Hoxa2- and rhombomere-dependent development of the mouse facial somatosensory map.

In the mouse trigeminal pathway, sensory inputs from distinct facial structures, such as whiskers or lower jaw and lip, are topographically mapped onto the somatosensory cortex through relay stations in the thalamus and hindbrain. In the developing hindbrain, the mechanisms generating such maps remain elusive. We found that in the principal sensory nucleus, the whisker-related map is contributed by rhombomere 3-derived neurons, whereas the rhombomere 2-derived progeny supply the lower jaw and lip representation. Moreover, early Hoxa2 expression in neuroepithelium prevents the trigeminal nerve from ectopically projecting to the cerebellum, whereas late expression in the principal sensory nucleus promotes selective arborization of whisker-related afferents and topographic connectivity to the thalamus. Hoxa2 inactivation further results in the absence of whisker-related maps in the postnatal brain. Thus, Hoxa2- and rhombomere 3-dependent cues determine the whisker area map and are required for the assembly of the whisker-to-barrel somatosensory circuit.

Putative membrane receptor for 1,25(OH)2 vitamin D3 in human mineralized tissues during prenatal development.

The calciotropic hormone, 1,25(OH)2vitamin D3[1,25(OH)2D3], controls the formation of dental and bone mineralized tissues. The role of nuclear 1,25(OH)2D3 receptor has been extensively studied in the diverse secretory cells, i.e., osteoblasts, chondrocytes, ameloblasts, and odontoblasts. A nongenomic pathway also has been characterized and related to the interactions of 1,25(OH)2D3 ligand with a putative cell membrane receptor. This recognition moiety called 1,25(OH)2vitamin D3 membrane-associated, rapid-response steroid-binding [1,25D3-MARRS] protein is investigated here in the craniofacial skeleton of human embryos and fetuses. Immunolocalization using a specific Ab099 against chick intestinal basolateral 1,25D3-MARRS protein was performed. The data show a complementary expression pattern of the membrane receptor when compared with published data on the nuclear receptor, notably during amelogenesis. In mandible, membrane receptors for 1,25(OH)2D3 were identified in the heterogenous bone cell population, including osteoblasts and osteoclasts. Differential 1,25D3-MARRS protein levels were observed in distinct developmental stages and anatomical sites of tooth and bone, suggesting the existence of cross-talk between local factors and 1,25D3-MARRS protein expression.

YY1 activates Msx2 gene independent of bone morphogenetic protein signaling.

Msx2 is a homeobox gene expressed in multiple embryonic tissues which functions as a key mediator of numerous developmental processes. YY1 is a bi-functional zinc finger protein that serves as a repressor or activator to a variety of promoters. The role of YY1 during embryogenesis remains unknown. In this study, we report that Msx2 is regulated by YY1 through protein-DNA interactions. During embryogenesis, the expression pattern of YY1 was observed to overlap in part with that of Msx2. Most notably, during first branchial arch and limb development, both YY1 and Msx2 were highly expressed, and their patterns were complementary. To test the hypothesis that YY1 regulates Msx2 gene expression, P19 embryonal cells were used in a number of expression and binding assays. We discovered that, in these cells, YY1 activated endogenous Msx2 gene expression as well as Msx2 promoter-luciferase fusion gene activity. These biological activities were dependent on both the DNA binding and activation domains of YY1. In addition, YY1 bound specifically to three YY1 binding sites on the proximal promoter of Msx2 that accounted for this transactivation. Mutations introduced to these sites reduced the level of YY1 transactivation. As bone morphogenetic protein type 4 (BMP4) regulates Msx2 expression in embryonic tissues and in P19 cells, we further tested whether YY1 is the mediator of this BMP4 activity. BMP4 did not induce the expression of YY1 in early mouse mandibular explants, nor in P19 cells, suggesting that YY1 is not a required mediator of the BMP4 pathway in these tissues at this developmental stage. Taken together, these findings suggest that YY1 functions as an activator for the Msx2 gene, and that this regulation, which is independent of the BMP4 pathway, may be required during early mouse craniofacial and limb morphogenesis.

Spatiotemporal assessment of fetal bovine osteoblast culture differentiation indicates a role for BSP in promoting differentiation.

