RESUMO
A subpopulation of deeply quiescent, so-called dormant hematopoietic stem cells (dHSCs) resides at the top of the hematopoietic hierarchy and serves as a reserve pool for HSCs. The state of dormancy protects the HSC pool from exhaustion throughout life; however, excessive dormancy may prevent an efficient response to hematological stresses. Despite the significance of dHSCs, the mechanisms maintaining their dormancy remain elusive. Here, we identify CD38 as a novel and broadly applicable surface marker for the enrichment of murine dHSCs. We demonstrate that cyclic adenosine diphosphate ribose (cADPR), the product of CD38 cyclase activity, regulates the expression of the transcription factor c-Fos by increasing the release of Ca2+ from the endoplasmic reticulum (ER). Subsequently, we uncover that c-Fos induces the expression of the cell cycle inhibitor p57Kip2 to drive HSC dormancy. Moreover, we found that CD38 ecto-enzymatic activity at the neighboring CD38-positive cells can promote human HSC quiescence. Together, CD38/cADPR/Ca2+/c-Fos/p57Kip2 axis maintains HSC dormancy. Pharmacological manipulations of this pathway can provide new strategies to improve the success of stem cell transplantation and blood regeneration after injury or disease.
Assuntos
ADP-Ribosil Ciclase 1 , ADP-Ribose Cíclica , Animais , Humanos , Camundongos , Cálcio/metabolismo , ADP-Ribose Cíclica/metabolismo , Células-Tronco Hematopoéticas , ADP-Ribosil Ciclase 1/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismoRESUMO
Mesoderm arises at gastrulation and contributes to both the mouse embryo proper and its extra-embryonic membranes. Two-photon live imaging of embryos bearing a keratin reporter allowed recording filament nucleation and elongation in the extra-embryonic region. Upon separation of amniotic and exocoelomic cavities, keratin 8 formed apical cables co-aligned across multiple cells in the amnion, allantois, and blood islands. An influence of substrate rigidity and composition on cell behavior and keratin content was observed in mesoderm explants. Embryos lacking all keratin filaments displayed a deflated extra-embryonic cavity, a narrow thick amnion, and a short allantois. Single-cell RNA sequencing of sorted mesoderm cells and micro-dissected amnion, chorion, and allantois, provided an atlas of transcriptomes with germ layer and regional information. It defined the cytoskeleton and adhesion expression profile of mesoderm-derived keratin 8-enriched cells lining the exocoelomic cavity. Those findings indicate a novel role for keratin filaments in the expansion of extra-embryonic structures and suggest mechanisms of mesoderm adaptation to the environment.
Assuntos
Gastrulação , Mesoderma , Animais , Embrião de Mamíferos , Membranas Extraembrionárias , Queratinas/genética , Queratinas/metabolismo , Mesoderma/metabolismo , CamundongosRESUMO
Regeneration-competent species possess the ability to reverse the progression of severe diseases by restoring the function of the damaged tissue. However, the cellular dynamics underlying this capability remain unexplored. Here, we have used single-cell transcriptomics to map de novo ß-cell regeneration during induction and recovery from diabetes in zebrafish. We show that the zebrafish has evolved two distinct types of somatostatin-producing δ-cells, which we term δ1- and δ2-cells. Moreover, we characterize a small population of glucose-responsive islet cells, which share the hormones and fate-determinants of both ß- and δ1-cells. The transcriptomic analysis of ß-cell regeneration reveals that ß/δ hybrid cells provide a prominent source of insulin expression during diabetes recovery. Using in vivo calcium imaging and cell tracking, we further show that the hybrid cells form de novo and acquire glucose-responsiveness in the course of regeneration. The overexpression of dkk3, a gene enriched in hybrid cells, increases their formation in the absence of ß-cell injury. Finally, interspecies comparison shows that plastic δ1-cells are partially related to PP cells in the human pancreas. Our work provides an atlas of ß-cell regeneration and indicates that the rapid formation of glucose-responsive hybrid cells contributes to the resolution of diabetes in zebrafish.
