Biomedical subjects

John Klingensmith

Publications and source records attributed to John Klingensmith.

14 recordsLinked to original sources

PubMed2007

The bone morphogenetic protein antagonist noggin regulates mammalian cardiac morphogenesis.

Bone morphogenetic proteins (BMPs) play many roles in mammalian cardiac development. Here we address the functions of Noggin, a dedicated BMP antagonist, in the developing mouse heart. In early cardiac tissues, the Noggin gene is mainly expressed in the myocardial cells of the outflow tract, atrioventricular canal, and future right ventricle. The major heart phenotypes of Noggin mutant embryos are thicker myocardium and larger endocardial cushions. Both defects result from increased cell number. Cell proliferation is increased and cell cycle exit is decreased in the myocardium. Although we find evidence of increased BMP signal transduction in the myocardium and endocardium, we show that the cardiac defects of Noggin mutants are rescued by halving the gene dosage of Bmp4. In culture, BMP increases the epithelial-to-mesenchymal transformation (EMT) of endocardial explant cells. Increased EMT likely accounts for the enlarged atrioventricular cushion. In the outflow tract cushion, we observed an increased contribution of cardiac neural crest cells to the mutant cushion mesenchyme, although many cells of the cushion were not derived from neural crest. Thus the enlarged outflow tract cushion of Noggin mutants likely arises by increased contributions both of endocardial cells that have undergone EMT as well as cells that have migrated from the neural crest. These data indicate that antagonism of BMP signaling by Noggin plays a critical role in ensuring proper levels of cell proliferation and EMT during cardiac morphogenesis in the mouse.

Animals↗

PubMed2006

BMP signaling in the epiblast is required for proper recruitment of the prospective paraxial mesoderm and development of the somites.

Bmpr1a encodes the BMP type IA receptor for bone morphogenetic proteins (BMPs), including 2 and 4. Here, we use mosaic inactivation of Bmpr1a in the epiblast of the mouse embryo (Bmpr-MORE embryos) to assess functions of this gene in mesoderm development. Unlike Bmpr1a-null embryos, which fail to gastrulate, Bmpr-MORE embryos initiate gastrulation, but the recruitment of prospective paraxial mesoderm cells to the primitive streak is delayed. This delay causes a more proximal distribution of cells with paraxial mesoderm character within the primitive streak, resulting in a lateral expansion of somitic mesoderm to form multiple columns. Inhibition of FGF signaling restores the normal timing of recruitment of prospective paraxial mesoderm and partially rescues the development of somites. This suggests that BMP and FGF signaling function antagonistically during paraxial mesoderm development.

Animals↗

PubMed2006

Roles of organizer factors and BMP antagonism in mammalian forebrain establishment.

A critical question in mammalian development is how the forebrain is established. In amphibians, bone morphogenetic protein (BMP) antagonism emanating from the gastrula organizer is key. Roles of BMP antagonism and the organizer in mammals remain unclear. Anterior visceral endoderm (AVE) promotes early mouse head development, but its function is controversial. Here, we explore the timing and regulation of forebrain establishment in the mouse. Forebrain specification requires tissue interaction through the late streak stage of gastrulation. Foxa2(-/-) embryos lack both the organizer and its BMP antagonists, yet about 25% show weak forebrain gene expression. A similar percentage shows ectopic AVE gene expression distally. The distal VE may thus be a source of forebrain promoting signals in these embryos. In wild-type ectoderm explants, AVE promoted forebrain specification, while anterior mesendoderm provided maintenance signals. Embryological and molecular data suggest that the AVE is a source of active BMP antagonism in vivo. In prespecification ectoderm explants, exogenous BMP antagonists triggered forebrain gene expression and inhibited posterior gene expression. Conversely, BMP inhibited forebrain gene expression, an effect that could be antagonized by anterior mesendoderm, and promoted expression of some posterior genes. These results lead to a model in which BMP antagonism supplied by exogenous tissues promotes forebrain establishment and maintenance in the murine ectoderm.

Animals↗

PubMed2006

The BMP antagonist Noggin promotes cranial and spinal neurulation by distinct mechanisms.

