RESUMEN
Metanephric kidney development is orchestrated by the iterative branching morphogenesis of the ureteric bud. We describe an underlying patterning associated with the ramification of this structure and show that this pattern is conserved between developing kidneys, in different parts of the organ and across developmental time. This regularity is associated with a highly reproducible branching asymmetry that is consistent with locally operative growth mechanisms. We then develop a class of tip state models to represent elaboration of the ureteric tree and describe rules for 'half-delay' branching morphogenesis that describe almost perfectly the patterning of this structure. Spatial analysis suggests that the observed asymmetry may arise from mutual suppression of bifurcation, but not extension, between the growing ureteric tips, and demonstrates that disruption of patterning occurs in mouse mutants in which the distribution of tips on the surface of the kidney is altered. These findings demonstrate that kidney development occurs by way of a highly conserved reiterative pattern of asymmetric bifurcation that is governed by intrinsic and locally operative mechanisms.
Asunto(s)
Riñón/embriología , Morfogénesis/fisiología , Uréter/embriología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Proteína Morfogenética Ósea 7/deficiencia , Proteína Morfogenética Ósea 7/genética , Proteína Morfogenética Ósea 7/fisiología , Imagenología Tridimensional , Conceptos Matemáticos , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Modelos Biológicos , Morfogénesis/genética , Mutación , Fenotipo , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Fosfoproteínas/fisiología , Factor de Crecimiento Transformador beta2/deficiencia , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/fisiologíaRESUMEN
BACKGROUND: Wnt5a and Mrfzb1 genes are involved in the regulation of tooth size, and their expression levels are similar to that of Bmp7 during morphogenesis, including during the cap and early bell stages of tooth formation. We previously reported that Usag-1-deficient mice form supernumerary maxillary incisors. Thus, we hypothesized that BMP7 and USAG-1 signaling molecules may play important roles in tooth morphogenesis. In this study, we established double genetically modified mice to examine the in vivo inter-relationships between Bmp7 and Usag-1. RESULTS: We measured the volume and cross-sectional areas of the mandibular incisors using micro-computed tomography (micro-CT) in adult Bmp7- and Usag-1-LacZ knock-in mice and their F2 generation upon interbreeding. The mandibular incisors of adult Bmp7+/- mice were significantly larger than those of wild-type (WT) mice. The mandibular incisors of adult Usag-1-/- mice were the largest of all genotypes examined. In the F2 generation, the effects of these genes were additive; Bmp7+/- was most strongly associated with the increase in tooth size using generalized linear models, and the total area of mandibular supernumerary incisors of Usag-1-/-Bmp7+/- mice was significantly larger than that of Usag-1-/-Bmp7 +/+ mice. At embryonic day 15 (E15), BrdU assays demonstrated that the labeling index of Bmp7+/- embryos was significantly higher than that of WT embryos in the cervical loop. Additionally, the labeling index of Usag-1-/- embryos was significantly the highest of all genotypes examined in dental papilla. CONCLUSIONS: Bmp7 heterozygous mice exhibited significantly increased tooth sizes, suggesting that tooth size was controlled by specific gene expression. Our findings may be useful in applications of regenerative medicine and dentistry.
Asunto(s)
Proteína Morfogenética Ósea 7/deficiencia , Proteínas Morfogenéticas Óseas/deficiencia , Morfogénesis , Diente/embriología , Proteínas Adaptadoras Transductoras de Señales , Envejecimiento , Animales , Apoptosis , Proteína Morfogenética Ósea 7/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Bromodesoxiuridina/metabolismo , Proliferación Celular , Cruzamientos Genéticos , Embrión de Mamíferos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Etiquetado Corte-Fin in Situ , Incisivo/diagnóstico por imagen , Incisivo/metabolismo , Modelos Lineales , Masculino , Mandíbula/diagnóstico por imagen , Mandíbula/metabolismo , Ratones Endogámicos C57BL , Diente Molar/metabolismo , Tamaño de los Órganos , Fenotipo , Coloración y Etiquetado , Diente/diagnóstico por imagen , Diente/metabolismo , Microtomografía por Rayos X , beta-Galactosidasa/metabolismoRESUMEN
AIMS: TGF-ß regulates tissue fibrosis: TGF-ß promotes fibrosis, whereas bone morphogenetic protein (BMP)-7 is antifibrotic. To demonstrate that (i) left ventricular (LV) remodelling after pressure overload is associated with disequilibrium in the signalling mediated by these cytokines, and (ii) BMP-7 exerts beneficial effects on LV remodelling and reverse remodelling. METHODS AND RESULTS: We studied patients with aortic stenosis (AS) and mice subjected to transverse aortic constriction (TAC) and TAC release (de-TAC). LV morphology and function were assessed by echocardiography. LV biopsies were analysed by qPCR, immunoblotting, and histology. Pressure overload reduced BMP-7 and pSmad1/5/8 and increased TGF-ß and pSmad2/3 in AS patients and TAC mice. BMP-7 correlated inversely with collagen, fibronectin, and ß-MHC expressions, and with hypertrophy and diastolic dysfunction, and directly with the systolic function. Multiple linear regression disclosed BMP-7 and TGF-ß as hypertrophy predictors, negative and positive, respectively. BMP-7 prevented TGF-ß-elicited hypertrophic program in cardiomyocytes, and Col1A1 promoter activity in NIH-3T3 fibroblasts. The treatment of TAC mice with rBMP-7 attenuated the development of structural damage and dysfunction, and halted ongoing remodelling. The reverse remodelling after pressure overload release was facilitated by rBMP-7, and hampered by disrupting BMP-7 function using a neutralizing antibody or genetic deletion. CONCLUSION: The disequilibrium between BMP-7 and TGF-ß signals plays a relevant role in the LV remodelling response to haemodynamic stress in TAC mice and AS patients. Our observations may provide new important insights aimed at developing novel therapies designed to prevent, halt, or reverse LV pathological remodelling in pressure overload cardiomyopathy.
