Experimental induction of palate shelf elevation in glutamate decarboxylase 67-deficient mice with cleft palate due to vertically oriented palatal shelf

BACKGROUND: Gamma-aminobutyric acid is an inhibitory neurotransmitter, synthesized by two isoforms of glutamate decarboxylase (GAD), GAD65 and -67. Unexpectedly, inactivation of GAD67 induces cleft palate in mice. Reduction of spontaneous tongue movement resulting from decreased motor nerve activity has been related to the development of cleft palate in GAD67(-/-) fetuses. In the present study, development of cleft palate was examined histologically and manipulated with culture of the maxilla and partial resection of fetal tongue. METHODS: GAD67(-/-) mice and their littermates were used. Histological examination and immunohistochemistry were performed conventionally. Organ culture of the maxilla was carried out as reported previously. Fetuses were maintained alive under anesthesia and tips of their tongues were resected. RESULTS: Elevation of palatal shelves, the second step of palate formation, was not observed in GAD67(-/-) mice. In wild-type mice, GAD67 and gamma-aminobutyric acid were not expressed in the palatal shelves, except in the medial edge epithelium. During 2 days of culture of maxillae dissected from E13.5-E14.0 GAD67(-/-) fetuses, elevation and fusion of the palatal shelves were induced. When E13.5-15.5 mutant fetuses underwent partial tongue resection, the palatal shelves became elevated within 30 min. CONCLUSIONS: These results suggest that the potential for palate formation is maintained in the palatal shelves of GAD67(-/-) fetuses, but it is obstructed by other, probably neural, factors, resulting in cleft palate.     

Ultrastructural evidence of contractile systems in mouse palates prior to rotation.

Previous studies have shown that the palatal shelves of mouse embryos synthesize the contractile proteins actin and myosin at a rate equal to that of tongue just prior to shelf movement (day 14.5). The purpose of this study was to examine the morphology of the palatal shelves for evidence of a contractile system. Myosin ATPase histochemistry was performed on frozen sections of day-14.5 fetal mouse heads. Three areas of the palatal shelves gave a positive reaction: 1) A reaction product typical of skeletal muscle on the oral side of the posterior palate (region 1); 2) a “heavy-diffuse” reaction product on the tongue side extending from the top mid-palate to the posterior end (region 2); and 3) a “light-diffuse” reaction product along the oral epithelium in the mid-palate (region 3). Electron microscopy of excised day-14.5 palates was carried out after fixation in glutaraldehyde or an acrolein-dichromate solution. Region 1 contained a large area of developing and adultlike skeletal muscle. In the area of region 2 a large population of filamentous-rich mesenchymal cells was observed. In addition, large neurons coursing through both contractile systems were noted. Preliminary observations in region 3 indicated the possibility of a primitive (nonmuscle) contractile system in that area. The contractile and nervous systems in the palate, prior to rotation, indicate the possibility that an innervated embryonic muscle system may provide the “intrinsic shelf force” to rotate the shelves.     

Synthesis and distribution of collagen in the rat palate during shelf elevation.

Previous studies have shown that drugs which block the crosslinking of collagen prevent formation of the secondary palate by inhibiting shelf elevation. In this paper, the authors establish that collagen synthesis increases significantly just prior to shelf elevation and that type I collagen is synthesized throughout this developmental period (gestational Days 14–16 in the rat embryo) by isolated shelves in vitro. The largest accumulation of collagen fibers, predominantly oriented in a rostral-caudal plane, was observed adjacent to the basement membrane of the oral palatal epithelium. The unique location and orientation of these collagen fibers suggest that they play a structural role in the elevation of the palate.     

Development of the fetal mouse palate in suspension organ culture

Explanted palates of day 12 and day 13 mouse fetuses were cultured in a chemically defined serumless medium for 48-72 h by a suspension culture technique. The palate of day 12 fetuses closed successfully within 72 h and that of day 13 fetuses within 48 h. Both macroscopically and histologically, the in vitro fusion of palatal shelves simulated the palatogenetic process in vivo. This novel technique for culturing the fetal mouse palate may be of potential use for the study of palatogenesis and in developmental toxicology.     

