RESUMEN
Tooth formation requires complex signaling interactions both within the oral epithelium and between the epithelium and the underlying mesenchyme. Previous studies of the Wnt/ß-catenin pathway have shown that tooth formation is partly inhibited in loss-of-function mutants, and gain-of-function mutants have perturbed tooth morphology. However, the stage at which Wnt signaling is first important in tooth formation remains unclear. Here, using an Fgf8-promoter-driven, and therefore early, deletion of ß-catenin in mouse molar epithelium, we found that loss of Wnt/ß-catenin signaling completely deletes the molar tooth, demonstrating that this pathway is central to the earliest stages of tooth formation. Early expression of a dominant-active ß-catenin protein also perturbs tooth formation, producing a large domed evagination at early stages and supernumerary teeth later on. The early evaginations are associated with premature mesenchymal condensation marker, and are reduced by inhibition of condensation-associated collagen synthesis. We propose that invagination versus evagination morphogenesis is regulated by the relative timing of epithelial versus mesenchymal cell convergence regulated by canonical Wnt signaling. Together, these studies reveal new aspects of Wnt/ß-catenin signaling in tooth formation and in epithelial morphogenesis more broadly.
Asunto(s)
Diente Molar/crecimiento & desarrollo , Diente Molar/metabolismo , Odontogénesis/fisiología , Vía de Señalización Wnt/fisiología , Animales , Proliferación Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Epitelio/metabolismo , Mesodermo/metabolismo , Ratones , Diente Molar/citología , Morfogénesis/fisiología , Odontogénesis/genética , beta Catenina/metabolismoRESUMEN
During both embryonic development and adult tissue regeneration, changes in chromatin structure driven by master transcription factors lead to stimulus-responsive transcriptional programs. A thorough understanding of how stem cells in the skeleton interpret mechanical stimuli and enact regeneration would shed light on how forces are transduced to the nucleus in regenerative processes. Here we develop a genetically dissectible mouse model of mandibular distraction osteogenesis-which is a process that is used in humans to correct an undersized lower jaw that involves surgically separating the jaw bone, which elicits new bone growth in the gap. We use this model to show that regions of newly formed bone are clonally derived from stem cells that reside in the skeleton. Using chromatin and transcriptional profiling, we show that these stem-cell populations gain activity within the focal adhesion kinase (FAK) signalling pathway, and that inhibiting FAK abolishes new bone formation. Mechanotransduction via FAK in skeletal stem cells during distraction activates a gene-regulatory program and retrotransposons that are normally active in primitive neural crest cells, from which skeletal stem cells arise during development. This reversion to a developmental state underlies the robust tissue growth that facilitates stem-cell-based regeneration of adult skeletal tissue.
Asunto(s)
Regeneración Ósea , Mandíbula/citología , Mandíbula/fisiología , Cresta Neural/citología , Osteogénesis por Distracción , Células Madre/citología , Animales , Cromatina/genética , Cromatina/metabolismo , Modelos Animales de Enfermedad , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Regulación de la Expresión Génica , Masculino , Mandíbula/cirugía , Ratones , Ratones Endogámicos C57BL , Retroelementos/genética , Transducción de Señal , Células Madre/metabolismo , Transcripción GenéticaRESUMEN
Genital malformations are among the most common human birth defects, and both genetic and environmental factors can contribute to these malformations. Development of the external genitalia in mammals relies on complex signaling networks, and disruption of these signaling pathways can lead to genital defects. Islet-1 (ISL1), a member of the LIM/Homeobox family of transcription factors, has been identified as a major susceptibility gene for classic bladder exstrophy in humans, a common form of the bladder exstrophy-epispadias complex (BEEC), and is implicated in a role in urinary tract development. We report that deletion of Isl1 from the genital mesenchyme in mice led to hypoplasia of the genital tubercle and prepuce, with an ectopic urethral opening and epispadias-like phenotype. These mice also developed hydroureter and hydronephrosis. Identification of ISL1 transcriptional targets via ChIP-Seq and expression analyses revealed that Isl1 regulates several important signaling pathways during embryonic genital development, including the BMP, WNT, and FGF cascades. An essential function of Isl1 during development of the external genitalia is to induce Bmp4-mediated apoptosis in the genital mesenchyme. Together, these studies demonstrate that Isl1 plays a critical role during development of the external genitalia and forms the basis for a greater understanding of the molecular mechanisms underlying the pathogenesis of BEEC and urinary tract defects in humans.
