Sonic hedgehog from pharyngeal arch 1 epithelium is necessary for early mandibular arch cell survival and later cartilage condensation differentiation.

BACKGROUND: Morphogenesis of vertebrate craniofacial skeletal elements is dependent on a key cell population, the cranial neural crest cells (NCC). Cranial NCC are formed dorsally in the cranial neural tube and migrate ventrally to form craniofacial skeletal elements as well as other tissues. Multiple extracellular signaling pathways regulate the migration, survival, proliferation, and differentiation of NCC. RESULTS: In this study, we demonstrate that Shh expression in the oral ectoderm and pharyngeal endoderm is essential for mandibular development. We show that a loss of Shh in these domains results in increased mesenchymal cell death in pharyngeal arch 1 (PA1) after NCC migration. This increased cell death can be rescued in utero by pharmacological inhibition of p53. Furthermore, we show that epithelial SHH is necessary for the early differentiation of mandibular cartilage condensations and, therefore, the subsequent development of Meckel's cartilage, around which the dentary bone forms. Nonetheless, a rescue of the cell death phenotype does not rescue the defect in cartilage condensation formation. CONCLUSIONS: Our results show that SHH produced by the PA1 epithelium is necessary for the survival of post-migratory NCC, and suggests a key role in the subsequent differentiation of chondrocytes to form Meckel's cartilage.

One shall become two: Separation of the esophagus and trachea from the common foregut tube.

The alimentary and respiratory organ systems arise from a common endodermal origin, the anterior foregut tube. Formation of the esophagus from the dorsal region and the trachea from the ventral region of the foregut primordium occurs by means of a poorly understood compartmentalization process. Disruption of this process can result in severe birth defects, such as esophageal atresia and tracheo-esphageal fistula (EA/TEF), in which the lumina of the trachea and esophagus remain connected. Here we summarize the signaling networks known to be necessary for regulating dorsoventral patterning within the common foregut tube and cellular behaviors that may occur during normal foregut compartmentalization. We propose that dorsoventral patterning serves to establish a lateral region of the foregut tube that is capable of undergoing specialized cellular rearrangements, culminating in compartmentalization. We review established as well as new rodent models that may be useful in addressing this hypothesis. Finally, we discuss new experimental models that could help elucidate the mechanism behind foregut compartmentalization. An integrated approach to future foregut morphogenesis research will allow for a better understanding of this complex process.

Multiple tissue-specific requirements for the BMP antagonist Noggin in development of the mammalian craniofacial skeleton.

Proper morphogenesis is essential for both form and function of the mammalian craniofacial skeleton, which consists of more than twenty small cartilages and bones. Skeletal elements that support the oral cavity are derived from cranial neural crest cells (NCCs) that develop in the maxillary and mandibular buds of pharyngeal arch 1 (PA1). Bone Morphogenetic Protein (BMP) signaling has been implicated in most aspects of craniofacial skeletogenesis, including PA1 development. However, the roles of the BMP antagonist Noggin in formation of the craniofacial skeleton remain unclear, in part because of its multiple domains of expression during formative stages. Here we used a tissue-specific gene ablation approach to assess roles of Noggin (Nog) in two different tissue domains potentially relevant to mandibular and maxillary development. We found that the axial midline domain of Nog expression is critical to promote PA1 development in early stages, necessary for adequate outgrowth of the mandibular bud. Subsequently, Nog expression in NCCs regulates craniofacial cartilage and bone formation. Mice lacking Nog in NCCs have an enlarged mandible that results from increased cell proliferation in and around Meckel׳s cartilage. These mutants also show complete secondary cleft palate, most likely due to inhibition of posterior palatal shelf elevation by disrupted morphology of the developing skull base. Our findings demonstrate multiple roles of Noggin in different domains for craniofacial skeletogenesis, and suggest an indirect mechanism for secondary cleft palate in Nog mutants that may be relevant to human cleft palate as well.

