Cyclin B3 Deficiency Impairs Germline Stem Cell Maintenance and Its Overexpression Delays Cystoblast Differentiation in Drosophila Ovary
Abstract
:1. Introduction
2. Results
2.1. Deficiency of cycB3 Impairs GSCs Maintenance in Drosophila Ovary
2.2. The Gene cycB3 Functions as an Intrinsic Factor in Controlling GSC Maintenance
2.3. cycB3 Does Not Influence Bmp/dpp-Mediated Dad Expression
2.4. cycB3 Is Not Required for Bam Transcriptional Silencing and Acts Probably in a Bam-Dependent Manner, in Ovarian GSCs of Drosophila
2.5. Deficiency of cycB3 Fails to Cause Apoptosis in Ovarian GSCs, Nor Influences CB Differentiation into Oocytes in Drosophila Ovary
2.6. Over-Expression of cycB3 Fails to Increases the Number of GSCs, but Dramatically Enhances the Number of CBs
3. Discussion
4. Materials and Methods
4.1. Constructs
4.2. Drosophila Stocks
4.3. Immunohistochemistry and Microscopy for Drosophila Ovary
4.4. Quantitative Real-Time PCR (qPCR)
4.5. TUNEL Apoptotic Cell Detection
4.6. Generating and Analyzing Marked Germline Clones
4.7. Statistical Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
cycB3 | cyclin B3 |
GSC | germline stem cell |
GFP | gfp fluorescent protein |
CB | Cystoblast |
Df | Deficiency strain of cycB3 gene |
qPCR | quantitative real-time PCR |
nosP-gvp | nanosP-gal4:vp16 |
AHST | after heat-shock treatment |
UASp | pattB-UAS |
hsP | pattB-hsP |
nosP | pattB-nosP |
Dad | Daughters agaist Dpp |
TUNEL | Terminal deoxynucleotidyl transferase(TdT)-mediated dUTP Nick End Labeling |
WT | wild-type |
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Genotype | Age 1 | Germaria | ||||
---|---|---|---|---|---|---|
Empty | 0 Germline Stem Cell (GSC) (Cysts Only) | 1 GSC | 2–3 GSCs | Total | ||
Oregon | Day 1 | 0 | 0 | 1.4% | 98.6% | 146 |
Day 7 | 0 | 0.6% | 3.9% | 95.5% | 178 | |
Day 14 | 0.7% | 1.4% | 7.4% | 90.5% | 148 | |
cycB32/cycB32 | Day 1 | 2.9% | 2.2% | 5.8% | 89.1% | 274 |
Day 7 | 26.6% | 4.4% | 29.6% | 39.4% | 203 | |
Day 14 | 42.1% | 10.5% | 24.8% | 22.6% | 420 * | |
cycB32/cycB3EY08012 | Day 1 | 1.9% | 0.5% | 3.3% | 94.3% | 209 |
Day 7 | 22.5% | 6.0% | 25.8% | 45.7% | 267 | |
Day 14 | 38.8% | 5.6% | 28.0% | 27.6% | 340 * | |
cycB32/cycB3L6540 | Day 1 | 3.1% | 3.6% | 6.7% | 86.6% | 194 |
Day 7 | 8.0% | 1.9% | 26.5% | 63.6% | 162 | |
Day 14 | 29.5% | 15.8% | 26.5% | 28.2% | 444 * | |
cycB3EY08012/cycB3L6540 | Day 1 | 6.9% | 0.9% | 11.4% | 80.8% | 219 |
Day 7 | 15.9% | 6.0% | 47.5% | 30.6% | 183 | |
Day 14 | 35.2% | 13.5% | 32.9% | 18.4% | 207 * | |
cycB32/Df | Day 1 | 4.3% | 3.8% | 6.9% | 85.0% | 160 |
Day 7 | 5.6% | 11.7% | 24.8% | 57.9% | 214 | |
Day 14 | 32.9% | 20.8% | 25.9% | 20.4% | 216 * |
Genotype | The Average Number of GSCs (Mean ± SD) | p Value | The Average Number of CBs (Mean ± SD) | p-Value |
---|---|---|---|---|
bamP-GFP (WT) | 2.1 ± 0.4 (n = 87) | - | 1.1 ± 0.4 (n = 87) | - |
bamP-GFP; attB-cycB3-gDNA | 2.6 ± 0.9 (n = 83) | p < 0.05 | 2.8 ± 1.3 (n = 83) | p < 0.01 |
bamP-GFP; nosP-cycB3 | 3.0 ± 1.4 (n = 113) | p < 0.05 | 3.1 ± 1.7 (n = 113) | p < 0.01 |
bamP-GFP; hsP-cycB3 (RT) | 2.1 ± 0.5 (n = 89) | p > 0.05 | 1.1 ± 0.5 (n = 89) | p < 0.01 |
bamP-GFP; hsP-cycB3 (HS) | 2.2 ± 0.8 (n = 98) | 3.2 ± 1.4 (n = 98) |
Primer Names | Sequences of Primers |
---|---|
cycB3 | 5’-CAGTGCCCGAGGAAGTAGAG-3′ (sense) |
5’-GCATATAGTCGGCAATGGGG-3′ (antisense) | |
rp49 | 5’-CACTTCATCCGCCACCAGTC-3′ (sense) |
5’-CGCTTGTTCGATCCGTAACC-3′ (antisense) |
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Chen, D.; Zhou, L.; Sun, F.; Sun, M.; Tao, X. Cyclin B3 Deficiency Impairs Germline Stem Cell Maintenance and Its Overexpression Delays Cystoblast Differentiation in Drosophila Ovary. Int. J. Mol. Sci. 2018, 19, 298. https://doi.org/10.3390/ijms19010298
Chen D, Zhou L, Sun F, Sun M, Tao X. Cyclin B3 Deficiency Impairs Germline Stem Cell Maintenance and Its Overexpression Delays Cystoblast Differentiation in Drosophila Ovary. International Journal of Molecular Sciences. 2018; 19(1):298. https://doi.org/10.3390/ijms19010298
Chicago/Turabian StyleChen, Dongsheng, Lijuan Zhou, Fuling Sun, Mingzhong Sun, and Xiaoqian Tao. 2018. "Cyclin B3 Deficiency Impairs Germline Stem Cell Maintenance and Its Overexpression Delays Cystoblast Differentiation in Drosophila Ovary" International Journal of Molecular Sciences 19, no. 1: 298. https://doi.org/10.3390/ijms19010298