Eclosion muscles secrete ecdysteroids to initiate asymmetric intestinal stem cell division in Drosophila.

During organ development, tissue stem cells first expand via symmetric divisions and then switch to asymmetric divisions to minimize the time to obtain a mature tissue. In the Drosophila midgut, intestinal stem cells switch their divisions from symmetric to asymmetric at midpupal development to produce enteroendocrine cells. However, the signals that initiate this switch are unknown. Here, we identify the signal as ecdysteroids. In the presence of ecdysone, EcR and Usp promote the expression of E93 to suppress Br expression, resulting in asymmetric divisions. Surprisingly, the primary source of pupal ecdysone is not from the prothoracic gland but from dorsal internal oblique muscles (DIOMs), a group of transient skeletal muscles that are required for eclosion. Genetic analysis shows that DIOMs secrete ecdysteroids during mTOR-mediated muscle remodeling. Our findings identify sequential endocrine and mechanical roles for skeletal muscle, which ensure the timely asymmetric divisions of intestinal stem cells.

A live-imaging protocol for tracking intestinal stem cell divisions in the Drosophila melanogaster pupal midgut.

Establishing a long-term ex vivo observation of the intestinal stem cell (ISC) is crucial to help understand the formation and homeostasis of the intestinal epithelium. Here, we present a protocol for tracking the division of Drosophila pupal ISCs during pupal midgut development. We describe steps for dissecting, mounting, and live imaging the pupal midgut. We then detail procedures for fluorescence quantification of each cell. This protocol can be applied to other fluorescently tagged proteins. For complete details on the use and execution of this protocol, please refer to Wu et al.1.

Apical-basal polarity precisely determines intestinal stem cell number by regulating Prospero threshold.

Apical-basal polarity and cell-fate determinants are crucial for the cell fate and control of stem cell numbers. However, their interplay leading to a precise stem cell number remains unclear. Drosophila pupal intestinal stem cells (pISCs) asymmetrically divide, generating one apical ISC progenitor and one basal Prospero (Pros)+ enteroendocrine mother cell (EMC), followed by symmetric divisions of each daughter before adulthood, providing an ideal system to investigate the outcomes of polarity loss. Using lineage tracing and ex vivo live imaging, we identify an interlocked polarity regulation network precisely determining ISC number: Bazooka inhibits Pros accumulation by activating Notch signaling to maintain stem cell fate in pISC apical daughters. A threshold of Pros promotes differentiation to EMCs and avoids ISC-like cell fate, and over-threshold of Pros inhibits miranda expression to ensure symmetric divisions in pISC basal daughters. Our work suggests that a polarity-dependent threshold of a differentiation factor precisely controls stem cell number.

Monitoring Gut Acidification in the Adult Drosophila Intestine.

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the proper functioning of the gut. One such region, the copper cell region (CCR), is localized to the middle midgut and consists, in part, of a group of cells known as copper cells. Copper cells are involved in gastric acid secretion, an evolutionarily conserved process whose precise role is poorly understood. This paper describes improvements in the current protocol used to assay for acidification of the adult Drosophila melanogaster gut and demonstrates that it can be used on other species of flies. In particular, this paper demonstrates that gut acidification is dependent on the fly's nutritional status and presents a protocol based on this new finding. Overall, this protocol demonstrates the potential usefulness of studying Drosophila copper cells to uncover general principles underlying the mechanisms of gut acidification.

Intestinal renewal across the animal kingdom: comparing stem cell activity in mouse and Drosophila.

The gastrointestinal (GI) tract renews frequently to sustain nutrient digestion and absorption in the face of consistent tissue stress. In many species, proliferative intestinal stem cells (ISCs) are responsible for the repair of the damage arising from chemical and mechanical aspects of food breakdown and exposure to pathogens. As the cellular source of all mature cell types of the intestinal epithelium throughout adulthood, ISCs hold tremendous therapeutic potential for understanding and treating GI disease in humans. This review focuses on recent advances in our understanding of ISC identity, behavior, and regulation during homeostasis and injury-induced repair, as revealed by two major animal models used to study regeneration of the small intestine: Drosophila melanogaster and Mus musculus. We emphasize recent findings from Drosophila that are likely to translate to the mammalian GI system, as well as challenging topics in mouse ISC biology that may be ideally suited for investigation in flies. For context, we begin by reviewing major physiological similarities and distinctions between the Drosophila midgut and mouse small intestine.

Amitosis of Polyploid Cells Regenerates Functional Stem Cells in the Drosophila Intestine.

Organ fitness depends on appropriate maintenance of stem cell populations, and aberrations in functional stem cell numbers are associated with malignancies and aging. Symmetrical division is the best characterized mechanism of stem cell replacement, but other mechanisms could also be deployed, particularly in situations of high stress. Here, we show that after severe depletion, intestinal stem cells (ISCs) in the Drosophila midgut are replaced by spindle-independent ploidy reduction of cells in the enterocyte lineage through a process known as amitosis. Amitosis is also induced by the functional loss of ISCs coupled with tissue demand and in aging flies, underscoring the generality of this mechanism. However, we also found that random homologous chromosome segregation during ploidy reduction can expose deleterious mutations through loss of heterozygosity. Together, our results highlight amitosis as an unappreciated mechanism for restoring stem cell homeostasis, but one with some associated risk in animals carrying mutations.

