Germ cell connectivity enhances cell death in response to DNA damage in Drosophila testis

Two broadly known characteristics of germ cells in many organisms are their development as a ‘cyst’ of interconnected cells and their high sensitivity to DNA damage. Here we provide evidence that these characteristics are linked, and that interconnectivity is a mechanism that confers to the Drosophila testis a high sensitivity to DNA damage. We show that all germ cells within a cyst die simultaneously even when only a subset of them exhibit detectable DNA damage. Compromising connectivity results in cysts in which only a subset of germ cells die upon DNA damage, lowering overall germ cell death. Our data indicate that a death-promoting signal is shared through the intercellular connections of germ cells. Taken together, we propose that intercellular connectivity is a mechanism that uniquely increases the sensitivity of the germline to DNA damage, thereby protecting the integrity of gamete genomes that are passed on to the next generation.


21
A prevalent feature of germ cell development across species is their proliferation as an 22 interconnected cluster of cells, widely known as a germ cell cyst. In many organisms from 23 insects to humans, germ cells divide with incomplete cytokinesis that results in interconnected 24 cells with shared cytoplasm, leading to cyst formation (Greenbaum,Iwamori,Buchold,& 25 Matzuk, 2011; Haglund, Nezis, & Stenmark, 2011; Pepling, de Cuevas, & Spradling, 1999). 26 During oogenesis, this intercellular connectivity is critical for the process of oocyte specification, 27 allowing only some of the developing germ cells to become oocytes while the others adopt a determination, spermatogenesis is a process where all germ cells within a cyst are considered to 36 be equivalent and become mature gametes (Fuller, 1993;Yoshida, 2016). Despite the lack of a 37 'nursing mechanism' during spermatogenesis, intercellular connectivity is widely observed in 38 spermatogenesis from insects to humans (Greenbaum et al., 2011;Yoshida, 2016). While a 39 function for this connectivity has been proposed in post-meiotic spermatids (Braun, Behringer, Oakberg, 1955). It has been postulated that the high sensitivity of the germline to DNA 46 damage is part of a quality control mechanism for the germ cell genome, which is passed on to 47 the next generation (Gunes, Al-Sadaan, & Agarwal, 2015). However, the means by which the 48 germline achieves such a high sensitivity to DNA damage remains unclear. 49 Here we provide evidence that germ cell connectivity serves as a mechanism to sensitize 50 the germline in response to DNA damage, inducing cell death in the Drosophila testis. We show 51 that an entire germ cell cyst undergoes synchronized cell death as a unit even when only a subset 52 of cells within the cyst exhibit detectable DNA damage. Disruption of germ cell connectivity in 53 mutants of the fusome, an organelle that connects the germ cells within a cyst, leads to death of 54 individual germ cells within a cyst in response to DNA damage, reducing overall germ cell 55 death. The sensitivity of a germ cell cyst to DNA damage increases as the number of 56 interconnected germ cells within increases, demonstrating that connectivity serves as a 57 mechanism to confer higher sensitivity to DNA damage. Taken together, we propose that germ 58 4 cell cyst formation serves as a mechanism to increase the sensitivity of genome surveillance, 59 ensuring the quality of the genome that is passed on to the next generation.

62
Ionizing radiation induces spermatogonial death preferentially at the 16-cell stage. 63 The Drosophila testis serves as an excellent model to study germ cell development owing 64 to its well-defined spatiotemporal organization, with spermatogenesis proceeding from the apical 65 tip down the length of the testis. Germline stem cells (GSCs) divide to produce gonialblasts 66 (GBs), which undergo transit-amplifying divisions to become a cyst of 16 interconnected 67 spermatogonia (16-SG) before entering meiosis (Fig. 1A) (Yang & Yamashita, 2015).

76
In search of the cause of this 16-SG death, we discovered that it can be induced by 77 ionizing radiation (see methods). When adult flies were exposed to ionizing radiation that causes 78 DNA double strand breaks (DSBs), a dramatic induction of SG death was observed (Fig. 1B, C).  1B). Importantly, in contrast to starvation-induced SG death, which was dependent on 84 somatic cyst cell apoptosis, radiation-induced SG death was not suppressed by inhibiting cyst 85 cell apoptosis ( Fig. 1 -Figure supplement 1), suggesting that radiation-induced SG death is a 86 germ cell-intrinsic response.

