Characterizing expression pattern of Six2Cre during mouse craniofacial development.

Craniofacial development is a complex process involving diverse cell populations. Various transgenic Cre lines have been developed to facilitate studying gene function in specific tissues. In this study, we have characterized the expression pattern of Six2Cre mice at multiple stages during craniofacial development. Our data revealed that Six2Cre lineage cells are predominantly present in frontal bone, mandible, and secondary palate. Using immunostaining method, we found that Six2Cre triggered reporter is co-expressed with Runx2. In summary, our data showed Six2Cre can be used to study gene function during palate development and osteogenesis in mouse models.

Characterizing the role of Pdgfra in calvarial development.

BACKGROUND: Mammalian calvarium is composed of flat bones developed from two origins, neural crest, and mesoderm. Cells from both origins exhibit similar behavior but express distinct transcriptomes. It is intriguing to ask whether genes shared by both origins play similar or distinct roles in development. In the present study, we have examined the role of Pdgfra, which is expressed in both neural crest and mesoderm, in specific lineages during calvarial development. RESULTS: We found that in calvarial progenitor cells, Pdgfra is needed to maintain normal proliferation and migration of neural crest cells but only proliferation of mesoderm cells. Later in calvarial osteoblasts, we found that Pdgfra is necessary for both proliferation and differentiation of neural crest-derived cells, but not for differentiation of mesoderm-derived cells. We also examined the potential interaction between Pdgfra and other signaling pathway involved in calvarial osteoblasts but did not identify significant alteration of Wnt or Hh signaling activity in Pdgfra genetic models. CONCLUSIONS: Pdgfra is required for normal calvarial development in both neural crest cells and mesoderm cells, but these lineages exhibit distinct responses to alteration of Pdgfra activity.

Deregulated Rac1 Activity in Neural Crest Controls Cell Proliferation, Migration and Differentiation During Midbrain Development.

Mutations in RAC1 allele are implicated in multiple brain tumors, indicating a rigorous control of Rac1 activity is required for neural tissue normal development and homeostasis. To understand how elevated Rac1 activity affects neural crest cells (NCCs) development, we have generated Rac1 CA ;Wnt1-Cre2 mice, in which a constitutively active Rac1 G12V mutant is expressed specifically in NCCs derivatives. Our results revealed that augmented Rac1 activity leads to enlarged midbrain and altered cell density, accompanied by increased NCCs proliferation rate and misrouted cell migration. Interestingly, our experimental data also showed that elevated Rac1 activity in NCCs disrupts regionalization of dopaminergic neuron progenitors in the ventral midbrain and impairs their differentiation. These findings shed light on the mechanisms of RAC1 mutation correlated brain tumor at the cellular and molecular level.

Pdgfra regulates multipotent cell differentiation towards chondrocytes via inhibiting Wnt9a/beta-catenin pathway during chondrocranial cartilage development.

The mammalian skull is composed of the calvarial bones and cartilages. Malformation of craniofacial cartilage has been identified in multiple human syndromes. However, the mechanisms of their development remain largely unknown. In the present study, we identified Pdgfra as a novel player of chondrocranial cartilage development. Our data show that Pdgfra is required for normal chondrocranial cartilage development. Using tissue-specific genetic tools, we demonstrated that Pdgfra is essential for chondrocyte progenitors formation, but not in mature chondrocytes. Further analysis revealed that Pdgfra regulates chondrocytes progenitors development at two stages: in embryonic mesenchymal stem cells (eMSCs), Pdgfra directs their differentiation toward chondrocyte progenitors; in chondrocytes progenitors, Pdgfra activation promotes cell proliferation. We also found that excessive Pdgfra activity causes ectopic cartilage formation. Our data show that Pdgfra directs eMSCs differentiation via inhibiting Wnt9a transcription and its downstream signaling, and activating Wnt signaling rescues ectopic cartilage phenotype caused by excessive Pdgfra activity. In summary, our study dissected the role of Pdgfra signaling in chondrocranial cartilage formation, and illustrated the underlying mechanisms at multiple stages.

Expression pattern of Kmt2d in murine craniofacial tissues.

