Influence of retinoids and EGF on growth of embryonic mouse palatal epithelia in culture

Summary Retinoids and growth factors seem to be important for normal mammalian reproduction and development. High levels of retinoic acid are teratogenic and induce cleft palate in the mouse. Little is known concerning the mechanisms through which retinoids induce cleft palate. Palatal epithelia from CD-1 embryonic mice on Day 12 of gestation were isolated from the mesenchyme and cultured in serum-free media, with all-trans retinoic acid or 13-cis retinoic acid, with or without epidermal growth factor (EGF). The epithelia attached and grew, and the cells differentiated over a 72-h culture period. Binding of [¹²⁵I]EGF was observed in all cultures in a pattern that correlated with thymidine (TdR) uptake by the epithelia. EGF enhanced growth and [³H]TdR incorporation of the oral cells, but nasal cells generally did not proliferate. In this culture system, both retinoids suppressed [³H]TdR incorporation in a concentration-dependent manner for epithelia cultured with or without EGF. Medial cells are important to normal palatogenesis as they play a role in fusion of opposing shelves and subsequently many of these cells undergo programmed cell death. Death of medial cells in vitro is prevented by EGF and by the retinoids, either with or without EGF. This response occurs in the absence of a mesenchymal interaction, suggesting that the medial cell response to EGF and retinoids is not mediated by or dependent on the mesenchymal tissues. The survival of medial cells may be responsible for the failure of opposing shelves to fuse.     

Antisense oligonucleotides to crabp I and II alter the expression of TGF-β3, RAR-β, and tenascin in primary cultures of embryonic palate cells

Summary The cellular retinoic acid-binding proteins (CRABPs) are thought to modulate the responsiveness of cells to retinoic acid (RA). We have previously shown that primary cultures of murine embryonic palate mesenchymal (MEPM) cells express both CRABP-I and CRABP-II genes and that this expression is regulated by RA and transforming growth factor β (TGF-β). These cells also express high levels of TGF-β3, which is also regulated by RA and TGF-β. We have used an antisense strategy to investigate the role of the CRABPs in retinoid-induced gene expression. Subconfluent cultures of MEPM cells were treated for several days with phosphorothioate modified 18-mer oligonucleotides antisense to CRABP-I or CRABP-II and then with all-trans-retinoic acid at a concentration of 3.3 μM or 0.33 μM for 5 or 22 h. Total RNA was then extracted and the expression of TGF-β3, retinoic acid receptor β (RAR-β), and tenascin was assessed by northern blot analysis. Antisense oligonucleotides to CRABP-I partially inhibited the RA-induced TGF-β3, RAR-β, and tenascin mRNA expression. The corresponding mis-sense oligonucleotides were without effect. Antisense oligonucleotides to CRABP-II also partially inhibited RA-induced expression of these genes. As with the CRABP-I antisense, mis-sense oligonucleotides to CRABP-II had no effect. These data suggest that both CRABPs modulate the responsiveness of MEPM cells to retinoic acid. Inhibition of endogenous CRABP expression renders MEPM cells less responsive to RA with respect to induction of TGF-β3, RAR-β, and tenascin gene expression. These results have important implications for our understanding of the role of the CRABPs in retinoid teratology.     

Physical Anthropology and the dental and medical specialties

The rise of two sub-specialties in Physical Anthropology traces back to the Anatomy Departments of Schools of Medicine in Germany and France during the nineteenth century. The study of human diversity in bones and bodies was largely by medically-trained anatomists. There developed Medical Anthropology and Dental Anthropology, employing osteometry and craniometry on the skeleton, somatometry and cephalometry on the living body. As a result cross-sectional studies gave way to longitudinal studies and X-ray techniques were added to purely mensurational procedures. In Medical Anthropology the specialties most directly concerned are pediatrics, plastic surgery, endocrinology, and orthopaedics. In Dental Anthropology the specialties most directly concerned are pedodontics, orthodontics, oral surgery, and prosthodontics. The contributions of Physical Anthropology to each is discussed.     

