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Keywords:

  • anamorsin;
  • CIAPIN1;
  • Drosophila;
  • follicle cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Background: The conserved cytokine-induced apoptosis inhibitor-1 (CIAPIN1) gene has been implicated in several processes, such as apoptosis, cell division, angiogenesis and Fe/S protein biogenesis. In this study, we identified the Drosophila CIAPIN1 homologue (D-CIAPIN1) and studied its role in ovarian development. Results: We found that D-CIAPIN1 is conserved as it can complement the nonviability of the yeast CIAPIN1-deletion strain. Several D-CIAPIN1 alleles were identified, including one allele in which that codon encoding the highly conserved twin cysteine CX2C motif is mutated, demonstrating for the first time the importance of this motif to protein function. We demonstrated D-CIAPIN1 is an essential gene required for ovarian development. We found that D-CIAPIN1 female mutants are sterile, containing rudimentary ovaries. We noted a decrease in follicle cell numbers in D-CIAPIN1 mutant egg chambers. We further demonstrated that the decrease in follicle cell numbers in D-CIAPIN1 mutants is due to a reduced mitotic index and enhanced cell death. Conclusions: Our study reveals that D-CIAPIN1 is essential for egg chamber development and is required for follicle cell proliferation and survival. Developmental Dynamics 242:731–737, 2013. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. ACKNOWLEDGMENTS
  7. REFERENCES

The cytokine-induced apoptosis inhibitor-1 (CIAPIN1), also termed anamorsin (meaning anti-death molecule in Latin), was identified as an anti-apoptotic molecule (Shibayama et al., 2004). CIAPIN1 does not show any homology to known apoptosis regulatory molecules, such as Bcl-2 or caspase family members, or signal transduction molecules. The expression of mouse CIAPIN1 (mCIAPIN1) was completely dependent upon stimulation by growth factors, with forced expression of mCIAPIN1 conferring resistance to apoptosis resulting from growth factor deprivation in vitro. Furthermore, mCIAPIN1 was found to act as an anti-apoptotic molecule in vivo as mCIAPIN1-deficient mice die in late gestation due to defective definitive hematopoiesis in the fetal liver (Shibayama et al., 2004). Since its identification, it was also shown that CIAPIN1 plays several roles in cancer cells, including involvement in multi-drug resistance (Hao et al., 2006; Li et al., 2007; Zhang et al., 2011; Lu et al., 2012), controlling the cell cycle (Li et al., 2007, 2008; He et al., 2009; Hao et al., 2009), and angiogenesis (Yan et al., 2009). Recent studies have also suggested that human CIAPIN1 is expressed at low levels in some malignant tumors (Shi et al., 2010; Zheng et al., 2010; Chen et al., 2012).

The functional conservation of the human and yeast CIAPIN1 genes is reflected in the ability of human CIAPIN1 to complement the nonviability of mutants of the yeast CIAPIN1 homologue, DRE2 (Zhang et al., 2008). Moreover, a novel role for DRE2 in Fe/S cluster biogenesis was reported (Zhang et al., 2008; Vernis et al., 2009; Netz et al., 2010). Also, it was found that human CIAPIN1 is a Fe/S cluster-containing substrate and may play a role in cytosolic Fe/S cluster biogenesis, once trapped in the mitochondrial inter-membrane space (Banci et al., 2011). Recently, it was shown that the highly conserved C-terminal region of Dre2, which contain two Fe-S clusters, also possess a S-adenosylmethionine methyltransferase-like domain (Soler et al., 2012).

