RESUMO
BMP signaling plays an essential role in second heart field-derived heart and arterial trunk development, including myocardial differentiation, right ventricular growth, and interventricular, outflow tract and aortico-pulmonary septation. It is mediated by a number of different BMP ligands, and receptors, many of which are present simultaneously. The mechanisms by which they regulate morphogenetic events and degree of redundancy amongst them have still to be elucidated. We therefore assessed the role of BMP Type I receptor AcvR1 in anterior second heart field-derived cell development, and compared it with that of BmpR1a. By removing Acvr1 using the driver Mef2c[AHF]-Cre, we show that AcvR1 plays an essential role in arterial pole morphogenesis, identifying defects in outflow tract wall and cushion morphology that preceded a spectrum of septation defects from double outlet right ventricle to common arterial trunk in mutants. Its absence caused dysregulation in gene expression important for myocardial differentiation (Isl1, Fgf8) and regional identity (Tbx2, Tbx3, Tbx20, Tgfb2). Although these defects resemble to some degree those in the equivalent Bmpr1a mutant, a novel gene knock-in model in which Bmpr1a was expressed in the Acvr1 locus only partially restored septation in Acvr1 mutants. These data show that both BmpR1a and AcvR1 are needed for normal heart development, in which they play some non-redundant roles, and refine our understanding of the genetic and morphogenetic processes underlying Bmp-mediated heart development important in human congenital heart disease.
Assuntos
Receptores de Ativinas Tipo I/metabolismo , Artérias/embriologia , Padronização Corporal/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Coração/embriologia , Morfogênese/fisiologia , Receptores de Ativinas Tipo I/genética , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Diferenciação Celular/fisiologia , Técnicas de Introdução de Genes , Vetores Genéticos/genética , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Transgênicos , Morfogênese/genética , Miocárdio/citologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: The role of Smad-independent TGF-ß signaling in craniofacial development is poorly elucidated. RESULTS: In craniofacial mesenchymal cells, Tak1 regulates both R-Smad C-terminal and linker region phosphorylation in TGF-ß signaling. CONCLUSION: Tak1 plays an irreplaceable role in craniofacial ecto-mesenchyme during embryogenesis. SIGNIFICANCE: Understanding the mechanisms of TGF-ß signaling contributes to knowledge of pathogenetic mechanisms underlying common craniofacial birth defects. Although the importance of TGF-ß superfamily signaling in craniofacial growth and patterning is well established, the precise details of its signaling mechanisms are still poorly understood. This is in part because of the concentration of studies on the role of the Smad-dependent (so-called "canonical") signaling pathways relative to the Smad-independent ones in many biological processes. Here, we have addressed the role of TGF-ß-activated kinase 1 (Tak1, Map3k7), one of the key mediators of Smad-independent (noncanonical) TGF-ß superfamily signaling in craniofacial development, by deleting Tak1 specifically in the neural crest lineage. Tak1-deficient mutants display a round skull, hypoplastic maxilla and mandible, and cleft palate resulting from a failure of palatal shelves to appropriately elevate and fuse. Our studies show that in neural crest-derived craniofacial ecto-mesenchymal cells, Tak1 is not only required for TGF-ß- and bone morphogenetic protein-induced p38 Mapk activation but also plays a role in agonist-induced C-terminal and linker region phosphorylation of the receptor-mediated R-Smads. Specifically, we demonstrate that the agonist-induced linker region phosphorylation of Smad2 at Thr-220, which has been shown to be critical for full transcriptional activity of Smad2, is dependent on Tak1 activity and that in palatal mesenchymal cells TGFßRI and Tak1 kinases mediate both overlapping and distinct TGF-ß2-induced transcriptional responses. To summarize, our results suggest that in neural crest-derived ecto-mesenchymal cells, Tak1 provides a critical point of intersection in a complex dialogue between the canonical and noncanonical arms of TGF-ß superfamily signaling required for normal craniofacial development.
