RESUMO
Critical early steps in human embryonic development include polarization of the inner cell mass, followed by formation of an expanded lumen that will become the epiblast cavity. Recently described three-dimensional (3D) human pluripotent stem cell-derived cyst (hPSC-cyst) structures can replicate these processes. To gain mechanistic insights into the poorly understood machinery involved in epiblast cavity formation, we interrogated the proteomes of apical and basolateral membrane territories in 3D human hPSC-cysts. APEX2-based proximity bioinylation, followed by quantitative mass spectrometry, revealed a variety of proteins without previous annotation to specific membrane subdomains. Functional experiments validated the requirement for several apically enriched proteins in cyst morphogenesis. In particular, we found a key role for the AP-1 clathrin adaptor complex in expanding the apical membrane domains during lumen establishment. These findings highlight the robust power of this proximity labeling approach for discovering novel regulators of epithelial morphogenesis in 3D stem cell-based models.
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The hedgehog (Hh) signaling pathway plays several diverse regulatory and patterning roles during organogenesis of the intestine and in the regulation of adult intestinal homeostasis. In the embryo, fetus, and adult, intestinal Hh signaling is paracrine: Hh ligands are expressed in the endodermally derived epithelium, while signal transduction is confined to the mesenchymal compartment, where at least a dozen distinct cell types are capable of responding to Hh signals. Epithelial Hh ligands not only regulate a variety of mesenchymal cell behaviors, but they also direct these mesenchymal cells to secrete additional soluble factors (e.g., Wnts, Bmps, inflammatory mediators) that feed back to regulate the epithelial cells themselves. Evolutionary conservation of the core Hh signaling pathway, as well as conservation of epithelial/mesenchymal cross talk in the intestine, has meant that work in many diverse model systems has contributed to our current understanding of the role of this pathway in intestinal organogenesis, which is reviewed here.
Assuntos
Proteínas Hedgehog/metabolismo , Homeostase/fisiologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiologia , Intestinos/fisiologia , Transdução de Sinais/fisiologia , Animais , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , HumanosRESUMO
Neural rosettes (NPC rosettes) are radially arranged groups of cells surrounding a central lumen that arise stochastically in monolayer cultures of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPC). Since NPC rosette formation is thought to mimic cell behavior in the early neural tube, these rosettes represent important in vitro models for the study of neural tube morphogenesis. However, using current protocols, NPC rosette formation is not synchronized and results are inconsistent among different hPSC lines, hindering quantitative mechanistic analyses and challenging live cell imaging. Here, we report a rapid and robust protocol to induce rosette formation within 6 h after evenly-sized "colonies" of NPC are generated through physical cutting of uniformly polarized NESTIN+/PAX6+/PAX3+/DACH1+ NPC monolayers. These NPC rosettes show apically polarized lumens studded with primary cilia. Using this assay, we demonstrate reduced lumenal size in the absence of PODXL, an important apical determinant recently identified as a candidate gene for juvenile Parkinsonism. Interestingly, time lapse imaging reveals that, in addition to radial organization and apical lumen formation, cells within cut NPC colonies initiate rapid basally-driven spreading. Further, using chemical, genetic and biomechanical tools, we show that NPC rosette morphogenesis requires this basal spreading activity and that spreading is tightly regulated by Rho/ROCK signaling. This robust and quantitative NPC rosette platform provides a sensitive system for the further investigation of cellular and molecular mechanisms underlying NPC rosette morphogenesis.
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Between embryonic days 10.5 and 14.5, active proliferation drives rapid elongation of the murine midgut epithelial tube. Within this pseudostratified epithelium, nuclei synthesize DNA near the basal surface and move apically to divide. After mitosis, the majority of daughter cells extend a long, basally oriented filopodial protrusion, building a de novo path along which their nuclei can return to the basal side. WNT5A, which is secreted by surrounding mesenchymal cells, acts as a guidance cue to orchestrate this epithelial pathfinding behavior, but how this signal is received by epithelial cells is unknown. Here, we have investigated two known WNT5A receptors: ROR2 and RYK. We found that epithelial ROR2 is dispensable for midgut elongation. However, loss of Ryk phenocopies the Wnt5a-/- phenotype, perturbing post-mitotic pathfinding and leading to apoptosis. These studies reveal that the ligand-receptor pair WNT5A-RYK acts as a navigation system to instruct filopodial pathfinding, a process that is crucial for continuous cell cycling to fuel rapid midgut elongation.