In this study, we analyzed the function of the Drosophila CIAPIN1 (D-CIAPIN1) gene during ovarian development. Based on bioinformatics analysis, we first identified the Drosophila gene, l(2)35Bg, as D-CIAPIN1. Several alleles of D-CIAPIN1 were identified, revealing that D-CIAPIN1 is an essential gene that also serves a role in gonad development. We focused our analysis on ovarian development in a D-CIAPIN1 mutant and found the gene to be required for follicle cell proliferation and survival.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Drosophila CIAPIN1 is an Essential Gene Required for Gonad Development

We found that the product of the Drosophila gene l(2)35Bg (also called CG4180) shows 52% similarity at the amino acid level to mammalian CIAPIN1, especially in the C-terminal region, which is highly conserved from yeast to mammals. Moreover, the highly conserved cysteine motifs (CX2CXC and twin CX2C) of CIAPIN1 found near the C-terminus of the human protein are also found in D-CIAPIN1 (Fig. 1A). Thus, to investigate the role of D-CIAPIN1 in Drosophila development, we searched for D-CIAPIN1 mutant strains. Several D-CIAPIN1 mutant alleles were reported previously and in this study, we focused our analysis on two of them, namely l(2)35Bg2 (O'Donnell et al., 1977) and l(2)35BgNP5152 (FlyBase Curators, 2008), both of which are homozygous and also hemizygous lethal. l(2)35Bg2 was generated with ethyl methanesulfonate (EMS), while the l(2)35BgNP5152 allele has a P-element insertion in the 5′ UTR, 37 bp upstream of the start codon of the gene. We determined that in both alleles, the second instar larval stage of the homozygous and hemizygous organism is the lethal phase. We also found that l(2)35BgNP5152/l(2)35Bg2 trans-heterozygous flies are viable but are female- and male-sterile. In consideration of the reader, we will subsequently refer to l(2)35Bg2 as D-CIAPIN12 and l(2)35BgNP5152 as D-CIAPIN1NP.

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Figure 1. Females trans-heterozygous for D-CIAPIN1 are characterized by undeveloped ovaries. A: Multiple sequence alignment of amino acid sequences of the C-terminal region of CIAPIN1 from different organisms was performed using the ClustalW program. Eight conserved cysteine residues are marked with black rectangles. The mutated cysteine at position 212 encoded by the D-CIAPIN12 allele is marked with an arrow. B,C: Confocal images of DNA staining of female ovaries from wild-type (B) and D-CIAPIN12/D-CIAPIN1NP (C) flies. D-CIAPIN1 mutant ovaries are much smaller than are wild-type ovaries. D: UAS-D-CIAPIN1 /Act5C-Gal4; D-CIAPIN12/D-CIAPIN1NP. Ubiquitous expression of D-CIAPIN1 rescued defects in ovarian development seen in the D-CIAPIN1 mutants. Scale bars = 100 μm.

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Next, we studied the molecular nature of each D-CIAPIN1 mutant allele. Because D-CIAPIN12 was generated by EMS, we sequenced the D-CIAPIN1 coding region from genomic DNA of the D-CIAPIN12 mutant stock and compared the sequence to that of the wild-type. We found a missense mutation in D-CIAPIN1 (G635A), resulting in an amino acid change of cysteine to tyrosine at position 212 (C212Y) in the C terminal portion of the protein (Fig. 1A). This mutation affects one of the twin CX2C cysteine motifs of the protein. This is the first report showing the importance of this motif to the function of the CIAPIN1 protein.

We then tested whether excision of the P-element from the D-CIAPIN1 gene in the D-CIAPIN1NP allele could eliminate both lethality and the sterility of the mutant flies. As expected, excision of the P-element reverted both traits. Moreover, after P-element excision, this fly line was able to complement the D-CIAPIN12 female and male sterility phenotypes.

To understand the nature of female sterility, we dissected ovaries from female flies trans-heterozygous for D-CIAPIN1 and found them to be characterized by rudimentary ovaries. Closer examination revealed that ovaries from the trans-heterozygous combination alleles (Fig. 1C) did not contain egg chambers beyond stage eight of development. Next, we performed rescue experiments by expressing the D-CIAPIN1 protein in the D-CIAPIN1mutant background. Accordingly, we created transgenic flies expressing the D-CIAPIN1 protein under the control of the UAS/Gal4 system. We addressed the ability of D-CIAPIN1 to rescue the D-CIAPIN1 lethality and sterility phenotype. We found that ubiquitous expression of D-CIAPIN1 using the actin-Gal4 promoter rescued lethality in both homozygous alleles as well as female and male sterility in all trans-heterozygous mutant allelic combinations (Fig. 1D), demonstrating that the two alleles used in this study (D-CIAPIN1NP and D-CIAPIN12) are alleles of D-CIAPIN1.

