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Larval optic nerve and adult extra-retinal photoreceptors sequentially associate with clock neurons during Drosophila brain development

Institut de Neurobiologie Alfred Fessard, CNRS UPR 2216 (NGI), 91198 Gif-sur-Yvette, France

View larger version (91K): [in a new window]   Fig. 1. Anatomical contact between the Bolwig nerve and the lateral neurons in wild-type third instar larvae. (A-C) Whole-mounted CNS of a third instar w; GMR-gal4/UAS-gfp larva, with one eye imaginal disc (epifluorescent microscopy). The white box indicates the region shown in greater detail in B and C. Green (A,C): staining of the visual system with GMR-gal4-driven GFP expression. Red (A,B): anti-ß-PDF staining of the lateral neurons. (D,E) Whole-mount of third instar larval CNS (epifluorescent microscopy). The LNs are visualized by anti-ß-PDF antibody (red). Expression of the rhodopsin genes in the Bolwig organ is detected through reporters driven by specific promoters. (D) Green: GFP staining of the BN in a w; UAS-gfp/+; rh5-gal 4/+ larva. (E) Green: ß-galactosidase expression in the BN revealed by anti-ß-gal antibody in a w; rh6-lacZ larva. (F) Confocal reconstruction. Double staining of the BN in a w; rh6-gfp/rh5-lacZ larva shows that rh5 and rh6 are expressed in different axons (and therefore different cells) of the BN. Green, GFP staining of rh6-expressing fibers; red, anti- ß-gal staining of rh5-expressing fibers. No rh1 expression was found in the BN using either rh1-gal4 (with a UAS-gfp reporter) or rh1-norpA (with anti-NORPA antibody) constructs. (G-I) Whole-mounts of third instar larval CNS (confocal microscopy) (G,H, 1 µm single optical section; I, five projected optical sections). (G) Anti- ß-PDF staining (red) reveals the LNs and anti-chaoptin staining (green) reveals the BN in a wild-type larva (w). Like the GMR-gal4 transgene used in A-C, the chaoptin gene is expressed in both larval and adult photoreceptors. (H,I) LNs are visualized by GFP expression (green) in w; UAS-gfp; gal1118 larvae. Anti-ChAT (H) or anti-NORPA (I) immunoreactivities (red) label the BN. No anti-NORPA labeling was observed in the BN of norpAP24 mutant larvae (not shown). APP, adult photoreceptors projections; BN, Bolwig nerve; DA, dendritic arborization of LNs; DP, dorsal projections of LNs; EID, eye-antenna imaginal disc; LNs, lateral neurons. Arrowheads indicate contact between BN terminus and LNs dendritic arborization. Scale bars: 10 µm.

View larger version (109K): [in a new window]   Fig. 2. Anatomical contact between the Bolwig nerve and the lateral neurons in wild type embryo. (A) Whole-mount of a stage-17 w; UAS-lacZ; gal1118 embryo stained with a monoclonal anti-ß-gal antibody to detect the LNs (see Materials and Methods). Broken line indicates the limit between different focal planes. Neurites extending from the labeled cells were seen in 10 out of 17 hemispheres. (B-E) Whole-mount of a stage-17 w; UAS-lacZ; gal1118 embryo double-stained with a polyclonal anti-ß-gal antibody and the anti-chaoptin antibody to reveal the visual system. We also found embryos with only the BO/BN labeled and no detectable gal1118 expression in the LNs (data not shown). This is consistent with the start of gal1118 expression at the beginning of stage 17 that we observed with gal1118-driven GFP in live embryos (not shown). (B) The two Bolwig organs are visible near the oral armature and their projections run around the CNS towards their targets. (C-E) detail of the contact zone (white box in B) from the same embryo at higher magnification. [C,D (green)] Anti-chaoptin staining of the BN termination. [D (red), E] Anti-ß-gal staining of the LNs and their projections. The BN ending appeared to contact the smaller one of the two main neuritic branches, which presumably represents an early stage of development of the dendritic arborization illustrated in Fig. 1. BN, Bolwig nerve; BOs, Bolwig organs; DA, dendritic arborization of LNs; DP, dorsal projections of LNs; LNs, lateral neurons. Arrowheads indicate the contact zone. Scale bars: 10 µm.

