Pride in STEM worldwide.

Cell asked LGBTQ+ scientists around the world about how their identity shapes their experiences in STEM. Here we share six unique perspectives of researchers highlighting how their area of expertise, research focus, institutions, and geographical location have played a role in this regard. We thank them for sharing their voices and continued efforts toward making science more inclusive.

Differential effects of commensal bacteria on progenitor cell adhesion, division symmetry and tumorigenesis in the Drosophila intestine.

Microbial factors influence homeostatic and oncogenic growth in the intestinal epithelium. However, we know little about immediate effects of commensal bacteria on stem cell division programs. In this study, we examined the effects of commensal Lactobacillus species on homeostatic and tumorigenic stem cell proliferation in the female Drosophila intestine. We identified Lactobacillus brevis as a potent stimulator of stem cell divisions. In a wild-type midgut, L.brevis activates growth regulatory pathways that drive stem cell divisions. In a Notch-deficient background, L.brevis-mediated proliferation causes rapid expansion of mutant progenitors, leading to accumulation of large, multi-layered tumors throughout the midgut. Mechanistically, we showed that L.brevis disrupts expression and subcellular distribution of progenitor cell integrins, supporting symmetric divisions that expand intestinal stem cell populations. Collectively, our data emphasize the impact of commensal microbes on division and maintenance of the intestinal progenitor compartment.

Mechanisms of epithelial growth and development in the zebrafish intestine.

The intestinal epithelium is a complex tissue monolayer composed of regionally and functionally specialized intestinal epithelial cells. Given epithelial exposure to harsh and varied luminal conditions, epithelial cells continuously regenerate to sustain the barrier against environmental factors, including microbial invaders. Multipotent intestinal stem cells are essential to epithelial regenerative capacity, generating a programed mixture of absorptive and secretory cell types. Mechanisms of epithelial growth and differentiation in response to endogenous or external stressors remain under investigation. In this review, we highlight the zebrafish, Danio rerio, as a potent model of intestinal epithelial development and function. We describe epithelial composition and key regulators of epithelial renewal to promote the zebrafish as an investigative tool to study epithelial development and growth. We also highlight areas for discovery, particularly in the context of stress-dependent regulation of epithelial function.

The Immune Deficiency Pathway Regulates Metabolic Homeostasis in Drosophila.

Immune and metabolic pathways collectively influence host responses to microbial invaders, and mutations in one pathway frequently disrupt activity in another. We used the Drosophila melanogaster model to characterize metabolic homeostasis in flies with modified immune deficiency (IMD) pathway activity. The IMD pathway is very similar to the mammalian TNF-α pathway, a key regulator of vertebrate immunity and metabolism. We found that persistent activation of IMD resulted in hyperglycemia, depleted fat reserves, and developmental delays, implicating IMD in metabolic regulation. Consistent with this hypothesis, we found that imd mutants weigh more, are hyperlipidemic, and have impaired glucose tolerance. To test the importance of metabolic regulation for host responses to bacterial infection, we challenged insulin pathway mutants with lethal doses of several Drosophila pathogens. We found that loss-of-function mutations in the insulin pathway impacted host responses to infection in a manner that depends on the route of infection and the identity of the infectious microbe. Combined, our results support a role for coordinated regulation of immune and metabolic pathways in host containment of microbial invaders.

Commensal pathogen competition impacts host viability.

While the structure and regulatory networks that govern type-six secretion system (T6SS) activity of Vibrio cholerae are becoming increasingly clear, we know less about the role of T6SS in disease. Under laboratory conditions, V. cholerae uses T6SS to outcompete many Gram-negative species, including other V. cholerae strains and human commensal bacteria. However, the role of these interactions has not been resolved in an in vivo setting. We used the Drosophila melanogaster model of cholera to define the contribution of T6SS to V. cholerae pathogenesis. Here, we demonstrate that interactions between T6SS and host commensals impact pathogenesis. Inactivation of T6SS, or removal of commensal bacteria, attenuates disease severity. Reintroduction of the commensal, Acetobacter pasteurianus, into a germ-free host is sufficient to restore T6SS-dependent pathogenesis in which T6SS and host immune responses regulate viability. Together, our data demonstrate that T6SS acts on commensal bacteria to promote the pathogenesis of V. cholerae.

