Fenglei He

Wnt5a regulates directional cell migration and cell proliferation via Ror2-mediated noncanonical pathway in mammalian palate development

Tissue and molecular heterogeneities are present in the developing secondary palate along the anteroposterior (AP) axis in mice. Here, we show that Wnt5a and its receptor Ror2 are expressed in a graded manner along the AP axis of the palate. Wnt5a deficiency leads to a complete cleft of the secondary palate, which exhibits distinct phenotypic alterations at histological, cellular and molecular levels in the anterior and posterior regions of the palate. We demonstrate that there is directional cell migration within the developing palate. In the absence of Wnt5a, this directional cell migration does not occur. Genetic studies and in vitro organ culture assays further demonstrate a role for Ror2 in mediating Wnt5a signaling in the regulation of cell proliferation and migration during palate development. Our results reveal distinct regulatory roles for Wnt5a in gene expression and cell proliferation along the AP axis of the developing palate, and an essential role for Wnt5a in the regulation of directional cell migration.

Shox2 is essential for the differentiation of cardiac pacemaker cells by repressing Nkx2-5

The pacemaker is composed of specialized cardiomyocytes located within the sinoatrial node (SAN), and is responsible for originating and regulating the heart beat. Recent advances towards understanding the SAN development have been made on the genetic control and gene interaction within this structure. Here we report that the Shox2 homeodomain transcription factor is restrictedly expressed in the sinus venosus region including the SAN and the sinus valves during embryonic heart development. Shox2 null mutation results in embryonic lethality due to cardiovascular defects, including an abnormal low heart beat rate (bradycardia) and severely hypoplastic SAN and sinus valves attributed to a significantly decreased level of cell proliferation. Genetically, the lack of Tbx3 and Hcn4 expression, along with ectopic activation of Nppa, Cx40, and Nkx2-5 in the Shox2(-/-) SAN region, indicates a failure in SAN differentiation. Furthermore, Shox2 overexpression in Xenopus embryos results in extensive repression of Nkx2-5 in the developing heart, leading to a reduced cardiac field and aberrant heart formation. Reporter gene expression assays provide additional evidence for the repression of Nkx2-5 promoter activity by Shox2. Taken together our results demonstrate that Shox2 plays an essential role in the SAN and pacemaker development by controlling a genetic cascade through the repression of Nkx2-5.

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.

Hand2 is required in the epithelium for palatogenesis in mice

The basic helix-loop-helix (bHLH) transcription factor Hand2 has been implicated in the development of multiple organs, including craniofacial organs. Mice carrying Hand2 hypomorphic alleles (Hand2(LoxP/-)) display a cleft palate phenotype. A specific deletion of the Hand2 branchial arch-specific enhancer also leads to a hypoplastic mandible and cleft palate formation in mice. However, the underlying mechanism of Hand2 regulation of palate development remains unknown. Here we show that Hand2 is expressed in both the epithelium and mesenchyme of the developing palate. While mesenchymal specific inactivation of Hand2 has no impact on palate development, epithelial specific deletion of Hand2 creates a cleft palate phenotype. Hand2 appears to exert distinct roles in the anterior and posterior palate. In the anterior palate of Hand2(LoxP/-) mice, premature death of periderm cells and a down-regulation of Shh are observed in the medial edge epithelium (MEE), accompanied by a decreased level of cell proliferation in the palatal mesenchyme. In the posterior palate, a lower dose of Hand2 causes aberrant periderm cell death on the surface of the epithelium, triggering abnormal fusion between the palatal shelf and mandible and preventing palatal shelf elevation. We further demonstrate that BMP activities are essential for the expression of Hand2 in the palate. We conclude that Hand2 is an intrinsic regulator in the epithelium and is required for palate development.

Wnt5a regulates growth, patterning, and odontoblast differentiation of developing mouse tooth

Wnt/β‐catenin signaling is essential for tooth development beyond the bud stage, but little is known about the role of non‐canonical Wnt signaling in odontogenesis. Here we compared the expression of Wnt5a, a representative of noncanonical Wnts, with that of Ror2, the Wnt5a receptor for non‐canonical signaling, in the developing tooth, and analyzed tooth phenotype in Wnt5a mutants. Wnt5a‐deficient mice exhibit retarded tooth development beginning from E16.5, leading to the formation of smaller and abnormally patterned teeth with a delayed odontoblast differentiation at birth. These defects are associated with upregulated Axin2 and Shh expression in the dental epithelium and reduced levels of cell proliferation in the dental epithelium and mesenchyme. Retarded tooth development and defective odontoblast differentiation were also observed in Ror2 mutant mice. Our results suggest that Wnt5a regulates growth, patterning, and odontoblast differentiation during odontogenesis, at least partially by modulating Wnt/β‐catenin canonical signaling. Developmental Dynamics 240:432–440, 2011.

