(W1130-03-16) Homologous Recombination Is Essential for DNA Damage-Induced Regeneration of Germline Stem Cells in the Drosophila Testis Niche

Purpose: Stem cells are integral to the development and maintenance of tissues. Adult stem cells occupy tissue-specific niches and have the unique ability to produce additional stem cells and differentiated daughter cells¹. As stem cells are vital for tissue homeostasis, studying their response to genotoxic stress like gamma-radiation will advance our understanding of how stem cells survive severe damage². Exposure to irradiation causes DNA double stranded breaks that result in cell cycle arrest and cell death if not repaired³. There are multiple double strand break repair pathways, but the homologous recombination (HR) and nonhomologous end joining (NHEJ) DNA damage response pathways predominate³. HR is error-free and accurately repairs double strand breaks using the sister chromatid as a template, while NHEJ is an error prone pathway that ligates the broken ends together, often resulting in mutations³. Adult testes contain sperm-producing germline stem cells (GSCs), and a conserved feature of GSC biology is their ability to resist levels of DNA damage that kills their differentiated progeny (spermatogonia)^4,5,6. The well characterized Drosophila testis stem cell niche is an excellent model system to investigate GSC survival due to the ease of determining cell identities and the wide variety of tools available for analysis⁷. In this tissue, a small cluster of somatic niche cells creates a local microenvironment, or niche, sustaining two stem cell populations: germline stem cells and somatic stem cells (Figure 1). The GSCs in this tissue are all mitotically active, dividing asymmetrically to produce a stem cell and a differentiated daughter cell, that will undergo several rounds of transit-amplifying divisions to become a 16-cell spermatogonial cyst, and eventually becomes sperm⁷. Under normal conditions, 20%-30% of spermatogonia undergo cell death, but GSC death has never been seen^5,8. We hypothesize that the GSCs have a preferential bias towards the more high-fidelity double strand break repair pathway, HR, due to their vital function in protecting the genome and ensuring continuity of the species in the presence of genotoxic stressors.

Methods: We first performed a dose curve of gamma-radiation to identify a dose that induces significant GSC loss since they are extremely resistant to death^4,5,6. We used null mutants and germline specific knockdowns of DNA damage repair pathways members to identify the pathways required for survival. Additionally, we leveraged our ex-vivo, whole tissue live imaging technique to visualize the cellular behaviors of the GSCs at various time points after irradiation.

Results: We found that 75 Gy of irradiation significantly reduces the GSC population within 24 hours (avg.=3.5 GSCs/testis, N=22) as compared to non-irradiated controls (avg.=7.8 GSCs/testis, N=28, p< 0.001) (Figure 2). Ex-vivo live imaging of testes immediately post irradiation revealed that instead of undergoing apoptosis, the GSCs detach and displace from the niche (N=18/73 GSCs), a phenomenon that does not occur in non-irradiated testes (N=0/52 GSCs). The niche becomes repopulated 7 days post irradiation (avg.=7.71 GSCs/testis, N=27), which was indistinguishable from non-irradiated controls (Figure 2A). Shifting the ex-vivo live imaging timepoint to 2.5 days post irradiation showed that GSC repopulation not only occurs through self-renewal of the remaining GSCs (as we expected), but also through dedifferentiation of spermatogonia back into stem cells. Figure 3 contains still images of maximum intensity projections of a testis 2.5 days after exposure to 75 Gy of irradiation, where a 2-cell spermatogonial cyst, derived from a fragmented four-cell cyst, contacts the niche and dedifferentiates in (Figure 3B). Dedifferentiation is observed in all irradiated testes (N=12/12) but is undetectable in non-irradiated control testes (N=0/8). Surprisingly, we found that GSCs require the HR pathway for regeneration post irradiation, and do not require the NHEJ pathway. Null mutants of HR pathway member, Blm, failed to recover their GSCs by 7 days post irradiation (avg.=0 GSCs/testis, N=37), compared to controls (avg.=8.3 GSCs/testis, N=29 testes, p< 0.0001, Figure 2B). Testes with a germline specific knockdown of HR pathway member, Rad51, also failed to recover their GSCs by 7 days post irradiation (avg.=1.9 GSCs/testis, N=32) compared to controls (avg.=7.7 GSCs/testis, N=16, p< 0.001, Figure 2C). Full body null mutants of the NHEJ pathway member, Ku70, fully recover their GSCs 7 days post irradiation (avg.=7.6 GSCs/testis, N=21), compared to controls (avg.=9 GSCs/testis, N=13, p= 0.2784, Figure 2D), indicating that this pathway is not necessary for GSC recovery. Male Drosophila GSCs require the more accurate HR over the error prone NHEJ for DNA double strand break repair, which could speak to their essential role in genome preservation and maintenance.