Formation of the calvaria is a multi-staged process and is regulated by multiple genetic factors. Disruption of normal calvarial development usually causes craniosynostosis, a prevalent birth defect characterized by premature fusion of calvarial bone. Recent studies have identified mutations of KMT2D allele in patients with craniosynostosis, indicating a potential role for Kmt2d in calvarial development. KMT2D mutations have also been implicated in Kabuki syndrome, which features a distinct facial appearance, skeletal abnormality, growth retardation and intellectual disability. However, the expression pattern of Kmt2d has not been fully elucidated. In the present study we examined the expression pattern of Kmt2d at multiple stages of embryo development in mice, with a focus on the craniofacial tissues. Our in situ hybridization results showed that Kmt2d mRNA is expressed in the developing calvarial osteoblasts, epithelia and neural tissues. Such an expression pattern is in line with the phenotypes of Kabuki syndrome, suggesting that Kmt2d plays an intrinsic role in normal development and homeostasis of these craniofacial tissues.

Pten regulates neural crest proliferation and differentiation during mouse craniofacial development.

BACKGROUND: The phosphatase and tensin homolog deleted on chromosome TEN (Pten) is implicated in a broad range of developmental events and diseases. However, its role in neural crest and craniofacial development has not been well illustrated. RESULTS: Using genetically engineered mouse models, we showed that inactivating Pten specifically in neural crest cells causes malformation of craniofacial structures. Pten conditional knockout mice exhibit perinatal lethality with overgrowth of craniofacial structures. At the cellular level, Pten deficiency increases cell proliferation rate and enhances osteoblast differentiation. Our data further revealed that inactivating Pten elevates PI3K/Akt signaling activity in neural crest derivatives, and confirmed that attenuation of PI3K/Akt activity led to decreased neural crest cell proliferation and differentiation both in vitro and in vivo. CONCLUSIONS: Our study revealed that Pten is essential for craniofacial morphogenesis in mice. Inactivating Pten in neural crest cells increases proliferation rate and promotes their differentiation toward osteoblasts. Our data further indicate that Pten acts via modulating PI3K/Akt activity during these processes. Developmental Dynamics 247:304-314, 2018. © 2017 Wiley Periodicals, Inc.

Dysregulated PDGFRα signaling alters coronal suture morphogenesis and leads to craniosynostosis through endochondral ossification.

Craniosynostosis is a prevalent human birth defect characterized by premature fusion of calvarial bones. In this study, we show that tight regulation of endogenous PDGFRα activity is required for normal calvarium development in the mouse and that dysregulated PDGFRα activity causes craniosynostosis. Constitutive activation of PDGFRα leads to expansion of cartilage underlying the coronal sutures, which contribute to suture closure through endochondral ossification, in a process regulated in part by PI3K/AKT signaling. Our results thus identify a novel mechanism underlying calvarial development in craniosynostosis.

[Effect of Zhuangyao Jianshen Wan (ZYJCW) on P2X1 and P2X3 mRNA expressions in rats with diuresis caused by kidney deficiency].

To investigate the urination-reducing effect and mechanism of Zhuangyao Jianshen Wan (ZYJCW). In this study, SI rats were subcutaneously injected with 150 mg · kg(-1) dose of D-galactose to prepare the sub-acute aging model and randomly divided into the model group, the Suoquan Wan group (1.17 g · kg(-1) · d(-1)), and ZYJCW high, medium and low dose groups (2.39, 1.20, 0.60 g · kg(-1) · d(-1)) , with normal rats in the blank group. They were continuously administered with drugs for eight weeks. The metabolic cage method was adopted to measure the 24 h urine volume and 5 h water load urine volume in rats. The automatic biochemistry analyzer was adopted to detect urine concentrations of Na+, Cl-, K+. The ELISA method was used to determine serum aldosterone (ALD) and antidiuretic hormone (ADH). The changes in P2X1 and P2X3 mRNA expressions in bladder tissues of rats were detected by RT-PCR. According to the results, both ZYJCW high and medium dose groups showed significant down-regulations in 24 h urine volume and 5 h water load urine volume in (P <0.05, P <0.01), declines in Na+ and Cl- concentrations in urine (P <0.01), notable rises in plasma ALD and ADH contents (P <0.05, P <0.01) and remarkable down-regulations in the P2X1 and P2X3 mRNA expressions in bladder tissues (P <0.01). The ZYJCW low dose group revealed obvious reductions in Na+ and Cl- concentrations in urine (P <0.01). The results indicated that ZYJCW may show the urination-reducing effect by down-regulating the P2X1 and P2X3 mRNA expressions in bladder tissues of rats with diuresis caused by kidney deficiency.

Sox10ER(T2) CreER(T2) mice enable tracing of distinct neural crest cell populations.

