RESUMO
Genotype-phenotype associations for common diseases are often compounded by pleiotropy and metabolic state. Here, we devised a pooled human organoid-panel of steatohepatitis to investigate the impact of metabolic status on genotype-phenotype association. En masse population-based phenotypic analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH)-genetic factors including the glucokinase regulatory protein (GCKR)-rs1260326:C>T. Analysis of NASH clinical cohorts revealed that GCKR-rs1260326-T allele elevates disease severity only under diabetic state but protects from fibrosis under non-diabetic states. Transcriptomic, metabolomic, and pharmacological analyses indicate significant mitochondrial dysfunction incurred by GCKR-rs1260326, which was not reversed with metformin. Uncoupling oxidative mechanisms mitigated mitochondrial dysfunction and permitted adaptation to increased fatty acid supply while protecting against oxidant stress, forming a basis for future therapeutic approaches for diabetic NASH. Thus, "in-a-dish" genotype-phenotype association strategies disentangle the opposing roles of metabolic-associated gene variant functions and offer a rich mechanistic, diagnostic, and therapeutic inference toolbox toward precision hepatology. VIDEO ABSTRACT.
Assuntos
Predisposição Genética para Doença , Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/genética , Organoides , Estudos de Associação Genética , Alelos , FígadoRESUMO
The in vitro differentiation of pluripotent stem cells into human intestinal organoids (HIOs) has served as a powerful means for creating complex three-dimensional intestinal structures. Owing to their diverse cell populations, transplantation into an animal host is supported with this system and allows the temporal formation of fully laminated structures, including crypt-villus architecture and smooth muscle layers that resemble native human intestine. Although the endpoint of HIO engraftment has been well described, here we aim to elucidate the developmental stages of HIO engraftment and establish whether it parallels fetal human intestinal development. We analyzed a time course of transplanted HIOs histologically at 2, 4, 6 and 8â weeks post-transplantation, and demonstrated that HIO maturation closely resembles key stages of fetal human intestinal development. We also utilized single-nuclear RNA sequencing to determine and track the emergence of distinct cell populations over time, and validated our transcriptomic data through in situ protein expression. These observations suggest that transplanted HIOs do indeed recapitulate early intestinal development, solidifying their value as a human intestinal model system.
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Intestinos , Células-Tronco Pluripotentes , Animais , Humanos , Mucosa Intestinal/metabolismo , Organoides , Diferenciação CelularRESUMO
Primary cilia are nearly ubiquitous organelles that transduce molecular and mechanical signals. Although the basic structure of the cilium and the cadre of genes that contribute to ciliary formation and function (the ciliome) are believed to be evolutionarily conserved, the presentation of ciliopathies with narrow, tissue-specific phenotypes and distinct molecular readouts suggests that an unappreciated heterogeneity exists within this organelle. Here, we provide a searchable transcriptomic resource for a curated primary ciliome, detailing various subgroups of differentially expressed genes within the ciliome that display tissue and temporal specificity. Genes within the differentially expressed ciliome exhibited a lower level of functional constraint across species, suggesting organism and cell-specific function adaptation. The biological relevance of ciliary heterogeneity was functionally validated by using Cas9 gene-editing to disrupt ciliary genes that displayed dynamic gene expression profiles during osteogenic differentiation of multipotent neural crest cells. Collectively, this novel primary cilia-focused resource will allow researchers to explore longstanding questions related to how tissue and cell-type specific functions and ciliary heterogeneity may contribute to the range of phenotypes associated with ciliopathies.
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Ciliopatias , Osteogênese , Humanos , Cílios/genética , Cílios/metabolismo , Ciliopatias/genética , Desenvolvimento Embrionário/genética , Diferenciação Celular/genéticaRESUMO
Notch signaling promotes maturation of nephron epithelia, but its proposed contribution to nephron segmentation into proximal and distal domains has been called into doubt. We leveraged single cell and bulk RNA-seq, quantitative immunofluorescent lineage/fate tracing, and genetically modified human induced pluripotent stem cells (iPSCs) to revisit this question in developing mouse kidneys and human kidney organoids. We confirmed that Notch signaling is needed for maturation of all nephron lineages, and thus mature lineage markers fail to detect a fate bias. By contrast, early markers identified a distal fate bias in cells lacking Notch2, and a concomitant increase in early proximal and podocyte fates in cells expressing hyperactive Notch1 was observed. Orthogonal support for a conserved role for Notch signaling in the distal/proximal axis segmentation is provided by the demonstration that nicastrin (NCSTN)-deficient human iPSC-derived organoids differentiate into TFA2B+ distal tubule and CDH1+ connecting segment progenitors, but not into HNF4A+ or LTL+ proximal progenitors.
