RESUMEN
Craniosynostosis is a congenital anomaly characterized by the premature fusion of cranial sutures. Sutures are a critical connective tissue that regulates bone growth; their aberrant fusion results in abnormal shapes of the head and face. The molecular and cellular mechanisms have been investigated for a long time, but knowledge gaps remain between genetic mutations and mechanisms of pathogenesis for craniosynostosis. We previously demonstrated that the augmentation of bone morphogenetic protein (BMP) signaling through constitutively active BMP type 1A receptor (caBmpr1a) in neural crest cells (NCCs) caused the development of premature fusion of the anterior frontal suture, leading to craniosynostosis in mice. In this study, we demonstrated that ectopic cartilage forms in sutures prior to premature fusion in caBmpr1a mice. The ectopic cartilage is subsequently replaced by bone nodules leading to premature fusion with similar but unique fusion patterns between two neural crest-specific transgenic Cre mouse lines, P0-Cre and Wnt1-Cre mice, which coincides with patterns of premature fusion in each line. Histologic and molecular analyses suggest that endochondral ossification in the affected sutures. Both in vitro and in vivo observations suggest a greater chondrogenic capacity and reduced osteogenic capability of neural crest progenitor cells in mutant lines. These results suggest that the augmentation of BMP signaling alters the cell fate of cranial NCCs toward a chondrogenic lineage to prompt endochondral ossification to prematurely fuse cranial sutures. By comparing P0-Cre;caBmpr1a and Wnt1-Cre;caBmpr1a mice at the stage of neural crest formation, we found more cell death of cranial NCCs in P0-Cre;caBmpr1a than Wnt1-Cre;caBmpr1a mice at the developing facial primordia. These findings may provide a platform for understanding why mutations of broadly expressed genes result in the premature fusion of limited sutures. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
RESUMEN
The bending of epithelial tubes is a fundamental process in organ morphogenesis, driven by various multicellular behaviours. The cochlea in the mammalian inner ear is a representative example of spiral tissue architecture where the continuous bending of the duct is a fundamental component of its morphogenetic process. Although the cochlear duct morphogenesis has been studied by genetic approaches extensively, it is still unclear how the cochlear duct morphology is physically formed. Here, we report that nuclear behaviour changes are associated with the curvature of the pseudostratified epithelium during murine cochlear development. Two-photon live-cell imaging reveals that the nuclei shuttle between the luminal and basal edges of the cell is in phase with cell-cycle progression, known as interkinetic nuclear migration, in the flat region of the pseudostratified epithelium. However, the nuclei become stationary on the luminal side following mitosis in the curved region. Mathematical modelling together with perturbation experiments shows that this nuclear stalling facilitates luminal-basal differential growth within the epithelium, suggesting that the nuclear stalling would contribute to the bending of the pseudostratified epithelium during the cochlear duct development. The findings suggest a possible scenario of differential growth which sculpts the tissue shape, driven by collective nuclear dynamics.
RESUMEN
A notable example of spiral architecture in organs is the mammalian cochlear duct, where the morphology is critical for hearing function. Genetic studies have revealed necessary signaling molecules, but it remains unclear how cellular dynamics generate elongating, bending, and coiling of the cochlear duct. Here, we show that extracellular signal-regulated kinase (ERK) activation waves control collective cell migration during the murine cochlear duct development using deep tissue live-cell imaging, Förster resonance energy transfer (FRET)-based quantitation, and mathematical modeling. Long-term FRET imaging reveals that helical ERK activation propagates from the apex duct tip concomitant with the reverse multicellular flow on the lateral side of the developing cochlear duct, resulting in advection-based duct elongation. Moreover, model simulations, together with experiments, explain that the oscillatory wave trains of ERK activity and the cell flow are generated by mechanochemical feedback. Our findings propose a regulatory mechanism to coordinate the multicellular behaviors underlying the duct elongation during development.