Assuntos
Sistema Digestório/crescimento & desenvolvimento , Sistema Digestório/metabolismo , Pseudópodes/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Apoptose , Núcleo Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Feminino , Masculino , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismoRESUMO
Early human embryonic development involves extensive lineage diversification, cell-fate specification and tissue patterning1. Despite its basic and clinical importance, early human embryonic development remains relatively unexplained owing to interspecies divergence2,3 and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast and amniotic ectoderm parts of the conceptus, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and primitive streak cells. We further show that amniotic ectoderm-like cells function as a signalling centre to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model could assist in the rational design of differentiation protocols of hPSCs for disease modelling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects.
Assuntos
Âmnio/embriologia , Camadas Germinativas/embriologia , Modelos Biológicos , Células-Tronco Pluripotentes/citologia , Âmnio/citologia , Diferenciação Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Feminino , Camadas Germinativas/citologia , Humanos , Gravidez , Linha Primitiva/citologiaRESUMO
During early post-implantation human embryogenesis, the epiblast (EPI) within the blastocyst polarizes to generate a cyst with a central lumen. Cells at the uterine pole of the EPI cyst then undergo differentiation to form the amniotic ectoderm (AM), a tissue essential for further embryonic development. While the causes of early pregnancy failure are complex, improper lumenogenesis or amniogenesis of the EPI represent possible contributing factors. Here we report a novel AM microtissue array platform that allows quantitative phenotyping of lumenogenesis and amniogenesis of the EPI and demonstrate its potential application for embryonic toxicity profiling. Specifically, a human pluripotent stem cell (hPSC)-based amniogenic differentiation protocol was developed using a two-step micropatterning technique to generate a regular AM microtissue array with defined tissue sizes. A computer-assisted analysis pipeline was developed to automatically process imaging data and quantify morphological and biological features of AM microtissues. Analysis of the effects of cell density, cyst size and culture conditions revealed a clear connection between cyst size and amniogenesis of hPSC. Using this platform, we demonstrated that pharmacological inhibition of ROCK signaling, an essential mechanotransductive pathway, suppressed lumenogenesis but did not perturb amniogenic differentiation of hPSC, suggesting uncoupled regulatory mechanisms for AM morphogenesis vs. cytodifferentiation. The AM microtissue array was further applied to screen a panel of clinically relevant drugs, which successfully detected their differential teratogenecity. This work provides a technological platform for toxicological screening of clinically relevant drugs for their effects on lumenogenesis and amniogenesis during early human peri-implantation development, processes that have been previously inaccessible to study.
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Âmnio/citologia , Avaliação Pré-Clínica de Medicamentos , Ectoderma/citologia , Células-Tronco Pluripotentes/citologia , Análise Serial de Tecidos , Âmnio/efeitos dos fármacos , Âmnio/metabolismo , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Ectoderma/efeitos dos fármacos , Ectoderma/metabolismo , Humanos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Análise Serial de Tecidos/métodos , Engenharia Tecidual/métodos , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
The adult gastrointestinal tract (GI) is a series of connected organs (esophagus, stomach, small intestine, colon) that develop via progressive regional specification of a continuous tubular embryonic organ anlage. This chapter focuses on organogenesis of the small intestine. The intestine arises by folding of a flat sheet of endodermal cells into a tube of highly proliferative pseudostratified cells. Dramatic elongation of this tube is driven by rapid epithelial proliferation. Then, epithelial-mesenchymal crosstalk and physical forces drive a stepwise cascade that results in convolution of the tubular surface into finger-like projections called villi. Concomitant with villus formation, a sharp epithelial transcriptional boundary is defined between stomach and intestine. Finally, flask-like depressions called crypts are established to house the intestinal stem cells needed throughout life for epithelial renewal. New insights into these events are being provided by in vitro organoid systems, which hold promise for future regenerative engineering of the small intestine.
Assuntos
Padronização Corporal/genética , Intestino Delgado/embriologia , Organogênese/genética , Transdução de Sinais/genética , Animais , Proliferação de Células/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Microvilosidades/metabolismoRESUMO
Proper development of the human embryo following its implantation into the uterine wall is critical for the successful continuation of pregnancy. However, the complex cellular and molecular changes that occur during this post-implantation period of human development are not amenable to study in vivo. Recently, several new embryo-like human pluripotent stem cell (hPSC)-based platforms have emerged, which are beginning to illuminate the current black box state of early human post-implantation biology. In this review, we will discuss how these experimental models are carving a way for understanding novel molecular and cellular mechanisms during early human development.
