Transcription factors regulate the molecular, morphological, and physiological characters of neurons and govern their impressive cell type diversity. Determining how these transcription factors (TFs) are activated during neuron differentiation will help us understand how neurons develop and establish better protocols for in vitro differentiation of pluripotent cells towards specific neuronal types. I propose to study this process from a molecular, developmental, and evolutionary perspective using the Drosophila optic lobes as a model system.
My first aim i s to bridge the gap between cell fate specification and terminal differentiation. How do neurons acquire their characters after they are specified? During the K99 part of the award, I will use a large- scale single-cell sequencing protocol to access the transcriptome of single cells in the optic lobes at different stages of development. By using new and established algorithms, I will draw developmental trajectories for every neuronal cell type and annotate them with the TFs that are important for a cell to acquire each trajectory.
My second aim i s to identify the cis-elements and trans-acting factors that regulate terminal characters. I will first scan the enhancers of neurotransmitter-related genes to identify cis-elements and, during the R00 phase of the award, I will use a prediction algorithm, knockout approaches, and the trajectories of the first Aim to identify the trans-acting factors and their onset of expression. Finally, in an approach that will lead to my future research, the above pipeline will be expanded to cell adhesion molecules and morphological features.
My third aim will be performed during the R00 period. I will study how neurons evolve and incorporate into circuits to control distinct behaviors in different species. I will first focus on a neuronal circuit that controls looming behavior in flies to find the genetic mutations that led to the evolution of this circuit. Second, I will sequence single neurons from the optic lobes of different arthropods. I will compare neurons amongst different brains and use CRISPR to knock crucial TFs out to identify their role in the evolution of novel cell types. The collective results of this project will generate a deep understanding of the developmental and evolutionary history of optic lobe neurons. I will identify basic principles of neuronal differentiation that will be of fundamental importance for the enhancement of in vitro neuronal differentiation techniques for regenerative therapies. A K99 Award would allow me to receive additional training in informatics and CRISPR and provide time to extend my expertise in neurobiology and generate a single-cell atlas for the Drosophila optic lobes. The training phase of this proposal will be performed in the laboratory Dr. Claude Desplan, which is ideal for exchanging exciting ideas about different aspects of the development of the Drosophila visual system and for tool development. The NYU Center for Developmental Genetics and Center for Genomics and Systems Biology provide imaging and sequencing facilities necessary for the proposed work. My scientific experiences have encouraged me to plan my career towards establishing an independent academic research laboratory.
The potential of regenerative therapies is limited by our poor knowledge of the developmental trajectories of different cell types. This proposal seeks to discover how a) neuronal diversity is generated by the implementation of a differentiation program during development, b) how different transcription factors are activated and act on the enhancer elements of terminal effecor genes to generate this diversity, and c) how diverse neurons evolve over time to accommodate the specific needs of different animals. Ultimately, understanding the developmental and evolutionary history of most neurons of the optic lobe will have profound implications for the treatment of neurodegenerative diseases.
