Fig. 7. The HB eyelet contacts the LNvs in wild type and so1 mutants, and expresses rh5, rh6 and the norpA gene product. (A-D) Whole mount of wild-type adult brains in epifluorescent microscopy. (A) Double staining of retinal R8 and eyelet projections with GFP (green) and of the LNvs with anti-PDF (red) in a w; rh6-gfp/+ brain. (B) The same brain with GFP staining only. (C) Double staining of retinal R8 and eyelet projections with GFP (green) and anti-PDF (red) in a w; UAS-gfp/+; rh5-gal4/+ brain. (D) The same brain with GFP staining only. Staining of LNvs-contacting fibers has been detected in 6 out of 26 hemispheres. No such staining was observed with either rh5-lacZ or rh5-gfp constructs (not shown). As a control for rh5-gal4 and rh6-gfp expression, staining of retinal photoreceptors projections [
70% of R8 fibers for rh6-gfp and 30%, for rh5-gal4 (Pichaud et al., 1999)] indicated that both constructions were specifically expressed in these cells. (E-G) Whole-mount of mutant w; so1 rh6-gfp/so1 late pupal brains. Pupal brains were used here because their HB eyelet somata remained attached to the optic lobes more frequently than in adult brains. (E,F) HB eyelet is labeled with rh6-driven GFP (green) and LNvs are labeled with anti-PDF (red). (E) Whole mount brain in epifluorescent microscopy. (F) Confocal projection of another sample shows the whole pathway of the HB fiber, from the somata outside of the PDF-labeled arborization in the medulla, to its target area inside the brain. (G) rh6-driven GFP expression in the HB somata of another sample at higher magnification (confocal projection). (H) Presence of the eyelet in the somda mutant. A w; somda mutant brain is stained with anti-chaoptin antibody, which reveals the eyelet cell bodies and projections (confocal projection). Such staining was observed in five out of 39 brain hemispheres, with clearly recognizable cell bodies in two of them. The space corresponding to the optic lobes was always filled with unorganized material (not shown). The star indicates an autofluorescent tracheal structure. (I-K) Expression of the ro-tauZ transgene in the eyelet of wild-type flies (whole mounted w; ro-tauZ adult brains, confocal projections). That construct drives tau-lacZ expression from an artificial promoter comprising rough and Krüppel enhancers (F. Pichaud and U. Gaul, personal communication). (I) Horizontal view of the HB pathway stained with anti-ß-gal antibody. Stained fibers in the larger part of the medulla are from unknown origin. (J,K) Doubly stained brain with anti-ß-gal (J, green) and anti-NORPA (K, red) antibodies. The ro-tauZ expressing eyelet is labeled by anti-NORPA. Similar results were obtained in so1 brains (not shown). No such staining was observed in a norpAP24 mutant context (not shown). The arrowheads indicate HB eyelet pathway. The V-shaped arrowheads indicate HB eyelet somata. HB, termination of the Hofbauer-Buchner eyelet; LNvs, ventral lateral neurons; l-LNvs, large ventral lateral neurons; s-LNvs, small ventral lateral neurons; L, lamina; M, medulla;. Scale bars: 10 µm.