87
The frequency of dying SG cysts peaked around 3 to 6 hours after irradiation and 88 decreased by 24 hours post-irradiation (Fig. 1C). Interestingly, we found that ionizing radiation   The fusome is required for synchronized all-or-none SG death within the cysts. 122 The above results led us to hypothesize that all SGs within a cyst might be triggered to   The mitochondrial protease HtrA2/Omi is required for all-or-none SG death 156 The above results suggest that intercellular connectivity mediated by the fusome plays a 157 critical role in allowing for all-or-none commitment of SGs to death or survival. Based on these 158 results, we hypothesized that a signal to promote cell death exists that is rapidly transmitted from 159 damaged SGs to others via their intercellular connections. 160 It has previously been shown that germ cell death in the Drosophila testis depends on  In HtrA2/Omi mutant flies, we frequently observed a mix of Lysotracker-positive and -168 negative SGs within a single cyst, similar to what was seen in fusome mutants (Fig. 4A). 169 Disruption of the all-or-none mode of SG death within the cyst was observed at any dose of 170 radiation tested (Fig. 4B). Disrupted all-or-none SG death occurred in both heterozygous 171 (Omi Δ1 /+) and transheterozygous (Omi Δ1 /Omi Df1 ) conditions, consistent with the previous report of radiation (Fig. 5 -Figure supplement 2). Even at a high dose, the reporter expression was 200 observed only at 24 hours after irradiation, much later than the peak of SG death, which typically 201 happens within a few hours. Collectively, these data indicate that differential expression of p53 Increasing connectivity of SGs inherently increases sensitivity to DNA damage 206 The above results suggest that the robust ability of SGs to trigger cell death in response to 207 DNA damage is facilitated by the sharing of signals to die amongst SGs within a cyst, killing 208 SGs that are not sufficiently damaged to commit to cell death on their own. This sharing of death 209 signals is mediated by and dependent on germ cell connectivity. If this is the case, it would be 210 predicted that increasing the connectivity of a SG cyst (the number of interconnected SGs within 211 the cyst) would increase its sensitivity to DNA damage. Indeed, as mentioned above, we 212 observed a trend of 16-SG cysts dying more frequently than 2-, 4-, or 8-SGs (Fig. 1C, Fig 1 - to radiation dose, which is reminiscent of somatic imaginal disc cells (Fig. 1D). These results 219 further support the idea that germ cell connectivity plays a key role in increasing the sensitivity 220 of the germline to DNA damage.  According to this model, higher connectivity would confer higher sensitivity to DNA 239 damage: as the connectivity increases, more cells would contribute to detecting any DNA 240 damage the germline may be experiencing. Indeed, our data show a direct correlation between 241 sensitivity to radiation and the increasing connectivity of SG cysts (Fig. 6A). Remarkably, the 242 9 fact that single-celled, unconnected GBs exhibit an essentially linear death response to increasing 243 radiation suggests that individual germ cells do not have an intrinsically different DNA damage 244 response that accounts for their high sensitivity to DNA damage (Fig. 6A).  Thus, a multicellular organism would require differential sensitivities to DNA damage between 252 the soma and the germline. The germline would require a more sensitive genome surveillance 253 mechanism to produce gametes with the highest genome quality, whereas the priority of the 254 soma shifts toward survival in order to support development and maintenance of somatic organs.

255
A connectivity-based increase in sensitivity to DNA damage would be a simple method for 256 multicellular organisms to achieve drastically different sensitivities to DNA damage between the 257 soma and germline without having to alter intrinsic damage response pathways. Thus, we 258 speculate that one reason germ cell connectivity has arisen during evolution might be to confer 259 higher sensitivity to DNA damage specifically in the germline.    assessed by number of Hoechst-stained nuclei only when using unfixed samples. Note that the 317 scoring of dying SGs is not directly comparable between fixed and unfixed samples (for 318 example, results shown in Fig. 1 vs. Fig. 2), due to the difference in the method of SG staging