Assuntos
MAP Quinase Quinase Quinases/fisiologia , Crista Neural/citologia , Processamento de Proteína Pós-Traducional , Proteínas Smad/metabolismo , Motivos de Aminoácidos , Animais , Células Cultivadas , Fissura Palatina/enzimologia , Fissura Palatina/genética , Ectoderma/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Cabeça/embriologia , MAP Quinase Quinase Quinases/deficiência , MAP Quinase Quinase Quinases/genética , Masculino , Mandíbula/anormalidades , Camundongos , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Proteínas Smad Reguladas por Receptor/metabolismo , Proteínas da Superfamília de TGF-beta/fisiologia , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
The p75(NTR) neurotrophin receptor has been implicated in multiple biological and pathological processes. While significant advances have recently been made in understanding the physiologic role of p75(NTR) , many details and aspects remain to be determined. This is in part because the two existing knockout mouse models (Exons 3 or 4 deleted, respectively), both display features that defy definitive conclusions. Here we describe the generation of mice that carry a conditional p75(NTR) (p75(NTR-FX) ) allele made by flanking Exons 4-6, which encode the transmembrane and all cytoplasmic domains, by loxP sites. To validate this novel conditional allele, both neural crest-specific p75(NTR) /Wnt1-Cre mutants and conventional p75(NTR) null mutants were generated. Both mutants displayed abnormal hind limb reflexes, implying that loss of p75(NTR) in neural crest-derived cells causes a peripheral neuropathy similar to that seen in conventional p75(NTR) mutants. This novel conditional p75(NTR) allele will offer new opportunities to investigate the role of p75(NTR) in specific tissues and cells.
Assuntos
Alelos , Camundongos Knockout/genética , Receptores de Fator de Crescimento Neural/genética , Animais , Clonagem Molecular , Cruzamentos Genéticos , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Desenvolvimento Embrionário , Éxons , Feminino , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Genótipo , Imuno-Histoquímica , Extremidade Inferior/fisiologia , Masculino , Camundongos , Camundongos Knockout/embriologia , Camundongos Knockout/metabolismo , Crista Neural/embriologia , Crista Neural/metabolismo , Crista Neural/patologia , Doenças do Sistema Nervoso Periférico/patologia , Receptores de Fator de Crescimento Neural/metabolismo , Reflexo AnormalRESUMO
The small GTP-binding protein Rac1, a member of the Rho family of small GTPases, has been implicated in regulation of many cellular processes including adhesion, migration and cytokinesis. These functions have largely been attributed to its ability to reorganize cytoskeleton. While the function of Rac1 is relatively well known in vitro, its role in vivo has been poorly understood. It has previously been shown that in neural crest cells (NCCs) Rac1 is required in a stage-specific manner to acquire responsiveness to mitogenic EGF signals. Here we demonstrate that mouse embryos lacking Rac1 in neural crest cells (Rac1/Wnt1-Cre) showed abnormal craniofacial development including regional ectodermal detachment associated with mesenchymal acellularity culminating in cleft face at E12. Rac1/Wnt1-Cre mutants also displayed inappropriate remodelling of pharyngeal arch arteries and defective outflow tract septation resulting in the formation of a common arterial trunk ('persistent truncus arteriosus' or PTA). The mesenchyme around the aortic sac also developed acellular regions, and the distal aortic sac became grossly dysmorphic, forming a pair of bilateral, highly dilated arterial structures connecting to the dorsal aortas. Smooth muscle cells lacking Rac1 failed to differentiate appropriately, and subpopulations of post-migratory NCCs demonstrated aberrant cell death and attenuated proliferation. These novel data demonstrate that while Rac1 is not required for normal NCC migration in vivo, it plays a critical cell-autonomous role in post-migratory NCCs during craniofacial and cardiac development by regulating the integrity of the craniofacial and pharyngeal mesenchyme.
