Characterizing the Gene Regulatory Network Required to Specify the Excitatory Neurotransmitter Phenotype of V0v Spinal Interneurons

The majority of neurons within the spinal cord are interneurons. Interneurons make important connections with other cells that allow signals to be relayed between the brain and the rest of the body, and aid in functions such as locomotion. One key functional characteristic of interneurons is the neurotransmitter that they use to communicate with other cells. Interneurons that use GABA or glycine neurotransmitters to communicate have an inhibitory phenotype, while interneurons that use glutamate neurotransmitters have an excitatory neurotransmitter phenotype. Without the correct neurotransmitter phenotype, connections within the central nervous system will malfunction. It is therefore important to understand how specific neurotransmitter phenotypes are specified during spinal cord development.There are several specific populations of interneurons arranged along the dorsal-ventral axis of the zebrafish spinal cord; each of these populations has distinct properties such as the unique combination of transcription factor genes that they express. V0v interneurons are a class of excitatory neurons that form in the middle of the dorsal-ventral axis of the spinal cord and have been implicated in correct left-right alternation during hindlimb locomotion. The Lewis Lab has previously shown that in zebrafish, two transcription factor genes, evx1 and evx2, are required to specify the excitatory neurotransmitter phenotype and repress an inhibitory neurotransmitter phenotype in V0v interneurons. Following this discovery, the Lewis Lab identified several transcription factor (TF) genes and neuronal intermediate filament (NIF) genes as candidate members of a potential gene regulatory network required to specify the excitatory neurotransmitter phenotype of V0v interneurons. In this thesis, I test if several of these genes are expressed downstream of Evx1 and Evx2 in the zebrafish spinal cord at 30 hours post fertilization (hpf). To do this, I used in situ hybridization to label spinal cord cells that express these candidate genes in evx1-/-;evx2-/- mutants and wild-type (WT) siblings at 30 hpf. I then counted the number of cells expressing these genes in a specific region of the spinal cord in evx1-/-;evx2-/- mutants and wild-type embryos to test for any statistically significant differences. The TF genes I analyzed are skor1a, skor1b, sox12, dmrt3a, ebf3a, and hmx3a. The NIF genes I analyzed are nefma, nefmb, zgc:65851, nelfa, neflb, inaa, and inab. I was unable to count cells expressing sox12 and ebf3a due to experimental difficulties which I will explain in my thesis, and my data show that inaa is not expressed in the zebrafish spinal cord at 30 hpf. However, my data show that statistically significantly fewer cells express skor1a, skor1b, nemfa, and zgc:65851 in evx1-/-;evx2-/- mutants compared to WTs, which suggests that these genes are expressed downstream of Evx1 and Evx2. My data also show that statistically significantly more cells express hmx3a in evx1-/-;evx2-/- mutants compared to WTs which suggests that Evx1 and Evx2 are required to repress hmx3a in V0v cells. My data do not suggest that dmrt3a, inab, inaa, nefla, neflb, and nefmb are downstream of Evx1 and Evx2, however many of these experiments were only performed once and should be repeated in the future to confirm these findings. The Lewis lab has also previously shown that the transcription factor gene skor2 is expressed downstream of Evx1 and Evx2. Preliminary data obtained by previous members of the Lewis Lab suggest that there is a statistically significant reduction in the number of excitatory spinal cord cells in skor1b-/- and skor2-/- single mutants and skor1a-/-;skor1b-/- double mutants compared to WTs at 30 hpf. Based on these data, as well as my data which suggest skor1a and skor1b are expressed downstream of Evx1 and Evx2, I also tested the hypothesis that skor1a, skor1b, and skor2 are required to specify an excitatory phenotype in V0v interneurons. To test this hypothesis, I performed in situ hybridization which labeled cells expressing the excitatory marker vglut in skor1a, skor1b, and skor2 single, double, and triple homozygous mutants and WTs. My data show no statistically significant difference in the number of excitatory spinal cord cells in a specific region of the spinal cord in any of the mutants compared to WTs. As shown by the Lewis Lab, Evx1 and Evx2 are not only required to specify an excitatory phenotype, but to suppress an inhibitory phenotype. Together, these results do not suggest that skor1a, skor1b, and skor2 are required to specify the V0v excitatory neurotransmitter phenotype, but they also do not eliminate the possibility that they are instead required to repress an inhibitory neurotransmitter phenotype in V0v cells. This thesis contributes to the understanding of how V0v excitatory neurotransmitter phenotypes are specified in zebrafish. Most neuronal characteristics are conserved between vertebrates, so these results will also advance our understanding of the nervous system circuitry essential for locomotion in a broad range of vertebrates