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Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Animais , Embrião de Mamíferos/citologia , Humanos , Células-Tronco Pluripotentes/citologiaRESUMO
The early midgut undergoes intensive elongation, but the underlying cellular and molecular mechanisms are unknown. The early midgut epithelium is pseudostratified, and its nuclei travel between apical and basal surfaces in concert with cell cycle. Using 3D confocal imaging and 2D live imaging, we profiled behaviors of individual dividing cells. As nuclei migrate apically for mitosis, cells maintain a basal process (BP), which splits but is inherited by only one daughter. After mitosis, some daughters directly use the inherited BP as a "conduit" to transport the nucleus basally, while >50% of daughters generate a new basal filopodium and use it as a path to return the nucleus. Post-mitotic filopodial "pathfinding" is guided by mesenchymal WNT5A. Without WNT5A, some cells fail to tether basally and undergo apoptosis, leading to a shortened midgut. Thus, these studies reveal previously unrecognized strategies for efficient post-mitotic nuclear trafficking, which is critical for early midgut elongation.
Assuntos
Pseudópodes/fisiologia , Proteína Wnt-5a/fisiologia , Animais , Ciclo Celular , Movimento Celular , Núcleo Celular/metabolismo , Sistema Digestório/metabolismo , Fenômenos Fisiológicos do Sistema Digestório , Endoderma , Epitélio/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitose/fisiologia , Proteína Wnt-5a/metabolismoRESUMO
Efficient absorption of nutrients by the intestine is essential for life. In mammals and birds, convolution of the intestinal surface into finger-like projections called villi is an important adaptation that ensures the massive surface area for nutrient contact that is required to meet metabolic demands. Each villus projection serves as a functional absorptive unit: it is covered by a simple columnar epithelium that is derived from endoderm and contains a mesodermally derived core with supporting vasculature, lacteals, enteric nerves, smooth muscle, fibroblasts, myofibroblasts, and immune cells. In cross section, the consistency of structure in the billions of individual villi of the adult intestine is strikingly beautiful. Villi are generated in fetal life, and work over several decades has revealed that villus morphogenesis requires substantial "crosstalk" between the endodermal and mesodermal tissue components, with soluble signals, cell-cell contacts, and mechanical forces providing specific dialects for sequential conversations that orchestrate villus assembly. A key part of this process is the formation of subepithelial mesenchymal cell clusters that act as signaling hubs, directing overlying epithelial cells to cease proliferation, thereby driving villus emergence and simultaneously determining the location of future stem cell compartments. Interestingly, distinct species-specific differences govern how and when tissue-shaping signals and forces generate mesenchymal clusters and control villus emergence. As the details of villus development become increasingly clear, the emerging picture highlights a sophisticated local self-assembled cascade that underlies the reproducible elaboration of a regularly patterned field of absorptive villus units. This article is categorized under: Vertebrate Organogenesis > From a Tubular Primordium: Non-Branched Comparative Development and Evolution > Organ System Comparisons Between Species Early Embryonic Development > Development to the Basic Body Plan.
Assuntos
Mucosa Intestinal/fisiologia , Microvilosidades/fisiologia , Organogênese/fisiologia , Transdução de Sinais , Animais , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/embriologia , Camundongos , Ratos , Especificidade da EspécieRESUMO
Human pluripotent stem cells (hPSCs) self-organize into apicobasally polarized cysts, reminiscent of the lumenal epiblast stage, providing a model to explore key morphogenic processes in early human embryos. Here, we show that apical polarization begins on the interior of single hPSCs through the dynamic formation of a highly organized perinuclear apicosome structure. The membrane surrounding the apicosome is enriched in apical markers and displays microvilli and a primary cilium; its lumenal space is rich in Ca2+ Time-lapse imaging of isolated hPSCs reveals that the apicosome forms de novo in interphase, retains its structure during mitosis, is asymmetrically inherited after mitosis, and relocates to the recently formed cytokinetic plane, where it establishes a fully polarized lumen. In a multicellular aggregate of hPSCs, intracellular apicosomes from multiple cells are trafficked to generate a common lumenal cavity. Thus, the apicosome is a unique preassembled apical structure that can be rapidly used in single or clustered hPSCs to initiate self-organized apical polarization and lumenogenesis.
