Polyploid cells, which contain more than two genome copies, occur throughout nature. Beyond well-established roles in increasing cell size/metabolic output, polyploidy can also promote nonuniform genome, transcriptome, and metabolome alterations. Polyploidy also frequently confers resistance to environmental stresses not tolerated by diploid cells. Recent progress has begun to unravel how this fascinating phenomenon contributes to normal physiology and disease.

The endocycle is a modified cell cycle that lacks M phase. Endocycles are well known for enabling continued growth of post-mitotic tissues. By contrast, we discovered pre-mitotic endocycles in precursors of Drosophila rectal papillae ( papillar cells). Unlike all known proliferative Drosophila adult precursors, papillar cells endocycle before dividing. Furthermore, unlike diploid mitotic divisions, these polyploid papillar divisions are frequently error prone, suggesting papillar structures may accumulate long-term aneuploidy. Here, we demonstrate an indispensable requirement for pre-mitotic endocycles during papillar development and also demonstrate that such cycles seed papillar aneuploidy. We find blocking pre-mitotic endocycles disrupts papillar morphogenesis and causes organismal lethality under high-salt dietary stress. We further show that pre-mitotic endocycles differ from post-mitotic endocycles, as we find only the M-phase-capable polyploid cells of the papillae and female germline can retain centrioles. In papillae, this centriole retention contributes to aneuploidy, as centrioles amplify during papillar endocycles, causing multipolar anaphase. Such aneuploidy is well tolerated in papillae, as it does not significantly impair cell viability, organ formation or organ function. Together, our results demonstrate that pre-mitotic endocycles can enable specific organ construction and are a mechanism that promotes highly tolerated aneuploidy.

Epithelial stem cells are routinely lost or damaged during adult life and must therefore be replaced to maintain homeostasis. Recent studies indicate that stem cell replacement occurs through neutral competition in many types of epithelial tissues, but little is known about the factors that determine competitive outcome. The epithelial follicle stem cells (FSCs) in the Drosophila ovary are regularly lost and replaced during normal homeostasis, and we show that FSC replacement conforms to a model of neutral competition. In addition, we found that FSCs mutant for the basolateral junction genes, lethal giant larvae (lgl) or discs large (dlg), undergo a biased competition for niche occupancy characterized by increased invasion of neighboring FSCs and reduced loss. Interestingly, FSCs mutant for a third basolateral junction gene, scribble (scrib), do not exhibit biased competition, suggesting that Lgl and Dlg regulate niche competition through a Scrib-independent process. Lastly, we found that FSCs have a unique cell polarity characterized by broadly distributed adherens junctions and the lack of a mature apical domain. Collectively, these observations indicate that Lgl and Dlg promote the differentiation of FSC progeny to a state in which they are less prone to invade the neighboring niche. In addition, we demonstrate that the neutral drift model can be adapted to quantify non-neutral behavior of mutant clones.

Multiple nuclei sharing a common cytoplasm are found in diverse tissues, organisms, and diseases. Yet, multinucleation remains a poorly understood biological property. Cytoplasm sharing invariably involves plasma membrane breaches. In contrast, we discovered cytoplasm sharing without membrane breaching in highly resorptive Drosophila rectal papillae. During a six-hour developmental window, 100 individual papillar cells assemble a multinucleate cytoplasm, allowing passage of proteins of at least 62kDa throughout papillar tissue. Papillar cytoplasm sharing does not employ canonical mechanisms such as incomplete cytokinesis or muscle fusion pore regulators. Instead, sharing requires gap junction proteins (normally associated with transport of molecules <1kDa), which are positioned by membrane remodeling GTPases. Our work reveals a new role for apical membrane remodeling in converting a multicellular epithelium into a giant multinucleate cytoplasm.

ONE SENTENCE SUMMARYApical membrane remodeling in a resorptive Drosophila epithelium generates a shared multinuclear cytoplasm. ABSTRACTMultiple nuclei sharing a common cytoplasm are found in diverse tissues, organisms, and diseases. Yet, multinucleation remains a poorly understood biological property. Cytoplasm sharing invariably involves plasma membrane breaches. In contrast, we discovered cytoplasm sharing without membrane breaching in highly resorptive Drosophila rectal papillae. During a sixhour developmental window, 100 individual papillar cells assemble a multinucleate cytoplasm, allowing passage of proteins of at least 27kDa throughout papillar tissue. Papillar cytoplasm sharing does not employ canonical mechanisms such as failed cytokinesis or muscle fusion pore regulators. Instead, sharing requires gap junction proteins (normally associated with transport of molecules <1kDa), which are positioned by membrane remodeling GTPases. Our work reveals a new role for apical membrane remodeling in converting a multicellular epithelium into a giant multinucleate cytoplasm. MAIN TEXTSharing of cytoplasm in a multinucleate tissue or organism is an important and recurring adaptation across evolution. Multinucleate structures include animal skeletal muscle, mammalian osteoclasts, and mammalian syncytial placental trophoblasts (1-3). In disease, cytoplasm sharing facilitates the spread of pathogens (4), oncogenic factors (5,6), and prion-like proteins (7).Cytoplasm sharing can occur through cytokinesis failure, or through plasma membrane breaches such as fusion pores, tunneling nanotubes, or plasmodesmata. Such clearly visible breaches enable exchange of cytoplasmic components such as RNA, proteins, and even organelles (8,9).The ubiquity and importance of cytoplasm sharing led us to seek out novel examples in the tractable animal model Drosophila melanogaster. Here, we report an animal-wide screen for tissues that share cytoplasm. We identify a novel mechanism of cytoplasm sharing in the rectal papilla, a resorptive intestinal epithelium (10) and known site of pathogen localization (11). Unlike all known examples of multinucleation, cytoplasm sharing in rectal papillae involves developmentally programmed apical membrane remodeling. To identify new examples of adult tissues in Drosophila that share cytoplasm, we ubiquitously expressed UAS-dBrainbow (12) (Fig1A), a Cre-Lox-based system that randomly labels cells with only one of three fluorescent proteins. Multi-labeled cells should only arise by fusion of cells not related by cell division/cytokinesis failure (Fig1B). We examined a wide range of tissues (FigS1A). From our screen, we discovered that the rectal papilla is a new example of a tissue with cytoplasm sharing. Adult Drosophila contain four papillae, each with 100 nuclei, that reside in the posterior hindgut (Fig1C). Using both fixed and live imaging of whole organs, we found that at 62 hours post puparium formation (HPPF), each papillar cell contains only one dBrainbow label (Fig1D). By contrast, at 69HPPF, mu...

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