Assuntos
Diabetes Mellitus/metabolismo , Células Secretoras de Insulina/citologia , Regeneração , Células Secretoras de Somatostatina/citologia , Animais , Cálcio/metabolismo , Diabetes Mellitus/patologia , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Análise de Célula Única , Células Secretoras de Somatostatina/metabolismo , Peixe-ZebraRESUMO
BACKGROUND AND AIMS: Hepatocytes were the first cell type for which oscillations of cytoplasmic calcium levels in response to hormones were described. Since then, investigation of calcium dynamics in liver explants and culture has greatly increased our understanding of calcium signaling. A bottleneck, however, exists in observing calcium dynamics in a noninvasive manner because of the optical inaccessibility of the mammalian liver. Here, we aimed to take advantage of the transparency of the zebrafish larvae to image hepatocyte calcium dynamics in vivo at cellular resolution. APPROACH AND RESULTS: We developed a transgenic model expressing a calcium sensor, GCaMP6s, specifically in zebrafish hepatocytes. Using this, we provide a quantitative assessment of intracellular calcium dynamics during multiple contexts, including growth, feeding, ethanol-induced stress, and cell ablation. Specifically, we show that synchronized calcium oscillations are present in vivo , which are lost upon starvation. Starvation induces lipid accumulation in the liver. Feeding recommences calcium waves in the liver, but in a spatially restricted manner, as well as resolves starvation-induced hepatic steatosis. By using a genetically encoded scavenger for calcium, we show that dampening of calcium signaling accelerates the accumulation of starvation-related lipid droplets in the liver. Furthermore, ethanol treatment, as well as cell ablation, induces calcium flux, but with different dynamics. The former causes asynchronous calcium oscillations, whereas the latter leads to a single calcium spike. CONCLUSIONS: We demonstrate the presence of oscillations, waves, and spikes in vivo . Calcium waves are present in response to nutrition and negatively regulate starvation-induced accumulation of lipid droplets.
Assuntos
Inanição , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Cálcio/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Etanol/farmacologia , Sinalização do Cálcio , Inanição/metabolismo , Mamíferos/metabolismoRESUMO
BACKGROUND: Tetraspanin-7 (Tspan7) is an islet autoantigen involved in autoimmune type 1 diabetes and known to regulate ß-cell L-type Ca2+ channel activity. However, the role of Tspan7 in pancreatic ß-cell function is not yet fully understood. METHODS: Histological analyses were conducted using immunostaining. Whole-body metabolism was tested using glucose tolerance test. Islet hormone secretion was quantified using static batch incubation or dynamic perifusion. ß-cell transmembrane currents, electrical activity and exocytosis were measured using whole-cell patch-clamping and capacitance measurements. Gene expression was studied using mRNA-sequencing and quantitative PCR. RESULTS: Tspan7 is expressed in insulin-containing granules of pancreatic ß-cells and glucagon-producing α-cells. Tspan7 knockout mice (Tspan7y/- mouse) exhibit reduced body weight and ad libitum plasma glucose but normal glucose tolerance. Tspan7y/- islets have normal insulin content and glucose- or tolbutamide-stimulated insulin secretion. Depolarisation-triggered Ca2+ current was enhanced in Tspan7y/- ß-cells, but ß-cell electrical activity and depolarisation-evoked exocytosis were unchanged suggesting that exocytosis was less sensitive to Ca2+ . TSPAN7 knockdown (KD) in human pseudo-islets led to a significant reduction in insulin secretion stimulated by 20 mM K+ . Transcriptomic analyses show that TSPAN7 KD in human pseudo-islets correlated with changes in genes involved in hormone secretion, apoptosis and ER stress. Consistent with rodent ß-cells, exocytotic Ca2+ sensitivity was reduced in a human ß-cell line (EndoC-ßH1) following Tspan7 KD. CONCLUSION: Tspan7 is involved in the regulation of Ca2+ -dependent exocytosis in ß-cells. Its function is more significant in human ß-cells than their rodent counterparts.
Assuntos
Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Humanos , Camundongos , Exocitose/fisiologia , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Tetraspaninas/genética , Tetraspaninas/metabolismoRESUMO
At gastrulation, a subpopulation of epiblast cells constitutes a transient posteriorly located structure called the primitive streak, where cells that undergo epithelial-mesenchymal transition make up the mesoderm and endoderm lineages. Mouse embryo epiblast cells were labelled ubiquitously or in a mosaic fashion. Cell shape, packing, organization and division were recorded through live imaging during primitive streak formation. Posterior epiblast displays a higher frequency of rosettes, some of which associate with a central cell undergoing mitosis. Cells at the primitive streak, in particular delaminating cells, undergo mitosis more frequently than other epiblast cells. In pseudostratified epithelia, mitosis takes place at the apical side of the epithelium. However, mitosis is not restricted to the apical side of the epiblast, particularly on its posterior side. Non-apical mitosis occurs specifically in the streak even when ectopically located. Posterior non-apical mitosis results in one or two daughter cells leaving the epiblast layer. Cell rearrangement associated with mitotic cell rounding in posterior epiblast, in particular when non-apical, might thus facilitate cell ingression and transition to a mesenchymal phenotype.