Conclusion: Overall, findings from these experiments have revealed the unexpected mechanisms contributing to the survival of germline stem cells. Future experiments will investigate the mechanisms involved in dedifferentiation, and the response of the somatic stem cell linage to irradiation. A deeper understanding of stem cell resistance to irradiation has implications in other radio-resistant stem cells, like cancer stem cells, which contribute to the regrowth of tumors after radiotherapy.

References: 1) Rossi, D. J., Jamieson, C. H., & Weissman, I. L. (2008). Stems cells and the pathways to aging and cancer. Cell, 132(4), 681–696. https://doi.org/10.1016/j.cell.2008.01.036
2) Mandal, P. K., Blanpain, C., & Rossi, D. J. (2011). DNA damage response in adult stem cells: pathways and consequences. Nature reviews. Molecular cell biology, 12(3), 198–202. https://doi.org/10.1038/nrm3060
3) Blanpain, C., Mohrin, M., Sotiropoulou, P. A., & Passegué, E. (2011). DNA-damage response in tissue-specific and cancer stem cells. Cell stem cell, 8(1), 16–29. https://doi.org/10.1016/j.stem.2010.12.012
4) Bloom, J. C., Loehr, A. R., Schimenti, J. C., & Weiss, R. S. (2019). Germline genome protection: implications for gamete quality and germ cell tumorigenesis. Andrology, 7(4), 516–526. https://doi.org/10.1111/andr.12651
5) Hasan, S., Hétié, P., & Matunis, E. L. (2015). Niche signaling promotes stem cell survival in the Drosophila testis via the JAK-STAT target DIAP1. Developmental biology, 404(1), 27–39. https://doi.org/10.1016/j.ydbio.2015.04.017
6) Xing, Y., Su, T. T., & Ruohola-Baker, H. (2015). Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster. Nature communications, 6, 7058. https://doi.org/10.1038/ncomms8058
7) de Cuevas, M., & Matunis, E. L. (2011). The stem cell niche: lessons from the Drosophila testis. Development (Cambridge, England), 138(14), 2861–2869. https://doi.org/10.1242/dev.056242
8) Yacobi-Sharon, K., Namdar, Y., & Arama, E. (2013). Alternative germ cell death pathway in Drosophila involves HtrA2/Omi, lysosomes, and a caspase-9 counterpart. Developmental cell, 25(1), 29–42. https://doi.org/10.1016/j.devcel.2013.02.002


Figure 1: Experimental design and summary of results. 1) Vials of adult male flies are irradiated with 75 Gy, then testes are dissected and imaged via confocal microscopy. The Drosophila testis stem cell niche is created by niche cells that signal to the attached germline and somatic stem cells. 2) GSCs autonomously require members of the Homologous Recombination repair pathway, Blm and Rad51, to repair their DNA double stranded breaks. 3) In response to irradiation, single GSCs detach and displace from the niche, a phenomenon unseen in non-irradiated testes. 4a). Germline stem cell numbers are replenished post irradiation from spermatogonia that dedifferentiate and revert into stem cells. 4b) Germline stem cells also recover through self-renewal, as expected because this also occurs in non-irradiated testes.


Figure 2: Regeneration of germline stem cells occurs at a specific radiation dose, fails with loss of homologous recombination pathway, and does not require non-homologous end joining pathway.
All graphs are scatter plots showing the number of GSCs per testis apex. In all graphs, bars indicate mean and S.D. Sample size for each treatment set indicated under the respective plot.
A) In wild-type males, 75 Gy of IR causes significant reduction in GSC count one day after IR exposure compared to non-irradiated controls. Interestingly, the GSC count recovers to that of un-irradiated controls one week after IR exposure. *** p<0.001, **** p<0.0001, 2-tailed Kruskal-Wallis test with multiple comparisons.
B) While the irradiated controls recover their GSC count 7 days after exposure to 75 Gy, GSC counts per testis are significantly reduced in testes null for Blm. Unpaired t-test.
C) While the irradiated controls recover their GSC count seven days after exposure to 75 Gy, GSC counts per testis are significantly reduced in testes that have a knockdown of Rad51 in the germline. Unpaired t-test.
D) The irradiated controls and the Ku70 null mutants recover their GSC count seven days after exposure to 75 Gy of irradiation. Unpaired t-test.


Figure 3: Live imaging of gamma-irradiation induced spermatogonial dedifferentiation. (A-B) Maximum intensity projections of a testis expressing a cortical cytoskeleton marker in germ cells (GMA-GFP, green) 2 days after exposure to 75 Gy of gamma-irradiation. Hub nuclei are rendered in white; spermatogonial nuclei are indicated with magenta spheres. Two 2-cell cysts, derived from a fragmented four-cell cyst, are outlined in yellow and blue; one (blue) contacts the niche (dedifferentiates) in (B).