The intestinal mucosa undergoes a dynamic process of continual proliferation, differentiation, and apoptosis. Delineating the mechanisms involved in intestinal epithelial cell (IEC) differentiation is crucial to our understanding of not only normal gut adaptation but also aberrant intestinal growth. Bone morphogenetic protein (BMP) signaling is a pivotal regulator of intestinal proliferation and differentiation. However, the molecular underpinnings of the BMP pathway in this context are not entirely known. Here, we show a key role for the BMP4/microRNA (miR)-181/glycolysis signaling pathway in the maintenance of intestinal epithelial cell proliferation and differentiation. Treatment with BMP4 increased the expression of enterocyte markers and decreased proliferation of IECs, and importantly, decreased the expression of miR-181a-5p in mouse and human intestinal organoids. miR-181a-5p is a member of the miR-181 family with the highest expression in IECs. Treatment with locked nucleic acid (LNA) miR-181a-5p inhibitor significantly increased enterocyte differentiation as noted by increased expression of enterocyte markers in human and mouse intestinal organoids. In addition, LNA miR-181a-5p inhibitor repressed intestinal stem cell self-renewal as noted by the decreased organoid forming efficiency and expression of Ki67, cyclin D1, OLFM4 in human and mouse intestinal organoids. Moreover, in vivo administration of LNA miR-181a-5p inhibitor enhanced increased intestinal enterocyte differentiation and repressed intestinal cell proliferation. In contrast, overexpression of miR-181a-5p mimic decreased basal and BMP4-induced expression of enterocyte markers. Moreover, BMP4 treatment or inhibition of miR-181a-5p repressed hexokinase (HK) 1 expression and inhibited glycolysis. Consistently, knockdown of HK1 or inhibition of glycolysis using 2-deoxyglucose (2-DG) promoted enterocyte maturation and inhibited proliferation of IECs. Together, we provide evidence showing that miR-181a-5p inhibits intestinal enterocyte differentiation and promotes IEC proliferation through HK1-dependent glycolysis. Importantly, our findings identify miR-181a-5p as downstream in mediating BMP4 induction of enterocyte differentiation and inhibition of proliferation in IECs.

Malaria control faces challenges from widespread insecticide resistance in major Anopheles species. This study, employing a cross-species approach, integrates RNA-Sequencing, whole-genome sequencing, and microarray data to elucidate drivers of insecticide resistance in Anopheles gambiae complex and An. funestus. Here we show an inverse relationship between genetic diversity and gene expression, with highly expressed genes experiencing stronger purifying selection. Gene expression clusters physically in the genome, revealing potential coordinated regulation, and we find that highly over-expressed genes are associated with selective sweep loci. We identify known and novel candidate insecticide resistance genes, enriched for metabolic, cuticular, and behavioural functioning. We also present AnoExpress, a Python package, and an online interface for user-friendly exploration of resistance candidate expression. Despite millions of years of speciation, convergent gene expression responses to insecticidal selection pressures are observed across Anopheles species, providing crucial insights for malaria vector control.

Elucidating the expression of microRNAs in developing single cells is critical for functional discovery. Here, we construct scCAMERA (single-cell cartography of microRNA expression based on reporter assay), utilizing promoter-driven fluorescent reporters in conjunction with imaging and lineage tracing. The cartography delineates the transcriptional activity of 54 conserved microRNAs in lineage-resolved single cells throughout C. elegans embryogenesis. The combinatorial expression of microRNAs partitions cells into fine clusters reflecting their function and anatomy. Notably, the expression of individual microRNAs exhibits high cell specificity and divergence among family members. Guided by cellular expression patterns, we identify developmental functions of specific microRNAs, including miR-1 in pharynx development and physiology, miR-232 in excretory canal morphogenesis by repressing NHR-25/NR5A, and a functional synergy between miR-232 and miR-234 in canal development, demonstrating the broad utility of scCAMERA. Furthermore, integrative analysis reveals that tissue-specific fate determinants activate microRNAs to repress protein production from leaky transcripts associated with alternative, especially neuronal, fates, thereby enhancing the fidelity of developmental fate differentiation. Collectively, our study offers rich opportunities for multidimensional expression-informed analysis of microRNA biology in metazoans.

