RESUMO
Climate change is predicted to increase atmospheric vapor pressure deficit, exacerbating soil drought, and thus enhancing tree evaporative demand and mortality. Yet, few studies have addressed the longer-term drought acclimation strategy of trees, particularly the importance of morphological versus hydraulic plasticity. Using a long-term (20 years) irrigation experiment in a natural forest, we investigated the acclimation of Scots pine (Pinus sylvestris) morpho-anatomical traits (stomatal anatomy and crown density) and hydraulic traits (leaf water potential, vulnerability to cavitation (Ψ50), specific hydraulic conductivity (Ks), and tree water deficit) to prolonged changes in soil moisture. We found that low water availability reduced twig water potential and increased tree water deficit during the growing season. Still, the trees showed limited adjustments in most branch-level hydraulic traits (Ψ50 and Ks) and needle anatomy. In contrast, trees acclimated to prolonged irrigation by increasing their crown density and hence the canopy water demand. This study demonstrates that despite substantial canopy adjustments, P. sylvestris may be vulnerable to extreme droughts because of limited adjustment potential in its hydraulic system. While sparser canopies reduce water demand, such shifts take decades to occur under chronic water deficits and might not mitigate short-term extreme drought events.
Assuntos
Aclimatação , Florestas , Pinus sylvestris , Água , Pinus sylvestris/fisiologia , Pinus sylvestris/anatomia & histologia , Pinus sylvestris/crescimento & desenvolvimento , Água/metabolismo , Água/fisiologia , Folhas de Planta/fisiologia , Folhas de Planta/anatomia & histologia , Irrigação Agrícola , Secas , Árvores/fisiologia , Árvores/anatomia & histologiaRESUMO
Drought is a major limitation for survival and growth in plants. With more frequent and severe drought episodes occurring due to climate change, it is imperative to understand the genomic and physiological basis of drought tolerance to be able to predict how species will respond in the future. In this study, univariate and multitrait multivariate genome-wide association study methods were used to identify candidate genes in two iconic and ecosystem-dominating species of the western USA, coast redwood and giant sequoia, using 10 drought-related physiological and anatomical traits and genome-wide sequence-capture single nucleotide polymorphisms. Population-level phenotypic variation was found in carbon isotope discrimination, osmotic pressure at full turgor, xylem hydraulic diameter, and total area of transporting fibers in both species. Our study identified new 78 new marker × trait associations in coast redwood and six in giant sequoia, with genes involved in a range of metabolic, stress, and signaling pathways, among other functions. This study contributes to a better understanding of the genomic basis of drought tolerance in long-generation conifers and helps guide current and future conservation efforts in the species.
Assuntos
Adaptação Fisiológica/genética , Genoma de Planta/genética , Sequoia/genética , Sequoiadendron/genética , Transdução de Sinais/genética , Isótopos de Carbono/análise , Conservação dos Recursos Naturais , Secas , Estudo de Associação Genômica Ampla , Herança Multifatorial/genética , Pressão Osmótica , Fenótipo , Estômatos de Plantas/genética , Estômatos de Plantas/fisiologia , Sequoia/fisiologia , Sequoiadendron/fisiologia , Xilema/genética , Xilema/fisiologiaRESUMO
Tracheid buckling may protect leaves in the dynamic environments of forest canopies, where rapid intensifications of evaporative demand, such as those brought on by changes in light availability, can result in sudden increases in transpiration rate. While treetop leaves function in reliably direct light, leaves below the upper crown must tolerate rapid, thermally driven increases in evaporative demand. Using synchrotron-based X-ray microtomography, we visualized impacts of experimentally induced water stress and subsequent fogging on living cells in redwood leaves, adding ecological and functional context through crown-wide explorations of variation in leaf physiology and microclimate. Under drought, leaf transfusion tracheids buckle, releasing water that supplies sufficient temporal reserves for leaves to reduce stomatal conductance safely while stopping the further rise of tension. Tracheid buckling fraction decreases with height and is closely coordinated with transfusion tissue capacity and stomatal conductance to provide temporal reserves optimized for local variation in microclimate. Foliar water uptake fully restores collapsed and air-filled transfusion tracheids in leaves on excised shoots, suggesting that trees may use aerial water sources for recovery. In the intensely variable deep-crown environment, foliar water uptake can allow for repetitive cycles of tracheid buckling and unbuckling, protecting the tree from damaging levels of hydraulic tension and supporting leaf survival.
