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 Tazuke, S. I. Articles by Fuller, M. T. Search for Related Content PubMed PubMed Citation Articles by Tazuke, S. I. Articles by Fuller, M. T.

A germline-specific gap junction protein required for survival of differentiating early germ cells

¹ Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305-5329, USA
² Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, CA 94305-5317, USA
³ Developmental Genetics Program, Skirball Institute, New York University Medical Center, 540 First Avenue, 4th Floor, New York, NY 10016, USA
⁴ Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th St, Chicago, IL 60637-1432, USA
⁵ Laboratoire de Biologie du Developpement, Institut Jacques Monod, 2, place Jussieu, 75251 Paris, Cedex 05, France
⁶ EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany
⁷ Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5329, USA

View larger version (73K): [in a new window]   Fig. 1. zpg mutant animals have tiny testes and ovaries. (A,B) Whole testes from (A) wild type and (B) zpgz–5352/Df(3L)Zn47 mutant male (2 days old after eclosion), all shown at the same magnification: early germ cells and spermatocytes stained with anti-Vasa (green); apical hub (arrow) stained with anti-Drosophila E-Cadherin (red); anti--Spectrin (blue). sv, seminal vesicle. Scale bar: 100 µm. (C) Whole wild-type ovary, and (D) a pair of ovaries from a zpgz-5352/Df(3L)CH12 female (2 days old after eclosion), at same magnification.

View larger version (85K): [in a new window]   Fig. 2. Wild-type function of zpg is required for differentiation and survival of male germ cells. Apical tips of testes from (A,C,E,G) wild type and (B,D,F,H) zpgz-5352/Df(3L)Zn47 males. (A,B) Germ cells at the testis apical tip labeled by staining with anti-Vasa. (A) Wild type. Rosette of single germline stem cells (arrowhead) surrounding the apical hub (*); cysts of spermatogonia or young spermatocytes (arrows). (B) zpgz-5352/Df(3L)Zn47. Single germ cells (arrowheads) away from the hub (*); small cluster of germ cells resembling early spermatogonia (arrow). (C,D) Same samples as in A,B stained with anti--Spectrin to label spectrosomes in stem cells (arrowhead in C) and gonialblasts (small arrow in C, arrowhead in D), and the linear branched fusomes in wild-type spermatogonia (large arrows in C). (C) Wild type. (D) zpgz-5352/Df(3L)Zn47. Germ cells resembling gonialblasts with spectrosomes (arrowheads); clusters of germ cells with abnormally large spectrosomes instead of fusomes (arrow). (E,F) Hub cells at the testes apical tip (arrows) labeled with anti-Drosophila E-Cadherin. (E) Wild type. (F) zpgz-5352/Df(3L)Zn47. Note the expanded number of hub cells. Scale bar: 50 µm. (G,H) Somatic cyst progenitor and cyst cells marked by ptc-GAL4; UAS-GFP. (G) Wild type. Note ‘lacy’ network of cyst cells enclosing developing germ cell clusters. (H) zpgz-5352/Df(3L)Zn47. Cyst cells are present but mostly either enclose single germ cells or no germ cells.

View larger version (68K): [in a new window]   Fig. 3. Wild-type function of zpg is required for survival of differentiating female germ cells. (A,B) Ovarioles and (C-F) germaria from newly eclosed (A,C,E) wild type and (B,D,F) zpgz-5352/Df(3L)Zn47 females. (A-D) Germ cells labeled by immunofluorescence with anti-Vasa. (B,D) zpg mutant ovaries contained only a few early germ cells, usually located at the tip of the germarium. (C,D) Higher magnification views of individual germaria. (E,F) Same samples as in C,D stained with anti--Spectrin to show spectrosomes and fusomes and with anti-Fasciclin III to outline somatic cells. Germ cells (arrows in C-F) with a spherical spectrosome at the stem cell position in (C,E) wild type and (D,F) zpgz-5352/Df(3L)Zn47 germaria.

View larger version (72K): [in a new window]   Fig. 4. zpg encodes a Drosophila gap junction protein – Innexin-4. (A) zpg genomic region in 65B5-65C1 (top) with position of P element insert in zpg3. Predicted transcripts (horizontal arrows); 6.15 kb genomic fragment that rescued zpg (bold bottom line). (B) Predicted topology of the Zpg protein, showing four transmembrane domains and conserved extracellular cysteine residues. Asterisks indicate sites of mutations in EMS-induced zpg alleles. (C) Multiple alignment and comparison (ClustalW) of eight innexin family members in Drosophila. Predicted transmembrane domains (unbroken bars); conserved predicted extracellular cysteine residues (asterisks); the oligopeptide used to generate the anti-sera is based on residues 345-367 (broken line). Amino acid changes in zpg alleles indicated above the protein sequence. ($) Stop codon; (#) base pair change at the splice acceptor site in zpgz-2679 allele leads to an altered predicted amino acid sequence starting at amino acid residue D(93).

