Fig. 2: CG7101/Mirana downregulation causes significant reduction of DCN axons in medulla.
A–G’ Temporal expression pattern of the tagged Mirana protein (67655BDSC combined with ato-Gal414A,UAS-CD4::tdTomato transgenes) during development DCNs shows highest peaks of protein detection at 30–40 h after puparium formation (P30-P40 APF) with subsequent decrease, scale bar – 5 µm. H–L Constitutive downregulation of CG7101/Mirana using two independent RNAi lines (100127VDRC and 27849VDRC) and CRISPR/Cas9 approach (Mirana_sgRNA341305 and UAS-UM-Cas934007 from Heidelberg CFD CRISPR library) in DCNs (driven by ato-Gal414A) leads to a significant decrease in the number of DCNs axons in the medulla, detected at the late pupal stage (P72, H–J) and adult (K–M). N–R Temporal depletion of Mirana in DCNs using Mirana-RNAi100127;ato-Gal414A,UAS-CD4::tdTomato/tub-Gal80ts flies. N, O Mirana depletion in L3-P48 developmental window is sufficient to recapitulate loss of medulla innervation (R). Flies were kept at 18 °C until wondering L3 larvae, and then transferred for 48 h to 29 °C for heat shock inactivation of Gal80 repressor. After heat shock flies were transferred back to 18 °C and were kept there until 14 days old adults. In contrast, Mirana depletion in later developmental stages (from P48 until eclosion) did not cause loss of in medulla innervation (P–R). Flies were kept at 18 °C until P48, and then transferred to 29 °C for heat shock inactivation of Gal80 repressor and kept there until eclosion. Adult flies were transferred into 18 °C and were kept there until 14 days old adults before dissection. OC optic chiasm, Me medulla, scale bar – 20 µm. We examined 12 P72 Control optic lobes, 29 P72 Mirana-RNAi100127, 126 optic lobes of control group adult flies (males and females), 83 of RNAi100127, 42 of RNAi27849, 14 of CRISPR/Cas9. In temporal depletion experiment in development: 34 optic lobes of control flies, 26 of Mirana-RNAi100127, in adult – 12 optic lobes of control flies and 12 of Mirana-RNAi100127. Statistical analysis was done using two-tailed Mann–Whitney test. Exact P values, *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns not significant. Schematics created in BioRender. Hassan, B. (2025) https://BioRender.com/fma66dn.
