Analysis of the presence of cell proliferation-related molecules in the Tgf-ß3 null mutant mouse palate reveals misexpression of EGF and Msx-1.

The Tgf-ß(3) null mutant mouse palate presents several cellular anomalies that lead to the appearance of cleft palate. One of them concerns the cell proliferation of both the palatal medial edge epithelium and mesenchyme. In this work, our aim was to determine whether there was any variation in the presence/distribution of several cell proliferation-related molecules that could be responsible for the cell proliferation defects observed in these palates. Our results showed no difference in the presence of EGF-R, PDGF-A, TGF-ß(2), Bmp-2, and Bmp-4, and differences were minimal for FGF-10 and Shh. However, the expression of EGF and Msx-1 changed substantially. The shift of the EGF protein expression was the one that most correlated with that of cell proliferation. This molecule is regulated by TGF-ß(3), and experiments blocking its activity in culture suggest that EGF misexpression in the Tgf-ß(3) null mutant mouse palate plays a role in the cell proliferation defect observed.

Chondroitin sulphate-mediated fusion of brain neural folds in rat embryos.

Previous studies have demonstrated that during neural fold fusion in different species, an apical extracellular material rich in glycoconjugates is involved. However, the composition and the biological role of this material remain undetermined. In this paper, we show that this extracellular matrix in rat increases notably prior to contact between the neural folds, suggesting the dynamic behaviour of the secretory process. Immunostaining has allowed us to demonstrate that this extracellular matrix contains chondroitin sulphate proteoglycan (CSPG), with a spatio-temporal distribution pattern, suggesting a direct relationship with the process of adhesion. The degree of CSPG involvement in cephalic neural fold fusion in rat embryos was determined by treatment with specific glycosidases.In vitro rat embryo culture and microinjection techniques were employed to carry out selective digestion, with chondroitinase AC, of the CSPG on the apical surface of the neural folds; this was done immediately prior to the bonding of the cephalic neural folds. In all the treated embryos, cephalic defects of neural fold fusion could be detected. These results show that CSPG plays an important role in the fusion of the cephalic neural folds in rat embryos, which implies that this proteoglycan could be involved in cellular recognition and adhesion.

Effect of butyl benzyl phthalate on early postnatal mortality in rats.

ABSTRACT The present study was undertaken to examine early postnatal mortality in rat pups following exposure to butyl benzyl phthalate (BBP) during pregnancy. Seventeen pregnant rats were given 750 mg/kg bw/day of BBP by oral gavage on gestation days 13, 14, and 15, and the volume of each dose was adjusted to 5 ml/kg body weight. Four rats were given olive oil only and served as control. Natural birth was allowed to take place. One hundred and eighty-three pups were born to the experimental rats and 46 pups to the control group. Close observation of the newborn pups during the first 3 h of life revealed that all the pups in both the control and experimental groups were born alive. Only six pups from the experimental group (3.2%) died within this time period. These and four control pups were fixed and decalcified. Histological examination of the thoracic cavity of the newborn rats in both groups revealed no differences in the position or size of any of the heart chambers, ductus arteriosus, or great vessels. However, the lungs of the six experimental pups that died showed athelectasia and bronchi dilatation. The results therefore suggest that exposure to BBP of rats during pregnancy does not produce significant postnatal mortality in their offspring.

TGF-beta(3)-induced chondroitin sulphate proteoglycan mediates palatal shelf adhesion.

In mammals, the adhesion and fusion of the palatal shelves are essential mechanisms in the development of the secondary palate. Failure of any of these processes leads to the formation of cleft palate. The mechanisms underlying palatal shelf adhesion are poorly understood, although the presence of filopodia on the apical surfaces of the superficial medial edge epithelial (MEE) cells seems to play an important role in the adhesion of the opposing MEE. We demonstrate here the appearance of chondroitin sulphate proteoglycan (CSPG) on the apical surface of MEE cells only immediately prior to contact between the palatal shelves. This apical CSPG has a functional role in palatal shelf adhesion, as either the alteration of CSPG synthesis by beta-D-Xyloside or its specific digestion by chondroitinase AC strikingly alters the in vitro adhesion of palatal shelves. We also demonstrate the absence of this apical CSPG in the clefted palates of transforming growth factor beta 3 (TGF-beta(3)) null mutant mice, and its induction, together with palatal shelf adhesion, when TGF-beta(3) is added to TGF-beta(3) null mutant palatal shelves in culture. When chick palatal shelves (that do not adherein vivo nor express TGF-beta(3), nor CSPG in the MEE) are cultured in vitro, they do not express CSPG and partially adhere, but when TGF-beta(3) is added to the media, they express CSPG and their adhesion increases strikingly. We therefore conclude that the expression of CSPG on the apical surface of MEE cells is a key factor in palatal shelf adhesion and that this expression is regulated by TGF-beta(3).

Basic sciences education in the dental curriculum in Southern Europe.

