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Signaling from germ cells mediated by the rhomboid homolog stet organizes encapsulation by somatic support cells

Cordula Schulz, Cricket G. Wood*, D. Leanne Jones, Salli I. Tazuke and Margaret T. Fuller{dagger}

Departments of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford, CA 94305-5329, USA
* Present address: Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, CA 93106, USA



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  		Fig. 1. Early germ cells accumulate in stet mutant testes. (A,C,E,G,I) Wild-type and (B,D,F,H,J) stet mutant testes, apical tips towards the left. Scale bars: 0.1 mm. (A,B) Phase-contrast images of adult (A) wild-type and (B) stet 871 mutant testes; small early germ cells (arrowheads) at the apical tip of wild-type testes and throughout stet mutant testes. Larger spermatocytes (black arrow) displaced away from the tip, differentiating round spermatids (white arrow) along the coil of the testis, bundles of elongated spermatids (ST), in wild type as indicated. (C,D) Third instar larval testes showing cells expressing the S3-46 marker (C) at the tip of wild-type testis (arrowhead) and (D) filling almost the entire stet mutant testis. (E,F) Third instar larval testes from (E) wild-type and (F) stet stained with anti-FasIII (red) and anti-phosphorylated Histone-H3 (green). Single anti-phosphorylated H3-positive cells are indicated by arrowheads. Dividing spermatogonia (arrow) are seen as clusters of eight anti-phosphorylated Histone-H3-positive cells in wild type. (G,H) Third instar larval testes stained with anti-FasIII (green) and anti-{alpha}-spectrin (red) of (G) wild-type and (H) stet with {alpha}-spectrin staining in spectrosomes (arrowheads) and in branched fusomes (arrows). Note slightly enlarged apical hub (green) in stet mutant testis compared with wild type. (I,J) In situ hybridization with esg mRNA in adult (G) wild-type and (H) stet mutant testes. Arrows indicate esg-positive cells at tip in wild type.

 


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  		Fig. 2. Somatic cyst cells do not correctly surround germ cells in stet mutant testes. (A) Diagram of a cross-section through the apical tip of a wild-type testis. Germ cells in blue, somatic cyst cells in yellow. Germline stem cells (S) and cyst progenitor cells (CP) next to the apical somatic hub (red); gonialblast (G), spermatogonia (SP) and somatic cyst cells (SC), as indicated. (B-G) Testes from (B,D,F) wild type and (C,E,G) stet mutants. Asterisks indicate apical tips. Scale bars: 0.1 mm. (B,C) Third instar larval (B) wild-type and (C) stet mutant testis tip stained with anti-FasIII (red) to label the hub and anti-Tj (green) to label cyst progenitor nuclei next to the hub (arrowheads). Cyst cell nuclei are displaced away from the hub (arrows). (D,E) Third instar larval wild-type and stet mutant testis tip stained for ß-galactosidase activity from the 17-en-40 marker. (D) Wild-type: cyst cells extend to surround the germ cells (arrows). (E) stet: ß-galactosidase is detected in dots (arrows) and small cytoplasmic extensions (arrowheads). (F,G) Adult wild-type and stet mutant testis tip expressing UAS-GFP under control of the ptc-GAL4 activator. (F) Wild-type: GFP is detected in extensions (arrows) surrounding the germ cells. (G) stet: GFP is mostly detected in dots (arrows) and a few extensions (arrowheads).

 


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  		Fig. 3. Wild-type function of stet is required in the male germline. (A) Apical tip of a testis expressing nuclear ubiquitin-GFP. Nuclei of early germ cells at the apical tip and spermatocytes are seen as round GFP-signals (arrowheads), while nuclei of somatic cyst cells appear triangular (arrows). (B) Wild-type control clones (outlined) marked by lack of nuclear targeted GFP. Somatic cyst cell nuclei were detected associated with these clones out of focus in the image shown. (C) Same testes as in (B) double labeled for GFP and DAPI (red). GFP-negative cells in control clones differentiate normally and have large size DAPI-stained spermatocyte nuclei (arrowheads). (D) stet mutant germline clone (outlined) marked by lack of nuclear targeted GFP. (E) Same clone as in D double labeled for GFP and DAPI (red), GFP-negative cells contain small bright DAPI-stained nuclei. (F) stet mutant germline clone. No triangular GFP-positive nuclei were detected on top, under or beside the clone; dying cells in stet mutant testes autofluoresce (arrowhead). (G) Cells in a stet mutant clone (arrowheads) express piwi-RNA, a marker usually expressed only in early germ cells at the apical tip (arrow). (H-L) Immunofluorescence staining with anti-Tj. (H) Cyst cell nuclei (arrows) at the tip of a stet/+ testes. (I) Clone of stet mutant germ cells located further basally in the testes shown in H; stet mutant germ cells appear slightly brighter than the surrounding stet/+ spermatogonia because of background staining. No Tj-positive cyst cell nuclei were detected associated with the stet mutant germ cells. (J,K) Examples of stet mutant germ cell clones associated with (J) one and (K) two Tj-positive nuclei (arrows). (L) Cyst cell nuclei (arrows) stained for the late cyst cell marker Eya were associated with neighboring stet/+ spermatocytes, but not with the clone of stet mutant germ cells in the center of the image. (M-P) DAPI images of clones corresponding to images in I-L, respectively. Note the many small brightly stained nuclei. Scale bars: in A, 0.1 mm for A-F; in G, 0.1 mm; in H, 0.1 mm; in I, 0.1 mm for I-P.