Asunto(s)
Proteína Morfogenética Ósea 4/genética , Factor 10 de Crecimiento de Fibroblastos/genética , Genitales/anomalías , Genitales/embriología , Proteínas con Homeodominio LIM/genética , Factores de Transcripción/genética , Proteína Wnt-5a/genética , Animales , Extrofia de la Vejiga/genética , Extrofia de la Vejiga/metabolismo , Proteína Morfogenética Ósea 4/biosíntesis , Proteína Morfogenética Ósea 4/metabolismo , Desarrollo Embrionario , Femenino , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genitales/metabolismo , Proteínas con Homeodominio LIM/biosíntesis , Proteínas con Homeodominio LIM/metabolismo , Masculino , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Ratones Noqueados , Organogénesis/genética , Transducción de Señal , Factores de Transcripción/biosíntesis , Factores de Transcripción/metabolismo , Anomalías Urogenitales/genética , Anomalías Urogenitales/metabolismo , Proteína Wnt-5a/biosíntesis , Proteína Wnt-5a/metabolismoRESUMEN
One of the major challenges that developing organs face is scaling, that is, the adjustment of physical proportions during the massive increase in size. Although organ scaling is fundamental for development and function, little is known about the mechanisms that regulate it. Bone superstructures are projections that typically serve for tendon and ligament insertion or articulation and, therefore, their position along the bone is crucial for musculoskeletal functionality. As bones are rigid structures that elongate only from their ends, it is unclear how superstructure positions are regulated during growth to end up in the right locations. Here, we document the process of longitudinal scaling in developing mouse long bones and uncover the mechanism that regulates it. To that end, we performed a computational analysis of hundreds of three-dimensional micro-CT images, using a newly developed method for recovering the morphogenetic sequence of developing bones. Strikingly, analysis revealed that the relative position of all superstructures along the bone is highly preserved during more than a 5-fold increase in length, indicating isometric scaling. It has been suggested that during development, bone superstructures are continuously reconstructed and relocated along the shaft, a process known as drift. Surprisingly, our results showed that most superstructures did not drift at all. Instead, we identified a novel mechanism for bone scaling, whereby each bone exhibits a specific and unique balance between proximal and distal growth rates, which accurately maintains the relative position of its superstructures. Moreover, we show mathematically that this mechanism minimizes the cumulative drift of all superstructures, thereby optimizing the scaling process. Our study reveals a general mechanism for the scaling of developing bones. More broadly, these findings suggest an evolutionary mechanism that facilitates variability in bone morphology by controlling the activity of individual epiphyseal plates.
