Role of hemocytes in the regeneration of germline stem cells in Drosophila

Cellular regeneration, which relies on extensive restructuring of cytoplasmic materials, is an essential process to restore tissues and organs lost during aging, degenerative diseases and injury. At early stages of Drosophila spermatogenesis, when cellular constituents are intensely remodeled, there are two different populations of stem cells, the somatic stem cells and the germline stem cells (GSCs). GSCs divide by asymmetric division to give rise two distinct daughter cells. One of them will leave the stem cells’ niche and differentiate into spermatogonial cells (SCs). Both aging and cellular stress can lead to the loss of GSCs. Lost GSCs can be restored by dedifferentiation of SCs into functional GSCs. In other tissues, macrophages provide specific conditions for cellular transformation. Here we examined the potential role of immune surveillance cells called hemocytes during dedifferentiation of SGs into GSCs. We found an elevated number of hemocytes during this dedifferentiation process. Immune depletion of hemocytes decreased the regeneration capacity of germline. We also show that autophagy, which plays a pivotal role in cellular differentiation by eliminating unwanted, superfluous parts of the cytoplasm, becomes upregulated in dedifferentiating SCs upon JAK-STAT signaling emitted by hemocytes. Furthermore, these immune cells regulate expression of Omi/HtrA2, a key regulator of apoptosis in early spermatogenesis. Together, we suggest that hemocytes have important functions in the dedifferentiation process of GSCs.


Introduction
Regeneration is a cellular process by which cells lost during aging, injury and various cellular insults can be effectively restored to maintain tissue functioning. The regeneration process is based on cell fate transformation mediated by remodeling of cellular constituents 1 . Cellular regeneration can be achieved by three distinct major mechanisms: 1) dedifferentiation of a terminally differentiated somatic cell into an unspecialized blastemal cell having the capacity to further proliferate and the daughter cells differentiate into the lost terminally differentiated cell type, 2) direct transdifferentiation of a specialized somatic cell into another specialized cell type, and 3) reprogramming from local somatic stem cells. During dedifferentiation, the affected specialized somatic cell regains its potency to differentiate into a cell type that characterizes the original lineage when differentiating into the blastema 2 . A tractable tissue model for studying dedifferentiation is represented by the zebrafish caudal fin, which can effectively be regenerated followed by amputation within a week. At the site of amputation, unipotent progenitor cells are generated which constitute the blastema (a mesenchymal cell mass serving the source of the regenerating tissue) 3 .
According to the literature, macrophages, which are specific immune cells, play an important role in tissue regeneration mediated by dedifferentiation. For example, these cells participate in the regeneration process of zebrafish caudal fin and amphibian limb 4,5 . It was also shown that residual macrophages affect the proliferation of somatic stem cells. For further studying the role of macrophages in tissue regeneration by dedifferentiation, the Drosophila testis may also serve as an ideal in vivo model system, since the Drosophila melanogaster male germline stem cell niche is one of the best characterised stem cell system 6,7 . In the fruit fly, specific immune cells called hemocytes involve plasmatocytes, lamellatocytes and crystal cells 8 . Plasmatocytes are the dominating hematocyte population during all developmental stages, and capable of phagocyting. Regarding their function, these cells are most similar to mammalian macrophages. Plasmocytes, together with crystal cells, generate the von Willebrand factor, also called hemolectin (Hml), which we used here as a marker for detecting these cells in the regeneration process 9 .
Hemocytes also function to eliminate apoptotic bodies of spermatogonial origin through phagocytosis 10 . Plasmocytes are able to emit a ligand, Upd3 protein, which serves as a ligand for the JAK-STAT signaling pathway 11 . Hence, hemocytes play a role not only in humoral immune response but also in processes associated with JAK-STAT signaling 12 . It has been demonstrated that during proliferation of Drosophila intestinal stem cells (ISCs) hemocytes produce Upd ligands and Dpp (Decapantaplegic), thereby affecting both JAK-STAT and BMP (bone morphogenetic factor) signaling 13 . According to results provided by Van de Bor and colleagues, hemocytes participate in shaping and maintaining the microenvironment of female GSCs, and regulating their number and differentiation by producing signaling factors and cell adhesion molecules 14 .
The number of GSCs can be lowered in response to certain insults such as stress and aging. The lost cells can be restored by dedifferentiation of SGs under favorable conditions 15 . In the apical part of the Drosophila testis, Hub serves as a regulatory center for the stem cell niche that consists of somatic origin, postmitotic cells. Hub binds to GSCs and somatic cyst stem cells (CySCs) through cell adhesion molecules, and the coordinated operation of the three cell populations is required for the continuous production of sperms. In this region, Hub produces Upd ligand for JAK-STAT signaling, hence primordial germ cells found only at the apical part of the gonad can adapt GSC fate, and bind directly to Hub 16 .
We performed experiments on the Drosophila testis, which is an ideal model for studying the regeneration process via dedifferentiation because GSC regeneration can be activated under laboratory conditions. In a transgenic fly strain. bam driven by a heat inducible promoter ectopically expresses the main germline regeneration factor Bam (bag of marbles) 17 . Bam is thus expressed in GSCs and induces their leaving the germ line niche and differentiation into SGs.
By finishing ectopic expression of Bam, dedifferentiation of SGs into GSCs can be activated.
These newly produced GSCs then initiate to divide asymmetrically and generate daughter cells that eventually create sperms. This process is termed as the regeneration of early spermatogenesis 18 . To assay the process, we applied an immunohistochemical method that indicates hub cells by red (anti-FasciclinIII) and germline cells by green (anti-Vasa) signals (