Epithelial Wnt/β-catenin signaling regulates palatal shelf fusion through regulation of Tgfβ3 expression

The canonical Wnt/β-catenin signaling plays essential role in development and diseases. Previous studies have implicated the canonical Wnt/β-catenin signaling in the regulation of normal palate development, but functional Wnt/β-catenin signaling and its tissue-specific activities remain to be accurately elucidated. In this study, we show that functional Wnt/β-catenin signaling operates primarily in the palate epithelium, particularly in the medial edge epithelium (MEE) of the developing mouse palatal shelves, consistent with the expression patterns of β-catenin and several Wnt ligands and receptors. Epithelial specific inactivation of β-catenin by the K14-Cre transgenic allele abolishes the canonical Wnt signaling activity in the palatal epithelium and leads to an abnormal persistence of the medial edge seam (MES), ultimately causing a cleft palate formation, a phenotype resembling that in Tgfβ3 mutant mice. Consistent with this phenotype is the down-regulation of Tgfβ3 and suppression of apoptosis in the MEE of the β-catenin mutant palatal shelves. Application of exogenous Tgfβ3 to the mutant palatal shelves in organ culture rescues the midline seam phenotype. On the other hand, expression of stabilized β-catenin in the palatal epithelium also disrupts normal palatogenesis by activating ectopic Tgfβ3 expression in the palatal epithelium and causing an aberrant fusion between the palate shelf and mandible in addition to severely deformed palatal shelves. Collectively, our results demonstrate an essential role for Wnt/β-catenin signaling in the epithelial component at the step of palate fusion during palate development by controlling the expression of Tgfβ3 in the MEE.

Expression survey of genes critical for tooth development in the human embryonic tooth germ

In the developing murine tooth, the expression patterns of numerous regulatory genes have been examined and their roles have begun to be revealed. To unveil the molecular mechanisms that regulate human tooth morphogenesis, we examined the expression patterns of several regulatory genes, including BMP4, FGF8, MSX1, PAX9, PITX2, and SHOX2, and compared them with that found in mice. All of these genes are known to play critical roles in murine tooth development. Our results show that these genes exhibit basically similar expression patterns in the human tooth germ compared with that in the mouse. However, slightly different expression patterns were also observed for some of the genes at certain stages. For example, MSX1 expression was detected in the inner enamel epithelium in addition to the dental mesenchyme at the bell stage of the human tooth. Moreover, FGF8 expression remained in the dental epithelium at the cap stage, while PAX9 and SHOX2 expression was detected in both dental epithelium and mesenchyme of the human tooth germ. Our results indicate that, although slight differences exist in the gene expression patterns, the human and mouse teeth not only share considerable homology in odontogenesis but also use similar underlying molecular networks. Developmental Dynamics 236:1307–1312, 2007.

A critical role for PDGFRα signaling in medial nasal process development.

The primitive face is composed of neural crest cell (NCC) derived prominences. The medial nasal processes (MNP) give rise to the upper lip and vomeronasal organ, and are essential for normal craniofacial development, but the mechanism of MNP development remains largely unknown. PDGFRα signaling is known to be critical for NCC development and craniofacial morphogenesis. In this study, we show that PDGFRα is required for MNP development by maintaining the migration of progenitor neural crest cells (NCCs) and the proliferation of MNP cells. Further investigations reveal that PI3K/Akt and Rac1 signaling mediate PDGFRα function during MNP development. We thus establish PDGFRα as a novel regulator of MNP development and elucidate the roles of its downstream signaling pathways at cellular and molecular levels.

BMP activity is required for tooth development from the lamina to bud stage.

Several Bmp genes are expressed in the developing mouse tooth germ from the initiation to the late-differentiation stages, and play pivotal roles in multiple steps of tooth development. In this study, we investigated the requirement of BMP activity in early tooth development by transgenic overexpression of the extracellular BMP antagonist Noggin. We show that overexpression of Noggin in the dental epithelium at the tooth initiation stage arrests tooth development at the lamina/early-bud stage. This phenotype is coupled with a significantly reduced level of cell proliferation rate and a down-regulation of Cyclin-D1 expression, specifically in the dental epithelium. Despite unaltered expression of genes known to be implicated in early tooth development in the dental mesenchyme and dental epithelium of transgenic embryos, the expression of Pitx2, a molecular marker for the dental epithelium, became down-regulated, suggesting the loss of odontogenic fate in the transgenic dental epithelium. Our results reveal a novel role for BMP signaling in the progression of tooth development from the lamina stage to the bud stage by regulating cell proliferation and by maintaining odontogenic fate of the dental epithelium.

Wnt Signaling in Lip and Palate Development

Wnt signaling regulates a variety of cell behaviors and represents a major pathway in development and disease. Mutations in Wnt genes and their downstream targets have been implicated in human craniofacial abnormalities, including the most prevalent birth defect, cleft lip with or without palate. Formation of the upper lip and palate is a complicated process and is composed of a series of highly coordinated steps during tissue morphogenesis, which are rigorously controlled by genetic networks. While genetic controls of lip/palate development have been extensively studied, the roles of Wnt signaling in these processes remained poorly understood. Within the cell, Wnt signaling is transduced in a β-catenin-dependent (canonical) or -independent (non-canonical) fashion. Recent studies have demonstrated that the canonical and non-canonical pathways play differential roles but both are essential in lip/palate development. Here we review these studies that have substantially advanced our knowledge by elucidating the function of Wnt signaling in upper lip formation, secondary palate development and their disease settings. These advances are important to delineate the genetic networks controlling craniofacial development and to develop personalized therapeutic strategies in related human birth defects in the future.