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Byline: Kyoko Oka (a), Shoji Oka (a), Tomoyo Sasaki (a), Yoshihiro Ito (a), Pablo Bringas (a), Kazuaki Nonaka (b), Yang Chai (a) Keywords: Cranial neural crest (CNC); Cell proliferation; Differentiation; Mandible and Meckel's cartilage development; CTGF; Msx1; TGF-[beta] Abstract: During craniofacial development, Meckel's cartilage and the mandible bone derive from the first branchial arch, and their development depends upon the contribution of cranial neural crest (CNC) cells. We previously demonstrated that conditional inactivation of Tgfbr2 in the neural crest of mice (Tgfbr2 .sup. fl/fl ;Wnt1-Cre) results in severe defects in mandibular development, although the specific cellular and molecular mechanisms by which TGF-[beta] signaling regulates the fate of CNC cells during mandibular development remain unknown. We show here that loss of Tgfbr2 does not affect the migration of CNC cells during mandibular development. TGF-[beta] signaling is specifically required for cell proliferation in Meckel's cartilage and the mandibular anlagen and for the formation of the coronoid, condyle and angular processes. TGF-[beta]-mediated connective tissue growth factor (CTGF) signaling is critical for CNC cell proliferation. Exogenous CTGF rescues the cell proliferation defect in Meckel's cartilage of Tgfbr2 .sup. fl/fl ;Wnt1-Cre mutants, demonstrating the biological significance of this signaling cascade in chondrogenesis during mandibular development. Furthermore, TGF-[beta] signaling controls Msx1 expression to regulate mandibular osteogenesis as Msx1 expression is significantly reduced in Tgfbr2 .sup. fl/fl ;Wnt1-Cre mutants. Collectively, our data suggest that there are differential signal cascades in response to TGF-[beta] to control chondrogenesis and osteogenesis during mandibular development. Author Affiliation: (a) Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033, USA (b) Section of Pediatric Dentistry, Division of Oral Health, Growth and Development, Kyushu University, Maidashi 3-1-1, Higashi-Ku, Fukuoka 812-8582, Japan Article History: Received 29 July 2006; Revised 12 November 2006; Accepted 15 November 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.05.014 Byline: Xun Xu, Jun Han, Yoshihiro Ito, Pablo Bringas, Mark M. Urata, Yang Chai Keywords: Epithelial-mesenchymal transformation (EMT); Irf6; Medial edge epithelium (MEE); Palatogenesis; TGF-[beta] signaling; Proliferation; Apoptosis Abstract: Palatal fusion is a complex, multi-step developmental process; the consequence of failure in this process is cleft palate, one of the most common birth defects in humans. Previous studies have shown that regression of the medial edge epithelium (MEE) upon palatal fusion is required for this process, and TGF-[beta] signaling plays an important role in regulating palatal fusion. However, the fate of the MEE and the mechanisms underlying its disappearance are still unclear. By using the Cre/lox system, we are able to label the MEE genetically and to ablate Tgfbr2 specifically in the palatal epithelial cells. Our results indicate that epithelial-mesenchymal transformation does not occur in the regression of MEE cells. Ablation of Tgfbr2 in the palatal epithelial cells causes soft palate cleft, submucosal cleft and failure of the primary palate to fuse with the secondary palate. Whereas wild-type MEE cells disappear, the mutant MEE cells continue to proliferate and form cysts and epithelial bridges in the midline of the palate. Our study provides for the first time an animal model for soft palate cleft and submucous cleft. At the molecular level, Tgfb3 and Irf6 have similar expression patterns in the MEE. Mutations in IRF6 disrupt orofacial development and cause cleft palate in humans. We show here that Irf6 expression is downregulated in the MEE of the Tgfbr2 mutant. As a recent study shows that heterozygous mutations in TGFBR1 or TGFBR2 cause multiple human congenital malformations, including soft palate cleft, we propose that TGF-[beta] mediated Irf6 expression plays an important, cell-autonomous role in regulating the fate of MEE cells during palatogenesis in both mice and humans. Author Affiliation: Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033, USA Article History: Received 21 February 2006; Revised 28 April 2006; Accepted 10 May 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.11.008 Byline: Bibha Choudhary (a), Yoshihiro Ito (b), Takako Makita (a), Tomoyo Sasaki (b), Yang Chai (b), Henry M. Sucov (a) Keywords: Neural crest; TGF[beta]; Type II receptor; Persistent truncus arteriosus; Interrupted aortic arch; DiGeorge syndrome Abstract: Previous studies have demonstrated that TGF[beta] induces a smooth muscle fate in primary neural crest cells in culture. By crossing a conditional allele of the type II TGF[beta] receptor with the neural crest-specific Wnt1cre transgene, we have addressed the in vivo requirement for TGF[beta] signaling in smooth muscle specification and differentiation. We find that elimination of the TGF[beta] receptor does not alter neural crest cell specification to a smooth muscle fate in the cranial or cardiac domains, and that a smooth muscle fate is not realized by trunk neural crest cells in either control or mutant embryos. Instead, mutant embryos exhibit with complete penetrance two very specific and mechanistically distinct cardiovascular malformations -- persistent truncus arteriosus (PTA) and interrupted aortic arch (IAA-B). Pharyngeal organ defects such as those seen in models of DiGeorge syndrome were not observed, arguing against an early perturbation of the cardiac neural crest cell lineage. We infer that TGF[beta] is an essential morphogenic signal for the neural crest cell lineage in specific aspects of cardiovascular development, although one that is not required for smooth muscle differentiation. Author Affiliation: (a) Institute for Genetic Medicine, Keck School of Medicine, School of Dentistry, University of Southern California, 2250 Alcazar St., IGM240, Los Angeles, CA 90033, USA (b) Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, CA 90033, USA Article History: Received 14 July 2005; Revised 26 October 2005; Accepted 8 November 2005

