Over- and ectopic expression of Wnt3 causes progressive loss of ameloblasts in postnatal mouse incisor teeth.

Intercellular signaling is essential for the development of teeth during embryogenesis and in maintenance of the continuously growing incisor teeth in postnatal rodents. WNT intercellular signaling molecules have been implicated in the regulation of tooth development, and the Wnt3 gene shows specific expression in the enamel knot at the cap stage. We demonstrate here that Wnt3 also is expressed in specific epithelial cell layers in postnatal incisor teeth. To begin to delineate the functions of Wnt3 in developing and postnatal teeth, we determined the effects of over- and ectopic expression of Wnt3 in the tooth epithelium of mice carrying a keratin 14-Wnt3 transgene. Expression of the transgene caused a progressive loss of ameloblasts from postnatal lower incisor teeth. Loss of ameloblasts may be due to defective proliferation or differentiation of ameloblast precursors, progressive apoptosis of ameloblasts, or loss of ameloblast stem cells.

The small bovine amelogenin LRAP fails to rescue the amelogenin null phenotype.

Amelogenins are the most abundant secreted proteins in developing dental enamel. These evolutionarily-conserved proteins have important roles in enamel mineral formation, as mutations within the amelogenin gene coding region lead to defects in enamel thickness or mineral structure. Because of extensive alternative splicing of the primary RNA transcript and proteolytic processing of the secreted proteins, it has been difficult to assign functions to individual amelogenins. To address the function of one of the amelogenins, we have created a transgenic mouse that expresses bovine leucine-rich amelogenin peptide (LRAP) in the enamel-secreting ameloblast cells of the dental organ. Our strategy was to breed this transgenic mouse with the recently generated amelogenin knockout mouse, which makes none of the amelogenin proteins and has a severe hypoplastic and disorganized enamel phenotype. It was found that LRAP does not rescue the enamel defect in amelogenin null mice, and enamel remains hypoplastic and disorganized in the presence of this small amelogenin. In addition, LRAP overexpression in the transgenic mouse (wildtype background) leads to pitting in the enamel surface, which may result from excess protein production or altered protein processing due to minor differences between the amino acid compositions of murine and bovine LRAP. Since introduction of bovine LRAP into the amelogenin null mouse does not restore normal enamel structure, it is concluded that other amelogenin proteins are essential for normal appearance and function.

Amelogenin-deficient mice display an amelogenesis imperfecta phenotype.

Dental enamel is the hardest tissue in the body and cannot be replaced or repaired, because the enamel secreting cells are lost at tooth eruption. X-linked amelogenesis imperfecta (MIM 301200), a phenotypically diverse hereditary disorder affecting enamel development, is caused by deletions or point mutations in the human X-chromosomal amelogenin gene. Although the precise functions of the amelogenin proteins in enamel formation are not well defined, these proteins constitute 90% of the enamel organic matrix. We have disrupted the amelogenin locus to generate amelogenin null mice, which display distinctly abnormal teeth as early as 2 weeks of age with chalky-white discoloration. Microradiography revealed broken tips of incisors and molars and scanning electron microscopy analysis indicated disorganized hypoplastic enamel. The amelogenin null phenotype reveals that the amelogenins are apparently not required for initiation of mineral crystal formation but rather for the organization of crystal pattern and regulation of enamel thickness. These null mice will be useful for understanding the functions of amelogenin proteins during enamel formation and for developing therapeutic approaches for treating this developmental defect that affects the enamel.

Expression of the elastin promoter in novel tissue sites in transgenic mouse embryos.

We have previously shown in a transgenic mouse line, in which 5.2 kb of the elastin promoter was linked to the reporter enzyme chloramphenicol acetyltransferase (CAT), that the highest levels of expression were found in embryonic lungs and aorta, while lower levels were detected in other elastin-containing tissues. Furthermore, in general, expression of the transgene showed developmental regulation similar to that of the endogenous gene. However, the precise location of cellular expression could not be determined in this model. To overcome this limitation, we have developed a similar model, but replaced CAT with the reporter enzyme beta-galactosidase. Enzyme activity was readily detected in the transgenic mouse embryos in expected regions of tissue forming elastic fibers, including the dermis and elastic cartilage. Of considerable interest, however, was the novel finding of expression in specific areas of neuroepithelium of the brain and in the perichondrium surrounding areas destined to form hyaline cartilage in endochondral bone formation. These latter areas included all the bones of the limbs, the spine and rib cage. It appeared that these segments of elastin expression demarcated the border between the developing cartilage and the surrounding mesenchymal tissue. Elastin promoter expression was also found in developing somites, in the mesenchymal layer of the forming cornea of the eye, in the genital tubercle and in the epithelium destined to form the olfactory epithelium. These findings indicate that the elastin promoter is activated during embryonic development in a variety of tissues, suggesting that elastin gene expression may play a role in organizing cutaneous, skeletal and neural structures.

