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doi: 10.1242/10.1242/dev.00510

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Requirement for two nearly identical TGIF-related homeobox genes in Drosophila spermatogenesis

Zhaohui Wang and Richard S. Mann

Department of Biochemistry and Molecular Biophysics, Columbia University, 701 West 168th Street, HHSC 1104, New York, NY 10032, USA



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  		Fig. 1. vis and achi encode two highly related TGIF-like proteins. (A) Sequence alignment of the predicted VisS and AchiL proteins with other members of the TGIF family. Residues conserved in all family members are highlighted in pink; residues conserved in all but one sequence are highlighted in yellow; residues conserved in a smaller subset of sequences are highlighted in blue; and similarities to Vis/Achi are shown in purple. The alternatively spliced exon in AchiL is indicated by the green arrowheads (also in B). * marks the predicted end of each protein. hTGIF, human TGIF; hTGIF2, human TGIF2; hTGIF2LX, human TGIF2-like on X; mTex1, mouse Tex1; fTGIF, Fugu TGIF; AgTGIF, Anopheles gambiae TGIF. The extent of the homeodomain and three alpha helices (1, 2, 3) is indicated above the sequence by the black and grey bars; the red box marks the three TALE residues in between helices 1 and 2. The residues `AYP', also in the loop between helices 1 and 2, are one of the hallmarks of the TGIF family. (B) Genomic organization of the vis/achi locus. cDNAs predicted to encode visS (SD01238), achiS2 (LD25085) and achiL (LP02076) have been sequenced; transcripts for visL and achiS1 are predicted from the analysis of the genome sequence. The alternatively spliced exons are indicated with an asterisk. Lighter shading indicates non-coding sequences, and the black boxes indicate the homeoboxes in vis and achi. (C) Df(2R)pingpong is a deletion (indicated by the dashed line) of vis, achi and four neighboring genes. The extent of the two rescue P elements for vis (P{vis}) and achi (P{achi}) are indicated.

 


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  		Fig. 3. Vis/Achi expression during male meiosis. All images are of wild-type testes stained for Vis/Achi (green), Aly (blue or white) and DNA (red). (A) Low magnification view of an entire testis. Vis/Achi and Aly are most highly expressed in the primary spermatocytes. (B) High magnification view of the apical tip and immature primary spermatocyte stages. Weak Aly and Vis/Achi staining is observed in the nuclei of the mitotically dividing cells (yellow arrows) and in the nuclei of immature primary spermatocytes (white arrows). (C) A cyst with mature primary spermatocytes. The DNA is present as three diffusely staining regions per nucleus (white arrows), which correspond to the three main chromosomes bivalents in Drosophila. Both Aly and Vis/Achi are highly expressed in these nuclei. (D) A cyst in metaphase of meiosis I. The DNA is compacted into 1-3 dots per nucleus. Aly appears associated with the spindle (white arrow) and Vis/Achi appears to be primarily cytoplasmic (yellow arrow). (E) A cyst in metaphase of meiosis II. The DNA, Aly and Vis/Achi signals are similar to that seen at metaphase of meiosis I (D). (F) An `onion stage' cyst. Meiosis is completed, and spermatid differentiation has begun by the 64 haploid progeny. Nuclei (white arrows) are closely paired to round, specialized mitochondria (yellow arrows). At this stage, the DNA is present in one dot per nucleus, Aly is tightly associated with the DNA (also in one dot/cell) and Vis/Achi is observed throughout the cell, but is excluded from the mitochondria. (G) At a later stage in spermatid differentiation, the DNA is less compact and is associated with Vis/Achi (arrow) whereas only background Aly staining is observed.

 


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  		Fig. 2. Vis and Achi are widely expressed proteins. Immunofluorescent images of Vis/Achi expression (green) in an embryo (A), a wing imaginal disc (C), an egg chamber (D), and a testis (E). All images are of wild-type tissues except for B which shows a Df(2R)pingpong homozygous mutant embryo. Only background staining is observed, confirming the specificity of the antibody. In C, the inset shows detail of wing disc expression co-stained with propidium iodide (red) to highlight the nuclei. In D, the nurse cell (N), follicle cell (F) and oocyte (O) nuclei are indicated (red arrowhead, oocyte nucleus). In E, the testes are co-stained for Comr (red), which has a very similar expression pattern to Vis/Achi in the testes. Three stages observed in wild-type testes, primary (1°) spermatocytes, elongating spermatids (s) and mitotically dividing germ cells (m) are indicated.

 


