In the zebrafish (Danio rerio), regeneration and fibrosis after cardiac injury are not mutually exclusive responses. Upon cardiac cryoinjury, collagen and other extracellular matrix (ECM) proteins ...accumulate at the injury site. However, in contrast to the situation in mammals, fibrosis is transient in zebrafish and its regression is concomitant with regrowth of the myocardial wall. Little is known about the cells producing this fibrotic tissue or how it resolves. Using novel genetic tools to mark periostin b- and collagen 1alpha2 (col1a2)-expressing cells in combination with transcriptome analysis, we explored the sources of activated fibroblasts and traced their fate. We describe that during fibrosis regression, fibroblasts are not fully eliminated but become inactivated. Unexpectedly, limiting the fibrotic response by genetic ablation of col1a2-expressing cells impaired cardiomyocyte proliferation. We conclude that ECM-producing cells are key players in the regenerative process and suggest that antifibrotic therapies might be less efficient than strategies targeting fibroblast inactivation.
Mutant KRAS is a common driver in epithelial cancers. Nevertheless, molecular changes occurring early after activation of oncogenic KRAS in epithelial cells remain poorly understood. We compared ...transcriptional changes at single-cell resolution after KRAS activation in four sample sets. In addition to patient samples and genetically engineered mouse models, we developed organoid systems from primary mouse and human induced pluripotent stem cell-derived lung epithelial cells to model early-stage lung adenocarcinoma. In all four settings, alveolar epithelial progenitor (AT2) cells expressing oncogenic KRAS had reduced expression of mature lineage identity genes. These findings demonstrate the utility of our in vitro organoid approaches for uncovering the early consequences of oncogenic KRAS expression. This resource provides an extensive collection of datasets and describes organoid tools to study the transcriptional and proteomic changes that distinguish normal epithelial progenitor cells from early-stage lung cancer, facilitating the search for targets for KRAS-driven tumors.
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•Alveolar epithelial progenitor cells are transcriptionally distinct upon KRAS expression•Alveolar epithelial organoids recapitulate early-stage lung adenocarcinoma•Oncogenic KRAS leads to loss of lineage identity in AT2 cells•Bulk, scRNA-seq, and proteomics data from murine and human KRAS mutant AT2 cells
Early-stage lung cancer is poorly understood. Dost, Moye et al. introduce new organoid systems to model lung cancer. KRAS-expressing alveolar progenitor cells had reduced expression of lineage genes in mouse and organoid models and stage IA cancers. This is the first report of loss of differentiation in early-stage lung cancer.
Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Although infection initiates in the proximal ...airways, severe and sometimes fatal symptoms of the disease are caused by infection of the alveolar type 2 (AT2) cells of the distal lung and associated inflammation. In this study, we develop primary human lung epithelial infection models to understand initial responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface (ALI) cultures of proximal airway epithelium and alveosphere cultures of distal lung AT2 cells are readily infected by SARS-CoV-2, leading to an epithelial cell-autonomous proinflammatory response with increased expression of interferon signaling genes. Studies to validate the efficacy of selected candidate COVID-19 drugs confirm that remdesivir strongly suppresses viral infection/replication. We provide a relevant platform for study of COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and emergent respiratory pathogens.
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•Human alveospheres are composed of renewing AT2 cells and AT1-like cells•Alveolar epithelial cells are efficiently infected by SARS-CoV-2 in vitro•Interferon signaling is activated in SARS-CoV-2-infected alveolar epithelial cells•Lung organoid models provide a platform for drug discovery and disease modeling
In vitro models of human lung epithelium, including diverse cell types of the proximo-distal axis, are critical for modeling infection. Mulay et al. show that alveospheres, with epithelial type 2- and type 1-like cells, are infected by SARS-CoV-2, initiating an interferon response, and serve as a platform for screening antiviral drugs.
The Notch signalling pathway plays fundamental roles in diverse developmental processes in metazoans, where it is important in driving cell fate and directing differentiation of various cell types. ...However, we still have limited knowledge about the role of Notch in early preimplantation stages of mammalian development, or how it interacts with other signalling pathways active at these stages such as Hippo. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos and pluripotent cell culture, we have found that Notch is active from the 4-cell stage. Transcriptomic analysis in single morula identified novel Notch targets, such as early naïve pluripotency markers or transcriptional repressors such as TLE4. Our results reveal a previously undescribed role for Notch in driving transitions during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions.
Idiopathic pulmonary fibrosis is a fatal disease involving destruction of the lung alveolar structure. In this issue of Cell, Wu et al. (2020) show that impaired alveolar (AT2) stem cells produce ...mechanical tension that leads to spatially regulated fibrosis, initiating a new chapter in understanding what underlies the periphery to center progression of this lung disease.
Pulmonary fibrosis is a fatal disease involving destruction of the lung alveolar structure. In this issue of Cell, Wu et al. (2020) show that impaired alveolar (AT2) stem cells produce mechanical tension that leads to spatially regulated fibrosis, initiating a new chapter in understanding what underlies the periphery to center progression of this lung disease.
