Extracellular Matrix in Secondary Palate Development.

The secondary palate arises from outgrowths of epithelia-covered embryonic mesenchyme that grow from the maxillary prominence, remodel to meet over the tongue, and fuse at the midline. These events require the coordination of cell proliferation, migration, and gene expression, all of which take place in the context of the extracellular matrix (ECM). Palatal cells generate their ECM, and then stiffen, degrade, or otherwise modify its properties to achieve the required cell movement and organization during palatogenesis. The ECM, in turn, acts on the cells through their matrix receptors to change their gene expression and thus their phenotype. The number of ECM-related gene mutations that cause cleft palate in mice and humans is a testament to the crucial role the matrix plays in palate development and a reminder that understanding that role is vital to our progress in treating palate deformities. This article will review the known ECM constituents at each stage of palatogenesis, the mechanisms of tissue reorganization and cell migration through the palatal ECM, the reciprocal relationship between the ECM and gene expression, and human syndromes with cleft palate that arise from mutations of ECM proteins and their regulators. Anat Rec, 2019. © 2019 American Association for Anatomy.

Cdc42 activation by endothelin regulates neural crest cell migration in the cardiac outflow tract.

BACKGROUND: Congenital cardiovascular malformations are the most common birth defects affecting children. Several of these defects occur in structures developing from neural crest cells. One of the key signaling pathways regulating cardiac neural crest cell (CNCC) development involves the endothelin-A receptor (Ednra). However, the exact function of Ednra signaling in CNCC is unknown. RESULTS: The fate mapping of CNCC in Ednra embryos indicated that the migration of these cells is aberrant in the cardiac outflow tract (OFT), but not in the pharyngeal arches. This premature arrest of CNCC migration occurs independently of CNCC proliferation and apoptosis changes and major gene expression changes. Analysis of the Rho family of small GTPases in the mutant embryos revealed that Cdc42 failed to localize normally in the CNCC migrating in the OFT. The inhibition of Cdc42 activity in cultured embryos recapitulated the migratory phenotype observed in Ednra mice. Further analyses revealed that Cdc42 is part of the signaling pathway activated by endothelin specifically in OFT CNCC to control their migration. CONCLUSIONS: These results indicated that the activation of Cdc42 by endothelin signaling is important for CNCC migration in the OFT but this pathway is not involved in mandibular or pharyngeal arch artery patterning.

Activation of Receptor Activator of Nuclear Factor-κB Ligand and Matrix Metalloproteinase Production in Periodontal Fibroblasts by Endothelin Signaling.

BACKGROUND: Periodontitis is a group of inflammatory diseases affecting the tissues supporting the teeth that will progressively cause the loss of alveolar bone and periodontal ligaments and eventually the dentition. Activation of osteoclast activity by receptor activator of nuclear factor-κB ligand (RANKL) and released enzymes such as matrix metalloproteinases (MMPs) are among the factors involved in the breakdown of the periodontium. However, the mechanisms regulating their production in periodontitis are poorly understood. Endothelin signaling via the activation of the endothelin-A receptor (EDNRA) by endothelin-1 may play a role in the disease because the expression of the receptor and ligand is elevated in the periodontal tissues of patients with periodontitis. METHODS: Cultured primary human periodontal fibroblasts were treated with 20 and 100 nM endothelin-1 for 6 and 24 hours and then collected to assess MMP and RANKL production by immunoblotting. Inhibitors were used to identify the molecular pathways activated by EDNRA in these cells. RESULTS: Endothelin-1 stimulated the production of MMP1, MMP8, and RANKL in a dose- and time-dependent manner; blocking EDNRA function with the antagonist TBC3214 inhibited the response, although EDNRA activation had no effects on osteoprotegerin production. These mechanistic studies indicate that EDNRA activates phospholipase C, which then 1) increases the MMP1 protein levels through activation of the extracellular signal-regulated kinase mitogen-activated protein kinase-dependent pathway and 2) upregulates RANKL by a different pathway. CONCLUSION: These results suggest that EDNRA may function in the breakdown of the periodontal tissues associated with periodontitis by promoting the protein expression of MMPs and RANKL via the phospholipase C pathway.

Expression analysis of α-smooth muscle actin and tenascin-C in the periodontal ligament under orthodontic loading or in vitro culture.