Asunto(s)
Movimiento Celular , Conducto Coclear/embriología , Sistema de Señalización de MAP Quinasas , Animales , Embrión de Mamíferos , Transferencia Resonante de Energía de Fluorescencia , Ratones Endogámicos ICR , Ratones Transgénicos , Modelos Teóricos , MorfogénesisRESUMEN
Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.
Asunto(s)
Técnicas de Cultivo de Célula , Células Madre Multipotentes/citología , Cresta Neural/citología , Animales , Sistemas CRISPR-Cas , Diferenciación Celular , Línea Celular , Ratones , Miocitos del Músculo Liso , Osteoblastos , ARN Interferente Pequeño/genéticaRESUMEN
Primordial germ cells (PGCs) are a highly migratory cell population that gives rise to eggs and sperm. Much is known about PGC specification, but less about the processes that control PGC migration. In this study, we document a deficiency in PGC development in embryos carrying global homozygous null mutations in Msx1 and Msx2, both immediate downstream effectors of Bmp signaling pathway. We show that Msx1(-/-);Msx2(-/-) mutant embryos have defects in PGC migration as well as a reduced number of PGCs. These phenotypes are also evident in a Mesp1-Cre-mediated mesoderm-specific mutant line of Msx1 and Msx2. Since PGCs are not marked in Mesp1-lineage tracing, our results suggest that Msx1 and Msx2 function cell non-autonomously in directing PGC migration. Consistent with this hypothesis, we noted an upregulation of fibronectin, well known as a mediator of cell migration, in tissues through which PGCs migrate. We also noted a reduction in the expression of Wnt5a and an increase in the expression in Bmp4 in such tissues in Msx1(-/-);Msx2(-/-) mutants, both known effectors of PGC development.
Asunto(s)
Movimiento Celular/genética , Células Germinales Embrionarias/citología , Proteínas de Homeodominio/genética , Factor de Transcripción MSX1/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteína Morfogenética Ósea 4/biosíntesis , Células Germinales Embrionarias/metabolismo , Fibronectinas/biosíntesis , Proteínas de Homeodominio/metabolismo , Factor de Transcripción MSX1/metabolismo , Mesodermo/citología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteína Wnt-5a/biosíntesisRESUMEN
The role of the Hippo signaling pathway in cranial neural crest (CNC) development is poorly understood. We used the Wnt1(Cre) and Wnt1(Cre2SOR) drivers to conditionally ablate both Yap and Taz in the CNC of mice. When using either Cre driver, Yap and Taz deficiency in the CNC resulted in enlarged, hemorrhaging branchial arch blood vessels and hydrocephalus. However, Wnt1(Cre2SOR) mutants had an open cranial neural tube phenotype that was not evident in Wnt1(Cre) mutants. In O9-1 CNC cells, the loss of Yap impaired smooth muscle cell differentiation. RNA-sequencing data indicated that Yap and Taz regulate genes encoding Fox transcription factors, specifically Foxc1. Proliferation was reduced in the branchial arch mesenchyme of Yap and Taz CNC conditional knockout (CKO) embryos. Moreover, Yap and Taz CKO embryos had cerebellar aplasia similar to Dandy-Walker spectrum malformations observed in human patients and mouse embryos with mutations in Foxc1. In embryos and O9-1 cells deficient for Yap and Taz, Foxc1 expression was significantly reduced. Analysis of Foxc1 regulatory regions revealed a conserved recognition element for the Yap and Taz DNA binding co-factor Tead. ChIP-PCR experiments supported the conclusion that Foxc1 is directly regulated by the Yap-Tead complex. Our findings uncover important roles for Yap and Taz in CNC diversification and development.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cara/embriología , Cresta Neural/embriología , Fosfoproteínas/metabolismo , Cráneo/embriología , Animales , Apoptosis/genética , Proteínas de Ciclo Celular , Diferenciación Celular , Proliferación Celular , Pérdida del Embrión/patología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Hemorragia/patología , Hidrocefalia/embriología , Hidrocefalia/patología , Mandíbula/patología , Ratones Noqueados , Miocitos del Músculo Liso/citología , Defectos del Tubo Neural/patología , Fenotipo , Análisis de Secuencia de ARN , Transducción de Señal , Transactivadores , Proteínas Señalizadoras YAPRESUMEN