Maintenance of the adult Drosophila intestine: all roads lead to homeostasis.

Maintenance of tissue homeostasis is critical in tissues with high turnover such as the intestinal epithelium. The intestinal epithelium is under constant cellular assault due to its digestive functions and its function as a barrier to chemical and bacterial insults. The resulting high rate of cellular turnover necessitates highly controlled mechanisms of regeneration to maintain the integrity of the tissue over the lifetime of the organism. Transient increase in stem cell proliferation is a commonly used and elaborate mechanism to ensure fast and efficient repair of the gut. However, tissue repair is not limited to regulating ISC proliferation, as emerging evidence demonstrates that the Drosophila intestine uses multiple strategies to ensure proper tissue homeostasis that may also extend to other tissues.

Stem cell regulation. Bidirectional Notch signaling regulates Drosophila intestinal stem cell multipotency.

Drosophila intestinal stem cells (ISCs) generate enterocytes (ECs) and enteroendocrine (ee) cells. Previous work suggests that different levels of the Notch ligand Delta (Dl) in ISCs unidirectionally activate Notch in daughters to control multipotency. However, the mechanisms driving different outcomes remain unknown. We found that during ee cell formation, the ee cell marker Prospero localizes to the basal side of dividing ISCs. After asymmetric division, the ee daughter cell acts as a source of Dl that induces low Notch activity in the ISC to maintain identity. Alternatively, ISCs expressing Dl induce high Notch activity in daughter cells to promote EC formation. Our data reveal a conserved role for Notch in Drosophila and mammalian ISC maintenance and suggest that bidirectional Notch signaling may regulate multipotency in other systems.

Specification of regional intestinal stem cell identity during Drosophila metamorphosis.

In the adult Drosophila midgut the bone morphogenetic protein (BMP) signaling pathway is required to specify and maintain the acid-secreting region of the midgut known as the copper cell region (CCR). BMP signaling is also involved in the modulation of intestinal stem cell (ISC) proliferation in response to injury. How ISCs are able to respond to the same signaling pathway in a regionally different manner is currently unknown. Here, we show that dual use of the BMP signaling pathway in the midgut is possible because BMP signals are only capable of transforming ISC and enterocyte identity during a defined window of metamorphosis. ISC heterogeneity is established prior to adulthood and then maintained in cooperation with regional signals from surrounding tissue. Our data provide a conceptual framework for how other tissues maintained by regional stem cells might be patterned and establishes the pupal and adult midgut as a novel genetic platform for identifying genes necessary for regional stem cell specification and maintenance.

Injury-induced BMP signaling negatively regulates Drosophila midgut homeostasis.

Although much is known about injury-induced signals that increase rates of Drosophila melanogaster midgut intestinal stem cell (ISC) proliferation, it is largely unknown how ISC activity returns to quiescence after injury. In this paper, we show that the bone morphogenetic protein (BMP) signaling pathway has dual functions during midgut homeostasis. Constitutive BMP signaling pathway activation in the middle midgut mediated regional specification by promoting copper cell differentiation. In the anterior and posterior midgut, injury-induced BMP signaling acted autonomously in ISCs to limit proliferation and stem cell number after injury. Loss of BMP signaling pathway members in the midgut epithelium or loss of the BMP signaling ligand decapentaplegic from visceral muscle resulted in phenotypes similar to those described for juvenile polyposis syndrome, a human intestinal tumor caused by mutations in BMP signaling pathway components. Our data establish a new link between injury and hyperplasia and may provide insight into how BMP signaling mutations drive formation of human intestinal cancers.

The Drosophila midgut: a model for stem cell driven tissue regeneration.

The Drosophila and mammalian digestive systems bear striking similarities in genetic control and cellular composition, and the Drosophila midgut has emerged as an amenable model for dissecting the mechanisms of tissue homeostasis. The Drosophila midgut is maintained by multipotent intestinal stem cells (ISCs) that give rise to all cell types in the intestinal epithelium and are required for long-term tissue homeostasis. ISC proliferation rate increases in response to a myriad of chemical and bacterial insults through the release of JAK-STAT and EGFR ligands from dying enterocytes that activate the JAK-STAT and EGFR pathways in ISCs. The Hippo and JNK pathways converge upon JAK-STAT and EGFR signaling, presumably in response to specific stresses, and JNK and insulin signaling have been shown to be critical in response to age-related stresses. This review details these emerging mechanisms of tissue homeostasis and the proliferative response of ISCs to epithelial damage, environmental stresses, and aging.

Nonautonomous regulation of Drosophila midgut stem cell proliferation by the insulin-signaling pathway.