BMP antagonism by Noggin is required in presumptive notochord cells for mammalian foregut morphogenesis.

Esophageal atresia with tracheoesophageal fistula (EA/TEF) is a serious human birth defect, in which the esophagus ends before reaching the stomach, and is aberrantly connected with the trachea. Several mouse models of EA/TEF have recently demonstrated that proper dorsal/ventral (D/V) patterning of the primitive anterior foregut endoderm is essential for correct compartmentalization of the trachea and esophagus. Here we elucidate the pathogenic mechanisms underlying the EA/TEF that occurs in mice lacking the BMP antagonist Noggin, which display correct dorsal/ventral patterning. To clarify the mechanism of this malformation, we use spatiotemporal manipulation of Noggin and BMP receptor 1A conditional alleles during foregut development. Surprisingly, we find that the expression of Noggin in the compartmentalizing endoderm is not required to generate distinct tracheal and esophageal tubes. Instead, we show that Noggin and BMP signaling attenuation are required in the early notochord to correctly resolve notochord cells from the dorsal foregut endoderm, which in turn, appears to be a prerequisite for foregut compartmentalization. Collectively, our findings support an emerging model for a mechanism underlying EA/TEF in which impaired notochord resolution from the early endoderm causes the foregut to be hypo-cellular just prior to the critical period of compartmentalization. Our further characterizations suggest that Noggin may regulate a cell rearrangement process that involves reciprocal E-cadherin and Zeb1 expression in the resolving notochord cells.

Compartmentalization of the foregut tube: developmental origins of the trachea and esophagus.

The mammalian trachea and esophagus share a common embryonic origin. They arise by compartmentalization of a single foregut tube, composed of foregut endoderm (FGE) and surrounding mesenchyme, around midgestation. Aberrant compartmentalization is thought to lead to relatively common human birth defects, such as esophageal atresia (EA) and tracheoesophageal fistula (EA/TEF), which can prevent or disrupt a newborn infant's ability to feed and breathe. Despite its relevance to human health, morphogenesis of the anterior foregut is still poorly understood. In this article, we provide a comprehensive review of trachea and esophagus formation from a common precursor, including the embryonic origin of the FGE, current models for foregut morphogenesis, relevant human birth defects, insights from rodent models, and the emerging picture of the mechanisms underlying normal and abnormal foregut compartmentalization. Recent research suggests that a number of intercellular signaling pathways and several intracellular effectors are essential for correct formation of the trachea and esophagus. Different types of defects in the formation of either ventral or dorsal foregut tissues can disrupt compartmentalization in rodent models. This implies that EA/TEF defects in humans may also arise by multiple mechanisms. Although our understanding of foregut compartmentalization is growing rapidly, it is still incomplete. Future research should focus on synthesizing detailed information gleaned from both human patients and rodent models to further our understanding of this enigmatic process.

RNA-binding protein FXR2 regulates adult hippocampal neurogenesis by reducing Noggin expression.

In adult mammalian brains, neurogenesis persists in the subventricular zone of the lateral ventricles (SVZ) and the dentate gyrus (DG) of the hippocampus. Although evidence suggest that adult neurogenesis in these two regions is subjected to differential regulation, the underlying mechanism is unclear. Here, we show that the RNA-binding protein FXR2 specifically regulates DG neurogenesis by reducing the stability of Noggin mRNA. FXR2 deficiency leads to increased Noggin expression and subsequently reduced BMP signaling, which results in increased proliferation and altered fate specification of neural stem/progenitor cells in DG. In contrast, Noggin is not regulated by FXR2 in the SVZ, because Noggin expression is restricted to the ependymal cells of the lateral ventricles, where FXR2 is not expressed. Differential regulation of SVZ and DG stem cells by FXR2 may be a key component of the mechanism that governs the different neurogenic processes in these two adult germinal zones.

Bone morphogenetic protein signaling is required in the dorsal neural folds before neurulation for the induction of spinal neural crest cells and dorsal neurons.