The skull vault is a complex, exquisitely patterned structure that plays a variety of key roles in vertebrate life, ranging from the acquisition of food to the support of the sense organs for hearing, smell, sight, and taste. During its development, it must meet the dual challenges of protecting the brain and accommodating its growth. The bones and sutures of the skull vault are derived from cranial neural crest and head mesoderm. The frontal and parietal bones develop from osteogenic rudiments in the supraorbital ridge. The coronal suture develops from a group of Shh-responsive cells in the head mesoderm that are collocated, with the osteogenic precursors, in the supraorbital ridge. The osteogenic rudiments and the prospective coronal suture expand apically by cell migration. A number of congenital disorders affect the skull vault. Prominent among these is craniosynostosis, the fusion of the bones at the sutures. Analysis of the pathophysiology underling craniosynostosis has identified a variety of cellular mechanisms, mediated by a range of signaling pathways and effector transcription factors. These cellular mechanisms include loss of boundary integrity, altered sutural cell specification in embryos, and loss of a suture stem cell population in adults. Future work making use of genome-wide transcriptomic approaches will address the deep structure of regulatory interactions and cellular processes that unify these seemingly diverse mechanisms.
Asunto(s)
Suturas Craneales/embriología , Craneosinostosis/embriología , Morfogénesis , Cráneo/embriología , Animales , Suturas Craneales/metabolismo , Craneosinostosis/genética , Craneosinostosis/fisiopatología , Regulación del Desarrollo de la Expresión Génica , Humanos , Mesodermo/embriología , Mesodermo/metabolismo , Cresta Neural/embriología , Cresta Neural/metabolismo , Transducción de Señal/genética , Cráneo/metabolismoRESUMEN
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.
Asunto(s)
Quinasas Quinasa Quinasa PAM/fisiología , Cresta Neural/citología , Procesamiento Proteico-Postraduccional , Proteínas Smad/metabolismo , Secuencias de Aminoácidos , Animales , Células Cultivadas , Fisura del Paladar/enzimología , Fisura del Paladar/genética , Ectodermo/citología , Femenino , Regulación del Desarrollo de la Expresión Génica , Cabeza/embriología , Quinasas Quinasa Quinasa PAM/deficiencia , Quinasas Quinasa Quinasa PAM/genética , Masculino , Mandíbula/anomalías , Ratones , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteínas Smad Reguladas por Receptores/metabolismo , Proteínas de la Superfamilia TGF-beta/fisiología , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMEN
The mammalian skull vault consists of several intricately patterned bones that grow in close coordination. The growth of these bones depends on the precise regulation of the migration and differentiation of osteogenic cells from undifferentiated precursor cells located above the eye. Here, we demonstrate a role for Foxc1 in modulating the influence of Bmp signaling on the expression of Msx2 and the specification of these cells. Inactivation of Foxc1 results in a dramatic reduction in skull vault growth and causes an expansion of Msx2 expression and Bmp signaling into the area occupied by undifferentiated precursor cells. Foxc1 interacts directly with a Bmp responsive element in an enhancer upstream of Msx2, and acts to reduce the occupancy of P-Smad1/5/8. We propose that Foxc1 sets a threshold for the Bmp-dependent activation of Msx2, thus controlling the differentiation of osteogenic precursor cells and the rate and pattern of calvarial bone development.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Factores de Transcripción Forkhead/fisiología , Hueso Frontal/embriología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Animales , Desarrollo Óseo/genética , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 2/farmacología , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Proteína Morfogenética Ósea 4/farmacología , Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular/genética , Células Cultivadas , Embrión de Mamíferos , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Hueso Frontal/crecimiento & desarrollo , Hueso Frontal/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteogénesis/genética , Osteogénesis/fisiología , Cráneo/embriología , Cráneo/metabolismoRESUMEN
