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. 2001 Sep;21(18):6280-91.
doi: 10.1128/MCB.21.18.6280-6291.2001.

RotundRacGAP functions with Ras during spermatogenesis and retinal differentiation in Drosophila melanogaster

E Bergeret  1 , I Pignot-Paintrand, A Guichard, K Raymond, M O Fauvarque, M Cazemajor, R Griffin-Shea
Affiliations

RotundRacGAP functions with Ras during spermatogenesis and retinal differentiation in Drosophila melanogaster

E Bergeret et al. Mol Cell Biol. 2001 Sep.

Abstract

Our analysis of rotund (rn) null mutations in Drosophila melanogaster revealed that deletion of the rn locus affects both spermatid and retinal differentiation. In the male reproductive system, the absence of RnRacGAP induced small testes, empty seminal vesicles, short testicular cysts, reduced amounts of interspermatid membrane, the absence of individualization complexes, and incomplete mitochondrial condensation. Flagellar growth continued within the short rn null cysts to produce large bulbous terminations of intertwined mature flagella. Organization of the retina was also severely perturbed as evidenced by grossly misshapen ommatidia containing reduced numbers of photoreceptor and pigment cells. These morphological phenotypes were rescued by genomic rnRacGAP transgenes, demonstrating that RnRacGAP function is critical to spermatid and retinal differentiation. The testicular phenotypes were suppressed by heterozygous hypomorphic mutations in the Dras1 and drk genes, indicating cross talk between RacGAP-regulated signaling and that of the Ras pathway. The observed genetic interactions are consistent with a model in which Rac signaling is activated by Ras and negatively regulated by RnRacGAP during spermatid differentiation. RnRacGAP and Ras cross talk also operated during retinal differentiation; however, while the heterozygous hypomorphic drk mutation continued to act as a suppressor of the rn null mutation, the heterozygous hypomorphic Dras1 mutation induced novel retinal phenotypes.