Asunto(s)
Huesos del Brazo/embriología , Huesos del Brazo/crecimiento & desarrollo , Desarrollo Óseo/fisiología , Huesos de la Pierna/embriología , Huesos de la Pierna/crecimiento & desarrollo , Animales , Huesos del Brazo/diagnóstico por imagen , Imagenología Tridimensional , Huesos de la Pierna/diagnóstico por imagen , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Modelos Estadísticos , Microtomografía por Rayos XRESUMEN
Increasing evidence implicates abnormal Ras signaling as a major contributor in neurodevelopmental disorders, yet how such signaling causes cortical pathogenesis is unknown. We examined the consequences of aberrant Ras signaling in the developing mouse brain and uncovered several critical phenotypes, including increased production of cortical neurons and morphological deficits. To determine whether these phenotypes are recapitulated in humans, we generated induced pluripotent stem (iPS) cell lines from patients with Costello syndrome (CS), a developmental disorder caused by abnormal Ras signaling and characterized by neurodevelopmental abnormalities, such as cognitive impairment and autism. Directed differentiation toward a neuroectodermal fate revealed an extended progenitor phase and subsequent increased production of cortical neurons. Morphological analysis of mature neurons revealed significantly altered neurite length and soma size in CS patients. This study demonstrates the synergy between mouse and human models and validates the use of iPS cells as a platform to study the underlying cellular pathologies resulting from signaling deficits. SIGNIFICANCE STATEMENT: Increasing evidence implicates Ras signaling dysfunction as a major contributor in psychiatric and neurodevelopmental disorders, such as cognitive impairment and autism, but the underlying cortical cellular pathogenesis remains unclear. This study is the first to reveal human neuronal pathogenesis resulting from abnormal Ras signaling and provides insights into how these phenotypic abnormalities likely contribute to neurodevelopmental disorders. We also demonstrate the synergy between mouse and human models, thereby validating the use of iPS cells as a platform to study underlying cellular pathologies resulting from signaling deficits. Recapitulating human cellular pathologies in vitro facilitates the future high throughput screening of potential therapeutic agents that may reverse phenotypic and behavioral deficits.
Asunto(s)
Síndrome de Costello/metabolismo , Síndrome de Costello/patología , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Proteínas ras/metabolismo , Adolescente , Adulto , Diferenciación Celular , Células Cultivadas , Niño , Preescolar , Femenino , Humanos , Células Madre Pluripotentes Inducidas/patología , Lactante , Masculino , Persona de Mediana Edad , Regulación hacia ArribaRESUMEN
RASopathies are syndromes caused by gain-of-function mutations in the Ras signaling pathway. One of these conditions, Costello syndrome (CS), is typically caused by an activating de novo germline mutation in HRAS and is characterized by a wide range of cardiac, musculoskeletal, dermatological and developmental abnormalities. We report that a majority of individuals with CS have hypo-mineralization of enamel, the outer covering of teeth, and that similar defects are present in a CS mouse model. Comprehensive analysis of the mouse model revealed that ameloblasts, the cells that generate enamel, lacked polarity, and the ameloblast progenitor cells were hyperproliferative. Ras signals through two main effector cascades, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways. To determine through which pathway Ras affects enamel formation, inhibitors targeting either PI3K or MEK 1 and 2 (MEK 1/2), kinases in the MAPK pathway, were utilized. MEK1/2 inhibition rescued the hypo-mineralized enamel, normalized the ameloblast polarity defect and restored normal progenitor cell proliferation. In contrast, PI3K inhibition only corrected the progenitor cell proliferation phenotype. We demonstrate for the first time the central role of Ras signaling in enamel formation in CS individuals and present the mouse incisor as a model system to dissect the roles of the Ras effector pathways in vivo.
