mTORC1 is required for differentiation of germline stem cells in the Drosophila melanogaster testis.

Metabolism participates in the control of stem cell function and subsequent maintenance of tissue homeostasis. How this is achieved in the context of adult stem cell niches in coordination with other local and intrinsic signaling cues is not completely understood. The Target of Rapamycin (TOR) pathway is a master regulator of metabolism and plays essential roles in stem cell maintenance and differentiation. In the Drosophila male germline, mTORC1 is active in germline stem cells (GSCs) and early germ cells. Targeted RNAi-mediated downregulation of mTor in early germ cells causes a block and/or a delay in differentiation, resulting in an accumulation of germ cells with GSC-like features. These early germ cells also contain unusually large and dysfunctional autolysosomes. In addition, downregulation of mTor in adult male GSCs and early germ cells causes non-autonomous activation of mTORC1 in neighboring cyst cells, which correlates with a disruption in the coordination of germline and somatic differentiation. Our study identifies a previously uncharacterized role of the TOR pathway in regulating male germline differentiation.

The septate junction component bark beetle is required for Drosophila intestinal barrier function and homeostasis.

Age-related loss of intestinal barrier function has been documented across species, but the causes remain unknown. The intestinal barrier is maintained by tight junctions (TJs) in mammals and septate junctions (SJs) in insects. Specialized TJs/SJs, called tricellular junctions (TCJs), are located at the nexus of three adjacent cells, and we have shown that aging results in changes to TCJs in intestines of adult Drosophila melanogaster. We now demonstrate that localization of the TCJ protein bark beetle (Bark) decreases in aged flies. Depletion of bark from enterocytes in young flies led to hallmarks of intestinal aging and shortened lifespan, whereas depletion of bark in progenitor cells reduced Notch activity, biasing differentiation toward the secretory lineage. Our data implicate Bark in EC maturation and maintenance of intestinal barrier integrity. Understanding the assembly and maintenance of TCJs to ensure barrier integrity may lead to strategies to improve tissue integrity when function is compromised.

Culture-Associated DNA Methylation Changes Impact on Cellular Function of Human Intestinal Organoids.

BACKGROUND & AIMS: Human intestinal epithelial organoids (IEOs) are a powerful tool to model major aspects of intestinal development, health, and diseases because patient-derived cultures retain many features found in vivo. A necessary aspect of the organoid model is the requirement to expand cultures in vitro through several rounds of passaging. This is of concern because the passaging of cells has been shown to affect cell morphology, ploidy, and function. METHODS: Here, we analyzed 173 human IEO lines derived from the small and large bowel and examined the effect of culture duration on DNA methylation (DNAm). Furthermore, we tested the potential impact of DNAm changes on gene expression and cellular function. RESULTS: Our analyses show a reproducible effect of culture duration on DNAm in a large discovery cohort as well as 2 publicly available validation cohorts generated in different laboratories. Although methylation changes were seen in only approximately 8% of tested cytosine-phosphate-guanine dinucleotides (CpGs) and global cellular function remained stable, a subset of methylation changes correlated with altered gene expression at baseline as well as in response to inflammatory cytokine exposure and withdrawal of Wnt agonists. Importantly, epigenetic changes were found to be enriched in genomic regions associated with colonic cancer and distant to the site of replication, indicating similarities to malignant transformation. CONCLUSIONS: Our study shows distinct culture-associated epigenetic changes in mucosa-derived human IEOs, some of which appear to impact gene transcriptomic and cellular function. These findings highlight the need for future studies in this area and the importance of considering passage number as a potentially confounding factor.

Escargot controls somatic stem cell maintenance through the attenuation of the insulin receptor pathway in Drosophila.

Adult stem cells coordinate intrinsic and extrinsic, local and systemic, cues to maintain the proper balance between self-renewal and differentiation. However, the precise mechanisms stem cells use to integrate these signals remain elusive. Here, we show that Escargot (Esg), a member of the Snail family of transcription factors, regulates the maintenance of somatic cyst stem cells (CySCs) in the Drosophila testis by attenuating the activity of the pro-differentiation insulin receptor (InR) pathway. Esg positively regulates the expression of an antagonist of insulin signaling, ImpL2, while also attenuating the expression of InR. Furthermore, Esg-mediated repression of the InR pathway is required to suppress CySC loss in response to starvation. Given the conservation of Snail-family transcription factors, characterizing the mechanisms by which Esg regulates cell-fate decisions during homeostasis and a decline in nutrient availability is likely to provide insight into the metabolic regulation of stem cell behavior in other tissues and organisms.