Drosophila adult midgut intestinal stem cells (ISCs) maintain tissue homeostasis by producing progeny that replace dying enterocytes and enteroendocrine cells. ISCs adjust their rates of proliferation in response to enterocyte turnover through a positive feedback loop initiated by secreted enterocyte-derived ligands. However, less is known about whether ISC proliferation is affected by growth of the progeny as they differentiate. Here we show that nutrient deprivation and reduced insulin signaling results in production of growth-delayed enterocytes and prolonged contact between ISCs and newly formed daughters. Premature disruption of cell contact between ISCs and their progeny leads to increased ISC proliferation and rescues proliferation defects in insulin receptor mutants and nutrient-deprived animals. These results suggest that ISCs can indirectly sense changes in nutrient and insulin levels through contact with their daughters and reveal a mechanism that could link physiological changes in tissue growth to stem cell proliferation.

Cell renewal in adjoining intestinal and tracheal epithelia of Manduca.

Cell renewal continuously replaces dead or dying cells in organs such as human and insect intestinal (midgut) epithelia; in insects, control of self-renewal determines insects' responses to any of the myriad pathogens and parasites of medical and agricultural importance that enter and cross their midgut epithelia. Regenerative cells occur in the midgut epithelia of many, if not all, insects and are probably derived from a distinctive population of stem cells. The control of proliferation and differentiation of these midgut regenerative cells is assumed to be regulated by an environment of adjacent cells that is referred to as a regenerative cell niche. An antibody to fasciclin II marks cell surfaces of tracheal regenerative cells associated with rapidly growing midgut epithelia. Tracheal regenerative cells and their neighboring midgut regenerative cells proliferate and differentiate in concert during the coordinated growth of the midgut and its associated muscles, nerves and tracheal cells.

A transient niche regulates the specification of Drosophila intestinal stem cells.

Stem cell niches are locations where stem cells reside and self-renew. Although studies have shown how niches maintain stem cell fate during tissue homeostasis, less is known about their roles in establishing stem cells. The adult Drosophila midgut is maintained by intestinal stem cells (ISCs); however, how they are established is unknown. Here, we show that an ISC progenitor generates a niche cell via Notch signaling. This niche uses the bone morphogenetic protein 2/4 homolog, decapentaplegic, to allow progenitors to divide in an undifferentiated state and subsequently breaks down and dies, resulting in the specification of ISCs in the adult midgut. Our results demonstrate a paradigm for stem cell-niche biology, where progenitors generate transient niches that determine stem cell fate and may give insights into stem cell specification in other tissues.

Multipotent Drosophila intestinal stem cells specify daughter cell fates by differential notch signaling.

The adult Drosophila midgut contains multipotent intestinal stem cells (ISCs) scattered along its basement membrane that have been shown by lineage analysis to generate both enterocytes and enteroendocrine cells. ISCs containing high levels of cytoplasmic Delta-rich vesicles activate the canonical Notch pathway and down-regulate Delta within their daughters, a process that programs these daughters to become enterocytes. ISCs that express little vesiculate Delta, or are genetically impaired in Notch signaling, specify their daughters to become enteroendocrine cells. Thus, ISCs control daughter cell fate by modulating Notch signaling over time. Our studies suggest that ISCs actively coordinate cell production with local tissue requirements by this mechanism.

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

The adult Drosophila posterior midgut is maintained by pluripotent stem cells.

Vertebrate and invertebrate digestive systems show extensive similarities in their development, cellular makeup and genetic control. The Drosophila midgut is typical: enterocytes make up the majority of the intestinal epithelial monolayer, but are interspersed with hormone-producing enteroendocrine cells. Human (and mouse) intestinal cells are continuously replenished by stem cells, the misregulation of which may underlie some common digestive diseases and cancer. In contrast, stem cells have not been described in the intestines of flies, and Drosophila intestinal cells have been thought to be relatively stable. Here we use lineage labelling to show that adult Drosophila posterior midgut cells are continuously replenished by a distinctive population of intestinal stem cells (ISCs). As in vertebrates, ISCs are multipotent, and Notch signalling is required to produce an appropriate fraction of enteroendocrine cells. Notch is also required for the differentiation of ISC daughter cells, a role that has not been addressed in vertebrates. Unlike previously characterized stem cells, which reside in niches containing a specific partner stromal cell, ISCs adjoin only the basement membrane, differentiated enterocytes and their most recent daughters. The identification of Drosophila intestinal stem cells with striking similarities to their vertebrate counterparts will facilitate the genetic analysis of normal and abnormal intestinal function.

The stem cell niche: theme and variations.

Stem cells in animal tissues are often located and controlled by special tissue microenvironments known as niches. Studies of stem cell niches in model systems such as Drosophila have revealed adhesive interactions, cell cycle modifications and intercellular signals that operate to control stem cell behavior. Candidate niches and regulatory molecules have also been identified in many mammalian tissues, including bone marrow, skin, gut and brain. While niches are an ancient evolutionary device with conserved features across diverse organisms, we suggest that certain niches display important differences in their organization and function.