Asunto(s)
Proteína Morfogenética Ósea 7/análisis , Proteína Morfogenética Ósea 7/metabolismo , Hipertrofia Ventricular Izquierda/prevención & control , Miocitos Cardíacos/metabolismo , Disfunción Ventricular Izquierda/prevención & control , Función Ventricular Izquierda , Remodelación Ventricular , Anciano , Anciano de 80 o más Años , Animales , Estenosis de la Válvula Aórtica/complicaciones , Proteína Morfogenética Ósea 7/administración & dosificación , Proteína Morfogenética Ósea 7/deficiencia , Proteína Morfogenética Ósea 7/genética , Estudios de Casos y Controles , Colágeno/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibronectinas/metabolismo , Fibrosis , Humanos , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Cadenas Pesadas de Miosina/metabolismo , Células 3T3 NIH , Ratas Wistar , Proteínas Recombinantes/administración & dosificación , Transducción de Señal , Proteínas Smad/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta1/metabolismo , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/metabolismo , Disfunción Ventricular Izquierda/fisiopatología , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacosRESUMEN
The cause of posterior urethral valves (PUV) is unknown, but genetic factors are suspected given their familial occurrence. We examined cases of isolated PUV to identify novel copy number variants (CNVs). We identified 56 cases of isolated PUV from all live-births in New York State (1998-2005). Samples were genotyped using Illumina HumanOmni2.5 microarrays. Autosomal and sex-linked CNVs were identified using PennCNV and cnvPartition software. CNVs were prioritized for follow-up if they were absent from in-house controls, contained ≥ 10 consecutive probes, were ≥ 20 Kb in size, had ≤ 20% overlap with variants detected in other birth defect phenotypes screened in our lab, and were rare in population reference controls. We identified 47 rare candidate PUV-associated CNVs in 32 cases; one case had a 3.9 Mb deletion encompassing BMP7. Mutations in BMP7 have been associated with severe anomalies in the mouse urethra. Other interesting CNVs, each detected in a single PUV case included: a deletion of PIK3R3 and TSPAN1, duplication/triplication in FGF12, duplication of FAT1--a gene essential for normal growth and development, a large deletion (>2 Mb) on chromosome 17q that involves TBX2 and TBX4, and large duplications (>1 Mb) on chromosomes 3q and 6q. Our finding of previously unreported novel CNVs in PUV suggests that genetic factors may play a larger role than previously understood. Our data show a potential role of CNVs in up to 57% of cases examined. Investigation of genes in these CNVs may provide further insights into genetic variants that contribute to PUV.