Assuntos
Gastrulação , Camadas Germinativas , Animais , Transição Epitelial-Mesenquimal/genética , Mesoderma , Camundongos , MitoseRESUMO
Natural models of heart regeneration in lower vertebrates such as zebrafish are based on invasive surgeries causing mechanical injuries that are limited in size. Here, we created a genetic cell ablation model in zebrafish that facilitates inducible destruction of a high percentage of cardiomyocytes. Cell-specific depletion of over 60% of the ventricular myocardium triggered signs of cardiac failure that were not observed after partial ventricular resection, including reduced animal exercise tolerance and sudden death in the setting of stressors. Massive myocardial loss activated robust cellular and molecular responses by endocardial, immune, epicardial and vascular cells. Destroyed cardiomyocytes fully regenerated within several days, restoring cardiac anatomy, physiology and performance. Regenerated muscle originated from spared cardiomyocytes that acquired ultrastructural and electrophysiological characteristics of de-differentiation and underwent vigorous proliferation. Our study indicates that genetic depletion of cardiomyocytes, even at levels so extreme as to elicit signs of cardiac failure, can be reversed by natural regenerative capacity in lower vertebrates such as zebrafish.
Assuntos
Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Coração/fisiologia , Miócitos Cardíacos/citologia , Regeneração , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Morte CelularRESUMO
Coordination of cellular activity through Ca2+ enables ß cells to secrete precise quantities of insulin. To explore how the Ca2+ response is orchestrated in space and time, we implement optogenetic systems to probe the role of individual ß cells in the glucose response. By targeted ß cell activation/inactivation in zebrafish, we reveal a hierarchy of cells, each with a different level of influence over islet-wide Ca2+ dynamics. First-responder ß cells lie at the top of the hierarchy, essential for initiating the first-phase Ca2+ response. Silencing first responders impairs the Ca2+ response to glucose. Conversely, selective activation of first responders demonstrates their increased capability to raise pan-islet Ca2+ levels compared to followers. By photolabeling and transcriptionally profiling ß cells that differ in their thresholds to a glucose-stimulated Ca2+ response, we highlight vitamin B6 production as a signature pathway of first responders. We further define an evolutionarily conserved requirement for vitamin B6 in enabling the Ca2+ response to glucose in mammalian systems.
Assuntos
Cálcio , Glucose , Células Secretoras de Insulina , Optogenética , Peixe-Zebra , Animais , Células Secretoras de Insulina/metabolismo , Glucose/metabolismo , Cálcio/metabolismo , Sinalização do CálcioRESUMO
Starvation causes the accumulation of lipid droplets in the liver, a somewhat counterintuitive phenomenon that is nevertheless conserved from flies to humans. Much like fatty liver resulting from overfeeding, hepatic lipid accumulation (steatosis) during undernourishment can lead to lipotoxicity and atrophy of the liver. Here, we found that although surface populations of Astyanax mexicanus undergo this evolutionarily conserved response to starvation, the starvation-resistant cavefish larvae of the same species do not display an accumulation of lipid droplets upon starvation. Moreover, cavefish are resistant to liver atrophy during starvation, providing a unique system to explore strategies for liver protection. Using comparative transcriptomics between zebrafish, surface fish, and cavefish, we identified the fatty acid transporter slc27a2a/fatp2 to be correlated with the development of fatty liver. Pharmacological inhibition of slc27a2a in zebrafish rescues steatosis and atrophy of the liver upon starvation. Furthermore, down-regulation of FATP2 in Drosophila larvae inhibits the development of starvation-induced steatosis, suggesting the evolutionarily conserved importance of the gene in regulating fatty liver upon nutrition deprivation. Overall, our study identifies a conserved, druggable target to protect the liver from atrophy during starvation.