Asunto(s)
Síndrome de Costello/metabolismo , Esmalte Dental/patología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Adolescente , Adulto , Ameloblastos/metabolismo , Ameloblastos/patología , Animales , Estudios de Casos y Controles , Polaridad Celular , Niño , Preescolar , Estudios de Cohortes , Síndrome de Costello/genética , Esmalte Dental/efectos de los fármacos , Esmalte Dental/metabolismo , Esmalte Dental/ultraestructura , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Lactante , Quinasa 1 de Quinasa de Quinasa MAP/antagonistas & inhibidores , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , Masculino , Ratones , Ratones Mutantes , Microscopía Electrónica de Rastreo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal/genética , Adulto JovenRESUMEN
The assembly of the musculoskeletal system requires the formation of an attachment unit between a bone and a tendon. Tendons are often inserted into bone eminences, superstructures that improve the mechanical resilience of the attachment of muscles to the skeleton and facilitate movement. Despite their functional importance, little is known about the development of bone eminences and attachment units. Here, we show that bone eminence cells are descendants of a unique set of progenitors and that superstructures are added onto the developing long bone in a modular fashion. First, we show that bone eminences emerge only after the primary cartilage rudiments have formed. Cell lineage analyses revealed that eminence cells are not descendants of chondrocytes. Moreover, eminence progenitors were specified separately and after chondroprogenitors of the primary cartilage. Fields of Sox9-positive, Scx-positive, Col2a1-negative cells identified at presumable eminence sites confirm the identity and specificity of these progenitors. The loss of eminences in limbs in which Sox9 expression was blocked in Scx-positive cells supports the hypothesis that a distinct pool of Sox9- and Scx-positive progenitors forms these superstructures. We demonstrate that TGFß signaling is necessary for the specification of bone eminence progenitors, whereas the SCX/BMP4 pathway is required for the differentiation of these progenitors to eminence-forming cells. Our findings suggest a modular model for bone development, involving a distinct pool of Sox9- and Scx-positive progenitor cells that form bone eminences under regulation of TGFß and BMP4 signaling. This model offers a new perspective on bone morphogenesis and on attachment unit development during musculoskeletal assembly.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Huesos/citología , Factor de Transcripción SOX9/metabolismo , Células Madre/citología , Tendones/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Huesos/metabolismo , Cartílago/citología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Condrocitos/citología , Femenino , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Ratones , Ratones Noqueados , Microscopía Fluorescente , Factor de Transcripción SOX9/genética , Células Madre/metabolismo , Tamoxifeno/farmacología , Tendones/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Microtomografía por Rayos XRESUMEN
The vertebrate skeleton consists of over 200 individual bones, each with its own unique shape, size and function. We study the role of intrauterine muscle-induced mechanical loads in determining the three-dimensional morphology of developing bones. Analysis of the force-generating capacity of intrauterine muscles in mice revealed that developing bones are subjected to significant and progressively increasing mechanical challenges. To evaluate the effect of intrauterine loads on bone morphogenesis and the contribution of the emerging shape to the ability of bones to withstand these loads, we monitored structural and mineral changes during development. Using daily micro-CT scans of appendicular long bones we identify a developmental program, which we term preferential bone growth, that determines the specific circumferential shape of each bone by employing asymmetric mineral deposition and transient cortical thickening. Finite element analysis demonstrates that the resulting bone structure has optimal load-bearing capacity. To test the hypothesis that muscle forces regulate preferential bone growth in utero, we examine this process in a mouse strain (mdg) that lacks muscle contractions. In the absence of mechanical loads, the stereotypical circumferential outline of each bone is lost, leading to the development of mechanically inferior bones. This study identifies muscle force regulation of preferential bone growth as the module that shapes the circumferential outline of bones and, consequently, optimizes their load-bearing capacity during development. Our findings invoke a common mechanism that permits the formation of different circumferential outlines in different bones.
Asunto(s)
Huesos/anatomía & histología , Huesos/fisiología , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/fisiología , Desarrollo Embrionario/fisiología , Soporte de Peso/fisiología , Adaptación Fisiológica/fisiología , Animales , Densidad Ósea/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Contracción Muscular/fisiología , Músculo Liso/fisiología , Periostio/citología , Periostio/crecimiento & desarrollo , Embarazo , Estrés Mecánico , Útero/anatomía & histología , Útero/fisiologíaRESUMEN
The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal stem cells and tooth regeneration. These progenitor populations actively divide, move, and differentiate to maintain tissue homeostasis and regenerate lost cells in a responsive manner. However, traditional analyses using fixed tissue sections could not capture the dynamic processes of cellular movements and interactions, limiting our ability to study their regulations. This paper describes a protocol to maintain whole mouse incisors in an explant culture system and live-track dental epithelial cells using multiphoton timelapse microscopy. This technique adds to our existing toolbox for dental research and allows investigators to acquire spatiotemporal information on cell behaviors and organizations in a living tissue. We anticipate that this methodology will help researchers further explore mechanisms that control the dynamic cellular processes taking place during both dental renewal and regeneration.
