QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Micchelli, C. A. Articles by Perrimon, N. Search for Related Content PubMed PubMed Citation Articles by Micchelli, C. A. Articles by Perrimon, N.

rasp, a putative transmembrane acyltransferase, is required for Hedgehog signaling

Craig A. Micchelli^1,2,*, Inge The^1,, Erica Selva¹, Vladic Mogila¹ and Norbert Perrimon^1,2

¹ Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA
² Howard Hughes Medical Institute, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA
Present address: Department of Molecular Medicine, NRB Floor 6, Office 621, 364 Plantation Street, Worcester, MA 01615, USA

View larger version (105K): [in a new window]   Fig. 1. Identification of a novel gene with a segment polarity phenotype. (A-C) Cuticle preparations were visualized under dark field optics; anterior is towards the left, dorsal is upwards. (A) Wild-type embryos display a ventral cuticle with a segmentally repeated pattern of denticle belts and naked cuticle. (B) Segment polarity phenotype of a rasp GLC-derived embryo lacking both maternal and zygotic contributions. Note the reduction in naked cuticle and the lawn of denticles. To identify unambiguously the genotype of the embryos that have received a paternal wild-type copy of rasp, we recombined the rasp chromosome with a mutation in trachealess (trh). trh results in embryos with a mutant Filtzkorper as described previously (Haecker et al., 1997). Note the Filtzkorper in rasp, trh/rasp, trh mutant embryos. (C) Cuticle preparation of rasp, trh/+ GLC-derived embryos that carry a wild-type paternal chromosome. The paternal rescue was often complete and cuticles were indistinguishable from those of wild-type embryos (compare A with C).

View larger version (109K): [in a new window]   Fig. 2. Loss of rasp activity does not affect Wg signaling. (A-F) Confocal micrographs of late third instar wing imaginal discs containing rasp7F21 (rasp–) clones. In this and all subsequent micrographs, anterior is upwards and dorsal is on the left. Large rasp– clones that cross the DV boundary were generated in a Minute heterozygous (M–/+) background and are marked by the absence of green fluorescent protein (GFP). (C,F) Broken line marks the approximate position of the DV boundary. (A) The absence of GFP in green marks the tissue homozygous for rasp. (B) The double row (DR) of anti-Sc staining shown in red marks the anterior proneural region along the presumptive wing margin. Activation of proneural gene expression along the anterior margin depends on Wg signaling. (C) Overlay of A and B. Note that anti-Sc levels and pattern within the clone are not affected by loss of rasp. (D) Absence of GFP in green marks tissue homozygous for rasp. (E) Dll is normally expressed throughout the majority of the presumptive wing pouch and is delimited by a broken line. Activation of Dll expression in the wing pouch depends on Wg signaling. (F) Overlay of D,E. Note that anti-Dll levels within the clone are not affected by loss of rasp.

View larger version (58K): [in a new window]   Fig. 3. Hh-dependent gene expression along the AP boundary. (A) Anterior cells are marked by the expression of ci, shown in red. Posterior cells are marked by anti-En/Inv staining shown in blue. A broken line marks the approximate position of the AP boundary. en is required in posterior cells for hh expression; hhGal4 UAS-GFP is shown in green. Anterior cells adjacent to the AP boundary respond to Hh by activating target gene expression. A narrow, Hh-dependent stripe of anti-En/Inv staining can be detected in anterior cells by late third instar (purple). The expression of two additional Hh target genes can be detected in anterior cells; (B) ptc-lacZ, is shown in blue and (C) dpp-lacZ, is shown in yellow. Brackets highlight the domains of anterior Hh target gene expression.

View larger version (114K): [in a new window]   Fig. 4. rasp is required for Hh target gene activation. (A-D) Comparison of Hh target gene expression in wing discs heterozygous and homozygous for rasp. (A) ptc-lacZ; rasp9B15/TM6 discs stained with anti-ß-gal (blue) and anti-En (brown). The domain of elevated ptc expression is detectable in a narrow stripe of anterior cells along the AP boundary (arrow). (B) ptc-lacZ; rasp9B15/rasp9B15 discs stained with anti-ß-gal (blue) and anti-En (brown). Levels of ptc-lacZ are reduced in this mutant background (compare A with B). The anti-ß-gal stain was amplified to detect low levels of ptc-lacZ reporter signal (see Materials and Methods). We failed to detect ptc-lacZ expression using fluorescent secondary antibodies. Note the reduced size of the homozygous wing disc. (C) ptc-lacZ; rasp9B15/TM6 discs hybridized to dpp RNA probes (blue). Like ptc-lacZ, dpp is also expressed in a stripe of anterior cells along the AP boundary (arrow). (D) ptc-lacZ; rasp9B15/rasp9B15 discs hybridized to dpp RNA probes (blue). Levels of dpp transcripts are reduced in this mutant background (compare C with D).

