Cell turnover in the vomeronasal epithelium: evidence for differential migration and maturation of subclasses of vomeronasal neurons in the adult opossum

Previous investigations of cell turnover in the mammalian vomeronasal sensory epithelium (VN-SE) raised two issues. First, if, in addition to the already demonstrated vertical migration, horizontal migration from the edges of the VN-SE participates in neuronal replacement. Second, whether or not migration and maturation is differential in upper and lower populations of vomeronasal neurons, since these two cell populations are chemically, physiologically, functionally, and perhaps evolutionarily different. By injecting bromodeoxyuridine (BrdU) into adult opossum (Monodelphis domestica) and permitting different survival times, the pattern of distribution of BrdU-labeled cells was analyzed. No evidence of horizontal migration in neuronal replacement was found. To investigate vertical migration and maturation of subclasses of vomeronasal neurons, double immunohistochemistry of BrdU and markers of the lower (G(oalpha) protein) and upper [G(i2alpha) protein and olfactory marker protein (OMP)] cell populations were performed. Three days after administration of BrdU, some mature neurons were observed in both lower and upper layers of the VN-SE, as demonstrated by coexpression of BrdU with G(oalpha) protein and OMP, respectively. The data on vertical distribution, however, indicate that most of the daughter cells enter the G(oalpha)-protein-expressing zone of the VN-SE by day 5, whereas most daughter cells do not reach the G(i2alpha)-protein-expressing zone until day 7, suggesting that these two populations mature at slightly different rates. These results are the first evidence of differential neurogenesis of subclasses of vomeronasal neurons.     

Neurogenesis, migration, and apoptosis in the vomeronasal epithelium of adult mice

The location of neurogenesis and the direction of migration of neurons in the adult mouse vomeronasal organ is controversial. Cell division occurs at the center, and particularly, at the edges of the epithelium. Newly generated cells at the center of the epithelium participate in neurogenesis, however, it is unknown to what extent dividing cells at the edges participate in growth, become apoptotic or mature into neurons. Premitotic cells were labeled with bromodeoxyuridine (BrdU) in adult mice and animals allowed to survive for different postinjection periods. The terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end-labeling (TUNEL) method was used to show the distribution of apoptotic cells. The vertical and horizontal position of BrdU-labeled cells was analyzed as a function of postinjection survival time. Vertical and horizontal migration of BrdU-labeled cells were detected. Cells in the central portions of the epithelium migrated vertically to become neurons as demonstrated by co-expression of olfactory marker protein. Cells at the edges migrated horizontally very slowly (less than 10% of the distance from the edge to the center of the epithelium per month), thus indicating that these cells participate in cell renewal exclusively in marginal regions. Neural turnover in the mouse vomeronasal epithelium, therefore appears to occur through a process of vertical migration. Data on the distribution of apoptotic cells indicate that a number of dividing cells throughout the epithelium, but particularly at the edges, die before becoming functional neurons. Accordingly, most dividing cells at the edges probably constitute a reservoir of stem cells dying before differentiation.     

Cell Proliferation in the Adult Rat Rostral Migratory Stream Following Exposure to Gamma Irradiation

Summary One of the few areas of the adult CNS, that are known to be competent for neuronal proliferation, is the subventricular zone (SVZ) lining the brain lateral ventricles. Cells proliferating in the SVZ migrate along a defined pathway, the rostral migratory stream (RMS), where their proliferation continues until reaching the olfactory bulb.1. In relation to the fact that brain is, in general, regarded as a radioresistant organ composed from non dividing cells, the aim of the present study was to investigate effect of ionizing radiation on proliferating cell numbers in the RMS of adult rats.2. Male Wistar rats were investigated 25 and 80 days after whole body gamma irradiation with the dose of 3 Gy. Dividing cells were labeled by bromodeoxyuridine (BrdU). BrdU-positive cells were counted by Disector program. The mean number of BrdU⁺ cells in the whole RMS and in its individual parts (vertical arm, elbow, and horizontal arm) was evaluated.3. Temporary increase in proliferating cell number (by 30%) was seen in the whole RMS at the 25th day after irradiation.4. The most expressive increase occurred in the vertical arm (by 60%) and elbow (about 37%). The values reduced till the 80th day after exposure.Our results show that ionizing irradiation significantly influences the extent of cell proliferation and migration in the adult rat RMS.     