Fetal bovine mandible-derived osteoblasts were cultured for the purpose of obtaining a spatiotemporal assessment of bone matrix protein expression during in vitro differentiation. The results obtained from electron microscopic, immunohistological, biochemical, and molecular biological analyses indicated that these primary cultured osteoblasts produce an abundant extracellular matrix which mineralizes during a 14-day culture period. During this process, a restricted, spatiotemporal pattern of bone sialoprotein expression was indicated by immunohistological and molecular evaluations. To test the possibility that bone sialoprotein promoted the continued morphodifferentiation of osteoblastic cells, cultures were grown in the presence of anti-bone sialoprotein antibodies known to interfere with cell-bone sialoprotein attachment. Compared with cultures grown in the presence of normal rabbit serum (1:150), cultures grown in the media containing anti-bone sialoprotein antibody (1:150) failed to mineralize as demonstrated by von Kossa staining and failed to express osteocalcin and osteopontin as shown by the reverse transcription polymerase chain reaction. These results contribute to the growing evidence that bone sialoprotein is an important determinant of osteoblast differentiation and bone formation. Matrix protein-cell interactions may be examined using this spatiotemporally defined model.

Prenatal fluoride for growth and development: Part X.

Examinations of prenatal fluoride supplemented (PNF) teeth in an animal model and in a five-month human fetus find these teeth to be more developed than the non-supplemented controls. The fact that PNF allows teeth to develop to their full potential suggests that PNF could be an essential nutrient for the entire human and this could be demonstrated most easily during rapid fetal growth. A review of the recent literature, including trials by NIH and The World Health Organization, provide evidence that fluoride (F) does allow the fetus to grow and develop to its full potential. The authors conclude that PNF must be supplied in at least a 2 mg/day pulse dose, and then F must be given from shortly after birth in a daily amount appropriate for the weight of the child with some consideration for the amount of F water utilized.

Chemical and X-ray analysis of fluoride, phosphorus, and calcium in human foetal blood and hard tissues.

To evaluate the beneficial effect of prenatal fluoride supplementation, the presence of fluoride in hard tissues in two populations of human foetuses coming from fluoridated (> or = 0.7 parts/10(6) F in drinking water) and non-fluoridated areas (< or = 0.1 parts/10(6) F in drinking water) were compared by chemical analysis and X-ray microanalysis. The fluoride concentrations measured in maternal and venous cord blood confirmed that placental transfer of fluoride was passive when fluoride intake was low. Total fluoride contents of tooth germs and mandibular bone appeared to increase with fluoride level in drinking water. However, these concentrations were too low to be detected by X-ray microanalysis. Phosphorus and calcium total contents were identical in mandibular and femoral bone of both populations. In incisor germs, phosphorus and calcium concentrations in enamel and dentine close to the amelodentinal junction did not differ significantly between the two populations. It is suggested that the low fluoride concentrations in enamel and dentine formed in utero would not have a significant effect on acid solubility.

Combined effects of caffeine and alcohol during pregnancy on bones in newborn rats.

The combined effects of caffeine and alcohol on mineral contents of fetal mandibles and femurs were studied. Pregnant rats were divided into four groups: group 1, control; group 2, caffeine; group 3, alcohol; and group 4, caffeine-plus-alcohol. Alcohol (1.0 g ethanol/kg body weight) was intubated twice daily, beginning at day 9 of gestation. Caffeine (2 mg/100 g body weight) was given as a dietary supplement. Groups 1 and 2 were intubated with isocaloric sucrose solution. At birth, randomly selected pups were killed and the mandible and femur were removed and dried. Ca, P, Mg, Zn and hydroxyproline in these bones were measured. Notwithstanding the dams' intake of caffeine and alcohol administered separately, the present results suggest that the combination of caffeine and alcohol exhibited the most detrimental effects.

Alterations in the expression of osteonectin, osteopontin and osteocalcin mRNAs during the development of skeletal tissues in vivo.

Heterogeneity in the expression of three members of non-collagenous matrix proteins in osteogenic and chondrogenic development in vivo were investigated by in situ hybridization. Sections of several skeletal tissues from mice at various stages of development were hybridized with digoxigenin-labeled complementary RNA probes encoding osteonectin (Osn), osteopontin (Osp) and osteocalcin (Osc). In calvariae and mandibulae, Osn messenger RNA (mRNA) was detected in cells in pre-osseous and osseous tissues before mineralization. Osp mRNA was found in cells attached to the mineralized bone matrix together with Osn mRNA followed by the expression Osc mRNA. In long bones, mRNAs for Osn, Osp and Osc were sequentially expressed with bone development from primary spongiosa to diaphyseal bone. In growth cartilage, Osn mRNA was observed in chondrocytes in non-mineralized cartilage, whereas Osp mRNA was detected in hypertrophic chondrocytes in mineralized cartilage matrix with a characteristic switch in expression. Osc mRNA was not detected in any chondrocytes. These results indicate that osteogenic differentiation in bone development in vivo is characterized by the sequential expression of these three genes, and suggest that these genes are expressed differentially and specifically, in association with extra-cellular matrix mineralization.