Presence of the major progesterone-induced proteins of the sheep endometrium in fetal fluids

Allantoic and amniotic fluids were collected on Days 60 (n = 3), 100 (n = 4), and 140 (n = 3) of pregnancy. The presence of uterine milk proteins (UTM-proteins) in these samples was evaluated by Ouchterlony immunodiffusion and enzyme-linked immunoabsorbant assay (ELISA). Eight of ten samples of allantoic fluid and three of ten samples of amniotic fluid produced one or two immunoprecipitin bands against antiserum to UTM-proteins. Each band fused with immunoprecipitin bands from UTM-proteins purified from uterine fluid. Data from a semi-quantitative ELISA indicated that allantoic fluid from all ewes and amniotic fluid from six of ten ewes contained immunoreactive UTM-proteins. Concentrations of UTM-proteins in these fluids were not statistically affected by day of gestation (p greater than 0.10), but tended to decline as gestation advanced. Greater concentrations of UTM-proteins were detected in allantoic fluid than in amniotic fluid (p less than 0.05). The physical characteristics of the immunoreactive material in allantoic and amniotic fluids were examined by polyacrylamide gel electrophoresis and Western blotting. The immunoreactive material was found to possess pIs and molecular weights identical to UTM-proteins. These results indicate that fetal fluids contain material that reacts with antiserum to UTM-proteins and has physical properties similar to UTM-proteins. It is likely, therefore, that the UTM-proteins are transported across the placenta during gestation, perhaps to serve some function in the fetal compartment.     

Synthesis of shock proteins in cultured fetal mouse myocardial cells

We examined the synthesis of shock proteins in cultured fetal mouse myocytes. The preparation is free from fibroblasts, and the cells are vital and morphologically intact with respect to beat frequency and electron microscopy. Cultured myocytes from fetal mouse heart respond to heat shock and cadmium chloride, H2O2, allylamine, cyclosporine, and azathioprine exposure with the synthesis of shock proteins. Heat shock induces the de novo synthesis of two proteins of 71 and 68 kDa; cadmium chloride induces, in addition, a protein of 30 kDa. The other substances tested provoke the synthesis only of the 30-kDa polypeptide. The formation of heat shock proteins is concentration-dependent: Cyclosporine provokes the de novo synthesis of the 30-kDa polypeptide at concentrations above 10 ng/ml, whereas azathioprine causes the same effect at concentrations above 50 micrograms/ml. Hence cyclosporine might be cardiotoxic already at concentrations below the pharmacological dosages while azathioprine influences the myocytes only at concentrations much higher than the therapeutic level. Our results indicate that heat shock protein expression in cultured myocytes may be a useful tool to monitor cardiotoxicity.     

Influence of H-2-linked genes on glucocorticoid receptors in the fetal mouse palate

The binding of ³H-dexamethasone to cytosolic receptors in fetal jaws and in cytosols and nuclei of primary cell cultures of fetal palates was studied in various congenic strains of mice. The amount of specific binding was greater in palatal tissues from B10.A and BlO.A(2R) mice than in B10 or B10.A(5R) preparations. These differences were not observed in the liver. Since the strains with higher levels of glucocorticoid receptor are known to be more susceptible to cortisone-induced cleft palate than the strains with low receptor levels, it is suggested that quantitative variation in receptor levels may be involved in determining H-2-linked differences in cleft-palate susceptibility. Whether or not this is the case, it appears that an H-2-linked gene affects the quantity of a cytosolic glucocorticoid-binding protein which translocates to the nucleus.     

Presence of cdc2(+)-like proteins in the preimplantation mouse embryo

An antibody raised against a portion of the human equivalent of the yeast cdc2+ protein reacts with a 34K protein in mouse cell lines and early embryonic cells. Western blot analysis coupled with phosphatase treatment of material collected from the early preimplantation embryo has shown that the murine cdc2+ homologue does not correspond to the previously described newly synthesised proteins that are phosphorylated in a cell-cycle-dependent fashion [Howlett, 1986]. The cdc2(+)-like protein is converted into a slower migrating form on entry into S-phase and is further modified during G2 prior to mitosis. Studies of embryos that are held in extended periods of M-phase, i.e. unfertilised eggs or 1-cell embryos treated with nocodazole, demonstrate that the cdc2(+)-like protein becomes demodified in these cells.     

Retinoic acid induction of stress proteins in fetal mouse limb buds

Retinoic acid (RA) is teratogenic in rodent embryos. Several teratogens have been shown to induce the synthesis of a subset of heat shock proteins (stress proteins) in Drosophila. To determine if RA induces the synthesis of these proteins in rodent embryos, pregnant ICR mice were dosed with 100 mg/kg RA on Day 11 of gestation. Forelimb buds were removed from embryos 2.5 hr post-RA-treatment and nuclei were isolated, stained, and sorted from stages of the cell cycle. Nuclear proteins were extracted and analyzed by two-dimensional polyacrylamide gel electrophoresis. Nuclear proteins with molecular weights of approximately 84 and 25 kDa were synthesized in embryos in the G0 + G1 phase after pregnant dams were treated with RA. Isoelectric points, molecular weights, immunochemical blotting, and polypeptide mapping demonstrated that these proteins are indistinguishable from stress proteins isolated under a variety of conditions from rat submaxillary glands and mouse lymphoma cells. These results suggest that treatment with RA induces the synthesis of a subset of stress proteins; the role of these proteins in the teratogenic effects of RA is not known.     