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Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes Induzidas , Animais , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/metabolismo , Camundongos , Néfrons/metabolismo , Organogênese/genética , Receptores Notch/genética , Receptores Notch/metabolismoRESUMO
Cooperative DNA binding is a key feature of transcriptional regulation. Here we examined the role of cooperativity in Notch signaling by CRISPR-mediated engineering of mice in which neither Notch1 nor Notch2 can homo- or heterodimerize, essential for cooperative binding to sequence-paired sites (SPS) located near many Notch-regulated genes. Although most known Notch-dependent phenotypes were unaffected in Notch1/2 dimer-deficient mice, a subset of tissues proved highly sensitive to loss of cooperativity. These phenotypes include heart development, compromised viability in combination with low gene dose, and the gut, developing ulcerative colitis in response to 1% dextran sulfate sodium (DSS). The most striking phenotypes-gender imbalance and splenic marginal zone B-cell lymphoma-emerged in combination with gene dose reduction or when challenged by chronic fur mite infestation. This study highlights the role of the environment in malignancy and colitis and is consistent with Notch-dependent anti-parasite immune responses being compromised in Notch dimer-deficient animals.
Assuntos
Linfócitos B/imunologia , Dosagem de Genes , Coração/embriologia , Homeostase , Intestinos/patologia , Infestações por Ácaros/imunologia , Receptores Notch/genética , Células-Tronco/patologia , Alelos , Animais , Sequência de Bases , Proliferação de Células , Cromatina/metabolismo , Sulfato de Dextrana , Ventrículos do Coração/embriologia , Ventrículos do Coração/patologia , Camundongos , Ácaros/fisiologia , Modelos Biológicos , Multimerização Proteica , Receptores Notch/metabolismo , Baço/imunologia , Esplenomegalia/imunologia , Esplenomegalia/parasitologia , Células-Tronco/metabolismoRESUMO
BACKGROUND: Ontologies of precisely defined, controlled vocabularies are essential to curate the results of biological experiments such that the data are machine searchable, can be computationally analyzed, and are interoperable across the biomedical research continuum. There is also an increasing need for methods to interrelate phenotypic data easily and accurately from experiments in animal models with human development and disease. RESULTS: Here we present the Xenopus phenotype ontology (XPO) to annotate phenotypic data from experiments in Xenopus, one of the major vertebrate model organisms used to study gene function in development and disease. The XPO implements design patterns from the Unified Phenotype Ontology (uPheno), and the principles outlined by the Open Biological and Biomedical Ontologies (OBO Foundry) to maximize interoperability with other species and facilitate ongoing ontology management. Constructed in Web Ontology Language (OWL) the XPO combines the existing uPheno library of ontology design patterns with additional terms from the Xenopus Anatomy Ontology (XAO), the Phenotype and Trait Ontology (PATO) and the Gene Ontology (GO). The integration of these different ontologies into the XPO enables rich phenotypic curation, whilst the uPheno bridging axioms allows phenotypic data from Xenopus experiments to be related to phenotype data from other model organisms and human disease. Moreover, the simple post-composed uPheno design patterns facilitate ongoing XPO development as the generation of new terms and classes of terms can be substantially automated. CONCLUSIONS: The XPO serves as an example of current best practices to help overcome many of the inherent challenges in harmonizing phenotype data between different species. The XPO currently consists of approximately 22,000 terms and is being used to curate phenotypes by Xenbase, the Xenopus Model Organism Knowledgebase, forming a standardized corpus of genotype-phenotype data that can be directly related to other uPheno compliant resources.
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Ontologias Biológicas , Animais , Ontologia Genética , Humanos , Fenótipo , Xenopus laevisRESUMO
Xenbase (www.xenbase.org) is a knowledge base for researchers and biomedical scientists that employ the amphibian Xenopus as a model organism in biomedical research to gain a deeper understanding of developmental and disease processes. Through expert curation and automated data provisioning from various sources Xenbase strives to integrate the body of knowledge on Xenopus genomics and biology together with the visualization of biologically significant interactions. Most current studies utilize next generation sequencing (NGS) but until now the results of different experiments were difficult to compare and not integrated with other Xenbase content. Xenbase has developed a suite of tools, interfaces and data processing pipelines that transforms NCBI Gene Expression Omnibus (GEO) NGS content into deeply integrated gene expression and chromatin data, mapping all aligned reads to the most recent genome builds. This content can be queried and visualized via multiple tools and also provides the basis for future automated 'gene expression as a phenotype' and gene regulatory network analyses.