Here we characterize the consequences of elevated bone morphogenetic protein (BMP) signaling on neural tube morphogenesis by analyzing mice lacking the BMP antagonist, Noggin. Noggin is expressed dorsally in the closing neural folds and ventrally in the notochord and somites. All Noggin-/- pups are born with lumbar spina bifida; depending on genetic background, they may also have exencephaly. The exencephaly is due to a primary failure of neurulation, resulting from a lack of mid/hindbrain dorsolateral hinge point (DLHP) formation. Thus, as previously shown for Shh signaling at spinal levels, BMP activity may inhibit cranial DLHP morphogenesis. However, the increased BMP signaling observed in the Noggin-/- dorsal neural tube is not sufficient to cause exencephaly; it appears to also depend on the action of a genetic modifier, which may act to increase dorsal Shh signaling. The spinal neural tube defect results from a different mechanism: increased BMP signaling in the mesoderm between the limb buds leads to abnormal somite differentiation and axial skeletal malformation. The resulting lack of mechanical support for the neural tube causes spina bifida. We show that this defect is due to elevated BMP4 signaling. Thus, Noggin is required for mammalian neurulation in two contexts, dependent on position along the rostrocaudal axis.

Animals↗

PubMed2006

Endogenous bone morphogenetic protein antagonists regulate mammalian neural crest generation and survival.

We demonstrate here that Chordin and Noggin function as bone morphogenetic protein (BMP) antagonists in vivo to promote mammalian neural crest development. Using Chrd and Nog single and compound mutants, we find that Noggin has a major role in promoting neural crest formation, in which Chordin is partially redundant. BMP signaling is increased in dorsal tissues lacking Noggin and is further increased when Chordin is also absent. The early neural crest domain is expanded with decreased BMP antagonism in vivo. Noggin and Chordin also regulate subsequent neural crest cell emigration from the neural tube. However, reduced levels of these BMP antagonists ultimately result in perturbation of neural crest cell derived peripheral nervous system and craniofacial skeletal elements. Such defects reflect, at least in part, a function to limit apoptosis in neural crest cells. Noggin and Chordin, therefore, function together to regulate both the generation and survival of neural crest cells in mammalian development.

Animals↗

PubMed2006

Morphogenesis of the trachea and esophagus: current players and new roles for noggin and Bmps.

The development of the anterior foregut of the mammalian embryo involves changes in the behavior of both the epithelial endoderm and the adjacent mesoderm. Morphogenetic processes that occur include the extrusion of midline notochord cells from the epithelial definitive endoderm, the folding of the endoderm into a foregut tube, and the subsequent separation of the foregut tube into trachea and esophagus. Defects in foregut morphogenesis underlie the constellation of human birth defects known as esophageal atresia (EA) and tracheoesophageal fistula (TEF). Here, we review what is known about the cellular events in foregut morphogenesis and the gene mutations associated with EA and TEF in mice and humans. We present new evidence that about 70% of mouse embryos homozygous null for Nog, the gene encoding noggin, a bone morphogenetic protein (Bmp) antagonist, have EA/TEF as well as defects in lung branching. This phenotype appears to correlate with abnormal morphogenesis of the notochord and defects in its separation from the definitive endoderm. The abnormalities in foregut and lung morphogenesis of Nog null mutant can be rescued by reducing the gene dose of Bmp4 by 50%. This suggests that normal foregut morphogenesis requires that the level of Bmp4 activity is carefully controlled by means of antagonists such as noggin. Several mechanisms are suggested for how Bmps normally function, including by regulating the intercellular adhesion and behavior of notochord and foregut endoderm cells. Future research must determine how Noggin/Bmp antagonism fits into the network of other factors known to regulate tracheal and esophagus development, both in mouse or humans.

Animals↗

PubMed2006

Fgf8 is required for anterior heart field development.

In the mouse embryo, the splanchnic mesodermal cells of the anterior heart field (AHF) migrate from the pharynx to contribute to the early myocardium of the outflow tract (OT) and right ventricle (RV). Recent studies have attempted to distinguish the AHF from other precardiac populations, and to determine the genetic and molecular mechanisms that regulate its development. Here, we have used an Fgf8lacZ allele to demonstrate that Fgf8 is expressed within the developing AHF. In addition, we use both a hypomorphic Fgf8 allele (Fgf8neo) and Cre-mediated gene ablation to show that Fgf8 is essential for the survival and proliferation of the AHF. Nkx2.5Cre is expressed in the AHF, primary heart tube and pharyngeal endoderm, while TnT-Cre is expressed only within the specified heart tube myocardium. Deletion of Fgf8 by Nkx2.5Cre results in a significant loss of the Nkx2.5Cre lineage and severe OT and RV truncations by E9.5, while the remaining heart chambers (left ventricle and atria) are grossly normal. These defects result from significant decreases in cell proliferation and aberrant cell death in both the pharyngeal endoderm and splanchnic mesoderm. By contrast, ablation of Fgf8 in the TnT-Cre domain does not result in OT or RV defects, providing strong evidence that Fgf8 expression is crucial in the pharyngeal endoderm and/or overlying splanchnic mesoderm of the AHF at a stage prior to heart tube elongation. Analysis of downstream signaling components, such as phosphorylated-Erk and Pea3, identifies the AHF splanchnic mesoderm itself as a target for Fgf8 signaling.