Assuntos
Sequoia , Árvores , Secas , Folhas de Planta/fisiologia , Transpiração Vegetal , Sequoia/fisiologia , Árvores/fisiologia , Tempo (Meteorologia)RESUMO
PREMISE: Trees in wet forests often have features that prevent water films from covering stomata and inhibiting gas exchange, while many trees in drier environments use foliar water uptake to reduce water stress. In forests with both wet and dry seasons, evergreen trees would benefit from producing leaves capable of balancing rainy-season photosynthesis with summertime water absorption. METHODS: Using samples collected from across the vertical gradient in tall redwood (Sequoia sempervirens) crowns, we estimated tree-level foliar water uptake and employed physics-based causative modeling to identify key functional traits that determine uptake potential by setting hydraulic resistance. RESULTS: We showed that Sequoia has two functionally distinct shoot morphotypes. While most shoots specialize in photosynthesis, the axial shoot type is capable of much greater foliar water uptake, and its within-crown distribution varies with latitude. A suite of leaf surface traits cause hydraulic resistance, leading to variation in uptake capacity among samples. CONCLUSIONS: Shoot dimorphism gives tall Sequoia trees the capacity to absorb up to 48 kg H2 O h-1 during the first hour of leaf wetting, ameliorating water stress while presumably maintaining high photosynthetic capacity year round. Geographic variation in shoot dimorphism suggests that plasticity in shoot-type distribution and leaf surface traits helps Sequoia maintain a dominate presence in both wet and dry forests.
Assuntos
Sequoia , Desidratação , Fotossíntese , Folhas de Planta , Caracteres Sexuais , ÁrvoresRESUMO
The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.
Assuntos
Adenoma/metabolismo , Colo/metabolismo , Neoplasias do Colo/metabolismo , Mucosa Intestinal/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adenoma/genética , Linhagem Celular Tumoral , Colo/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Citometria de Fluxo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Mucosa Intestinal/citologia , Organoides/metabolismo , Transdução de SinaisRESUMO
The intestine plays a central role in digestion, nutrient absorption and metabolism, with individual regions of the intestine having distinct functional roles. Many examples of region-specific gene expression in the adult intestine are known, but how intestinal regional identity is established during development is a largely unresolved issue. Here, we have identified several genes that are expressed in a region-specific manner in the developing human intestine. Using human embryonic stem cell-derived intestinal organoids, we demonstrate that the duration of exposure to active FGF and WNT signaling controls regional identity. Short-term exposure to FGF4 and CHIR99021 (a GSK3ß inhibitor that stabilizes ß-catenin) resulted in organoids with gene expression patterns similar to developing human duodenum, whereas longer exposure resulted in organoids similar to ileum. When region-specific organoids were transplanted into immunocompromised mice, duodenum-like organoids and ileum-like organoids retained their regional identity, demonstrating that regional identity of organoids is stable after initial patterning occurs. This work provides insights into the mechanisms that control regional specification of the developing human intestine and provides new tools for basic and translational research.
Assuntos
Padronização Corporal , Desenvolvimento Embrionário , Feto/embriologia , Intestinos/embriologia , Células-Tronco Pluripotentes/citologia , Animais , Biomarcadores/metabolismo , Padronização Corporal/genética , Diferenciação Celular/genética , Biologia Computacional , Desenvolvimento Embrionário/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Organoides/metabolismo , Organoides/transplante , Células-Tronco Pluripotentes/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Transdução de Sinais/genética , Proteínas Wnt/metabolismoRESUMO
Parenchyma cells in the xylem store nonstructural carbohydrates (NSC), providing reserves of energy that fuel woody perennials through periods of stress and/or limitations to photosynthesis. If the capacity for storage is subject to selection, then the fraction of wood occupied by living parenchyma should increase towards stressful environments. Ray parenchyma fraction (RPF) and seasonal NSC dynamics were quantified for 12 conifers and three oaks along a transect spanning warm dry foothills (500 m above sea level) to cold wet treeline (3250 m asl) in California's central Sierra Nevada. Mean RPF was lower for both conifer and oak species with warmer dryer ranges. RPF variability increased with elevation or in relation to associated climatic variables in conifers - treeline-dominant Pinus albicaulis had the lowest mean RPF measured (c. 3.7%), but the highest environmentally standardized variability index. Conifer RPF variability was explained by environment, increasing predominantly towards cooler wetter range edges. In oaks, NSC was explained by environment - values increasing for evergreen and decreasing for deciduous oaks with elevation. Lastly, all species surveyed appear to prioritize filling available RPF with sugar to achieve molarities that balance reasonable tensions over starch to maximize stored carbon. RPF responds to environment but is unlikely to spatially constrain NSC storage.