Assuntos
Fígado Gorduroso , Inanição , Animais , Humanos , Peixe-Zebra , Fígado Gorduroso/genética , Inanição/complicações , Larva , AtrofiaRESUMO
Starvation causes the accumulation of lipid droplets in the liver, a somewhat counterintuitive phenomenon that is nevertheless conserved from flies to humans. Much like fatty liver resulting from overfeeding, hepatic lipid accumulation (steatosis) during undernourishment can lead to lipotoxicity and atrophy of the liver. Here, we found that while surface populations of Astyanax mexicanus undergo this evolutionarily conserved response to starvation, the starvation-resistant cavefish larvae of the same species do not display an accumulation of lipid droplets upon starvation. Moreover, cavefish are resistant to liver atrophy during starvation, providing a unique system to explore strategies for liver protection. Using comparative transcriptomics between zebrafish, surface fish, and cavefish, we identified the fatty acid transporter slc27a2a/fatp2 to be correlated with the development of fatty liver. Pharmacological inhibition of slc27a2a in zebrafish rescues steatosis and atrophy of the liver upon starvation. Further, down-regulation of FATP2 in drosophila larvae inhibits the development of starvation-induced steatosis, suggesting the evolutionary conserved importance of the gene in regulating fatty liver upon nutrition deprivation. Overall, our study identifies a conserved, druggable target to protect the liver from atrophy during starvation.
RESUMO
An altered gut microbiota is associated with type 1 diabetes (T1D), affecting the production of short-chain fatty acids (SCFA) and glucose homeostasis. We previously demonstrated that enhancing serum acetate and butyrate using a dietary supplement (HAMSAB) improved glycemia in non-obese diabetic (NOD) mice and patients with established T1D. The effects of SCFA on immune-infiltrated islet cells remain to be clarified. Here, we performed single-cell RNA sequencing on islet cells from NOD mice fed an HAMSAB or control diet. HAMSAB induced a regulatory gene expression profile in pancreas-infiltrated immune cells. Moreover, HAMSAB maintained the expression of ß-cell functional genes and decreased cellular stress. HAMSAB-fed mice showed preserved pancreatic endocrine cell identity, evaluated by decreased numbers of poly-hormonal cells. Finally, SCFA increased insulin levels in human ß-like cells and improved transplantation outcome in NOD/SCID mice. Our findings support the use of metabolite-based diet as attractive approach to improve glucose control in T1D.
RESUMO
Calcium in interstitial fluids is central to systemic physiology and a crucial ion pool for entry into cells through numerous plasma membrane channels. Its study has been limited by the scarcity of methods that allow monitoring in tight inter-cell spaces of living tissues. Here we present high performance ultra-low affinity genetically encoded calcium biosensors named GreenT-ECs. GreenT-ECs combine large fluorescence changes upon calcium binding and binding affinities (Kds) ranging from 0.8 mM to 2.9 mM, making them tuned to calcium concentrations in extracellular organismal fluids. We validated GreenT-ECs in rodent hippocampal neurons and transgenic zebrafish in vivo, where the sensors enabled monitoring homeostatic regulation of tissue interstitial calcium. GreenT-ECs may become useful for recording very large calcium transients and for imaging calcium homeostasis in inter-cell structures in live tissues and organisms.
Assuntos
Cálcio , Peixe-Zebra , Animais , Cálcio/metabolismo , Peixe-Zebra/metabolismo , Fluorescência , Sinalização do Cálcio/fisiologia , Diagnóstico por Imagem , CorantesRESUMO
The transcription factor nuclear factor-κB (NF-κB) has a key role in the pathogenesis of diabetes and its complications. Although activation of the canonical NF-κB pathway in ß-cells is generally deleterious, little is known about the role of the non-canonical NF-κB signalling and its main regulator, the NF-κB-inducing kinase (NIK), on pancreatic ß-cell survival and function. Previous studies based on models of NIK overexpression in pancreatic islet cells showed that NIK induced either spontaneous ß-cell death due to islet inflammation or glucose intolerance during diet-induced obesity (DIO) in mice. Therefore, NIK has been proposed as a potential target for diabetes therapy. However, no clear studies showed whether inhibition of NIK improves diabetes development. Here we show that genetic silencing of NIK in pancreatic ß-cells neither modifies diabetes incidence nor inflammatory responses in a mouse model of immune-mediated diabetes. Moreover, NIK silencing in DIO mice did not influence body weight gain, nor glucose metabolism. In vitro studies corroborated the in vivo findings in terms of ß-cell survival, function, and downstream gene regulation. Taken together, our data suggest that NIK activation is dispensable for the development of diabetes.