A deregulated intestinal cell cycle program disrupts tissue homeostasis without affecting longevity in Drosophila.

Recent studies illuminate a complex relationship between the control of stem cell division and intestinal tissue organization in the model system Drosophila melanogaster. Host and microbial signals drive intestinal proliferation to maintain an effective epithelial barrier. Although it is widely assumed that proliferation induces dysplasia and shortens the life span of the host, the phenotypic consequences of deregulated intestinal proliferation for an otherwise healthy host remain unexplored. To address this question, we genetically isolated and manipulated the cell cycle programs of adult stem cells and enterocytes. Our studies revealed that cell cycle alterations led to extensive cell death and morphological disruptions. Despite the extensive tissue damage, we did not observe an impact on longevity, suggesting a remarkable degree of plasticity in intestinal function.

The Drosophila platelet-derived growth factor and vascular endothelial growth factor-receptor related (Pvr) protein ligands Pvf2 and Pvf3 control hemocyte viability and invasive migration.

Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) family members are essential and evolutionary conserved determinants of blood cell development and dispersal. In addition, VEGFs are integral to vascular growth and permeability with detrimental contributions to ischemic diseases and metastatic cancers. The PDGF/VEGF-receptor related (Pvr) protein is implicated in the migration and trophic maintenance of macrophage-like hemocytes in Drosophila melanogaster embryos. pvr mutants have a depleted hemocyte population and a breakdown in hemocyte distribution. Previous studies suggested redundant functions for the Pvr ligands, Pvf2 and Pvf3 in the regulation of hemocyte migration, proliferation, and size. However, the precise roles that Pvf2 and Pvf3 play in hematopoiesis remain unclear due to the lack of available mutants. To determine Pvf2 and Pvf3 functions in vivo, we generated a genomic deletion that simultaneously disrupts Pvf2 and Pvf3. From our studies, we identified contributions of Pvf2 and Pvf3 to the Pvr trophic maintenance of hemocytes. Furthermore, we uncovered a novel role for Pvfs in invasive migrations. We showed that Pvf2 and Pvf3 are not required for the directed migration of hemocytes, but act locally in epithelial cells to coordinate trans-epithelial migration of hemocytes. Our findings redefine Pvf roles in hemocyte migration and highlight novel Pvf roles in hemocyte invasive migration. These new parallels between the Pvr and PDGF/VEGF pathways extend the utility of the Drosophila embryonic system to dissect physiological and pathological roles of PDGF/VEGF-like growth factors.

Vibrio cholerae arrests intestinal epithelial proliferation through T6SS-dependent activation of the bone morphogenetic protein pathway.

To maintain an effective barrier, intestinal progenitor cells must divide at a rate that matches the loss of dead and dying cells. Otherwise, epithelial breaches expose the host to systemic infection by gut-resident microbes. Unlike most pathogens, Vibrio cholerae blocks tissue repair by arresting progenitor proliferation in the Drosophila model. At present, we do not understand how V. cholerae circumvents such a critical antibacterial defense. We find that V. cholerae blocks epithelial repair by activating the growth inhibitor bone morphogenetic protein (BMP) pathway in progenitors. Specifically, we show that interactions between V. cholerae and gut commensals initiate BMP signaling via host innate immune defenses. Notably, we find that V. cholerae also activates BMP and arrests proliferation in zebrafish intestines, indicating an evolutionarily conserved link between infection and failure in tissue repair. Our study highlights how enteric pathogens engage host immune and growth regulatory pathways to disrupt intestinal epithelial repair.

The emergence of circadian timekeeping in the intestine.