Assuntos
Anormalidades Cardiovasculares/genética , Anormalidades Craniofaciais/genética , Crista Neural/metabolismo , Deleção de Sequência , Proteínas rac1 de Ligação ao GTP/genética , Animais , Região Branquial/citologia , Separação Celular , Células Cultivadas , Embrião de Mamíferos , Matriz Extracelular/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Crista Neural/citologiaRESUMO
BACKGROUND: Approximately 66% of college students gain weight in their first year of college and continue to gain weight at a higher rate than the general population. On-campus clinics are often the only contact this generally healthy population has with healthcare providers. OBJECTIVE: The aim of this quality improvement project was to increase screening, diagnosis, and intervention of overweight (OW) and obesity by healthcare providers in a university-based student health center. METHODS: Manual audits of electronic health records were conducted 6 weeks before and 14 weeks after two educational seminars to increase provider awareness of evidence-based practice recommendations and available online and on-campus resources related to OW and obesity of young adult college students. Two hundred and forty-one pre-intervention and 520 post-intervention EHRs were reviewed to collect data on screening, diagnosis, and intervention by providers in the health center. RESULTS: Assessment of OW and obesity, diagnosis, and intervention all significantly (p = .00) increased following nurse practitioner (NP) education. CONCLUSIONS: Following two continuing education seminars, provider adherence to evidence-based guidelines in the health center significantly increased. IMPLICATIONS FOR PRACTICE: Education on evidence-based recommendations and available resources may increase health promotion, screening, diagnosis, and intervention of OW and obesity by healthcare providers in university-based health centers.
RESUMO
BACKGROUND AND AIM OF THE STUDY: Human mesenchymal stem cells (MSCs) are a potential cell source for the tissue engineering of biological structures, including cardiac valves. A comprehensive, phenotypic analysis of MSCs and, for the latter, their comparison with valve interstitial cells (ICs) is therefore essential. METHODS: Isolates of bone marrow-derived human MSCs and human cardiac valve ICs were extensively phenotyped for their expression of membrane proteins involved in adhesion and cell-cell communication, cytoskeletal components, extracellular matrix (ECM) proteins and gene expression of WNT/FZD/SFRP/DKK/LRP family members. RESULTS: MSCs and valve ICs (>80%) expressed fibroblast surface antigen, smooth muscle alpha-actin, vimentin and CD44; expression of MHC class I and II and calponin was inconsistent, and a small proportion expressed desmin and smooth muscle myosin. CD105 was weakly expressed by a low percentage of valve ICs (<10%) compared to MSCs (>90%). ECM components made by both cell types demonstrated similar levels and patterns of staining, although expression of elastin was not detected by both cell types. Adhesion molecule expression was highly variable among the MSC isolates and between the two cell types, with the predominant integrins being alphal, alpha3, alpha5, and beta1 by both cell types. PCR analysis of WNT/FZD/SFRP/LRP family members revealed a greater range of the WNT family of genes being expressed in MSCs compared to ICs. CONCLUSION: The study results provided an extensive fingerprint of valve ICs and of MSCs for the tissue engineering of biological structures and for the manipulation of their desired phenotype. MSCs represent a promising cell type for valve tissue engineering, and will require extensive phenotyping after differentiation.
Assuntos
Valvas Cardíacas/citologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Células-Tronco Mesenquimais/fisiologia , Adulto , Idoso , Diferenciação Celular , Expressão Gênica , Humanos , Pessoa de Meia-Idade , Fenótipo , Engenharia TecidualRESUMO
Matrix remodeling, which involves proteolytic enzymes, such as the matrix metalloproteinases (MMPs), is of significant importance with respect to tissue engineering a heart valve construct. The ability of valve interstitial cells (ICs) to release these enzymes in biological scaffolds and to synthesize their own matrix has not been adequately studied, and this has important implications for tissue engineering. Cultured human aortic valve ICs were seeded onto a 3-dimensional type I collagen matrix for 28 days, whereby the presence of the remodeling enzymes, MMPs, were determined using immunohistochemistry, and detection of extracellular matrix (ECM) gene expression was performed using in situ hybridization. The collagenases, stromelysins, and membrane-type MMPs were expressed in 1%, 2%, and 5% collagen scaffolds after 28 days, whereas gelatinase expression was not observed. In situ hybridization revealed the presence of the ECM messenger ribonucleic acid (mRNA) in cells cultured in collagen scaffolds however, an increase in all three mRNAs was only detected in the 1% collagen scaffolds. The presence of collagenases, stromelysins, and membrane-type MMPs indicate that human valve ICs have the capacity to remodel type I collagen scaffold and that the genes necessary for synthesizing matrix have been turned on within the cells themselves. Scaffold composition also demonstrated differential effects onMMPexpression. These observations are of relevance with respect to the development of tissue-engineered heart valves.
