A Comprehensive Cell-Type-Specific Developmental Genetic Toolkit for the Drosophila Visual System - Project Summary (R21) Neural progenitors produce an enormous diversity of neuronal and glial cell types to form a functional nervous system. With the advance of single-cell genomic technologies, more and more cell types with distinct transcriptomic signatures have been defined; yet there is a significant lack of genetic tools to identify, label, and manipulate specific cell types for developmental and functional studies. To efficiently and reliably identify useful GAL4 drivers expressed in a single cell type throughout development, we will take advantage of single- cell mRNA sequencing (scRNA-seq) and single nucleus RNA/ATACseq data to produce GAL4/split-GAL4 lines that provide targeted genetic access throughout development to any and all optic lobe neurons. Aim 1: Developing a collection of gene-specific split-GAL4 lines expressed in single cell types in the developing optic lobe. Our scMarco pipeline allows us to identify marker gene pairs in scRNA-seq data whose overlap selectively labels one specific optic lobe cell type. We will generate split-GAL4s inserted in these gene pairs that reproduce the expression of the genes. Their overlap will generate cell-type specific drivers. We have identified 123 genes to generate split-GAL4s hemi-drivers whose pair combinations can selectively target 81% of all optic lobe cell types. Aim 2: Using single-cell multiomics to identify enhancer fragments to generate cell-type-specific drivers. The expression pattern of most genes is controlled by multiple enhancers that each regulate the same gene in different cell types: These enhancers offer high cell-type-specificity. We will use our chromatin accessibility single-cell multiomics (RNA+ATAC-seq) datasets to identify enhancers individually specific to almost any neuron during development that will be cloned to generate GAL4 lines targeting these neuron types. Aim3: Combining gene-specific and enhancer-based split-GAL4 lines for complete neuron coverage. As not all cell types can be marked by a combination of gene specific split-GAL4 drivers or by specific enhancers, we will combine the two approaches above to produce tools targeting all cell types in the optic lobe. We will generate split-GAL4 lines with enhancer fragments that will be used in combination with the gene-specific- split-GAL4 lines. This will provide the cellular morphology and thus the identity of the neuron throughout development and will allow manipulation of their function of each neuron. Altogether, this collection of developmental cell-type-specific driver lines will be a valuable resource to study any cell type in the optic lobe, thus powering further developmental and functional studies. The gene- specific split-GAL4 tools (Aim 1) can also be used in any tissue of Drosophila where single cell transcriptomics is available, thereby expanding the power of this genetic approach to the whole fly community.