The circadian clock is a molecular timekeeper, present from cyanobacteria to mammals, that coordinates internal physiology with the external environment. The clock has a 24-h period however development proceeds with its own timing, raising the question of how these interact. Using the intestine of Drosophila melanogaster as a model for organ development, we track how and when the circadian clock emerges in specific cell types. We find that the circadian clock begins abruptly in the adult intestine and gradually synchronizes to the environment after intestinal development is complete. This delayed start occurs because individual cells at earlier stages lack the complete circadian clock gene network. As the intestine develops, the circadian clock is first consolidated in intestinal stem cells with changes in Ecdysone and Hnf4 signalling influencing the transcriptional activity of Clk/cyc to drive the expression of tim, Pdp1, and vri. In the mature intestine, stem cell lineage commitment transiently disrupts clock activity in differentiating progeny, mirroring early developmental clock-less transitions. Our data show that clock function and differentiation are incompatible and provide a paradigm for studying circadian clocks in development and stem cell lineages.

Autocrine platelet-derived growth factor-vascular endothelial growth factor receptor-related (Pvr) pathway activity controls intestinal stem cell proliferation in the adult Drosophila midgut.

A dynamic pool of undifferentiated somatic stem cells proliferate and differentiate to replace dead or dying mature cell types and maintain the integrity and function of adult tissues. Intestinal stem cells (ISCs) in the Drosophila posterior midgut are a well established model to study the complex genetic circuitry that governs stem cell homeostasis. Exposure of the intestinal epithelium to environmental toxins results in the expression of cytokines and growth factors that drive the rapid proliferation and differentiation of ISCs. In the absence of stress signals, ISC homeostasis is maintained through intrinsic pathways. In this study, we uncovered the PDGF- and VEGF-receptor related (Pvr) pathway as an essential regulator of ISC homeostasis under unstressed conditions in the posterior midgut. We found that Pvr is coexpressed with its ligand Pvf2 in ISCs and that hyperactivation of the Pvr pathway distorts the ISC developmental program and drives intestinal dysplasia. In contrast, we show that mutant ISCs in the Pvf/Pvr pathway are defective in homeostatic proliferation and differentiation, resulting in a failure to generate mature cell types. Additionally, we determined that extrinsic stress signals generated by enteropathogenic infection are epistatic to the hypoplasia generated in Pvf/Pvr mutants, making the Pvr pathway unique among all previously studied intrinsic pathways. Our findings illuminate an evolutionarily conserved signal transduction pathway with essential roles in metazoan embryonic development and direct involvement in numerous disease states.

The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing.

Many metazoan cells can take up exogenous double-stranded (ds) RNA and use it to initiate an RNA silencing response, however, the mechanism for this uptake is ill-defined. Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses.

Single-cell resolution of the adult zebrafish intestine under conventional conditions and in response to an acute Vibrio cholerae infection.

Vibrio cholerae is an aquatic bacterium that causes severe and potentially deadly diarrheal disease. Despite the impact on global health, our understanding of host mucosal responses to Vibrio remains limited, highlighting a knowledge gap critical for the development of effective prevention and treatment strategies. Using a natural infection model, we combine physiological and single-cell transcriptomic studies to characterize conventionally reared adult zebrafish guts and guts challenged with Vibrio. We demonstrate that Vibrio causes a mild mucosal immune response characterized by T cell activation and enhanced antigen capture; Vibrio suppresses host interferon signaling; and ectopic activation of interferon alters the course of infection. We show that the adult zebrafish gut shares similarities with mammalian counterparts, including the presence of Best4+ cells, tuft cells, and a population of basal cycling cells. These findings provide important insights into host-pathogen interactions and emphasize the utility of zebrafish as a natural model of Vibrio infection.

The protein Dredd is an essential component of the c-Jun N-terminal kinase pathway in the Drosophila immune response.

