Our immediate research goals are:

• Dissect the function of transcriptional regulagtory networks on neural circuit formation.
• Investigate the role of neuronal lineage in regulating fly behavior.
• Create a library of split-GAL4 library that can uniquely target each hemilineage in the VNC.

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Dissect the function of transcriptional regulatory networks on neural circuit formation.

Our long-term goal is to use our lineage-specific split-GAL4 libraries to dissect the transcriptional regulatory networks that govern neuronal differentiation, circuit formation, and behavior in the adult CNS. We have identified over 50 transcription factors that are each expressed in discrete subsets of hemilineages in the adult fly VNC. Most of these factors are conserved; many, like the homeodomain-containing proteins Unc-4 and Hb9 and the Pou domain-containing protein Acj6, are known to regulate neuronal fate acquisition in worms, the Drosophila embryo, and vertebrates [1-6]. We expect most of them to act in the adult VNC to govern neuronal differentiation and/or connectivity and behavior. Our proposed studies will clarify the function of select transcription factors such as hb9 and acj6 in the CNS adding to our understanding of how transcriptional regulatory networks dictate neuronal fate, connectivity, and behavior, while addressing larger models of how transcription factors create functionally distinct sets of neurons.

Our approach for characterizing the function of select transcription factor will mirror the one we just completed for Unc-4 [7]. Here, I outline this approach to illustrate its logic, resolution, and power. To dissect the function of Unc-4, we used CRISPR-based methods tools to introduce FRT sites that flank exons 2 and 3 of Unc-4 as well as an attp site just 5’ of exon 2 (Fig. 1A) [7]. Using this line, we created unc-4 GAL4 and split-GAL4 lines as well as null and conditional alleles. Using our split-GAL4 system, we removed unc-4 function throughout the CNS and in individual VNC hemilineages and found that unc-4 acts in hemilineage 11A to promote the cholinergic fate and cell-autonomously in 7B to ensure it innervates leg neuropils, especially the second thoracic (t2) segments (Fig. 1B)[7]. As 7B neurons regulates flight take-off, we used ey-GAL4^AD and dbx-GAL4^DBD lines to remove unc-4 function uniquely in 7B neurons and assessed flight take-off behavior. By eliciting an escape response via a looming visual stimulus, we found that flies lacking unc-4 function only in the 7B lineage processed the visual stimulus, initiated flight take-off behavior by adjusting their posture and raising their wings, but failed to extend their middle legs to drive lift-off (Fig. 1C), a phenotype perfectly recapitulated by cell ablation of 7B neurons with the same driver lines. The T2 specific TTMN muscle-TTMn motor neuron pair drive this lift-off behavior [8]. , We found via retrograde labeling that the processes of 7B interneurons interact at multiple contact sites with TTMn motor neurons throughout t2 leg neuropils, indicating 7B neurons send direct inputs to TTMn motor neurons to execute the TTMn-powered flight take-off behavior (Fig. 1D, E). Our work on Unc-4 illustrates the power of our split-GAL4 system to dissect the function of any gene in the VNC at high resolution.

Another long-term goal of our research is to generate a comrehensive lineage-behavior map. First, we will map individual neuronal hemilineages in the adult fly VNC to neural circuits. To understand how a neural circuit is built, one must first identify its elements and uncover how they interconnect. To connect and place individual neural hemilineages within functional neural circuits in the adult fly VNC, we will be taking multiple complementary approaches (Fig. 2). One approach integrates our adult-specific split-GAL4 library with the genetic-based trans-synaptic labeling tools, such as trans-Tango^[9], to label and reveal the morphology of the postsynaptic partners of the same subsets of neurons. As we have mapped the morphology, marker expression, and fiber tract pattern of all neuronal hemilineages, this approach allows quick identification of the likely post-synaptic partners of individual subclasses of neurons. Another approach uses serial electron microscopy (EM) volume of the adult VNC generated by W. Lee’s lab at Harvard university [10] to reveal how information flows among neuronal lineages. Tracing via the EM volume is the gold standard at identifying synaptic connectivity between neurons at a single synapse level. I recently became part of an EM tracing/proofreading community of about 20 labs across the world who collaborate to map connectivity across the entire adult VNC. Our efforts are greatly assisted by machine learning approaches, which have already generated an initial skeleton for all neurons in the EM volume and detected potential synapse among all neurons. We have already used this approach to identify synaptic connections between 7B neurons and TTMn jump motor neurons.hemilineages in the adult fly VNC to neural circuits.

The adult fly VNC acts as a sensory motor center to sense and respond appropriately to external cues by triggering specific behaviors, such as walking, retreat, take-off, and flight. Our nascent studies support the hypothesis that each neuronal hemilineage or subclass of neurons within a hemilineage controls a specific component of a defined behavior. In the second part of this aim, we will link each neuronal lineage to the behaviors they control by using our lineage specific GAL4 library to ablate, artificially activate, or silence the activity of all neurons in each neuronal hemilineage as well as in subclasses of neurons within each lineage and assess the resulting effect on behavior via high-speed, high-resolution videography and machine learning techniques. . As illustrated in Fig.3, I used these approaches to characterize the function of neurons in the 21A hemilineage. I found that loss of 21A neurons reduced walking speed due to a change in walking gait patterns and geometry of leg joints. In agreement with this, optogenetic activation of all 21A neurons led to flexion of tibia-femur joints and an immediate cessation of walking.

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Create a library of split-GAL4 library that can uniquely target each hemilineage in the VNC.

As explained earlier, our long-term goals are to map each lineage to its neural circuit and associated behaviors and investigate the role of transcription factors on neural circuit formation. To accomplish these goals beyond lineages expressing Hb9 and Acj6, we must first create a complete split-GAL4 library ( Fig.4 ) that can uniquely target gene and cell function in each hemilineage in the adult VNC. Through searches of publicly available single cell RNAseq databases, we have identified 42 genes the expression of which when combined in a binary manner allows us to uniquely mark 32 of the 34 hemilineages in the adult VNC.