Diabetes mellitus affects prostaglandin E2 levels in mouse embryos during neurulation.

The arachidonic acid cascade leading to prostaglandins has been implicated in diabetic embryopathy. Both arachidonic acid and prostaglandin E2 reverse the teratogenic effects of high glucose concentrations on neural tube development in mouse embryos in culture. Arachidonic acid supplementation also protects against diabetes-induced neural tube defects in vivo. In the present study, prostaglandin E2 was measured directly in embryos from normal and diabetic mice. In normal mice a clear developmental pattern was seen. Prostaglandin E2 levels were high during early formation of the cranial neural folds (day 8), declined during convergence and fusion of the cranial neural folds to form the neural tube (day 9), and were low after neurulation was complete (days 10 and 11). In addition, evidence in this study indicates that embryos have cyclooxygenase activity capable of generating prostaglandin E2 during a brief developmental period preceding neural tube closure. In embryos from mice made diabetic (> 13.9 mmol/l glucose) with streptozotocin, prostaglandin E2 levels were significantly lower than normal during early development of the cranial neural folds (day 8), but similar to normal after the cranial neural tube had closed (late day 9 and day 10). The findings suggest that diabetes mellitus, as ascertained by high blood glucose, promotes cranial neural tube malformations by causing a functional deficiency of prostaglandin E2 during early neurulation. Whether the altered PGE2 pattern in the embryo indicates a diabetic effect on the arachidonic acid-prostaglandin cascade in cells of the embryo or in cells of extraembryonic or maternal tissues is uncertain.

Analysis of the regulatory region of the bovine X-chromosomal amelogenin gene.

The amelogenin proteins, which are crucial for normal enamel mineral formation, are secreted by ameloblasts during development of tooth enamel. In order to better understand the mechanisms involved in regulation of expression of the amelogenin genes, the bovine X-chromosomal amelogenin gene was cloned and a 3.5 KB fragment upstream of exon 1 was inserted into a beta galactosidase (beta gal) expression vector for production of transgenic mice. When tissues from these mice were treated with Xgal, a substrate for beta gal, only ameloblasts and some of the adjacent stratum intermedium cells contained blue stain. To obtain further information concerning regulation of expression, the 3.5 KB amelogenin gene fragment was evaluated in transfection experiments. Nonoverlapping 1.9 and 1.5 KB fragments of the upstream region were subcloned separately into a vector that contains the SV40 promoter and the CAT reporter gene. Each amelogenin gene fragment was able to suppress CAT activity driven by the heterologous SV40 promoter in transfected HeLa cells. We theorize that each of these gene fragments contains regulatory elements important for the tissue-specific and developmentally-regulated pattern of expression of the X-chromosomal amelogenin gene.

Regulation of amelogenin gene expression during tooth development.

The amelogenins are the predominant matrix proteins in developing enamel and are crucial for proper enamel mineralization. Transgenic mice were constructed in order to identify the segment of the amelogenin gene required for specific expression in enamel organ cells. A 3.5 kb fragment of the bovine X-chromosomal amelogenin gene that includes a TATA box, the transcription initiation site, and 32 bp of exon 1 was linked to the beta galactosidase gene and injected into fertilized mouse eggs. Newborn transgene positive mice expressed beta galactosidase activity in developing teeth treated with the chromogenic substrate Xgal. Foci of ameloblasts were positive in newborn mice; stain intensity and number of positive ameloblasts increased in 1-day and 2-day postnatal mice. Some of the adjacent stratum intermedium cells also were positive in the later stages. Targeting of the transgene to the enamel organ was specific; the only other cells observed to be positive were macrophages, which have endogenous beta galactosidase activity.

Myo-inositol and prostaglandins reverse the glucose inhibition of neural tube fusion in cultured mouse embryos.

Neural tube defects in infants of diabetic mothers constitute an important and frequent cause of neonatal mortality/morbidity and long-term chronic handicaps. The mechanism by which normal neural tube fusion occurs is not known. The failure of rostral neural tube fusion seen in mouse embryos incubated in the presence of excess-D-glucose can be significantly prevented by the supplementation of myo-inositol to the culture medium. This protective effect of myo-inositol is reversed by indomethacin, an inhibitor of arachidonic acid metabolism leading to prostaglandin synthesis. Prostaglandin E2 added to the culture medium completely protects against the glucose-induced neural tube defect. These data suggest that the failure of neural tube fusion seen in diabetic embryopathy is mediated through a mechanism involving abnormalities in both the myo-inositol and arachidonic acid pathways, resulting in a functional deficiency of prostaglandins at a critical time of neural tube fusion.

Inhibition of embryonic palatal shelf horizontalization and medial edge epithelial breakdown by cortisol: role of H-2 in the mouse.