Objectives
Our aim was to uncover novel cellular and molecular factors required for GSC regeneration in the Drosophila testis. We also examined whether hemocytes have a role in this process because our preliminary results have indicated that inhibiting hemocyte functions decreasess the number of GSCs. We monitored a mitochondrial serine-protease involved in cell death during spermatogenesis, Omi/HtrA 19

Results
We previously examined lysosomal degradation during germline regeneration in the Drosophila testis. These experiments revealed large, multipodial cellular structures that are characteristics of hemocytes (Fig. 2). It was previously described that residual macrophages can be found in the Drosophila testis and these are identified as hemocytes 20 .We confirmed the presence of these immune cells in the testis by using hemocyte-specific cell adhesion markers such as hemolectin (Hml) 21 . Applying a Hml-GFP transgene, we observed that hemocytes are recruited to the site GSC regeneration ( Fig. S1). We also wondered whether inhibiting hemocyte function affects the regeneration capacity of GSCs. For that, germline cells were indicated by anti-Vasa (green), while hub cells were labeled by anti-FasciclinIII (red) staining (Fig. 2). Elimination of immune cells was established by overexpressing a proapoptotic gene, reaper (rpr) 22 . According to our results, in the absence of hemocyte function GSC regeneration becomes compromised (Fig. 3). It was shown that hemocytes regulates processes via JAK-STAT signaling 23 . This prompted us to analyze their potential function in GSC regeneration. The expression of chinmo serving as a target gene for JAK-STAT signaling was examined by RT-qPCR 24 . Relative mRNA levels were found to gradually increase during the regeneration process (Fig. 4). According to our results JAK-STAT signaling is active during GSC regeneration. We next asked whether hemocytes contribute to the activation of the signaling pathway in this process.
Upd1 and Udp3 genes encode ligands for JAK-STAT signaling and were downregulated by RNA interference specifically in immune cells using HmlGal4 (hemocyte-specific) driver. Using immunohistochemistry, we showed that Upd1 and Upd3 deficiency interferes with the regeneration capacity of GSCs (Fig. 5).

