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First published online 30 June 2004
doi: 10.1242/dev.01242

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Rhomboid 3 orchestrates Slit-independent repulsion of tracheal branches at the CNS midline

¹ Department of Developmental Biology, Wenner-Gren Institute, Stockholm University, S-106 96 Stockholm, Sweden
² Department of Medical Nutrition, Karolinska Institute, Stockholm, Sweden
³ Department of Natural Sciences, Södertörns Högskola, S-141 04 Huddinge, Sweden
⁴ Umeå Centre for Molecular Pathogenesis, Umeå University, S-901 87, Umeå, Sweden

View larger version (69K): [in a new window]   Fig. 1. rho3 affects CNS midline repulsion of tracheal ganglionic branches (GBs). (A-F) Three dimensional confocal reconstructions of late stage16 embryos double-stained to reveal the tracheal lumen (by mAb2A12, A-F in red) and longitudinal fascicles (by mAb1D4, B, E in green) or CNS glia (with the exception of midline glia, by anti-Repo C, F in green). All panels show ventral views, anterior towards the left. In wild-type embryos, GBs (A, red) and longitudinal fascicles (B, green) never cross the midline. In rho3, an average of two GBs per embryo cross the midline (D-F, arrows). In the same embryos the longitudinal fascicles appear unaffected (green in E and compare with B), as does the pattern of glial cells (F, compare with C). Scale bar: 20 µm.

View larger version (62K): [in a new window]   Fig. 2. rho3 acts independently of slit. (A-D) Slit protein levels are not changed in rho3 mutants. An anti-Slit antibody (in green; red is the tracheal lumen, mAb2A12) detects similar levels of Slit in early (C) and late (D) rho3 stage 16 embryos when compared with wild type (A,B), even in the vicinity of branches that cross the midline in the mutant (D, arrows). A-D are ventral views, anterior towards the left. Scale bar in B: 20 µm. (E-G) A ventral view of late stage 16 embryos stained for the tracheal lumen (by mAb2A12) reveals GB pathfinding defects in slit mutants (F) and slit, rho3 double mutants (G) when compared with wild type (E, anterior is towards the left). (H) Quantification of the midline cross phenotype shows that the effect of the two mutations is additive.

View larger version (92K): [in a new window]   Fig. 3. rho3 is expressed by the ventral unpaired group of midline neurons (VUM). (A) Ventral view of late stage 16 inga heterozygote embryo triple stained to reveal the tracheal lumen (mAb2A12 in blue), a subset of PNS and CNS axons (mAb22C10 in green), and the expression of ß-Gal in the rho3inga enhancer trap (anti-ß gal in red). The panel shows a 3D reconstructions deriving from a confocal stack, anterior is towards the left. Strong ß-gal expression is detected in each segment in ventral clusters of cells at the midline. (B) A similar expression pattern is detected by in situ hybridisation with a specific rho3 cDNA probe at stage 15, in clusters of cells at the VNC ventral midline (B, arrow; B-E are lateral views of the VNC, anterior towards the left, ventral downwards; B is a stage 15 embryo, C-E are late stage 16). (C-E) 3D confocal reconstruction allows the identification of the rho3-expressing cells (in red, stained by anti-ß-gal) as the VUM neurons. VUM cell bodies are readily stained by mAb22C10 (C, in green), which also allows identification of the characteristic VUM axonal tract (C, arrowhead). Slit staining (E, in green) and Wrapper staining (D, in green) of midline glia, shows little overlap with ß-gal expression in rho3inga (D,E, in red). Scale bars: 20 µm.

View larger version (56K): [in a new window]   Fig. 5. Rhomboid is required in the CNS to prevent GB midline crossing. (A) The inga enhancer trap showed weak, but reproducible, reporter gene expression in GB1. Ventral view of an inga/+ embryo, double stained for ß-gal and the tracheal lumen (mAb2A12, both in brown). GB1 expression is shown (arrowheads in A). (B-D) RicinA ablation of most of all GB1 cells (in a SRF>RicinA embryo) did not affect longitudinal fascicles (stained by anti-Fas2 in green, B) and glial populations (green in C,D; stained by anti-Repo and anti-Wrapper respectively, all panels show ventral views). Scale bar in D: 20 µm. In addition, isolated surviving GBs migrate correctly (B-D arrows). (E) Rhomboid expression by CNS cells, but not by GB1, rescues the rho3 GB midline cross phenotype. The table represents the frequency of midline crosses for the different genotypes. Expression of Rho1 in midline glia (by slit-Gal4) or in midline cells (by sim-Gal4) halved the frequency of GB midline crosses in rho3, whereas expression in all CNS neurons (by elav-Gal4) substantially rescued the rho3 midline cross phenotype. Expression of the same transgene in GB1 (by SRF-Gal4), did not produce convincing rescue.

View larger version (70K): [in a new window]   Fig. 4. The Egfr mediates midline repulsion of GB1. (A,B) A ventral view of late stage 16 embryos stained for the tracheal lumen (by mAb2A12). Many GB midline crosses are observed in spi mutants (A). (C,D) spi mutants also lack a functional midline. (C) In spi, longitudinal fascicles collapse on the midline (stained by mAb1D4; compare with D, wild type; all panels show ventral views, anterior towards the left). (B,E) SRF-Gal4 drives gene expression specifically in tracheal tip cells. (E) A single GB is shown that expresses UAS-NLS:lacZ in the GB1 nucleus (arrow) under the control of SRF-Gal4 (mAb2A12 stains the tracheal lumen in red, anti-ß-gal is green). (B) A ventral view of a late stage 16 embryo expressing dominant-negative Egfr, showing misroutings and midline crosses.

View larger version (84K): [in a new window]   Fig. 6. Dominant-negative Ras, but not Raf or activated Yan, can cause GB1 midline crosses. (A-D) SRF driven expression of dominant-negative Egfr and Ras, but not dominant-negative Raf, PI3K or activated Yan produced midline crosses. Ventral views of embryos expressing dominant-negative constructs stained by mAb2A12. Dominant-negative Ras (A), Raf (B) and PI3K (p60, see Materials and methods, C) or activated Yan (YanACT, D) had similar effects on GB migration, causing many GBs to fail to enter the VNC, or to arrest (arrowheads in A-D) or meander inside the VNC (white arrowheads in A,B,D). Only dominant-negative Ras caused midline crosses (arrow in A). In all panels, anterior is towards the left. Scale bars: 20 µm. (E) Quantification of the frequency of midline crosses for the different genotypes.

View larger version (83K): [in a new window]   Fig. 7. Egfr pathway overactivation in GB1 causes premature turns. (A-E) Ventral views of late stage 16 embryos stained for tracheal lumen (mAb2A12) and double-stained for lumen and longitudinal fascicles (B, by mAb2A12 in black and anti-Fas2 in brown, respectively). SRF driven expression of Rho1 (A,B), two different forms of activated Egfr (C,D) and activated Ras (E) caused GBs to turn posteriorly prematurely, before reaching the midline (arrowheads in A-E). (F) The overall frequency of early turns for each genotype (black bar) and the fraction of affected branches that was classified as early turns (white bar). As an example, dominant-negative Egfr had a strong effect on GB migration but only about 10% of the affected branches classified as early turns (F). We interpret those early turns as a result of randomised migration. By contrast, close to 30% of the branches affected by activated Egfr were early turns (F). In SRF-Rho1 50% of the GBs were turning prematurely (F), yet the longitudinal fascicles appear unaffected (B, anti-Fas2 in brown).