We have investigated the effect of glucocorticoids on the development of the embryonic palate in vivo and of glucocorticoids and diphenylhydantoin (DPH) in culture in the cleft palate-sensitive B10.A strain and its resistant congenic partner strain, B10, as well as the effect of glucocorticoids in vivo in the sensitive A/J strain. The B10.A (H-2a) strain differs from its congenic partner strain, B10 (H-2b), only in the H-2 region of chromosome 17, and thus, differences between the two strains in the responses to the drugs can be ascribed to H-2-linked genes. The degree of corticoid-induced inhibition of shelf horizontalization in vivo is only slightly (if at all) greater in B10.A than in B10, but the degree of corticoid-induced inhibition of fusion following contact in vivo and the inhibition by cortisol and DPH of programmed cell death and breakdown of the medial edge epithelium (MEE) in vitro are much greater in B10.A than in B10. The corticoid-induced delay of shelf horizontalization produced in vivo in the A/J (H-2a) strain is considerably greater than that produced in either B10.A or B10. Thus, H-2-linked genes appear to influence slightly, if at all, the degree of corticoid-induced delay of shelf elevation, but they have a major effect on the corticoid-induced inhibition of fusion via the inhibition of breakdown of the medial edge epithelia. The delay of corticoid-induced shelf horizontalization appears to be a trait influenced primarily by non H-2-linked genes.

Hyperglycemia-induced teratogenesis is mediated by a functional deficiency of arachidonic acid.

Congenital malformations now represent the largest single cause of mortality in the infant of the diabetic mother. The mechanism by which diabetes exerts its teratogenic effects is not known. This study evaluated whether arachidonic acid might be involved, a possibility raised by the role of arachidonic acid in palatal elevation and fusion, processes analogous to neural tube folding and fusion. This hypothesis was tested in two animal models of diabetic embryopathy, the in vivo pregnant diabetic rat and the in vitro hyperglycemic mouse embryo culture. The subcutaneous injection of arachidonic acid (200-400 mg/kg per day) into pregnant diabetic rats during the period of organ differentiation (days 6-12) did not alter the maternal glucose concentration, the maternal weight gain, or the weight of the embryos. However, the incidence of neural tube fusion defects was reduced from 11% to 3.8% (P less than 0.005), the frequency of cleft palate was reduced from 11% to 4% (P less than 0.005), and the incidence of micrognathia was reduced from 7% to 0.8% (P less than 0.001). The addition of arachidonic acid to B10.A mouse embryos in culture also resulted in a reversal of hyperglycemia-induced teratogenesis. The teratogenic effect of D-glucose (8 mg/ml) in the medium resulted in normal neural tube fusion in only 32% of the embryos (P less than 0.006 when compared to controls). Arachidonic acid supplementation (1 or 10 micrograms/ml) produced a rate of neural tube fusion (67%) that was not significantly different from that observed in controls. The evidence presented indicates that arachidonic acid supplementation exerts a significant protective effect against the teratogenic action of hyperglycemia in both in vivo (rat) and in vitro (mouse) animal models. These data therefore suggest that the mechanism mediating the teratogenic effect of an increased glucose concentration involves a functional deficiency of arachidonic acid at a critical stage of organogenesis.

Palatal development and the arachidonic acid cascade.

The production of cleft palate by glucocorticoids and phenytoin is a complicated interference of a complex developmental program involving many genetic and biochemical processes. The H-2 histocompatibility region includes genes which affect susceptibility to glucocorticoid- and phenytoin-induced cleft palate; glucocorticoid receptor level in a variety of tissues including maternal and embryonic palates, adult thymuses and lungs; and the degree of inhibition of prostaglandin and thromboxane production by glucocorticoids and phenytoin in thymocytes. A gene linked to a minor histocompatibility locus (H-3) on the second chromosome also influences susceptibility to glucocorticoid and phenytoin-induced cleft palate. Phenytoin is an alternate ligand for the glucocorticoid receptor affecting prostaglandin and/or thromboxane production. The capacity of glucocorticoids to induce cleft palate is correlated with their anti-inflammatory potency. At least some of the anti-inflammatory effects of glucocorticoids can be explained by the inhibition of prostaglandin and/or thromboxane release, which in turn could be caused by inhibition of arachidonic acid release from phospholipids. Similar mechanisms may be involved in cleft palate induction, as exogenous arachidonic acid injected into pregnant rats and mice at the same time as glucocorticoids reduces the teratogenic potency of the steroids and indomethacin, and inhibitor of cyclooxygenase, blocks the corrective action of arachidonic acid. Glucocorticoids and phenytoin cause a delay in shelf elevation, and this delay is promoted by fetal membranes and the tongue. However, the cells of the medial edge epithelium are programmed to die whether contact is made with the apposing shelf or not. Glucocorticoids and phenytoin interfere with this programmed cell death, and this interference by both drugs seems to be a glucocorticoid receptor mediated event, to require protein synthesis and to be related to arachidonic acid release.

