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First published online 14 March 2007
doi: 10.1242/dev.001578

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Soma-dependent modulations contribute to divergence of rhomboid expression during evolution of Drosophila eggshell morphology

¹ Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
² Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama, Myodaiji, Okazaki 444-8787, Japan.
³ Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.

View larger version (34K): [in this window] [in a new window]   Fig. 1. Evolutionary divergence of eggshell morphology and rho expression pattern between D. melanogaster and D. virilis. (A) The egg of D. virilis has four DAs, an ancestral trait, as found in the Hirtodrosophila outgroup of the genus Drosophila. The two DAs found in D. melanogaster is a more recently evolved trait. D. virilis and D. melanogaster diverged 40-60 million years ago (mya). (B,C) The eggshells of D. melanogaster (B) and D. virilis (C) are shown with anterior to the left (dorsal view). (D,E) Schematic comparison of the rho expression patterns between D. melanogaster and D. virilis during oogenesis. Dorsal lateral views of stage 10A- to 12-egg chambers are shown with rho expression (blue). Anterior is to the left. (D) D. melanogaster rho was expressed in a saddle-shaped zone at the dorsal anterior at stage 10A. This expression was refined into an L-shaped stripe on either side of the dorsal midline at stage 10B. Expression was then restricted to the anterior row lacking the midline region at stage 11, as it continued until stage 12. (E) D. virilis rho was expressed in two domains at the dorsal-lateral sides at stage 10A. No expression occurred in the midline, unlike in D. melanogaster. The expression was repressed at the posterior region and refined into a Vshaped stripe missing its apex at stage 10B. Expression was upregulated at the front and rear of each stripe, and expression in the rear moved anteriorly at stage 11. Subsequently, the expression was divided into four domains at stage 12, corresponding to the positions of the four DAs.

View larger version (77K): [in this window] [in a new window]   Fig. 2. Dvir rho4.2 carries a minimal enhancer activity accounting for the endogenous rho expression pattern during DA formation. (A,B) D. virilis rho mRNA was detected in a V-shaped stripe missing its apex at stage 10B (A), and at stage 12 its distribution was restricted to the four domains where the four DAs later form (B). (C,D) The Dvir rho4.2 enhancer drove the expression of GFP in a pattern similar to that of the endogenous rho in D. virilis. At stage 10B, a Vshaped stripe missing its apex was observed (C), and then at stage 12, the GFP reporter was expressed where the four DAs would later form (D). White arrowheads and brackets indicate the cells forming the anterior and posterior DAs, respectively (B,D). (E-G) Confocal images of the GFP reporter expression driven by the Dvir rho4.2 enhancer and of endogenous Broad-Complex (BR-C) expression. The expression of Dvir rho4.2-GFP (green in E and G) and BR-C (magenta in F and G) was mutually exclusive at stage 10B. G is a merged image of E and F.

View larger version (45K): [in this window] [in a new window]   Fig. 3. The trans-regulatory landscape controlling rho expression diverged between D. virilis and D. melanogaster. (A,B) In D. virilis, the Dmel rho2.2 enhancer drove expression of the reporter in a pattern resembling that of the endogenous Dvir rho4.2 enhancer during DA formation; the V-shaped stripe at stage 10B (A) and the pattern corresponding to the four clusters of DA-forming cells at stage 12 (B) were observed. The cluster of cells forming the anterior DA is indicated by a white arrowhead (B). (C-F) Dmel rho2.2 and Dvir rho4.2 expressed in D. melanogaster were activated in a largely similar pattern, with some exceptions. The expression of Dmel rho2.2-GFP was detected as the L-shaped stripe on either side of the midline from stage 10B (C) to 12 (D) in D. melanogaster. When expressed in D. melanogaster, Dvir rho4.2-GFP showed the L-shaped expression pattern, similar to Dmel rho2.2 in this species, but with a slightly posterior expansion (E). This extra expression of Dvir rho4.2-GFP further expanded posteriorly until stage 12 (F). Anterior is to the left; (A,C-F) dorsal view; (B) dorso-lateral view.