Drosophila CIAPIN1 Can Complement the Nonviability of the Yeast dre2 Deletion Strain

Because it was shown that human CIAPIN1 was able to complement the nonviability of the yeast CIAPIN1 deletion strain (Δdre) (Zhang et al., 2008), we decided to examine the ability of Drosophila CIAPIN1 to complement the lethality phenotype of this yeast strain. As such, the Δdre2 shuffle strain containing the DRE2 plasmid carrying wild-type DRE2 and a CYH2 counter-selectable marker was transformed with various constructs based on plasmid YCplac22-TRP1 and driven by the GPD promoter (Fig. 2). The covering plasmid was removed by plating on cycloheximide (CHX), leaving only the test YCplac22-TRP1 plasmid (Fig. 2). As reported by Zhang et al. (2008), transformation with plasmid YCplac22-TRP1 alone does not rescue the nonviability of the Δdre2 strain, although the YCplac22-TRP1 plasmid containing the yeast dre2 gene does (Fig. 2). To examine whether the nonviability of the dre2 deletion strain can also be rescued by the Drosophila CIAPIN1 homologue, we cloned the homologous gene into the YCplac22-TRP1 plasmid so as to perform a counter-selection assay (Fig. 2). We found that the Drosophila CIAPIN1 homologue completely rescued lethality of the Δdre2 strain. As described above, we showed that, in the D-CIAPIN12 mutant, one of the conserved cysteines, namely Cys-212, was changed to tyrosine. Thus, we decided to analyze the ability of this mutant protein to rescue the nonviability of the Δdre2 strain. We found that the dre2 deletion strain grows poorly when containing this Drosophila mutant allele (Fig. 2).

image

Figure 2. Drosophila CIAPIN1 can complement the nonviablity of the yeast Δdre2 strain. The yeast shuffle strain with the dre::kanMX deletion covered by a DRE2 plasmid carrying wild-type DRE2 and the CYH2 counter-selectable marker was transformed with various constructs carried on plasmid YCplac22 and driven by the GPD promoter, including YCplac22 alone, YCplac22-Dre2, YCplac22-D-CIAPIN1, and YCplac22- D-CIAPIN1 (C212Y). A: Transformants containing both the counter-selectable Dre2-CYH2 and the test YCplac22-TRP1 markers were grown as controls. B: The covering plasmid was removed by plating on cycloheximide (CHX), leaving only the test YCplac22-TRP1 plasmid. As reported by Zhang et al. (2008), DRE2 is required for viability, and the deletion can be rescued by expression of the wild-type yeast protein. We show that the deletion can also be rescued by the Drosophila homolog (YCplac22- D-CIAPIN1) and that the deletion strain grows poorly when containing the Drosophila mutant allele, D-Ciapin12, YCplac22-D-CIAPIN1 (C212Y).

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Mutations in D-CIAPIN1 Lead to Elevated Levels of Follicle Cell Death

