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First published online 15 November 2006
doi: 10.1242/dev.02674

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Sprinter: a novel transmembrane protein required for Wg secretion and signaling

¹ University of Delaware, Department of Biological Sciences, Newark DE, USA.
² Duke University, DCMB Group, Biology Department Durham, NC, USA.

View larger version (124K): [in this window] [in a new window]   Fig. 1. sprinter mutations yield a `lawn of denticles' phenotype in germline clone embryos. Embryos orientated anterior left, posterior right and ventral or ventrolateral surface facing out. (A) A typical cuticle for wild-type embryos has a regular pattern of denticles and naked cuticle. (B) A cuticle from srt7E4 germline clones that are paternally rescued show minor segmentation defects and often survive to adult stages of development. Non-paternally rescued srt7E4 germline clone embryos show a `lawn of denticles' phenotype that is typical of loss of Wg- or Hh-signaling if the father contributes the srt7E4 chromosome (C) or srt (D) from Df(3L)vin5.

View larger version (61K): [in this window] [in a new window]   Fig. 2. Wg accumulates in the cells that express the ligand in srt7E4 embryos. Stage-9 (A-C) and stage-13 (D-F) embryos oriented anterior up, posterior down and ventral or ventrolateral surface facing out. (A,D) Expression of Wg in wild-type embryos shows a low level of Wg in Wg-expressing cells, and numerous Wg punctate endocytic vesicles in the receiving cells at both stages of development. (B) Wg accumulates in expressing cells in non-paternally rescued embryos at stage 9. Notice that punctate vesicles are absent and Wg fills expressing cells. (C) Similar retention of Wg is observed in srt7E4 paternally rescued embryos, although a few endocytic vesicles can be detected in receiving cells (arrowheads). (E) By stage 13, Wg has completely faded from the epidermal surface of nonpaternally rescued srt7E4 embryos and can be detected only in neuroblasts. (F) In paternally rescued stage-13 srt7E4 embryos, there is still a significant level of Wg within the cells that express it, suggesting that the Wg-Hh-feedback signaling loop is still intact. Wg vesicles are also detected (arrowheads). The difference in background between wild-type and srt7E4 embryos is due to the higher laser intensity used to collect data from wild-type embryos, which is not required for the mutant. For a direct comparison of Wg levels between wild-type and srt7E4 mutant tissues see Fig. 6. All images are projections of three optical sections acquired at 1 µm z-intervals, except E, which is a single optical section. Scale bar: 20 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 3. Engrailed expression fades in srt7E4 embryos. (A) En expression in the non-paternally rescued srt7E4 embryos at stage 9. The expression of En has been lost from many of the target cells that normally express En in response to Wg signaling in the ventral epidermis. (B) En expression in paternally rescued srt7E4 embryos at stage 9 is indistinguishable from wild type. Paternal rescue, embryo orientation and data collection from three optical sections are exactly as described in Fig. 2. Scale bar: 20 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 4. srt7E4 mitotic clones cause wing nicks in adult wings. (A) Wild-type adult wing. (B-E) Wing phenotypes resulting from homozygous srt7E4 clones during wing development. (B,D) Loss of sensory bristles at the anterior margin; (C,D) loss of sensory bristles and wing vein at the anterior margin. Homozygous-mutant tissues are identified in the adult wing blade by the presence multiple wing hair (mwh).

