Concepción Martínez-Álvarez

Alteration of medial-edge epithelium cell adhesion in two Tgf-β3 null mouse strains

Although palatal shelf adhesion is a crucial event during palate development, little work has been carried out to determine which molecules are responsible for this process. Furthermore, whether altered palatal shelf adhesion causes the cleft palate presented by Tgf-β3 null mutant mice has not yet been clarified. Here, we study the presence/distribution of some extracellular matrix and cell adhesion molecules at the time of the contact of palatal shelves in both wild-type and Tgf-β3 null mutant palates of two strains of mice (C57/BL/6J (C57), and MF1) that develop cleft palates of different severity. We have performed immunohistochemistry with antibodies against collagens IV and IX, laminin, fibronectin, the α5- and β1-integrins, and ICAM-1; in situ hybridization with a Nectin-1 riboprobe; and palatal shelf cultures treated or untreated with TGF-β3 or neutralizing antibodies against fibronectin or the α5-integrin. Our results show the location of these molecules in the wild-type mouse medial edge epithelium (MEE) of both strains at the time of the contact of palatal shelves; the heavier (C57) and milder (MF1) alteration of their presence in the Tgf-β3 null mutants; the importance of TGF-β3 to restore their normal pattern of expression; and the crucial role of fibronectin and the α5-integrin in palatal shelf adhesion. We thus provide insight into the molecular bases of this important process and the cleft palate presented by Tgf-β3 null mutant mice.

Folic acid deficiency induces premature hearing loss through mechanisms involving cochlear oxidative stress and impairment of homocysteine metabolism

Nutritional imbalance is emerging as a causative factor of hearing loss. Epidemiologic studies have linked hearing loss to elevated plasma total homocysteine (tHcy) and folate deficiency, and have shown that folate supplementation lowers tHcy levels potentially ameliorating age‐related hearing loss. The purpose of this study was to address the impact of folate deficiency on hearing loss and to examine the underlying mechanisms. For this purpose, 2‐mo‐old C57BL/6J mice (Animalia Chordata Mus musculus) were randomly divided into 2 groups (n = 65 each) that were fed folate‐deficient (FD) or standard diets for 8 wk. HPLC analysis demonstrated a 7‐fold decline in serum folate and a 3‐fold increase in tHcy levels. FD mice exhibited severe hearing loss measured by auditory brainstem recordings and TUNEL‐positive‐apoptotic cochlear cells. RT‐quantitative PCR and Western blotting showed reduced levels of enzymes catalyzing homocysteine (Hcy) production and recycling, together with a 30% increase in protein homocysteinylation. Redox stress was demonstrated by decreased expression of catalase, glutathione peroxidase 4, and glutathione synthetase genes, increased levels of manganese superoxide dismutase, and NADPH oxidase‐complex adaptor cytochrome b‐245, α‐polypeptide (p22phox) proteins, and elevated concentrations of glutathione species. Altogether, our findings demonstrate, for the first time, that the relationship between hyperhomocysteinemia induced by folate deficiency and premature hearing loss involves impairment of cochlear Hcy metabolism and associated oxidative stress.—Martínez‐Vega, R., Garrido, F., Partearroyo, T., Cediel, R., Zeisel, S. H., Martínez‐Álvarez, C., Varela‐Moreiras, G., Varela‐Nieto, I., and Pajares, M. A. Folic acid deficiency induces premature hearing loss through mechanisms involving cochlear oxidative stress and impairment of homocysteine metabolism. FASEB J. 29, 418‐432 (2015). www.fasebj.org

Interactions between TGF-β1 and TGF-β3 and their role in medial edge epithelium cell death and palatal fusion in vitro