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Citocinese , Camadas Germinativas/ultraestrutura , Morfogênese/genética , Células-Tronco Pluripotentes/ultraestrutura , Actinas/genética , Actinas/metabolismo , Biomarcadores/metabolismo , Cálcio/metabolismo , Calnexina/genética , Calnexina/metabolismo , Linhagem Celular , Polaridade Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Expressão Gênica , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Humanos , Interfase , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Mitose , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Análise de Célula Única , Imagem com Lapso de TempoRESUMO
Development of the asymmetric amniotic sac-with the embryonic disc and amniotic ectoderm occupying opposite poles-is a vital milestone during human embryo implantation. Although essential to embryogenesis and pregnancy, amniotic sac development in humans remains poorly understood. Here, we report a human pluripotent stem cell (hPSC)-based model, termed the post-implantation amniotic sac embryoid (PASE), that recapitulates multiple post-implantation embryogenic events centered around amniotic sac development. Without maternal or extraembryonic tissues, the PASE self-organizes into an epithelial cyst with an asymmetric amniotic ectoderm-epiblast pattern that resembles the human amniotic sac. Upon further development, the PASE initiates a process that resembles posterior primitive streak development in a SNAI1-dependent manner. Furthermore, we observe asymmetric BMP-SMAD signaling concurrent with PASE development, and establish that BMP-SMAD activation/inhibition modulates stable PASE development. This study reveals a previously unrecognized fate potential of human pluripotent stem cells and provides a platform for advancing human embryology.Early in human embryonic development, it is unclear how amniotic sac formation is regulated. Here, the authors use a human pluripotent stem cell-based model, termed the post-implantation amniotic sac embryoid, to recapitulate early embryogenic events of human amniotic sac development.
Assuntos
Âmnio/embriologia , Implantação do Embrião , Desenvolvimento Embrionário , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Âmnio/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Ectoderma/embriologia , Ectoderma/metabolismo , Feminino , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Humanos , Gravidez , Transdução de Sinais , Proteínas Smad/metabolismo , Fatores de Transcrição da Família Snail/metabolismoRESUMO
Amniogenesis-the development of amnion-is a critical developmental milestone for early human embryogenesis and successful pregnancy. However, human amniogenesis is poorly understood due to limited accessibility to peri-implantation embryos and a lack of in vitro models. Here we report an efficient biomaterial system to generate human amnion-like tissue in vitro through self-organized development of human pluripotent stem cells (hPSCs) in a bioengineered niche mimicking the in vivo implantation environment. We show that biophysical niche factors act as a switch to toggle hPSC self-renewal versus amniogenesis under self-renewal-permissive biochemical conditions. We identify a unique molecular signature of hPSC-derived amnion-like cells and show that endogenously activated BMP-SMAD signalling is required for the amnion-like tissue development by hPSCs. This study unveils the self-organizing and mechanosensitive nature of human amniogenesis and establishes the first hPSC-based model for investigating peri-implantation human amnion development, thereby helping advance human embryology and reproductive medicine.
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Âmnio/metabolismo , Materiais Biomiméticos , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Nicho de Células-Tronco , Engenharia Tecidual/métodos , Âmnio/citologia , Linhagem Celular , Humanos , Células-Tronco Pluripotentes/citologia , Medicina Reprodutiva/métodosRESUMO
During late gestation, villi extend into the intestinal lumen to dramatically increase the surface area of the intestinal epithelium, preparing the gut for the neonatal diet. Incomplete development of the intestine is the most common gastrointestinal complication in neonates, but the causes are unclear. We provide evidence in mice that Yin Yang 1 (Yy1) is crucial for intestinal villus development. YY1 loss in the developing endoderm had no apparent consequences until late gestation, after which the intestine differentiated poorly and exhibited severely stunted villi. Transcriptome analysis revealed that YY1 is required for mitochondrial gene expression, and ultrastructural analysis confirmed compromised mitochondrial integrity in the mutant intestine. We found increased oxidative phosphorylation gene expression at the onset of villus elongation, suggesting that aerobic respiration might function as a regulator of villus growth. Mitochondrial inhibitors blocked villus growth in a fashion similar to Yy1 loss, thus further linking oxidative phosphorylation with late-gestation intestinal development. Interestingly, we find that necrotizing enterocolitis patients also exhibit decreased expression of oxidative phosphorylation genes. Our study highlights the still unappreciated role of metabolic regulation during organogenesis, and suggests that it might contribute to neonatal gastrointestinal disorders.
