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First published online 26 January 2006
doi: 10.1242/dev.02253

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The highly ordered assembly of retinal axons and their synaptic partners is regulated by Hedgehog/Single-minded in the Drosophila visual system

¹ Laboratory of Pattern Formation, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan.
² Graduate Program in Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.

View larger version (67K): [in a new window]   Fig. 1. smo mutant clones are not recovered between R axon bundles. Unless otherwise noted, all images are late third instar, oriented with anterior left and dorsal up. (A) Developing visual system of the third instar larva. R axons (white) and Dac-positive neural cell precursors (green) are labeled; the developing retina, optic stalk and lamina are shown. (B) A schematic drawing of the R axon projection. Photoreceptor cells differentiate as a row in a synchronized stepwise fashion and in a posterior to anterior order, and extend their axons to the lamina via the optic stalk. R axon projection induces lamina neuron differentiation; hence lamina neurons differentiate coordinately in the same manner. Colored bars represent rows of developing photoreceptors and lamina neurons; the same colors roughly represent axonal connections. (C) Schematic illustration of lamina column assembly from the horizontal perspective (left) and lateral perspective (right). As R axon innervations trigger the differentiation of lamina precursor cells in a posterior-to-anterior direction in a stepwise manner, the lamina furrow progresses anteriorly. The G1-phase lamina precursor cells (yellow) contact the most recently arriving R axons, receive Hh provided by the axons, and differentiate into the lamina neurons (light blue) at the trough of the lamina furrow. In older columns (at the posterior of the lamina), a subset of lamina neurons express neuronal markers (pink) as they become mature lamina neurons L1-L5. The R1-R6 axons (orange lines in horizontal and dots in lateral) stop between rows of glial cells (light green), whereas the R7 and R8 axons proceed to deeper target regions in the medulla. We have divided the lamina into two domains, one anterior to the newly arriving R axons and one posterior, and refer to the domains as pre-assembling and assembling, respectively. (D-I) Clones were induced by NP6099-GAL4 UAS-flp and marked by the absence of GFP. The optic lobe with wild-type (D-F) and smo (G-I) clones is shown. The developing lamina was demarcated by white dots (D,G). Developing lamina neurons express Dac (cyan in E,H; blue in F,I). R axons are visualized by the anti-HRP antibody (white in E,F,H,I). (G) smo clones are recovered in OPC (arrow) and in the pre-assembling domains (arrowhead). GFP-negative clones are outlined in F and I (yellow). (G,H) Dac expression is abolished in smo1 clones cell autonomously. (I) In contrast to control (F), smo1 clones were not recovered in the assembling domain.

View larger version (106K): [in a new window]   Fig. 2. Development of lamina neurons and R axon projection. (A-H) Positional relationship between R axons and lamina neurons in early to mid third instar larval brains. Single optical sections (A-D) and 3D reconstructions (E-H) of distinct developmental timing through the third instar larval stage. Lamina neurons visualized by Dac (magenta) and R axons visualized by anti-HRP (white) are shown. In early stages of four to five (A,E), six to seven (B, F) and 11 to 12 (C,G) rows of ommatidial differentiation, lamina neurons are not present in the assembling domain (encircled by yellow dots in F,G). Lamina neurons are detected in the assembling domain when 13 to 14 rows of ommatidia have differentiated (D,H), and at all later stages. (I-K') Newly arriving axons make contact with existing axons. R axons are visualized by GMR-GAL4 UAS-GFP (green), whereas only anterior-most (most recently arriving) R axon bundles are detected by anti-Flamingo antibody (magenta). Images are from a lateral perspective (I,J,J') and a horizontal perspective (K,K'). A magnified image of the dorsal area (white-lined box) in I is shown in J with Fmi signal, and in J' without Fmi signal. Fmi-positive R axon bundles (arrow in K) make contact with Fmi-negative bundles (arrowhead in K) all along the entire lamina layer.

View larger version (99K): [in a new window]   Fig. 3. Incorporation of lamina neurons into the assembling domain is independent of dac or egfr. (A-F) dac (A-C) and egfr (D-F) mutant clones are normally recovered in the assembling domain. Clones are visualized by the absence of GFP (A,D; green), R axons by anti-HRP (A-F; white) and lamina neurons by anti-Dac (C,F; cyan). Mutant clones in lamina are shown (B,E; yellow).

View larger version (101K): [in a new window]   Fig. 4. Single-minded is specifically expressed in lamina under the control of Hh signaling. (A,B) Single-minded (Sim) is expressed specifically in lamina neurons. Sim (white) and Dac (blue) protein are visualized by immunofluorescence microscopy. (C) A magnified image of the boxed area of B. Nuclei of the lamina neurons were visualized by NP6099-GAL4 UAS-nlsGFP in C'. Note that nuclear localization of Sim is obscure in the anterior (left side) and obvious in the posterior (right side) region. (D) A marginal level of Sim expression (white) is detected in the eye disc. Dac (blue) is expressed at the morphogenetic furrow. (E-G) Sim expression is cell-autonomously abolished in smo mutant clones. smo mutant clones (arrowheads) are visualized by the absence of GFP (green); Sim (magenta) and Dac (blue in E; cyan in G) were visualized by immunofluorescence microscopy.

