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doi: 10.1242/10.1242/dev.00153

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hephaestus encodes a polypyrimidine tract binding protein that regulates Notch signalling during wing development in Drosophila melanogaster

¹ Genes and Development Research Group and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada
² Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6E 4G2, Canada

View larger version (32K): [in a new window]   Fig. 1. The hephaestus locus. (A) Five heph alleles map to a single, complex transcription unit predicted to encode an RNA-binding protein with four RNA-recognition motifs (RRM1-4). Three P-element alleles (heph03429, hephj11B9 and heph2) map to heph introns and two EMS-induced alleles (hephe1 and hephe2) alter conserved RNA-binding domains. Alternative splicing produces predicted protein variants: isoforms A and B include a bipartite nuclear localization signal (NLS). Only isoform A includes an N-terminal glutamine-rich domain (Q-rich). (B) The heph protein is highly related to the previously characterized RNA-binding proteins PTB/hnRNPI (human, NM_002819) and Vg1RBP60 (Xenopus, AF091370). Percent amino acid identity is indicated and, in brackets, percent similarity.

View larger version (112K): [in a new window]   Fig. 2. hephaestus genetic mosaic wing phenotypes. (A) A dorsal anterior Df(3R)G45, f mitotic clone associated with ectopic dorsal anterior wing margin bristles. Df(3R)G45 mutant tissue is marked with forked (red arrowheads), and the adjacent twin is marked with bald (outlined in blue). Most of the ectopic margin is formed in heph+ tissue next to the clone. (B) A ventral anterior Df(3R)G45, M+ f mitotic clone associated with ectopic ventral anterior wing margin bristles (outlined in red). The growth disadvantage of Df(3R)G45 mutant tissue has been rescued with the Minute technique. The ectopic margin is induced next to the Df(3R)G45, M+ f mitotic clone when the clone boundary is close to the normal margin. (C) The margin inducing effect of heph clones is mainly non-autonomous. Df(3R)G45 mutant tissue has been genetically marked with yellow (arrowheads), which make up a minority of the ectopic bristles in this typical example. (D) heph mitotic clones induce ectopic wing margin only within a competent region close to the normal margin. Each red circle represents one of 129 patches of ectopic margin from 300 heph genetic mosaic wings. Only a fraction of the ectopic patches of bristles included Df(3R)G45, y marked ectopic bristles (12/58 patches from the anterior compartment where y can be reliably scored). (E,F,H) Examples of heph03429 clones marked with pwn, outlined in red (dorsal) or blue (ventral). Loss of heph from dorsal or ventral clones is associated with ectopic wing margin (E,F) and/or wing margin nicks (F). Occasionally, heph clones that induce ectopic margin also induce small outgrowths and overgrowth of adjacent tissue.

View larger version (37K): [in a new window]   Fig. 4. Notch feedback mechanisms operating along the DV boundary and presumptive veins of the wing disc. (A) A model representing asymmetric intercellular communication that refine and maintain the DV boundary cell fate during the third larval instar (Micchelli et al., 1997). The red domain represents Notch-activated boundary cells that express wg and cut, and the blue domains represent the flanking cells where Notch is repressed by DL and SER. The feedback loop represented here restricts and maintains the boundary cell fate through autonomous inhibition of Notch in the flanking cells, and non-autonomous maintenance of Notch activation in the boundary cells. (B) A model representing the feedback loop operating during pupal development that refines EGFR activation and thus vein differentiation to a subset of the vein competent provein cells (de Celis et al., 1997). The blue domain represents a presumptive vein where EGFR is active, and the red domains represent the lateral proveins where Notch is active. DL is required in the presumptive veins to activate Notch in the lateral proveins. Active Notch signalling then represses EGFR signalling, allowing lateral provein cells to differentiate as intervein.

View larger version (74K): [in a new window]   Fig. 5. Delta and Serrate expression are reduced in heph clones. Normal DL (A) and SER (D) expression in heph03429 heterozygous wing discs. heph03429 mosaic wing discs stained for DL (B,C) or SER (E,F) protein expression. The relative levels of DL and SER are reduced within heph03429 clones (arrows). Although not dependent on proximity to the normal margin, this effect on DL and SER levels can be observed most clearly in cells flanking the boundary cells, where DL and SER expression is normally highest.