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Bases de Dados Genéticas , Redes Reguladoras de Genes/genética , Genômica , Software , Xenopus/genética , Animais , Sequenciamento de Cromatina por Imunoprecipitação , Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , RNA-Seq , Interface Usuário-ComputadorRESUMO
The N-ethyl-N-nitrosourea (ENU) âforward genetic screen is a useful tool for the unbiased discovery of novel mechanisms regulating developmental processes. We recovered the dorothy mutation in such a screen designed to recover recessive mutations affecting craniofacial development in the mouse. Dorothy embryos die prenatally and exhibit many striking phenotypes commonly associated with ciliopathies, including a severe midfacial clefting phenotype. We used exome sequencing to discover a missense mutation in nucleotide binding protein 2 (Nubp2) to be causative. This finding was confirmed by a complementation assay with the dorothy allele and an independent Nubp2 null allele (Nubp2null). We demonstrated that Nubp2 is indispensable for embryogenesis. NUBP2 is implicated in both the cytosolic iron/sulfur cluster assembly pathway and negative regulation of ciliogenesis. Conditional ablation of Nubp2 in the neural crest lineage with Wnt1-cre recapitulates the dorothy craniofacial phenotype. Using this model, we found that the proportion of ciliated cells in the craniofacial mesenchyme was unchanged, and that markers of the SHH, FGF, and BMP signaling pathways are unaltered. Finally, we show evidence that the phenotype results from a marked increase in apoptosis within the craniofacial mesenchyme.
Assuntos
Proteínas de Ligação ao GTP/genética , Crista Neural/embriologia , Animais , Desenvolvimento Embrionário/genética , Etilnitrosoureia , Feminino , Proteínas de Ligação ao GTP/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese/genética , Mutação/genética , Crista Neural/metabolismo , Fenótipo , Transdução de Sinais/fisiologia , Crânio/metabolismo , Proteína Wnt1/metabolismoRESUMO
Digestive system development is orchestrated by combinatorial signaling interactions between endoderm and mesoderm, but how these signals are interpreted in the genome is poorly understood. Here we identified the transcriptomes of Xenopus foregut and hindgut progenitors, which are conserved with mammals. Using RNA-seq and ChIP-seq we show that BMP/Smad1 regulates dorsal-ventral gene expression in both the endoderm and mesoderm, whereas Wnt/ß-catenin acts as a genome-wide toggle between foregut and hindgut programs. Unexpectedly, ß-catenin and Smad1 binding were associated with both transcriptional activation and repression, with Wnt-repressed genes often lacking canonical Tcf DNA binding motifs, suggesting a novel mode of direct repression. Combinatorial Wnt and BMP signaling was mediated by Smad1 and ß-catenin co-occupying hundreds of cis-regulatory DNA elements, and by a crosstalk whereby Wnt negatively regulates BMP ligand expression in the foregut. These results extend our understanding of gastrointestinal organogenesis and of how Wnt and BMP might coordinate genomic responses in other contexts.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Sistema Digestório/metabolismo , Genoma , Proteína Smad1/metabolismo , Transcrição Gênica , Via de Sinalização Wnt/genética , Xenopus laevis/genética , Animais , Sequência de Bases , Padronização Corporal/genética , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ligação Proteica , Transcriptoma/genética , Xenopus laevis/embriologia , beta Catenina/metabolismoRESUMO
Xenbase (www.xenbase.org) is an online resource for researchers utilizing Xenopus laevis and Xenopus tropicalis, and for biomedical scientists seeking access to data generated with these model systems. Content is aggregated from a variety of external resources and also generated by in-house curation of scientific literature and bioinformatic analyses. Over the past two years many new types of content have been added along with new tools and functionalities to reflect the impact of high-throughput sequencing. These include new genomes for both supported species (each with chromosome scale assemblies), new genome annotations, genome segmentation, dynamic and interactive visualization for RNA-Seq data, updated ChIP-Seq mapping, GO terms, protein interaction data, ORFeome support, and improved connectivity to other biomedical and bioinformatic resources.