Asunto(s)
Proteína Morfogenética Ósea 7/genética , Cadherinas/genética , Variaciones en el Número de Copia de ADN , Factores de Crecimiento de Fibroblastos/genética , Fosfatidilinositol 3-Quinasas/genética , Eliminación de Secuencia , Tetraspaninas/genética , Estrechez Uretral/genética , Secuencia de Bases , Proteína Morfogenética Ósea 7/deficiencia , Cadherinas/deficiencia , Estudios de Casos y Controles , Preescolar , Cromosomas Humanos Par 17 , Cromosomas Humanos Par 3 , Cromosomas Humanos Par 6 , Hibridación Genómica Comparativa , Factores de Crecimiento de Fibroblastos/deficiencia , Expresión Génica , Genotipo , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , New York/epidemiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Fosfatidilinositol 3-Quinasas/deficiencia , Polimorfismo de Nucleótido Simple , Tetraspaninas/deficiencia , Uretra/metabolismo , Uretra/patología , Estrechez Uretral/diagnóstico , Estrechez Uretral/epidemiología , Estrechez Uretral/patologíaRESUMEN
Human Langerhans cell (LC) precursors populate the epidermis early during prenatal development and thereafter undergo massive proliferation. The prototypic antiproliferative cytokine TGF-ß1 is required for LC differentiation from human CD34(+) hematopoietic progenitor cells and blood monocytes in vitro. Similarly, TGF-ß1 deficiency results in LC loss in vivo. However, immunohistology studies revealed that human LC niches in early prenatal epidermis and adult basal (germinal) keratinocyte layers lack detectable TGF-ß1. Here we demonstrated that these LC niches express high levels of bone morphogenetic protein 7 (BMP7) and that Bmp7-deficient mice exhibit substantially diminished LC numbers, with the remaining cells appearing less dendritic. BMP7 induces LC differentiation and proliferation by activating the BMP type-I receptor ALK3 in the absence of canonical TGF-ß1-ALK5 signaling. Conversely, TGF-ß1-induced in vitro LC differentiation is mediated via ALK3; however, co-induction of ALK5 diminished TGF-ß1-driven LC generation. Therefore, selective ALK3 signaling by BMP7 promotes high LC yields. Within epidermis, BMP7 shows an inverse expression pattern relative to TGF-ß1, the latter induced in suprabasal layers and up-regulated in outer layers. We observed that TGF-ß1 inhibits microbial activation of BMP7-generated LCs. Therefore, TGF-ß1 in suprabasal/outer epidermal layers might inhibit LC activation, resulting in LC network maintenance.
Asunto(s)
Proteína Morfogenética Ósea 7/metabolismo , Células de Langerhans/citología , Células de Langerhans/metabolismo , Animales , Proteína Morfogenética Ósea 7/deficiencia , Proteína Morfogenética Ósea 7/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Citocinas/biosíntesis , Células Epidérmicas , Epidermis/embriología , Epidermis/metabolismo , Expresión Génica , Humanos , Células de Langerhans/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Transducción de Señal , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/fisiologíaRESUMEN
Palatogenesis is a complex process implying growth, elevation and fusion of the two lateral palatal shelves during embryogenesis. This process is tightly controlled by genetic and mechanistic cues that also coordinate the growth of other orofacial structures. Failure at any of these steps can result in cleft palate, which is a frequent craniofacial malformation in humans. To understand the etiology of cleft palate linked to the BMP signaling pathway, we studied palatogenesis in Bmp7-deficient mouse embryos. Bmp7 expression was found in several orofacial structures including the edges of the palatal shelves prior and during their fusion. Bmp7 deletion resulted in a general alteration of oral cavity morphology, unpaired palatal shelf elevation, delayed shelf approximation, and subsequent lack of fusion. Cell proliferation and expression of specific genes involved in palatogenesis were not altered in Bmp7-deficient embryos. Conditional ablation of Bmp7 with Keratin14-Cre or Wnt1-Cre revealed that neither epithelial nor neural crest-specific loss of Bmp7 alone could recapitulate the cleft palate phenotype. Palatal shelves from mutant embryos were able to fuse when cultured in vitro as isolated shelves in proximity, but not when cultured as whole upper jaw explants. Thus, deformations in the oral cavity of Bmp7-deficient embryos such as the shorter and wider mandible were not solely responsible for cleft palate formation. These findings indicate a requirement for Bmp7 for the coordination of both developmental and mechanistic aspects of palatogenesis.
Asunto(s)
Proteína Morfogenética Ósea 7/genética , Fisura del Paladar/etiología , Fisura del Paladar/metabolismo , Animales , Proteína Morfogenética Ósea 7/deficiencia , Proliferación Celular , Fisura del Paladar/genética , Embrión de Mamíferos/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Sirenomelia is a severe congenital malformation of the lower body characterized by the fusion of the legs into a single lower limb. This striking external phenotype consistently associates severe visceral abnormalities, most commonly of the kidneys, intestine, and genitalia that generally make the condition lethal. Although the causes of sirenomelia remain unknown, clinical studies have yielded two major hypotheses: i) a primary defect in the generation of caudal mesoderm, ii) a primary vascular defect that leaves the caudal part of the embryo hypoperfused. Interestingly, Sirenomelia has been shown to have a genetic basis in mice, and although it has been considered a sporadic condition in humans, recently some possible familial cases have been reported. Here, we report that the removal of one or both functional alleles of Shh from the Bmp7-null background leads to a sirenomelia phenotype that faithfully replicates the constellation of external and internal malformations, typical of the human condition. These mutants represent an invaluable model in which we have analyzed the pathogenesis of sirenomelia. We show that the signaling defect predominantly impacts the morphogenesis of the hindgut and the development of the caudal end of the dorsal aortas. The deficient formation of ventral midline structures, including the interlimb mesoderm caudal to the umbilicus, leads to the approximation and merging of the hindlimb fields. Our study provides new insights for the understanding of the mechanisms resulting in caudal body malformations, including sirenomelia.