As described above, we found that female flies trans-heterozygous for D-CIAPIN1 alleles are characterized by rudimentary ovaries. To determine whether these defects in cyst formation reflect a role for CIAPIN1 in the germline or in the somatic cells that surround them, namely follicle cells, we specifically expressed D-CIAPIN1 in the germline using the nosGal4-VP16 driver line. We found that expressing D-CIAPIN1 in the germline in the D-CIAPIN1NP/D-CIAPIN12 trans-heterozygous mutant background failed to rescue ovarian development, suggesting that D-CIAPIN1 is probably required in the somatic cells for development of the egg chamber. To better understand the nature of female sterility in D-CIAPIN1 mutant flies, we focused our analysis on follicle cell development. Using DNA staining to mark the nucleus of follicle cells, we showed that the number of follicle cells is reduced in D-CIAPIN1 mutants. In comparison to wild-type egg chambers (Fig. 3A,B), loss of follicle cells is especially seen in cross-sections of the mutant egg chambers (Fig. 3C,D). Next, we followed cyst formation in D-CIAPIN1 mutant flies using antibodies against the adducin-like molecule, 1B1, a marker of the follicle cell membrane (Lin et al., 1994). We found that in the mutants, follicle cell tissue failed to cover the entire egg chamber and the tissue membrane seemed to be torn, creating holes in the tissue (Fig. 3C). As such, these results suggest that D-CIAPIN1 plays a role in follicle cell development. Next, we analyzed follicle cell development in D-CIAPIN1NP/D-CIAPIN12 trans-heterozygous flies. We counted the number of follicle cells in a stage 4 egg chamber from wild-type and mutant flies. We found that, whereas the wild-type stage 4 egg chamber contains 172 ± 35 follicle cells (mean ± standard deviation, number of egg chambers = 60), as demonstrated both by DNA analysis and follicle cell membrane staining, a significant reduction (t119 = 26.46; P < 0.001) in follicle cell numbers (43 ± 35; n = 60) was found in the D-CIAPIN1NP/D-CIAPIN12 stage 4 egg chamber. We next considered follicle cell proliferation in D-CIAPIN1 mutants. Follicle cells are generated from 2–3 somatic stem cells, called follicle cell stem cells. Follicle cells divide during egg chamber development until the end of stage 6. At this point, the follicle cells undergo endo-replication. The decrease in follicle cell number in the D-CIAPIN1NP/D-CIAPIN12 trans-heterozygous mutants could be a result of excessive cell death and/or defects in follicle cell proliferation. To determine whether loss of follicle cells in these flies is indeed due to apoptosis, we marked apoptotic cells using anti-cleaved caspase-3 antibodies. We showed that mutation in D-CIAPIN1 resulted in an elevated level of follicle cell apoptosis, starting in the early stages of follicle cell development (Fig. 4). We demonstrated that, in 90% of stage 2 to stage 5 wild-type egg chambers, no anti-cleaved caspase-3 antibody-labeled follicle cells were evident (Fig. 4A–C), while in the remaining 10%, only one to two apoptotic cells were found (Fig. 4D–F). In D-CIAPIN1 mutant flies, anti-cleaved caspase-3 antibody-stained follicle cells were evident from D-CIAPIN1 mutant egg chambers as early as stage 2 and onward (Fig. 4G–I). We found that in D-CIAPIN1 stage 2 to stage 5 mutant egg chambers, the number of anti-cleaved caspase-3 antibody-labeled follicle cells was 26 ± 8 (n = 15 ovarioles). Closer examination of follicle cells marked with the antibodies showed that their DNA is highly condensed (Fig. 4J), a further indicator of cell death. Although the loss of follicle cells due to apoptosis is not massive, the loss of follicle cells at an early stage of egg chamber development may nonetheless lead to a massive reduction in follicle cell numbers at the end of the stage of follicle cell proliferation.

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Figure 3. Defects in follicle cell organization in D-CIAPIN1 mutants. Confocal images of egg chambers stained with antibodies against the adducin-like molecule, 1B1 (green), and DAPI (blue or white). A–D: Wild-type (A,B) and D-CIAPIN12/D-CIAPIN1NP mutants (C,D). C,D: Closer examination of the egg chamber from D-CIAPIN12/D-CIAPIN1NP mutants shows a lack in follicle cells that surround the egg chamber, leading to defects in follicle cell membrane tissue organization (green). Scale bars = 10 μm in A,B, 30 μm in C–H.

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Figure 4. D-CIAPIN1 is required for follicle cell survival. Confocal images of egg chambers stained with antibodies against cleaved-caspase 3 (red), and DAPI (4′,6-diamidine-2-phenylidole-dihydrochloride; white). A–L: Wild-type (A–F) and D-CIAPIN12/D-CIAPIN1NP mutants (G–L). B,E: In 90% of wild-type ovarioles, no cleaved-caspase 3 antibody-stained follicle cells were found (B), while in the remaining 10%, one to two cleaved-caspase 3 antibody-stained follicle cells were found (arrow in E). In D-CIAPIN12/D-CIAPIN1NP mutants, positive anti-cleaved caspase-3 antibody-stained follicle cells were evident as early as stage 2 and onward. J: Closer examination of follicle cells marked with the anti-cleaved caspase-3 antibodies revealed highly condensed DNA (arrows) unevenly distributed, as compared to nonapoptotic follicle cells (arrowhead). A: S2, S3, and S4 in refer to egg chambers at stage 2, 3 and 4, respectively. Scale bars = 20μm in C, 30μm in F, 40μm in I, 5 μm, in L.

