Spermatogonial dedifferentiation into germline stem cells in Drosophila testes

Abstract

Stem cell pools are dynamic and capable of reacting to insults like injury and starvation. Recent work has highlighted the key role of dedifferentiation as a conserved mechanism for replenishing stem cell pools after their loss, thereby maintaining tissue homeostasis. The testis of the fruit fly Drosophila melanogaster offers a simple but powerful system to study dedifferentiation, the process by which differentiating spermatogonia can revert their fate to become fully functional germline stem cells (GSCs). Dedifferentiated GSCs show interesting characteristics, such as being more proliferative than their wild type sibling GSCs. To facilitate the study of the cellular and molecular mechanisms underlying the process of germline dedifferentiation in the Drosophila testis, here we describe techniques for inducing high rates of dedifferentiation and for unambiguously labelling dedifferentiated GSCs.

1. Introduction

Homeostasis of highly proliferative tissues relies on the robustness of pools of resident tissue stem cells. The depletion of these stem pools causes tissue atrophy, and stem cell exhaustion is a hallmark of aging [1, 2]. During the life of the animal, stem cell pools are dynamic, capable of responding to insults like starvation and injury, replenishing depleted pools with new stem cells. Dedifferentiation has proven to be a key mechanism enabling this recovery, where cells on a path to terminal differentiation can revert their trajectory and regain stemness [3].

Germline dedifferentiation has been observed in Drosophila gonads after transiently forcing all GSCs to differentiate [4–6]. The Drosophila testis in particular is a suitable tissue to study this process, thanks to extensive knowledge about precise anatomical positions and molecular markers of every cell type and every stage of differentiation. In the testis, a pool of 8–14 GSCs resides attached to a niche of quiescent cells, termed the “hub” [7]. A GSC undergoes oriented mitosis that results in the production of one daughter GSC and one gonialblast. The latter begins differentiation through four rounds of mitosis with incomplete cytokinesis that results in 2-, 4-, 8- and 16-cell spermatogonial cysts. The 16-cell cyst then undergoes meiosis and ultimately produces 64 spermatids (Figure 1).

Figure 1:

Diagram of a testis, showing the different cell types and differentiation stages of the spermatogonia, up to the pre-meiotic stage. Germline cells are represented in red, the hub cells in blue and other somatic cells in grey. The expression domain of the differentiation factor bam is shown as purple nuclei.

Dedifferentiation of 4- and 8-spermatogonia occurs as a result of aging, mating, and after starvation [8–11]. While not essential to maintain the GSC pool during aging in standard laboratory conditions, dedifferentiation is essential for rapid recovery of the GSC pool after acute stress and for preserving robust spermatogenesis during chronic stress [10]. Furthermore, dedifferentiated GSCs display higher proliferative rates than non-dedifferentiated, “wild type”, sibling GSCs in the same testis [10].

Dedifferentiated GSCs can be monitored by indelibly labelling germline cells that have already started the differentiation process and should otherwise not be present at the stem cell niche. This can be achieved by lineage-tracing spermatogonial cells expressing bam-Gal4, a key factor in the germline differentiation that becomes active in 4- and 8-cell spermatogonial cysts [8, 10]. As with other lineage-tracing strategies, this Gal4 line is used to drive the expression of a recombinase, UAS-Flp, which in turn induces the excision of a stop codon flanked by FRT recombination sites on a cassette, in our case ubiP63E-FRT-STOP-FRT-Stinger.GFP (abbreviated ubi>STOP>GFP) [12]. Once recombined, this GFP mark remains under the genetic control of the ubiquitous promoter ubiP63E and persists even if the marked cell turns off the bam promoter (Figure 2). A limitation of this methodology is that it cannot detect cases of dedifferentiation among goniablasts and 2-cell cysts, as bam-Gal4 is not expressed at these stages. The technique, however, induces consistent and unambiguous labelling of dedifferentiation events among 4- and 8-cell spermatogonial cysts.

Figure 2:

Genetic methodology and crossing scheme for labelling dedifferentiated GSCs. A female expressing a bam-Gal4 transgene is mated with a male carrying a ubiP63E>STOP>GFP and UAS-Flp transgenes. In the F1 progeny, the bam-Gal4 driver line is used to drive the expression of a UAS-Flp recombinase. Flp in turn recombines the FRT sites flanking a stop codon on a ubiP63E>STOP>GFP cassette. The excision of the codon places GFP under direct control of the ubiquitous ubiP63E promoter, thereby indelibly and permanently marking germline cells. The GFP-labeling persists even if these cells dedifferentiate into GSCs, which turn off bam-Gal4 expression.

