Comparison of growth and cell proliferation kinetics during mouse molar odontogenesis in vivo and in vitro

Abstract. The growth of embryonic first lower mouse molars in vitro was slow and reduced in comparison with in vivo development: the volume of teeth removed on day 15 and 16 of gestation and cultured for 6 days did not exceed the volume reached at day 18 in vivo . The volume of teeth removed on day 17 and 18 and cultured for 6 days either remained constant or decreased. The appearance of post-mitotic odontoblasts and ameloblasts was delayed in vitro . This behaviour might be correlated with a lengthening of the cell cycle ( T _c). In vitro , the average durations of T _c (established by the percentage labelled mitoses technique) were 17.4–20.2 hr and 19.1–19.4 hr for pre-odontoblasts and pre-ameloblasts respectively. In vivo , the corresponding T _c values were 13.9 hr and 13.5 hr. The coordination of mitotic activities of pre-odontoblasts and pre-ameloblasts existing in vivo was maintained in vitro , and therefore seemed to require intra-dental control mechanisms. Non-specific extra-dental serum factors may affect the duration of T _c.     

Comparison of growth and cell proliferation kinetics during mouse molar odontogenesis in vivo and in vitro

The growth of embryonic first lower mouse molars in vitro was slow and reduced in comparison with in vivo development: the volume of teeth removed on day 15 and 16 of gestation and cultured for 6 days did not exceed the volume reached at day 18 in vivo. The volume of teeth removed on day 17 and 18 and cultured for 6 days either remained constant or decreased. The appearance of post-mitotic odontoblasts and ameloblasts was delayed in vitro. This behaviour might be correlated with a lengthening of the cell cycle (Tc). In vitro, the average durations of Tc (established by the percentage labelled mitoses technique) were 17.4-20.2 hr and 19.1-19.4 hr for pre-odontoblasts and pre-ameloblasts respectively. In vivo, the corresponding Tc values were 13.9 hr and 13.5 hr. The coordination of mitotic activities of pre-odontoblasts and pre-ameloblasts existing in vivo was maintained in vitro, and therefore seemed to require intra-dental control mechanisms. Non-specific extra-dental serum factors may affect the duration of Tc.     

Mouse odontogenesis in vitro: the cap-stage mesenchyme controls individual molar crown morphogenesis.

Day 14 ICR mouse first lower (M1) and upper molars (M1) as well as heterotopic recombinations of M1 epithelium/M1 mesenchyme and M1 epithelium/M1 mesenchyme were cultured for 6, 8 and 10 days on semi-solid medium. Computer-assisted 3D reconstructions were performed to follow the in vitro development of these explants. In vitro culture of cap-stage molars allowed for the emergence of unequivocal morphological features distinctive for M1 versus M1 including the cusp pattern, cusp inclination and tooth specific chronology for odontoblast and ameloblast terminal differentiations. Both M1 epithelium/M1 mesenchyme and M1 epithelium/M1 mesenchyme recombinations developed according to the known developmental fate of the mesenchyme. Our data demonstrate that the cap-stage dental ecto-mesenchyme not only directs tooth class specific morphogenesis, but also individual molar crown features. Furthermore, the mesenchyme apparently also controls the typical mirror symmetry of right and left handed teeth.     

Seasonal variation in molar outline of bank voles: An effect of wear?

