ABSTRACT
Retinoic acid (RA), a metabolite of vitamin A, plays a key role in a variety of biological processes and is essential for normal embryonic development. On the other hand, exogenous RA could cause cleft palate in offspring when it is given to pregnant animals at either the early or late phases of palatogenesis, but the pathogenetic mechanism of cleft palate caused by excess RA remains not fully elucidated. The aim of the present study was to investigate the effects of excess of RA on early palatogenesis in mouse fetuses and analyze the teratogenic mechanism, especially at the stage prior to palatal shelf elevation. We gave all-trans RA (100 mg/kg) orally to E11.5 ICR pregnant mice and observed the changes occurring in the palatal shelves of their fetuses. It was found that apoptotic cell death increased not only in the epithelium of the palatal shelves but also in the tongue primordium, which might affect tongue withdrawal movement during palatogenesis and impair the horizontal elevation of palatal shelves. In addition, RA was found to prevent the G(1)/S progression of palatal mesenchymal cells through upregulation of p21(Cip1), leading to Rb hypophospholylation. Thus, RA appears to cause G(1) arrest in palatal mesenchymal cells in a similar manner as in various cancer and embryonic cells. It is likely that apoptotic cell death and cell cycle disruption are involved in cleft palate formation induced by RA.
Subject(s)
Apoptosis/drug effects , Cleft Palate/pathology , G1 Phase/drug effects , S Phase/drug effects , Tretinoin/toxicity , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/toxicity , Blotting, Western , Cell Cycle Proteins/metabolism , Cleft Palate/chemically induced , Cleft Palate/ultrastructure , Embryo, Mammalian/abnormalities , Embryo, Mammalian/metabolism , Embryo, Mammalian/ultrastructure , Epithelial Cells/drug effects , Epithelial Cells/pathology , Epithelial Cells/ultrastructure , Female , Fetal Development/drug effects , In Situ Nick-End Labeling , Intubation, Gastrointestinal , Jaw/drug effects , Jaw/physiopathology , Male , Maxilla/abnormalities , Maxilla/ultrastructure , Mice , Mice, Inbred ICR , Microscopy, Electron, Scanning , Movement/drug effects , Palate/abnormalities , Palate/ultrastructure , Pregnancy , Tongue/abnormalities , Tongue/drug effects , Tretinoin/administration & dosageABSTRACT
Mouse ENU mutagenesis programmes have yielded a series of independent mutations on proximal chromosome 4 leading to dominant head-bobbing and circling behaviour due to truncations of the lateral semicircular canal of the inner ear. Here, we report the identification of mutations in the Chd7 gene in nine of these mutant alleles including six nonsense and three splice site mutations. The human CHD7 gene is known to be involved in CHARGE syndrome, which also shows inner ear malformations and a variety of other features with varying penetrance and appears to be due to frequent de novo mutation. We found widespread expression of Chd7 in early development of the mouse in organs affected in CHARGE syndrome including eye, olfactory epithelium, inner ear and vascular system. Closer inspection of heterozygous mutant mice revealed a range of defects with reduced penetrance, such as cleft palate, choanal atresia, septal defects of the heart, haemorrhages, prenatal death, vulva and clitoral defects and keratoconjunctivitis sicca. Many of these defects mimic the features of CHARGE syndrome. There were no obvious features of the gene that might make it more mutable than other genes. We conclude that the large number of mouse mutants and human de novo mutations may be due to the combination of the Chd7 gene being a large target and the fact that many heterozygous carriers of the mutations are viable individuals with a readily detectable phenotype.
Subject(s)
DNA Helicases/genetics , DNA-Binding Proteins/genetics , Ear, Inner/abnormalities , Mutation , Animals , Cardiovascular Abnormalities/genetics , Choanal Atresia/genetics , Choanal Atresia/ultrastructure , Cleft Palate/genetics , Cleft Palate/ultrastructure , Disease Models, Animal , Ear, Inner/embryology , Eye Abnormalities/genetics , Genitalia/abnormalities , Humans , Mice , Mice, Mutant Strains , SyndromeABSTRACT
Palatogenesis is a complex developmental processthat requires two main events: elevation andthen fusion of the palatal shelves. These processesare disrupted by teratogens such as retinoicacid (RA) and genetic defects, resulting in variousmalformations (including cleft palate). Usinghistological and immunohistochemical techniques,the effects of different isomers of RA, administeredin various concentrations to pregnantrats on different gestational days (GD), were assessedfrom observations of the state of palataldevelopment on GD 18 in foetuses without exencephaly.Varying degrees of clefting of thepalate were observed, from failure of elevationof the palatal shelves to failure of fusion in themidline. This study shows that all-trans-RA isthe most teratogenic RA isomer in terms of ratpalatal abnormalities. It also supports previousfindings that the timing of administration of alltrans-RA is more critical than the concentration,with treatment between GD 10 and 10.5 havingthe most severe effects. Previous histologicalstudies also suggested that RA is associatedwith the appearance of ectopic cartilages withinthe developing palate of foetuses showing exencephaly.In this investigation, immunohistochemicallabelling of the foetal material with antibodiesthat recognise epitopes present in linkproteins 1, 2, and 3 (8A4), chondroitin-4-sulphatestubs (2B6), and G1 and chondroitin sulphateattachments (7D1) present in aggrecan (associatedwith hyaluronan in cartilage) showedno signs of ectopic cartilage formation within the palate at GD18. Internal controls of the cartilagesof the nasal septum, vomeronasal cartilage, andMerkels cartilage labelled intensely and appearedmorphologically normal (AU)
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