Assuntos
Pinus , Árvores , Metabolismo dos Carboidratos , Carboidratos , XilemaRESUMO
The intestine is a vital organ responsible for nutrient absorption, bile and waste excretion, and a major site of host immunity. In order to keep up with daily demands, the intestine has evolved a mechanism to expand the absorptive surface area by undergoing a morphogenetic process to generate finger-like units called villi. These villi house specialized cell types critical for both absorbing nutrients from food, and for protecting the host from commensal and pathogenic microbes present in the adult gut. In this review, we will discuss mechanisms that coordinate intestinal development, growth, and maturation of the small intestine, starting from the formation of the early gut tube, through villus morphogenesis and into early postnatal life when the intestine must adapt to the acquisition of nutrients through food intake, and to interactions with microbes.
Assuntos
Endoderma/crescimento & desenvolvimento , Intestinos/embriologia , Intestinos/crescimento & desenvolvimento , Morfogênese/genética , Diferenciação Celular , HumanosRESUMO
PREMISE OF THE STUDY: Leaves are the sites of greatest water stress in trees and a key means of acclimation to the environment. We considered phenotypic plasticity of Pseudotsuga menziesii leaves in their ecological context, exploring responsiveness to natural gradients in water stress (indicated by sample height) and light availability (measured from hemispherical photos) to understand how leaf structure is controlled by abiotic factors in tall tree crowns. METHODS: After measuring anatomy, morphology, and carbon isotope composition (δ13 C) of leaves throughout crowns of P. menziesii >90 m tall, we compared structural plasticity of leaves among the three tallest conifer species using equivalent data from past work on Sequoia sempervirens and Picea sitchensis. KEY RESULTS: Leaf mass per projected area (LMA) and δ13 C increased and mesoporosity (airspace/area) decreased along the water-stress gradient, while light did not play a detectable role in leaf development. Overall, leaves of P. menziesii were far less phenotypically responsive to within-crown abiotic gradients than either P. sitchensis, whose leaves responded strongly to light availability, or S. sempervirens, whose leaves responded equally strongly to water stress. CONCLUSIONS: P. menziesii maintain remarkably consistent leaf structure despite pronounced vertical gradients in abiotic factors. Contrasting patterns of leaf structural plasticity underlie divergent ecological strategies of the three tallest conifer species, which coexist in Californian rainforests.
Assuntos
Adaptação Fisiológica , Folhas de Planta/crescimento & desenvolvimento , Pseudotsuga/crescimento & desenvolvimento , Água/fisiologia , Luz , Folhas de Planta/anatomia & histologia , Pseudotsuga/anatomia & histologia , Pseudotsuga/efeitos da radiaçãoRESUMO
PREMISE OF THE STUDY: Leaves respond to environmental signals and acclimate to local conditions until their ecological limits are reached. Understanding the relationships between anatomical variation in leaves and the availability of water and light improves our ability to predict ecosystem-level impacts of foliar response to climate change, as it expands our knowledge of tree physiology. METHODS: We examined foliar anatomy and morphology of the largest plant species, Sequoiadendron giganteum, from leafy shoot samples collected throughout crowns of trees up to 95 m tall and assessed the functionality of within-crown variation with a novel drought/recovery experiment. KEY RESULTS: We found phenotypic variation in response to water availability in 13 anatomical traits of Sequoiadendron leaves. Shoot expansion was constrained by the hydrostatic gradient of maximum water potential, while functional traits supporting succulence and toughness were associated with sites of peak hydraulic limitation. Water-stress tolerance in experimental shoots increased dramatically with height. CONCLUSION: We propose a heat-sink function for transfusion tissue and uncover a suite of traits suggesting rapid hydraulic throughput and flexibility in water-stress tolerance investments as strategies that help this montane species reach such enormous size. Responses to water stress alter the amount of carbon stored in foliage and the rate of the eventual release of carbon.