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Bases de Dados Genéticas , Epigenômica , Genoma , Transcriptoma , Xenopus/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Imunoprecipitação da Cromatina , Biologia Computacional/organização & administração , Bases de Dados de Ácidos Nucleicos , Ontologia Genética , Genômica , MicroRNAs/genética , Anotação de Sequência Molecular , Fases de Leitura Aberta/genética , RNA/genética , Software , Interface Usuário-Computador , Navegador , Xenopus laevis/genéticaRESUMO
During early fetal development, paracrine Hedgehog (HH) ligands secreted from the foregut epithelium activate Gli transcription factors in the surrounding mesenchyme to coordinate formation of the respiratory system, digestive track and the cardiovascular network. Although disruptions to this process can lead to devastating congenital defects, the underlying mechanisms and downstream targets, are poorly understood. We show that the zinc finger transcription factor Osr1 is a novel HH target as Osr1 expression in the foregut mesenchyme depends on HH signaling and the effector of HH pathway Gli3 binds to a conserved genomic loci near Osr1 promoter region. Molecular analysis of mouse germline Osr1 mutants reveals multiple functions of Osr1 during foregut development. Osr1 mutants exhibit fewer lung progenitors in the ventral foregut. Osr is then required for the proper branching of the primary lung buds, with mutants exhibiting miss-located lung lobes. Finally, Osr1 is essential for proper mesenchymal differentiation including pulmonary arteries, esophageal and tracheal smooth muscle as well as tracheal cartilage rings. Tissue specific conditional knockouts in combination with lineage tracing indicate that Osr1 is required cell autonomously in the foregut mesenchyme. We conclude that Osr1 is a novel downstream target of HH pathway, required for lung specification, branching morphogenesis and foregut mesenchymal differentiation.
Assuntos
Sistema Digestório/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Organogênese/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Animais , Sistema Digestório/embriologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Proteínas Hedgehog/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Pulmão/embriologia , Pulmão/metabolismo , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de ZincoRESUMO
ETS transcription factor ETV2/Etsrp functions as a key regulator of embryonic vascular development in multiple vertebrates. However, its role in pathological vascular development has not been previously investigated. To analyze its role in tumor angiogenesis, we utilized a zebrafish xenotransplantation model. Using a photoconvertible kdrl:NLS-KikGR line, we demonstrated that all tumor vessels originate from the existing embryonic vasculature by the mechanism of angiogenesis. Xenotransplantation of mouse B16 melanoma cells resulted in a significant increase in expression of the ETS transcription factors etv2 and fli1b expression throughout the embryonic vasculature. etv2 null mutants which undergo significant recovery of embryonic angiogenesis during later developmental stages displayed a strong inhibition of tumor angiogenesis. We utilized highly specific and fully validated photoactivatable morpholinos to inhibit Etv2 function after embryonic vasculogenesis has completed. Inducible inhibition of Etv2 function resulted in a significant reduction of tumor angiogenesis and inhibition of tumor growth. Furthermore, inducible inhibition of Etv2 function in fli1b mutant embryos resulted in even stronger reduction in tumor angiogenesis and growth, demonstrating that Etv2 and Fli1b have a partially redundant requirement during tumor angiogenesis. These results demonstrate the requirement for Etv2 and Fli1b in tumor angiogenesis and suggest that inhibition of these ETS factors may present a novel strategy to inhibit tumor angiogenesis and reduce tumor growth.
Assuntos
Neoplasias/irrigação sanguínea , Neoplasias/genética , Neovascularização Patológica/genética , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/patologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Melanoma Experimental/patologia , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/patologia , Fatores de Transcrição/genética , Regulação para Cima/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
The fetal liver is a hematopoietic organ, hosting a diverse and evolving progenitor population. While human liver organoids derived from pluripotent stem cells (PSCs) mimic aspects of embryonic and fetal development, they typically lack the complex hematopoietic niche and the interaction between hepatic and hematopoietic development. We describe the generation of human Fetal Liver-like Organoids (FLOs), that model human hepato-hematopoietic interactions previously characterized in mouse models. Developing FLOs first integrate a yolk sac-like hemogenic endothelium into hepatic endoderm and mesoderm specification. As the hepatic and hematopoietic lineages differentiate, the FLO culture model establishes an autonomous niche capable of driving subsequent progenitor differentiation without exogenous factors. Consistent with yolk sac-derived waves, hematopoietic progenitor cells (HPCs) within FLOs exhibit multipotency with a preference for myeloid lineage commitment, while retaining fetal B and T cell differentiation potential. We reconstruct in FLOs the embryonic monocyte-to-macrophage and granulocyte immune trajectories within the FLO microenvironment and assess their functional responses in the liver niche. In vivo, FLOs demonstrate a liver engraftment bias of hematopoietic cells, recapitulating a key phenomenon of human hematopoietic ontogeny. Our findings highlight the intrinsic capacity of liver organoids to support hematopoietic development, establishing FLOs as a platform for modeling and manipulating human blood-liver niche interactions during critical stages of development and disease.