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Because we found that mutations in D-CIAPIN1 led to follicle cell death, we asked whether blocking cell death by expression of a caspase inhibitor, such as p35, could affect follicle cell development in the mutant flies. Accordingly, we expressed p35 specifically in follicle cells using the GR1-Gal4 promoter that was shown to expresses GAL4 in follicle cells throughout development, starting in the stage 3 egg chambers. We found that ovaries from flies expressing p35 specifically in the follicle cells of D-CIAPIN1 mutants showed the same defects in follicle cell development as when D-CIAPIN1 appeared alone.

Mutations in D-CIAPIN1 Lead to a Reduced Mitotic Index

Next, we decided to follow replication and mitosis progression in mutant follicle cells during egg chamber development. Follicle cells replication was followed using bromodeoxyuridine (BrdU) staining and mitotic cells were marked using anti-phosphorylated histone-3 (PH3) and anti-CycB antibodies (Shcherbata et al., 2004).

Using BrdU to pulse label sites of active DNA replication, we followed staining in egg chambers from stage 2 to stage 6 in wild-type ovaries (Fig. 5A–C), as compared to such staining of follicle cells from the egg chambers of D-CIAPIN1 mutants (Fig. 5D–F). We found that mutations in D-CIAPIN1 had no effect on follicle cell replication, as revealed by the similar patterns of replication seen.

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Figure 5. Follicle cell replication is not affected in D-CIAPIN1 mutants. Confocal images of egg chambers stained with antibodies against BrdU (bromodeoxyuridine; red), and DAPI (4′,6-diamidine-2-phenylidole-dihydrochloride; white). A–F: Wild-type (A–C) and D-CIAPIN12/D-CIAPIN1NP mutants (D–F). Similar replication of follicle cells was found throughput wild-type and in D-CIAPIN12/D-CIAPIN1NP mutants. C: S2, S3, S4, and S5 refer to egg chambers at stage 2, 3, 4 and 5, respectively. Scale bars = 40μm in C, 50 μm in D.

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Using anti-PH3 antibodies, we found that in egg chambers from both wild-type (Fig. 6A) and D-CIAPIN1NP/D-CIAPIN12 mutant flies (Fig. 6B,C), follicle cells divided mitotically until stage 6. However, in D-CIAPIN1NP/D-CIAPIN12 mutants, there was a sharp decrease in the number of follicle cells that were stained with anti-PH3 antibodies (Fig. 6B,C). A similar reduction in anti-CycB antibody staining was detected in D-CIAPIN1NP/D-CIAPIN12 flies upon analysis of labeling of wild-type (Fig. 6D) and mutant egg chambers (Fig. 6E). To better quantify the defects in follicle cell mitosis in the mutant flies, we focused our analysis on the anti-PH3 marker. Because the number of follicle cells in D-CIAPIN1NP/D-CIAPIN12 flies was significantly lower than in the wild-type, we compared the mitotic index (i.e., the percentage of mitosis-surviving follicle cells) of wild-type and mutant flies. Apoptotic follicle cells (as revealed by their highly condensed DNA [Fig. 4J]) were not counted. We found that in wild-type flies, the mitotic index was 4.2±1.9 (n = 60), a value that was significantly (t119 = 11.06; P < 0.001) reduced to 1.0 ± 2.5 (n = 60) in D-CIAPIN1NP/D-CIAPIN12 flies. Closer examination revealed that, whereas 70% of stage 6 wild-type egg chambers had a mitotic index between 2 to 4, in D-CIAPIN1NP/D-CIAPIN12 flies, the mitotic index was zero in more than 90% of the egg chambers (Fig. 6C). These results suggest that D-CIAPIN1 may also regulate follicle cell proliferation.