Bone Morphogenetic Protein (BMP) activity has been implicated as a key regulator of multiple aspects of dorsal neural tube development. BMP signaling in the dorsal-most neuroepithelial cells presumably plays a critical role. We use tissue-specific gene ablation to probe the roles of BMPR1A, the type 1 BMP receptor that is seemingly the best candidate to mediate the activities of BMPs on early dorsal neural development. We use two different Cre lines expressed in the dorsal neural folds, one prior to spinal neurulation and one shortly afterward, together with a Bmpr1a conditional null mutation. Our findings indicate that BMPR1A signaling in the dorsal neural folds is important for hindbrain neural tube closure, but suggest it is dispensable for spinal neurulation. Our results also demonstrate a requirement for BMP signaling in patterning of dorsal neural tube cell fate and in neural crest cell formation, and imply a critical period shortly before neural tube closure.

The actin nucleator Cordon-bleu is required for development of motile cilia in zebrafish.

The cordon-bleu (Cobl) gene is widely conserved in vertebrates, with developmentally regulated axial and epithelial expression in mouse and chick embryos. In vitro, Cobl can bind monomeric actin and nucleate formation of unbranched actin filaments, while in cultured cells it can modulate the actin cytoskeleton. However, an essential role for Cobl in vivo has yet to be determined. We have used zebrafish as a model to assess the requirements for Cobl in embryogenesis. We find that cobl shows enriched expression in ciliated epithelial tissues during zebrafish organogenesis. Cobl protein is enriched in the apical domain of ciliated cells, in close proximity to the apical actin cap. Reduction of Cobl by antisense morpholinos reveals an essential role in development of motile cilia in organs such as Kupffer's vesicle and the pronephros. In Kupffer's vesicle, the reduction in Cobl coincides with a reduction in the amount of apical F-actin. Thus, Cobl represents a molecular activity that couples developmental patterning signals with local intracellular cytoskeletal dynamics to support morphogenesis of motile cilia.

Modulation of BMP signaling by Noggin is required for the maintenance of palatal epithelial integrity during palatogenesis.

BMP signaling plays many important roles during organ development, including palatogenesis. Loss of BMP signaling leads to cleft palate formation. During development, BMP activities are finely tuned by a number of modulators at the extracellular and intracellular levels. Among the extracellular BMP antagonists is Noggin, which preferentialy binds to BMP2, BMP4 and BMP7, all of which are expressed in the developing palatal shelves. Here we use targeted Noggin mutant mice as a model for gain of BMP signaling function to investigate the role of BMP signaling in palate development. We find prominent Noggin expression in the palatal epithelium along the anterior-posterior axis during early palate development. Loss of Noggin function leads to overactive BMP signaling, particularly in the palatal epithelium. This results in disregulation of cell proliferation, excessive cell death, and changes in gene expression, leading to formation of complete palatal cleft. The excessive cell death in the epithelium disrupts the palatal epithelium integrity, which in turn leads to an abnormal palate-mandible fusion and prevents palatal shelf elevation. This phenotype is recapitulated by ectopic expression of a constitutively active form of BMPR-IA but not BMPR-IB in the epithelium of the developing palate; this suggests a role for BMPR-IA in mediating overactive BMP signaling in the absence of Noggin. Together with the evidence that overexpression of Noggin in the palatal epithelium does not cause a cleft palate defect, we conclude from our results that Noggin mediated modulation of BMP signaling is essential for palatal epithelium integrity and for normal palate development.

BMP antagonism protects Nodal signaling in the gastrula to promote the tissue interactions underlying mammalian forebrain and craniofacial patterning.

