Papers by Devanjali Dutta
Nature Protocols, 2021
Adult-stem-cell-derived organoids model human epithelial tissues ex vivo, which enables the study... more Adult-stem-cell-derived organoids model human epithelial tissues ex vivo, which enables the study of host-microbe interactions with great experimental control. This protocol comprises methods to coculture organoids with microbes, particularly focusing on human small intestinal and colon organoids exposed to individual bacterial species. Microinjection into the lumen and periphery of 3D organoids is discussed, as well as exposure of organoids to microbes in a 2D layer. We provide detailed protocols for characterizing the coculture with regard to bacterial and organoid cell viability and growth kinetics. Spatial relationships can be studied by fluorescence live microscopy, as well as scanning electron microscopy. Finally, we discuss considerations for assessing the impact of bacteria on gene expression and mutations through RNA and DNA sequencing. This protocol requires equipment for standard mammalian tissue culture, or bacterial or viral culture, as well as a microinjection device.
Cell reports, Jan 24, 2014
One promising approach for in vivo studies of cell proliferation is the FUCCI system (fluorescent... more One promising approach for in vivo studies of cell proliferation is the FUCCI system (fluorescent ubiquitination-based cell cycle indicator). Here, we report the development of a Drosophila-specific FUCCI system (Fly-FUCCI) that allows one to distinguish G1, S, and G2 phases of interphase. Fly-FUCCI relies on fluorochrome-tagged degrons from the Cyclin B and E2F1 proteins, which are degraded by the ubiquitin E3-ligases APC/C and CRL4(Cdt2), during mitosis or the onset of S phase, respectively. These probes can track cell-cycle patterns in cultured Drosophila cells, eye and wing imaginal discs, salivary glands, the adult midgut, and probably other tissues. To support a broad range of experimental applications, we have generated a toolkit of transgenic Drosophila lines that express the Fly-FUCCI probes under control of the UASt, UASp, QUAS, and ubiquitin promoters. The Fly-FUCCI system should be a valuable tool for visualizing cell-cycle activity during development, tissue homeostasis...
Current Protocols in Stem Cell Biology, 2007
The adult Drosophila midgut is built of five distinct cell types, including stem cells, enterobla... more The adult Drosophila midgut is built of five distinct cell types, including stem cells, enteroblasts, enterocytes, enteroendocrine cells, and visceral muscles, and is divided into five major regions (R1 to R5), which are morphologically and functionally distinct from each other. This unit describes a protocol for the isolation of Drosophila intestinal cell populations for the purpose of cell type-specific transcriptome profiling from the five different regions. A method to select a cell type of interest labeled with green or yellow fluorescent protein (GFP, YFP) by making use of the GAL4-UAS bipartite system and fluorescent-activated cell sorting (FACS) is presented. Total RNA is isolated from the sorted cells of each region, and linear RNA amplification is used to obtain sufficient amounts of high-quality RNA for analysis by microarray, RT-PCR, or RNA sequencing. This method will be useful for quantitative transcriptome comparison across intestinal cell types in the different regions under normal and various experimental conditions. © 2015 by John Wiley & Sons, Inc.
Nature Cell Biology, 2015
Mutations that inhibit differentiation in stem cell lineages are a common early step in cancer de... more Mutations that inhibit differentiation in stem cell lineages are a common early step in cancer development, but precisely how a loss of differentiation initiates tumorigenesis is unclear. We investigated Drosophila intestinal stem cell (ISC) tumours generated by suppressing Notch (N) signalling, which blocks differentiation. Notch-defective ISCs require stress-induced divisions for tumour initiation and an autocrine EGFR ligand, Spitz, during early tumour growth. On achieving a critical mass these tumours displace surrounding enterocytes, competing with them for basement membrane space and causing their detachment, extrusion and apoptosis. This loss of epithelial integrity induces JNK and Yki/YAP activity in enterocytes and, consequently, their expression of stress-dependent cytokines (Upd2, Upd3). These paracrine signals, normally used within the stem cell niche to trigger regeneration, propel tumour growth without the need for secondary mutations in growth signalling pathways. The appropriation of niche signalling by differentiation-defective stem cells may be a common mechanism of early tumorigenesis.
Cell Reports, 2015
Graphical Abstract Highlights d Drosophila intestinal cells are transcriptionally diverse across ... more Graphical Abstract Highlights d Drosophila intestinal cells are transcriptionally diverse across regions R1-R5 d GATAe, sna, and Ptx1 are key players in global and regional ISC regulation d Digestive enzymes are expressed cell type specifically in regions d P. entomophila infection causes a massive transcriptional change in midgut cells
Developmental Biology
Maintaining tissue homeostasis is a critical process during infection and inflammation. Tissues w... more Maintaining tissue homeostasis is a critical process during infection and inflammation. Tissues with a high intrinsic turnover, such as the intestinal epithelium, must launch a rapid response to infections while simultaneously coordinating cell proliferation and differentiation decisions. In this study, we searched for genes required for regeneration of the Drosophila intestine, and thereby affecting overall organism survival after infection with pathogenic bacteria. We found that Dpp/Gbb (BMP) signaling is essential for normal midgut regeneration, and that infection induces the BMP signaling ligands Dpp and Gbb. We demonstrate that Dpp is induced in visceral muscle and required for signaling activation. Subsequently, Gbb is induced in enterocytes after oral infection. Loss-of Dpp signaling in ISCs and transient committed progenitors called enteroblasts (EBs) led to ISC hyperproliferation. This treatment also resulted in an increased number of abnormally small Pdm1-positive cells, s...
