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First published online 5 November 2003
doi: 10.1242/dev.00874

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The glypican Dally-like is required for Hedgehog signalling in the embryonic epidermis of Drosophila

Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, UK

View larger version (82K): [in a new window]   Fig. 1. RNAi silencing of dally-like but not dally generates a segment polarity phenotype in Drosophila embryos. (A) Ventral view of a wild-type larval cuticle. Unless specified, the head of embryos is to the left. Eight belts of denticles are visible in the abdomen. (B and C) Segment polarity phenotype of null mutations in wingless (wgCX4) and hedgehog (hhAC). The areas of naked cuticle are replaced by denticles, generating a lawn of denticles without clear polarity. (D) Position of the dsRNA sequences (in red) and the RT-PCR sequences (in blue) used in dally and dally-like RNAi experiments. (E) Agarose gel electrophoresis of semiquantitative RT-PCR reactions performed on extracts of embryos injected by either buffer, dlp 3'dsRNA or dally 3'dsRNA. There is at least a fourfold decrease in dlp mRNA following dlp RNAi, and at least a fivefold decrease in dally mRNA following dally RNAi. By contrast mRNA levels of dally and dlp following dlp and dally RNAi, respectively, are identical to the levels after injection of buffer. (F) Weak segmentation defects exhibited by dally dsRNA-injected embryos. The same type of defects are found in buffer-injected embryos (not shown), and thus are a consequence of the injection process. There is no clear transformation of naked cuticle into denticles in these embryos, but rather a loss of tissue that leads to the fusion of denticle belts. (G-I) Segment polarity defects found in dlp dsRNA injected embryos. (G) The weak segment polarity phenotypes are distinct from the phenotypes observed in buffer and dally-injected embryos: the naked cuticle is transformed into denticles without much tissue loss. (H) Strong segment polarity phenotype and (I) lawn of denticles identical to the phenotype of wingless or hedgehog null mutants. (See also Table 1.)

View larger version (48K): [in a new window]   Fig. 3. RNAi of dally-like and dally do not inhibit Wingless signalling. (A-C) Late stage 11 embryos. en transcription revealed by in situ hybridisation in wild type (A) and in embryos ectopically expressing wg (B,C). In armGal4/UASwg embryos, the ubiquitous activation of wg signalling stimulates en transcription in a competence domain spanning half a segment (B). The ectopic transcription of en is unaffected in embryos of the same genotype injected with dlp dsRNA (n=83), showing that dlp is not required for Wg signalling (C). (D-F) Cuticle preparations of simGal4/UASwg embryos, where the cells of the ventral midline express wg. (D) In response to Wg signalling, the cells in the ventral-most portion of the denticle belts secrete a naked cuticle. (E) In embryos of the same genotype injected with dlp dsRNA, a segment polarity phenotype appears in the ventral-lateral portions of the abdomen (87%, n=119), showing that dlp has been silenced efficiently. However, naked cuticle is still produced at the midline, indicating that ectopic Wg signalling is unaffected by the loss of dlp. (F) The same proportion of segment polarity phenotypes is found when dlp and dally dsRNA are injected together in simGal4/UASwg embryos (81%, n=67). As occurs with dlp single injection, naked cuticle secretion is unaffected at the midline, showing that these genes are not required in a redundant manner for Wg signalling.

View larger version (50K): [in a new window]   Fig. 4. Dally-like is required for Hedgehog signalling. (A-C,E,F) wg transcription revealed by in situ hybridisation in wild type (A) and in embryos ectopically expressing hh (B,C,E,F). (B) In armGal4/UAShh embryos, hh is expressed in every epidermal cells. In response to Hh signalling, wg transcription is activated in half a segment, which corresponds to its competence domain. (C) In embryos of the same genotype injected with dlp dsRNA, ectopic and endogenous expression of wg disappears completely (72%, n=47), showing that dlp is required for Hh signalling. (D) In situ hybridisation against lacZ in simGal4/UASnuclacZ embryos showing that simGal4 drives expression in the ventral midline. (E) In simGal4/UAShh embryos, hh is expressed in the ventral midline. In response, wg transcription is activated over a few cell diameters on either side of the midline, in each segmental domain of competence. (F) In simGal4/UAShh embryos injected with dlp dsRNA, both ectopic and endogenous wg expression are gone (97%, n=29), confirming the requirement of dlp for Hh signalling (F). All embryos shown are late stage 11.