Assuntos
Diabetes Mellitus , Células Secretoras de Insulina , Animais , Diabetes Mellitus/patologia , Células Secretoras de Insulina/metabolismo , Camundongos , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/fisiologia , Quinase Induzida por NF-kappaBRESUMO
The thyroid gland captures iodide in order to synthesize hormones that act on almost all tissues and are essential for normal growth and metabolism. Low plasma levels of thyroid hormones lead to hypothyroidism, which is one of the most common disorder in humans and is not always satisfactorily treated by lifelong hormone replacement. Therefore, in addition to the lack of in vitro tractable models to study human thyroid development, differentiation and maturation, functional human thyroid organoids could pave the way to explore new therapeutic approaches. Here we report the generation of transplantable thyroid organoids derived from human embryonic stem cells capable of restoring plasma thyroid hormone in athyreotic mice as a proof of concept for future therapeutic development.
Assuntos
Hipotireoidismo , Organoides , Humanos , Animais , Camundongos , Hipotireoidismo/terapia , Células-Tronco Embrionárias , Hormônios TireóideosRESUMO
Type 1 diabetes (T1D) results from autoimmune destruction of ß-cells in the pancreas. Protein tyrosine phosphatases (PTPs) are candidate genes for T1D and play a key role in autoimmune disease development and ß-cell dysfunction. Here, we assessed the global protein and individual PTP profiles in the pancreas from nonobese mice with early-onset diabetes (NOD) mice treated with an anti-CD3 monoclonal antibody and interleukin-1 receptor antagonist. The treatment reversed hyperglycemia, and we observed enhanced expression of PTPN2, a PTP family member and T1D candidate gene, and endoplasmic reticulum (ER) chaperones in the pancreatic islets. To address the functional role of PTPN2 in ß-cells, we generated PTPN2-deficient human stem cell-derived ß-like and EndoC-ßH1 cells. Mechanistically, we demonstrated that PTPN2 inactivation in ß-cells exacerbates type I and type II interferon signaling networks and the potential progression toward autoimmunity. Moreover, we established the capacity of PTPN2 to positively modulate the Ca2+-dependent unfolded protein response and ER stress outcome in ß-cells. Adenovirus-induced overexpression of PTPN2 partially protected from ER stress-induced ß-cell death. Our results postulate PTPN2 as a key protective factor in ß-cells during inflammation and ER stress in autoimmune diabetes.
Assuntos
Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Animais , Apoptose/genética , Diabetes Mellitus Tipo 1/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Células Secretoras de Insulina/metabolismo , Interferon gama/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Proteína Tirosina Fosfatase não Receptora Tipo 2/genéticaRESUMO
The thyroid gland regulates metabolism and growth via secretion of thyroid hormones by thyroid follicular cells (TFCs). Loss of TFCs, by cellular dysfunction, autoimmune destruction or surgical resection, underlies hypothyroidism. Recovery of thyroid hormone levels by transplantation of mature TFCs derived from stem cells in vitro holds great therapeutic promise. However, the utilization of in vitro derived tissue for regenerative medicine is restricted by the efficiency of differentiation protocols to generate mature organoids. Here, to improve the differentiation efficiency for thyroid organoids, we utilized single-cell RNA-Seq to chart the molecular steps undertaken by individual cells during the in vitro transformation of mouse embryonic stem cells to TFCs. Our single-cell atlas of mouse organoid systematically and comprehensively identifies, for the first time, the cell types generated during production of thyroid organoids. Using pseudotime analysis, we identify TGF-beta as a negative regulator of thyroid maturation in vitro. Using pharmacological inhibition of TGF-beta pathway, we improve the level of thyroid maturation, in particular the induction of Nis expression. This in turn, leads to an enhancement of iodide organification in vitro, suggesting functional improvement of the thyroid organoid. Our study highlights the potential of single-cell molecular characterization in understanding and improving thyroid maturation and paves the way for identification of therapeutic targets against thyroid disorders.