Regeneration, Rejuvenation, and Replacement: Turning Back the Clock on Tissue Aging.

While some animals, such as planaria and hydra, appear to be capable of seemingly endless cycles of regeneration, most animals experience a gradual decline in fitness and ultimately die. The progressive loss of cell and tissue function, leading to senescence and death, is generally referred to as aging. Adult ("tissue") stem cells maintain tissue homeostasis and facilitate repair; however, age-related changes in stem cell function over time are major contributors to loss of organ function or disease in older individuals. Therefore, considerable effort is being invested in restoring stem cell function to counter degenerative diseases and age-related tissue dysfunction. Here, we will review strategies that could be used to restore stem cell function, including the use of environmental interventions, such as diet and exercise, heterochronic approaches, and cellular reprogramming. Maintaining optimal stem cell function and tissue homeostasis into late life will likely extend the amount of time older adults are able to be independent and lead healthy lives.

Neuroglian regulates Drosophila intestinal stem cell proliferation through enhanced signaling via the epidermal growth factor receptor.

The Drosophila intestine is an excellent system for elucidating mechanisms regulating stem cell behavior. Here we show that the septate junction (SJ) protein Neuroglian (Nrg) is expressed in intestinal stem cells (ISCs) and enteroblasts (EBs) within the fly intestine. SJs are not present between ISCs and EBs, suggesting Nrg plays a different role in this tissue. We reveal that Nrg is required for ISC proliferation in young flies, and depletion of Nrg from ISCs and EBs suppresses increased ISC proliferation in aged flies. Conversely, overexpression of Nrg in ISC and EBs promotes ISC proliferation, leading to an increase in cells expressing ISC/EB markers; in addition, we observe an increase in epidermal growth factor receptor (Egfr) activation. Genetic epistasis experiments reveal that Nrg acts upstream of Egfr to regulate ISC proliferation. As Nrg function is highly conserved in mammalian systems, our work characterizing the role of Nrg in the intestine has implications for the treatment of intestinal disorders that arise due to altered ISC behavior.

Redox signaling as a modulator of germline stem cell behavior: Implications for regenerative medicine.

Germline stem cells (GSCs) are crucial for the generation of gametes and propagation of the species. Both intrinsic signaling pathways and environmental cues are employed in order to tightly control GSC behavior, including mitotic divisions, the choice between self-renewal or onset of differentiation, and survival. Recently, oxidation-reduction (redox) signaling has emerged as an important regulator of GSC and gamete behavior across species. In this review, we will highlight the primary mechanisms through which redox signaling acts to influence GSC behavior in different model organisms (Caenorhabditis elegans, Drosophila melanogaster and Mus musculus). In addition, we will summarize the latest research on the use of antioxidants to support mammalian spermatogenesis and discuss potential strategies for regenerative medicine in humans to enhance reproductive fitness.

The impact of ageing on lipid-mediated regulation of adult stem cell behavior and tissue homeostasis.

Adult stem cells sustain tissue homeostasis throughout life and provide an important reservoir of cells capable of tissue repair in response to stress and tissue damage. Age-related changes to stem cells and/or the specialized niches that house them have been shown to negatively impact stem cell maintenance and activity. In addition, metabolic inputs have surfaced as another crucial layer in the control of stem cell behavior (Chandel et al., 2016; Folmes and Terzic, 2016; Ito and Suda, 2014; Mana et al., 2017; Shyh-Chang and Ng, 2017). Here, we will present a brief review of how lipid metabolism influences adult stem cell behavior under homeostatic conditions and speculate on how changes in lipid metabolism may impact stem cell ageing. This review considers the future of lipid metabolism research in stem cells, with the long-term goal of identifying mechanisms that could be targeted to counter or slow the age-related decline in stem cell function.

Lipid Mediated Regulation of Adult Stem Cell Behavior.