The role of transforming growth factor [beta] in formation of neural crest cells in some aspects of cardiovascular development in mouse embryos is studied.

Transforming growth factor (TGF)-[beta]3 is an important contributor to the regulation of medial edge epithelium (MEE) disappearance during palatal fusion. SMAD2 phosphorylation in the MEE has been shown to be directly regulated by TGF-[beta]3. No phospho-SMAD2 was identified in the MEE in Tgf-[beta]3-null mutant mice ([Tgf-[beta]3.sup.-/-]), which was correlated with the persistence of the MEE and failure of palatal fusion. In the present study, the cleft palate phenotype in [Tgf-[beta]3.sup.-/-] mice was rescued by overexpression of a Smad2 transgene in Keratin 14-synthesizing MEE cells following mating Tgf-[beta]3 heterozygous mice with Keratin 14 promoter directed Smad2 transgenic mice (K14-Smad2). Success of the rescue could be attributed to the elevated phospho-SMAD2 level in the MEE, demonstrated by two indirect evidences. The rescued palatal fusion in [Tgf-[beta]3.sup.-/-]/K14-Smad2 mice, however, never proceeded to the junction of primary and secondary palates and the most posterior border of the soft palate, despite phospho-SMAD2 expression in these regions at the same level as in the middle portion of the secondary palate. The K14-Smad2 transgene was unable to restore all the functional outcomes of TGF-[beta]3. This may indicate an anterior-posterior patterning in the palatal shelves with respect to TGF[beta]3 signaling and the mechanism of secondary palatalfusion. Keywords: TGF-[beta]3; Smad2; Keratin 14; Overexpression; Mouse; Cleft palate; Palatal fusion; Medial edge epithelium; Epithelial-mesenchymal transformation; Rescue

LEF1 is a cell-type-specific transcription factor and mediates Wnt signaling pathway by association with its co-activator [beta]-catenin. Wnt signaling is known to be critical for the specification of cranial neural crest (CNC) cells and may regulate the late diversity of the CNC during craniofacial morphogeuesis. Loss of Lef1 results in arrested tooth development at the late bud stage and LEF1 is required for a relay of a Wnt signaling to a cascade of FGF signaling activities to mediate the epithelial--mesenchymal interaction during tooth morphogenesis. It remains unclear, however, what is the cellular mechanism of LEF1 signaling in regulating tooth morphogenesis. To test the hypothesis that LEF1 signaling regulates the late of the dental epithelial and the CNC-derived mesenchymal cells during tooth morphogenesis, we investigated and compared the cellular migration, proliferation, and apoptotic activity within the tooth germ between the wild-type and Lef1 null mutant mice. Using the Wnt1-Cre/R26R transgenic system for indelibly marking the progenies of CNC cells, we show that there is no CNC migration defect in the Lef1 null mutant mice, indicating that the arrest in tooth development is not the result of shortage of the CNC contribution into the first branchial arch in the Lef1 mutant. Furthermore, there is no alteration in cell proliferation or condensation of the CNC-derived dental mesenchyme in the Lef1 null mutant, suggesting that LEF1 may not affect the cell cycle progression of the multipotential CNC cells during tooth morphogenesis. Importantly, apoptotic activity is significantly increased within the dental epithelium in the Lef1 null mutant mice. As the result of this increased cell death, the bud stage tooth germ Fails to advance to the cap stage in the absence of Lef1. Inhibition of apoptotic activity by FGF4 rescues the tooth development in the Lef1 null mutant. Our studies suggest that LEF1 is a critical survival factor for the dental epithelial cells during tooth morphogenesis. Keywords: Apoptosis of dental epithelium; Cranial neural crest (CNC); LEF1 signaling; Tooth morphogenesis