The signaling environment, or niche, often governs the initial difference in behavior of an adult stem cell and a derivative that initiates a path towards differentiation. The transition between an instructive stem cell niche and differentiation niche must generally have single-cell resolution, suggesting that multiple mechanisms might be necessary to sharpen the transition. Here, we examined the Drosophila ovary and found that Cap cells, which are key constituents of the germline stem cell (GSC) niche, express a conserved microRNA (miR-124). Surprisingly, loss of miR-124 activity in Cap cells leads to a defect in differentiation of GSC derivatives. We present evidence that the direct functional target of miR-124 in Cap cells is the epidermal growth factor receptor (EGFR) and that failure to limit EGFR expression leads to the ectopic expression of a key anti-differentiation BMP signal in neighboring somatic escort cells (ECs), which constitute a differentiation niche. We further found that Notch signaling connects EFGR activity in Cap cells to BMP expression in ECs. We deduce that the stem cell niche communicates with the differentiation niche through a mechanism that begins with the selective expression of a specific microRNA and culminates in the suppression of the major anti-differentiation signal in neighboring cells, with the functionally important overall role of sharpening the spatial distinction between self-renewal and differentiation environments.

Tissues such as the intestine harbor stem cells that have remarkable functional plasticity in response to a dynamic environment. To adapt to the environment, stem cells constantly receive information from their surrounding microenvironment (also called the 'niche') that instructs them how to adapt to changes. The Drosophila midgut shows morphological and functional similarities to the mammalian small intestine and has been a useful model system to study signaling events in stem cells and tissue homeostasis. In this review, we summarize the current understanding of the Drosophila midgut regarding how stem cells communicate with microenvironmental niches including enteroblasts, enterocytes, enteroendocrine cells and visceral muscles to coordinate tissue regeneration and homeostasis. In addition, distant cells such as hemocytes or tracheal cells have been shown to interact with stem cells and influence the development of intestinal diseases. We discuss the contribution of stem cell niches in driving or counteracting disease progression, and review conceptual advances derived from the Drosophila intestine as a model for stem cell biology.

The growing interest in microRNAs (miRNAs) over recent years has led to their characterization in numerous organisms. However, there is currently a lack of data available on miRNAs from triatomine bugs (Reduviidae: Triatominae), which are the vectors of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. A comprehensive understanding of the molecular biology of vectors provides new insights into insect-host interactions and insect control approaches, which are key methods to prevent disease incidence in endemic areas. In this work, we describe the miRNome profiles from gut, hemolymph, and salivary gland tissues of the Rhodnius prolixus triatomine. Small RNA sequencing data revealed abundant expression of miRNAs, along with tRNA- and rRNA-derived fragments. Fifty-two mature miRNAs, previously reported in Ecdysozoa, were identified, including 39 ubiquitously expressed in the three tissues. Additionally, 112, 73, and 78 novel miRNAs were predicted in the gut, hemolymph, and salivary glands, respectively. In silico prediction showed that the top eight most highly expressed miRNAs from salivary glands potentially target human blood-expressed genes, suggesting that R. prolixus may modulate the host's gene expression at the bite site. This study provides the first characterization of miRNAs in a Triatominae species, shedding light on the role of these crucial regulatory molecules.

Previous studies have shown that high levels of reactive oxygen species (ROS) and low tricarboxylic acid (TCA) activity in the brain promote pupal diapause, which is characterized by metabolic depression and lifespan extension. However, it is unclear whether ROS are associated with TCA activity. In this study, we demonstrate that ROS downregulate TCA activity and acetyl-CoA and pyruvate levels in the brains of diapause-destined pupae in the moth Helicoverpa armigera, as well as the protein levels of acetyl-CoA synthetase (ACS) and pyruvate kinase (PK), two proteins involved in the biosynthesis of acetyl-CoA and pyruvate, respectively. Interestingly, miR-34, which is highly expressed in the brains of diapause-destined pupae, can respond to ROS signaling. Furthermore, we show that miR-34 can reduce the expression of ACS and PK by directly targeting their mRNAs. Additionally, hypoxia-inducible factor (HIF), a transcription factor, can be activated by ROS and then promotes miR-34 transcription by binding a cis-element in its promoter. Moreover, we observed delayed pupal development after treatment with a ROS activator paraquat and a HIF activator dimethyloxallyl glycine. Taken together, these results suggest that a novel pathway ROS/HIF/miR-34/ACS-PK was found to negatively regulate TCA activity to promote insect diapause for lifespan extension.