Glucocorticoid-induced phospholipase A2-inhibitory proteins mediate glucocorticoid teratogenicity in vitro.

Dexamethasone induces the synthesis of a phospholipase A2-inhibitory protein (PLIP) of molecular weight approximately equal to 55,000 from calf thymus and PLIPs of molecular weights 55,000, 40,000, 28,000, and 15,000 from A/J mouse thymus and from 12-day embryonic B10. A mouse palates. Sufficient quantities of calf thymus PLIP and of the 15,000 molecular weight mouse thymus and palate PLIPs were prepared and tested as inhibitors of programmed cell death in the medial-edge epithelium of single mouse embryonic palatal shelves in culture. All of the proteins tested prevent the loss of the medial-edge epithelium and, thus, produce the teratogenic effects of glucocorticoids in the palatal culture model. This teratogenic action of both PLIP and glucocorticoids is reversed by arachidonic acid, the precursor of prostaglandins and thromboxanes, suggesting that PLIP mediates the effects of glucocorticoids by inhibiting phospholipase A2.

Further evidence for a role of arachidonic acid in glucocorticoid teratogenic action in the palate.

Arachidonic acid produces a significant reversal of the production of cleft palate by cortisone in the offspring of sensitive strains of mice in vivo. Arachidonic acid in nanogram per milliliter concentrations also produces a significant reversal of the cortisol inhibition of the programmed cell death of the medial edge epithelium of palatal shelves in vitro. This corrective action of arachidonic acid in vitro is significantly blocked by indomethacin at a nanogram per milliliter concentration which selectively inhibits the conversion of arachidonic acid to prostaglandins and/or thromboxanes at the level of cyclooxygenase. These results support the hypothesis that the inhibition of arachidonic acid release and subsequent prostaglandin and/or thromboxane production by glucocorticoids is involved in the teratogenic action of glucocorticoids and demonstrate that one site of this action is the inhibition of epithelial loss.

Inhibition of programmed cell death in mouse embryonic palate in vitro by cortisol and phenytoin: receptor involvement and requirement of protein synthesis.

In an in vitro model cortisol and phenytoin inhibit the precisely timed process of palatal development, the lysosomally mediated cell death of the medial edge palatal epithelium. This inhibition of programmed cell death of the palatal midline epithelium by each drug is virtually completely blocked by the antiglucocorticoid, cortexolone, whose blocking action results from competitive binding of the glucocorticoid receptor site. The inhibition produced by each of these drugs is prevented by the protein synthesis blocker, cycloheximide. Thus, blockade of programmed cell death by each of these drugs involves the glucocorticoid receptor site and requires protein synthesis.

Cortisol inhibition of development of various lysosomal enzymes in cultured palatal shelves from mouse embryos.

The lysosomal enzymes, non-specific esterases, beta-glucuronidase, N-acetyl-beta-glucosaminidase and aryl sulphatases A and B, were examined histochemically in medial-edge epithelia (MEE) of single palatal shelves in vitro. Activities of most enzymes increased gradually in MEE with a peak at 24-26 h of culture. Aryl sulphatase B activities were lower than the others and aryl sulphatase A activities could not be detected in the palatal cells during the entire culture period. By 48 h, MEE cells degenerated and were lost. Cortisol suppressed increased activities of these hydrolytic enzymes and prevented programmed breakdown of the palatal epithelium.

Subcellular distribution of glutamyltransferase activities in embryonic cerebral hemispheres.

Glutamyltransferase (GT) activities were examined in cell groups from embryonic cerebral hemispheres. During normal development, GT activities were highest in a mixed population of small neurons and non-neuronal cells (small cell group). In culture, in response to hydrocortisone, activities exceeded normal levels only in this small cell group. In view of these findings, the small cell population was used to study the subcellular distribution of the enzyme. At the subcellular level, GT activities in the small cell group were distributed differentially but increased generally during normal development. In vitro, hydrocortisone promoted a precocious increase in GT activity, under experimental conditions regarded as optimum, only in the subcellular fraction characterized by synaptic endings.

Photon correlation analysis of cytoplasmic streaming.

Laser light scattering has been used to investigate particle movements in a plant cell. Intensity autocorrelation functions are obtained by digital photon correlation of laser light scattered from cells of Nitella opaca both during cytoplasmic streaming and during the transitory cessation of streaming induced by electrical stimulation. The average velocity computed from the periodic oscillation in the intensity autocorrelation function during streaming corresponds to the velocity estimated using light microscopy. An estimate of the distribution of streaming velocities has been obtained from the decay in the amplitude of the envelope of the autocorrelation function derived from a streaming cell.