View larger version (120K): [in a new window]   Fig. 5. rasp is required in posterior cells to activate Hh target gene expression in anterior cells. (A-L) Analysis of rasp7F21 (rasp–) clones in wing imaginal discs. Clones were generated in a M–/+ background and marked by the absence of GFP shown in green. (B,E,H,K) Brackets highlight the domain of Hh-dependent gene expression in anterior cells. (C,F,I,L) The broken line marks the approximate position of the AP boundary. (A) A large anterior rasp– clone is marked by the absence of GFP. This clone defines the position of the AP boundary from the anterior side. (B) The pattern of Ptc protein is shown in red and is indistinguishable from wild type. (C) Overlay of A and B. (D) A large posterior rasp– clone is marked by the absence of GFP. This clone defines the position of the AP boundary from the posterior side. (E) The elevated domain of anterior Ptc protein is reduced in this disc (compare with B). (F) Overlay of D and E. (G) A large anterior rasp– clone is marked by the absence of GFP. This clone defines the position of the AP boundary from the anterior side. (H) The domain of both anterior and posterior En protein is shown in red and is indistinguishable from wild type. The brackets mark the approximate position of anterior cells expressing En protein. (I) Overlay of G and H. The domain of anterior En protein is clearly detectable in this micrograph in relation to the AP boundary. Anterior En protein is not detectably altered by the absence of rasp–. (J) A large posterior rasp– clone is marked by the absence of GFP. This clone defines the position of the AP boundary from the posterior side. (K) The domain of anterior En protein is reduced in this disc (compare with H). (L) Overlay of J and K.

View larger version (115K): [in a new window]   Fig. 6. Hh transcription and protein levels do not require rasp. (A-F) rasp7F21 (rasp–) clones in wing imaginal discs. (A-C) Analysis of hh transcription in rasp– clones. (A) Clone is marked by the absence of GFP. (B) Anti-ß-gal staining in red. The hhP30 enhancer trap is a reporter of hh transcription. The level of hh transcription in posterior cells is not altered by the absence of rasp. (C) Overlay of A and B. (D-F) Analysis of Hh protein levels in rasp– clones. (D) Clones are marked by the absence of GFP. (E) Anti-Hh protein shown in red. There is no detectable increase or decrease in Hh protein levels in the absence of rasp. (F) Overlay of D and F.

View larger version (84K): [in a new window]   Fig. 7. rasp is required to produce active Hh protein. (A-F) A comparison of Hh target gene expression in wing discs that express different Hh transgenes and which are either heterozygous or homozygous for rasp9B15. (A,B,E) A broken line marks the approximate position of the AP boundary. (A-C) en Gal4, UAS-GFP/UAS-Hh F HA; rasp9B15/TM6 discs. (A) The domain of en Gal4 expression shown in green. The domain of anterior en expression can be detected (brackets). (B) Elevated Ptc protein levels can be detected in a narrow stripe anterior to the AP boundary (brackets). (C) Overlay of A and B. (D,E) en Gal4, UAS-GFP/UAS-Hh F HA; rasp9B15/rasp9B15 discs stained with anti-Ptc (red); en Gal4 expression shown in green. (D) High levels of anterior Ptc protein are not detected (compare C with D). Note that the anterior expression of en is also not detectable in these discs. (E) Higher magnification of disc shown in D. (F) UAS-Hh N/+; en Gal4, UAS-GFP/+; rasp9B15/ rasp9B15 discs stained with anti-Ptc (red); en Gal4 expression shown in green. High levels of anterior Ptc protein are not detected.

View larger version (54K): [in a new window]   Fig. 8. rasp encodes a putative multipass transmembrane protein with homology to acyltransferases. (A) Sequence analysis of rasp. Hydrophobicity plot shows that Rasp encodes a predicted protein that is highly hydrophobic. At least eleven transmembrane regions can be detected. (B) Linear sequence of amino acids predicted by the BDGP rasp sequence. Hydrophobic regions are indicated in bold. Sequencing genomic DNA from rasp7F21 homozygous embryos led to the identification of a single base pair change that converts a tryptophan to a premature stop codon at amino acid 52 (asterisk). An invariant histidine residue (underline) conserved among MBOAT family members is a likely candidate for the active site of Rasp.