DEV02212 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Hh-Np sets both the anterior and posterior boundaries of ser expression independently of Wg. (A) Wild-type embryo. (B,C) hh^AC, engal4 UAS-hh-Np embryos. (D-D¢¢) hh^AC, wgGal4 UAS-wg embryo. Hh is in green, Wg in blue and ser mRNA in red. At stage 12, the Hh-Np LPS distribution is restricted to two or three rows of cells correlating with an expansion of ser expression (A). Moderate overexpression of Hh-Np increases the spreading of Hh-LPS and restricts ser expression (B). Strong Hh-Np overexpression allows wide LPS spreading and completely abolishes ser expression (C). Previous work has suggested that ser expression is repressed by Wg (Alexandre et al., 1999; Gritzan et al., 1999; Wiellette and McGinnis, 1999). Strikingly, in embryos in which Wg was widely activated by Hh overexpression (B), the limits of ser expression were not changed and were adjacent to Wg-expressing cells in the middle of the Hh inter-stripes. Furthermore, when Wg was overexpressed in hh-mutant embryos, ser expression covered most of the segment (D-D¢¢), similar to what is observed in hh loss-of-function embryos (Gallet et al., 2003). Some cells clearly expressed both Wg and ser (arrows in D-D¢¢), strongly suggesting that Hh, and not Wg, is the main repressor of ser expression. (E) A model to describe Hh function during embryonic development and ventral cuticle specification. Hh is secreted from two rows of cells per segment (green). In juxtaposed anterior cells, Hh activates wg (blue), which allows naked cuticle differentiation. In posterior cells, Hh activates rho (gray) expression, which is involved in denticle diversity. We would like to modify the model suggested by Alexandre et al. (1999) and Hatini and DiNardo (2001), and propose that the Hh signal  restricts ser (red) expression to three rows of cells and thus defines both the anterior and posterior edges of ser expression.

  Additional references

  Gritzan, U., Hatini, V. and DiNardo, S. (1999). Mutual antagonism between signals secreted by adjacent wingless and engrailed cells leads to specification of complementary regions of the drosophila parasegment. Development 126, 4107-4115.

  Hatini, V. and DiNardo, S. (2001). Divide and conquer: pattern formation in Drosophila embryonic epidermis. Trends Genet. 17, 574-579.

  Wiellette, E. L. and McGinnis, W. (1999). Hox genes differentially regulate Serrate to generate segment-specific structures. Development 126, 1985-1995.

• Supplemental Figure 2 -

  Fig. S2. Quantification of Hh-Np and Hh-N transgenes expression. (A) Western blot analysis of extracts from embryos expressing one UAS-hh-N transgene (lane 1) or one UAS-hh-Np transgene (lane 2) under the control of the ubiquitous driver 69BGal4 at 25°C. Upper panel was blotted with Hh-recognizing antibody and lower panel with a tubulin-recognizing antibody. (B) Graph representing the tubulin-based normalization of the amount of Hh protein detected by western blot in A.

• Supplemental Figure 3 -

  Fig. S3. Lack of palmitoylated cysteine residues abolishes Hh-N activity. Analysis of hh loss-of-function embryonic rescue using different UAS-hh constructs under the control of the enGAL4Gal4 driver: (A-C) hh, UAS-hh-Np; (D-F) hh, UAS-hh-N-CD2; (G-I) hh, UAS-hh-N; and (J-L) hh, UAS-C85S-hh-N. Blue indicates mRNA in situ hybridization for wg (A,D,G,J), rho (B,E,H,K) and ser (C,F,I,L). En-expressing cells are stained in brown. The hh-Np transgene rescues the loss of target genes regulation caused by the loss of hh gene function (A-C), while the absence of cholesterol on Hh-N affects wg activation (arrows in G) and ser repression (I) without affecting rho expression (H). Anchoring Hh to the plasma membrane only rescues rho expression (D-F). An absence of both lipidic modifications on Hh completely abolishes Hh function (J-L).

• Supplemental Figure 4 -

  Fig. S4. Autocrine activities of Hh-Np and Hh-N. Flip-out clones of cells (green) expressing Hh-Np (A-C) or Hh-N (D-F) stained for En in blue (A-F) and dpplacz in red (B,C,E,F). Hh-Np (A,A¢) and Hh-N (D,D¢) activate En inside and around the clone (arrows), although half of the Hh-N clones (the smaller ones) do not activate En (arrow heads). Both Hh-Np and Hh-N are unable to activate En in the hinge region (bracket), suggesting that this domain is more refractory to Hh activity. (B,C,E,F) Magnified views of clones showing that dpp and en are activated at a longer range around Hh-Np-expressing clones than around those expressing Hh-N.