α-smooth muscle actin (α-SMA) and tenascin-C are stress-induced phenotypic features of myofibroblasts. The expression levels of these two proteins closely correlate with the extracellular mechanical microenvironment. We investigated how the expression of α-SMA and tenascin-C was altered in the periodontal ligament (PDL) under orthodontic loading to indirectly reveal the intrinsic mechanical microenvironment in the PDL. In this study, we demonstrated the synergistic effects of transforming growth factor-ß1 (TGF-ß1) and mechanical tensile or compressive stress on myofibroblast differentiation from human periodontal ligament cells (hPDLCs). The hPDLCs under higher tensile or compressive stress significantly increased their levels of α-SMA and tenascin-C compared with those under lower tensile or compressive stress. A similar trend was observed in the tension and compression areas of the PDL under continuous light or heavy orthodontic load in rats. During the time-course analysis of expression, we observed that an increase in α-SMA levels was matched by an increase in tenascin-C levels in the PDL under orthodontic load in vivo. The time-dependent variation of α-SMA and tenascin-C expression in the PDL may indicate the time-dependent variation of intrinsic stress under constant extrinsic loading.

Control of endothelin-a receptor expression by progesterone is enhanced by synergy with Gata2.

The endothelin-A receptor (Ednra) is involved in several physiological, pathological, and developmental pathways. Known for its function in vasoconstriction after being activated by endothelin-1, Ednra also controls cephalic neural crest cell development and appears to play a role in several pathologies, including cancer and periodontitis. However, the mechanisms regulating Ednra expression have not been identified despite its important functions. In this study, we investigated the role progesterone plays in Ednra gene expression in vivo and in vitro. In mice, pregnancy promotes Ednra expression in the heart, kidney, lung, uterus, and placenta, and the up-regulation is mediated by progesterone. We determined that the conserved region between -5.7 and -4.2 kb upstream of the mouse Ednra gene is necessary for the progesterone response. We also found that progesterone mediates Ednra activation through progesterone receptor B activation by its recruitment to PRE6, one of the 6 progesterone response elements found in that locus. However, gene activation by means of a GATA2 site was also necessary for the progesterone response. The Gata2 transcription factor enhances the progesterone response mediated by the progesterone receptor B. Together these results indicate that progesterone regulates Ednra expression by synergizing with Gata2 activity, a previously unknown mechanism. This mechanism may have an impact on pathologies involving the endothelin signaling.

Analysis of Snail1 function and regulation by Twist1 in palatal fusion.

Palatal fusion is a tightly controlled process which comprises multiple cellular events, including cell movement and differentiation. Midline epithelial seam (MES) degradation is essential to palatal fusion. In this study, we analyzed the function of Snail1 during the degradation of the MES. We also analyzed the mechanism regulating the expression of the Snail1 gene in palatal shelves. Palatal explants treated with Snail1 siRNA did not degrade the MES and E-cadherin was not repressed leading to failure of palatal fusion. Transforming growth factor beta 3 (Tgfß3) regulated Snail1 mRNA, as Snail1 expression decreased in response to Tgfß3 neutralizing antibody and a PI-3 kinase (PI3K) inhibitor. Twist1, in collaboration with E2A factors, regulated the expression of Snail1. Twist1/E47 dimers bond to the Snail1 promoter to activate expression. Without E47, Twist1 repressed Snail1 expression. These results support the hypothesis that Tgfß3 may signal through Twist1 and then Snail1 to downregulate E-cadherin expression during palatal fusion.

The rescue of dentin matrix protein 1 (DMP1)-deficient tooth defects by the transgenic expression of dentin sialophosphoprotein (DSPP) indicates that DSPP is a downstream effector molecule of DMP1 in dentinogenesis.

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are essential for the formation of dentin. Previous in vitro studies have indicated that DMP1 might regulate the expression of DSPP during dentinogenesis. To examine whether DMP1 controls dentinogenesis through the regulation of DSPP in vivo, we cross-bred transgenic mice expressing normal DSPP driven by a 3.6-kb rat Col1a1 promoter with Dmp1 KO mice to generate mice expressing the DSPP transgene in the Dmp1 KO genetic background (referred to as "Dmp1 KO/DSPP Tg mice"). We used morphological, histological, and biochemical techniques to characterize the dentin and alveolar bone of Dmp1 KO/DSPP Tg mice compared with Dmp1 KO and wild-type mice. Our analyses showed that the expression of endogenous DSPP was remarkably reduced in the Dmp1 KO mice. Furthermore, the transgenic expression of DSPP rescued the tooth and alveolar bone defects of the Dmp1 KO mice. In addition, our in vitro analyses showed that DMP1 and its 57-kDa C-terminal fragment significantly up-regulated the Dspp promoter activities in a mesenchymal cell line. In contrast, the expression of DMP1 was not altered in the Dspp KO mice. These results provide strong evidence that DSPP is a downstream effector molecule that mediates the roles of DMP1 in dentinogenesis.