In recent decades, studies have shown that both TGF-beta(1) and TGF-beta(3) play an important role in the induction of medial edge epithelium (MEE) cell death and palatal fusion. Many of these experiments involved the addition or blockage of one of these growth factors in wild-type (WT) mouse palate cultures, where both TGF-beta(1) and TGF-beta(3) are present. Few studies have addressed the existence of interactions between TGF-beta(1) and TGF-beta(3), which could modify their individual roles in MEE cell death during palatal fusion. We carried out several experiments to test this possibility, and to investigate how this could influence TGF-beta(1) and TGF-beta(3) actions on MEE cell death and palatal shelf fusion. We double-immunolabelled developing mouse palates with anti-TGF-beta(1) or anti-TGF-beta(3) antibodies and TUNEL, added rhTGF-beta(1) or rhTGF-beta(3) or blocked the TGF-beta(1) and TGF-beta(3) action at different concentrations to WT or Tgf-beta(3) null mutant palate cultures, performed in situ hybridizations with Tgf-beta(1) or Tgf-beta(3) riboprobes, and measured the presence of TUNEL-positive midline epithelial seam (MES) cells and MES disappearance (palatal shelf fusion) in the different in vitro conditions. By combining all these experiments, we demonstrate great interaction between TGF-beta(1) and TGF-beta(3) in the developing palate and confirm that TGF-beta(3) has a more active role in MES cell death than TGF-beta(1), although both are major inductors of MES disappearance. Finally, the co-localization of TGF-beta(1), but not TGF-beta(3), with TUNEL in the MES allows us to suggest a possible role for TGF-beta(1) in MES apoptotic clearance.

Maternal folic acid–deficient diet causes congenital malformations in the mouse eye

BACKGROUND The eye is a very complex structure derived from the neural tube, surface ectoderm, and migratory mesenchyme from a neural crest origin. Because structures that evolve from the neural tube may be affected by a folate/folic acid (FA) deficiency, the aim of this work was to investigate whether a maternal folic acid-deficient diet may cause developmental alterations in the mouse eye. METHODS Female C57BL/6J mice (8 weeks old) were assigned into two different folic acid groups for periods ranging between 2 and 16 weeks. Animals were killed at gestation day 17. Hepatic folate was analyzed, and the eyes from 287 fetuses were macroscopically studied, sectioned and immunolabeled with anti-transforming growth factor (TGF)-β2 and anti-TGF-βRII. RESULTS Mice exposed to a FA-deficient diet exhibited numerous eye macroscopic anomalies, such as anophthalmia and microphthalmia. Microscopically, the eye was the most affected organ (43.7% of the fetuses). The highest incidence of malformations occurred from the 8th week onward. A statistically significant linear association between the number of maternal weeks on the FA-deficient diet and embryonic microscopic eye malformations was observed. The optic cup derivatives and structures forming the eye anterior segment showed severe abnormalities. In addition, TGF-β2 and TGF-βRII expression in the eye was also altered. CONCLUSION This study suggests that an adequate folic acid/folate status plays a key role in the formation of ocular tissues and structures, whereas a vitamin deficiency is negatively associated with a normal eye development even after a short-term exposure.

Occurrence of Cleft-Palate and Alteration of Tgf-β3 Expression and the Mechanisms Leading to Palatal Fusion in Mice following Dietary Folic-Acid Deficiency

Folic acid (FA) is essential for numerous bodily functions. Its decrease during pregnancy has been associated with an increased risk of congenital malformations in the progeny. The relationship between FA deficiency and the appearance of cleft palate (CP) is controversial, and little information exists on a possible effect of FA on palate development. We investigated the effect of a 2–8 weeks’ induced FA deficiency in female mice on the development of CP in their progeny as well as the mechanisms leading to palatal fusion, i.e. cell proliferation, cell death, and palatal-shelf adhesion and fusion. We showed that an 8 weeks’ maternal FA deficiency caused complete CP in the fetuses although a 2 weeks’ maternal FA deficiency was enough to alter all the mechanisms analyzed. Since transforming growth factor-β3 (TGF-β3) is crucial for palatal fusion and since most of the mechanisms impaired by FA deficiency were also observed in the palates of Tgf-β3null mutant mice, we investigated the presence of TGF-β3 mRNA, its protein and phospho-SMAD2 in FA-deficient (FAD) mouse palates. Our results evidenced a large reduction in Tgf-β3 expression in palates of embryos of dams fed an FAD diet for 8 weeks; Tgf-β3 expression was less reduced in palates of embryos of dams fed an FAD diet for 2 weeks. Addition of TGF-β3 to palatal-shelf cultures of embryos of dams fed an FAD diet for 2 weeks normalized all the altered mechanisms. Thus, an insufficient folate status may be a risk factor for the development of CP in mice, and exogenous TGF-β3 compensates this deficit in vitro.

Osteoinduction in the palatal submucosa by injecting BMP-2 on 2 different carriers.