View larger version (63K): [in a new window]   Fig. 3. Atrophy of the dendritic arborization of the lateral neurons in the absence of the BN. (A-F) Whole-mounts of third instar larval CNS; LNs are stained with GFP (green) and the visual system with anti-chaoptin (red). (A,B) Visual system (A) and LNs (B) in a w; UAS-gfp/+; gal1118/+ control CNS. (C) Apparently normal visual system in a w UAS-hid UAS-rpr/w; UAS-gfp/+; gal1118/+ CNS (as observed in 10 out of 10 brains). (D) BN-depleted visual system and LNs without dendritic arborization in a w; GMR-hid/UAS-gfp; gal1118/+ CNS. Although the BN was always undetectable in these third instar larval brains, the developing adult photoreceptors were present. They only degenerated during pupal life (data not shown), presumably when the apoptotic pathways were fully activated by HID expression. (E) Absence of visual system, and LNs without any detectable dendritic arborization in a w; UAS-gfp/+; gl60J gal1118/ gl60J CNS. (F) Visual system without adult photoreceptors and LNs with their dendritic arborization in a w; eya2 UAS-gfp/ eya2; gal1118/+ mutant. (G,H) Whole-mounts of third instar larval CNS. LNs are stained with anti-PDF. (G) LNs without dendritic tree in a somda mutant. Broken line indicates the limit between different focal planes. (H) Wild-type LNs. A dendritic arborization was observed in only 9% of somda brain hemispheres (n=34), versus 92% of controls (n=13). APP, adult photoreceptors projections; BN, Bolwig nerve; DA, dendritic arborization of LNs; DP, dorsal projections of LNs; LNs, lateral neurons. Scale bar: 10 µm.

View larger version (91K): [in a new window]   Fig. 4. Extension of dendritic arborization of the larval LNs, caused by Kir2.1 expression in the visual system, and in a wild-type prepupa. (A,B) Whole-mount of a doubly stained CNS of w; GMR-gal4 UAS-gfp/+; UAS-Kir2.1/+ third instar larval brain. (A) Anti-PDF staining of the LNs. Arrowheads indicate the long PDF-immunoreactive extension (seen in 9/10 hemispheres, versus 0/6 for the third instar controls in the same experiment, not shown). Similar alterations were already detectable in the first larval stage (not shown). (B) GFP staining of the visual system. The BN ending is much thinner than normal (compare with Fig. 1C). (C,D) Whole-mount of a doubly stained CNS of control w; UAS-cd8-gfp; gal1118 at 4 hours after puparium formation (APF). CD8-GFP is used instead of GFP to label the processes of the LNs better. (C) CD8-GFP staining of LNs. The same kind of extension has been seen in about 10% of hemispheres from three independent experiments (also with pdf-gal4 driven UAS-gfp expression in the LNs) performed within approximately 6 hours around puparium formation. (D) Anti-chaoptin staining of the visual system. No morphological alterations were observed. APP, adult photoreceptors projections; BN, Bolwig nerve; DA, dendritic arborization of LNs; DP, dorsal projections of LNs; LNs, lateral neurons. Scale bar: 10 µm.

View larger version (84K): [in a new window]   Fig. 5. Changes in the visual system and the LNs during pupation. Whole mounts of w; pdf-gal4 UAS-gfp pupal brains doubly stained with anti-chaoptin (red) for the visual system and GFP (green) for the LNvs. Similar results were obtained with both gal1118-driven GFP labeling and anti-PDF labeling of the LNvs (not shown). However, most of the experiments were performed with pdf-gal4-driven GFP labeling, because it remained restricted to the s-LNvs for the longest developmental time, thus ensuring that the arborization observed up to 32 hours APF indeed originated from the s-LNvs and not from the l-LNvs. Time APF is indicated in hours. Thin arrows indicate the BN that is detected up to 8 hours APF (A-C). (D,E) Thick arrows indicate the reduced dendritic arborization of the LNs at 16 hours (D) and 32 hours (E) APF. Arrowheads show the newly appeared chaoptin-expressing fiber (F-H). G,H are the same sample, but H shows only chaoptin staining for better visualization. APP, adult photoreceptors projections; BN, Bolwig nerve; LNs, lateral neurons; DP, dorsal projections of LNs; l-LNvs, large ventral lateral neurons; s-LNvs, small ventral lateral neurons; M, medulla. Scale bar: 10 µm.

View larger version (11K): [in a new window]   Fig. 6. The dendritic arborization of the s-LNvs regresses during early metamorphosis. The ordinate shows the average relative size of the dendritic arborization of the LNs where 100 is the mean value for the L3 larvae used as reference. The x-axis indicates time spent APF. Bars indicate s.e.m. Quantification was performed on GFP labeling in s-LNvs of w; pdf-gal4 UAS-gfp at times when GFP expression could not yet be detected in the l-LNvs (see also legend to Fig. 5).

View larger version (97K): [in a new window]   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.