Assuntos
Adaptação Fisiológica , Folhas de Planta/fisiologia , Estresse Fisiológico , Traqueófitas/fisiologia , Água/fisiologia , Secas , Fenótipo , Brotos de Planta/anatomia & histologia , Brotos de Planta/fisiologia , Sequoiadendron/fisiologia , Traqueófitas/anatomia & histologiaRESUMO
Premise: To improve forest conservation monitoring, we developed a protocol to automatically count and identify the seeds of plant species with minimal resource requirements, making the process more efficient and less dependent on human operators. Methods and Results: Seeds from six North American conifer tree species were separated from leaf litter and imaged on a flatbed scanner. In the most successful species-classification approach, an ImageJ macro automatically extracted measurements for random forest classification in the software R. The method allows for good classification accuracy, and the same process can be used to train the model on other species. Conclusions: This protocol is an adaptable tool for efficient and consistent identification of seed species or potentially other objects. Automated seed classification is efficient and inexpensive, making it a practical solution that enhances the feasibility of large-scale monitoring projects in conservation biology.
RESUMO
Analysis of functional traits is a cornerstone of ecology, yet individual traits seldom explain useful amounts of variation in species distribution or climatic tolerance, and their functional significance is rarely validated experimentally. Multivariate suites of interacting traits could build an understanding of ecological processes and improve our ability to make sound predictions of species success in our rapidly changing world. We use foliar water uptake capacity as a case study because it is increasingly considered to be a key functional trait in plant ecology due to its importance for stress-tolerance physiology. However, the traits behind the trait, that is, the features of leaves that determine variation in foliar water uptake rates, have not been assembled into a widely applicable framework for uptake prediction. Focusing on trees, we investigated relationships among 25 structural traits, leaf osmotic potential (a source of free energy to draw water into leaves), and foliar water uptake in 10 diverse angiosperm and conifer species. We identified consistent, multitrait "uptake syndromes" for both angiosperm and conifer trees, with differences in key traits revealing suspected differences in the water entry route between these two clades and an evolutionarily significant divergence in the function of homologous structures. A literature review of uptake-associated functional traits, which largely documents similar univariate relationships, provides additional support for our proposed "uptake syndrome." Importantly, more than half of shared traits had opposite-direction influences on the capacity of leaves to absorb water in angiosperms and conifers. Taxonomically targeted multivariate trait syndromes provide a useful tool for trait selection in ecological research, while highlighting the importance of micro-traits and the physiological verification of their function for advancing trait-based ecology.
Assuntos
Magnoliopsida , Traqueófitas , Árvores/fisiologia , Água/análise , Ecologia , Traqueófitas/fisiologia , Folhas de Planta/químicaRESUMO
Absorption of water across the surfaces of leaves is an ecologically important aspect of tree physiology. Variation in foliar water uptake capacity depends on environmental conditions when traits associated with the uptake pathway respond to climatic signals. Using a series of experiments, we verify that water enters Sequoia sempervirens (D. Don) Endl. leaves by crossing the cuticle, and show that surface-trait acclimation alters the kinetic parameters of foliar water uptake. Under our experimental conditions, the cuticle was the primary pathway for water entry into the leaf. Exposure to climatic variation may induce surface acclimations, such as increased waxiness, that reduce water-film formation over stomata at the expense of dry-season foliar uptake rates. We found that water uptake is negatively associated with the interaction of leaf-surface wax coverage and stomatal density, and provide an accessible protocol to measure these key traits in Sequoia. Linking absorptive pathways and trait acclimation to physiological performance can provide a foundation for range-wide or genomic investigations of forest interactions with water and a mechanism-centered means to monitor canopy hydraulic parameters over time.
Assuntos
Aclimatação , Água , Água/fisiologia , Folhas de Planta/fisiologia , Estações do Ano , Florestas , Árvores/fisiologiaRESUMO
The current study aimed to understand the developmental mechanisms regulating bud tip progenitor cells in the human fetal lung, which are present during branching morphogenesis, and to use this information to induce a bud tip progenitor-like population from human pluripotent stem cells (hPSCs) in vitro. We identified cues that maintained isolated human fetal lung epithelial bud tip progenitor cells in vitro and induced three-dimensional hPSC-derived organoids with bud tip-like domains. Bud tip-like domains could be isolated, expanded, and maintained as a nearly homogeneous population. Molecular and cellular comparisons revealed that hPSC-derived bud tip-like cells are highly similar to native lung bud tip progenitors. hPSC-derived epithelial bud tip-like structures survived in vitro for over 16 weeks, could be easily frozen and thawed, maintained multilineage potential, and successfully engrafted into the airways of immunocompromised mouse lungs, where they persisted for up to 6 weeks and gave rise to several lung epithelial lineages.
