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Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA-Seq]

ABSTRACT: Background: V0v spinal interneurons are highly-conserved, glutamatergic, commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However, we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks, we FAC-sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and scRNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild-type siblings. Results: Our data reveal two molecularly-distinct subtypes of V0v spinal interneurons at 48 h and suggest that, by this stage of development, evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuronal expression of skor1a and nefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly-distinct subsets of V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.

ORGANISM(S): Danio rerio

PROVIDER: GSE240238 | GEO | 2023/08/09

REPOSITORIES: GEO

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Project description:Background: V0v spinal interneurons are highly-conserved, glutamatergic, commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However, we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks, we FAC-sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and scRNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild-type siblings. Results: Our data reveal two molecularly-distinct subtypes of V0v spinal interneurons at 48 h and suggest that, by this stage of development, evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuronal expression of skor1a and nefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly-distinct subsets of V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.

Project description:Summary: We build a model for the molecular and cellular events underlying phenotypic discordance in glycine receptor defects (beta subunit). Some mice progress and die, while their littermates recover and get better, despite the same mutation on an inbred genetic background. We find evidence for glycine neurotransmitter toxicity and loss of glycinergic interneurons early in the disease, but some mice are able to keep things going until they can over-express homomeric alpha1 channels, whereupon they recover. In the mice progressing towards lethality, neurotransmitter toxicity too quickly extends to GABAergic interneurons and motorneurons, and they lose their window of time to upregulate the alpha1 glycine receptor, and they crash and burn. Importantly, human patients with glycine receptor defects typically show a resolution of their phenotype with age, and we propose that the same remodeling events are occuring in human patients. Hypothesis: Our data suggests that functional recovery of GlyRb mutant mice is likely due to expression of homomeric glycine receptors, rescue from excitotoxicity, and subsequent neuronal remodeling. We propose that human patients with hyperekplexia (mutations of glycine receptors) show remodeling similar to that of the recovering spastic mice, as human patients also show a lessening of symptoms as a function of age. Specific Aim: Murine models for human Startle Disease show clinical variability between littermates. Here, we determined the molecular remodeling of spastic GlyRb mutant spinal cord through the course of the disease, and develop a model for clinical disparity between littermates. At young ages, all animals were spastic, showed loss of glycine receptors, increased expression of vesicular glycine/GABA transporter and NMDA receptors, induction of activated caspase3, and preferential loss of glycinergic interneurons consistent with neurotransmitter toxicity model. Those littermates that recovered from symptoms showed strong over-expression of the GlyRa1 subunit, and increased myelination and synaptic plasticity. Littermates that showed a deteriorating clinical course failed to over-express GlyRa1, and also showed relative loss of gephyrin. These molecular changes were associated with preferential loss of GABAergic interneurons, and extensive motorneuron loss.

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Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA-Seq]

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