How temporal transcription factors regulate guidance cues and dendrite morphogenesis in Drosophila somatosensory circuits

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ABSTRACT Properly developed somatosensory circuits are critical for any animal to sense and interact with their environment. The development of somatosensory circuits requires that each neuron within the circuit to correctly position its axons and dendrites. This allows them to establish specific synaptic connections and for circuits to assemble. Within somatosensory circuits, interneurons represent are the most numerous neuron type. The other types are sensory neurons of the peripheral nervous system. In comparison to sensory neurons, interneurons are under investigated due to lack of tools and knowledge about somatosensory circuit architecture. Molecular factors, like temporal transcription factors and guidance cues, are known to regulate different aspects of neuronal development, however the role of these molecular factors has not been deeply investigated in interneuron dendrite development, synaptic partner selection, and circuit formation. I will test the central hypothesis that temporal transcription factors regulate guidance receptor expression, and this establishes specific dendritic morphologies and synaptic connections. To investigate this central hypothesis, in the Drosophila nerve cord, I will be using the neuroblast 3-3 stem cell and the EL interneurons it generates as a model. In Aim 1, I will test the working hypothesis in NB3-3, temporal transcription factors, Cas and Nab, activate different sets of guidance cue receptors in its early-born and late-born EL daughter interneurons, and that this determines differential dendrite positioning. I use single cell RNA-sequencing, NB3-3 specific gene manipulation of Cas and Nab, and confocal microscopy to examine dendrite morphology. I expect to connect the role of temporal transcription factors to the role of guidance cue receptor expression. Additionally, I expect to introduce an innovative sequencing approach to our current analysis of somatosensory circuit development. In Aim 2, I will test the working hypothesis that the guidance receptor Roundabout3 (Robo3) is necessary in early-born EL dendrites and sufficient in late-born EL dendrites to regulate dendrite morphogenesis and position. I will perform Robo3 subcellular localization assays alongside gain and loss-of-function assays of Robo3 to examine the function of Robo3 in EL dendrites. This aim will serve to identify the requirement of Robo3 in interneurons and develop new assays to analyze Robo3 localization. Overall, this proposal will connect to previous disparate areas of somatosensory circuit development. This work will also enable future research to investigate the broad conservation of neuronal circuit specification and development of therapies for neurodevelopmental pathologies in which circuit wiring is disrupted.