Oncogene, 2014
Src non-receptor kinases have been implicated in events late in tumor progression. Here, we study... more Src non-receptor kinases have been implicated in events late in tumor progression. Here, we study the role of Src kinases in the Drosophila intestinal stem cell (ISC) lineage, during tissue homeostasis and tumor onset. The adult Drosophila intestine contains only two progenitor cell types, division-capable ISCs and their daughters, postmitotic enteroblasts (EBs). We found that Drosophila Src42a and Src64b were required for optimal regenerative ISC division. Conversely, activation of Src42a, Src64b or another nonreceptor kinase, Ack, promoted division of quiescent ISCs by coordinately stimulating G1/S and G2/M cell cycle phase progression. Prolonged Src kinase activation caused tissue overgrowth owing to cytokine receptor-independent Stat92E activation. This was not due to increased symmetric division of ISCs, but involved accumulation of weakly specified Notch + but division-capable EB-like cells. Src activation triggered expression of a mitogenic module consisting of String/Cdc25 and Cyclin E that was sufficient to elicit division not only of ISCs but also of EBs. A small pool of similarly division-capable transit-amplifying Notch + EBs was also identified in the wild type. Expansion of intermediate cell types that do not robustly manifest their transit-amplifying potential in the wild type may also contribute to regenerative growth and tumor development in other tissues in other organisms.
Cell reports, Jan 24, 2014
One promising approach for in vivo studies of cell proliferation is the FUCCI system (fluorescent... more One promising approach for in vivo studies of cell proliferation is the FUCCI system (fluorescent ubiquitination-based cell cycle indicator). Here, we report the development of a Drosophila-specific FUCCI system (Fly-FUCCI) that allows one to distinguish G1, S, and G2 phases of interphase. Fly-FUCCI relies on fluorochrome-tagged degrons from the Cyclin B and E2F1 proteins, which are degraded by the ubiquitin E3-ligases APC/C and CRL4(Cdt2), during mitosis or the onset of S phase, respectively. These probes can track cell-cycle patterns in cultured Drosophila cells, eye and wing imaginal discs, salivary glands, the adult midgut, and probably other tissues. To support a broad range of experimental applications, we have generated a toolkit of transgenic Drosophila lines that express the Fly-FUCCI probes under control of the UASt, UASp, QUAS, and ubiquitin promoters. The Fly-FUCCI system should be a valuable tool for visualizing cell-cycle activity during development, tissue homeostasis...
The EMBO Journal, 2014
Snail family transcription factors are expressed in various stem cell types, but their function i... more Snail family transcription factors are expressed in various stem cell types, but their function in maintaining stem cell identity is unclear. In the adult Drosophila midgut, the Snail homolog Esg is expressed in intestinal stem cells (ISCs) and their transient undifferentiated daughters, termed enteroblasts (EB). We demonstrate here that loss of esg in these progenitor cells causes their rapid differentiation into enterocytes (EC) or entero-endocrine cells (EE). Conversely, forced expression of Esg in intestinal progenitor cells blocks differentiation, locking ISCs in a stem cell state. Cell type-specific transcriptome analysis combined with Dam-ID binding studies identified Esg as a major repressor of differentiation genes in stem and progenitor cells. One critical target of Esg was found to be the POU-domain transcription factor, Pdm1, which is normally expressed specifically in differentiated ECs. Ectopic expression of Pdm1 in progenitor cells was sufficient to drive their differentiation into ECs. Hence, Esg is a critical stem cell determinant that maintains stemness by repressing differentiation-promoting factors, such as Pdm1.
Developmental Biology, 2014
Maintaining tissue homeostasis is a critical process during infection and inflammation. Tissues w... more Maintaining tissue homeostasis is a critical process during infection and inflammation. Tissues with a high intrinsic turnover, such as the intestinal epithelium, must launch a rapid response to infections while simultaneously coordinating cell proliferation and differentiation decisions. In this study, we searched for genes required for regeneration of the Drosophila intestine, and thereby affecting overall organism survival after infection with pathogenic bacteria. We found that Dpp/Gbb (BMP) signaling is essential for normal midgut regeneration, and that infection induces the BMP signaling ligands Dpp and Gbb. We demonstrate that Dpp is induced in visceral muscle and required for signaling activation. Subsequently, Gbb is induced in enterocytes after oral infection. Loss-of Dpp signaling in ISCs and transient committed progenitors called enteroblasts (EBs) led to ISC hyperproliferation. This treatment also resulted in an increased number of abnormally small Pdm1-positive cells, suggesting that EBs defective for receiving Dpp/Gbb signaling produce immature enterocytes (ECs) and are therefore defective in EC maturation. Tkv-Mad/Med/Shn activity also promotes EC differentiation and growth, which is epistatic to Notch pathway, and is required for differentiation in normal midgut. Our results suggest that Dpp/BMP signaling plays an important role in EBs to maintain tissue integrity and homeostasis during pathogenic infections.
Current Protocols in Stem Cell Biology, 2007
This unit describes a protocol for the isolation of Drosophila intestinal cell populations for th... more This unit describes a protocol for the isolation of Drosophila intestinal cell populations for the purpose of cell type-specific transcriptome profiling. A method to select a cell type of interest labeled with green or yellow fluorescent protein (GFP, YFP) by making use of the GAL4-UAS bipartite system and fluorescent-activated cell sorting (FACS) is presented. Total RNA is isolated from the sorted cells and linear RNA amplification is used to obtain sufficient amounts of high-quality RNA for analysis by microarray, RT-PCR, or RNA sequencing. This method will be useful for quantitative transcriptome comparison across intestinal cell types under normal and various experimental conditions.
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Papers by Devanjali Dutta