Adult stem cells constitute an important reservoir of self-renewing progenitor cells and are crucial for maintaining tissue and organ homeostasis. The capacity of stem cells to self-renew or differentiate can be attributed to distinct metabolic states, and it is now becoming apparent that metabolism plays instructive roles in stem cell fate decisions. Lipids are an extremely vast class of biomolecules, with essential roles in energy homeostasis, membrane structure and signaling. Imbalances in lipid homeostasis can result in lipotoxicity, cell death and diseases, such as cardiovascular disease, insulin resistance and diabetes, autoimmune disorders and cancer. Therefore, understanding how lipid metabolism affects stem cell behavior offers promising perspectives for the development of novel approaches to control stem cell behavior either in vitro or in patients, by modulating lipid metabolic pathways pharmacologically or through diet. In this review, we will first address how recent progress in lipidomics has created new opportunities to uncover stem-cell specific lipidomes. In addition, genetic and/or pharmacological modulation of lipid metabolism have shown the involvement of specific pathways, such as fatty acid oxidation (FAO), in regulating adult stem cell behavior. We will describe and compare findings obtained in multiple stem cell models in order to provide an assessment on whether unique lipid metabolic pathways may commonly regulate stem cell behavior. We will then review characterized and potential molecular mechanisms through which lipids can affect stem cell-specific properties, including self-renewal, differentiation potential or interaction with the niche. Finally, we aim to summarize the current knowledge of how alterations in lipid homeostasis that occur as a consequence of changes in diet, aging or disease can impact stem cells and, consequently, tissue homeostasis and repair.

EGFR signaling promotes basal autophagy for lipid homeostasis and somatic stem cell maintenance in the Drosophila testis.

In contrast to stress-induced macroautophagy/autophagy that happens during nutrient deprivation and other environmental challenges, basal autophagy is thought to be an important mechanism that cells utilize for homeostatic purposes. For instance, basal autophagy is used to recycle damaged and malfunctioning organelles and proteins to provide the building blocks for the generation of new ones throughout life. In addition, specialized autophagic processes, such as lipophagy, the autophagy-induced breakdown of lipid droplets (LDs), and glycophagy (breakdown of glycogen), are employed to maintain proper energy levels in the cell. The importance of autophagy in the regulation of stem cell behavior has been the focus of recent studies. However, the upstream signals that control autophagic activity in stem cells and the precise role of autophagy in stem cells are only starting to be elucidated. In a recent publication, we described how the Egfr (epidermal growth factor receptor) pathway stimulates basal autophagy to support the maintenance of somatic cyst stem cells (CySCs) and to control lipid levels in the Drosophila testis.

EGFR Signaling Stimulates Autophagy to Regulate Stem Cell Maintenance and Lipid Homeostasis in the Drosophila Testis.

Although typically upregulated upon cellular stress, autophagy can also be utilized under homeostatic conditions as a quality control mechanism or in response to developmental cues. Here, we report that autophagy is required for the maintenance of somatic cyst stem cells (CySCs) in the Drosophila testis. Disruption of autophagy in CySCs and early cyst cells (CCs) by the depletion of autophagy-related (Atg) genes reduced early CC numbers and affected CC function, resembling decreased epidermal growth factor receptor (EGFR) signaling. Indeed, our data indicate that EGFR acts to stimulate autophagy to preserve early CC function, whereas target of rapamycin (TOR) negatively regulates autophagy in the differentiating CCs. Finally, we show that the EGFR-mediated stimulation of autophagy regulates lipid levels in CySCs and CCs. These results demonstrate a key role for autophagy in regulating somatic stem cell behavior and tissue homeostasis by integrating cues from both the EGFR and TOR signaling pathways to control lipid metabolism.

DNA Methylation Analysis Validates Organoids as a Viable Model for Studying Human Intestinal Aging.

BACKGROUND & AIMS: The epithelia of the intestine and colon turn over rapidly and are maintained by adult stem cells at the base of crypts. Although the small intestine and colon have distinct, well-characterized physiological functions, it remains unclear if there are fundamental regional differences in stem cell behavior or region-dependent degenerative changes during aging. Mesenchyme-free organoids provide useful tools for investigating intestinal stem cell biology in vitro and have started to be used for investigating age-related changes in stem cell function. However, it is unknown whether organoids maintain hallmarks of age in the absence of an aging niche. We tested whether stem cell-enriched organoids preserved the DNA methylation-based aging profiles associated with the tissues and crypts from which they were derived. METHODS: To address this, we used standard human methylation arrays and the human epigenetic clock as a biomarker of age to analyze in vitro-derived, 3-dimensional, stem cell-enriched intestinal organoids. RESULTS: We found that human stem cell-enriched organoids maintained segmental differences in methylation patterns and that age, as measured by the epigenetic clock, also was maintained in vitro. Surprisingly, we found that stem cell-enriched organoids derived from the small intestine showed striking epigenetic age reduction relative to organoids derived from colon. CONCLUSIONS: Our data validate the use of organoids as a model for studying human intestinal aging and introduce methods that can be used when modeling aging or age-onset diseases in vitro.