The regulation of stem cell survival, self-renewal, and differentiation is critical for the maintenance of tissue homeostasis. Although the involvement of signaling pathways and transcriptional control mechanisms in stem cell regulation have been extensively investigated, the role of post-transcriptional control is still poorly understood. Here we show that the nuclear activity of the RNA-binding protein Second Mitotic Wave Missing (Swm) is critical for Drosophila melanogaster intestinal stem cells (ISCs) and their daughter cells, enteroblasts (EBs), to maintain their progenitor cell properties and functions. Loss of swm causes ISCs and EBs to stop dividing and instead detach from the basement membrane, resulting in severe progenitor cell loss. swm loss is further characterized by nuclear accumulation of poly(A)+ RNA in progenitor cells. Swm associates with transcripts involved in epithelial cell maintenance and adhesion, and the loss of swm, while not generally affecting the levels of these Swm-bound mRNAs, leads to elevated expression of proteins encoded by some of them, including the fly ortholog of Filamin. Taken together, this study indicates a nuclear role for Swm in adult stem cell maintenance, raising the possibility that nuclear post-transcriptional regulation of mRNAs encoding cell adhesion proteins ensures proper attachment of progenitor cells.

Since the widespread discovery of microRNAs (miRNAs) 20 years ago, the Drosophila melanogaster model system has made important contributions to understanding the biology of this class of noncoding RNAs. These contributions are based on the amenability of this model system not only for biochemical analysis but molecular, genetic, and cell biological analyses as well. Nevertheless, while the Drosophila genome is now known to encode 258 miRNA precursors, the function of only a small minority of these have been well characterized. In this review, we summarize the current resources and methods that are available to study miRNA function in Drosophila with a particular focus on the large-scale resources that enable systematic analysis. Application of these methods will accelerate the discovery of ways that miRNAs are embedded into genetic networks that control basic features of metazoan cells.

Embryonic stem cells (ESCs) have the extraordinary properties to indefinitely proliferate and self-renew in culture to produce different cell progeny through differentiation. This latter process recapitulates embryonic development and requires rounds of the epithelial-mesenchymal transition (EMT). EMT is characterized by the loss of the epithelial features and the acquisition of the typical phenotype of the mesenchymal cells. In pathological conditions, EMT can confer stemness or stem-like phenotypes, playing a role in the tumorigenic process. Cancer stem cells (CSCs) represent a subpopulation, found in the tumor tissues, with stem-like properties such as uncontrolled proliferation, self-renewal, and ability to differentiate into different cell types. ESCs and CSCs share numerous features (pluripotency, self-renewal, expression of stemness genes, and acquisition of epithelial-mesenchymal features), and most of them are under the control of microRNAs (miRNAs). These small molecules have relevant roles during both embryogenesis and cancer development. The aim of this review was to recapitulate molecular mechanisms shared by ESCs and CSCs, with a special focus on the recently identified classes of microRNAs (noncanonical miRNAs, mirtrons, isomiRs, and competitive endogenous miRNAs) and their complex functions during embryogenesis and cancer development.

Adult organisms must sense and adapt to environmental fluctuations. In high-turnover tissues such as the intestine, these adaptive responses require rapid changes in gene expression that, in turn, likely involve posttranscriptional gene control. However, intestinal-tissue-specific microRNA (miRNA)-mediated regulatory pathways remain unexplored. Here, we report the role of an intestinal-specific miRNA, miR-958, that non-cell autonomously regulates stem cell numbers during tissue homeostasis and regeneration in the Drosophila adult midgut. We identify its downstream target cabut, the Drosophila ortholog of mammalian KLF10/11 transcription factors, which mediates this miR-958 function by promoting paracrine enterocyte-to-stem-cell bone morphogenetic protein (BMP) signaling. We also show that mature miR-958 levels transiently decrease in response to stress and that this decrease is required for proper stem cell expansion during tissue regeneration. In summary, we have identified a posttranscriptional mechanism that modulates BMP signaling activity within Drosophila adult intestinal tissue during both normal homeostasis and tissue regeneration to regulate intestinal stem cell numbers.