Identification of Split-GAL4 Drivers and Enhancers That Allow Regional Cell Type Manipulations of the Drosophila melanogaster Intestine

Abstract

The Drosophila adult midgut is a model epithelial tissue composed of a few major cell types with distinct regional identities. One of the limitations to its analysis is the lack of tools to manipulate gene expression based on these regional identities. To overcome this obstacle, we applied the intersectional split-GAL4 system to the adult midgut and report 653 driver combinations that label cells by region and cell type. We first identified 424 split-GAL4 drivers with midgut expression from over 7,300 drivers screened, and then evaluated the expression patterns of each of these 424 when paired with three reference drivers that report activity specifically in progenitor cells, enteroendocrine cells, or enterocytes. We also evaluated a subset of the drivers expressed in progenitor cells for expression in enteroblasts using another reference driver. We show that driver combinations can define novel cell populations by identifying a driver that marks a distinct subset of enteroendocrine cells expressing genes usually associated with progenitor cells. The regional cell type patterns associated with the entire set of driver combinations are documented in a freely available website, providing information for the design of thousands of additional driver combinations to experimentally manipulate small subsets of intestinal cells. In addition, we show that intestinal enhancers identified with the split-GAL4 system can confer equivalent expression patterns on other transgenic reporters. Altogether, the resource reported here will enable more precisely targeted gene expression for studying intestinal processes, epithelial cell functions, and diseases affecting self-renewing tissues.

Deterministic Genetic Barcoding for Multiplexed Behavioral and Single-Cell Transcriptomic Studies

“…In summary, combined with the large collections of Gal4 and split-Gal4 lines that have been established in Drosophila that enable precise targeting of a high proportion of cell types (Ariyapala et al, 2020;Aso et al, 2014;Davis et al, 2020;Gohl et al, 2011;Namiki et al, 2018;Pfeiffer et al, 2010Pfeiffer et al, , 2008Venken et al, 2011), TaG-EM provides a means to target and label cells in vivo for subsequent detection in single-cell sequencing. Moreover, these genetic barcodes can be used to multiplex behavioral or other phenotypic measurements.…”

“…To test whether we could detect TaG-EM barcodes in single-cell sequencing data, we crossed three TaG-EM barcode lines to two different gut Gal4 driver lines (Ariyapala et al, 2020), one expressing in the enterocytes (EC-Gal4) and the other in intestinal precursor cells (PC-Gal4), including stem cells and enteroblasts (EBs). Due to weak GFP expression with the EC-Gal4 driver, we did not see visible GFP positive cells for this driver line.…”

“…Thus, most of these cell classifications relied upon inference as opposed to direct positive labeling. Recently, a large collection of split-Gal4 lines were screened for expression in the adult and larval gut (Ariyapala et al, 2020). These include pan-midgut driver lines, split-Gal4 lines specific for the EBs, ECs, EECs, and ISC/EBs, as well as driver lines with regionalized gene expression.…”

“…These include pan-midgut driver lines, split-Gal4 lines specific for the EBs, ECs, EECs, and ISC/EBs, as well as driver lines with regionalized gene expression. We crossed four different TaG-EM barcode lines with the pan-midgut driver (PMG-Gal4), and one barcode line to each of the precursor cell (PC-Gal4), enterocyte (EC-Gal4), enteroblast (EB-Gal4) and enteroendocrine (EE-Gal4) drivers (Ariyapala et al, 2020). Larval guts were dissected from these lines and cells were dissociated, flow sorted as described above to select live, GFP positive cells, and approximately 30,000 cells were loaded into a 10x Genomics droplet generator for single-cell sequencing.…”

split-intein Gal4 provides intersectional genetic labeling that is fully repressible by Gal80

“…amino acids 2-93), split intein GeneSwitch constructs were generated using as the splice junction the same C 21 cysteine residue. The split intein Gal4 and split intein GeneSwitch constructs thus share the same N-terminal Gal4 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] component.…”