 


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  		Fig. 4. Defects in stet mutant ovarioles. DAPI staining of (A) wild-type and (B-D) stet mutant ovarioles in the same magnification, apical tips towards the left, germarium (g) at the apical tip. Scale bar: 0.1 mm. (A) Wild-type ovariole showing egg chambers at different developmental stages; nurse cells (nc) and oocyte (oc) as indicated, egg chambers contain 16 germ cells. (B) Ovariole from a young stet mutant female showing egg chambers at many stages. Note egg chambers with different sized nurse cell nuclei (arrowheads). (C) Egg chambers from a young stet mutant female showing abnormal numbers of germ cells, as indicated. (D) Ovariole from a 10-day-old stet mutant female showing germ cells at the apical tip (arrow) and degenerating egg chamber (arrowhead).

 


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  		Fig. 5. Early germ cells accumulate in stet mutant germaria. (A) Diagram of a wild-type germarium. Germ cells in dark gray, somatic cells in light gray. Note spectrosomes (sp) in germline stem cells (gsc) and cystoblasts (cb) in region 1, and branched fusomes (fu) in cystocytes (cys). In region 1 and 2A, germ cells are surrounded by cytoplasmic extensions (light gray) from inner sheath cells (isc). In region 2B, germ cells become enclosed by somatic follicle cells (fc). Region 3 contains one egg chamber. Terminal filament cells (tfc), cap cells (cc), follicle precursor cells (fpc), as indicated. (B,H) Scale bars: 5 µm. (B-G) Germaria from (B,D,F) wild-type and (C,E,G) stet mutant females; apical tips towards the left. (B,C) Immunofluorescense staining with anti-Sxl. (B) Wild type: stem cells and cystoblasts at the tip have high level of cytoplasmic Sxl (arrow). (C) stet: many early germ cells with high level of cytoplasmic Sxl (arrows). (D,E) Immunofluorescense staining with anti-{alpha}-spectrin. (D) Wild-type: {alpha}-spectrin in spectrosome (arrows) in stem cells and cystoblasts and the branching fusome (arrowhead) in cystocytes. (E) stet: many cells with spectrosomes (arrows). (F,G) Expression of UAS-GFP under control of the en-GAL4 activator in inner sheath cells in region 1 and 2A. (F) Wild type: extensions from inner sheath cells extend among germ cells in region 1 and 2A (arrows). (G) stet: inner sheath cells are present and express GFP (arrows), but no cytoplasmic extensions were detected. (H) Electron microscope image showing a cytoplasmic extension (arrowheads) originating from an inner sheath cell (n, nucleus of inner sheath cell) and extending between a germline stem cell (s) and a cystoblast (c). Cap cell (cc), spectrosome (sp), as indicated. The image was taken by A. T. Carpenter from the wild-type germarium analyzed by Carpenter (Carpenter, 1975Go).

 


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  		Fig. 6. stet encodes a predicted transmembrane protein homolog of rho. (A) Predicted structure of the stet transmembrane protein, showing seven transmembrane domains, placing the N terminus and the C terminus on opposite sites of the membrane (TMpred, ExPASy Molecular Biology Server). Amino acid substitutes in sequenced stet alleles (Table 1) are indicated. (B) Intron-exon structure based on the sequence of the testis cDNA, with predicted start codon in exon 2, stop codon in exon 4, and indicating the splice site mutation between exon 3 and 4 in stet 871 leading to a premature stop codon.

 


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  		Fig. 7. stet is expressed in testes and ovaries. (A) Northern blot showing stet transcript in testes, ovaries and early embryos and rp49 loading control in all lanes. Testes lane, 5 µg poly(A)+ RNA loaded; ovary and embryo lanes, 1 µg poly(A)+ RNA loaded. (B) Wild-type ovariole hybridized in situ with stet mRNA. Note expression in the germarium (arrowhead) and localization of the transcript to the oocyte in egg chambers (arrows).

 


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  		Fig. 8. stet may function through Egfr signaling. (A-C) Phase contrast images. (A) stet mutant testis filled with small cells (arrows). (B,C) Spermatogenesis is restored in stet mutant testes expressing (B) a UAS-stet construct and (C) a UAS-rho construct in germ cells, note elongated sperm bundles (arrows). Occasionally, clusters of stet mutant germ cells can still be observed (arrowheads). (D,E) In situ hybridization to adult wild-type testes. (D) Star mRNA at high levels in germ cells at the apical tip (arrow). (E) Sense-control: no staining. (F,G) Immunocytochemical stain with anti-activated MAP-kinase (DP-ERK) to adult testes. (F) Wild-type: activated MAP-kinase in the somatic hub cells (arrow) and in the cytoplasm of cyst cells (arrowheads). (G) stet: activated MAP-kinase in somatic hub cells (arrow) and a few cyst cells (arrowhead) next to the hub. Scale bars: in A, 0.1 mm for A-C; in D, 0.1 mm for D,E; in F, 0.1 mm for F,G.

 

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© The Company of Biologists Ltd 2002