Mitochondrial fission regulates germ cell differentiation by suppressing ROS-mediated activation of Epidermal Growth Factor Signaling in the Drosophila larval testis.

Mitochondria are essential organelles that have recently emerged as hubs for several metabolic and signaling pathways in the cell. Mitochondrial morphology is regulated by constant fusion and fission events to maintain a functional mitochondrial network and to remodel the mitochondrial network in response to external stimuli. Although the role of mitochondria in later stages of spermatogenesis has been investigated in depth, the role of mitochondrial dynamics in regulating early germ cell behavior is relatively less-well understood. We previously demonstrated that mitochondrial fusion is required for germline stem cell (GSC) maintenance in the Drosophila testis. Here, we show that mitochondrial fission is also important for regulating the maintenance of early germ cells in larval testes. Inhibition of Drp1 in early germ cells resulted in the loss of GSCs and spermatogonia due to the accumulation of reactive oxygen species (ROS) and activation of the EGFR pathway in adjacent somatic cyst cells. EGFR activation contributed to premature germ cell differentiation. Our data provide insights into how mitochondrial dynamics can impact germ cell maintenance and differentiation via distinct mechanisms throughout development.

Mitochondrial fusion regulates lipid homeostasis and stem cell maintenance in the Drosophila testis.

The capacity of stem cells to self-renew or differentiate has been attributed to distinct metabolic states. A genetic screen targeting regulators of mitochondrial dynamics revealed that mitochondrial fusion is required for the maintenance of male germline stem cells (GSCs) in Drosophila melanogaster. Depletion of Mitofusin (dMfn) or Opa1 led to dysfunctional mitochondria, activation of Target of rapamycin (TOR) and a marked accumulation of lipid droplets. Enhancement of lipid utilization by the mitochondria attenuated TOR activation and rescued the loss of GSCs that was caused by inhibition of mitochondrial fusion. Moreover, constitutive activation of the TOR-pathway target and lipogenesis factor Sterol regulatory element binding protein (SREBP) also resulted in GSC loss, whereas inhibition of SREBP rescued GSC loss triggered by depletion of dMfn. Our findings highlight a critical role for mitochondrial fusion and lipid homeostasis in GSC maintenance, providing insight into the potential impact of mitochondrial and metabolic diseases on the function of stem and/or germ cells.

Heterochromatin Protein 1 (HP1) inhibits stem cell proliferation induced by ectopic activation of the Jak/STAT pathway in the Drosophila testis.

Stem cells can divide asymmetrically with respect to cell fate, producing a copy of themselves (self-renewal), while giving rise to progeny that will differentiate along a specific lineage. Mechanisms that bias the balance towards self-renewal or extend the proliferative capacity of the differentiating progeny can result in tissue overgrowth and, eventually, the formation of tumors. Recent work has explored the role of heterochromatin and heterochromatin-associated proteins in the regulation of stem cell behavior under homeostatic conditions, but less is known about their possible roles in potentiating or suppressing stem cell overproliferation. Here we used ectopic activation of the Jak/STAT pathway in germline and somatic stem cells of the D. melanogaster testis as an in vivo model to probe the function of Heterochromatin Protein 1 (HP1) in stem cell overproliferation. Forced expression of HP1 in either early germ or somatic cells suppressed the overgrowth of testes in response to ectopic Jak/STAT activation. Interestingly, HP1 expression led to distinct phenotypes, depending on whether it was overexpressed in somatic or germ cells, possibly reflecting different cell-autonomous and non-autonomous effects in each cell type. Our results provide a new framework for further in vivo studies aimed at understanding the interactions between heterochromatin and uncontrolled stem cell proliferation, as well as the complex cross-regulatory interactions between the somatic and germline lineages in the Drosophila testis.

Intestinal Snakeskin Limits Microbial Dysbiosis during Aging and Promotes Longevity.