±S.D. indicates mean deviations.
When an immune-depleted environment was induced, a TUNEL assay was applied to uncover increases levels of cell death, which occurred exclusively during the regeneration process (Fig.   6). Similar results were obtained when we inhibited the JAK-STAT signaling pathway. This Both HS and UAS-rpr lead to defects in the regeneration process. GSCs indicated by dotted suggests a link between the number of apoptotic SGs and JAK-STAT signaling. In the absence of hemocytes, there was no increase in apoptosis even under normal condition. Similar to that, there was no TUNEL-positive structure during the normal regeneration process (control). How-ever, when hemocytes were eliminated, the number of TUNEL-positive cells increased significantly under regeneration-inducing conditions, indicating higher levels of apoptotic cell death.
Similar results were obtained when upd3 was specifically silenced in hemocytes. Together, these results suggest that hemocytes contribute to the regeneration process and their absence leads to the activation of caspase-independent apoptosis. The multifunctional hemocytes participate in the removal of apoptotic corpses of SG origin, in which Omi/HtrA2 plays a key role 25 .
We could uncover a link between Omi and JAK-STAT signaling; stat92e encodes a transcription factor for JAK-STAT signaling and its downregulation in the germline resulted in enhanced levels of omi expression (Fig. 7). During GSC regeneration, omi/HtrA2 expression was changed as revealed by a semi-qPCR analysis (Fig. 8). At the beginning of regeneration (0-2 h after induction), omi expression became lowered, while it was increased after 24 h of regeneration (Fig. 8). Then, we examined the potential role of omi in GSC regeneration, by using fluorescent microscopy. We found that inhibiting omi transcription in the germline blocks the dedifferentiation process. Based on these results, Omi is required for the regeneration of GSCs (Fig. 9).

Diagram showing the number of GSCs during the regeneration process. After 24 h, a significant level of difference was observed (**: p<0.005). Blue indicates control, green labels Omi-depleted states. bars indicate ±S.D.
Using a TUNEL assay, we tested changes in programmed cell death in Omi defective background. The results showed that omi downregulation leads to an increase in the number of cells undergoing apoptosis (Fig. 10). However, the increase was evident under both normal and regeneration-inducing conditions, but the latter was more significant as compared with the former condition. During regeneration of GSCs, we performed a LysoTracker Red staining and could observed an increased level of acidic compartments, which indicates elevated autophagic activity (Fig.   S2). These results may result from two processes. Either autophagy became hyperactivated or a later stage of the process was inhibited, causing the accumulation of early autophagic structures (phagophores and autophagosomes). To distinguish between the two alternatives, we quantify Ref(2)P levels, which orthologous to mammalian p62 26 . Ref(2)P/p62 serves as a substrate for autophagy, so its levels inversely correlate with autophagyic activity. We found that when omi is downregulated under normal conditions, Ref (2)

Conlusions
During the dedifferentiation process, hemocytes play multifunctional roles. First, these cells are capable of inducing signaling pathways required for the dedifferentiation of SGs into GSCs.
Upd1 and Upd3 function as ligands for JAK-STAT signaling, and our results obtained here indicate that they are omitted by hemocytes. Thus, JAK-STAT signaling is activated in SGs by hemocytes. Upds can transmit through either Cyst cells (in which JAK-STAT signaling becomes activated to cause Upd expression toward SGs) or directly reach SGs. It is possible that at this time Cyst cells do not surround dedifferentiating germline cells.
During regeneration, JAK-STAT signaling may play a dual role in SGs. First, it may activate autophagy, which is required for cytoplasmic remodeling of SGs undergoing dedifferentiation.
Second, the pathway may inhibit apoptosis through repressing a mitochondrial serine protease, HrtA2. In the presence of JAK-STAT signaling Omi expression becomes lowered, and its low levels are required for regeneration. Omi may play an important role in maintaining the balance between autophagy and programmed cell death (Fig. 12). Together, results obtained by this study may contribute to a better understanding of mechanisms underlying tissue regeneration, and its regulation by immune cells. The signaling pathway also acts as a relay of whether dedifferentiation occurs through autophagy, or caspase-independent apoptosis occurs through Omi.