Asunto(s)
Células Madre Mesenquimatosas , Células Madre , Ratones , Animales , Células Madre Mesenquimatosas/fisiología , Incisivo , Células Epiteliales , División Celular , Diferenciación CelularRESUMEN
Amelogenesis, the formation of dental enamel, is driven by specialized epithelial cells called ameloblasts, which undergo successive stages of differentiation. Ameloblasts secrete enamel matrix proteins (EMPs), proteases, calcium, and phosphate ions in a stage-specific manner to form mature tooth enamel. Developmental defects in tooth enamel are common in humans, and they can greatly impact the well-being of affected individuals. Our understanding of amelogenesis and developmental pathologies is rooted in past studies using epithelial Cre driver and knockout alleles. However, the available mouse models are limited, as most do not allow targeting different ameloblast sub-populations, and constitutive loss of EMPs often results in severe phenotype in the mineral, making it difficult to interpret defect mechanisms. Herein, we report on the design and verification of a toolkit of twelve mouse alleles that include ameloblast-stage specific Cre recombinases, fluorescent reporter alleles, and conditional flox alleles for the major EMPs. We show how these models may be used for applications such as sorting of live stage specific ameloblasts, whole mount imaging, and experiments with incisor explants. The full list of new alleles is available at https://dev.facebase.org/enamelatlas/mouse-models/ .
RESUMEN
The Hedgehog (HH) pathway is critical for development and adult tissue homeostasis. Aberrant HH signaling can lead to congenital malformations and diseases including cancer. Although cholesterol and several oxysterol lipids have been shown to play crucial roles in HH activation, the molecular mechanisms governing their regulation remain unresolved. Here, we identify Canopy4 (CNPY4), a Saposin-like protein, as a regulator of the HH pathway that modulates levels of membrane sterol lipids. Cnpy4-/- embryos exhibit multiple defects consistent with HH signaling perturbations, most notably changes in digit number. Knockdown of Cnpy4 hyperactivates the HH pathway in vitro and elevates membrane levels of accessible sterol lipids, such as cholesterol, an endogenous ligand involved in HH activation. Our data demonstrate that CNPY4 is a negative regulator that fine-tunes HH signal transduction, revealing a previously undescribed facet of HH pathway regulation that operates through control of membrane composition.
Asunto(s)
Proteínas Hedgehog , Esteroles , Colesterol , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Lípidos de la Membrana , Transducción de Señal/fisiologíaRESUMEN
Bone is the most widespread mineralized tissue in vertebrates and its formation is orchestrated by specialized cells - the osteoblasts. Crystalline carbonated hydroxyapatite, an inorganic calcium phosphate mineral, constitutes a substantial fraction of mature bone tissue. Yet key aspects of the mineral formation mechanism, transport pathways and deposition in the extracellular matrix remain unidentified. Using cryo-electron microscopy on native frozen-hydrated tissues we show that during mineralization of developing mouse calvaria and long bones, bone-lining cells concentrate membrane-bound mineral granules within intracellular vesicles. Elemental analysis and electron diffraction show that the intracellular mineral granules consist of disordered calcium phosphate, a highly metastable phase and a potential precursor of carbonated hydroxyapatite. The intracellular mineral contains considerably less calcium than expected for synthetic amorphous calcium phosphate, suggesting the presence of a cellular mechanism by which phosphate entities are first formed and thereafter gradually sequester calcium within the vesicles. We thus demonstrate that in vivo osteoblasts actively produce disordered mineral packets within intracellular vesicles for mineralization of the extracellular developing bone tissue. The use of a highly disordered precursor mineral phase that later crystallizes within an extracellular matrix is a strategy employed in the formation of fish fin bones and by various invertebrate phyla. This therefore appears to be a widespread strategy used by many animal phyla, including vertebrates.