Prenatal effects of maternal caffeine intake and dietary high protein on mandibular development in fetal rats.

The purpose of the present study was to determine the effects of caffeine on the mandibles of newborn rats whose dams were given a normal diet (200 g protein/kg diet) compared with those given a high-protein diet (400 g protein/kg diet) during gestation. A total of twenty pregnant Sprague-Dawley rats were randomly divided into four groups of five each. Starting on day 7 of gestation, groups 1 and 2 were fed on control and high-protein diets respectively, and groups 3 and 4 were pair-fed with groups 1 and 2 respectively, but with caffeine added to their diets. The caffeine supplement was 20 mg/kg body-weight. At birth, pups were killed and various measurements of their mandibles were made. The mandibular weights, calcium contents, and alkaline (EC 3.1.3.1) and acid (EC 3.1.3.2) phosphatase activities of the group given the caffeine-supplemented control diet were significantly lower than those of the corresponding unsupplemented group. Alkaline and acid phosphatase activities, collagen synthesis and hydroxyproline contents of the caffeine-supplemented high-protein group were greater than those of the corresponding unsupplemented group, whereas Ca and protein contents of the caffeine-supplemented high-protein group were lower than those of the corresponding unsupplemented group. There were no significant differences in plasma caffeine levels for either dams or pups between the caffeine-supplemented control and high-protein groups. The effects of caffeine on the development of fetal mandibles are apparently modified by different levels of maternal dietary protein.

The effects of maternal caffeine intake during pregnancy on mineral contents of fetal rat bone.

Various levels of maternal caffeine ingestion during pregnancy were investigated to determine whether caffeine will affect the mineral contents of the growing bones of fetal rats. On day 8 of gestation, rat dams were fed with a 20% protein diet supplemented with 0.5 mg, 1 mg, or 2 mg caffeine/100 g of dams body weight as an experimental group and the same without caffeine as a control until day 22 of gestation. Fetuses were removed by cesarean section on day 22 and mandibular bones were removed to study the mineral contents of calcium, phosphorus, magnesium, and zinc. Although the mandible weighed more in the 0.5-mg caffeine group as compared to the controls, an additional increase of caffeine resulted in a decrease in weight. All calcium, magnesium, and zinc contents per bone decreased in the 1- and 2-mg caffeine groups as compared to either controls or 0.5-mg caffeine group, whereas phosphorus stayed relatively constant regardless of the different levels of caffeine intake. When data are expressed as per gram of bone tissue, most of the mineral contents among the groups disappeared suggesting that normal growth and development of the fetal bone are impaired as a results of maternal caffeine intake. Caffeine intake during gestation in the present study indicates that different levels of caffeine intake may exert not only different effects on mineral contents in bone development, but also affect the growth of the fetal bone.

Chondroid bone arises from mesenchymal stem cells in organ culture of mandibular condyles.

Mandibular condyles of fetal mice 19 to 20 days in utero comprising clean cartilage and its perichondrium were cultured for up to 14 days, and their capacity to develop osteoid and to mineralize in vitro was examined. After 3 days in culture the cartilage of the mandibular condyle appeared to have lost its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic cells. At that time interval no chondroblasts could be seen; instead, most of the cartilage consisted of hypertrophic chondrocytes. By that time, the surrounding perichondrium, which contains pluripotential mesenchymal stem cells, revealed the first signs of extracellular matrix enclosing type I collagen, bone alkaline phosphatase, osteonection, fibronectin, and bone sialoprotein as demonstrated by immunofluorescent techniques. Electron microscopic examinations of the newly formed matrix revealed foci of mineralization within and along collagen fibers as is normally observed during bone development. The composition of the latter mineral deposits resembled calcium pyrophosphate crystals. Following 14 days in culture larger portions of the condyle revealed signs of osseous matrix, yet the tissue reacted positively for type II collagen. Hence, the condylar cartilage, a genuine representative of secondary-type cartilage, elaborated in vitro a unique type of bone that would be most appropriately defined as chondroid bone. Biochemical assays indicated that the de novo formation of chondroid bone was correlated with changes in alkaline phosphatase activity and 45Ca incorporation. The findings of the present study imply that mesenchymal stem cells that ordinarily differentiate into cartilage possess the capacity to differentiate into osteogenic cells and form chondroid bone.