Assuntos
Feto , Pulmão , Células-Tronco Pluripotentes , Animais , Feto/citologia , Feto/metabolismo , Xenoenxertos , Humanos , Pulmão/citologia , Pulmão/embriologia , Camundongos , Camundongos Transgênicos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/transplanteRESUMO
Leaf-level anatomical variation is readily apparent within tall tree crowns, yet the relative importance of water and light availability in controlling this variation remains unclear. Sitka spruce (Picea sitchensis, (Bong.) Carr.) thrives in temperate rainforests of the Pacific Northwest, where it has historically reached heights >100 m, despite rarely living more than 400 years alongside redwoods that are five times older. We examined leaves of trees up to 97 m tall using a combination of transverse sections, longitudinal sections, epidermal imprints and whole-leaf measurements to explore the combined effects of water stress and light availability on leaf development in P. sitchensis. In contrast to the situation in tall Cupressaceae, light availability-not hydraulic limitation-is the primary ecological driver of leaf-level anatomical variation in P. sitchensis. While height-associated decreases in leaf length and mesoporosity are best explained by hydrostatic constraints on leaf elongation, the majority of anatomical traits we measured reflect acclimation to light availability, including increases in leaf width and vascular tissue areas in the brightest parts of the crown. Along with these changes, the appearance of abaxial stomata in the bright upper crown, and the arrangement of mesophyll in uniseriate, transverse plates-with radially arranged apoplastic pathways leading directly to stomata before bridging them with a V-shaped cell-may enhance gas exchange and hydraulic conductivity. This suite of leaf traits suggests an adaptive strategy that maximizes photosynthesis at the expense of water-stress tolerance. Anatomical investigations spanning the height gradient in tall tree crowns build our understanding of mechanisms underlying among-species variation in growth rates, life spans, and potential responses to climate change.
Assuntos
Picea/fisiologia , Folhas de Planta/fisiologia , Árvores/fisiologia , Aclimatação , Luz , Picea/crescimento & desenvolvimento , Árvores/crescimento & desenvolvimento , Água/metabolismoRESUMO
The human gastrointestinal tract is immature at birth, yet must adapt to dramatic changes such as oral nutrition and microbial colonization. The confluence of these factors can lead to severe inflammatory disease in premature infants; however, investigating complex environment-host interactions is difficult due to limited access to immature human tissue. Here, we demonstrate that the epithelium of human pluripotent stem-cell-derived human intestinal organoids is globally similar to the immature human epithelium and we utilize HIOs to investigate complex host-microbe interactions in this naive epithelium. Our findings demonstrate that the immature epithelium is intrinsically capable of establishing a stable host-microbe symbiosis. Microbial colonization leads to complex contact and hypoxia driven responses resulting in increased antimicrobial peptide production, maturation of the mucus layer, and improved barrier function. These studies lay the groundwork for an improved mechanistic understanding of how colonization influences development of the immature human intestine.
Assuntos
Escherichia coli/fisiologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/fisiologia , Estresse Fisiológico , Simbiose , Peptídeos Catiônicos Antimicrobianos/metabolismo , Humanos , Muco/metabolismo , Técnicas de Cultura de Órgãos , Células-Tronco Pluripotentes/fisiologiaRESUMO
BACKGROUND & AIMS: The Lgr family of transmembrane proteins (Lgr4, 5, 6) act as functional receptors for R-spondin proteins (Rspo 1, 2, 3, 4), and potentiate Wnt signaling in different contexts. Lgr5 is arguably the best characterized of the Lgr family members in a number of adult and embryonic contexts in mice. However, the function of LGR family members in early embryonic development is unclear, and has not been explored during human development or tissue differentiation in detail. METHODS: We interrogated the function and expression of LGR family members using human pluripotent stem cell-derived tissues including definitive endoderm, mid/hindgut, and intestinal organoids. We performed embryonic lineage tracing in Lgr5-GFP-IRES-CreERT2 mice. RESULTS: We show that LGR5 is part of the human definitive endoderm (DE) gene signature, and LGR5 transcripts are induced robustly when human pluripotent stem cells are differentiated into DE. Our results show that LGR4 and 5 are functionally required for efficient human endoderm induction. Consistent with data in human DE, we observe Lgr5 reporter (eGFP) activity in the embryonic day 8.5 mouse endoderm, and show the ability to lineage trace these cells into the adult intestine. However, gene expression data also suggest that there are human-mouse species-specific differences at later time points of embryonic development. CONCLUSIONS: Our results show that LGR5 is induced during DE differentiation, LGR receptors are functionally required for DE induction, and that they function to potentiate WNT signaling during this process.
