RESUMO
The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis, erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis1 to define the anatomy of normal and stress haematopoiesis. In the steady state, across the skeleton, single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels, where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells, which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults, as it was maintained after haemorrhage, systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment, and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF, and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis, define the anatomy of normal and stress responses, identify discrete microanatomical production sites that confer plasticity to haematopoiesis, and uncover unprecedented heterogeneity of stress responses across the skeleton.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Estresse Fisiológico , Animais , Feminino , Masculino , Camundongos , Envelhecimento/fisiologia , Infecções Bacterianas/patologia , Infecções Bacterianas/fisiopatologia , Vasos Sanguíneos/citologia , Linhagem da Célula , Eritropoese , Fator Estimulador de Colônias de Granulócitos/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Hemorragia/patologia , Hemorragia/fisiopatologia , Linfopoese , Megacariócitos/citologia , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Mielopoese , Crânio/irrigação sanguínea , Crânio/patologia , Crânio/fisiopatologia , Esterno/irrigação sanguínea , Esterno/citologia , Esterno/metabolismo , Estresse Fisiológico/fisiologia , Tíbia/irrigação sanguínea , Tíbia/citologia , Tíbia/metabolismoRESUMO
In contrast to nearly all other tissues, the anatomy of cell differentiation in the bone marrow remains unknown. This is owing to a lack of strategies for examining myelopoiesis-the differentiation of myeloid progenitors into a large variety of innate immune cells-in situ in the bone marrow. Such strategies are required to understand differentiation and lineage-commitment decisions, and to define how spatial organizing cues inform tissue function. Here we develop approaches for imaging myelopoiesis in mice, and generate atlases showing the differentiation of granulocytes, monocytes and dendritic cells. The generation of granulocytes and dendritic cells-monocytes localizes to different blood-vessel structures known as sinusoids, and displays lineage-specific spatial and clonal architectures. Acute systemic infection with Listeria monocytogenes induces lineage-specific progenitor clusters to undergo increased self-renewal of progenitors, but the different lineages remain spatially separated. Monocyte-dendritic cell progenitors (MDPs) map with nonclassical monocytes and conventional dendritic cells; these localize to a subset of blood vessels expressing a major regulator of myelopoiesis, colony-stimulating factor 1 (CSF1, also known as M-CSF)1. Specific deletion of Csf1 in endothelium disrupts the architecture around MDPs and their localization to sinusoids. Subsequently, there are fewer MDPs and their ability to differentiate is reduced, leading to a loss of nonclassical monocytes and dendritic cells during both homeostasis and infection. These data indicate that local cues produced by distinct blood vessels are responsible for the spatial organization of definitive blood cell differentiation.
Assuntos
Rastreamento de Células/métodos , Células Mieloides/citologia , Mielopoese , Coloração e Rotulagem/métodos , Animais , Atlas como Assunto , Vasos Sanguíneos/citologia , Vasos Sanguíneos/metabolismo , Linhagem da Célula , Autorrenovação Celular , Células Dendríticas/citologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Feminino , Granulócitos/citologia , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Fator Estimulador de Colônias de Macrófagos/deficiência , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Masculino , Camundongos , Monócitos/citologia , Células Mieloides/metabolismoRESUMO
BACKGROUND: Current management of AKI, a potentially fatal disorder that can also initiate or exacerbate CKD, is merely supportive. Therefore, deeper understanding of the molecular pathways perturbed in AKI is needed to identify targets with potential to lead to improved treatment. METHODS: We performed single-cell RNA sequencing (scRNA-seq) with the clinically relevant unilateral ischemia-reperfusion murine model of AKI at days 1, 2, 4, 7, 11, and 14 after AKI onset. Using real-time quantitative PCR, immunofluorescence, Western blotting, and both chromogenic and single-molecule in situ hybridizations, we validated AKI signatures in multiple experiments. RESULTS: Our findings show the time course of changing gene expression patterns for multiple AKI stages and all renal cell types. We observed elevated expression of crucial injury response factors-including kidney injury molecule-1 (Kim1), lipocalin 2 (Lcn2), and keratin 8 (Krt8)-and of several novel genes (Ahnak, Sh3bgrl3, and Col18a1) not previously examined in kidney pathologies. AKI induced proximal tubule dedifferentiation, with a pronounced nephrogenic signature represented by Sox4 and Cd24a. Moreover, AKI caused the formation of "mixed-identity cells" (expressing markers of different renal cell types) that are normally seen only during early kidney development. The injured tubules acquired a proinflammatory and profibrotic phenotype; moreover, AKI dramatically modified ligand-receptor crosstalk, with potential pathologic epithelial-to-stromal interactions. Advancing age in AKI onset was associated with maladaptive response and kidney fibrosis. CONCLUSIONS: The scRNA-seq, comprehensive, cell-specific profiles provide a valuable resource for examining molecular pathways that are perturbed in AKI. The results fully define AKI-associated dedifferentiation programs, potential pathologic ligand-receptor crosstalk, novel genes, and the improved injury response in younger mice, and highlight potential targets of kidney injury.