RESUMO
Lifelong kidney function relies on the complement of nephrons generated during mammalian development from a mesenchymal nephron progenitor cell (NPC) population. Low nephron endowment confers increased susceptibility to chronic kidney disease. We asked whether reduced nephron numbers in the popular Six2TGC transgenic mouse line 1 was due to disruption of a regulatory gene at the integration site or to ectopic expression of a gene(s) contained within the transgene. Targeted locus amplification identified integration of the Six2TGC transgene within an intron of Cntnap5a on chr1. We generated Hi-C datasets from NPCs isolated from the Six2TGC tg/+ mice, the Cited1 CreERT2/+ control mice, and the Six2TGC tg/+ ; Tsc1 +/Flox,2 mice that exhibited restored nephron number compared with Six2TGC tg/+ mice, and mapped the precise integration of Six2TGC and Cited1 CreERT2 transgenes to chr1 and chr14, respectively. No changes in topology, accessibility, or expression were observed within the 50-megabase region centered on Cntnap5a in Six2TGC tg/+ mice compared with control mice. By contrast, we identified an aberrant regulatory interaction between a Six2 distal enhancer and the Six3 promoter contained within the transgene. Increasing the Six2TGC tg to Six2 locus ratio or removing one Six2 allele in Six2TGC tg/+ mice, caused severe renal hypoplasia. Furthermore, CRISPR disruption of Six3 within the transgene ( Six2TGC Δ Six3CT ) restored nephron endowment to wildtype levels and abolished the stoichiometric effect. Data from genetic and biochemical studies together suggest that in Six2TGC, SIX3 interferes with SIX2 function in NPC renewal through its C-terminal domain. Significance: Using high-resolution chromatin conformation and accessibility datasets we mapped the integration site of two popular transgenes used in studies of nephron progenitor cells and kidney development. Aberrant enhancer-promoter interactions drive ectopic expression of Six3 in the Six2TGC tg line which was correlated with disruption of nephrogenesis. Disruption of Six3 within the transgene restored nephron numbers to control levels; further genetic and biochemical studies suggest that Six3 interferes with Six2 -mediated regulation of NPC renewal.
RESUMO
Xenbase (https://www.xenbase.org/), the Xenopus model organism knowledgebase, is a web-accessible resource that integrates the diverse genomic and biological data from research on the laboratory frogs Xenopus laevis and Xenopus tropicalis. The goal of Xenbase is to accelerate discovery and empower Xenopus research, to enhance the impact of Xenopus research data, and to facilitate the dissemination of these data. Xenbase also enhances the value of Xenopus data through high-quality curation, data integration, providing bioinformatics tools optimized for Xenopus experiments, and linking Xenopus data to human data, and other model organisms. Xenbase also plays an indispensable role in making Xenopus data interoperable and accessible to the broader biomedical community in accordance with FAIR principles. Xenbase provides annotated data updates to organizations such as NCBI, UniProtKB, Ensembl, the Gene Ontology consortium, and most recently, the Alliance of Genomic Resources, a common clearing house for data from humans and model organisms. This article provides a brief overview of key and recently added features of Xenbase. New features include processing of Xenopus high-throughput sequencing data from the NCBI Gene Expression Omnibus; curation of anatomical, physiological, and expression phenotypes with the newly created Xenopus Phenotype Ontology; Xenopus Gene Ontology annotations; new anatomical drawings of the Normal Table of Xenopus development; and integration of the latest Xenopus laevis v10.1 genome annotations. Finally, we highlight areas for future development at Xenbase as we continue to support the Xenopus research community.
Assuntos
Bases de Dados Genéticas , Genômica , Animais , Humanos , Xenopus laevis/genética , Xenopus/genética , Biologia ComputacionalRESUMO
Human intestinal organoids (HIOs) derived from pluripotent stem cells provide a valuable model for investigating human intestinal organogenesis and physiology, but they lack the immune components required to fully recapitulate the complexity of human intestinal biology and diseases. To address this issue and to begin to decipher human intestinal-immune crosstalk during development, we generated HIOs containing immune cells by transplanting HIOs under the kidney capsule of mice with a humanized immune system. We found that human immune cells temporally migrate to the mucosa and form cellular aggregates that resemble human intestinal lymphoid follicles. Moreover, after microbial exposure, epithelial microfold cells are increased in number, leading to immune cell activation determined by the secretion of IgA antibodies in the HIO lumen. This in vivo HIO system with human immune cells provides a framework for future studies on infection- or allergen-driven intestinal diseases.