Cranial neural crest cells give rise to ectomesenchymal derivatives such as cranial bones, cartilage, smooth muscle, dentin, as well as melanocytes, corneal endothelial cells, and neurons and glial cells of the peripheral nervous system. Previous studies have suggested that although multipotent stem-like cells may exist during the course of cranial neural crest development, they are transient, undergoing lineage restriction early in embryonic development. We have developed culture conditions that allow cranial neural crest cells to be grown as multipotent stem-like cells. With these methods, we obtained 2 independent cell lines, O9-1 and i10-1, which were derived from mass cultures of Wnt1-Cre; R26R-GFP-expressing cells. These cell lines can be propagated and passaged indefinitely, and can differentiate into osteoblasts, chondrocytes, smooth muscle cells, and glial cells. Whole-genome expression profiling of O9-1 cells revealed that this line stably expresses stem cell markers (CD44, Sca-1, and Bmi1) and neural crest markers (AP-2α, Twist1, Sox9, Myc, Ets1, Dlx1, Dlx2, Crabp1, Epha2, and Itgb1). O9-1 cells are capable of contributing to cranial mesenchymal (osteoblast and smooth muscle) neural crest fates when injected into E13.5 mouse cranial tissue explants and chicken embryos. These results suggest that O9-1 cells represent multipotent mesenchymal cranial neural crest cells. The O9-1 cell line should serve as a useful tool for investigating the molecular properties of differentiating cranial neural crest cells.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Línea Celular , Cresta Neural/citología , Cráneo/citología , Animales , Biomarcadores/metabolismo , Movimiento Celular , Embrión de Pollo , Medios de Cultivo/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Receptores de Hialuranos/metabolismo , Ratones , Microinyecciones , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Osteogénesis , Cráneo/metabolismoRESUMEN
UNLABELLED: The knowledge concerning fetal hepatic stellate cells (HSCs) is scarce, and their cell lineage and functions are largely unknown. The current study isolated fetal liver mesenchymal cells from a mouse expressing beta-galactosidase under the control of Msx2 promoter by fluorescence-activated cell sorting (FACS) and surveyed marker genes by microarray analysis. Based on the location and immunostaining with conventional and newly disclosed markers, we have identified three distinct populations of fetal liver mesenchymal cells expressing both desmin and p75 neurotrophin receptor (p75NTR): HSCs in the liver parenchyma; perivascular mesenchymal cells expressing alpha-smooth muscle actin (alpha-SMA); and submesothelial cells associated with the basal lamina beneath mesothelial cells and expressing activated leukocyte cell adhesion molecule (ALCAM) and platelet-derived growth factor receptor alpha. A transitional cell type from the submesothelial cell phenotype to fetal HSCs was also identified near the liver surface. Mesothelial cells expressed podoplanin and ALCAM. Ki-67 staining showed that proliferative activity of the submesothelial cells is higher than that of mesothelial cells and transitional cells. Using anti-ALCAM antibodies, submesothelial and mesothelial cells were isolated by FACS. The ALCAM(+) cells expressed hepatocyte growth factor and pleiotrophin. In culture, the ALCAM(+) cells rapidly acquired myofibroblastic morphology and alpha-SMA expression. The ALCAM(+) cells formed intracellular lipid droplets when embedded in collagen gel and treated with retinol, suggesting the potential for ALCAM(+) cells to differentiate to HSCs. Finally, we demonstrated that fetal HSCs, submesothelial cells, and perivascular mesenchymal cells are all derived from mesoderm by using MesP1-Cre and ROSA26 reporter mice. CONCLUSION: Fetal HSCs, submesothelial cells, and perivascular mesenchymal cells are mesodermal in origin, and ALCAM(+) submesothelial cells may be a precursor for HSCs in developing liver.