Asunto(s)
Calcificación Fisiológica/fisiología , Fosfatos de Calcio/metabolismo , Microscopía por Crioelectrón/métodos , Gránulos Citoplasmáticos/química , Animales , Huesos/química , Huesos/citología , Huesos/fisiología , Fosfatos de Calcio/química , Gránulos Citoplasmáticos/metabolismo , Durapatita/química , Embrión de Mamíferos/fisiología , Embrión de Mamíferos/ultraestructura , Peces/anatomía & histología , Peces/fisiología , Ratones , Ratones Endogámicos C57BL , Cráneo/química , Cráneo/ultraestructura , Tomografía Computarizada por Rayos X/métodosRESUMEN
A fundamental question in biomineralization is the nature of the first-formed mineral phase. In vertebrate bone formation, this issue has been the subject of a long-standing controversy. We address this key issue using the continuously growing fin bony rays of the Tuebingen long-fin zebrafish as a model for bone mineralization. Employing high-resolution scanning and transmission electron microscopy imaging, electron diffraction, and elemental analysis, we demonstrate the presence of an abundant amorphous calcium phosphate phase in the newly formed fin bones. The extracted amorphous mineral particles crystallize with time, and mineral crystallinity increases during bone maturation. Based on these findings, we propose that this amorphous calcium phosphate phase may be a precursor phase that later transforms into the mature crystalline mineral.
Asunto(s)
Estructuras Animales/crecimiento & desarrollo , Fosfatos de Calcio/metabolismo , Osteogénesis , Pez Cebra/crecimiento & desarrollo , Estructuras Animales/ultraestructura , Animales , Calcificación Fisiológica , Masculino , Espectroscopía Infrarroja por Transformada de Fourier , Tomografía Computarizada por Rayos XRESUMEN
Ror1 is a member of the Ror-family receptor tyrosine kinases. Ror1 is broadly expressed in various tissues and organs during mouse embryonic development. However, so far little is known about its function. The closely related family member Ror2 was shown to play a crucial role in skeletogenesis and has been shown to act as a co-receptor for Wnt5a mediating non-canonical Wnt-signaling. Previously, it has been shown that during embryonic development Ror1 acts in part redundantly with Ror2 in the skeletal and cardiovascular systems. In this study, we report that loss of the orphan receptor Ror1 results in a variety of phenotypic defects within the skeletal and urogenital systems and that Ror1 mutant mice display a postnatal growth retardation phenotype.
Asunto(s)
Huesos/anomalías , Retardo del Crecimiento Fetal/etiología , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/deficiencia , Animales , Ratones , Fenotipo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/fisiología , Anomalías UrogenitalesRESUMEN
PKBalpha/Akt1, a protein kinase, is a major mediator of angiogenic signaling. The purpose of this study was to determine the role of PKB alpha/Akt1 in bone vascularization and development. For that aim, macromolecular dynamic contrast enhanced MRI was applied to examine in vivo vascular changes in long bones of 40-day-old growing PKB alpha/Akt1-deficient, heterozygous, and wild-type mice. Ex vivo microMRI and microCT were applied to monitor the impact of PKB alpha/Akt1 gene dosage on trabecular bone formation during endochondral bone growth. PKB alpha/Akt1-deficient mice and, remarkably, also heterozygous mice showed significantly reduced blood volume fraction in the humerus compared to wild-type mice. Moreover, PKB alpha/Akt1-deficient mice showed a more severe vascular deficiency with reduced permeability. microCT and microMRI of trabeculae revealed impaired bone formation in both PKB alpha/Akt1-deficient and heterozygous mice, whereas cortical bone parameters were only reduced in PKB alpha/Akt1-deficient mice. Reduction of metaphyseal blood vessel invasion, concomitant with aberrant trabeculae and shorter long bones, demonstrates a gene-dose-dependent role for PKB alpha/Akt1 in regulation of overall size and endochondral bone growth. MRI proved to provide high sensitivity for in vivo detection of subtle gene dose effects leading to impaired bone vascularity and for uncovering changes in trabecular bone.