BACKGROUND: Neural crest cells play an important role in craniofacial morphogenesis and many other developmental processes. The formation of neural crest cells (NCCs) in vivo is a highly dynamic process and remains to be fully understood. RESULTS: To investigate the spatiotemporal patterning of NCCs in vivo, we have generated Sox10ER(T2) CreER(T2) (SECE) mice, a transgenic line driving inducible Cre expression in NCCs. Inducing Cre activity at different stages triggered reporter expression in distinct NCC populations in SECE; R26R mice. By optimizing the timing and dosage of tamoxifen administration, we controlled Cre expression specifically in cranial NCCs. Using this approach, we demonstrate an important role for PDGFRα in cranial NCCs mitosis within the mandibular processes. Further reducing Cre activity within the cranial NCCs of SECE; R26R embryos revealed that SECE labels preferentially progenitors of medial nasal process (MNP) rather than the lateral nasal process (LNP), before their formation from the frontonasal prominence (FNP). CONCLUSIONS: Our results indicate that NCCs are formed sequentially from rostral to caudal regions along the neural tube. These findings also suggest that NCCs within the FNP become specified regionally and genetically before they divide into MNP and LNP.

Receptor tyrosine kinases modulate distinct transcriptional programs by differential usage of intracellular pathways.

Receptor tyrosine kinases (RTKs) signal through shared intracellular pathways yet mediate distinct outcomes across many cell types. To investigate the mechanisms underlying RTK specificity in craniofacial development, we performed RNA-seq to delineate the transcriptional response to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) signaling in mouse embryonic palatal mesenchyme cells. While the early gene expression profile induced by both growth factors is qualitatively similar, the late response is divergent. Comparing the effect of MEK (Mitogen/Extracellular signal-regulated kinase) and PI3K (phosphoinositide-3-kinase) inhibition, we find the FGF response is MEK dependent, while the PDGF response is PI3K dependent. Furthermore, FGF promotes proliferation but PDGF favors differentiation. Finally, we demonstrate overlapping domains of PDGF-PI3K signaling and osteoblast differentiation in the palate and increased osteogenesis in FGF mutants, indicating this differentiation circuit is conserved in vivo. Our results identify distinct responses to PDGF and FGF and provide insight into the mechanisms encoding RTK specificity.

Directed Bmp4 expression in neural crest cells generates a genetic model for the rare human bony syngnathia birth defect.

Congenital bony syngnathia, a rare but severe human birth defect, is characterized by bony fusion of the mandible to the maxilla. However, the genetic mechanisms underlying this birth defect are poorly understood, largely due to limitation of available animal models. Here we present evidence that transgenic expression of Bmp4 in neural crest cells causes a series of craniofacial malformations in mice, including a bony fusion between the maxilla and hypoplastic mandible, resembling the bony syngnathia syndrome in humans. In addition, the anterior portion of the palatal shelves emerged from the mandibular arch instead of the maxilla in the mutants. Gene expression assays showed an altered expression of several facial patterning genes, including Hand2, Dlx2, Msx1, Barx1, Foxc2 and Fgf8, in the maxillary and mandibular processes of the mutants, indicating mis-patterned cranial neural crest (CNC) derived cells in the facial region. However, despite of formation of cleft palate and ectopic cartilage, forced expression of a constitutively active form of BMP receptor-Ia (caBmprIa) in CNC lineage did not produce the syngnathia phenotype, suggesting a non-cell autonomous effect of the augmented BMP4 signaling. Our studies demonstrate that aberrant BMP4-mediated signaling in CNC cells leads to mis-patterned facial skeleton and congenital bony syngnathia, and suggest an implication of mutations in BMP signaling pathway in human bony syngnathia.

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.

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 ß-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.

Tissue interaction is required for glenoid fossa development during temporomandibular joint formation.

The mammalian temporomandibular joint (TMJ) develops from two distinct mesenchymal condensations that grow toward each other and ossify through different mechanisms, with the glenoid fossa undergoing intramembranous ossification while the condyle being endochondral in origin. In this study, we used various genetically modified mouse models to investigate tissue interaction between the condyle and glenoid fossa during TMJ formation in mice. We report that either absence or dislocation of the condyle results in an arrested glenoid fossa development. In both cases, glenoid fossa development was initiated, but failed to sustain, and became regressed subsequently. However, condyle development appears to be independent upon the presence of the forming glenoid fossa. In addition, we show that substitution of condyle by Meckel's cartilage is able to sustain glenoid fossa development. These observations suggest that proper signals from the developing condyle or Meckel's cartilage are required to sustain the glenoid fossa development.

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

Wnt/ß-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/ß-catenin canonical signaling.