View larger version (92K): [in this window] [in a new window]   Fig. 5. srt7E4 mitotic clones result in Wg accumulation at the dorsoventral boundary and blocks the activation of targets in Wg receiving cells in the wing disc. The wild-type expression pattern for Wg (red; A) and its downstream targets Senseless (Sen, blue; B) and Achaete (Ac, red; J) in third instar wing imaginal discs. (C) Images A and B merged with wild-type Green Fluorescent Protein (GFP). (D-I) The effects of srt7E4 homozygous mutant tissues on the expression of Wg and Sen at two different magnifications. (K-L) Anterior Ac expression. srt7E4-homozygous-mutant cells are marked by the absence of GFP shown in merged images (F,I,L,N). Retention of Wg within Wg-expressing cells at the dorsoventral boundary blocks dorsal and ventral expression of Sen (E,H). Sen is expressed when GFP-positive (heterozygous) cells are present at the dorsoventral boundary, but does not require wild-type srt because many srt7E4-homozygous cells are present (Senexpressing cells excluded from outlined region in E and H). Arrows in G and H show two srt7E4-mutant cells that express high levels of Sen, but not Wg. Similarly, anterior Achaete expression also requires a wild-type srt in Wg-expressing cells at the dorsoventral boundary (K,L). (M-O) A projection of optical sections of a Wg-stained (red) third instar wing disc shows the dramatic difference in the presence of Wg within the cells that express it between srt7E4-mutant (left side) and heterozygous tissue (right side). Homozygous mutant cells are identified by the absence of GFP (O). Punctate Wg endocytic vesicles (arrowheads) are present adjacent to Wg-expressing heterozygous cells and absent near srt7E4-homozygous tissue. There also appears to be an expansion in the number of cells that express Wg at the dorsoventral boundary when srt function is absent. All wing discs are orientated dorsal up and anterior left. Images A-I are single optical sections focusing on the Sen-expressing nuclei. (J-O) Projection of eight optical sections acquired at 1 µm z-intervals. Scale bars: 20 µm.

View larger version (122K): [in this window] [in a new window]   Fig. 6. Hh signaling in wing imaginal discs does not require wild-type srt. (A-F) Expression of the ligand Hh is shown in red (A,D) and Ci in blue (B,E) to mark the anterior compartment and anterior-posterior compartment boundary. (G-I) The expression of a sensitive anterior target of Hh in receiving cells, Patched (Ptc), is shown in red. Absence of GFP marks srt7E4-mutant cells in the merged images (C,F,I). The expression and localization of Hh in signaling cells and receiving cells appears wild type in the mutant clones regardless of whether Hh is synthesized in or received by srt7E4-mutant cells. Heterozygous tissue along the anteriorposterior boundary is outlined in A and D. Wildtype anterior Ptc expression is also unchanged in mutant clones (G) regardless of the location of homozygous mutants cells relative to the anteriorposterior boundary (H,I). Wing discs are orientated dorsal up and anterior left. All images are projections generated from eight optical sections acquired at 1 µm z-intervals, except G, which is a single optical section. Scale bars: 20 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 7. srt encodes an evolutionarily conserved multi-transmembrane protein. (A) Injection of srt mRNA into non-paternally rescued germline clone embryos rescues the segmentation defect (right). The buffer-injected control shows the null srt phenotype. (B) Injection of a 543 bp srt dsRNA into GFP+ paternally rescued srt embryos yields a cuticle phenotype (right) almost as severe as the buffer-injected non-paternal rescued embryo in A and Fig. 1C. (C) The srt genomic region located at 68A9 is a gene-dense region. The srt ORF is flanked closely by Alg10 (left) and shares its 5 ' proximal region with CG7616 (right; www.flybase.org). The srt ORF is composed of two or three exons to yield two splice variants. (D) One possible topological model for the protein encoded by srt: the luminal/extracellular region, above; the cytoplasmic region, below. Four putative transmembrane stretches are shown. The 7E4 mutation in srt results in a nonsense mutation in the fourth transmembrane span in our model. The location of the splice variant in the intracellular loop and possible sites of N-linked glycosylation are shown. (E) Sequence alignment of Drosophila srt B isoform (NCBI accession: NP_729681) with its homologs from C. elegans (NP_001022275), X. laevis (AAH81130) and human (NP_079187). The four transmembrane domains shown in D are underlined in black. Four possible additional transmembrane domains are underlined in gray. The asterisk under the last transmembrane marks the location of the srt7E4 mutation.

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