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Endotelio Vascular/citología , Células Epiteliales/citología , Células Estrelladas Hepáticas/citología , Hígado/citología , Hígado/embriología , Células Madre Mesenquimatosas/citología , Organogénesis/fisiología , Molécula de Adhesión Celular del Leucocito Activado/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Desmina/metabolismo , Endotelio Vascular/metabolismo , Células Epiteliales/metabolismo , Femenino , Células Estrelladas Hepáticas/metabolismo , Proteínas de Homeodominio/metabolismo , Operón Lac/genética , Hígado/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Transgénicos , Receptores de Factor de Crecimiento Nervioso/metabolismoRESUMEN
BACKGROUND: Msx1 and Msx2, which belong to the highly conserved Nk family of homeobox genes, display overlapping expression patterns and redundant functions in multiple tissues and organs during vertebrate development. Msx1 and Msx2 have well-documented roles in mediating epithelial-mesenchymal interactions during organogenesis. Given that both Msx1 and Msx2 are crucial downstream effectors of Bmp signaling, we investigated whether Msx1 and Msx2 are required for the Bmp-induced endothelial-mesenchymal transformation (EMT) during atrioventricular (AV) valve formation. RESULTS: While both Msx1-/- and Msx2-/- single homozygous mutant mice exhibited normal valve formation, we observed hypoplastic AV cushions and malformed AV valves in Msx1-/-; Msx2-/- mutants, indicating redundant functions of Msx1 and Msx2 during AV valve morphogenesis. In Msx1/2 null mutant AV cushions, we found decreased Bmp2/4 and Notch1 signaling as well as reduced expression of Has2, NFATc1 and Notch1, demonstrating impaired endocardial activation and EMT. Moreover, perturbed expression of chamber-specific genes Anf, Tbx2, Hand1 and Hand2 reveals mispatterning of the Msx1/2 double mutant myocardium and suggests functions of Msx1 and Msx2 in regulating myocardial signals required for remodelling AV valves and maintaining an undifferentiated state of the AV myocardium. CONCLUSION: Our findings demonstrate redundant roles of Msx1 and Msx2 in regulating signals required for development of the AV myocardium and formation of the AV valves.
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Cojinetes Endocárdicos/embriología , Proteínas de Homeodominio/genética , Factor de Transcripción MSX1/genética , Miocardio/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Tipificación del Cuerpo , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Cojinetes Endocárdicos/citología , Endocardio/citología , Endocardio/embriología , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Válvulas Cardíacas/citología , Válvulas Cardíacas/embriología , Mesodermo/citología , Mesodermo/embriología , Ratones , Ratones Endogámicos BALB C , Factores de Transcripción NFATC/genética , Organogénesis , Receptor Notch1/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The Bmp pathway is of critical importance in the development of the skull vault. Analysis of gain and loss of function phenotypes of Bmp pathway effectors, particularly Msx genes, has shown that the Bmp pathway functions in the growth of both mesodermal and neural crest-derived calvarial bones. It is required for the development of the frontal and parietal bones during the interval between the initial osteogenic mesenchymal condensations at E12.5 to the apposition of the paired frontal and parietal bones at E18.5. During postnatal development, forced expression of the Bmp inhibitor, noggin, maintains the patency of sutures, consistent with a role for the Bmp pathway in regulating suture development. The availability of conditional mutants of Bmp ligands, receptors and downstream effectors will make possible an increasingly high resolution analysis of precisely how the Bmp functions in these processes and how aberrations in its activity can contribute to pathological conditions such as familial parietal foramina and craniosynostosis.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Suturas Craneales/crecimiento & desarrollo , Cráneo/crecimiento & desarrollo , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/genética , Proteínas Portadoras/genética , Craneosinostosis/genética , Motivos Nodales de Cisteina/genética , Hueso Frontal/crecimiento & desarrollo , Humanos , Mesodermo/fisiología , Mutación/genética , Cresta Neural/fisiología , Hueso Parietal/crecimiento & desarrolloRESUMEN