Type 2 alveolar epithelial cells (AT2s), facultative progenitor cells of the lung alveolus, play a vital role in the biology of the distal lung. In vitro model systems that incorporate human cells, ...recapitulate the biology of primary AT2s, and interface with the outside environment could serve as useful tools to elucidate functional characteristics of AT2s in homeostasis and disease. We and others recently adapted human induced pluripotent stem cell-derived AT2s (iAT2s) for air-liquid interface (ALI) culture. Here, we comprehensively characterize the effects of ALI culture on iAT2s and benchmark their transcriptional profile relative to both freshly sorted and cultured primary human fetal and adult AT2s. We find that iAT2s cultured at ALI maintain an AT2 phenotype while upregulating expression of transcripts associated with AT2 maturation. We then leverage this platform to assay the effects of exposure to clinically significant, inhaled toxicants including cigarette smoke and electronic cigarette vapor.
Pluripotency is regulated by a network of transcription factors that maintain early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for ...maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However,
is expressed during gastrulation across all the posterior epiblast, and only later in development is its expression restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which
specifically represses genes involved in anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed
expression to be negatively correlated with that of
during the early stages of differentiation. We have functionally demonstrated
to be a direct target of NANOG by using a dual transgene system for the controlled expression of
Use of
null ES cells further demonstrated a role for
in repressing a subset of anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of
revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of
inhibiting neural regulatory networks by repressing
at the onset of gastrulation.This article has an associated First Person interview with the joint first authors of the paper.
Progenitors of the first hematopoietic cells in the mouse arise in the early embryo from Brachyury‐positive multipotent cells in the posterior‐proximal region of the epiblast, but the mechanisms that ...specify primitive blood cells are still largely unknown. Pluripotency factors maintain uncommitted cells of the blastocyst and embryonic stem cells in the pluripotent state. However, little is known about the role played by these factors during later development, despite being expressed in the postimplantation epiblast. Using a dual transgene system for controlled expression at postimplantation stages, we found that Nanog blocks primitive hematopoiesis in the gastrulating embryo, resulting in a loss of red blood cells and downregulation of erythropoietic genes. Accordingly, Nanog‐deficient embryonic stem cells are prone to erythropoietic differentiation. Moreover, Nanog expression in adults prevents the maturation of erythroid cells. By analysis of previous data for NANOG binding during stem cell differentiation and CRISPR/Cas9 genome editing, we found that Tal1 is a direct NANOG target. Our results show that Nanog regulates primitive hematopoiesis by directly repressing critical erythroid lineage specifiers.
Synopsis
Mechanisms that specify primitive blood cells during embryonic development are largely unknown. Here, the stem cell factor NANOG is shown to exert important roles during specification from mesoderm to the first hematopoietic cells, instructing haematopoiesis by direct repression of critical lineage specifiers.
NANOG blocks erythroid differentiation in the gastrulating embryo in a cell‐autonomous manner.
Loss of NANOG in embryonic stem cells increases expression of hematopoietic genes and shifts differentiation towards erythroid progenitors.
NANOG can block generation of megakaryocyte‐erythroid progenitors in the adult bone marrow.
Tal1, a key regulator of erythroid fate, is directly repressed by NANOG during gastrulation.
NANOG regulates the transition from mesoderm to primitive hematopoietic system by directly repressing erythroid lineage specification.
Pluripotent cells are a transient population of the mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while suppressing lineage specification. ...However, these factors are also expressed during early phases of differentiation, and their role in the transition from pluripotency to lineage specification is largely unknown. We found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that
Oct4
is necessary for activation of
HoxB
genes during differentiation of embryonic stem cells and in the embryo. In addition, we show that the
HoxB
cluster is coordinately regulated by OCT4 binding sites located at the 3′ end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the precommitted epiblast but are also necessary for the proper deployment of subsequent developmental programs.
The role of OCT4 extends beyond pluripotency to regulate in a dual fashion lineage determinants such as the Hox genes.
The trophectoderm (TE) is the first cell population to appear in the mammalian preimplantation embryo, as the result of the differentiation of totipotent blastomeres located on the outer surface of ...the late morula. Trophectodermal cells arrange in a monolayer covering the expanding blastocyst and acquire an epithelial phenotype with distinct apicobasal polarity and a basal lamina placed toward the blastocyst interior. During later development through the periimplantation and gastrulation stages, the TE gives rise to extraembryonic membranes and cell types that will eventually form most of the fetal placenta, the specialized organ through which the embryo obtains maternal nourishment necessary for subsequent exponential growth. The specification of the TE is controlled by the combination of morphological cues arising from cell polarity with differential activity of signaling pathways such as Hippo and Notch, and the restriction to outer cells of lineage specifiers such as CDX2. This is possibly the first symmetry-breaking decision undertaken by the uncommitted cells produced by a handful of mitosis divisions from the newly fertilized zygote. Understanding how this cell lineage is specified will therefore provide unique information about development, differentiation, and how the interplay between cellular morphology and signaling and regulatory factors results in a correctly 3D-patterned embryo.
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