View larger version (57K): [in a new window]   Fig. 5. A pre-processed form of Hedgehog, Hh-N, requires Dally-like for its activity. All panels show wg in situ hybridisation in late stage 11 embryos. (A-F) Ectopic expression of a pre-processed, cholesterol unmodified form of Hh, Hh-N. (A) In armGal4/UAShh-N embryos, ubiquitous Hh signalling activates wg transcription in the whole competence domain. (B) When the same experiment is repeated in a hh null background, ectopic wg expression is mostly unaffected. Endogenous wg expression is expected to disappear in absence of hh, which explains the slightly irregular pattern. (C) In armGal4/UAShh-N injected with dlp dsRNA, both ectopic and endogenous wg expression disappear (82%, n=39), showing that Hh-N requires Dlp for its activity. (D) In simGal4/UAShh-N embryos, Hh-N secretion from the midline activates wg transcription on both sides of the midline within each competence domain. (E) In simGal4/UAShh-N [hh-] embryos, Hh-N activates wg transcription less efficiently and at short distance from the source, suggesting that Hh-N is partially dependent on endogenous Hh for its non-autonomous activity. However, in simGal4/UAShh-N embryos injected with dlp dsRNA (F), all ectopic wg transcription is wiped out (93%, n=54), showing that Hh-N requires Dlp activity for both its autonomous and non-autonomous effects. Both sets of experiments indicate that Dlp acts downstream of Hh processing.

View larger version (55K): [in a new window]   Fig. 6. Dally-like is required upstream or at the level of Patched in the Hedgehog pathway. (A-C) Assays for the range of Hh signalling. (A) ArmGal4VP16/UASdlp embryos ubiquitously express Dlp at high levels, but wg transcription is activated in a single row of cells, as in wild type, showing that Dlp does not stimulate the range of Hh signalling in embryos. (B) In contrast, increasing the concentration of wild-type Hh in the cells that normally secrete it (the En cells) stimulates wg transcription over two to three cell diameters. (C) Repeating the same experiment with Hh-N leads to the stimulation of wg transcription over three to five cell diameters. (D-F) Epistatic relationship between patched (ptc) and dlp. (D) In ptc homozygous null mutant embryos, Hh signalling is constitutively activated and as a result wg transcription is stimulated in the whole competence domain. (E) In ptc- homozygous embryos injected for dlp dsRNA, ectopic wg transcription is unaffected showing that Dlp is not required for the constitutive activation of Hh signalling. (F) In sibling embryos which are heterozygous for ptc and thus wild type for Hh signalling, dlp RNAi leads to the disappearance of endogenous wg as expected. ptc heterozygous and homozygous embryos were separated in this experiment by the presence of lacZ expression in the heterozygous embryos (signal at the midline in F).

View larger version (42K): [in a new window]   Fig. 2. Engrailed and rhomboid expression in dally-like RNAi embryos. (A-D) Antibody staining against En in stage 11 embryos. Wild-type En expression (A). En expression in the ectoderm is completely gone in a wg null mutant (B), and has just started to fade in a hh null mutant (C) and dlp RNAi embryos (D). (E) Schema representing the autoregulatory loop between wg, en and hh at stage 9-10 in the ectoderm. As a consequence of this loop, En is lost earlier in wg mutants than in hh mutants. Note that at this stage, the gradient of Wg protein is symmetrical. PS: parasegment boundary. (F-I) Antibody staining against En in stage 12 embryos. En expression is mostly gone in a hh null mutant (H) and dlp RNAi embryos (I). Mutant phenotypes were found in dlp RNAi embryos in 87% of cases (n=140). (J) Schema showing the segmental gene regulation at stage 11-12. At stage 11, En expression becomes independent of Wg, and the Wg gradient becomes asymmetrical. The parasegment groove (PS) disappears around stage 12 when the segment boundary (S) groove starts to be visible. (K-N) In situ hybridisation against rhomboid (rho) in stage 14 embryos. In wild type, rho is expressed in one stripe per segment (K), whereas two stripes per segment are found in the wg null mutant (L). In dlp RNAi embryos (75%, n=24) (N), as in a hh null mutant (M), one irregular stripe is found per segment as in wild type, suggesting that loss of dlp mimics the loss of hh. (O) Schema depicting the intrasegmental patterning occurring in stage 12-14 embryos. Wg represses rho expression on the anterior side of the Engrailed domain, whereas hh and serrate (ser) maintain rho expression on the posterior side. S, segment boundary.

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