The homeobox genes Msx1 and Msx2 function as transcriptional regulators that control cellular proliferation and differentiation during embryonic development. Mutations in the Msx1 and Msx2 genes in mice disrupt tissue-tissue interactions and cause multiple craniofacial malformations. Although Msx1 and Msx2 are both expressed throughout the entire development of the frontal bone, the frontal bone defect in Msx1 or Msx2 null mutants is rather mild, suggesting the possibility of functional compensation between Msx1 and Msx2 during early frontal bone development. To investigate this hypothesis, we generated Msx1(-/-);Msx2(-/-) mice. These double mutant embryos died at E17 to E18 with no formation of the frontal bone. There was no apparent defect in CNC migration into the presumptive frontal bone primordium, but differentiation of the frontal mesenchyme and establishment of the frontal primordium was defective, indicating that Msx1 and Msx2 genes are specifically required for osteogenesis in the cranial neural crest lineage within the frontal bone primordium. Mechanistically, our data suggest that Msx genes are critical for the expression of Runx2 in the frontonasal subpopulation of cranial neural crest cells and for differentiation of the osteogenic lineage. This early function of the Msx genes is likely independent of the Bmp signaling pathway.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Unión al ADN/fisiología , Hueso Frontal/embriología , Proteínas de Homeodominio/fisiología , Factor de Transcripción MSX1/fisiología , Cresta Neural/citología , Animales , Diferenciación Celular/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Proteínas de Unión al ADN/genética , Hueso Frontal/citología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/genética , Factor de Transcripción MSX1/genética , Ratones , Ratones Noqueados , Osteogénesis/genéticaRESUMEN
The msh-related homeobox genes, Msx1 and Msx2, have a variety functions during murine organogenesis, Msx1 in the development of the palate and teeth, Msx2 in the skull, teeth, and skin. Msx1 mutants die perinatally. Compound Msx1-2 mutants do not survive past late gestation. The multiplicity of functions of Msx1 and 2, as well as the lethality of Msx1 and Msx1-2 mutants limits the utility of the conventional knockouts. We therefore produced conditional alleles of Msx1 and Msx2. We constructed targeting vectors with LoxP sites flanking the homeodomain-encoding second exons and Frt sites flanking a neo gene. These vectors were used to produce targeted ES cells and mice with floxed alleles. The functionality of the LoxP sites in the floxed alleles was established by crosses with K14-Cre mice (epidermis-specific), and with an Msx2-Cre line that produces a germline deletion. Analysis of progeny by PCR revealed correct Cre-mediated recombination, as well as expected phenotypes.
Asunto(s)
Alelos , Proteínas de Unión al ADN/genética , Marcación de Gen , Proteínas de Homeodominio/genética , Integrasas/metabolismo , Factor de Transcripción MSX1/genética , Animales , Southern Blotting , Cruzamientos Genéticos , Embrión de Mamíferos/citología , Células Madre Embrionarias/metabolismo , Células Epidérmicas , Epidermis/metabolismo , Exones , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos , Masculino , Ratones , Ratones Noqueados , Fenotipo , Reacción en Cadena de la Polimerasa , Recombinación GenéticaRESUMEN
Msx1 and Msx2 are highly conserved, Nk-related homeodomain transcription factors that are essential for a variety of tissue-tissue interactions during vertebrate organogenesis. Here we show that combined deficiencies of Msx1 and Msx2 cause conotruncal anomalies associated with malalignment of the cardiac outflow tract (OFT). Msx1 and Msx2 play dual roles in outflow tract morphogenesis by both protecting secondary heart field (SHF) precursors against apoptosis and inhibiting excessive proliferation of cardiac neural crest, endothelial and myocardial cells in the conotruncal cushions. During incorporation of SHF precursors into the OFT myocardium, ectopic apoptosis in the Msx1-/-; Msx2-/- mutant SHF is associated with reduced expression of Hand1 and Hand2, which from work on Hand1 and Hand2 mutants may be functionally important in the inhibition of apoptosis in Msx1/2 mutants. Later during aorticopulmonary septation, excessive proliferation in the OFT cushion mesenchyme and myocardium of Msx1-/-; Msx2-/- mutants is associated with premature down-regulation of p27(KIP1), an inhibitor of cyclin-dependent kinases. Diminished accretion of SHF precursors to the elongating OFT myocardium and excessive accumulation of mesenchymal cells in the conotruncal cushions may work together to perturb the rotation of the truncus arteriosus, leading to OFT malalignment defects including double-outlet right ventricle, overriding aorta and pulmonary stenosis.
