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. 2022 Jan 17;11:e73357. doi: 10.7554/eLife.73357

Figure 2. Hedgehog (Hh) signaling regulates nord expression in the Drosophila wing imaginal disc.

(A) Schematic diagram of the wild-type nord locus and the protein-trap alleles of nord. The EGFP-FlAsH-StrepII-3xFLAG (GFSTF) or TagRFP-T-3xHA (RFPHA) tag was inserted in the appropriate orientation and reading frame of nord, which permitted visualization of the Nord protein localization in vivo. (B) Wing imaginal discs from late third instar larvae carrying nord-GFP (Mi{PT-GFSTF.2}nordMI06414-GFSTF.2/+) were immunostained for GFP (green), Ptc (red), and Ci (blue). Maximum intensity z-projection and 3D reconstruction from a confocal image stack show nord expression in a representative wing imaginal disc. White brackets indicate the expression range of Ptc or Nord-GFP. (C, D) Wing imaginal discs from late third instar larvae carrying nord-RFP (Mi{PT-RFPHA.2}nordMI06414-RFPHA.2/+) and flip-out clones expressing the indicated UAS-transgenes were immunostained for HA (Nord-RFP, red), GFP (flip-out clones, green), and Ci (A compartment, blue). (D’–D”’) Zoomed view of the indicated area from panel (D). Note that ectopic nord-RFP is induced in UAS-SmoGlu-expressing clones located in the A compartment flanking the wing pouch (D’), but not in the P compartment (D”’). In the central wing pouch (D”), little (yellow star) or none (blue star) ectopic Nord-RFP was detected in SmoGlu-expressing flip-out clones. Dashed white lines indicate the clone boundary; dashed blue lines indicate the A/P compartment boundary, which is determined by the expression of endogenous Ci. Scale bar, 50 μm.

Figure 2.

Figure 2—figure supplement 1. Schematic diagram of the wild-type nord locus, the gene-trap, and the protein-trap alleles of nord.

Figure 2—figure supplement 1.

(A) Schematic diagram of the wild-type nord locus, the gene-trap, and the protein-trap alleles of nord. A Minos-Mediated Integration Cassette (MiMIC) consisting of a splice acceptor site followed by stop codons in all three reading frames was inserted into the first coding intron of nord in the nord gene-trap allele Mi{MIC}nordMI0641. The EGFP-FlAsH-StrepII-3xFLAG (GFSTF) or TagRFP-T-3xHA (RFPHA) protein-trap cassette was inserted into the first coding intron in the appropriate orientation and reading frame of nord, which resulted in the Nord-GFP or Nord-RFP fusion. Note that the gene Ir60a (light blue) is transcribed from the opposite strand. (B) Schematic diagram of the predicted protein or polypeptide products from the wild-type nord locus, the gene-trap, and the protein-trap alleles of nord. (C–F) RT-PCR confirmation of the predicted transcripts from the wild-type nord locus, the gene-trap, and the protein-trap alleles of nord. RNA is isolated and reverse-transcribed from the indicated fly lines. The location of PCR primers is marked in (A).
Figure 2—figure supplement 2. Alignment of Nord, Nord-GFP, and Nord-RFP encoded by transcripts from the wild-type or nord protein-trap alleles.

Figure 2—figure supplement 2.

Figure 2—figure supplement 3. Hedgehog (Hh) signaling regulates nord expression in the Drosophila wing disc.

Figure 2—figure supplement 3.

(A–C) Larvae carrying nord-GFP (Mi{PT-GFSTF.2}nordMI06414-GFSTF.2/+) and hs-Gal4 in combination with the indicated transgenes were heat shocked at 37°C for 30 min. 24–48 hr later, the wing imaginal discs were collected and immunostained for GFP (green), Ptc (red), and Ci (blue). Note that Nord-GFP expression domain is expanded anteriorly with ectopic expression of Hh (B), but reduced with ectopic expression of Ptc (C). White brackets indicate the expression range of Nord-GFP. Scale bar, 50 μm.