Programmed cell death is not a necessary prerequisite for fusion of the fetal mouse palate

It has been widely accepted that programmed cell death (PCD) is an essential event in palatogenesis and that its failure can result in cleft palate, one of the most common birth defects in the human. However, some conflicting results have been reported concerning the timing of cell death occurring in the fusing palate and therefore the role of PCD in palatal fusion is controversial. In order to clarify whether cell death is indispensable for mammalian palatogenesis, we cultivated the palates of day-13 mouse fetuses in vitro and prevented cell death by treating them with the inhibitors of caspases-1 and -3 or with aurintricarboxylic acid which inhibits the activity of caspase-activated DNase. Even when cell death was almost completely inhibited, palatal fusion took place successfully. Histological examination revealed that in the absence of apoptotic cell death, the medial edge epithelia of opposing palatal shelves adhered to each other and subsequently, the midline epithelial seam was disrupted and disappeared to bring about mesenchymal confluence across the palate. It seems that cell death is not a necessary prerequisite for palatal fusion but it may help to efficiently eliminate unnecessary cells which failed to migrate or differentiate properly.     

Immunoelectron microscopic evidence of contractile proteins in the cellular and acellular components of mouse kidney glomeruli

Actin and/or actin-like protein have been localized in the cellular and acellular components of the glomerular walls of mouse kidney by means of immunoelectron microscopy, employing human antibodies to smooth muscle (SMA). Contractile antigens have been confirmed to be present in the cytoplasm of podocytes and mesangial cells in association with fine filaments which are considered of importance in the control of blood flow, intravascular pressure, and filtration rate within the glomerulus. The extracellular presence of contractile proteins in the mesangial matrix and glomerular basement membrane can be related to cell movement in a frictional environment. This latter phenomenon, which is strictly interdependent with cell adhesion and aggregation, is most evident in the mesangial cells in a form of luminar pseudopodia, cytoplasmic projections, and phagocytosis.     

Histones bind to contractile proteins and inhibit actomyosin ATPase

Nuclear histones bind to and precipitate the major contractile proteins, actin and myosin. The binding of histone to actin seems to reach saturation at 2:1 ratio, the interaction may serve some regulatory function(s) in intranuclear events. The binding of histone to myosin is not saturable, and, although it inhibits the actin-activated Mg2+-dependent myosin ATPase activity, does therefore not seem of physiological importance.     

Presence of non-histone proteins in nucleosomes

It has been established that nucleosomes are made of histones and DNA fragments. The purpose of this work to establish whether some non-histone proteins are also present in these chromatin subunits. We have found that nucleosome preparations contain phosphorylated non-histone proteins and protein kinases by sucrose gradient analysis. In order to establish whether these proteins are actually bound to nucleosomes or if they represent unbound or aggregated proteins, the following experiments were performed. (a) Free non-histone proteins and proteins released from chromatin by DNase overdigestion were analyzed by sucrose gradient centrifugation. No phosphoproteins but some phosvitin kinase activity was found in the part of the gradient which contained the nucleosomes. It could be assumed that part of the phosphoproteins are bound to nucleosomes. (b) A digestion of nucleosomes with DNase I suppressed the phosvitin kinase activity in the 11-S region of the gradient. (c) High ionic strength, which extracted non-histone proteins, suppressed the phosvitin kinase activity in the nucleosome region. Part of phosvitin kinase and of nuclear phosphoproteins are therefore bound to nucleosomes and are released by nuclease digestion and by high ionic strength.     

Alterations in cardiac contractile proteins due to oxygen free radicals.

In view of the potential role of free radicals in the genesis of cardiac abnormalities under different pathophysiological conditions and the importance of contractile proteins in determining heart function, this study was undertaken to examine the effects of oxygen free radicals on the rat heart myofibrils. Xanthine plus xanthine oxidase (X + XO) which is known to generate superoxide anions (O2-) and hydrogen peroxide (H2O2), an activated species of oxygen, was found to decrease Ca(2+)-stimulated ATPase activity, increase Mg(2+)-ATPase activity and reduce sulfhydryl (SH) group contents in myofibrils; these effects were completely prevented by superoxide dismutase (SOD) plus catalase (CAT). Both H2O2 and hypochlorous acid (HOCl), an oxidant, produced actions on cardiac myofibrils similar to those observed by X + XO. The effects of H2O2 and HOCl were prevented by CAT and L-methionine, respectively. N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), inhibitors of SH groups, also produced effects similar to those seen with X + XO. Dithiothreitol (DTT), a well known sulfhydryl-reducing agent, prevented the actions of X + XO, H2O2, HOCl, NEM and DTNB. These results suggest that marked changes in myofibrillar ATPase activities by different species of oxygen free radicals may be mediated by the oxidation of SH groups.