This method can also be modified to block dedifferentiation, and so study the effects of its absence. As the key factor that drives germline differentiation in normal conditions, transient mis-expression of bam is sufficient to induce the differentiation of GSCs [6, 13]. By mis-expressing additional Bam protein (with a UAS-bam transgene) in these bam-Gal4-positive cells, spermatogonia are forced to follow irreversibly the differentiation path, and as a result dedifferentiation rates drop to baseline control levels [10]. Blocking dedifferentiation can be also achieved by inhibiting the Jun N-terminal kinase pathway, using pathway repressors such as UAS-puc or UAS-bsk^K53R [10].

Here, we describe a genetic methodology for labelling dedifferentiated GSCs, alternative methods for inducing high rates of dedifferentiation, and a detailed procedure for immunostaining testes and distinguishing dedifferentiated GSCs from their non-dedifferentiated siblings.

2. Materials

1. Phosphate-Buffered saline (PBS): NaCl at 137 mM, KCl at 2.7 mM, Na₂HPO₄ at 10 mM, KH₂PO₄ at 1.8 mM. Adjust the pH to 7.4 with HCl.

2. Triton X-100 detergent diluted with dH₂O to a final concentration of 10%.

3. PBS-Triton: PBS solution supplemented with both 0.2% and 0.5% Triton X-100.

4. PBTB: PBS-Triton 0.2% solution supplemented with 1% bovine serum albumin. This blocking solution can be aliquoted and frozen for better preservation (see Notes 4.1).

5. Paraformaldehyde (PFA). Dilute a 16% PFA commercial solution in PBS to a final concentration of 4%. This fixative solution can be aliquoted and frozen for better preservation (see Notes 4.1).

6. Dumont #5 forceps.

7. Optionally, a thin insect pin mounted on a syringe. Hold a syringe needle and very carefully place it close to/on top of a flame (but not directly inside the flame). Pull the metal needle from the plastic part using forceps when the plastic partially melts. With the plastic still molten, grab an insect pin with forceps and introduce it onto the plastic, replacing the discarded needle. Wait for the plastic to solidify, mount it on a syringe and replace the cap of the needle to protect the pin while not in use.

8. Dissecting microscope.

9. Dissecting plates: a small petri dish coated with a thin bed of Sylgard 184 Silicone Elastomer mixed with Indian ink.

10. 1.5 mL Eppendorf tubes.

11. Microscope slides and coverslips.

12. Vectashield plus DAPI mounting medium (Vector laboratories) (see Notes 4.9).

13. Nail polish.

14. Primary antibodies: rat anti-Vasa used at a 1:20 concentration (stains the cytoplasm of germline cells) and mouse anti-Fasciclin3 used 1:50 (stains the membrane of hub cells). Both antibodies can be obtained from the DSHB.

15. Secondary antibodies: donkey secondary antisera from Jackson ImmunoResearch. We recommend anti-rat conjugated with Alexa 555 for revealing Vasa and anti-mouse conjugated with Alexa 647 for Fasciclin3.

16. Fly food vials and plugs, filled with approximately 1 inch of standard fly food.

17. 25°C Incubator.

18. Sucrose.

19. Agarose.

20. Cheesecloth.

21. Confocal microscope.

22. Scientific imaging software. We suggest Fiji/ImageJ (https://imagej.net/software/fiji/) [14].

23. Fly stocks (see Table 1).

Table 1:

Fly stocks used

Fly line	Source	Purpose
Oregon-R	BDSC #2376	Source of virgins for mating
Ubi-p63E(FRT.STOP)Stinger, UAS-Flp	BDSC #28282	ubi>stop>GFP cassette
bam-Gal4:VP16	[15]	Driver for 4- and 8-cell spermatogonia
UAS-bam:GFP	[15]	Possible control for blocking dedifferentiation
UAS-puc	[16]	Possible control for blocking dedifferentiation
UAS-bsk K53R	BDSC #9311	Possible control for blocking dedifferentiation
UAS-LacZ	BDSC #3955	Control for normal dedifferentiation

3. Methods

3.1. Lineage-tracing genetics and husbandry

1. If the goal of the experiment is simply to observe dedifferentiated germ cells, the only stocks required will be bam-Gal4 for virgin females and ubi>STOP>GFP for males. If instead the goal is studying factors affecting the dedifferentiation process, we recommend using males of the genotype UAS-bam; ubi>STOP>GFP (or ubi>STOP>GFP; UAS-puc or ubi>STOP>GFP; UAS-bsk^K53R) for the positive control cross (where dedifferentiation will be blocked) and ubi>STOP>GFP; UAS-LacZ for the negative control (where a neutral UAS line is used to maintain the titration of the Gal4 protein). We can provide these stocks upon request, or they can be built easily through standard genetic crosses. When building these stocks, it is critical to ensure that the hs-Flp construct, present in many balancer stocks, does not exist on the final stock, as it would induce spurious recombination of the cassette.

2. Collect virgin females of the bam-Gal4 stock line and cross them with the males from the stocks indicated in the previous point.