Assuntos
Células-Tronco Pluripotentes , Transplantes , Humanos , Animais , Camundongos , Intestinos , Mucosa Intestinal , OrganoidesRESUMO
Mammalian nephron endowment is determined by the coordinated cessation of nephrogenesis in independent niches. Here we report that translatome analysis in Tsc1+/- nephron progenitor cells from mice with elevated nephron numbers reveals how differential translation of Wnt antagonists over agonists tips the balance between self-renewal and differentiation. Wnt agonists are poorly translated in young niches, resulting in an environment with low R-spondin and high Fgf20 promoting self-renewal. In older niches we find increased translation of Wnt agonists, including R-spondin and the signalosome-promoting Tmem59, and low Fgf20, promoting differentiation. This suggests that the tipping point for nephron progenitor exit from the niche is controlled by the gradual increase in stability and possibly clustering of Wnt/Fzd complexes in individual cells, enhancing the response to ureteric bud-derived Wnt9b inputs and driving synchronized differentiation. As predicted by these findings, removing one Rspo3 allele in nephron progenitors delays cessation and increases nephron numbers in vivo.
Assuntos
Organogênese/fisiologia , Percepção/fisiologia , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Animais , Diferenciação Celular , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Homeodomínio , Rim/citologia , Rim/patologia , Masculino , Proteínas de Membrana , Camundongos , Néfrons/citologia , Proteínas do Tecido Nervoso , Nicho de Células-Tronco , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/genética , Via de Sinalização WntRESUMO
The gene regulatory networks that coordinate the development of the cardiac and pulmonary systems are essential for terrestrial life but poorly understood. The T-box transcription factor Tbx5 is critical for both pulmonary specification and heart development, but how these activities are mechanistically integrated remains unclear. Here using Xenopus and mouse embryos, we establish molecular links between Tbx5 and retinoic acid (RA) signaling in the mesoderm and between RA signaling and sonic hedgehog expression in the endoderm to unveil a conserved RA-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary (CP) development. We demonstrate that Tbx5 directly maintains expression of aldh1a2, the RA-synthesizing enzyme, in the foregut lateral plate mesoderm via an evolutionarily conserved intronic enhancer. Tbx5 promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module to restrict FGF activity to the anterior. We find that Tbx5/Aldh1a2-dependent RA signaling directly activates shh transcription in the adjacent foregut endoderm through a conserved MACS1 enhancer. Hedgehog signaling coordinates with Tbx5 in the mesoderm to activate expression of wnt2/2b, which induces pulmonary fate in the foregut endoderm. These results provide mechanistic insight into the interrelationship between heart and lung development informing CP evolution and birth defects.
Assuntos
Família Aldeído Desidrogenase 1/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Coração/embriologia , Pulmão/embriologia , Retinal Desidrogenase/genética , Proteínas com Domínio T/genética , Proteínas de Xenopus/genética , Xenopus/embriologia , Família Aldeído Desidrogenase 1/metabolismo , Animais , Sequência de Bases , Mesoderma/embriologia , Camundongos , Retinal Desidrogenase/metabolismo , Alinhamento de Sequência , Proteínas com Domínio T/metabolismo , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Congenital tracheomalacia, resulting from incomplete tracheal cartilage development, is a relatively common birth defect that severely impairs breathing in neonates. Mutations in the Hedgehog (HH) pathway and downstream Gli transcription factors are associated with tracheomalacia in patients and mouse models; however, the underlying molecular mechanisms are unclear. Using multiple HH/Gli mouse mutants including one that mimics Pallister-Hall Syndrome, we show that excessive Gli repressor activity prevents specification of tracheal chondrocytes. Lineage tracing experiments show that Sox9+ chondrocytes arise from HH-responsive splanchnic mesoderm in the fetal foregut that expresses the transcription factor Foxf1. Disrupted HH/Gli signaling results in 1) loss of Foxf1 which in turn is required to support Sox9+ chondrocyte progenitors and 2) a dramatic reduction in Rspo2, a secreted ligand that potentiates Wnt signaling known to be required for chondrogenesis. These results reveal a HH-Foxf1-Rspo2 signaling axis that governs tracheal cartilage development and informs the etiology of tracheomalacia.