Assuntos
Injúria Renal Aguda/etiologia , Injúria Renal Aguda/patologia , Células Epiteliais/fisiologia , Túbulos Renais Proximais/patologia , Células Estromais/fisiologia , Animais , Comunicação Celular , Modelos Animais de Doenças , Masculino , Camundongos , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologia , Análise de Sequência de RNARESUMO
Vascular patterning is critical for organ function. In the eye, there is simultaneous regression of embryonic hyaloid vasculature (important to clear the optical path) and formation of the retinal vasculature (important for the high metabolic demands of retinal neurons). These events occur postnatally in the mouse. Here we have identified a light-response pathway that regulates both processes. We show that when mice are mutated in the gene (Opn4) for the atypical opsin melanopsin, or are dark-reared from late gestation, the hyaloid vessels are persistent at 8 days post-partum and the retinal vasculature overgrows. We provide evidence that these vascular anomalies are explained by a light-response pathway that suppresses retinal neuron number, limits hypoxia and, as a consequence, holds local expression of vascular endothelial growth factor (VEGFA) in check. We also show that the light response for this pathway occurs in late gestation at about embryonic day 16 and requires the photopigment in the fetus and not the mother. Measurements show that visceral cavity photon flux is probably sufficient to activate melanopsin-expressing retinal ganglion cells in the mouse fetus. These data thus show that light--the stimulus for function of the mature eye--is also critical in preparing the eye for vision by regulating retinal neuron number and initiating a series of events that ultimately pattern the ocular blood vessels.
Assuntos
Olho/irrigação sanguínea , Olho/crescimento & desenvolvimento , Feto/efeitos da radiação , Transdução de Sinal Luminoso/efeitos da radiação , Luz , Neurônios Retinianos/efeitos da radiação , Opsinas de Bastonetes/metabolismo , Animais , Contagem de Células , Hipóxia Celular/efeitos da radiação , Olho/metabolismo , Olho/efeitos da radiação , Feminino , Feto/citologia , Feto/embriologia , Feto/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica , Neovascularização Fisiológica/efeitos da radiação , Fótons , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/efeitos da radiação , Neurônios Retinianos/citologia , Neurônios Retinianos/metabolismo , Opsinas de Bastonetes/deficiência , Opsinas de Bastonetes/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Lung stretch is critical for normal lung development and for compensatory lung growth after pneumonectomy (PNX), but the mechanisms by which strain induces matrix remodeling are unclear. Our prior work demonstrated an association of chymotrypsin-like elastase 1 (Cela1) with lung elastin remodeling, and that strain triggered a near-instantaneous elastin-remodeling response. We sought to determine whether stretch regulates Cela1 expression and Cela1 binding to lung elastin. In C57BL/6J mice, Cela1 protein increased 176-fold during lung morphogenesis. Cela1 was covalently bound to serpin peptidase inhibitor, clade A, member 1, resulting in a higher molecular mass in lung homogenate compared to pancreas homogenate. Post-PNX, Cela1 mRNA increased 6-fold, protein 3-fold, and Cela1-positive cells 2-fold. Cela1 was expressed predominantly in alveolar type II cells in the embryonic lung and predominantly in CD90-positive lung fibroblasts postnatally. During compensatory lung growth, Cela1 expression was induced in nonproliferative mesenchymal cells. In ex vivo mouse lung sections, stretch increased Cela1 binding to lung tissue by 46%. Competitive inhibition with soluble elastin completely abrogated this increase. Areas of stretch-induced elastase activity and Cela1 binding colocalized. The stretch-dependent expression and binding kinetics of Cela1 indicate an important role in stretch-dependent remodeling of the peripheral lung during development and regeneration.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Pulmão/fisiologia , Elastase Pancreática/metabolismo , Sequência de Aminoácidos , Animais , Fenômenos Biomecânicos , Células Cultivadas , Quimases , Elastina/metabolismo , Fibroblastos/metabolismo , Rim/citologia , Rim/embriologia , Pulmão/citologia , Camundongos , Elastase Pancreática/genética , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismoRESUMO
Fibrosis is a common outcome of numerous pathologies, including chronic kidney disease (CKD), a progressive renal function deterioration. Current approaches to target activated fibroblasts, key effector contributors to fibrotic tissue remodeling, lack specificity. Here, we report Gucy1α1 as a specific kidney fibroblast marker. Gucy1α1 levels significantly increased over the course of two clinically relevant murine CKD models and directly correlated with established fibrosis markers. Immunofluorescent (IF) imaging showed that Gucy1α1 comprehensively labelled cortical and medullary quiescent and activated fibroblasts in the control kidney and throughout injury progression, respectively. Unlike traditionally used markers platelet derived growth factor receptor beta (Pdgfrß) and vimentin (Vim), Gucy1α1 did not overlap with off-target populations such as podocytes. Notably, Gucy1α1 labelled kidney fibroblasts in both male and female mice. Furthermore, we observed elevated GUCY1α1 expression in the human fibrotic kidney and lung. Studies in the murine models of cardiac and liver fibrosis revealed Gucy1α1 elevation in activated Pdgfrß-, Vim- and alpha smooth muscle actin (αSma)-expressing fibroblasts paralleling injury progression and resolution. Overall, we demonstrate Gucy1α1 as an exclusive fibroblast marker in both sexes. Due to its multiorgan translational potential, GUCY1α1 might provide a novel promising strategy to specifically target and mechanistically examine fibroblasts.
RESUMO
Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with "secretory", "contractile" and "vascular" transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8 and Vcam1, while the surviving proximal tubules (PTs) showed restored transcriptional signature. We also found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.
Assuntos
Túbulos Renais , Insuficiência Renal Crônica , Humanos , Túbulos Renais/patologia , Rim/patologia , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Fibroblastos/fisiologia , FibroseRESUMO
Fibrosis is a common outcome of numerous pathologies, including chronic kidney disease (CKD), a progressive renal function deterioration. Current approaches to target activated fibroblasts, key effector contributors to fibrotic tissue remodeling, lack specificity. Here, we report Gucy1α1 as a specific kidney fibroblast marker. Gucy1α1 levels significantly increased over the course of two clinically relevant murine CKD models and directly correlated with established fibrosis markers. Immunofluorescent (IF) imaging showed that Gucy1α1 comprehensively labelled cortical and medullary quiescent and activated fibroblasts in the control kidney and throughout injury progression, respectively. Unlike traditionally used markers platelet derived growth factor receptor beta (Pdgfrß) and vimentin (Vim), Gucy1α1 did not overlap with off-target populations such as podocytes. Notably, Gucy1α1 labelled kidney fibroblasts in both male and female mice. Furthermore, we observed elevated GUCY1α1 expression in the human fibrotic kidney and lung. Studies in the murine models of cardiac and liver fibrosis revealed Gucy1α1 elevation in activated Pdgfrß-, Vim- and alpha smooth muscle actin (αSma)-expressing fibroblasts paralleling injury progression and resolution. Overall, we demonstrate Gucy1α1 as an exclusive fibroblast marker in both sexes. Due to its multiorgan translational potential, GUCY1α1 might provide a novel promising strategy to specifically target and mechanistically examine fibroblasts.
RESUMO
Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with "secretory", "contractile" and "vascular" transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8, while the surviving proximal tubules (PTs) showed restored transcriptional signature. Furthermore, we found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.