View larger version (87K): [in this window] [in a new window]   Fig. 4. Function of the rho enhancer also diverged between D. melanogaster and D. virilis. (A-I) D. melanogaster transgenic flies carrying both Dmel rho2.2-ß-Gal (magenta in A,C,D,F,G,I) and Dvir rho4.2-GFP (green in B,C,E,F,H,I) reporters were generated. The activities of the two enhancers were then determined at different developmental stages: early in stage 10B (A-C), late in stage 10B (D-F) and stage 12 (G-I). (A-C) At early stage 10B, Dmel rho2.2-ß-Gal (A) was activated, but Dvir rho4.2-GFP (B) was not yet active (C). (D-F) Late in stage 10B, Dvir rho4.2-GFP expression began (E) in a pattern similar to that of Dmel rho2.2-ß-Gal (D). The domain of Dvir rho4.2-GFP activation (E) largely overlapped with that of Dmel rho2.2-ß-Gal (D), although a slight posterior expansion of Dvir rho4.2-GFP was seen (F). (G-I) The anterior boundaries of the Dmel rho2.2-ß-Gal (G) and Dvir rho4.2-GFP (H) activation domains were similar, but the posterior expansion of Dvir rho4.2-GFP expression became prominent (I). The right panels are merged images of the left and middle panels.

View larger version (57K): [in this window] [in a new window]   Fig. 5. Conservation of the Grk signal in D. melanogaster and D. virilis. (A) The experimental scheme for interspecific pole cell transplantation from D. virilis to D. melanogaster embryos. (Top) Pole cells were transplanted from D. virilis donor embryos into D. melanogaster host embryos carrying the EGFP-vasa construct. (Bottom) Left: a normal egg chamber composed of an EGFP-Vasapositive D. melanogaster oocyte and D. melanogaster somatic follicle cells. The D. melanogaster Grk activated D. melanogaster EGFR in this egg chamber. Right: a chimeric egg chamber. In the egg chamber of a D. melanogaster host female, the oocyte was replaced with one of D. virilis origin. In this chimeric egg chamber, the D. virilis Grk activated the D. melanogaster EGFR and directed the DA formation. (B-B'') A gonad of a D. melanogaster host embryo with transplanted D. virilis pole cells. The host embryos were stained with anti-GFP (B, green) and anti-Vasa (B', magenta) antibodies. The D. melanogaster host pole cells expressed both GFP and Vasa (B, green; B'', white), whereas the transplanted D. virilis pole cells expressed only Vasa (B',B'', magenta). The D. virilis pole cells were located within the gonad of the D. melanogaster host (B'', magenta). B'' is a merged image of B and B'. (C) Ovary of a D. melanogaster host female. GFP-positive host germline (green) and GFPnegative D. virilis germline (white arrowhead) cells are shown. The D. virilis germline was surrounded by D. melanogaster host follicle cells. (D,E) The chimeric egg (negative for EGFP, data not shown) had two DAs (E) identical to those of the wild-type egg (D). (F,G) The percentages of eggs in which the DA roots were separated by the distances indicated in the horizontal axes are shown. The two DAs were separated by a mean distance of 48.2±4.6 µm (n=57) in the wild-type (F) and 49.2±3.6 µm (n=12) in the chimeric (G) eggshells. The differences were not significant (0.5>P>0.2, Student's t-test).

View larger version (108K): [in this window] [in a new window]   Fig. 6. The expression pattern of mirr diverged evolutionarily between D. melanogaster and D. virilis. (A-H) The expression of mirr was detected by in situ hybridization during the DA formation in D. melanogaster (A-D) and D. virilis (E-H). (A) D. melanogaster mirr was expressed as a single broad domain at the dorsal-anterior region of the follicle cells at stage 9. (B) At stage 10A the D. melanogaster mirr expression was repressed in the dorsal midline (white arrowhead) and elevated in the cells at the anterior border of this expression domain (white arrow). By stage 10B, the mirr expression was restricted to the single row of anterior cells on either side of the midline (white arrow in C), and then was barely detected at stage 11 (D). (E) D. virilis mirr was expressed at stage 9 at the dorsal-anterior region of the follicle cells, like D. melanogaster mirr. (F) The mirr expression was gradually lost from the anterior region of its expression domain (white arrowhead) early in stage 10A. (G) By late in stage 10A, the D. virilis mirr expression was resolved into two dorso-lateral domains, similar to D. virilis rho expression at the same stage (see Fig. 1E). Note that the mirr expression was absent from a triangle at the anterior midline region of the follicle cells. (H) The expression was almost absent at stage 11.

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