Holoprosencephaly (HPE) is the most common forebrain and craniofacial malformation syndrome in humans. The genetics of HPE suggest that it often stems from a synergistic interaction of mutations in independent loci. In mice, several combinations of mutations in Nodal signaling pathway components can give rise to HPE, but it is not clear whether modest deficits of Nodal signaling along with lesions in other pathways might also cause such defects. We find that HPE results from simultaneous reduction of Nodal signaling and an organizer BMP (bone morphogenetic protein) antagonist, either Chordin or Noggin. These defects result from reduced production of tissues that promote forebrain and craniofacial development. Nodal promotes the expression of genes in the anterior primitive streak that are important for the development of these tissues, whereas BMP inhibits their expression. Pharmacological and transgenic manipulation of these signaling pathways suggests that BMP and Nodal antagonize each other prior to intracellular signal transduction. Biochemical experiments in vitro indicate that secreted Bmp2 and Nodal can form extracellular complexes, potentially interfering with receptor activation. Our results reveal that the patterning of forebrain and medial craniofacial elements requires a fine balance between BMP and Nodal signaling during primitive streak development, and provide a potential mechanistic basis for a new multigenic model of HPE.

Bone morphogenetic protein 4 mediates myocardial ischemic injury through JNK-dependent signaling pathway.

Bone morphogenetic protein (BMP) signaling regulates embryonic development of many organ systems and defective BMP signaling has been implicated in adult disorders of many of these systems. However, its relevance in cardiac disease has not been reported. Here we demonstrate for the first time that Bmp4 activity promotes cellular apoptosis following ischemia-reperfusion (I/R) injury induced myocardial infarction (MI). Bmp4 heterozygous null mice (Bmp4(+/)(-)) demonstrated reduced infarct size, less myocardial apoptosis and down-regulation of pro-apoptotic proteins relative to wild-type mice following I/R injury. This was associated with reduction in I/R induced BMP4 levels in the left ventricular infarcted region. Furthermore, treatment of neonatal cardiomyocytes with BMP4 resulted in time and dose-dependent increase in cellular apoptosis and activation of the JNK MAP kinase pathway. In contrast, while JNK activation was significantly attenuated in Bmp4(+/)(-) mice and following Smad1 inhibition in myocytes, inhibition of JNK with a specific inhibitory peptide, TAT-JBD(20,) blocked BMP4 induced apoptosis. In vivo treatment of mice with Noggin, an endogenous extracellular BMP antagonist, or dorsomorphin, a small molecule inhibitor of BMP signaling, reduced infarct size, and inhibited pro-apoptotic signaling accompanied by an inhibition of Smad1 phosphorylation and JNK activation. These studies identify a novel role for Bmp4 in the pathogenesis of myocardial infarction and illustrate the use of a small molecule inhibitor of BMP signaling for treatment of acute I/R injury.

Roles of bone morphogenetic protein signaling and its antagonism in holoprosencephaly.

Holoprosencephaly (HPE) is the most common malformation of the forebrain, resulting from a failure to completely septate the left and right hemispheres at the rostral end of the neural tube. Because of the tissue interactions that drive head development, these forebrain defects are typically accompanied by midline deficiencies of craniofacial structures. Early events in setting up tissue precursors of the head, as well as later interactions between these tissues, are critical for normal head formation. Defects in either process can result in HPE. Signaling by bone morphogenetic proteins (BMPs), a family of secreted cytokines, generally plays negative roles in early stages of head formation, and thus must be attenuated in multiple contexts to ensure proper forebrain and craniofacial development. Chordin and Noggin are endogenous, extracellular antagonists of BMP signaling that promote the normal organization of the forebrain and face. Mouse mutants with reduced levels of both factors display mutant phenotypes remarkably analogous to the range of malformations seen in human HPE sequence. Chordin and Noggin function in part by antagonizing the inhibitory effects of BMP signaling on the Sonic hedgehog and Nodal pathways, genetic lesions in each being associated with human HPE. Study of Chordin;Noggin mutant mice is helping us to understand the molecular, cellular, and genetic pathogenesis of HPE and associated malformations.

Chordin is a modifier of tbx1 for the craniofacial malformations of 22q11 deletion syndrome phenotypes in mouse.

