Molecular analyses of zebrafish V0v spinal interneurons and identification of transcriptional regulators downstream of Evx1 and Evx2 in these cells.

Acknowledgements: We thank the Zebrafish Information Network for providing information on nomenclature and other essential zebrafish resources, and Henry Putz, Jessica Bouchard, Paul Campbell, Leslie Vogt, Annika Swanson, and several Syracuse University undergraduate fish husbandry workers for help with maintaining zebrafish lines. We thank Roda Ntiranyibagira for help with uncx4.1 in situ hybridization in WT embryos and genotyping of evx1;evx2 mutant embryos with uncx4.1 in situ staining. We also thank Lisa Phelps for technical assistance with FACS, Sungmin Baek and Tatjana Piotrowski for advice on methanol fixation for single-cell experiments, Karen Gentile for RNA-seq library preparation, Frank Middleton for advice on Partek Flow, Elisabeth Busch-Nentwich and Ian Sealy for advice on bulk RNA-seq experimental design, Yasir Ahmed-Braimah for advice on scRNA-seq statistical analyses, and Francesca Pignoni for kindly providing lab space and equipment for experimental steps that needed to occur close to the sequencing facility.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 wild-type and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and single cell RNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 double mutants and wild-type siblings. RESULTS: Our data reveal two molecularly distinct subtypes of zebrafish 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 interneuron 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 zebrafish 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 these cells. 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 and spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type