Using single-cell RNA sequencing to generate predictive cell-type-specific split-GAL4 reagents throughout...

Area of Science:

• Neuroscience
• Genetics
• Developmental Biology

Background:

• Cell-type-specific genetic tools are crucial for dissecting complex neuronal circuits.
• Existing Drosophila enhancer-based GAL4 lines have limitations in accurately reflecting native gene expression.
• Current intersectional techniques like split-GAL4 require extensive screening.

Purpose of the Study:

• To develop an efficient and predictive pipeline (scMarco) for generating cell-type-specific split-GAL4 lines.
• To utilize developmental single-cell RNA sequencing (scRNAseq) data for selecting gene pairs for split-GAL4.
• To enable the generation of gene-specific split-GAL4 lines at any developmental stage.

Main Methods:

• Leveraged existing developmental scRNAseq datasets to identify gene pairs for split-GAL4 construction.
• Developed the scMarco pipeline for efficient and predictive generation of split-GAL4 lines.
• Utilized coding intronic MiMIC/CRIMIC lines or CRISPR knock-in for generating gene-specific split-GAL4 lines.
• Validated the approach using the developing Drosophila visual system.

Main Results:

• Demonstrated high predictive power of scRNAseq-guided gene-specific split-GAL4 lines in targeting known cell types.
• Successfully annotated cell clusters within scRNAseq datasets.
• Identified novel cell types within the developing Drosophila visual system.
• Showcased the broad applicability of generated lines to other Drosophila tissues.

Conclusions:

• The scMarco pipeline provides a highly efficient method for generating cell-type-specific split-GAL4 tools.
• This approach enables precise genetic manipulation of distinct cell types throughout development.
• The generated gene-specific split-GAL4 lines represent a valuable resource for the Drosophila research community.