AbstractIn this work, we investigated the ability of injected recombinant human bone morphogenetic protein 2 (rhBMP-2) on brushite cement (a &bgr;-tricalcium phosphate–based biomaterial) and collagen gel as carriers to induce osteogenic differentiation in the palatal submucosa of 10-day-old rats. This was part of a broader study aiming to create bone in the palatal submucosa at cleft palate edges in the search for a minimally invasive treatment. Thirteen treated animals, 7 with rhBMP-2/brushite cement and 6 with rhBMP-2/collagen gel, were injected with 5 to 10 &mgr;L of each biomaterial in the right palatal submucosa at the level between the second and third rugae. The contralateral site was uninjected and served as the control. Six weeks after injection, both brushite cement and collagen gel were histologically unrecognizable in all treated animals. New bone structures such as ossicles of woven bone were not detected. However, an augmentation in the thickness of the palatal fibromucosa was observed at the injection site of all palates. In addition, immunolabeling for osteopontin, proliferating cell nuclear antigen, and TUNEL revealed intense osteogenic induction at the injection site with both constructs, which was negative in the control site from the same specimens; no differences regarding cell proliferation and death were observed. The present study confirms the feasibility of generating osteogenic cells in the palatal submucosa by injecting low doses of rhBMP-2 in these 2 biomaterials, together with their inability to form bone.

Analysis of the Presence of Cell Proliferation-Related Molecules in the Tgf-β3 Null Mutant Mouse Palate Reveals Misexpression of EGF and Msx-1

The Tgf-β3 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-β2, 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-β3, and experiments blocking its activity in culture suggest that EGF misexpression in the Tgf-β3 null mutant mouse palate plays a role in the cell proliferation defect observed.

Epidermal growth factor impairs palatal shelf adhesion and fusion in the Tgf-β 3 null mutant.

The cleft palate presented by transforming growth factor-β3 (Tgf-β3) null mutant mice is caused by altered palatal shelf adhesion, cell proliferation, epithelial-to-mesenchymal transformation and cell death. The expression of epidermal growth factor (EGF), transforming growth factor-β1 (Tgf-β1) and muscle segment homeobox-1 (Msx-1) is modified in the palates of these knockout mice, and the cell proliferation defect is caused by the change in EGF expression. In this study, we aimed to determine whether this change in EGF expression has any effect on the other mechanisms altered in Tgf-β3 knockout mouse palates. We tested the effect of inhibiting EGF activity in vitro in the knockout palates via the addition of Tyrphostin AG 1478. We also investigated possible interactions between EGF, Tgf-β1 and Msx-1 in Tgf-β3 null mouse palate cultures. The results show that the inhibition of EGF activity in Tgf-β3 null mouse palate cultures improves palatal shelf adhesion and fusion, with a particular effect on cell death, and restores the normal distribution pattern of Msx-1 in the palatal mesenchyme. Inhibition of TGF-β1 does not affect either EGF or Msx-1 expression.

Tongue Abnormalities Are Associated to a Maternal Folic Acid Deficient Diet in Mice

It is widely accepted that maternal folic acid (FA) deficiency during pregnancy is a risk factor for abnormal development. The tongue, with multiple genes working together in a coordinated cascade in time and place, has emerged as a target organ for testing the effect of FA during development. A FA-deficient (FAD) diet was administered to eight-week-old C57/BL/6J mouse females for 2–16 weeks. Pregnant dams were sacrificed at gestational day 17 (E17). The tongues and heads of 15 control and 210 experimental fetuses were studied. In the tongues, the maximum width, base width, height and area were compared with width, height and area of the head. All measurements decreased from 10% to 38% with increasing number of weeks on maternal FAD diet. Decreased head and tongue areas showed a harmonic reduction (Spearman nonparametric correlation, Rho = 0.802) with respect to weeks on a maternal FAD diet. Tongue congenital abnormalities showed a 10.9% prevalence, divided in aglossia (3.3%) and microglossia (7.6%), always accompanied by agnathia (5.6%) or micrognathia (5.2%). This is the first time that tongue alterations have been related experimentally to maternal FAD diet in mice. We propose that the tongue should be included in the list of FA-sensitive birth defect organs due to its relevance in several key food and nutrition processes.

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.