View larger version (64K): [in a new window]   Fig. 5. Germline specific expression of zpg. (A) Northern blot showing 1.6 kb zpg transcript detected in adult males and females containing germ cells but not in agametic adults. (B) Expression of zpg mRNA in wild-type adult testes assayed by in situ hybridization: left testis probed with zpg antisense RNA. Right testis probed with sense strand control. zpg transcript is in spermatogonia at the testis apical tip (arrow). (C,D) Expression of zpg mRNA in wild-type embryos assayed by in situ hybridization with zpg antisense probe. Anterior is towards the left. (C) Blastoderm stage embryo showing zpg mRNA in pole cells, (D) zpg mRNA in primordial germ cells in coalescing gonads.

View larger version (166K): [in a new window]   Fig. 6. Zpg protein (green) concentrated at the interface between germ cells and surrounding somatic cells in spermatogonia and early spermatocytes. Confocal image of apical tip of wild-type third instar larval testis. Apical hub cells stained by immunofluorescence with anti-Fasciclin-III (red), plus spectrosomes and fusomes in germ cells stained by anti--Spectrin (red).

View larger version (65K): [in a new window]   Fig. 7. Zpg protein is localized on the surface of germ cells in the ovary. (A) Wild-type ovariole through stage 8-9, stained with anti-Zpg (green) and anti-Fasciclin-III (red) plus anti--Spectrin (red). (B-D) Wild-type egg chambers, (B) stage 6 and (C,D) stage 7, stained with (B,C) anti-Zpg or (D) anti-Zpg sera pre-adsorbed with the C-terminal Zpg peptide immunogen. Plaques of Zpg staining (arrows) at the nurse cell-follicle cell interface; diffuse Zpg staining (arrowheads) at the nurse cell-nurse cell interface. (E,F) Wild-type germaria stained with anti-Zpg antisera. In both cases, anti-Zpg antibody stained the surface of early germ cells in the germarium. Zpg staining (arrowheads) in the dividing cysts. (E) Anti-Zpg (green) and anti--Spectrin (red). (F,I,J,L,M) Under conditions with lower levels of overall staining, Zpg protein was detected in a discrete dot (arrow) adjacent and just apical to the spectrosome in female germline stem cells. (G) Wild-type germaria stained with anti-Zpg sera pre-adsorbed with the C-terminal Zpg peptide immunogen. (H-M) High magnification views of the apical tip of two different wild-type germaria co-stained with anti-Zpg (green) (I,J,L,M) and anti-Fasciclin III plus anti--Spectrin (H,J,K,M) (red). (J,M) Merged images of H,I and K,L respectively. Dots of Zpg staining at the germline stem cell-cap cell interface (arrows).

View larger version (149K): [in a new window]   Fig. 8. Gap junctions between germline stem cell and cap cell viewed by electron microscopy. Scale bar=100 nm. (A) Low-magnification infiltrated with lanthanum, showing the spectrosome (Sp) of the stem cell (lower cell) and the adjacent cap cell (upper cell) with a prominent Golgi apparatus (G). Gap junction (arrow). (B) Higher magnification of A. Gap junction with characteristic 2x10–9 m (20 Å) spacing (arrow). Wide lanthanum-filled intercellular space (arrowheads in B and C) (>200 Å; 2x10–8 m) adjacent to gap junction. (C) A second germarium, fixed without lanthanum, showing a gap junction (arrow) between a stem cell (below) and cap cell (above).

View larger version (37K): [in a new window]   Fig. 9. Loss of female germline cells in stem cell niche with age in zpg mutants. (A) Wild-type tip of an ovariole. (B,C) Whole ovaries from newly eclosed (B) and 3-week-old (C) zpgz–5352/Df(3L)Zn47 females labeled with anti-Vasa (green) and anti-Fasciclin-III (red). A-C are at same magnification. Terminal filament (arrow). (D) Percent of germaria containing germ cells at the tip for 0- to 3-day-old versus 21- to 24-day-old zpg/Df females. Blue bars: zpgz–5352/Df(3L)Zn47 (n=324 germaria from 0- to 3-day-old females, n=415 germaria from 21- to 24-day-old females). Black bars: zpgz–5352/Df(3L)CH12 (n=388 from 0- to 3-day-old females, n=430 from 21- to 24-day-old females). Yellow bars: zpgz–5352/Df(3L)CH20 (n=526 from 0- to 3-day-old females, n=389 from 21- to 24-day-old females).