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  		Fig. 4. Df(2R)pingpong spermatocytes arrest prior to meiosis I. (A,C) Low and high magnification phase contrast images, respectively, of a testis from pingpong; P{achi} males, which is indistinguishable from wild type. Several stages can be observed, including the mitotically dividing spermatogonia (m), large primary spermatocytes (1°), mature spermatids (s), and the onion stage (o), in which small white nuclei (arrow) and round, dark specialized mitochondria (arrowhead) can be seen. N, a primary spermatocyte nucleus. (B,D) Low and high magnification phase contrast images, respectively, of a testis from a pingpong mutant male. These images are at the same magnification as their wild-type counterparts (A,C). Note that the entire testis is filled with primary spermatocytes but that these cells are not as round or large as in the wild type (arrow in B). The testes shown in A-D were gently squashed with a cover slip to generate thin specimens. The `*' in B indicates the basal region in which cells are degenerating. (E,G) pingpong; P{achi} testes stained for Vis/Achi (green) and DNA (red). In the wild type, several stages can be observed, including cysts in which the DNA is in three diffuse regions per nucleus (mature primary spermatocytes, white arrows), cysts in which the DNA is partially condensed (white arrowhead), and cysts in which the DNA is fully condensed (blue arrowhead). The levels of nuclear Vis/Achi are highest in the mature primary spermatocyte stage and gradually decrease as the cells approach the G2/M transition. (F,H) pingpong mutant testes stained for DNA (red) and Aly (blue, H only). In the mutant, some chromosomes appear to be partially condensed and others appear fully condensed (*). Aly is still observed in nuclei in the mutant. The images in E and F, and G and H, are at the same magnification and are directly comparable. (I,J) pingpong/CyO (I) and pingpong (J) testes labeled for BrdU incorporation (green) during DNA synthesis. BrdU-labeled 16-cell cysts were observed in both the wild type and mutant. The insets show an example of a labeled cyst at higher magnification. Not all 16 cells are visible in a single focal plane.

 


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  		Fig. 5. Relationship between vis, achi and other meiotic arrest genes. (A,B) boule in situ hybridization in pingpong and pingpong/CyO (wild type) testes. boule is not expressed in pingpong testes. (C,D) twine in situ hybridization in pingpong and pingpong; P{achi} (wild type) testes. twine is not expressed in pingpong testes. (E,F) mst87F in situ hybridization in pingpong and

pingpong/CyO (wild type) testes. mst87F is not expressed in pingpong testes. (G,H) Aly protein expression in pingpong and pingpong; P{achi} (wild type) testes. Aly is expressed and is found in the nucleus in pingpong testes. The arrows point to high levels observed apically in the wild type and the more uniform levels observed throughout the testes in the mutant. (I,J) Comr protein expression in pingpong and pingpong; P{achi} (wild type) testes. Comr is expressed and is found in the nucleus in pingpong testes. The arrows point to high levels observed apically in the wild type and the more uniform levels observed throughout the testes in the mutant. (K-P) Vis/Achi protein expression. (K,L) aly1 and aly1/CyO (wild type) testes. Vis/Achi is expressed in aly testes. The aly1 flies were raised at 25°C, where no protein is observed (White-Cooper et al., 2000Go). The arrows point to high levels observed apically in the wild type and the more uniform levels observed throughout the testes in the mutant. (M,N) sa1 and sa1/CyO (wild type) testes. Vis/Achi is expressed in sa testes. The arrows point to high levels observed apically in the wild type and the more uniform levels observed throughout the testes in the mutant. (O,P) can12 and can12/CyO (wild type) testes. Vis/Achi is expressed in can testes. The arrows point to high levels observed apically in the wild type and the more uniform levels observed throughout the testes in the mutant.

 


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  		Fig. 6. Expression of cell cycle regulators in pingpong testes. (A,B) Wild-type (A) and pingpong (B) testes stained for DNA (red), CycB (green) and Vis/Achi (blue). In the pingpong mutant CycB is expressed throughout the testes at intermediate levels and is cytoplasmic. Loss of staining is only observed far from the apical tip of the testes where cells are degenerating. The arrows point to the high levels of CycB staining in wild type and more uniform staining in the mutants. (C,D) Wild-type (C) or pingpong (D) testes stained for DNA (red) and CycA (green). In the pingpong mutant CycA is expressed throughout the testes and is primarily cytoplasmic. Loss of staining is only observed at the basal end of the testes where the cells are degenerating. The arrows point to the high levels of CycA staining in wild type and more uniform staining in the mutants. (E,F) Wild-type (E) or pingpong (F) testes stained for DNA (red) and Polo (green). Staining is high at the apical tip in both wild type and the mutant. In the mutant, the levels gradually decrease in cells farther from the apical tip whereas in the wild type, Polo levels are high in older (metaphase) 16-cell cysts.

 


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  		Fig. 7. AchiL can rescue pingpong function. (A,B) Immunoblot analysis of Vis/Achi proteins in wild type (WT), pingpong, pingpong; P{vis}, pingpong; P{achi}, and pingpong; tub-AchiL. Germline (O, ovaries; T, testes) and somatic tissues (soma; F, female adult soma; M, male adult soma) were analyzed. In extracts from wild-type testes, two bands are observed, the lower of which is also observed in ovaries. In both male and female somatic tissues a single band is observed that appears to migrate slightly slower than the ~60 kDa germline band; the reason for this difference is not known. However, none of these bands are observed in extracts from the pingpong mutant, confirming that they are derived from these genes. These images were cut just above a background band that was present in all lanes. The lower panels in both A and B show each blot stained for total proteins to illustrate that similar amounts of lysate were loaded in each lane (see also Methods). (C,D) pingpong; tub-AchiL testes appear normal by phase contrast microscopy and by immunostaining for Vis/Achi (blue), DNA (red) and CycB (green).

 


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  		Fig. 8. Vis/Achi co-immunoprecipitates with Aly and Comr. Extracts from wild-type (WT) or pingpong testes were immunoprecipitated (IP) with anti-Achi antibody, run on SDS-PAGE gels, and sequentially probed with the anti-Aly antibody (top panel), anti-Comr antibody (lower panel), and anti-Achi antibody (not shown). The two lanes on the left show the input samples. Both Aly and Comr were observed in the IP from WT, but not pingpong, extracts (arrows).

 




© The Company of Biologists Ltd 2003