View larger version (40K): [in a new window]   Fig. 6. heph03429 partially rescues the fngD4 margin loss phenotype. (A) An example of a wing from fngD4/+ male. Most or all of the wing margin is missing. This phenotype is partially rescued in fngD4/heph03429 male (B) or in NAx-1/+; fngD4/+ transheterozygous females (C). All flies were cultured at 25°C. The fngD4 phenotype is variable and these images are representatives of the most common phenotype observed in flies of each genotype.

View larger version (112K): [in a new window]   Fig. 7. The amount of NICD is higher in hephaestus clones relative to normal surrounding tissue. (A,C) Normal NICD staining in a heterozygous heph03429 wing disc. The position of the DV boundary is noted in A (arrowhead). (B-B'',D-D'') A typical example of a heph03429 mosaic wing disc with elevated levels of NICD staining corresponding to the positions of heph mutant clones that are marked by loss of a GFP reporter transgene. (C,D-D'') Higher magnification view of the discs in A,B-B'', respectively. The positions of the XZ sections through C,D-D'' are marked by a broken line. Each image is a projection of 20 laser scanning fluorescent images (spanning a 30 µm total thickness) of GFP from a P{Ubi-GFP} transgene (green; B',D') and mouse anti-NICD primary antibody labelled with an anti-mouse Alexa-fluor594nm secondary antibody (red; A-D) in control flies of genotype y w P{hsFLP}; P{neoFRT} P{M} Sb heph03429/TM6B (A,C) or target class flies of genotype y w P{hsFLP}; P{neoFRT} P{M} Sb heph03429/P{neoFRT} P{hsGFP} (B-B'',D-D'').

View larger version (136K): [in a new window]   Fig. 3. heph clones autonomously misexpress WG and CT, and induce ectopic AC expression in surrounding tissue. Normal WG (A), CT (D), AC (G) and DLL (J) expression from heph03429 heterozygous wing discs. (B,C) A heph mosaic wing disc with ectopic WG (arrows) within heph03429, GFP- mitotic clones that are close to the normal WG-expressing cells. (E,F) A heph mosaic wing disc with ectopic CT (arrows) within heph03429, GFP- mitotic clones that are close to the normal CT-expressing boundary cells. Notice that CT is preferentially expressed along the heph clone border (F,F'). (H,I) A heph mosaic wing disc with ectopic AC (arrow) in cells adjacent to heph03429 clones that are close to the normal DV boundary (marked by the normal gap between domains of AC expression). Notice in I that some heph03429 mutant cells express AC. (K,L) Loss of heph in clones does not affect expression of the WG target gene Dll.

View larger version (100K): [in a new window]   Fig. 8. The intracellular localization of NICD is altered in hephaestus clones. A heph mosaic disc stained for NICD (A'-A''') or NECD (B'-B'''). Loss of heph is marked by loss of GFP from a P{Ubi-GFP} transgene (A,B). Each image is an XY projection representing one of three 10 µm apical (A',B') to basal (A''',B''') sections through each disc. The increased relative amount of NICD staining in heph03429 clones appears to be inside of the mutant cells, not bound to the apical membrane. No corresponding increase is apparent using the NECD antibody. The position of the DV boundary is noted with an arrowhead, the position of heph clones are marked with arrows.

View larger version (75K): [in a new window]   Fig. 9. The effects of heph clones on DL and NICD depend on Su(H). DL (A) and NICD (D) immunostaining in Su(H)8/Su(H)2 wing discs from heterozygous heph03429 larvae. DL (B,C) or NICD (E,F) expression from a Su(H)8 / Su(H)2 wing discs with heph03429 mutant clones marked by loss of a P{hs-GFP} transgene. Notice that staining for DL and NICD are comparable between heph03429 mutant and wild-type tissue within mosaic wing discs and when compared with non-mosaic discs. Clones are indicated with arrows.

View larger version (200K): [in a new window]   Fig. 10. Delta is epistatic to hephaestus. (A) An example of a wing with two heph03429 clones, one dorsal (red) and one ventral (blue), causing autonomous loss of the dorsal aspect of vein L4 and the region of the ventral aspect of vein L4 included in the ventral clone. (B) An example of two overlapping heph03429 clones, one dorsal (red) and one ventral (blue), that disrupt the posterior crossvein. Typical examples of Dlrev10 mutant clones (C,D) and Dlrev10 heph03429 double mutant clones (E,F). Like Dl clones, Dl heph clones cause an autonomous thick vein phenotype and are associated with non-autonomous vein differentiation in neighbouring tissue.

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