Morphometric characters can be of use for elucidating the evolutionary history of species by providing an insight into the selective pressure related to the character of interest, and by allowing integration of fossil specimens. This potential interest of phenotypic characters, however, depends on how much other sources of variation, such as the life-history of the animal, may blur an evolutionary signal. For instance, age structure varies along the year, causing in turn various assemblages of wear stages in the teeth sampled at a given place and time. In this context, we investigated the season of trapping as potential source of variation in the size and shape of the molar occlusal surface of the bank vole, Clethrionomys glareolus.The size and shape of the occlusal surface of the third upper molar was quantified using outline analysis in 60 bank voles from Finland, trapped at the same study site in successive spring and autumn. The occlusal surface clearly differed in size and shape between the two seasons of trapping. Using 3D imaging as a visual support, we interpret this difference as the result of differential wear. The population in autumn is dominated by young specimens with unworn teeth whereas spring populations are composed of old animals with worn down molars. The range of seasonal variation in tooth size and shape appears to be of the same order of magnitude as biogeographic variation, demonstrating that differential wear may have a strong impact on biogeographic and evolutionary studies. Yet, beyond the effect of trapping season, a biogeographic signal still emerged, related to the genetic lineages evidenced in other studies. In consequence, morphometric characters such as size and shape of molar occlusal surfaces appear as valuable tracers of biogeographic differentiation, but future studies should take seasonal variations into account for more robust interpretation.     

Rescue of odontogenesis in Dmp1-deficient mice by targeted re-expression of DMP1 reveals roles for DMP1 in early odontogenesis and dentin apposition in vivo

Dentin matrix protein 1 (DMP1) is expressed in both pulp and odontoblast cells and deletion of the Dmp1 gene leads to defects in odontogenesis and mineralization. The goals of this study were to examine how DMP1 controls dentin mineralization and odontogenesis in vivo. Fluorochrome labeling of dentin in Dmp1-null mice showed a diffuse labeling pattern with a 3-fold reduction in dentin appositional rate compared to controls. Deletion of DMP1 was also associated with abnormalities in the dentinal tubule system and delayed formation of the third molar. Unlike the mineralization defect in Vitamin D receptor-null mice, the mineralization defect in Dmp1-null mice was not rescued by a high calcium and phosphate diet, suggesting a different effect of DMP1 on mineralization. Re-expression of Dmp1 in early and late odontoblasts under control of the Col1a1 promoter rescued the defects in mineralization as well as the defects in the dentinal tubules and third molar development. In contrast, re-expression of Dmp1 in mature odontoblasts, using the Dspp promoter, produced only a partial rescue of the mineralization defects. These data suggest that DMP1 is a key regulator of odontoblast differentiation, formation of the dentin tubular system and mineralization and its expression is required in both early and late odontoblasts for normal odontogenesis to proceed.     

The structure of morphological variability (with the masticatory surface morphotypes of the lower first molar in voles as an example)

An analysis of morphotypical variability of the lower first molar in twelve species of Microtus genus revealed similarity in patterns of variability, consisting in two-three homologous series of three morphotypes of one class of rugosity in each series. In the majority of species, morphotypes are located in a single plane. In some species, morphotypes of other class of rugosity form the homologous series of a second plane so that the structure of the variability takes on a three-dimensional form. The variability spectrum is limited by molars design; at the different trends in complication of a masticatory surface have taxonomic weight of subgenus or species rank. Thus, incompleteness of homologous series is caused by taxonomic specificity and is nonrandom in essence.     

Proliferation, apoptosis and thymic involution.

The thymus reaches its maximal size at the age of 1 month in ICR mice and thereafter, the thymic cortex undergoes an exponential decline. This study was designed to compare the proliferation and apoptosis of thymocytes in different parts of the thymus of ICR female mice at the beginning and after the rapid phase of decline of the thymic cortical cellularity. The pattern of proliferation and apoptosis of the thymus was studied in situ in 1-month-old ICR female mice (10 mice) compared to mice at 7 months of age (10 mice). Staining for argyrophylic nucleolar organizer region by histochemistry was used to determine the proportion of type 2 thymocytes, which are considered as cells at S phase of the cell cycle. The mean number of type 2 cells in four random samples of 50 cells in each part of the thymus was defined as the proliferation index of this part of the thymus. In situ detection of apoptosis of thymocytes was carried out using the Apoptag kit, which can detect a single cell apoptosis. The mean number of apoptotic cells in five randomly selected fields of each part of the thymus was defined as the apoptotic index of this part of the thymus. The proliferation index of the peripheral cortex of the 1-month-old mice was 3.6 times higher than the proliferation index of the deep cortex and 5.8 times higher than the proliferation index of the medulla (P < 0.0001). The proliferation index of the peripheral cortex of the 7-month-old mice was reduced by 45% compared to the 1-month-old mice (P < 0.005). The apoptotic index of the corticomedullary junction of the 1-month-old mice was six times higher than the apoptotic index of the cortex and 18 times higher than the apoptotic index of the medulla. The apoptotic index of the thymic cortex was elevated by 66% in the 7-month-old mice compared to the 1-month-old mice (P < 0.0001). We conclude that there is a reduction of the proliferation index and an elevation of the apoptotic index of the thymic cortex in adult mice compared to young mice. These changes might account for the reduction of thymic cortical cellularity during thymic involution.     