Animals↗

PubMed2004

BMP receptor IA is required in the mammalian embryo for endodermal morphogenesis and ectodermal patterning.

BMPRIA is a receptor for bone morphogenetic proteins with high affinity for BMP2 and BMP4. Mouse embryos lacking Bmpr1a fail to gastrulate, complicating studies on the requirements for BMP signaling in germ layer development. Recent work shows that BMP4 produced in extraembryonic tissues initiates gastrulation. Here we use a conditional allele of Bmpr1a to remove BMPRIA only in the epiblast, which gives rise to all embryonic tissues. Resulting embryos are mosaics composed primarily of cells homozygous null for Bmpr1a, interspersed with heterozygous cells. Although mesoderm and endoderm do not form in Bmpr1a null embryos, these tissues are present in the mosaics and are populated with mutant cells. Thus, BMPRIA signaling in the epiblast does not restrict cells to or from any of the germ layers. Cells lacking Bmpr1a also contribute to surface ectoderm; however, from the hindbrain forward, little surface ectoderm forms and the forebrain is enlarged and convoluted. Prechordal plate, early definitive endoderm, and anterior visceral endoderm appear to be expanded, likely due to defective morphogenesis. These data suggest that the enlarged forebrain is caused in part by increased exposure of the ectoderm to signaling sources that promote anterior neural fate. Our results reveal critical roles for BMP signaling in endodermal morphogenesis and ectodermal patterning.

Animals↗

PubMed2004

BMP receptor IA is required in mammalian neural crest cells for development of the cardiac outflow tract and ventricular myocardium.

The neural crest is a multipotent, migratory cell population arising from the border of the neural and surface ectoderm. In mouse, the initial migratory neural crest cells occur at the five-somite stage. Bone morphogenetic proteins (BMPs), particularly BMP2 and BMP4, have been implicated as regulators of neural crest cell induction, maintenance, migration, differentiation and survival. Mouse has three known BMP2/4 type I receptors, of which Bmpr1a is expressed in the neural tube sufficiently early to be involved in neural crest development from the outset; however, earlier roles in other domains obscure its requirement in the neural crest. We have ablated Bmpr1a specifically in the neural crest, beginning at the five-somite stage. We find that most aspects of neural crest development occur normally; suggesting that BMPRIA is unnecessary for many aspects of early neural crest biology. However, mutant embryos display a shortened cardiac outflow tract with defective septation, a process known to require neural crest cells and to be essential for perinatal viability. Surprisingly, these embryos die in mid-gestation from acute heart failure, with reduced proliferation of ventricular myocardium. The myocardial defect may involve reduced BMP signaling in a novel, minor population of neural crest derivatives in the epicardium, a known source of ventricular myocardial proliferation signals. These results demonstrate that BMP2/4 signaling in mammalian neural crest derivatives is essential for outflow tract development and may regulate a crucial proliferation signal for the ventricular myocardium.

Animals↗

PubMed2004

The mammalian twisted gastrulation gene functions in foregut and craniofacial development.

Extracellular modulators of cell-cell signaling control numerous aspects of organismal development. The Twisted gastrulation (Twsg1) gene product is a small, secreted cysteine-rich protein that has the unusual property of being able to either enhance or inhibit signaling by the bone morphogenetic protein (BMP) subfamily of TGF-beta type factors in a context-dependent manner. In this report, we characterize the early embryonic and skeletal phenotypes associated with loss of Twsg1 function in mice. All Twsg1 mutant mice, irrespective of genetic background, exhibit deletions of neural arches in the cervical vertebrae. In a C57BL/6 background, we also observe pronounced forebrain defects including rostral truncations, holoprosencephaly, cyclopia, as well as alterations in the first branchial arch (BA1) leading to lack of jaw (agnathia). Characterization of marker expression suggests that these defects are attributable to loss of signaling from forebrain-organizing centers including Fgf8 from the anterior neural ridge (ANR) and Shh from the prechordal plate (PrCP). In addition, we find defects in the foregut endoderm and a reduction in Hex expression, which may contribute to both the forebrain and BA1 defects.

Animals↗

PubMed2003

Cordon-bleu is a conserved gene involved in neural tube formation.