3. Ten days after setting up the crosses, collect twice daily F1 virgin males, keeping them separate from females (see Note 4.6).

3.2. Aging and stress assays

The different methodologies presented below will promote high rates of spermatogonial dedifferentiation. Upon normal aging, protein deprivation (commonly referred to as starvation) and mating, dedifferentiated GSCs will comprise approximately 20–25% of the GSC pool. By contrast, after chronic stress, this percentage increases to approximately 45% of the GSC pool. Bear in mind that methodology involving chronic stress takes longer and requires more work.

If the experiment goal is to study spermatogonial cells during the dedifferentiation process, rather than observing the final result, we recommend the starvation methodology, as the spike of dedifferentiation takes place in a narrow window of time, with a sharp increase of dedifferentiated GSCs between the days 2 and 3 of refeeding [10].

3.3. Normal aging

1. Place young (preferentially no older than 3 days) adult males into regular food vials, maintaining not more than 20–30 males per vial (see Note 4.3).

2. Flip flies into fresh food vials every two days.

3. Dedifferentiation rates increase with aging [8, 10], but age-related mortality will negatively impact the recovery of flies for dissection, especially when approaching the 60-day mark. In our experience, 45 days of adulthood is a good compromise allowing for fly survival and high levels of dedifferentiation.

3.4. Starvation

1. Prepare starvation food by melting 1% agarose with 10% sucrose in an Erlenmeyer flask. Distribute it in empty fly vials (about 1 inch of food per vial), cover them with a cheesecloth while it solidifies and then plug the vials. The vials can be stored at 4°C for up to 2 weeks.

2. Place young (preferentially no older than 3 days) adult males on starvation vials, maintaining not more than 20–30 per vial (see Note 4.3). Flip them every two days into fresh vials with starvation food.

3. For refeeding, flip the males into vials with regular food. Flip them again every two days into fresh vials with regular food.

4. The maximum loss of GSCs is achieved after 6 days of starvation, while their complete recovery happens after 5 days of refeeding [9, 10]. The shortest protocol to obtain as many dedifferentiated GSCs as possible using this protocol should encompass 11 days, with 6 days of starvation and 5 of refeeding.

3.5. Stress through mating

1. Collect Oregon-R virgin females during the whole span of the experiment

2. Place young (preferentially no older than 3 days) adult males into regular food vials together with Oregon-R virgin females in a ratio 1:2 (male:female). The total number of flies per vial should not exceed 20–30, see Note 4.3.

3. Flip flies into fresh vials every two days.

4. Every 7 days, anesthetize the files, discard the old females and replace them with fresh virgins.

5. In our experience, good levels of dedifferentiation are achieved after 2–3 weeks.

3.6. Chronic stress

1. Prepare starvation food as explained in step 3.4.1 and collect Oregon-R virgin females during the whole span of the experiment.

2. Place young (preferentially no older than 3 days) adult males on a starvation regime of 6 days, as explained in step 3.4.2.

3. Refeed the flies by flipping them into a fresh vial of regular food for two days

4. Transfer flies into a fresh vial of regular food together with Oregon-R virgin females in a ratio 1:2 (male:female). The total number of flies per vial should not exceed 20–30, see Note 4.3.

5. After two days, anesthetize the files and discard the females.

6. The previous 4 steps represent a cycle of 10 days. Repeat this cycle 3 times for a total of 40 days.

7. After the last cycle, transfer the males into a fresh vial of regular food for one additional day, so the last refeeding period is 5 days in total.

3.7. Dissection of testes, imaging and cell characterization

1. Dissect flies in PBS on a dissection dish. Use a pair of forceps to tear apart the abdomen cuticle between segments (ideally at the anterior border of the A5, where the strong male pigmentation begins). Pull slowly and gently the posterior part of the abdomen and place it in an Eppendorf tube with PBS.

2. Remove the PBS from the tube and fix for 30 minutes at room temperature with gentle shaking in 0.5 mL of 4% paraformaldehyde + 10 μL of 10% Triton X-100.

3. Rinse once in PBS and then wash 2 × 30 minutes in PBS-Triton 0.5% with gentle shaking.

4. Block in PBTB for one hour.

5. Incubate overnight in primary antibodies, diluted in 50 μL of PBS-Triton 0.2%, at 4°C with gentle shaking.

6. Rinse once in PBS and then wash 2 × 30 minutes in PBS-Triton 0.2% with gentle shaking.

7. Incubate 2 hours in secondary antibodies at room temperature with gentle shaking. Dilute 1 μL of each Alexa-labelled antibody in 200 μL of PBS-Triton 0.2%. Protect the tubes from light from this step onward, for example by using aluminium foil.