Point mutations in TBX1 can recapitulate many of the structural defects of 22q11 deletion syndromes (22q11DS), usually associated with a chromosomal deletion at 22q1.2. 22q11DS often includes specific cardiac and pharyngeal organ anomalies, but the presence of characteristic craniofacial defects is highly variable. Even among family members with a single TBX1 point mutation but no cytological deletion, cleft palate and low-set ears may or may not be present. In theory, such differences could depend on an unidentified, second-site lesion that modifies the craniofacial consequences of TBX1 deficiency. We present evidence for such a locus in a mouse model. Null mutations of chordin have been reported to cause severe defects recapitulating 22q11DS, which we show are highly dependent on genetic background. In an inbred strain in which chordin(-/-) is fully penetrant, we found a closely linked, strong modifier--a mutation in a Tbx1 intron causing severe splicing defects. Without it, lack of chordin results in a low penetrance of mandibular hypoplasia but no cardiac or thoracic organ malformations. This hypomorphic Tbx1 allele per se results in defects resembling 22q11DS but with a low penetrance of hallmark craniofacial malformations, unless chordin is mutant. Thus, chordin is a modifier for the craniofacial anomalies of Tbx1 mutations, demonstrating the existence of a second-site modifier for a specific subset of the phenotypes associated with 22q11DS.

An FGF autocrine loop initiated in second heart field mesoderm regulates morphogenesis at the arterial pole of the heart.

In order to understand how secreted signals regulate complex morphogenetic events, it is crucial to identify their cellular targets. By conditional inactivation of Fgfr1 and Fgfr2 and overexpression of the FGF antagonist sprouty 2 in different cell types, we have dissected the role of FGF signaling during heart outflow tract development in mouse. Contrary to expectation, cardiac neural crest and endothelial cells are not primary paracrine targets. FGF signaling within second heart field mesoderm is required for remodeling of the outflow tract: when disrupted, outflow myocardium fails to produce extracellular matrix and TGFbeta and BMP signals essential for endothelial cell transformation and invasion of cardiac neural crest. We conclude that an autocrine regulatory loop, initiated by the reception of FGF signals by the mesoderm, regulates correct morphogenesis at the arterial pole of the heart. These findings provide new insight into how FGF signaling regulates context-dependent cellular responses during development.

BMP antagonism is required in both the node and lateral plate mesoderm for mammalian left-right axis establishment.

In mouse, left-right (L-R) patterning depends on asymmetric expression of Nodal around the node, leading to Nodal expression specifically in the left lateral plate mesoderm (LPM). Bone morphogenetic protein (BMP) signaling is also involved, but the mechanistic relationship with Nodal expression remains unclear. We find that BMP signal transduction is higher in the right LPM, although Bmp4, which is required for L-R patterning, is expressed symmetrically. By contrast, the BMP antagonists noggin (Nog) and chordin (Chrd) are expressed at higher levels in the left LPM. In Chrd;Nog double mutants, BMP signaling is elevated on both sides, whereas Nodal expression is absent. Ectopic expression of Nog in the left LPM of double mutants restores Nodal expression. Ectopic Bmp4 expression in the left LPM of wild-type embryos represses Nodal transcription, whereas ectopic Nog in the right LPM leads to inappropriate Nodal expression. These data indicate that chordin and noggin function to limit BMP signaling in the left LPM, thereby derepressing Nodal expression. In the node, they promote peripheral Nodal expression and proper node morphology, potentially in concert with Notch signaling. These results indicate that BMP antagonism is required in both the node and LPM to facilitate L-R axis establishment in the mammalian embryo.

Intracardiac septation requires hedgehog-dependent cellular contributions from outside the heart.