Assuntos
Mucosa Intestinal/metabolismo , Intestinos/citologia , Organogênese/fisiologia , Fator de Transcrição YY1/metabolismo , Aerobiose/genética , Aerobiose/fisiologia , Animais , Western Blotting , Genótipo , Imuno-Histoquímica , Masculino , Camundongos , Organogênese/genética , Fosforilação Oxidativa , Transcriptoma/genética , Fator de Transcrição YY1/genéticaRESUMO
Efficient digestion and absorption of nutrients by the intestine requires a very large apical surface area, a feature that is enhanced by the presence of villi, fingerlike epithelial projections that extend into the lumen. Prior to villus formation, the epithelium is a thick pseudostratified layer. In mice, villus formation begins at embryonic day (E)14.5, when clusters of mesenchymal cells form just beneath the thick epithelium. At this time, analysis of the flat lumenal surface reveals a regular pattern of short apical membrane invaginations that form in regions of the epithelium that lie in between the mesenchymal clusters. Apical invaginations begin in the proximal intestine and spread distally, deepening with time. Interestingly, mitotically rounded cells are frequently associated with these invaginations. These mitotic cells are located at the tips of the invaginating membrane (internalized within the epithelium), rather than adjacent to the apical surface. Further investigation of epithelial changes during membrane invagination reveals that epithelial cells located between mesenchymal clusters experience a circumferential compression, as epithelial cells above each cluster shorten and widen. Using a computational model, we examined whether such forces are sufficient to cause apical invaginations. Simulations and in vivo data reveal that proper apical membrane invagination involves intraepithelial compressive forces, mitotic cell rounding in the compressed regions and apico-basal contraction of the dividing cell. Together, these data establish a new model that explains how signaling events intersect with tissue forces to pattern apical membrane invaginations that define the villus boundaries.
Assuntos
Mucosa Intestinal/fisiologia , Mecanotransdução Celular/fisiologia , Microvilosidades/fisiologia , Microvilosidades/ultraestrutura , Mitose/fisiologia , Modelos Biológicos , Morfogênese/fisiologia , Animais , Tamanho Celular , Força Compressiva/fisiologia , Simulação por Computador , Humanos , Mucosa Intestinal/ultraestrutura , Camundongos , Estresse MecânicoRESUMO
The vertebrate small intestine requires an enormous surface area to effectively absorb nutrients from food. Morphological adaptations required to establish this extensive surface include generation of an extremely long tube and convolution of the absorptive surface of the tube into villi and microvilli. In this Review, we discuss recent findings regarding the morphogenetic and molecular processes required for intestinal tube elongation and surface convolution, examine shared and unique aspects of these processes in different species, relate these processes to known human maladies that compromise absorptive function and highlight important questions for future research.