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Figure 6. Defects in follicle cell proliferation in D-CIAPIN1 mutants. A–E: Confocal images of stage 5 egg chambers stained with anti-PH3 (A–C) and anti-cyclinB antibodies (D,E). A–E: DAPI (4′,6-diamidine-2-phenylidole-dihydrochloride) staining is blue; wild-type egg chambers (A,D) and egg chambers from a D-CIAPIN12/D-CIAPIN1NP mutant (B,C,E). A–C: There is reduced (B) or no (C) anti-PH3 antibody staining in D-CIAPIN12/D-CIAPIN1NP mutants, as compared to wild-type egg chambers (A). E: A similar reduction in anti-CycB antibody staining was observed in the D-CIAPIN12/D-CIAPIN1NP mutant. Scale bars = 30 μm.

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Next, progression through follicle cell mitosis was studied. To identify cells at different mitotic stages, ovaries were stained with the mitosis marker, anti-PH3 antibodies, along with anti-tubulin antibodies to follow microtubule organization. The different mitotic stages were identified by the following criteria: Prophase stage cells were anti-PH3 antibody-stained and showed interphase-like organization of microtubules without visible asters (Fig. 7A). Prometaphase/metaphase stage cells were anti-PH3 antibody-stained and showed visible asters (Fig. 7B,C). Finally, anaphase/telophase stage cells were anti-PH3 antibody-stained and presented chromosomes in different stages of separation (Fig. 7D). Initially, we found that all phases of mitosis were readily identified both in wild-type and D-CIAPIN1 mutants, with all showing typical mitosis patterns. Moreover, no significant differences (χ22 = 0.898; P = 0.638) in the different mitosis stages were found between wild-type and D-CIAPIN1 mutants (Fig. 7E).

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Figure 7. Follicle cell mitosis progression is not affected in D-CIAPIN1 mutants. A–D: Confocal images of wild-type follicle cells stained with anti-PH3 and (red) anti-tubulin (green) antibodies. A: Prophase. B: Prometaphase. C: Metaphase. D: Anaphase. Scale bars = 10 μm. E: Quantification of mitotic parameters in wild-type and D-CIAPIN1 mutants.

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Our results showed that D-CIAPIN1 is required for regulating cell division and survival of follicle cells. What could be the mechanism by which D-CIAPIN1 affects follicle cells development? One possible explanation is that D-CIAPIN1 is requiring for either cell division or cell survival. For example, D-CIAPIN1 may be require for cell division such that any cells that fail to enter mitosis might undergo apoptosis. Hence, apoptosis would be a secondary effect in response to the actions of D-CIAPIN1 in cell division. This hypothesis is supported by the finding that overexpression of caspase inhibitors, such as p35, in the follicle cells of D-CIAPIN1 mutants had no effect on the defects seen in D-CIAPIN1 flies. Another possible explanation is that D-CIAPIN1 may serve separate functions in cell division and survival. The finding that hCIAPIN1 could regulate the cell cycle (He et al., 2009; Saito et al., 2011), along with its initial identification as an anti-apoptotic molecule, further supports such a role for D-CIAPIN1.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Drosophila Stocks

The Oregon-R and relevant Gal4 driver Drosophila strains were used as wild-type controls. The following mutant flies were obtained from the Bloomington Stock Center: l(2)35Bg2, l(2)35BgNP5152 and Df(2L)TE35BC-7. Germline and ubiquitous expression was performed with: P{GAL4::VP16-nos.UTR}CG6325MVD1 and Act-Gal4, respectively. Both Gal4 drivers were obtained from the Bloomington Stock Center. Follicle cell expression was performed with GR1-Gal4 (kindly provided by T. Schüpbach).