A survey was conducted to determine the current status of the basic sciences education in Southern European dental schools. Responses were collected from schools in Malta, Greece, Portugal, Italy, France and Spain. The results show that there is some uniformity across Southern Europe, even if there are some variations among dental schools both within one country and among the different countries. The links with Medicine seem to be strong. Most basic sciences dental educators have a medical degree and usually have their main appointment in a medical school. The only exception to this is found in France, where the faculty, who are mostly dentists, have their main appointment in a dental school. In half of the countries, courses are given jointly to both dental and medical students. There is, in general, poor coordination between the basic science subjects and other subjects in the dental curriculum. All the surveyed schools maintain traditional curricula and teaching methodologies. However, there is an increased movement towards self-directed learning, computer-assisted learning and improved coordination with clinical subjects.

Chondroitin sulphate proteoglycan is involved in lens vesicle morphogenesis in chick embryos.

Proteoglycans have been implicated in the invagination and formation of various embryonal cavitied primordia. In this paper the expression of chondroitin sulphate proteoglycan (CSPG) is analysed in the lens primordium during lens vesicle formation, and demonstrate that this proteoglycan has a specific distribution pattern with regard to invagination and fusion processes in the transformation of placode into lens vesicle. More specifically, CSPG was detected in: (1) the apical surface of lens epithelial cells, where early CSPG expression was observed in the whole of the lens placode whilst in the vesicle phase it was restricted to the posterior epithelium; (2) intense CSPG expression in the basal lamina, which remained constant for the entire period under study; (3) CSPG expression in the intercellular spaces of the lens primordium epithelium, which increased during the invagination of the primordium and which at the vesicle stage was more evident in the posterior epithelium; and (4) CSPG expression on the edges of the lens placode both prior to and during fusion. Treatment with beta- D -xyloside causes significant CSPG depletion in the lens primordium together with severe alterations in the invagination and fusion of the lens vesicle; this leads to the formation of lens primordia which in some cases remain practically flat or show partial invagination defects or fusion disruption. Similar results were obtained by enzyme digestion with chondroitinase AC but not with type II heparinase, which indicates that alterations induced by beta- D -xyloside were due to interference in CSPG synthesis. The findings demonstrate that CSPG is a common component of the lens primordium at the earliest developmental stages during which it undergoes specific modifications. It also includes experimental evidence to show that 'in vivo' CSPG plays an important role in the invagination and fusion processes of the lens primordium.

Bulging medial edge epithelial cells and palatal fusion.

The surface of the medial edge epithelium of embryonic day 12, 13 and 14 mouse palatal shelves was observed utilising Environmental Scanning Electron Microscopy (ESEM). This technique offers the advantage of visualisation of biological samples after short fixation times in their natural hydrated state. Bulging epithelial cells were observed consistently on the medial edge epithelium prior to palatal shelf fusion. Additionally, we have used ESEM to compare the morphology and surface features of palatal shelves from embryonic day 13 to 16 mouse embryos that are homozygous null (TGF-beta3 -/-), heterozygous (TGF-beta3 +/-) or homozygous normal (TGF-beta3 +/+) for transforming growth factor beta-3 (TGF-beta3). At embryonic day 15 and 16 most TGF-beta3 +/- and +/+ embryos showed total palatal fusion, whilst all TGF-beta3 null mutants had cleft palate: the middle third of the palatal shelves had adhered, leaving an anterior and posterior cleft. From embryonic day 14 to 16 abundant cells were observed bulging on the medial edge epithelial surface of palates from the TGF-beta3 +/- and +/+ embryos. However, they were never seen in the TGF-beta3 null embryos, suggesting that these surface bulges might be important in palatal fusion and that their normal differentiation is induced by TGF-beta3. The expression pattern of E-Cadherin, beta-catenin, chondroitin sulphate proteoglycan, beta-Actin and vinculin as assayed by immunocytochemistry in these cells shows specific variations that suggest their importance in palatal shelf adhesion.

Medial edge epithelial cell fate during palatal fusion.

To explain the disappearance of medial edge epithelial (MEE) cells during palatal fusion, programmed cell death, epithelial-mesenchymal transformation, and migration of these cells to the oral and nasal epithelia have been proposed. However, MEE cell death has not always been accepted as a mechanism involved in midline epithelial seam disappearance. Similarly, labeling of MEE cells with vital lipophilic markers has not led to a clear conclusion as to whether MEE cells migrate, transform into mesenchyme, or both. To clarify these controversies, we first utilized TUNEL techniques to detect apoptosis in mouse palates at the fusion stage and concomitantly analyzed the presence of macrophages by immunochemistry and confocal microscopy. Second, we in vitro infected the MEE with the replication-defective helper-free retroviral vector CXL, which carries the Escherichia coli lacZ gene, and analyzed beta-galactosidase activity in cells after fusion to follow their fate. Our results demonstrate that MEE cells die and transform into mesenchyme during palatal fusion and that dead cells are phagocytosed by macrophages. In addition, we have investigated the effects of the absence of transforming growth factor beta(3) (TGF-beta(3)) during palatal fusion. Using environmental scanning electron microscopy and TUNEL labeling we compared the MEE of the clefted TGF-beta(3) null and wild-type mice. We show that MEE cell death in TGF-beta(3) null palates is greatly reduced at the time of fusion, revealing that TGF-beta(3) has an important role as an inducer of apoptosis during palatal fusion. Likewise, the bulging cells observed on the MEE surface of wild-type mice prior to palatal shelf contact are very rare in the TGF-beta(3) null mutants. We hypothesize that these protruding cells are critical for palatal adhesion, being morphological evidence of increased cell motility/migration.