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Figures

FIG. 1
FIG. 1
rn null mutant testicular and seminal vesicle phenotypes. The apical side is to the left, and the basal side is to the right. (A, B, E) Wild type. (C, D, F) Null mutant rn20 red e/rn20 pp. (A, C) Testes; the seminal vesicle protrudes from the basal end of the testes in panel A. (B, D) Seminal vesicles. Spermatozoa are visible as fine coils exterior to the vesicle in panel B. (E, F) Testes have been teased apart near the apical end at a point corresponding to about one-third of the length of the testes. Germaria are to the left. Boxes mark areas of the testes that have been enlarged (3×) as insets to show cyst morphology. The arrowhead in panel E points to a seminal vesicle which protrudes from the basal end of the testis, and spermatozoa are visible escaping from its free end. Bar, 80 μm (A); 25 μm (B); 100 μm (E). This and all following figures have been processed with Adobe Photoshop 5.0 software.
FIG. 2
FIG. 2
Rescue by genomic rnRacGAP transgene of testicular cyst length and termination defect phenotypes. (A, D) Wild type. (B, E, F, G) Null mutant rn20 red e/rn20 pp. (C) Transgenic fly P(rnRacGAP)13 rn20/rn20 pp. (A, B, C) Fluorescence microscopy of isolated DAPI-stained testicular cysts. Bar, 180 μm. Inset, gel analysis of RT-PCR products from isolated testis RNA, assayed with rnRacGAP sequence-specific primers. Lanes: 1, wild type; 2, null mutant rn20 red e/rn20 pp; 3, transgenic flies P(rnRacGAP)10/+; rn20/rn20 pp; 4, transgenic flies P(rnRacGAP)13 rn20/rn20 pp; 5, genomic DNA assayed with the same primers. For lanes 1, 3, and 4 a single band appears with the predicted length of 274 bp; for lane 5 the band at 394 bp reflects the presence of an intron of 120 bp. (D, E) Phase-contrast microscopy of cyst terminations. Bar, 7.5 μm. In panel E, rn null mutant cysts show the characteristic ball-of-yarn termination phenotype. Bracket F, terminal bulge; bracket G, area proximal to the terminal bulge. (F, G) Transmission electron microscopy of rn null mutant cysts. In panel F mature flagella are visible in various orientations within the terminations. Bar, 2.2 μm. In panel G arrows mark the membrane of the cyst cell, which is visible in its integrity over practically the entire field of view, confirming the continuity of the somatic cell contents and envelope. Bar, 1.2 μm.
FIG. 3
FIG. 3
Localized F-actin accumulation in testicular cysts depends upon RnRacGAP. Superposition of DAPI (blue) and rhodamine-coupled phalloidin (red) staining visualized by UV fluorescence microscopy. Asterisks mark the positions of the heads. (A, D) Wild type. The arrow marks the position of an actin-rich IC. (B) Null mutant rn20 red pp/rn20 red. (C, E) Transgenic strain P(rnRacGAP) 13 rn20/rn20 pp. For wild-type and transgenically rescued cysts, overlapping images taken along the 1.8-mm cyst length were superimposed and fused to create the full-length composites shown here.
FIG. 4
FIG. 4
Rescue by genomic rnRacGAP transgene of membrane and mitochondrial defects in rn null mutants. (A, D, G) Wild type. (B, E, H) Null mutant rn20 red pp/rn20 red. (C, F, I) Transgenic fly P(rnRacGAP)13 rn20/rn20 pp. (A, B, C) Optical microscopy of semithin sections of dissected reproductive organs stained with toluidine blue. White arrows, early-stage spermatocytes; black arrows, mature cysts in longitudinal section, with long, darkly staining flagella; black arrowheads, hexagonally shaped cysts in transverse section; white arrowhead, section of seminal vesicle; black-and-white arrowheads, abnormal vacuolated cysts. Bar, 10 μm. (D to I) Transmission electron microscopy of ultrathin sections of testes. (D to F) In comparison to wild-type cysts, rn null cysts are disorganized and developing spermatids are randomly oriented. The rnRacGAP transgene restores spermatid orientation, resulting in a more orderly distribution. Bar, 1.2 μm. (G to I) Magnified views of mature sperm from panels D to F, respectively. Bar, 230 nm. The white arrow in panel H points to a fully retracted minor mitochondrial derivative.
FIG. 5
FIG. 5
Defective membrane deposition during spermatid differentiation in rn null mutants. (A, C) Early and late-intermediate stages of wild-type spermatid differentiation. (B, D) Early stages of rn null mutant spermatid differentiation. (A) Arrows point to interspermatid membranes. Bar, 245 nm. (B) Black arrows point to a single membrane surrounding three developing spermatids; the white arrow points to a mitochondrion in which condensation has initiated at both poles. (C) Thin arrows point to interspermatid cytoplasmic bridges; thick arrows point to the single interspermatid membrane. Bar, 240 nm. (D) Multiple mature spermatids without intervening membrane. Developmental staging was based upon the extent of axonemal decoration and the staining intensity of condensed portions of the mitochondria.
FIG. 6
FIG. 6
Suppression of rn null cyst and sperm membrane phenotypes by heterozygous hypomorphic mutations in genes coding for Ras and Drk. (A, C) Double mutant Ras1E1B rn20/rn20 red. (B, D) Double mutant E(sev)EDA/CyO; rn20red/rn20 red. (A, B) Testicular cysts. (C, D) Magnified views of mature sperm from panels A and B, respectively. (A) rn null cysts suppressed by a heterozygous hypomorphic mutation in Dras1 are more compact, although spermatids are not perfectly oriented and the surrounding membrane is somewhat irregular. Bar, 520 nm. (C) rn null cysts suppressed by a heterozygous hypomorphic mutation in drk present the same aspect as cysts in panel B, but the interspermatid material is more condensed and the spermatid membrane is more regular. Bar, 260 nm.
FIG. 7
FIG. 7
Modification of rn null internal eye phenotypes by heterozygous hypomorphic mutations in genes coding for Ras and Drk. Optical microscopy of semithin sections of eyes stained with toluidine blue. (A, E) Wild type. (B, C, F, G) Null mutant rn20 red e/rn20 pp. (B, C) Sagittal and longitudinal sections through the rn null mutant eye. Note that even though ommatidia are malformed, they tend to span the entire retinal distance. (D, H) Transgenic fly P(rnRacGAP)10/+; rn20/rn20 pp. (I, M) Double mutant E(sev)EDA/CyO; rn20 red/rn20 red. (J, N) Double mutant Ras1E1B rn20/rn20 red. (K, O) Triple mutant E(sev)EDA/CyO; Ras1E1B rn20/rn20 red. (L, P) Transgenic double mutant P(rnRacGAP)10/+; Ras1E1B rn20/rn20 red. Arrow, folded rhabdomere; arrowhead, fused rhabdomere.
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References

    1. Agnel M, Kerridge S, Vola C, Griffin-Shea R. Two transcripts from the rotund region of Drosophila show similar positional specificities in imaginal disc tissues. Genes Dev. 1989;3:85–95. - PubMed
    1. Agnel M, Roder L, Griffin-Shea R, Vola C. The spatial expression of Drosophila rotund gene reveals that the imaginal discs are organized in domains along the proximal-distal axis. Roux's Arch Dev Biol. 1992;201:284–295. - PubMed
    1. Agnel M, Roder L, Vola C, Griffin-Shea R. A Drosophila rotund transcript expressed during spermatogenesis and imaginal disc morphogenesis encodes a protein which is similar to human Rac GTPase-activating (racGAP) proteins. Mol Cell Biol. 1992;12:5111–5122. - PMC - PubMed
    1. Ashburner M. Drosophila: a laboratory handbook. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1989. p. 198.
    1. Bahri S M, Yang X, Chia W. The Drosophila bifocal gene encodes a novel protein which colocalizes with actin and is necessary for photoreceptor morphogenesis. Mol Cell Biol. 1997;17:5521–5529. - PMC - PubMed

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