Immunocytochemical study of amelogenin deposition during the early odontogenesis of molars in alendronate-treated newborn rats.

Newborn rats were treated with sodium alendronate to study how enamel is formed and the effect of alendronate during early odontogenesis. Ultrastructural analysis combined with high-resolution immunocytochemistry for amelogenin was carried out. Twelve rats were subjected to daily SC injections of sodium alendronate (2.5 mg/kg/day) for 3 days on their dorsal region, whereas three rats were daily injected with saline solution as a control. Molar tooth germs from 3-day-old rats were fixed under microwave irradiation in 0.1% glutaraldehyde + 4% formaldehyde buffered at pH 7.2 with 0.1 M sodium cacodylate. The specimens were left undecalcified, postfixed with osmium tetroxide, dehydrated, and embedded in LR White resin. Ultrathin sections were incubated with a chicken anti-24-kDa rat amelogenin antibody, a secondary antibody, and finally with a protein A-gold complex. Large patches of amelogenin were present over the unmineralized mantle dentin and at early secretory ameloblasts. At more advanced stages, they were also detected at the enamel matrix, as well as in the mineralized dentin, at the periodontoblastic space of the dentinal tubules, and at the predentin. It is likely that the main effect of alendronate at early stages of odontogenesis is the increase of synthesis/secretion of amelogenin, promoting its deposition within the forming dentin and enamel.     

Intergenerational transmission of the behavioral consequences of early experience in prairie voles

We examined intergenerational and epigenetic effects of early handling manipulations on the social behavior of the prairie vole (Microtus ochrogaster), a monogamous rodent. Laboratory-born parents and their newborn pups were assigned to either a MAN0 “zero handling” manipulation (transfer with a cup during weekly cage changes) or a MAN1 “gloved handling” manipulation (transfer with a gloved hand). Previous studies from our laboratory (Bales et al., 2007) showed that MAN0 juvenile males that received this manipulation as pups are less alloparental and that MAN0 adult females that received this manipulation as pups display impaired pair-bonding. In the present study, when MAN0 and MAN1 pups reached adulthood, they were mated in three combinations (MAN1 female × MAN1 male; MAN0 female × MAN1 male; MAN1 female and MAN0 male). Once the pairs produced offspring, we examined their parental behavior towards their own pups. The offspring of these pairings (F2 generation) also were tested as juveniles for alloparental behavior. MAN1 females paired with a MAN0 male displayed higher levels of parenting behaviors. In the F2 generation, juvenile offspring with a MAN0 parent were less alloparental than were offspring from other pairs. These results suggest that early experiences can be transmitted intergenerationally.     