Ovary Staining

Ovaries were dissected in phosphate-buffered saline (PBS), fixed in 200 μl of 4% formaldehyde in PBS combined with 600 μl heptane for 20 min, and washed in PBST (PBS + 0.3% Triton X-100). The ovaries were incubated with primary antibodies overnight at 4°C, and then with secondary antibodies for one hour. The ovaries were applied to slides coated in 50% glycerol. The following antibodies were used at the indicated dilutions: rabbit anti-PH3 antibodies (1:100; Upstate Biotech), mouse anti-adducin-like (1B1) (1:100; Hybridoma Bank), mouse anti-cyclin B (CycB) (1:5; Hybridoma Bank), and rabbit polyclonal anti-cleaved Caspase-3 (Asp175) antibodies (1:100, CM1, Cell Signaling Technology). Cy2- and Cy3-conjugated secondary antibodies (Jackson Laboratories) were each used at 1:100 dilutions. For DNA staining, Hoechst stain (Molecular Probes) was used at a concentration of 1 μg/ml. Egg chambers were imaged on Olympus FV1000 Laser-scanning confocal microscope.

BrdU Staining

Ovarian BrdU staining was performed as described (Calvi and Lilly, 2004). Briefly, ovaries were dissected in Grace's medium and incubated in 10 μM BrdU for 1 hr. Following fixation in 6% formaldehyde for 20 min, ovaries were incubated with 12.5 units/ml of DNAse I (Sigma) for 30 min at 37°C. Mouse anti-BrdU antibodies (1:20, BD Biosciences Pharmigen) were used for BrdU detection.

Transgenic Flies

To create D-CIAPIN1 transgenic flies, the entire coding sequence of the gene was amplified by polymerase chain reaction (PCR) using modified primers to introduce XbaI restriction sites at the 5′ and 3′ ends of the sequence. The resulting PCR product was cloned into the pUASp vector. P-element-mediated germline transformation of this construct was carried out according to standard protocols (Spradling, 1986). Ten independent lines were established.

Yeast Strains and Culture

To express D-CIAPIN1 in yeast, the entire coding sequence of the gene was amplified by PCR using modified primers to introduce a NdeI restriction site at the 5′ end and a XbaI restriction site at the 3′ end of the sequence. The resulting PCR product was cloned into the YCplac22 vector. To generate the YCplac22-D-CIAPIN1 mutant construct (D-CIAPIN1 C212Y), we performed site-directed mutagenesis using overlapping primers introducing the guanine to adenine point mutation, thereby changing amino acid 212 from cysteine to tyrosine. The PCR reaction was performed using Turbo PFU (Stratagene, La Jolla, CA), according to the manufacturer's instructions.

The DRE2 shuffle strain, MATa ura3–52 lys2–801(amber) ade2–101(ochre) trp163 his3200 leu21 cyh2 dre2::kanMX [pRS318-DRE2] (Zhang et al. 2008), with the Δdre::kanMX deletion covered by the DRE2 plasmid carrying wild-type DRE2 and CYH2 counter-selectable markers, was transformed with various plasmids, including YCplac22-TRP1 (Zhang et al., 2008). The DRE2 shuffle strain with each of the following plasmids, YCplac22-GPD-Dre2 plasmid-GPD promoter-driven yeast Dre2 (Zhang et al., 2008), YCplac22-GPD-D-CIAPIN1 plasmid and YCplac22-GPD-D-CIAPIN1 (C212Y) was grown on plates with or without cycloheximide.

Statistical Analysis

To detect defects in follicle cell development, we compared the number of follicle cells from stage 3 egg chambers from wild-type and D-CIAPIN1 mutants using Student's t-test. The mitotic index was also compared by t-test but because the data consisted of proportions, we arcsine-transformed the data to better fit a normal distribution. We used the R*C test of independence to compare the distribution among the three groups of mitotic stages. The statistical analyses were performed using Systat 9.0 (Systat software, CITY, CA), and results were considered statistically significantly if α < 0.05.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. ACKNOWLEDGMENTS
  7. REFERENCES

We thank the Bloomington stock center and Trudi Schüpbach for generously providing fly strains. We also thank Andrew Dancis for providing the yeast strains.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. ACKNOWLEDGMENTS
  7. REFERENCES
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