Drosophila strains and their maintenance
For the maintenance of the used frutifly strains the compliants and characterications of the food were published in the article below. 28

Protocol of Dedifferentiation
We performed experiments on the Drosophila testis in order to observe regeneration via dedifferentiation because GSC regeneration can be activated under laboratory conditions. Based on the hs-bam, bamGal4 expression system, bam is driven by a heat inducible promoter (Hsp70) ectopically expresses the main germline regeneration factor Bam (bag of marbles) 17 . Under normal conditions, bam is silenced in the germ line. Due to ectopic expression of bam, it is expressed in GSCs and makes differentiation possible. By finishing ectopic expression of Bam, dedifferentiation of SGs (and GBs) into GSCs can be activated. This process is termed as the regeneration of early spermatogenesis. After the termination of ectopic expression recovering GSCs is possible by only spermatogonial dedifferentiation 7 .
Regeneration protocol step by step: Fruitflies were maintained at 25°C unless they were used to the regeneration protocol Day 1: 3x30 min heat shock by applying 37°C water bath. In between the water baths at least 2 hours needs to be elapsed and maintained at 29°C Day 2: Besides the water baths fruitflies are maintained at 32°C Day3: The fruitflies are taken back to the 29°C termostate. Applied control groups: Fruitflies were maintained at 25°C temperature in a termostate. 2 regeneration groups were used according to how many hours are passed after terminating the ectopic expression of bam: 1) 24h: After 3 days of heatshock then keeping them at 25°C temperature for 24 hours 2) 6h: Same as 24h but keeping them at 25°C temperature for 6 hours 3) bam overexpression (HS): maintaining this group at 29°C for 3 days. Maintaining a high level of bam expression, thus preventing regeneration.

Fluorescent microscopy
A fluoreszcens képek készítése: Images were captured with Zeiss Axioimager Z2 upright epifluorescent microscope equipped with ApoTome2 semi-confocal upright. We used the following softcovers: ZEN 3.1 Images were captured with Zeiss Axioimager Z2 upright epifluorescent microscope equipped with ApoTome2 semi-confocal upright. We used the following._programs for evaluation of images: ZEN 3.1 (blue edition) and Fiji ImageJ 1.8.0_77.

Electron Microscopy
Electron microscopy images were taken with JEOL (1011; JELOL, Tokyo Japan) transmission electron microscope equipped with Olypmos Morada digital camera, and we used iTEM software 30 We used the following solution during fixation: 2,4 ml 16% formaldehyde (final concentration: 3,2) 480 µl 25% glutaraldehyde (1%) 0,12 g sucrose 0,00435 g CaCl2 Na-cacodylate 0,1M 7,4pH qPCR reaction and applied primeres 1. RNA isolation and cDNA synthesis The whole amount of RNA was extracted from 20 testes using TRI reagent (Sigma, T9424), then purifying RNA was conducted by Directzol RNA MiniPrep kit-tel (Zymo Research, R2050) and was performed as described in manufacturer's protocol. The single strand cDNA synthesis was provided by RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, K1622).  ATT TCG CTT TG -3'; reverse -5' -TTC CCG  CAG GTA TAT TCG TC -3' PCR products were detected by a fluorescent, DNA-binding (double strand) SYBR Green dye (Roche FastStart Essential DNA Green Master 11560300). Verifying the specificity of the reaction of qPCR was provided by analyzing the melting curves. By normalizing the values of the PCR threshold cycles, it was possible to identify the average of relative mRNA level.

TUNELassay
After the pre-fixation protocol, we followed the manufacturer's instructions. 31 We used the following materials during TUNEL-assay: ApopTag kit(Chemicon/Millpore, Billerica, MA, USA, S7105, S7106, S7107). Testicles were fixed in 4% formaldehyde (diluted with PBS) for 20 minutes and washed 3 times in cold methanol. We incubated samples in -20°C cold methanol for 24 hours.

Statistics
We used the RStudio (Version 1.2.5033) program for statistical analysis.. Lilliefors test were used to know that the distribution of samples examined is normal or not. If it was normal, F test was performed to compare two variances. If the variances were equal, twosample Student's t-test was used otherwise t-test for unequal variances was applied. If the distribution of a sample is not normal, Mann-Whitney U test was performed