Mapping of the chicken cleft primary palate mutation on chromosome 11 and sequencing of the 4.9 Mb linked region

An embryonic lethal mutation in chicken named cleft primary palate (cpp) is inherited in an autosomal recessive mode and results in a severely truncated upper beak. In this study, genotyping and sequencing techniques were employed to advance our genetic and genomic knowledge of the mutation’s chromosomal location, candidate region and possible causative element using a congenic inbred line. Herein, the candidate region for the cpp developmental mutation was established as a ca. 5.1 Mb region of chicken chromosome 11 (GGA 11) through the use of a 600K Affymetrix SNP array. The SNPs identified from this array linked to cpp were used to genotype individuals from the congenic inbred line over several generations and thereby fine-map the causative region resulting in an approximately 200 kb size reduction. This candidate region (4.9 Mb) was sequenced via capture array in a cohort of 24 individuals, including carriers, mutants and their wild type (wt) siblings. Interestingly, the GGA 11 region for cpp encompasses the predicted centromere location and is thus unlikely to be highly disrupted by further recombination. Here we report on the variation unique to the cpp mutation, i.e. single-nucleotide variants and insertions or deletions. Although the candidate region contains several genes of interest with regard to the cpp phenotype, only one cpp-linked variant was predicted to have a significant physiological effect by causing a frameshift mutation in ESRP2, which has a role in tissue-specific splicing during development.     

Medial epithelial seam cell migration during palatal fusion

The mammalian secondary palate forms from two shelves of mesenchyme sheathed in a single-layered epithelium. These shelves meet during embryogenesis to form the midline epithelial seam (MES). Failure of MES degradation prevents mesenchymal confluence and results in a cleft palate. Previous studies indicated that MES cells undergo features of epithelial-to-mesenchymal transition (EMT) and may become migratory as part of the fusion mechanism. To detect MES cell movement over the course of fusion, we imaged the midline of fusing embryonic ephrin-B2/GFP mouse palates in real time using two-photon microscopy. These mice express an ephrin-B2-driven green fluorescent protein (GFP) that labels the palatal epithelium nuclei and persists in those cells through the time window necessary for fusion. We observed collective migration of MES cells toward the oral surface of the palatal shelf over 48 hr of imaging, and we confirmed histologically that the imaged palates had fused by the end of the imaged period. We previously reported that ephrin reverse signaling in the MES is required for palatal fusion. We therefore added recombinant EphA4/Fc protein to block this signaling in imaged palates. The blockage inhibited fusion, as expected, but did not change the observed migration of GFP-labeled cells. Thus, we uncoupled migration and fusion. Our data reveal that palatal MES cells undergo a collective, unidirectional movement during palatal fusion and that ephrin reverse signaling, though required for fusion, controls aspects of the fusion mechanism independent of migration.     

Retinoic acid alters the proliferation and survival of the epithelium and mesenchyme and suppresses Wnt/β-catenin signaling in developing cleft palate

Retinoic acid (RA) contributes to cleft palate; however, the cellular and molecular mechanisms responsible for the deleterious effects on the developing palate are unclear. Wnt signaling is a candidate pathway in the cleft palate and is associated with RA in organ development; thus, we aim to investigate whether RA-induced cleft palate also results from altered Wnt signaling. Administration of RA to mice altered cell proliferation and apoptosis in craniofacial tissues by regulating molecules controlling cell cycle and p38 MAPK signaling, respectively. This altered cell fate by RA is a crucial mechanism contributing to 100% incidence of cleft palate. Moreover, Wnt/β-catenin signaling was completely inhibited by RA in the early developing palate via its binding and activation with RA receptor (RAR) and is responsible for RA-induced cleft palate. Furthermore, PI3K/Akt signaling was also involved in actions of RA. Our findings help in elucidating the mechanisms of RA-induced cleft palate.     

Molecular control of secondary palate development

Compared with the embryonic development of other organs, development of the secondary palate is seemingly simple. However, each step of palatogenesis, from initiation until completion, is subject to a tight molecular control that is governed by epithelial-mesenchymal interactions. The importance of a rigorous molecular regulation of palatogenesis is reflected when loss of function of a single protein generates cleft palate, a frequent malformation with a complex etiology. Genetic studies in humans and targeted mutations in mice have identified numerous factors that play key roles during palatogenesis. This review highlights the current understanding of the molecular and cellular mechanisms involved in normal and abnormal palate development with special respect to recent advances derived from studies of mouse models.