Assuntos
Materiais Biocompatíveis , Bioprótese , Células do Tecido Conjuntivo/enzimologia , Proteínas da Matriz Extracelular/biossíntese , Matriz Extracelular , Metaloproteinases da Matriz/biossíntese , Valva Aórtica/citologia , Valva Aórtica/enzimologia , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Células do Tecido Conjuntivo/citologia , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Regulação Enzimológica da Expressão Gênica , Próteses Valvulares Cardíacas , Humanos , Engenharia Tecidual/métodosRESUMO
Cardiac valve interstitial cells (ICs) are a heterogeneous and dynamic population of specific cell types that have many unique characteristics. They are responsible for maintaining the extracellular scaffold that provides the mechanical characteristics vital for sustaining the unique dynamic behaviour of the valve. A number of cellular phenotypes can be distinguished: some are sparsely arranged throughout the valve leaflets, whilst others are arranged in thin bundles. These cells express molecular markers similar to those of skeletal, cardiac and smooth muscle cells (SMCs) and in particular, many ICs express smooth muscle (SM) alpha-actin, a marker of myofibroblasts. In this respect, these cells exhibit a profile unlike skin fibroblasts, which may allude to their role in valve function.
Assuntos
Valvas Cardíacas/citologia , Valvas Cardíacas/fisiologia , Actinas/metabolismo , Animais , Comunicação Celular , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Valvas Cardíacas/imunologia , Humanos , FenótipoRESUMO
Tgfb3 is strongly and specifically expressed in the epithelial tips of pre-fusion palatal shelves where it plays a critical non-redundant role in palatal fusion in both medial edge epithelial (MEE) cells and in a thin layer of flattened peridermal cells that covers the MEE. It is not known how Tgfb3 expression is regulated in these specific cell types. Using comparative genomics and transgenic reporter assays, we have identified cis-regulatory elements that could control Tgfb3 expression during palatogenesis. Our results show that a 61-kb genomic fragment encompassing the Tgfb3 gene drives remarkably specific reporter expression in the MEE and adjacent periderm. Within this fragment, we identified two small, non-coding, evolutionarily conserved regions in intron 2 of the neighboring Ift43 gene, and a larger region in the intervening sequence between the Ift43 and Tgfb3 genes, each of which could target reporter activity to the tips of pre-fusion/fusing palatal shelves. Identification of the cis-regulatory sequences controlling spatio-temporal Tgfb3 expression in palatal shelves is a key step toward understanding upstream regulation of Tgfb3 expression during palatogenesis and should enable the development of improved tools to investigate palatal epithelial fusion.
RESUMO
Bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly in humans. Despite recent advances, the molecular basis of BAV development is poorly understood. Previously it has been shown that mutations in the Notch1 gene lead to BAV and valve calcification both in human and mice, and mice deficient in Gata5 or its downstream target Nos3 have been shown to display BAVs. Here we show that tissue-specific deletion of the gene encoding Activin Receptor Type I (Alk2 or Acvr1) in the cushion mesenchyme results in formation of aortic valve defects including BAV. These defects are largely due to a failure of normal development of the embryonic aortic valve leaflet precursor cushions in the outflow tract resulting in either a fused right- and non-coronary leaflet, or the presence of only a very small, rudimentary non-coronary leaflet. The surviving adult mutant mice display aortic stenosis with high frequency and occasional aortic valve insufficiency. The thickened aortic valve leaflets in such animals do not show changes in Bmp signaling activity, while Map kinase pathways are activated. Although dysfunction correlated with some pro-osteogenic differences in gene expression, neither calcification nor inflammation were detected in aortic valves of Alk2 mutants with stenosis. We conclude that signaling via Alk2 is required for appropriate aortic valve development in utero, and that defects in this process lead to indirect secondary complications later in life.
