detailed protocols on the generation of gdnf morphants using vivo morpholino are outlined. This method can be applied for targeting of genes in the brain to determine specific spatiotemporal gene function in situ.

Craniofacial and cardiac defects in chd7 zebrafish mutants mimic CHARGE syndrome.

Congenital heart defects occur in almost 80% of patients with CHARGE syndrome, a sporadically occurring disease causing craniofacial and other abnormalities due to mutations in the CHD7 gene. Animal models have been generated to mimic CHARGE syndrome; however, heart defects are not extensively described in zebrafish disease models of CHARGE using morpholino injections or genetic mutants. Here, we describe the co-occurrence of craniofacial abnormalities and heart defects in zebrafish chd7 mutants. These mutant phenotypes are enhanced in the maternal zygotic mutant background. In the chd7 mutant fish, we found shortened craniofacial cartilages and extra cartilage formation. Furthermore, the length of the ventral aorta is altered in chd7 mutants. Many CHARGE patients have aortic arch anomalies. It should be noted that the aberrant branching of the first branchial arch artery is observed for the first time in chd7 fish mutants. To understand the cellular mechanism of CHARGE syndrome, neural crest cells (NCCs), that contribute to craniofacial and cardiovascular tissues, are examined using sox10:Cre lineage tracing. In contrast to its function in cranial NCCs, we found that the cardiac NCC-derived mural cells along the ventral aorta and aortic arch arteries are not affected in chd7 mutant fish. The chd7 fish mutants we generated recapitulate some of the craniofacial and cardiovascular phenotypes found in CHARGE patients and can be used to further determine the roles of CHD7.

gdnf affects early diencephalic dopaminergic neuron development through regulation of differentiation-associated transcription factors in zebrafish.

Glial cell line-derived neurotrophic factor (GDNF) has been reported to enhance dopaminergic neuron survival and differentiation in vitro and in vivo, although those results are still being debated. Glial cell line-derived neurotrophic factor (gdnf) is highly conserved in zebrafish and plays a role in enteric nervous system function. However, little is known about gdnf function in the teleost brain. Here, we employed clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 to impede gdnf function in the maintenance of dopaminergic neuron development. Genotyping of gdnf crispants revealed successful deletions of the coding region with various mutant band sizes and down-regulation of gdnf transcripts at 1, 3 and 7 day(s) post fertilization. Notably, ~20% reduction in ventral diencephalic dopaminergic neuron numbers in clusters 8 and 13 was observed in the gdnf-deficient crispants. In addition, gdnf depletion caused a modest reduction in dopaminergic neurogenesis as determined by 5-ethynyl-2'-deoxyuridine pulse chase assay. These deleterious effects could be partly attributed to deregulation of dopaminergic neuron fate specification-related transcription factors (otp,lmx1b,shha,and ngn1) in both crispants and established homozygous mutants with whole mount in-situ hybridization (WISH) on gdnf mutants showing reduced otpb and lmx1b.1 expression in the ventral diencephalon. Interestingly, locomotor function of crispants was only impacted at 7 dpf, but not earlier. Lastly, as expected, gdnf deficiency heightened crispants vulnerability to 1-methyl-4-phenylpyridinium toxic insult. Our results suggest conservation of teleost gdnf brain function with mammals and revealed the interactions between gdnf and transcription factors in dopaminergic neuron differentiation.

Cellular Localization of gdnf in Adult Zebrafish Brain.

Glial cell line-derived neurotrophic factor (GDNF) was initially described as important for dopaminergic neuronal survival and is involved in many other essential functions in the central nervous system. Characterization of GDNF phenotype in mammals is well described; however, studies in non-mammalian vertebrate models are scarce. Here, we characterized the anatomical distribution of gdnf-expressing cells in adult zebrafish brain by means of combined in situ hybridization (ISH) and immunohistochemistry. Our results revealed that gdnf was widely dispersed in the brain. gdnf transcripts were co-localized with radial glial cells along the ventricular area of the telencephalon and in the hypothalamus. Interestingly, Sox2 positive cells expressed gdnf in the neuronal layer but not in the ventricular zone of the telencephalon. A subset of GABAergic precursor cells labeled with dlx6a-1.4kbdlx5a/6a: green fluorescence protein (GFP) in the pallium, parvocellular preoptic nucleus, and the anterior and dorsal zones of the periventricular hypothalamus also showed expression with gdnf mRNA. In addition, gdnf signals were detected in subsets of dopaminergic neurons, including those in the ventral diencephalon, similar to what is seen in mammalian brain. Our work extends our knowledge of gdnf action sites and suggests a potential role for gdnf in adult brain neurogenesis and regeneration.

Comprehensive Analysis of Neurotoxin-Induced Ablation of Dopaminergic Neurons in Zebrafish Larvae.

Neurotoxin exposure of zebrafish larvae has been used to mimic a Parkinson's disease (PD) phenotype and to facilitate high-throughput drug screening. However, the vulnerability of zebrafish to various neurotoxins was shown to be variable. Here, we provide a direct comparison of ablative effectiveness in order to identify the optimal neurotoxin-mediated dopaminergic (DAnergic) neuronal death in larval zebrafish. Transgenic zebrafish, Tg(dat:eGFP), were exposed to different concentrations of the neurotoxins MPTP, MPP+, paraquat, 6-OHDA, and rotenone for four days, starting at three days post-fertilization. The LC50 of each respective neurotoxin concentration was determined. Confocal live imaging on Tg(dat:eGFP) showed that MPTP, MPP+, and rotenone caused comparable DAnergic cell loss in the ventral diencephalon (vDC) region while, paraquat and 6-OHDA caused fewer losses of DAnergic cells. These results were further supported by respective gene expression analyses of dat, th, and p53. Importantly, the loss of DAnergic cells from exposure to MPTP, MPP+, and rotenone impacted larval locomotor function. MPTP induced the largest motor deficit, but this was accompanied by the most severe morphological impairment. We conclude that, of the tested neurotoxins, MPP+ recapitulates a substantial degree of DAnergic ablation and slight locomotor perturbations without systemic defects indicative of a Parkinsonian phenotype.