Neural crest cells are multipotential progenitors that contribute to various cell and tissue types during embryogenesis. Here, we have investigated the molecular and cellular mechanism by which the fate of neural crest cell is regulated during tooth development. Using a two-component genetic system for indelibly marking the progeny of neural crest cells, we provide in vivo evidence of a deficiency of CNC-derived dental mesenchyme in Msx1 null mutant mouse embryos. The deficiency of the CNC results from an elevated CDK inhibitor p[19.sup.INK4d] activity and the disruption of cell proliferation. Interestingly, in the absence of Msx1, the CNC-derived dental mesenchyme misdifferentiates and possesses properties consistent with a neuronal fate, possibly through a default mechanism. Attenuation of p[19.sup.INK4d] in Msx1 null mutant mandibular explants restores mitotic activity in the dental mesenchyme, demonstrating the functional significance of Msx1-mediated p[19.sup.INK4d] expression in regulating CNC cell proliferation during odontogenesis. Collectively, our results demonstrate that homeobox gene Msx1 regulates the fate of CNC cells by controlling the progression of the cell cycle. Genetic mutation of Msx1 may alternatively instruct the fate of these progenitor cells during craniofacial development. Keywords: Cranial neural crest (CNC) cells; Cell cycle; Msx1; p[19.sup.INK4d]; Tooth; Proliferation; Differentiation; Apoptosis

Palatal fusion process in mammals and biochemical reactions associated with this are examined.

Members of the transforming growth factor-[Beta] (TGF-[Beta]) superfamily are critical regulators for epithelial growth and can alter the differentiation of keratinocytes. Transduction of TGF-[Beta] signaling depends on the phosphorylation and activation of Smad proteins by heteromeric complexes of ligand-specific type I and II receptors. To understand the function of TGF-[Beta] and activin-specific Smad, we generated transgenic mice that overexpress Smad2 in epidermis under the control of keratin 14 promoter. Overexpression of Smad2 increases endogenous Smad4 and TGF-[Beta]1 expression while heterozygous loss of Smad2 reduces their expression levels, suggesting a concerted action of Smad2 and -4 in regulating TGF-[Beta] signaling during skin development. These transgenic mice have delayed hair growth, underdeveloped ears, and shorter tails. In their skin, there is severe thickening of the epidermis with disorganized epidermal architecture, indistinguishable basement membrane, and dermal fibrosis. These abnormal phenotypes are due to increased proliferation of the basal epidermal cells and abnormalities in the program of keratinocyte differentiation. The ectodermally derived enamel structure is also abnormal. Collectively, our study presents the first in vivo evidence that, by providing an auto-feedback in TGF-[Beta] signaling, Smad2 plays a pivotal role in regulating TGF-[Beta]-mediated epidermal homeostasis. Key Words: skin; Smad2 transgenic; Smad4; TGF-[Beta] signaling; tooth.

Neural crest cells are multipotential progenitors that contribute to various cell and tissue types during embryogenesis. Here, we have investigated the molecular and cellular mechanism by which the fate of neural crest cell is regulated during tooth development. Using a two- component genetic system for indelibly marking the progeny of neural crest cells, we provide in vivo evidence of a deficiency of CNC-derived dental mesenchyme in Msx1 null mutant mouse embryos. The deficiency of the CNC results from an elevated CDK inhibitor p19INK4d activity and the disruption of cell proliferation. Interestingly, in the absence of Msx1, the CNC-derived dental mesenchyme misdifferentiates and possesses properties consistent with a neuronal fate, possibly through a default mechanism. Attenuation of p19INK4d in Msx1 null mutant mandibular explants restores mitotic activity in the dental mesenchyme, demonstrating the functional significance of Msx1-mediated p19INK4d expression in regulating CNC cell proliferation during odontogenesis. Collectively, our results demonstrate that homeobox gene Msx1 regulates the fate of CNC cells by controlling the progression of the cell cycle. Genetic mutation of Msx1 may alternatively instruct the fate of these progenitor cells during craniofacial development. [Copyright &y& Elsevier]