Characterization of a morphogenetic furrow specific Gal4 driver in the developing Drosophila eye

Abstract

The ability to express a gene of interest in a spatio-temporal manner using Gal4-UAS system has allowed the use of Drosophila model to study various biological phenomenon. During Drosophila eye development, a synchronous wave of differentiation called Morphogenetic furrow (MF) initiates at the posterior margin resulting in differentiation of retinal neurons. This synchronous differentiation is also observed in the differentiating retina of vertebrates. Since MF is highly dynamic, it can serve as an excellent model to study patterning and differentiation. However, there are not any Gal4 drivers available to observe the gain- of- function or loss- of- function of a gene specifically along the dynamic MF. The decapentaplegic (dpp) gene encodes a secreted protein of the transforming growth factor-beta (TGF-beta) superfamily that expresses at the posterior margin and then moves with the MF. However, unlike the MF associated pattern of dpp gene expression, the targeted dpp-Gal4 driver expression is restricted to the posterior margin of the developing eye disc. We screened GMR lines harboring regulatory regions of dpp fused with Gal4 coding region to identify MF specific enhancer of dpp using a GFP reporter gene. We employed immuno-histochemical approaches to detect gene expression. The rationale was that GFP reporter expression will correspond to the dpp expression domain in the developing eye. We identified two new dpp-Gal4 lines, viz., GMR17E04-Gal4 and GMR18D08-Gal4 that carry sequences from first intron region of dpp gene. GMR17E04-Gal4 drives expression along the MF during development and later in the entire pupal retina whereas GMR18D08-Gal4 drives expression of GFP transgene in the entire developing eye disc, which later drives expression only in the ventral half of the pupal retina. Thus, GMR18D08-Gal4 will serve as a new reagent for targeting gene expression in the ventral half of the pupal retina. We compared misexpression phenotypes of Wg, a negative regulator of eye development, using GMR17E04-Gal4, GMR18D08-Gal4 with existing dpp-Gal4 driver. The eye phenotypes generated by using our newly identified MF specific driver are not similar to the ones generated by existing dpp-Gal4 driver. It suggests that misexpression studies along MF needs revisiting using the new Gal4 drivers generated in our studies.

A soy protein Lunasin can ameliorate amyloid-beta 42 mediated neurodegeneration in Drosophila eye

“…Eye-antennal discs from wandering third instar larvae were dissected, and fixed in 4% paraformaldehyde in Phosphate Buffered Saline (PBS), and stained following the protocol 85 – 87 . The primary antibodies used were rabbit anti-Dlg (1:200; a gift from K. Cho), mouse anti-Wg [1:50,Developmental Studies Hybridoma Bank,(DSHB)], rat anti-Elav (1:50; DSHB), mouse anti-Dlg (1:100; DSHB), mouse anti-22C10 (1:100; DSHB), mouse anti-Chaoptin (MAb24B10) (1:100; DSHB 88 ), mouse anti-Ey (1:100, DSHB), mouse anti-Eya (1:100, DSHB), mouse anti-Dac (1:100, DSHB), mouse anti-β-galactosidase (1:100; DSHB), rabbit anti-β-galactosidase (1:200) (Cappel), and mouse anti-GFP (1:100, GFP-G1, DSHB).…”

Proximal fate marker homothorax marks the lateral extension of stalk‐eyed fly Cyrtodopsis whitei

“…Adult flies were prepared for imaging by freezing at −20°C for ~2 hr (Sarkar, Gogia, Farley, Payton, & Singh, ; Wittkorn, Sarkar, Garcia, Kango‐Singh, & Singh, ). Images were captured on a stereomicroscope attached to a camera.…”

A Positive Feedback Loop of Hippo- and c-Jun-Amino-Terminal Kinase Signaling Pathways Regulates Amyloid-Beta-Mediated Neurodegeneration

“…Eye-antennal imaginal discs were dissected from the wandering third-instar larvae in 1X phosphate buffered saline (PBS), fixed in 4% paraformaldehyde in PBS for 20 min, and washed in PBS. We stained the tissue with a combination of antibodies following a previously published protocol Sarkar et al, 2018a). The primary antibodies used were rat anti-Embryonic Lethal Abnormal Vision (ELAV) (1:50; Developmental Studies Hybridoma Bank, DSHB), mouse antidiscs large (Dlg) (1:100; DSHB), rabbit anti-Dlg (1:200; a gift from Dr. K. Cho), mouse anti-6E10 (1:100), rabbit anti-βgalactosidase (1:200; Cappel), mouse anti-22C10 (1:100; DSHB), and mouse anti-Chaoptin (MAb24B10) (1:100; DSHB) (Zipursky et al, 1984).…”