Septation of the mammalian heart into four chambers requires the orchestration of multiple tissue progenitors. Abnormalities in this process can result in potentially fatal atrioventricular septation defects (AVSD). The contribution of extracardiac cells to atrial septation has recently been recognized. Here, we use a genetic marker and novel magnetic resonance microscopy techniques to demonstrate the origins of the dorsal mesenchymal protrusion in the dorsal mesocardium, and its substantial contribution to atrioventricular septation. We explore the functional significance of this tissue to atrioventricular septation through study of the previously uncharacterized AVSD phenotype of Shh(-/-) mutant mouse embryos. We demonstrate that Shh signaling is required within the dorsal mesocardium for its contribution to the atria. Failure of this addition results in severe AVSD. These studies demonstrate that AVSD can result from a primary defect in dorsal mesocardium, providing a new paradigm for the understanding of human AVSD.

Cordon-bleu is an actin nucleation factor and controls neuronal morphology.

Despite the wealth of different actin structures formed, only two actin nucleation factors are well established in vertebrates: the Arp2/3 complex and formins. Here, we describe a further nucleator, cordon-bleu (Cobl). Cobl is a brain-enriched protein using three Wiskott-Aldrich syndrome protein homology 2 (WH2) domains for actin binding. Cobl promotes nonbundled, unbranched filaments. Filament formation relies on barbed-end growth and requires all three Cobl WH2 domains and the extended linker L2. We suggest that the nucleation power of Cobl is based on the assembly of three actin monomers in cross-filament orientation. Cobl localizes to sites of high actin dynamics and modulates cell morphology. In neurons, induction of both neurites and neurite branching is dramatically increased by Cobl expression-effects that critically depend on Cobl's actin nucleation ability. Correspondingly, Cobl depletion results in decreased dendritic arborization. Thus, Cobl is an actin nucleator controlling neuronal morphology and development.

Neural plate morphogenesis during mouse neurulation is regulated by antagonism of Bmp signalling.

Dorsolateral bending of the neural plate, an undifferentiated pseudostratified epithelium, is essential for neural tube closure in the mouse spinal region. If dorsolateral bending fails, spina bifida results. In the present study, we investigated the molecular signals that regulate the formation of dorsolateral hinge points (DLHPs). We show that Bmp2 expression correlates with upper spinal neurulation (in which DLHPs are absent); that Bmp2-null embryos exhibit premature, exaggerated DLHPs; and that the local release of Bmp2 inhibits neural fold bending. Therefore, Bmp signalling is necessary and sufficient to inhibit DLHPs. By contrast, the Bmp antagonist noggin is expressed dorsally in neural folds containing DLHPs, noggin-null embryos show markedly reduced dorsolateral bending and local release of noggin stimulates bending. Hence, Bmp antagonism is both necessary and sufficient to induce dorsolateral bending. The local release of Shh suppresses dorsal noggin expression, explaining the absence of DLHPs at high spinal levels, where notochordal expression of Shh is strong. DLHPs ;break through' at low spinal levels, where Shh expression is weaker. Zic2 mutant embryos fail to express Bmp antagonists dorsally and lack DLHPs, developing severe spina bifida. Our findings reveal a molecular mechanism based on antagonism of Bmp signalling that underlies the regulation of DLHP formation during mouse spinal neural tube closure.

Independent requirements for Hedgehog signaling by both the anterior heart field and neural crest cells for outflow tract development.

Cardiac outflow tract (OFT) septation is crucial to the formation of the aortic and pulmonary arteries. Defects in the formation of the OFT can result in serious congenital heart defects. Two cell populations, the anterior heart field (AHF) and cardiac neural crest cells (CNCCs), are crucial for OFT development and septation. In this study, we use a series of tissue-specific genetic manipulations to define the crucial role of the Hedgehog pathway in these two fields of cells during OFT development. These data indicate that endodermally-produced SHH ligand is crucial for several distinct processes, all of which are required for normal OFT septation. First, SHH is required for CNCCs to survive and populate the OFT cushions. Second, SHH mediates signaling to myocardial cells derived from the AHF to complete septation after cushion formation. Finally, endodermal SHH signaling is required in an autocrine manner for the survival of the pharyngeal endoderm, which probably produces a secondary signal required for AHF survival and for OFT lengthening. Disruption of any of these steps can result in a single OFT phenotype.

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.