Intestinal barrier dysfunction is an evolutionarily conserved hallmark of aging, which has been linked to microbial dysbiosis, altered expression of occluding junction proteins, and impending mortality. However, the interplay between intestinal junction proteins, age-onset dysbiosis, and lifespan determination remains unclear. Here, we show that altered expression of Snakeskin (Ssk), a septate junction-specific protein, can modulate intestinal homeostasis, microbial dynamics, immune activity, and lifespan in Drosophila. Loss of Ssk leads to rapid and reversible intestinal barrier dysfunction, altered gut morphology, dysbiosis, and dramatically reduced lifespan. Remarkably, restoration of Ssk expression in flies showing intestinal barrier dysfunction rescues each of these phenotypes previously linked to aging. Intestinal up-regulation of Ssk protects against microbial translocation following oral infection with pathogenic bacteria. Furthermore, intestinal up-regulation of Ssk improves intestinal barrier function during aging, limits dysbiosis, and extends lifespan. Our findings indicate that intestinal occluding junctions may represent prolongevity targets in mammals.

Keeping it tight: The relationship between bacterial dysbiosis, septate junctions, and the intestinal barrier in Drosophila.

Maladaptive changes in the intestinal flora, typically referred to as bacterial dysbiosis, have been linked to intestinal aging phenotypes, including an increase in intestinal stem cell (ISC) proliferation, activation of inflammatory pathways, and increased intestinal permeability1,2. However, the causal relationships between these phenotypes are only beginning to be unravelled. We recently characterized the age-related changes that occur to septate junctions (SJ) between adjacent, absorptive enterocytes (EC) in the fly intestine. Changes could be observed in the overall level of SJ proteins, as well as the localization of a subset of SJ proteins. Such age-related changes were particularly noticeable at tricellular junctions (TCJ)3. Acute loss of the Drosophila TCJ protein Gliotactin (Gli) in ECs led to rapid activation of stress signalling in stem cells and an increase in ISC proliferation, even under axenic conditions; a gradual disruption of the intestinal barrier was also observed. The uncoupling of changes in bacteria from alterations in ISC behaviour and loss of barrier integrity has allowed us to begin to explore the interrelationship of these intestinal aging phenotypes in more detail and has shed light on the importance of the proteins that contribute to maintenance of the intestinal barrier.

Pink1 and Parkin regulate Drosophila intestinal stem cell proliferation during stress and aging.

Intestinal stem cells (ISCs) maintain the midgut epithelium in Drosophila melanogaster Proper cellular turnover and tissue function rely on tightly regulated rates of ISC division and appropriate differentiation of daughter cells. However, aging and epithelial injury cause elevated ISC proliferation and decreased capacity for terminal differentiation of daughter enteroblasts (EBs). The mechanisms causing functional decline of stem cells with age remain elusive; however, recent findings suggest that stem cell metabolism plays an important role in the regulation of stem cell activity. Here, we investigate how alterations in mitochondrial homeostasis modulate stem cell behavior in vivo via RNA interference-mediated knockdown of factors involved in mitochondrial dynamics. ISC/EB-specific knockdown of the mitophagy-related genes Pink1 or Parkin suppresses the age-related loss of tissue homeostasis, despite dramatic changes in mitochondrial ultrastructure and mitochondrial damage in ISCs/EBs. Maintenance of tissue homeostasis upon reduction of Pink1 or Parkin appears to result from reduction of age- and stress-induced ISC proliferation, in part, through induction of ISC senescence. Our results indicate an uncoupling of cellular, tissue, and organismal aging through inhibition of ISC proliferation and provide insight into strategies used by stem cells to maintain tissue homeostasis despite severe damage to organelles.

Intestinal stem cell ablation reveals differential requirements for survival in response to chemical challenge.

The Drosophila intestine is maintained by multipotent intestinal stem cells (ISCs). Although increased intestinal stem cell (ISC) proliferation has been correlated with a decrease in longevity, there is some discrepancy regarding whether a decrease or block in proliferation also has negative consequences. Here we identify headcase (hdc) as a novel marker of ISCs and enteroblasts (EBs) and demonstrate that Hdc function is required to prevent ISC/EB loss through apoptosis. Hdc depletion was used as a strategy to ablate ISCs and EBs in order to test the ability of flies to survive without ISC function. While flies lacking ISCs showed no major decrease in survival under unchallenged conditions, flies depleted of ISCs and EBs exhibited decreased survival rates in response to damage to mature enterocytes (EC) that line the intestinal lumen. Our findings indicate that constant renewal of the intestinal epithelium is not absolutely necessary under normal laboratory conditions, but it is important in the context of widespread chemical-induced damage when significant repair is necessary.