The evolution of molar occlusion in the Cercopithecidae and early catarrhines

Those Eocene prosimians which are possible catarrhine ancestors have four blade-like crests on each lower molar. Each crest shears in sequence across two upper molar crests. Occluding crests are concavely curved to hold the foods being sheared. Each of two medial lower molar crests bordering the principal crushing surface shear past single upper molar crests at about the same time the lateral lower molar crests contact the second rank of upper molar crests. Grinding and crushing areas are restricted to hypoconid, trigonid, and protocone surfaces. Oligocene catarrhine molars have increased crushing-grinding capacities and maintained but modify their shearing. As the crushing surface of the protocone expands and a crushing hypocone is added, the “second rank” upper molar shearing crests are functionally reduced. At the same time medial crests are increasingly emphasized so that the total shearing capacity remains virtually unchanged. Marginal shearing blades are straight edged; leading edges of occluding blades are set at different angles to the occlusal plane so that blades contact at only one point at any given time. Early Primates have separate crushing basins surrounded by shearing blades. Catarrhines tend to link expanding crushing surfaces anteroposteriorly into a continuous surface between all molars. A cladistic analysis based on both new and previously recognized characters indicates that: 1, Apidium may be more closely related to Aegyptopithecus than to Parapithecus; 2, cercopithecids are derived from a Parapithecus-related stock; 3, Oreopithecus could equally well have come from an Apidium or Aegyptopithecus stock.     

Mitochondrial ROS burst as an early sign in sarsasapogenin-induced apoptosis in HepG2 cells

Sarsasapogenin is a steroidal sapogenin with antitumor properties. To explain the mechanism of its apoptotic effect, mitochondrial activity was assessed via a 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry (FCM) was used to estimate the changes in mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, and cellular-reduced glutathione (GSH) level. Laser scanning confocal microscope (LSCM) recorded instantaneous ROS burst after application of sarsasapogenin. Western blotting was used to determine the expression level and intracellular distribution of cytochrome c (cyt c). It is demonstrated that during apoptosis, ROS burst acted as an early event followed by depolarization of MMP, prolonged ROS generation, and significantly declined GSH level. Cyt c was upregulated and released from mitochondria to cytosol during the process. These findings show that a mitochondrial ROS burst is an early upstream apoptotic signal which may trigger the mitochondrial apoptotic pathway and play a vital role in sarsasapogenin-induced HepG2 cell apoptosis.     

Habitat use by singing voles and tundra voles in the southern Yukon

Summary We investigated how far competitive interactions influence the use of habitats and relative abundance of two species of Microtus in the southwestern Yukon. We worked in the ecotone between alpine tundra and subalpine shrub tundra where populations of singing voles (Microtus miurus) and tundra voles (M. oeconomus) overlap little.We removed tundra voles from shrub tundra on one live-trapping area to look at the effect on the contiguous population of singing voles in alpine tundra. The removal of tundra voles did not affect the distribution or relative abundance of singing voles. The spatial distribution of these species and their movements within habitats suggest that they have a strong habitat preference.Populations of small mammals in the area are extremely dynamic and the relative importance of competitive interactions may change as density varies. At present we have no evidence that competition affects habitat use in M. miurus.     

Aggregation of modified hexabenzocoronenes as models for early stage asphaltene self-assembly

Molecular dynamics simulations were performed on solvated systems containing three molecules of hexabenzocoronene (HBC) derivatives to model the initial self-association of asphaltenes. Specifically, the hexane-modified HBC (HBC6) and those with substitutions containing a side chain with sulphur at three positions (α-H6SA, β-H6SB, and γ-HBCS) and a nitrogen substitution (H6N1) are included. These molecules were solvated with 20 mol% heptane and 80% toluene. Intermediate aggregation states, partial (two molecule clusters), as well as full aggregation of trimer clusters were observed. The conformation of the associated clusters was characterised by π–π stacking, giving the cluster a pre-rod-like shape. The aggregates displayed stability and did not dissociate throughout the course of the simulation (100–530 ns). In particular, the H6SB system showed statistically significant difference in means for both the separation distance and overall interaction energy. H6N1 and H6SA systems differed from the others in terms of electrostatic contributions. To gain a thorough understanding of initial aggregation behaviour and conditions of asphaltenes, additional simulations need to be conducted, in particular those incorporating oxygen substitutions as oxygen is a common heteroatom in crude.     