Assuntos
Receptores de Ativinas Tipo I/deficiência , Valva Aórtica/anormalidades , Comunicação Interventricular/genética , Doenças das Valvas Cardíacas/genética , Receptores de Ativinas Tipo I/genética , Animais , Valva Aórtica/embriologia , Valva Aórtica/metabolismo , Proliferação de Células , Coxins Endocárdicos/metabolismo , Feminino , Fator de Transcrição GATA5/metabolismo , Fator de Transcrição GATA5/fisiologia , Expressão Gênica , Comunicação Interventricular/embriologia , Comunicação Interventricular/patologia , Doenças das Valvas Cardíacas/embriologia , Doenças das Valvas Cardíacas/patologia , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Recombinação Genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Proteínas com Domínio T/metabolismo , Tenascina/metabolismo , Versicanas/metabolismoRESUMO
In the developing heart, reciprocal interactions between the epicardium and myocardium drive further sublineage specification and ventricular chamber morphogenesis. Several observations suggest that the epicardium is a source of secreted factors that influence cardiomyocyte proliferation, and these factors may have other roles as well. However, the identity of these epicardial factors remains mostly unknown. We have identified platelet-derived growth factor-A (PDGF-A) as one of several mitogens expressed by the rat EMC epicardial cell line (epicardial mesothelial cells), by embryonic epicardium and myocardium during mouse heart development, and by adult epicardium. Expression of the cognate receptor gene Pdgfra was detected in the epicardium, although a low level of expression in myocardium could not be ruled out. To address the potential role of PDGF signaling in heart development, we mutated both PDGF receptor genes in the myocardial and mesodermal compartments of the heart; however, this did not result in an observable cardiac phenotype. This finding suggests that mesodermal PDGF signaling is not essential in heart development, although its role may be redundant with other signaling pathways. Indeed, our results demonstrate the presence of additional mitogens that may have such an overlapping role.
Assuntos
Coração/embriologia , Miocárdio/metabolismo , Pericárdio/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Linhagem Celular , Meios de Cultivo Condicionados , Camundongos , Mitógenos/metabolismo , Miocárdio/citologia , Organogênese , Pericárdio/citologia , Pericárdio/embriologia , RatosRESUMO
Developmental abnormalities in endocardial cushions frequently contribute to congenital heart malformations including septal and valvular defects. While compelling evidence has been presented to demonstrate that members of the TGF-beta superfamily are capable of inducing endothelial-to-mesenchymal transdifferentiation in the atrioventricular canal, and thus play a key role in formation of endocardial cushions, the detailed signaling mechanisms of this important developmental process, especially in vivo, are still poorly known. Several type I receptors (ALKs) for members of the TGF-beta superfamily are expressed in the myocardium and endocardium of the developing heart, including the atrioventricular canal. However, analysis of their functional role during mammalian development has been significantly complicated by the fact that deletion of the type I receptors in mouse embryos often leads to early embryonal lethality. Here, we used the Cre/loxP system for endothelial-specific deletion of the type I receptor Alk2 in mouse embryos. The endothelial-specific Alk2 mutant mice display defects in atrioventricular septa and valves, which result from a failure of endocardial cells to appropriately transdifferentiate into the mesenchyme in the AV canal. Endocardial cells deficient in Alk2 demonstrate decreased expression of Msx1 and Snail, and reduced phosphorylation of BMP and TGF-beta Smads. Moreover, we show that endocardial cells lacking Alk2 fail to delaminate from AV canal explants. Collectively, these results indicate that the BMP type I receptor ALK2 in endothelial cells plays a critical non-redundant role in early phases of endocardial cushion formation during cardiac morphogenesis.