The Drosophila immune deficiency (IMD) pathway mobilizes c-Jun N-terminal kinase (JNK), caspase, and nuclear factor-κB (NF-κB) modules to counter infection with gram-negative bacteria. Dredd is an essential caspase in the IMD pathway, and it is widely established that NF-κB activation depends on Dredd. More recent cell culture studies suggested a role for Dredd in the activation of dJNK (Drosophila JNK). However, there are no epistatic or mechanistic data on the involvement of Dredd in dJNK activation. More importantly, there is no in vivo evidence to demonstrate a physiological requirement for Dredd in the IMD/dJNK pathway. We performed a comprehensive analysis of the role of Dredd in the IMD/dJNK pathway, and we demonstrated that Dredd is essential for the activation of IMD/dJNK in cell culture. We positioned Dredd activity at an early point of the IMD/dJNK pathway and uncovered a series of interactions between Dredd and additional proximal IMD pathway molecules. Mechanistically, we showed that the caspase activity inhibitor p35 blocked dJNK activation and the induction of dJNK-dependent genes in cell culture and in vivo. Most importantly, we demonstrated that dredd mutant flies are completely inhibited in their ability to activate dJNK or express dJNK-responsive target genes after bacterial infection in vivo. In conclusion, we established Dredd as an essential component of the IMD pathway required for the full activation of IMD/dJNK in cell culture and in vivo. Our data enhance our appreciation of Dredd-dependent IMD signal transduction events.

Microbial recognition regulates intestinal epithelial growth in homeostasis and disease.

The intestine is constantly exposed to a dynamic community of microbes. Intestinal epithelial cells respond to microbes through evolutionarily conserved recognition pathways, such as the immune deficiency (IMD) pathway of Drosophila, the Toll-like receptor (TLR) response of flies and vertebrates, and the vertebrate nucleotide-binding oligomerization domain (NOD) pathway. Microbial recognition pathways are tightly controlled to respond effectively to pathogens, tolerate the microbiome, and limit intestinal disease. In this review, we focus on contributions of different model organisms to our understanding of how epithelial microbe recognition impacts intestinal proliferation and differentiation in homeostasis and disease. In particular, we compare how microbes and subsequent recognition by the intestine influences barrier integrity, intestinal repair and tumorigenesis in Drosophila, zebrafish, mice, and organoids. In addition, we discuss the importance of microbial recognition in homeostatic intestinal growth and discuss how immune pathways directly impact stem cell and crypt dynamics.

A cell atlas of microbe-responsive processes in the zebrafish intestine.

Gut microbial products direct growth, differentiation, and development in animal hosts. However, we lack system-wide understanding of cell-specific responses to the microbiome. We profiled cell transcriptomes from the intestine, and associated tissue, of zebrafish larvae raised in the presence or absence of a microbiome. We uncovered extensive cellular heterogeneity in the conventional zebrafish intestinal epithelium, including previously undescribed cell types with known mammalian homologs. By comparing conventional to germ-free profiles, we mapped microbial impacts on transcriptional activity in each cell population. We revealed intricate degrees of cellular specificity in host responses to the microbiome that included regulatory effects on patterning and on metabolic and immune activity. For example, we showed that the absence of microbes hindered pro-angiogenic signals in the developing vasculature, causing impaired intestinal vascularization. Our work provides a high-resolution atlas of intestinal cellular composition in the developing fish gut and details the effects of the microbiome on each cell type.

Immune regulation of intestinal-stem-cell function in Drosophila.

Intestinal progenitor cells integrate signals from their niche, and the gut lumen, to divide and differentiate at a rate that maintains an epithelial barrier to microbial invasion of the host interior. Despite the importance of evolutionarily conserved innate immune defenses to maintain stable host-microbe relationships, we know little about contributions of stem-cell immunity to gut homeostasis. We used Drosophila to determine the consequences of intestinal-stem-cell immune activity for epithelial homeostasis. We showed that loss of stem-cell immunity greatly impacted growth and renewal in the adult gut. In particular, we found that inhibition of stem-cell immunity impeded progenitor-cell growth and differentiation, leading to a gradual loss of stem-cell numbers with age and an impaired differentiation of mature enteroendocrine cells. Our results highlight the importance of immune signaling in stem cells for epithelial function in the adult gut.

The E3 ubiquitin ligase IDOL induces the degradation of the low density lipoprotein receptor family members VLDLR and ApoER2.