Asunto(s)
Dosificación de Gen , Húmero , Proteínas Proto-Oncogénicas c-akt/genética , Animales , Densidad Ósea , Húmero/irrigación sanguínea , Húmero/diagnóstico por imagen , Húmero/crecimiento & desarrollo , Inmunohistoquímica , Imagen por Resonancia Magnética , Ratones , Ratones Noqueados , RadiografíaRESUMEN
Somatic KRAS mutations are highly prevalent in many cancers. In addition, a distinct spectrum of germline KRAS mutations causes developmental disorders called RASopathies. The mutant proteins encoded by these germline KRAS mutations are less biochemically and functionally activated than those in cancer. We generated mice harboring conditional KrasLSL-P34Rand KrasLSL-T58I knock-in alleles and characterized the consequences of each mutation in vivo. Embryonic expression of KrasT58I resulted in craniofacial abnormalities reminiscent of those seen in RASopathy disorders, and these mice exhibited hyperplastic growth of multiple organs, modest alterations in cardiac valvulogenesis, myocardial hypertrophy, and myeloproliferation. By contrast, embryonic KrasP34R expression resulted in early perinatal lethality from respiratory failure due to defective lung sacculation, which was associated with aberrant ERK activity in lung epithelial cells. Somatic Mx1-Cre-mediated activation in the hematopoietic compartment showed that KrasP34R and KrasT58I expression had distinct signaling effects, despite causing a similar spectrum of hematologic diseases. These potentially novel strains are robust models for investigating the consequences of expressing endogenous levels of hyperactive K-Ras in different developing and adult tissues, for comparing how oncogenic and germline K-Ras proteins perturb signaling networks and cell fate decisions, and for performing preclinical therapeutic trials.
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Cardiomiopatías/patología , Craneosinostosis/patología , Enfermedades Hematológicas/patología , Enfermedades Pulmonares/patología , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Cardiomiopatías/etiología , Cardiomiopatías/metabolismo , Craneosinostosis/etiología , Craneosinostosis/metabolismo , Femenino , Enfermedades Hematológicas/etiología , Enfermedades Hematológicas/metabolismo , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , EmbarazoRESUMEN
Classical mechanical methods for testing whole bone have been critically assessed in a previous review where their limitations in terms of precision, accuracy and the amount of data yielded were described. This article describes the use of optical metrology methods and their novel adaptation to the study of whole bone response to mechanical load. Such methods overcome many of the limitations of mechanical testing: they do not require contact with the tested sample, are non-destructive, can be conducted on wet samples, and results comprise deformation maps of entire surfaces. The concepts upon which each method is based are reviewed, and examples of their use in biomechanical studies of bone are presented. Potential future applications that are expected to make significant contributions to the understanding of whole bone mechanics are outlined.
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Huesos/anatomía & histología , Huesos/fisiología , Estrés Mecánico , Animales , Fenómenos Biomecánicos , Fuerza Compresiva , Elasticidad , Interpretación de Imagen Radiográfica Asistida por Computador , Resistencia a la TracciónRESUMEN
The classical model of tissue renewal posits that small numbers of quiescent stem cells (SCs) give rise to proliferating transit-amplifying cells before terminal differentiation. However, many organs house pools of SCs with proliferative and differentiation potentials that diverge from this template. Resolving SC identity and organization is therefore central to understanding tissue renewal. Here, using a combination of single-cell RNA sequencing (scRNA-seq), mouse genetics and tissue injury approaches, we uncover cellular hierarchies and mechanisms that underlie the maintenance and repair of the continuously growing mouse incisor. Our results reveal that, during homeostasis, a group of actively cycling epithelial progenitors generates enamel-producing ameloblasts and adjacent layers of non-ameloblast cells. After injury, tissue repair was achieved through transient increases in progenitor-cell proliferation and through direct conversion of Notch1-expressing cells to ameloblasts. We elucidate epithelial SC identity, position and function, providing a mechanistic basis for the homeostasis and repair of a fast-turnover ectodermal appendage.
