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CRISPR/Cas9-induced zebrafish mutants for ebf3a

ABSTRACT: Mutations in the transcription factor EBF3 result in a neurodevelopmental disorder, and studies in animal models indicate that it has a critical role in neuronal differentiation. The molecular pathways and neuron types disrupted by its loss, however, have not been thoroughly investigated. Nor have the outcomes of these changes on behavior and brain activity. Here, we generated and characterized a zebrafish ebf3a loss-of-function mutant. We discovered morphological and neural phenotypes, including an overall smaller brain size, particularly in the hypothalamus, cerebellum, and hindbrain. Brain function was also compromised, with activity strongly increased in the cerebellum and abnormal behavior at baseline and in response to visual and acoustic stimuli. RNA-sequencing of developing larvae revealed significant downregulation of genes that mark olfactory sensory neurons, the lateral line, and cerebellar Purkinje neurons. Corroborating the RNA-sequencing, staining revealed fewer lateral line neuromasts and reduced Parvalbumin signal in the cerebellum. This study sets the stage for determining which downstream pathways underlie the emergence of the observed phenotypes and establishes multiple strong phenotypes that could form the basis of a drug screen.

ORGANISM(S): Danio rerio

PROVIDER: GSE276705 | GEO | 2024/09/09

REPOSITORIES: GEO

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Project description:Inherited mutations of calcium ion channels exhibit neurological defects, such as epilepsy, ataxia, and migraine, and these phenotypes are shared among humans and mouse models. Absence epilepsy and ataxic phenotypes are present in the calcium channelopathy mutants stargazer (stg-gamma2 subunit), tottering (tg-alpha1 subunit), and lethargic (lh-beta4 subunit). These mutations of high-voltage-activated (HVA) calcium channel subunits initiate increases in membrane excitability of low-voltage-activated (LVA) calcium channels in thalamic neurons, thus enhancing LVA currents. Elevated LVA currents, produced from T-type calcium channels, induce rhythmic thalamocortical burst firing and spike-wave seizures. The changes in gene expression originating from the different mutations result in similar T-type channel function; however, the network of gene modifications causing altered molecular plasticity and the emergence of pathological phenotypes remain unknown. We would like to understand how loss of 3 different subunits of a calcium ion channel differentially alter gene expression in the brain. Characterizing the gene expression profiles of the mutant stg, tg, and lh mice will provide evidence of the epileptic and ataxic mechanisms, which may identify potential therapeutic targets. We will compare the gene expression profiles among adult mutant stg, tg, and lh mice, which display the ataxic and epileptic phenotypes, along with their wildtype, litter-mate controls, in the cerebellum and brain (without cerebellum). We propose that the neuronal dysfunction associated with the ataxic and epileptic phenotypes develops from a common network of interacting gene alterations within the mutant stg, tg, and lh brain. The changes dictated by the initial mutations leading to the ataxic and epileptic phenotypes are hypothesized to be localized to the cerebellum and the remaining areas of the brain, including the thalamus and cortex, respectively. Adult mutant stg, tg, and lh mice, between 2 and 5 months of age, along with wildtype, litter-mate controls, will be sacrificed. Two brain regions responsible for two phenotypes will be examined. The cerebellum (ataxia) will be dissected from remainder of the forebrain (generalized epilepsy). Each tissue set will be collected in triplicate (3 separate mice per set) to account for biological variance, and total RNA isolated via standard Trizol procedure (Invitrogen) and purified with the RNeasy cleanup kit (Qiagen). RNA samples will be stored at -80 C until sent for analysis using the Affymetrix GeneChip Mouse Genome 430 2.0 whole genome array.

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CRISPR/Cas9-induced zebrafish mutants for ebf3a

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