Assuntos
Biomarcadores/metabolismo , Iodetos/metabolismo , Organoides/citologia , Análise de Célula Única/métodos , Simportadores/metabolismo , Glândula Tireoide/citologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Diferenciação Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Organoides/metabolismo , Simportadores/genética , Glândula Tireoide/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Strategies have not been available until recently to uncover interacting protein networks specific to key cell types, their subcellular compartments, and their major regulators during complex in vivo events. Here, we apply BioID2 proximity labeling to capture protein networks acting within cardiomyocytes during a key model of innate heart regeneration in zebrafish. Transgenic zebrafish expressing a promiscuous BirA2 localized to the entire myocardial cell or membrane compartment were generated, each identifying distinct proteomes in adult cardiomyocytes that became altered during regeneration. BioID2 profiling for interactors with ErbB2, a co-receptor for the cardiomyocyte mitogen Nrg1, implicated Rho A as a target of ErbB2 signaling in cardiomyocytes. Blockade of Rho A during heart regeneration, or during cardiogenic stimulation by the mitogenic influences Nrg1, Vegfaa, or vitamin D, disrupted muscle creation. Our findings reveal proximity labeling as a useful resource to interrogate cell proteomes and signaling networks during tissue regeneration in zebrafish.
Assuntos
Coração/fisiologia , Miócitos Cardíacos/metabolismo , Regeneração , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Regeneração/genética , Transdução de Sinais , Peixe-ZebraRESUMO
During embryogenesis, beta-cells arise from the dorsal and ventral bud originating in the endoderm germ layer. As the animal develops to adulthood, the beta-cell mass dramatically increases. The expansion of the beta-cell population is driven by cell division among the embryonic beta-cells and supplanted by neogenesis from post-embryonic progenitors. Here, we describe a protocol for multicolor clonal analysis in zebrafish to define the contribution of individual embryonic beta-cells to the increase in cell numbers. This technique provides insights into the proliferative history of individual beta-cells in an islet. This insight helps in defining the replicative heterogeneity among individual beta-cells during development. Additionally, the ability to discriminate individual cells based on unique color signatures helps quantify the volume occupied by beta-cells and define the contribution of cellular size to the beta-cell mass.
Assuntos
Proliferação de Células , Rastreamento de Células/métodos , Processamento de Imagem Assistida por Computador/métodos , Células Secretoras de Insulina/citologia , Microscopia Confocal/métodos , Coloração e Rotulagem/métodos , Animais , Animais Geneticamente Modificados , Linhagem da Célula , Clonagem Molecular/métodos , Cor , Genes Reporter , Células Secretoras de Insulina/química , Integrases , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Animais , Peixe-ZebraRESUMO
High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.
Assuntos
Fracionamento Celular/métodos , Núcleo Celular , Detergentes/farmacologia , Métodos Analíticos de Preparação de Amostras , Animais , Encéfalo/citologia , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Citometria de Fluxo , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Reprodutibilidade dos Testes , Peixe-ZebraRESUMO
Activation of the adrenal gland stress response is of utmost importance to survive sepsis. Experimental and clinical evidence exists demonstrating that adrenal gland often develops functional and structural damage due to sepsis with mechanisms remaining largely unknown. In the present study, we have used RNA Sequencing (RNA-Seq) technology to analyze changes in adrenal transcriptome elucidated by bacterial LPS. We aimed to find particularly alterations in genes that were previously not reported to be involved in the adrenal gland dysregulation in contexts of sepsis. Our results demonstrate that systemic administration of LPS significantly altered expression of 8458 genes as compared to saline injected animals. The subsequent quality and functional analysis of these gene signatures revealed that LPS-induced highly homogenous transcriptional response in total upregulating 4312 and downregulating 4146 genes. Furthermore, functional annotation analysis together with gene enrichment set analysis (GSEA) clearly demonstrated that adrenal response to LPS involved alterations in multiple pathways related to the inflammatory response along with previously unexplored activation of the hypoxia pathway. In addition, LPS strongly downregulated genes involved in the adrenal homeostasis, development, and regeneration. Those alterations were subsequently verified in clinically relevant cecal ligation and puncture (CLP)-induced sepsis model. Collectively, our study demonstrates that RNA-seq is a very useful method that can be applied to search for new unexplored pathways potentially involved in adrenal gland dysregulation during sepsis.