Asunto(s)
Ameloblastos/citología , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Ectodermo/citología , Incisivo/citología , Animales , División Celular/fisiología , Células Epiteliales/citología , Ratones Transgénicos , Transducción de Señal/fisiología , Células Madre/citologíaRESUMEN
INTRODUCTION: The relationship between load and the structure and mechanical properties of mature bones has been thoroughly described. In contrast, this relationship has been studied much less in immature bones, which consist of bony tissue and cartilaginous growth plate, during the postnatal period. This paper describes the effect of an externally applied load on the bones of young fast-growing chicks; in particular, we examine the effect on the growth plate, which regulates longitudinal bone growth, and the consequences in terms of bone structural and mechanical properties. MATERIALS AND METHODS: The tibial growth plates from chicks subjected to external load and control chicks, immediately after loading and following 5 days of load release, were studied by histological staining and quantitative PCR. The contralateral tibiae were mechanically tested by three-point bending and their structural features determined by micro-CT. RESULTS: At the end of the external loading period, the tibias of the experimental group were shorter and their growth plate narrower than in controls. However, at this time point, effects were not yet apparent in the bones' structural or mechanical parameters. After a further 5 days of no external load, bones and growth plates of the experimental group demonstrated the phenomenon of 'catch-up': the thickness of the growth plate exceeded that of the control; however the relative expression of genes controlling chondrocyte differentiation (collagen II and X) did not change, while the expression of factors related to growth-plate ossification (osteopontin, alkaline phosphatase) and cartilage and bone calcification (matrix and bone Gla proteins) was upregulated as a result of the catch-up process. At this time, however, the tibiae of the experimental group showed inferior mechanical and structural properties relative to the control group. CONCLUSION: External loading during bone elongation negatively affects the mechanical and structural properties of the skeleton. The effect is first noticeable in the growth plate, which regulates bone growth, and is exhibited in the bone phenotype after a lag period.
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Desarrollo Óseo/fisiología , Huesos/metabolismo , Placa de Crecimiento/metabolismo , Soporte de Peso/fisiología , Adaptación Fisiológica/fisiología , Fosfatasa Alcalina/genética , Animales , Animales Recién Nacidos , Huesos/citología , Huesos/diagnóstico por imagen , Proteínas de Unión al Calcio/genética , Pollos , Colágeno Tipo II/genética , Colágeno Tipo X/genética , Proteínas de la Matriz Extracelular/genética , Perfilación de la Expresión Génica , Placa de Crecimiento/citología , Placa de Crecimiento/diagnóstico por imagen , Hibridación in Situ , Osteocalcina/genética , Osteopontina/genética , Radiografía , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tibia/citología , Tibia/diagnóstico por imagen , Tibia/metabolismo , Proteína Gla de la MatrizRESUMEN
The mechanical behaviour of material bone can be completely described by a group of material properties. The mechanical behaviour of the entire bone organ, however, is much more difficult to predict; it is the result both of the properties of the material of which it is made, and of the geometric spatial architecture in which this is arranged. This review first describes material bone in terms of its complex, graded and hierarchical structure. Basic concepts used in the field of mechanics of materials are defined and explained and then used to describe the mechanical properties of whole bone. Some clinical implications of these properties are provided. Commonly used mechanical testing methods for the study of the mechanical behaviour of whole bone are reviewed and the technical difficulties associated with them are discussed.