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Proteínas de Unión al ADN/fisiología , Corazón Fetal/embriología , Proteínas de Homeodominio/fisiología , Factor de Transcripción MSX1/fisiología , Cresta Neural/citología , Animales , Apoptosis , Tipificación del Cuerpo , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/metabolismo , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Femenino , Corazón Fetal/citología , Corazón Fetal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Cardiopatías Congénitas/embriología , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Proteínas de Homeodominio/genética , Factor de Transcripción MSX1/deficiencia , Factor de Transcripción MSX1/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Mutación , Cresta Neural/metabolismo , Embarazo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Boundaries between cellular compartments often serve as signaling interfaces during embryogenesis. The coronal suture is a major growth center of the skull vault and develops at a boundary between cells derived from neural crest and mesodermal origin, forming the frontal and parietal bones, respectively. Premature fusion of these bones, termed coronal synostosis, is a common human developmental anomaly. Known causes of coronal synostosis include haploinsufficiency of TWIST1 and a gain of function mutation in MSX2. In Twist1(+/-) mice with coronal synostosis, we found that the frontal-parietal boundary is defective. Specifically, neural crest cells invade the undifferentiated mesoderm of the Twist1(+/-) mutant coronal suture. This boundary defect is accompanied by an expansion in Msx2 expression and reduction in ephrin-A4 distribution. Reduced dosage of Msx2 in the Twist1 mutant background restores the expression of ephrin-A4, rescues the suture boundary and inhibits craniosynostosis. Underlining the importance of ephrin-A4, we identified heterozygous mutations in the human orthologue, EFNA4, in three of 81 patients with non-syndromic coronal synostosis. This provides genetic evidence that Twist1, Msx2 and Efna4 function together in boundary formation and the pathogenesis of coronal synostosis.
Asunto(s)
Suturas Craneales/metabolismo , Craneosinostosis/metabolismo , Craneosinostosis/patología , Efrinas/metabolismo , Mesodermo/metabolismo , Cresta Neural/metabolismo , Receptores de la Familia Eph/metabolismo , Animales , Secuencia de Bases , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Efrina-A2/genética , Efrina-A2/metabolismo , Efrina-A4/genética , Efrina-A4/metabolismo , Regulación del Desarrollo de la Expresión Génica , Heterocigoto , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunohistoquímica , Mesodermo/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Cresta Neural/citología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Transducción de Señal , Células Tumorales Cultivadas , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
The neural crest is a multipotent, migratory cell population that contributes to a variety of tissues and organs during vertebrate embryogenesis. Here, we focus on the function of Msx1 and Msx2, homeobox genes implicated in several disorders affecting craniofacial development in humans. We show that Msx1/2 mutants exhibit profound deficiencies in the development of structures derived from the cranial and cardiac neural crest. These include hypoplastic and mispatterned cranial ganglia, dysmorphogenesis of pharyngeal arch derivatives and abnormal organization of conotruncal structures in the developing heart. The expression of the neural crest markers Ap-2alpha, Sox10 and cadherin 6 (cdh6) in Msx1/2 mutants revealed an apparent retardation in the migration of subpopulations of preotic and postotic neural crest cells, and a disorganization of neural crest cells paralleling patterning defects in cranial nerves. In addition, normally distinct subpopulations of migrating crest underwent mixing. The expression of the hindbrain markers Krox20 and Epha4 was altered in Msx1/2 mutants, suggesting that defects in neural crest populations may result, in part, from defects in rhombomere identity. Msx1/2 mutants also exhibited increased Bmp4 expression in migratory cranial neural crest and pharyngeal arches. Finally, proliferation of neural crest-derived mesenchyme was unchanged, but the number of apoptotic cells was increased substantially in neural crest-derived cells that contribute to the cranial ganglia and the first pharyngeal arch. This increase in apoptosis may contribute to the mispatterning of the cranial ganglia and the hypoplasia of the first arch.