The axial midline is an important source of patterning and morphogenesis cues in the vertebrate embryo. The midline derives from a small group of cells in the gastrulating embryo, known as "the organizer" in recognition of its ability to organize an entire body plan. The mammalian organizer, the node, gives rise to axial midline structures: the notochord, dorsal foregut, and part of the floor plate of the neural tube. Only some of the genes that direct midline development are known. In this study, we present the complete coding sequence for a novel gene, cordon-bleu (cobl), expressed specifically in the node and its derivatives until organogenesis stages. The deduced sequence does not resemble any gene of known function. However, cobl is widely conserved: apparent orthologs and paralogs are found in many vertebrate species, with several sequence domains of high conservation but unknown function. We find that chicken cordon-bleu is similarly expressed in the node and its derivatives, suggesting functional conservation. We also report the sequence and nonoverlapping expression of a related mouse gene, Coblr1. Finally, we show that cobl interacts with the neurulation gene Vangl2 to facilitate midbrain neural tube closure, demonstrating roles for both cobl and Vangl2 in midbrain neurulation.

Alleles↗

PubMed2003

The role of chordin/Bmp signals in mammalian pharyngeal development and DiGeorge syndrome.

The chordin/Bmp system provides one of the best examples of extracellular signaling regulation in animal development. We present the phenotype produced by the targeted inactivation of the chordin gene in mouse. Chordin homozygous mutant mice show, at low penetrance, early lethality and a ventralized gastrulation phenotype. The mutant embryos that survive die perinatally, displaying an extensive array of malformations that encompass most features of DiGeorge and Velo-Cardio-Facial syndromes in humans. Chordin secreted by the mesendoderm is required for the correct expression of Tbx1 and other transcription factors involved in the development of the pharyngeal region. The chordin mutation provides a mouse model for head and neck congenital malformations that frequently occur in humans and suggests that chordin/Bmp signaling may participate in their pathogenesis.

Animals↗

PubMed2002

Chordin and noggin promote organizing centers of forebrain development in the mouse.

In this study we investigate the roles of the organizer factors chordin and noggin, which are dedicated antagonists of the bone morphogenetic proteins (BMPs), in formation of the mammalian head. The mouse chordin and noggin genes (Chrd and Nog) are expressed in the organizer (the node) and its mesendodermal derivatives, including the prechordal plate, an organizing center for rostral development. They are also expressed at lower levels in and around the anterior neural ridge, another rostral organizing center. To elucidate roles of Chrd and Nog that are masked by the severe phenotype and early lethality of the double null, we have characterized embryos of the genotype Chrd(-/-);Nog(+/-). These animals display partially penetrant neonatal lethality, with defects restricted to the head. The variable phenotypes include cyclopia, holoprosencephaly, and rostral truncations of the brain and craniofacial skeleton. In situ hybridization reveals a loss of SHH expression and signaling by the prechordal plate, and a decrease in FGF8 expression and signaling by the anterior neural ridge at the five-somite stage. Defective Chrd(-/-);Nog(+/-) embryos exhibit reduced cell proliferation in the rostral neuroepithelium at 10 somites, followed by increased cell death 1 day later. Because these phenotypes result from reduced levels of BMP antagonists, we hypothesized that they are due to increased BMP activity. Ectopic application of BMP2 to wild-type cephalic explants results in decreased FGF8 and SHH expression in rostral tissue, suggesting that the decreased expression of FGF8 and SHH observed in vivo is due to ectopic BMP activity. Cephalic explants isolated from Chrd;Nog double mutant embryos show an increased sensitivity to ectopic BMP protein, further supporting the hypothesis that these mutants are deficient in BMP antagonism. These results indicate that the BMP antagonists chordin and noggin promote the inductive and trophic activities of rostral organizing centers in early development of the mammalian head.

Animals↗

PubMedDate not supplied

A novel mutation in the gene encoding noggin is not causative in human neural tube defects.

Neural tube defects (NTD) are a common birth defect, with both genetic and environmental contributions to their etiology. In mouse, null mutations in Noggin result in fully-penetrant NTDs. We investigated Noggin for mutations that may predispose to human NTDs in 202 NTD cases. One variant allele was identified in a male patient with myelomeningocele. The patient's father and a sibling also carried the variant allele, but neither was affected with an open NTD. DNA sequencing confirmed a C1064A missense mutation predicted to result in the conversion of residue 84 from proline to histidine. The variant found in the NTD patient is a newly identified variant, the role of which is uncertain.

Bone Morphogenetic Proteins↗