Asunto(s)
Proteína Morfogenética Ósea 7/genética , Modelos Animales de Enfermedad , Ectromelia/embriología , Embrión de Mamíferos/anomalías , Eliminación de Gen , Proteínas Hedgehog/genética , Fenotipo , Animales , Proteína Morfogenética Ósea 7/deficiencia , Huesos/anomalías , Huesos/embriología , Muerte Celular/genética , Proliferación Celular , Ectromelia/genética , Ectromelia/patología , Proteínas Hedgehog/deficiencia , Humanos , Extremidad Inferior/embriología , Extremidad Inferior/patología , RatonesRESUMEN
BACKGROUND: During normal development in human and other placental mammals, the embryonic cloacal cavity separates along the axial longitudinal plane to give rise to the urethral system, ventrally, and the rectum, dorsally. Defects in cloacal development are very common and present clinically as a rectourethral fistula in about 1 in 5,000 live human births. Yet, the cellular mechanisms of cloacal septation remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We previously detected Bone morphogenetic protein 7 (Bmp7) expression in the urorectal mesenchyme (URM), and have shown that loss of Bmp7 function results in the arrest of cloacal septation. Here, we present evidence that cloacal partitioning is driven by Bmp7 signaling in the cloacal endoderm. We performed TUNEL and immunofluorescent analysis on cloacal sections from Bmp7 null and control littermate embryos. We found that loss of Bmp7 results in a dramatic decrease in the endoderm survival and a delay in differentiation. We used immunological methods to show that Bmp7 functions by activating the c-Jun N-terminal kinase (JNK) pathway. We carried out confocal and 3D imaging analysis of mitotic chromosome bundles to show that during normal septation cells in the cloacal endoderm divide predominantly in the apical-basal direction. Loss of Bmp7/JNK signaling results in randomization of mitotic angles in the cloacal endoderm. We also conducted immunohistochemical analysis of human fetal sections to show that BMP/phospho-SMAD and JNK pathways function in the human cloacal region similar as in the mouse. CONCLUSION/SIGNIFICANCE: Our results strongly indicate that Bmp7/JNK signaling regulates remodeling of the cloacal endoderm resulting in a topological separation of the urinary and digestive systems. Our study points to the importance of Bmp and JNK signaling in cloacal development and rectourethral malformations.
Asunto(s)
Tipificación del Cuerpo , Proteína Morfogenética Ósea 7/metabolismo , Polaridad Celular , Cloaca/citología , Cloaca/embriología , Animales , Proteína Morfogenética Ósea 7/deficiencia , Diferenciación Celular , División Celular , Proliferación Celular , Supervivencia Celular , Cloaca/enzimología , Endodermo/citología , Epitelio/embriología , Feto/embriología , Feto/enzimología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Modelos Biológicos , Recto/embriología , Recto/enzimología , Proteínas Smad/metabolismo , Uretra/embriología , Uretra/enzimologíaRESUMEN
As malignant breast cancers progress, they acquire the ability to spread to other regions of the body, including bone and lung, but the molecular mechanism underlying the increase in metastatic potential is not fully understood. Here we studied murine 4T1E/M3 highly bone marrow metastatic breast cancer cells, which we established previously. These cells show upregulated expression of bone morphogenetic protein (BMP) 7 and BMP receptors, as well as augmented phosphorylation of Smad1/5/8. Both anchorage-independent cell growth measured in colony forming assays and cell migration measured in wound healing assays were suppressed in 4T1E/M3 cells following treatment with a neutralizing anti-BMP7 antibody or knockdown of BMP7 gene expression. In addition, metastasis of 4T1E/M3 cells to the spine and lung and intracellular levels of phosphorylated Smad1/5/8 were suppressed by knocking down BMP7. Conversely, overexpression of BMP7 in the weakly metastatic parental 4T1E cells augmented their anchorage-independent growth, migration and metastasis to spine and lung. Taken together, our results strongly suggest that augmented autocrine BMP7 signaling leads to increases in the anchorage-independent cell growth, migration and metastatic potential in our bone marrow metastatic breast cancer model.