Specific inactivation of Twist1 in the mandibular arch neural crest cells affects the development of the ramus and reveals interactions with hand2.

BACKGROUND: The basic helix-loop-helix (bHLH) transcription factor Twist1 fulfills an essential function in neural crest cell formation, migration, and survival and is associated with the craniosynostic Saethre-Chotzen syndrome in humans. However, its functions during mandibular development, when it may interact with other bHLH transcription factors like Hand2, are unknown because mice homozygous for the Twist1 null mutation die in early embryogenesis. To determine the role of Twist1 during mandibular development, we used the Hand2-Cre transgene to conditionally inactivate the gene in the neural crest cells populating the mandibular pharyngeal arch. RESULTS: The mutant mice exhibited a spectrum of craniofacial anomalies, including mandibular hypoplasia, altered middle ear development, and cleft palate. It appears that Twist1 is essential for the survival of the neural crest cells involved in the development of the mandibular ramal elements. Twist1 plays a role in molar development and cusp formation by participating in the reciprocal signaling needed for the formation of the enamel knot. This gene is also needed to control the ossification of the mandible, a redundant role shared with Hand2. CONCLUSION: Twist1, along with Hand2, is essential for the proximodistal patterning and development of the mandible and ossification.

Analysis of neural crest cell fate during cardiovascular development using Cre-activated lacZ/ß-galactosidase staining.

It is important to identify the mechanisms regulating cardiovascular development. However, complex genetic tools are often required, including transgenic animals that express the lacZ transgene encoding the ß-galactosidase enzyme under the control of a specific promoter or following recombination with the Cre recombinase. The latter can be useful for identifying specific cell populations of the developing cardiovascular system, including neural crest cells. The tracking of these cells can help clarify their fate in mutant embryos and elucidate the etiology of some congenital cardiovascular birth defects. This chapter highlights the methods used to stain embryonic tissues in whole mount or sections to detect the expression of the lacZ transgene with a focus on tracking cardiac neural crest cells using the Wnt1-Cre and R26R mouse lines. We also provide a protocol using fluorescence-activated cell sorting for collecting neural crest cells for further analysis. These protocols can be used with any embryos expressing Cre and lacZ.

Cleft lip and palate genetics and application in early embryological development.

The development of the head involves the interaction of several cell populations and coordination of cell signalling pathways, which when disrupted can cause defects such as facial clefts. This review concentrates on genetic contributions to facial clefts with and without cleft palate (CP). An overview of early palatal development with emphasis on muscle and bone development is blended with the effects of environmental insults and known genetic mutations that impact human palatal development. An extensive table of known genes in syndromic and non-syndromic CP, with or without cleft lip (CL), is provided. We have also included some genes that have been identified in environmental risk factors for CP/L. We include primary and review references on this topic.

Fetal alcohol syndrome (FAS) in C57BL/6 mice detected through proteomics screening of the amniotic fluid.

BACKGROUND: Fetal Alcohol Syndrome (FAS), a severe consequence of the Fetal Alcohol Spectrum Disorders, is associated with craniofacial defects, mental retardation, and stunted growth. Previous studies in C57BL/6J and C57BL/6N mice provide evidence that alcohol-induced pathogenesis follows early changes in gene expression within specific molecular pathways in the embryonic headfold. Whereas the former (B6J) pregnancies carry a high-risk for dysmorphogenesis following maternal exposure to 2.9 g/kg alcohol (two injections spaced 4.0 h apart on gestation day 8), the latter (B6N) pregnancies carry a low-risk for malformations. The present study used this murine model to screen amniotic fluid for biomarkers that could potentially discriminate between FAS-positive and FAS-negative pregnancies. METHODS: B6J and B6N litters were treated with alcohol (exposed) or saline (control) on day 8 of gestation. Amniotic fluid aspirated on day 17 (n = 6 replicate litters per group) was subjected to trypsin digestion for analysis by matrix-assisted laser desorption-time of flight mass spectrometry with the aid of denoising algorithms, statistical testing, and classification methods. RESULTS: We identified several peaks in the proteomics screen that were reduced consistently and specifically in exposed B6J litters. Preliminary characterization by liquid chromatography tandem mass spectrometry and multidimensional protein identification mapped the reduced peaks to alpha fetoprotein (AFP). The predictive strength of AFP deficiency as a biomarker for FAS-positive litters was confirmed by area under the receiver operating characteristic curve. CONCLUSIONS: : These findings in genetically susceptible mice support clinical observations in maternal serum that implicate a decrease in AFP levels following prenatal alcohol damage.