RESUMO
Much of our understanding about how intestinal stem and progenitor cells are regulated comes from studying the late fetal stages of development and the adult intestine. In this light, little is known about intestine development prior to the formation of stereotypical villus structures with columnar epithelium, a stage when the epithelium is pseudostratified and appears to be a relatively uniform population of progenitor cells with high proliferative capacity. Here, we investigated a role for WNT/ß-CATENIN signaling during the pseudostratified stages of development (E13.5, E14.5) and following villus formation (E15.5) in mice. In contrast to the well-described role for WNT/ß-CATENIN signaling as a regulator of stem/progenitor cells in the late fetal and adult gut, conditional epithelial deletion of ß-catenin or the Frizzled co-receptors Lrp5 and Lrp6 had no effect on epithelial progenitor cell proliferation in the pseudostratified epithelium. Mutant embryos displayed obvious developmental defects, including loss of proliferation and disruptions in villus formation starting only at E15.5. Mechanistically, our data suggest that WNT signaling-mediated proliferation at the time of villus formation is driven by mesenchymal, but not epithelial, WNT ligand secretion.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Animais , Morte Celular , Diferenciação Celular , Proliferação de Células , Expressão Gênica , Intestinos/citologia , Intestinos/embriologia , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Transgênicos , Morfogênese/genética , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Organogênese/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Human intestinal organoids (HIOs) are a tissue culture model in which small intestine-like tissue is generated from pluripotent stem cells. By carrying out unsupervised hierarchical clustering of RNA-sequencing data, we demonstrate that HIOs most closely resemble human fetal intestine. We observed that genes involved in digestive tract development are enriched in both fetal intestine and HIOs compared to adult tissue, whereas genes related to digestive function and Paneth cell host defense are expressed at higher levels in adult intestine. Our study also revealed that the intestinal stem cell marker OLFM4 is expressed at very low levels in fetal intestine and in HIOs, but is robust in adult crypts. We validated our findings using in vivo transplantation to show that HIOs become more adult-like after transplantation. Our study emphasizes important maturation events that occur in the intestine during human development and demonstrates that HIOs can be used to model fetal-to-adult maturation.
RESUMO
Bone morphogenetic protein (BMP) signaling pathways regulate multiple aspects of endochondral bone formation. The importance of extracellular antagonists as regulators of BMP signaling has been defined. In vitro studies reveal that the intracellular regulators, inhibitory Smads 6 and 7, can regulate BMP-mediated effects on chondrocytes. Although in vivo studies in which inhibitory Smads were overexpressed in cartilage have shown that inhibitory Smads have the potential to limit BMP signaling in vivo, the physiological relevance of inhibitory Smad activity in skeletal tissues is unknown. In this study, we have determined the role of Smad6 in endochondral bone formation. Loss of Smad6 in mice leads to defects in both axial and appendicular skeletal development. Specifically, Smad6-/- mice exhibit a posterior transformation of the seventh cervical vertebra, bilateral ossification centers in lumbar vertebrae, and bifid sternebrae due to incomplete sternal band fusion. Histological analysis of appendicular bones revealed delayed onset of hypertrophic differentiation and mineralization at midgestation in Smad6-/- mice. By late gestation, however, an expanded hypertrophic zone, associated with an increased pool of proliferating cells undergoing hypertrophy, was evident in Smad6 mutant growth plates. The mutant phenotype is attributed, at least in part, to increased BMP responsiveness in Smad6-deficient chondrocytes. Overall, our results show that Smad6 is required to limit BMP signaling during endochondral bone formation.