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A-D: data from N=96 8N and N=93 2N cells respectively, from 8 replicates. cycle. To survive mitosis with acentric chromosomes, papillar cells require Fanconi Anemia proteins FANCD2 and FANCI, and Blm helicase, but not canonical DDR signaling. FANCD2 acts independently of previous S-phases to promote alignment and segregation of acentric DNA produced by double-strand breaks, thus avoiding micronuclei and organ malformation. As polyploidy and impaired DDRs can promote cancer, our findings provide insight into disease-relevant DNA damage tolerance mechanisms. endocycled cells accumulate the ATM/ATR phosphorylation mark -H2AV (Mehrotra et al. 2008), a readout of double-strand DNA breaks (DSBs). In such DSB accumulation is likely due to low p53 (a core DNA damage-responsive transcription factor) levels and chromatin silencing at p53 pro-apoptotic target genes (Mehrotra et al. 2008; Hassel et al. 2014; Zhang et al. 2014). Similarly, in mice, differentiation of endocycling trophoblast giant cells involves decreased p53 (Soloveva and Linzer 2004), and suppression of the DDR kinase Chk1 (Ullah et al. 2008;2011). Thus, in both developmental and cancerous settings, endocycles promote impaired DDRs and tolerated DNA DSBs. However, many developmentally endocycled cells do not resume mitosis, and thus these Tepoxalin systems cannot be used to identify responses enabling continued mitosis of genome-damaged cells. We recently developed study of papillar cells as a developmentally and genetically tractable model of polyploid mitosis after endocycles. Here, using our model, we uncover mechanisms permitting these polyploid cells to undergo viable division with DNA damage. Similar to previous studies, we find endocycled papillar cells lack p53-mediated apoptosis. Further, we find papillar cells lack S-phase checkpoints and enter mitosis without undergoing high fidelity DNA Tepoxalin repair. Despite lacking these normally crucial DDRs, both papillar mitosis and organ development are highly resistant to DNA damage by DSBs. By live imaging pupal development, we show an important part of the papillar DDR involves alignment and segregation of broken, acentric chromosome fragments. This response does not depend on p53, or core DNA damage kinases. Instead, the Fanconi Anemia protein FANCD2, its frequent partner FANCI, and the Bloom helicase (Blm) are a crucial part of this non-canonical DDR. We show FANCD2 acts independently of S-phases prior to mitosis entry, and does not require its core complex partner FANCM to promote segregation of acentric fragments produced by DNA DSBs. This response ensures normal organ development by preventing acentric micronuclei. Our results pinpoint a mechanism enabling viable mitosis despite an impaired DDR. RESULTS Lack of apoptosis and S-phase checkpoints during pre-mitotic endocycles Previous study of endocycle-induced DDR inactivity focused on post-mitotic tissues. To understand the impact of endocycles on subsequent divisions, we turned to an accessible model: rectal papillar cells (hereafter: papillar cells or papillar precursors). During 2nd larval instar, papillar precursors endocycle, producing octoploid nuclei (Fox et al. 2010; Schoenfelder et al. 2014). Unlike previously studied examples of endocycled cells with an inactive DDR, papillar cells then undergo polyploid divisions. We thus asked if these mitotic endocycled cells also lack an apoptotic response to Tepoxalin damaged DNA. It is well established that exposure to Ionizing Radiation (IR) causes DNA damage and apoptotic cell death in diploid cells. Accordingly, we find induction of pycnotic nuclei and TUNEL labeling in diploid wing imaginal tissue after 20 Gy of X-ray induced IR (Fig1A,B,E, FigS1A,B, Methods). In contrast, IR does not induce pycnotic nuclei or TUNEL in endocycling 2nd instar papillar precursors (Fig1C-E, FigS1C,D). The lack of apoptosis KLF1 in papillar precursors is not due to lack of IR-induced DNA breakage, as IR causes robust -H2AV accumulation in endocycling papillar precursors one hour after IR (FigS1E,F). Open in a separate window Figure1 Lack of p53-dependent apoptosis.