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First published online 9 July 2008
doi: 10.1242/dev.017814

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Hedgehog and Wingless stabilize but do not induce cell fate during Drosophila dorsal embryonic epidermal patterning

¹ Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA.
² Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

View larger version (78K): [in this window] [in a new window]   Fig. 1. Groove markers in wild-type embryos. The figures are projections of confocal image stacks. Unless otherwise indicated, anterior is leftwards and dorsal is upwards. Embryos are 500 µm long and cell diameters after stage 11 are 4 µm. (A) Schematic representation of the groove. Groove cells have rectangular junctions and are located at the bottom of the groove. (B) Cross-section of a stage 14 embryo expressing β-gal (green) in the en domain. Odd (blue) marks groove cells and Dlg (red) reveals cell shapes. (C) Projection oriented as in A to display Crb accumulation (green) at the subapical domain of groove cells. Cadherin, red; Odd, blue. (D) Stage 12 wild-type embryo showing Crb (green), Odd (red) and cadherin (blue). Grooves are absent from the ventral domain, although Crb accumulation is visible there. (E-E''') En face view of rectangular groove cells in a stage 14 embryo showing Ena (green; E'), Cadherin (red; E") and aPKC (blue; E'''). Ena marks junctions between groove cells (arrowhead). Cadherin is uniformly expressed. aPKC is enriched in the subapical domain of groove cells. Yellow dots indicate groove cells. (F) Stage 12 cadherin-GFP-expressing embryo showing Cadherin-GFP (green), En (red) and Odd (blue).

View larger version (135K): [in this window] [in a new window]   Fig. 2. Groove markers in pair rule mutants. (A) Wild type. Seven additional stripes of Odd (blue) appear before germ band extension. En stripes (green) also appear at that stage. Cadherin, red. (B,B'') In stage 13 odd paired8 embryos, Ena (green; B") accumulates at junctions between Odd cells (blue). Cadherin, red. (B') Cross-section at the position of the yellow arrowheads in B. Slight depressions are present at the position of the Odd cells. (C) In runt3 stage 13 embryos, Crb (green; C') accumulates in Odd-expressing cells (blue). Cadherin, red. (D) Stage 13 Odd-lacZ/odd5 transheterozygous embryo. Residual Odd (red) correlates with remaining groove cells. Cadherin (green) and En (blue). (D') Cross-section at the position of the yellow arrowheads in D. No groove forms posterior to En-expressing cells where Odd is absent.

View larger version (129K): [in this window] [in a new window]   Fig. 3. Dynamics of Odd-expressing cells. (A) Odd cells (blue) dorsal and ventral to the tracheal placode, marked by β-gal (red) in stage 9 trachealess-lacZ embryos. (B,C) Images from a Cadherin-GFP time-lapse movie (see Movie 1 in the supplementary material). Cell shape and disposition allows the identification of the groove and tracheal cells. (C) The epithelium after tracheal invagination. Looking earlier in development shows that groove cells originate from the Odd domain. Tracheal precursors, red; groove cell precursors, blue, yellow, pink, burgundy. Some anterior cells (green) reveal that the groove cells abutting the tracheal placode have been displaced ventrally. Two pink sister cells follow different fates as only one becomes a groove cell. (D-E) runt3 embryos at stage 12 (D) and 14 (E) display refinement of Odd expression. First, Odd expression (blue; D') decreases away from the En cells (green); later a single cell wide stripe remains (E). (F-F'') Odd lacZ shows that β-gal remains visible (green; F') in cells that have lost Odd. (red; F").