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Absorção Intestinal , Intestinos/crescimento & desenvolvimento , Animais , Humanos , Microvilosidades/metabolismo , Modelos Biológicos , Morfogênese , Transdução de SinaisRESUMO
BACKGROUND: Digestion is facilitated by coordinated contractions of the intestinal muscularis externa, a bilayered smooth muscle structure that is composed of inner circular muscles (ICM) and outer longitudinal muscles (OLM). We performed transcriptome analysis of intestinal mesenchyme tissue at E14.5, when the ICM, but not the OLM, is present, to investigate the transcriptional program of the ICM. RESULTS: We identified 3967 genes enriched in E14.5 intestinal mesenchyme. The gene expression profiles were clustered and annotated to known muscle genes, identifying a muscle-enriched subcluster. Using publically available in situ data, 127 genes were verified as expressed in ICM. Examination of the promoter and regulatory regions for these co-expressed genes revealed enrichment for cJUN transcription factor binding sites, and cJUN protein was enriched in ICM. cJUN ChIP-seq, performed at E14.5, revealed that cJUN regulatory regions contain characteristics of muscle enhancers. Finally, we show that cJun is a target of Hedgehog (Hh), a signaling pathway known to be important in smooth muscle development, and identify a cJun genomic enhancer that is responsive to Hh. CONCLUSIONS: This work provides the first transcriptional catalog for the developing ICM and suggests that cJun regulates gene expression in the ICM downstream of Hh signaling. Developmental Dynamics 245:614-626, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Intestinos/embriologia , Músculo Liso/embriologia , Transcriptoma , Animais , Genes jun/fisiologia , Proteínas Hedgehog , CamundongosRESUMO
Gastric adenocarcinoma is the third most common cause of cancer-related death worldwide. Here we report a novel, highly-penetrant mouse model of invasive gastric cancer arising from deregulated Hedgehog/Gli2 signaling targeted to Lgr5-expressing stem cells in adult stomach. Tumor development progressed rapidly: three weeks after inducing the Hh pathway oncogene GLI2A, 65% of mice harbored in situ gastric cancer, and an additional 23% of mice had locally invasive tumors. Advanced mouse gastric tumors had multiple features in common with human gastric adenocarcinomas, including characteristic histological changes, expression of RNA and protein markers, and the presence of major inflammatory and stromal cell populations. A subset of tumor cells underwent epithelial-mesenchymal transition, likely mediated by focal activation of canonical Wnt signaling and Snail1 induction. Strikingly, mTOR pathway activation, based on pS6 expression, was robustly activated in mouse gastric adenocarcinomas from the earliest stages of tumor development, and treatment with rapamycin impaired tumor growth. GLI2A-expressing epithelial cells were detected transiently in intestine, which also contains Lgr5+ stem cells, but they did not give rise to epithelial tumors in this organ. These findings establish that deregulated activation of Hedgehog/Gli2 signaling in Lgr5-expressing stem cells is sufficient to drive gastric adenocarcinoma development in mice, identify a critical requirement for mTOR signaling in the pathogenesis of these tumors, and underscore the importance of tissue context in defining stem cell responsiveness to oncogenic stimuli.
Assuntos
Adenocarcinoma/patologia , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Neoplásicas/patologia , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Gástricas/patologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Nus , Proteínas Quinases S6 Ribossômicas/metabolismo , Sirolimo/uso terapêutico , Fatores de Transcrição da Família Snail/metabolismo , Via de Sinalização Wnt , Proteína Gli2 com Dedos de Zinco , beta Catenina/metabolismoRESUMO
BACKGROUND: The Hedgehog (Hh) signaling pathway, acting through three homologous transcription factors (GLI1, GLI2, GLI3) in vertebrates, plays multiple roles in embryonic organ development and adult tissue homeostasis. At the level of the genome, GLI factors bind to specific motifs in enhancers, some of which are hundreds of kilobases removed from the gene promoter. These enhancers integrate the Hh signal in a context-specific manner to control the spatiotemporal pattern of target gene expression. Importantly, a number of genes that encode Hh pathway molecules are themselves targets of Hh signaling, allowing pathway regulation by an intricate balance of feed-back activation and inhibition. However, surprisingly few of the critical enhancer elements that control these pathway target genes have been identified despite the fact that such elements are central determinants of Hh signaling activity. Recently, ChIP studies have been carried out in multiple tissue contexts using mouse models carrying FLAG-tagged GLI proteins (GLI(FLAG)). Using these datasets, we tested whether a meta-analysis of GLI binding sites, coupled with a machine learning approach, could reveal genomic features that could be used to empirically identify Hh-regulated enhancers linked to loci of the Hh signaling pathway. RESULTS: A meta-analysis of four existing GLI(FLAG) datasets revealed a library of GLI binding motifs that was substantially more restricted than the potential sites predicted by previous in vitro binding studies. A machine learning method (kmer-SVM) was then applied to these datasets and enriched k-mers were identified that, when applied to the mouse genome, predicted as many as 37,000 potential Hh enhancers. For functional analysis, we selected nine regions which were annotated to putative Hh pathway molecules and found that seven exhibited GLI-dependent activity, indicating that they are directly regulated by Hh signaling (78% success rate). CONCLUSIONS: The results suggest that Hh enhancer regions share common sequence features. The kmer-SVM machine learning approach identifies those features and can successfully predict functional Hh regulatory regions in genomic DNA surrounding Hh pathway molecules and likely, other Hh targets. Additionally, the library of enriched GLI binding motifs that we have identified may allow improved identification of functional GLI binding sites.