Proliferation and apoptosis in solid tumors. Analysis by laser scanning cytometry

OBJECTIVE: To examine the relationship between apoptosis and proliferation in a series of human solid malignant tumors, making use of objective, reproducible techniques newly developed for laser scanning cytometry (LSC). STUDY DESIGN: Apoptosis was detected by in situ end labeling of DNA strand breaks with FITC-conjugated nucleotide. Proliferation was detected by Ki-67 antibody. Two parameters were detected independently and simultaneously with DNA measurement on aliquots of cell suspensions obtained by mechanical dissociation of fresh tumors and placed on microscope slides. RESULTS: The number of cells undergoing apoptosis varied from 0.5% to 28.1% (average, 5.4 +/- 6.0). Aneuploid tumors showed a higher percentage of apoptotic cells (7.9 +/- 7.2) as compared to diploid tumors (3.4 +/- 4.0). Tumors with the greatest number of apoptotic cells on LSC also had the largest number of apoptotic cells on light microscopic examination. The number of cells labeled by Ki-67 ranged from 1.7% to 56.7% (average, 20.0 +/- 15.5). Aneuploid tumors were characterized by a higher Ki-67 index (average, 28.3 +/- 14.3%) than the diploid tumors (13.2 +/- 13.3%). CONCLUSION: Overall, there was a very weak or no correlation between apoptosis and proliferation. However, a subset of aneuploid tumors had a high percentage of cells positive for Ki-67 and low percentage of apoptotic cells. Diploid tumors did not show any correlation between apoptosis and proliferation, although many of those tumors had both low apoptotic and proliferative indices. Whether those differences are of prognostic significance remains to be determined in follow-up studies that include more cases and clinical data. Here we have shown that LSC is a powerful new tool of potential clinical value for fast, objective analysis of apoptosis, proliferation and DNA ploidy in solid malignant tumors.     

Analysis of laser scattering pattern as an early measure of apoptosis.

Light scattering pattern analysis (LSPA) was applied in the current study for accurate and sensitive detection of subtle changes in cell size, which occur in mouse thymocytes undergoing apoptosis. The decrease in cell diameter as measured by LSPA was found to be an early signal of apoptosis preceding the externalization of phosphatidylserine on the outer membrane. When apoptosis was induced by dexamethasone, the change in cell size was dose and time dependent, and could be blocked by pretreatment of the thymocytes with N-acetylcysteine (NAC). This implies that the scattering pattern, when combined with fluorescent markers such as annexine-V, may be a powerful tool for early detection of apoptosis. Another advantage gained by the use of this method is the ability to repeatedly trace the same cells and to monitor the kinetics of their size changes.     

Perturbation of membrane dynamics in nerve cells as an early event during bilirubin-induced apoptosis

Increased levels of unconjugated bilirubin, the end product of heme catabolism, impair crucial aspects of nerve cell function. In previous studies, we demonstrated that bilirubin toxicity may be due to cell death by apoptosis. To characterize the sequence of events leading to neurotoxicity, we exposed developing rat brain astrocytes and neurons to unconjugated bilirubin and investigated whether changes in membrane dynamic properties can mediate apoptosis. Bilirubin induced a rapid, dose-dependent increase in apoptosis, which was nevertheless preceded by impaired mitochondrial metabolism. Using spin labels and electron paramagnetic resonance spectroscopy analysis of whole cell and isolated mitochondrial membranes exposed to bilirubin, we detected major membrane perturbation. By physically interacting with cell membranes, bilirubin induced an almost immediate increase in lipid polarity sensed at a superficial level. The enhanced membrane permeability coincided with an increase in lipid fluidity and protein mobility and was associated with significant oxidative injury to membrane lipids. In conclusion, apoptosis of nerve cells induced by bilirubin is mediated by its primary effect at physically perturbing the cell membrane. Bilirubin directly interacts with membranes influencing lipid polarity and fluidity, protein order, and redox status. These data suggest that nerve cell membranes are primary targets of bilirubin toxicity.     

Proliferation and metabolism: it's as easy as APC

Proliferating cells must generate both ATP and biosynthetic precursors for macromolecular synthesis. While proliferative signals have long been known to regulate metabolism, Garedew et?al. now demonstrate that the?proliferation apparatus itself, in the form of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), directly controls mitochondrial biogenesis, morphology, and respiratory activity.