View larger version (103K): [in this window] [in a new window]   Fig. 4. Wg signaling regulates the number of En and Odd cells. (A-C'') wgIL114 mutant embryos. (A) Stage 10 stained for En (green), Odd (red) and Cadherin (blue). (B) Stage 11 stained for En (green), Odd (red) and Sloppy paired (blue). (C) Stage 15 wg mutant: some En cells (green; green arrow) remain in a thoracic segment, surrounded by Odd cells (red). Cadherin, blue. (C') Magnification of En cells from (C), with Cadherin (green) showing groove, En (blue) and Odd (red). (C'') 90° rotation of C'. For 3D rotation, see Movie 2 in the supplementary material. (D-F'') nkd2 mutant embryos. (D) Stage 10 stained for En (green), Odd (red) and Cadherin (blue). (E) Stage 11 stained for En (green), Odd (red) and Sloppy paired (blue), showing expansion of the En domain and few remaining Odd cells. During retraction, grooves form only where Odd (red) is detected (F; magnified in F'). Cadherin staining (blue) shows that grooves are absent along the En cells (green) not bordered by Odd cells. (F'') 90° rotation of F' (arrowhead). Odd, gray; Cadherin, red. For 3D rotation, see Movie 3 in the supplementary material. (G,H) Wild-type embryos. (G) Stage 10 stained for En (green), Odd (red) and Cadherin (blue). (H) Stage 11 stained for En (green), Odd (red) and Sloppy paired (blue). (I) pannier-Gal4 UAS-wg drives ectopic Wg (green; I') but does not affect Odd expression (blue; I') or groove formation (Cadherin, red in I'; I''). Yellow arrows, mesodermal Odd staining; white arrows, ectodermal Odd staining.

View larger version (146K): [in this window] [in a new window]   Fig. 5. Hh maintains Odd expression near the En cells. (A,A') Stage 11 hhAC embryo stained for En (green), Odd (red) and Slp (blue). Odd is still present, whereas En disappears. (B,B') Stage 12 hhAC embryo stained for En (green), Odd (red) and cadherin (blue). Some En-expressing cells remain, but no Odd cells are in the epidermis. Some Odd staining is visible in the nervous system. (C,C') Stage 12 ptcIN embryo stained for En (green), Odd (red) and cadherin (blue). The Odd domain is wider than in the wild type (compare with Fig. 4H). (D,D') Stage 13 En-gal4 UASHhWT embryo stained for En (green), Odd (red) and cadherin (blue). Odd is present in a four- or five-cell wide stripe. (E-E'') Detail of a stage 11 wild-type embryo (E) stained for Ptc (green), Cadherin (red) and Odd (blue). Both Ptc (E') and Odd (E'') domains are several cells wide. (F-F'') Detail of a stage 13 wild-type embryo (F) stained for Ptc (green), Cadherin (red) and Odd (blue). Both the Ptc (F') and the Odd (F'') domains are a single cell wide. (G-G'') Stage 13 ptcIN embryo stained for En (green), aPKC (red) and cadherin (blue). Crb is enriched in the widened grooves. En face (G') and z-section (G'') detail from G. The groove on the left has ectopic En posteriorly, whereas the groove on the right does not. For 3D rotation movie, see Movie 4 in the supplementary material.

View larger version (100K): [in this window] [in a new window]   Fig. 6. Hh cannot induce grooves in dedifferentiated En cells. (A) Stage 13 prd-Gal4 UAS-Yanact embryo stained for Yan (green), Cadherin (red) and Odd (blue; A'). Yan-expressing cells near the tracheal invagination move posteriorly between the two Odd populations. (B-B'') En-Gal4 UAS-Yanact embryo stained for Ptc (green; B'), cadherin (red) and Odd (blue; B''). Ptc is expressed between the separated Odd populations. (C-C'') En-Gal4 UAS-Yanact embryo stained for Yan (green), Cadherin (red, C') and aPKC (blue, C'') showing that the Yan-expressing cells do not accumulate aPKC and do not become rectangular. 3D visualization reveals an interruption in the groove (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 7. Model of progression of cell identity. Progression of cell identities (red) proceeds independently of the Wg and Hh patterning signals. Patterning signals (green) stabilize already specified cell identities and prevent their switch towards different identities. Differentiation acts on the cells according to their current cell identity (blue). The already differentiated second row of groove cells switch from groove cells to cells producing hairs, showing that differentiation does not block the other two mechanisms. Interplay between cell identity progression and patterning signals ensures that a cell identity is specified when the previous and adjacent one is patterned. Newly specified cells take the patterned cells as a reference, so the anterior and the posterior side of a group of cells is patterned at different stages by distinct signals. The progression of cell identity is therefore visible in the spatial sequence of cell types present in the wild-type embryo. Wild-type Odd cells normally receive Hh early. This early influence maintains the population that will later transition to hair-producing cells, explaining why these cells are missing in Hh mutants.

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