View larger version (114K): [in a new window]   Fig. 5. sim mutant lamina neurons are not incorporated into the assembling domain. (A-C) sim2 clones are not recovered in the assembling domain. Clones are visualized by the absence of GFP (green in A,B), R axons by anti-HRP (white) and lamina neurons by anti-Dac (blue in the merged image B). Mutant clones in lamina are shown in C (yellow). (D-M) Fewer lamina neurons are incorporated into the assembling domain in the mutant. Wild-type (D-F) and sim2/simry75 mutant (G-I) lamina neurons are marked by Dac (magenta in D,F,G,I) and R axons are visualized by anti-HRP (white in D,E,G,H). 3D reconstructions of Dac expression in D and G are shown in F and I, respectively. In contrast to wild type (D-F), in sim2/simry75 mutant brains, a smaller number of lamina neurons are present in the assembling domain (compare G with D, arrows), leaving more lamina neurons behind in the pre-assembling domain (compare G with D, arrowheads). In sim2/simry75 mutant brains, the assembling domain is smaller (H) than in wild type (E). (J-M) Wild-type (J) and sim2/simry75 mutant (L) lamina viewed from a horizontal perspective. Dac expression (magenta) and R axons (white) are visualized with the respective antibodies. Assembling domains are encircled by a yellow line. The number of lamina neurons in the assembling domain is reduced (arrows in J,L) and the number of lamina neurons in the pre-assembling domain is increased (arrowheads in J,L) in sim2/simry75. (K,M) Schematic illustration of J and L, respectively. Colors of cells are as noted in Fig. 1C.

View larger version (144K): [in a new window]   Fig. 6. Targeted expression of dominant-negative dARNT prevents lamina neurons from assembling lamina columns. (A-C) NP6099-GAL4 expression in the optic lobe visualized using UAS-nlsGFP (green) and anti-Dac (magenta in merged image B). NP6099-Gal4 is also expressed in a small part of the most posterior eye disc, including in about 10 ommatidia (arrowhead in C). R axons are visualized by 24B10 (magenta in C). (D-F) A dominant-negative form of dARNT inhibits the incorporation of lamina neurons into the assembling domain; Dac, magenta and R axons, white. 3D reconstruction of Dac expression in D is shown in F. Lamina neurons are almost never seen in the assembling domain (F, arrow in D). (G-I) Effect of a dominant-negative form of dARNT expression is suppressed by co-expression of sim; Dac, magenta and R axons, white. 3D reconstruction of Dac expression in G is shown in I. Lamina neurons are incorporated in the assembling domain (arrow in G) and lamina columns as in the wild type.

View larger version (121K): [in a new window]   Fig. 7. Overexpression of sim causes the premature incorporation of lamina neurons into the assembling domain. (A-C) Overexpression of sim in lamina neurons using NP6099-GAL4 (B,C). Lamina neurons are marked by Dac (magenta) and R axons are visualized by anti-HRP (white in A,B). NP6099-GAL4 driving GFP alone was used as a control (A). 3D reconstruction of Dac expression in B is shown in C. The number of lamina neurons in the pre-assembling domain is reduced (arrowheads in A-C). (D-I) Horizontal views of wild-type and mutant lamina at third larval instar. In contrast to wild type (D,G) and sim mutants (F,I), lamina neurons overexpressing sim (E,H) do not migrate apically; instead, they enter the assembling domain directly (arrowheads in D-F; yellow broken lines in G-I). The lamina precursor cells at the lamina furrow are visualized with tkv-lacZ (magenta). Lamina neurons are marked by NP6099-GAL4 UAS-nlsGFP (blue in D) or Dac (blue in E,F), and R axons by anti-HRP (white). G-I are single channel of tkv-lacZ for D-F, respectively. The migratory route is shown by arrows.

View larger version (46K): [in a new window]   Fig. 8. A model for the onset of lamina column formation, which depends on Single-minded induced by Hh delivered from R axons. (A) Schematic illustrations of the sim mutant phenotype. Wild-type (upper) and sim mutant (lower) lamina. sim mutant neurons (pale blue circles) fail to be incorporated into the assembling domain and accumulate in the pre-assembling domain. Lateral perspective of the wild-type and sim mutant lamina are also illustrated (right). (B) A model for the incorporation of the lamina neurons into the process of lamina column formation. Newly arriving axons project to the lamina layer along existing R axons (I). G1-phase lamina precursor cells receive Hh from R axons, which triggers Dac and Sim expression and differentiation to lamina neurons (II). Sim enables lamina neurons to associate with one of the paired R axon bundles (III). The R axon bundle is enveloped by lamina neurons and is incorporated into the lamina column. Lamina neurons become mature by receiving Spitz, an Egf ligand, which is also provided by R axons at a later stage. (C) Hh delivered from R axons induces dac, egfr and sim independently.