8. Rinse once in PBS and then wash 2 × 30 minutes in PBS-Triton 0.2% with gentle shacking.

9. Remove as much PBS-T as possible, add one drop of Vectashield plus DAPI.

10. Store at 4°C until mounting (fluorescence can last up to a month or more) or incubate at least 30 minutes and proceed to mounting.

11. For mounting, take the posterior abdomens from the tube and place them on a slide, together with about 20 μL of the Vectashield from the tube. Using forceps and the insect pin prepared in step 2.5 (or two forceps), separate the testes from the abdomens and move them gently towards the center of the slide. Testes can be identified as thick and coiled tubes with a fibrous content. The insect pin can be used to tear non-testis structures and to drag gently the dissected testes to the center of the slide. With the testes separated, carefully remove from the slide abdomens and other tissue debris. Put 10 μL of Vectashield on top of the testes, and carefully place a coverslip on top and use nail polish to seal.

12. To scan the testes on a confocal microscope, locate the hub at the apical tip of the testis through Fasciclin3 staining. Use at least 63x magnification and acquire stacks with a z-step of 2 μm.

13. GSCs can be identified as cells with a Vasa-positive cytoplasm directly in contact with the membranes of hub cells, labelled with Fas3 (see Figure 3A). Navigate through the image stack, as GSCs can be in a rosette around the hub or in some cases above or below the hub. Germline cells separated from the hub by a gap where another nucleus is present are gonialblasts and should not be scored as GSCs.

14. Among the GSCs, the dedifferentiated ones can be identified by GFP-positive nuclei (see Figure 3B).

Figure 3:

Representative confocal images of a testis without (A) or with (B) dedifferentiated GSCs. A’ and B’ are diagrams provided as an aid for interpreting the micrographs. GSCs can be identified as cells with Vasa-positive cytoplasm (red) in direct contact with the hub (which express Fasciclin 3 (blue membranes) at apical tip of the testis). Dedifferentiated GSCs can be distinguished by the GFP-positive nuclei (green). Spurious lineage cassette recombination can happen in somatic cells, but these can be distinguished from the dedifferentiated GSCs, by the expression of cytoplasmic Vasa surrounding the GFP nuclei in the latter.

4. Notes

1. Once thawed, PBT-BSA and paraformaldehyde aliquots can be preserved at 4°C. PBT-BSA must be discarded after a week and paraformaldehyde after 3 days.

2. Spurious recombination of the lineage-tracing FRT cassette is frequently observed in fly stocks in harboring both an FRT cassette and a UAS-Flp, such as the ones used on this protocol. If this happens in the germline, the recombined cassette will be transmitted to the next generations, rendering the stock useless. Periodically check the stocks containing the FRT cassette for presence of GFP-positive larvae, and make new stocks again if recombination is detected.

3. Overcrowding of vials can result on suboptimal nutritional intake and affect the results – 20–30 flies is the maximum number that should be housed in a 1 inch-width vial.

4. Always flip the F1 males into fresh food every two days for optimal nutrition and better reproducibility of the results.

5. Keep at all times the F1 males on a 25°C incubator with controlled humidity for better reproducibility. This is especially important in aging protocols.

6. Unless females are intentionally introduced to vials with adult males to induce stress, males need to be strictly separated at day 0 from females. The presence of females (and presumed mating between males and females) induces proliferative responses in GSCs [10], affecting the reproducibility of results.

7. Anticipate some lethality (in the case of chronic stress, up to 50%) after the treatments explained above. Plan to age more flies than those you strictly need for the experiment.

8. Dissection of abdomens should not take longer than 20 minutes before fix with paraformaldehyde. If longer times are required, PBS should be used ice-cold, while keeping the Eppendorf tube with the dissected abdomens in an ice container. Even if using ice, the total dissection time should not be longer than 30 minutes.

9. Staining the nuclei, while not strictly necessary, is very helpful for identifying the different cell types of the testis. If the used mounting medium does not contain DAPI, DNA can be stained alternatively by adding either DAPI or Hoechst at a 1:500 concentration during the incubation with secondary antibodies.

10. The Stinger (GFP) fluorescence from the lineage cassette shows a weaker signal in germline cells compared with somatic cells, but should be strong enough to be detected on confocal microscopes. If required, anti-GFP staining can be used to enhance the signal.

11. Occasionally the lineage cassette can spuriously recombine in some somatic cells and should not be confused with dedifferentiated GSCs. These cells can be identified by their lack of Vasa staining in their cytoplasm and denser nuclei.

12. A high variance in the proportion of dedifferentiated GSCs per testis not unusual, especially in aged flies. It is possible to have testes where none or all of the GSCs are positive for the dedifferentiation lineage mark.

13. If analyzing rates of dedifferentiation, always compare for each genotype the result of the chosen treatment with 0-day-old young males. A baseline of dedifferentiation of around 5% of GSCs on average is normal, possibly representing cases of dedifferentiation during development or spurious recombination of the cassette.