Asunto(s)
Tipificación del Cuerpo/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Homeodominio/genética , Factor de Transcripción MSX1/deficiencia , Factor de Transcripción MSX1/genética , Cresta Neural/embriología , Animales , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/metabolismo , Anomalías Cardiovasculares/genética , Movimiento Celular/genética , Cruzamientos Genéticos , Proteínas de Unión al ADN/fisiología , Proteína 2 de la Respuesta de Crecimiento Precoz/biosíntesis , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/fisiología , Factor de Transcripción MSX1/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Defectos del Tubo Neural/embriología , Defectos del Tubo Neural/genética , Receptor EphA4/biosíntesis , Receptor EphA4/genética , Rombencéfalo/embriología , Rombencéfalo/metabolismo , Cráneo/anomalías , Cráneo/embriología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
The flat bones of the vertebrate skull vault develop from two migratory mesenchymal cell populations, the cranial neural crest and paraxial mesoderm. At the onset of skull vault development, these mesenchymal cells emigrate from their sites of origin to positions between the ectoderm and the developing cerebral hemispheres. There they combine, proliferate and differentiate along an osteogenic pathway. Anomalies in skull vault development are relatively common in humans. One such anomaly is familial calvarial foramina, persistent unossified areas within the skull vault. Mutations in MSX2 and TWIST are known to cause calvarial foramina in humans. Little is known of the cellular and developmental processes underlying this defect. Neither is it known whether MSX2 and TWIST function in the same or distinct pathways. We trace the origin of the calvarial foramen defect in Msx2 mutant mice to a group of skeletogenic mesenchyme cells that compose the frontal bone rudiment. We show that this cell population is reduced not because of apoptosis or deficient migration of neural crest-derived precursor cells, but because of defects in its differentiation and proliferation. We demonstrate, in addition, that heterozygous loss of Twist function causes a foramen in the skull vault similar to that caused by loss of Msx2 function. Both the quantity and proliferation of the frontal bone skeletogenic mesenchyme are reduced in Msx2-Twist double mutants compared with individual mutants. Thus Msx2 and Twist cooperate in the control of the differentiation and proliferation of skeletogenic mesenchyme. Molecular epistasis analysis suggests that Msx2 and Twist do not act in tandem to control osteoblast differentiation, but function at the same epistatic level.
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Proteínas de Unión al ADN/metabolismo , Hueso Frontal/embriología , Proteínas de Homeodominio/metabolismo , Mesodermo/fisiología , Cresta Neural/citología , Proteínas Nucleares/metabolismo , Factores de Transcripción , Animales , Apoptosis , Biomarcadores , Proteínas de Unión al ADN/genética , Hueso Frontal/anomalías , Hueso Frontal/anatomía & histología , Hueso Frontal/fisiología , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Proteínas de Homeodominio/genética , Humanos , Mesodermo/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Cresta Neural/fisiología , Proteínas Nucleares/genética , Proteína 1 Relacionada con TwistRESUMEN
The neural crest plays a crucial part in cardiac development. Cells of the cardiac subpopulation of cranial neural crest migrate from the hindbrain into the outflow tract of the heart where they contribute to the septum that divides the pulmonary and aortic channels. In Splotch mutant mice, which lack a functional Pax3 gene, migration of cardiac neural crest is deficient and aorticopulmonary septation does not occur. Downstream genes through which Pax3 regulates cardiac neural crest development are unknown. Here, using a combination of genetic and molecular approaches, we show that the deficiency of cardiac neural crest development in the Splotch mutant is caused by upregulation of Msx2, a homeobox gene with a well-documented role as a regulator of BMP signaling. We provide evidence, moreover, that Pax3 represses Msx2 expression via a direct effect on a conserved Pax3 binding site in the Msx2 promoter. These results establish Msx2 as an effector of Pax3 in cardiac neural crest development.