The conserved transcription factor UNC-30/PITX1-3 coordinates synaptogenesis and cell identity in C. elegans motor neurons

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PROJECT SUMMARY The overall goal of this proposal is to determine the molecular mechanisms that take place in neurons and enable them to establish and maintain functional synapses. For this, the ability of presynaptic neurons to synthesize, package, and release a neurotransmitter must be coordinated with the ability of postsynaptic neurons to present the correct neurotransmitter receptors. Revealing the molecular mechanism that coordinates these processes could have biomedical implications since defects in the establishment and maintenance of functional synapses are linked to severe neurodegenerative disorders in humans. To address this knowledge gap, C. elegans represents an ideal model to study those mechanisms due to its known connectome, powerful genetics, and single-cell resolution analysis. Leveraging these tools, the evolutionarily conserved transcription factor (TF) UNC-30/PITX1-3 has been shown to control neuronal communication between nerve cord GABAergic (GABA) motor neurons (MNs) and body-wall muscle by directly activating the expression of GABA biosynthesis genes (e.g., unc-47/VGAT, unc-25/GAD). We obtained data that shows genetic loss of either unc-30 or madd-4S, a secreted synaptic organizer produced by GABA MNs, results in a dramatic reduction of GABA-Receptor (GABAR) clustering on the postsynaptic domain (muscle). Moreover, our preliminary data shows that animals lacking unc-30 gene activity show reduction of madd-4S expression in GABA MNs. Therefore, we hypothesize that UNC-30 controls both the establishment and maintenance of functional synapses by directly activating madd-4S and GABA biosynthesis genes. To evaluate this hypothesis, in Aim 1 I will use madd-4S specific reporters to determine whether UNC-30 directly regulates madd-4S expression. I will also conduct cross-species rescue experiments to determine whether UNC-30’s role in regulating madd-4S expression is conserved across phylogeny. In Aim 2 I will take advantage of the auxin-inducible degron system to selectively deplete UNC-30 at a particular stage throughout development and determine whether UNC-30 is required to maintain the functionality of synapses. Moreover, our preliminary data suggest UNC-30 is also required to prevent the adoption of alternative neuronal identities in GABA MNs, suggesting a dual role: activator of madd-4S and GABA biosynthesis genes, and repressor of alternative identity genes. In Aim 3, I will examine the hypothesis that distinct regulatory factors dictate UNC-30’s dual role. This proposal is significant because human mutations in PITX and GABA biosynthesis genes are linked to neurodegenerative disorders. Also, it addresses a long-standing question in the fields of neuron development and disease: how do neurons establish and maintain functional synapses. This work is technically innovative in its use of powerful genetic approaches and implementing novel methods to study TF function in vivo. This project will provide an excellent training opportunity to support my long-term goal of becoming a productive, independent researcher.