We have previously identified the E3 ubiquitin ligase-inducible degrader of the low density lipoprotein receptor (LDLR) (Idol) as a post-translational modulator of LDLR levels. Idol is a direct target for regulation by liver X receptors (LXRs), and its expression is responsive to cellular sterol status independent of the sterol-response element-binding proteins. Here we demonstrate that Idol also targets two closely related LDLR family members, VLDLR and ApoE receptor 2 (ApoER2), proteins implicated in both neuronal development and lipid metabolism. Idol triggers ubiquitination of the VLDLR and ApoER2 on their cytoplasmic tails, leading to their degradation. We further show that the level of endogenous VLDLR is sensitive to cellular sterol content, Idol expression, and activation of the LXR pathway. Pharmacological activation of the LXR pathway in mice leads to increased Idol expression and to decreased Vldlr levels in vivo. Finally, we establish an unexpected functional link between LXR and Reelin signaling. We demonstrate that LXR activation results in decreased Reelin binding to VLDLR and reduced Dab1 phosphorylation. The identification of VLDLR and ApoER2 as Idol targets suggests potential roles for this LXR-inducible E3 ligase in the central nervous system in addition to lipid metabolism.

A quantitative RNAi screen for JNK modifiers identifies Pvr as a novel regulator of Drosophila immune signaling.

Drosophila melanogaster responds to gram-negative bacterial challenges through the IMD pathway, a signal transduction cassette that is driven by the coordinated activities of JNK, NF-kappaB and caspase modules. While many modifiers of NF-kappaB activity were identified in cell culture and in vivo assays, the regulatory apparatus that determines JNK inputs into the IMD pathway is relatively unexplored. In this manuscript, we present the first quantitative screen of the entire genome of Drosophila for novel regulators of JNK activity in the IMD pathway. We identified a large number of gene products that negatively or positively impact on JNK activation in the IMD pathway. In particular, we identified the Pvr receptor tyrosine kinase as a potent inhibitor of JNK activation. In a series of in vivo and cell culture assays, we demonstrated that activation of the IMD pathway drives JNK-dependent expression of the Pvr ligands, Pvf2 and Pvf3, which in turn act through the Pvr/ERK MAP kinase pathway to attenuate the JNK and NF-kappaB arms of the IMD pathway. Our data illuminate a poorly understood arm of a critical and evolutionarily conserved innate immune response. Furthermore, given the pleiotropic involvement of JNK in eukaryotic cell biology, we believe that many of the novel regulators identified in this screen are of interest beyond immune signaling.

A glucose-supplemented diet enhances gut barrier integrity in Drosophila.

Dietary intervention has received considerable attention as an approach to extend lifespan and improve aging. However, questions remain regarding optimal dietary regimes and underlying mechanisms of lifespan extension. Here, we asked how an increase of glucose in a chemically defined diet extends the lifespan of adult Drosophilamelanogaster We showed that glucose-dependent lifespan extension is not a result of diminished caloric intake, or changes to systemic insulin activity, two commonly studied mechanisms of lifespan extension. Instead, we found that flies raised on glucose-supplemented food increased the expression of cell-adhesion genes, delaying age-dependent loss of intestinal barrier integrity. Furthermore, we showed that chemical disruption of the gut barrier negated the lifespan extension associated with glucose treatment, suggesting that glucose-supplemented food prolongs adult viability by enhancing the intestinal barrier. We believe our data contribute to understanding intestinal homeostasis, and may assist efforts to develop preventative measures that limit effects of aging on health.

Vibrio cholerae-Symbiont Interactions Inhibit Intestinal Repair in Drosophila.

Pathogen-mediated damage to the intestinal epithelium activates compensatory growth and differentiation repair programs in progenitor cells. Accelerated progenitor growth replenishes damaged tissue and maintains barrier integrity. Despite the importance of epithelial renewal to intestinal homeostasis, we know little about the effects of pathogen-commensal interactions on progenitor growth. We find that the enteric pathogen Vibrio cholerae blocks critical growth and differentiation pathways in Drosophila progenitors, despite extensive damage to epithelial tissue. We show that the inhibition of epithelial repair requires interactions between the Vibrio cholerae type six secretion system and a community of common symbiotic bacteria, as elimination of the gut microbiome is sufficient to restore homeostatic growth in infected intestines. This work highlights the importance of pathogen-symbiont interactions for intestinal immune responses and outlines the impact of the type six secretion system on pathogenesis.