GAL4 drivers for midgut study

Reference driver stocks

Reference drivers are available in the following stocks.


Additional stocks for midgut study were generated in subsequent studies.


Characterization of reference drivers

To assure that the reference drivers were expressed effectively in specific intestinal cell types, we compared the expression of UAS-GFP in the presence of driver pairs to staining with antibodies detecting cell type-specific protein expression. The results are presented in detail in Ariyapala et al. (2020), but the following summarizes our results.

In general, we saw that the reference drivers were expressed well in their target cells except 1) they were all expressed in fewer cells in R3 than in other regions and 2) drivers we expected to express in all Enteroendocrine cells (R57F07.A) were expressed in a fraction of EEs. 

Because EE identification was limited by our original reference drivers, we later identified and characterized a new pair of EE reference drivers (R20C06), which are described in detail in Holsopple et al. (2020). Details on both EE reference driver pairs are summarized on this page.

 

Pan-midgut drivers

To assess expression of P{CG10116-p65.AD}attP40 and P{CG10116-GAL4.DBD}su(Hw)attP6, we combined them in the same flies and compared P{UAS-GFP.nls}8 expression to DAPI or antibody staining. Results are shown in the graph and antibody details are given in the box. Note the relatively low EE expression.

Image displaying percentages of cells expressing GFP by region and cell subtype

CellAntibody staining criteria
Progenitor cellspositive for anti-Horseradish Peroxidase (HRP)
Enteroblastspositive for anti-HRP AND negative for anti-Delta
Enterocytesnegative for anti-HRP AND anti-Prospero
Enteroendocrine cellspositive for anti-Prospero

Progenitor cell drivers

Progenitor cells are intestinal stem cells and their daughter cells, the enteroblasts. We combined P{VT004241-p65.AD}attP40 and P{VT024642-GAL4.DBD}attP2 with P{UAS-Stinger}2, stained with antibodies and saw the results shown in the graph.

Graph displaying percentage of progenitor cells expressing GFP by region when progenitor reference drivers were crossed together

Enteroblast driver

We were able to generate only a DBD driver with enteroblast expression. We combined P{GBE-GAL4.DBD}attP2 with the pan-midgut driver P{CG10116-p65.AD}attP40 and P{UAS-GFP.nls}8, stained with antibodies and saw the results shown in the graph. Negligible expression was observed in R3.

Graph displaying percentages of EBs with GFP expression by region when crossed to the PM.AD driver.

Enterocyte drivers

Flies carrying both our enterocyte-specific AD and DBD drivers died, so we assessed the drivers in crosses to pan-midgut drivers. P{VT004958-p65.AD}attP40  was combined with P{CG10116-GAL4.DBD}su(Hw)attP6 and P{UAS-GFP.nls}8. P{VT004958-GAL4.DBD}attP2 was combined with P{CG10116-p65.AD}attP40 and P{UAS-GFP.nls}8. The graphs show the percentage of ECs with GFP expression in each midgut region.

Graph displaying number of ECs expressing GFP by region when EC.AD was crossed to PM.DBD

Graph displaying percentages of ECs expressing GFP by region when EC.DBD was crossed to the PM.AD driver.

Enteroendocrine drivers

P{R57F07-p65.AD.A}attP40 and P{R57F07-GAL4.DBD.A}attP2 were combined with P{UAS-Stinger}2. P{R20C06-p65.AD}attP40 and P{R20C06-GAL4.DBD}attP2 were also combined with P{UAS-Stinger}2. The R57F07.A drivers were created and characterized in Ariyapala et al. (2020) and further characterized by Holsopple et al. (2022), along with the R20C06 drivers. The figure below compares the relative percentages of EEs detected by each driver pair.

EE-driver-characterization-graph_web.png


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