Epithelial Wnt/ß-catenin signaling regulates palatal shelf fusion through regulation of Tgfß3 expression.

The canonical Wnt/ß-catenin signaling plays essential role in development and diseases. Previous studies have implicated the canonical Wnt/ß-catenin signaling in the regulation of normal palate development, but functional Wnt/ß-catenin signaling and its tissue-specific activities remain to be accurately elucidated. In this study, we show that functional Wnt/ß-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 ß-catenin and several Wnt ligands and receptors. Epithelial specific inactivation of ß-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 Tgfß3 mutant mice. Consistent with this phenotype is the down-regulation of Tgfß3 and suppression of apoptosis in the MEE of the ß-catenin mutant palatal shelves. Application of exogenous Tgfß3 to the mutant palatal shelves in organ culture rescues the midline seam phenotype. On the other hand, expression of stabilized ß-catenin in the palatal epithelium also disrupts normal palatogenesis by activating ectopic Tgfß3 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/ß-catenin signaling in the epithelial component at the step of palate fusion during palate development by controlling the expression of Tgfß3 in the MEE.

Overexpression of constitutively active BMP-receptor-IB in mouse skin causes an ichthyosis-vulgaris-like disease.

The skin is the outer layer of protection against the environment. The development and formation of the skin is regulated by several genetic cascades including the bone morphogenetic protein (BMP) signaling pathway, which has been suggested to play an important role during embryonic organ development. Several skin defects and diseases are caused by genetic mutations or disorders. Ichthyosis is a common genetic skin disorder characterized by dry scaly skin. Loss-of-function mutations in the filaggrin (FLG) gene have been identified as the cause of the ichthyosis vulgaris (IV) phenotype; however, the direct regulation of filaggrin expression in vivo is unknown. We present evidence that BMP signaling regulates filaggrin expression in the epidermis. Mice expressing a constitutively active form of BMP-receptor-IB in the developing epidermis exhibit a phenotype resembling IV in humans, including dry flaky skin, compact hyperkeratosis, and an attenuated granular layer associated with a significantly downregulated expression of filaggrin. Regulation of filaggrin expression by BMP signaling has been further confirmed by the application of exogenous BMP2 in skin explants and by a transgenic model overexpressing Noggin in the epidermis. Our results demonstrate that aberrant BMP signaling in the epidermis causes overproliferation and hyperkeratinization, leading to an IV-like skin disease.

Gsk3ß is required in the epithelium for palatal elevation in mice.

In Wnt/ß-catenin signaling pathway, Gsk3ß functions to facilitate ß-catenin degradation. Inactivation of Gsk3ß in mice causes a cleft palate formation, suggesting an involvement of Wnt/ß-catenin signaling during palatogenesis. In this study, we have investigated the expression pattern, tissue-specific requirement and function of Gsk3ß during mouse palatogenesis. We showed that Gsk3ß is primarily expressed in the palatal epithelium, particularly in the medial edge epithelium overlapping with ß-catenin. Tissue-specific gene inactivation studies demonstrated an essential role for Gsk3ß in the epithelium for palate elevation, and disruption of which contributes to cleft palate phenotype in Gsk3ß mutant. We observed that expression of Aixn2, a direct target gene of Wnt/ß-catenin signaling, is ectopically activated in the mutant tongue, but not in the palate. Our results indicate that Gsk3ß is an intrinsic regulator required in the epithelium for palate elevation, and could act through a pathway independent of Wnt/ß-catenin signaling to regulate palate development.

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.

Control of hair follicle cell fate by underlying mesenchyme through a CSL-Wnt5a-FoxN1 regulatory axis.

Epithelial-mesenchymal interactions are key to skin morphogenesis and homeostasis. We report that maintenance of the hair follicle keratinocyte cell fate is defective in mice with mesenchymal deletion of the CSL/RBP-Jkappa gene, the effector of "canonical" Notch signaling. Hair follicle reconstitution assays demonstrate that this can be attributed to an intrinsic defect of dermal papilla cells. Similar consequences on hair follicle differentiation result from deletion of Wnt5a, a specific dermal papilla signature gene that we found to be under direct Notch/CSL control in these cells. Functional rescue experiments establish Wnt5a as an essential downstream mediator of Notch-CSL signaling, impinging on expression in the keratinocyte compartment of FoxN1, a gene with a key hair follicle regulatory function. Thus, Notch/CSL signaling plays a unique function in control of hair follicle differentiation by the underlying mesenchyme, with Wnt5a signaling and FoxN1 as mediators.