Increased number of BrdU-labeled neurons in the rostral migratory stream of the estrous prairie vole

In the mammalian forebrain, most neurons originate from proliferating cells in the ventricular zone lining the lateral ventricles, including a discrete area of the subventricular zone in which neurogenesis continues into adulthood. The majority of the cells generated in the anterior portion of the subventricular zone (SVZa) are neuronal precursors with progeny that migrate to the olfactory bulb (OB) along a pathway known as the rostral migratory stream (RMS). The list of factors that influence the proliferation and survival of neurons in the adult brain remains incomplete, but previous studies have implicated neurotrophins in mammals and estrogen in birds. This study examined the effect of estrus induction on the proliferation of SVZa neurons in female prairie voles. Prairie voles, unlike many other rodents, are induced into estrus by chemosensory cues from a male. This olfactory-mediated process results in an increase in serum estrogen levels and the consequent induction of behavioral estrus (sexual receptivity). Female prairie voles induced into estrus by male exposure had a 92% increase in BrdU-labeled cells in the SVZa compared to females exposed to a female. Double-label immunocytochemical studies demonstrated that 80% of the BrdU-labeled cells in the RMS displayed a neuronal phenotype. Ovariectomized females exposed to a male did not show an increase in serum estrogen or BrdU labeling in the RMS. Conversely, ovariectomized females injected with estrogen were sexually receptive and had more BrdU-labeled cells in the RMS than oil-injected females. These data suggest that, in female prairie voles, estrus induction is associated with increased numbers of dividing cells in the RMS, possibly via an estrogen-mediated process.     

BrdU-, neuroD (nrd)- and Hu-studies reveal unusual non-ventricular neurogenesis in the postembryonic zebrafish forebrain

In the postembryonic zebrafish forebrain, subpial locations of neurogenesis do exist in the early cerebellar external granular layer, and--unusually among vertebrates--in the primordial pretectal (M1) and preglomerular (M2) Anlagen as shown here with 5-bromo-2'-deoxyuridine (BrdU)/Hu-immunocytochemistry and in situ hybridization of neuroD. An intermediate BrdU incubation time of 12-16 h reveals in addition to proliferative ventricularly located cells those in M1 and M2. This BrdU saturation-labeling shows--in conjunction with a Hu-assay demonstrating earliest neuronal differentiation--that proliferating cells in M1 and M2 represent neuronal progenitors. This is demonstrated by single BrdU-labeled and double BrdU-/Hu-labeled cells in these aggregates. Further, expression of NeuroD--a marker for freshly determined neuronal cells--confirms this unusual subpial postembryonic forebrain neurogenesis.     

Early determination and long-term persistence of adult-generated new neurons in the hippocampus of mice

New neurons are continually generated in the adult hippocampus, but the important question, whether adult neurogenesis is transient or leads to the lasting presence of new neurons, has not yet been answered. Dividing cells were labeled with bromodeoxyuridine (BrdU) and were investigated by means of immunofluorescence and confocal microscopy at several time-points 1 day to 11 months thereafter. BrdU-labeled neurons remained stable in number and in their relative position in the granule cell layer over at least 11 months. This finding implies that the addition of new neurons is not transient and that their final number and localization are determined early. By contrast, expression of immature markers beta-III-tubulin and doublecortin in BrdU-labeled cells, peaked early after division and was not detectable after 4 weeks. In transgenic mice expressing enhanced green fluorescent protein under the nestin promoter none of the BrdU/nestin-positive cells early after division expressed the mature marker NeuN, confirming that no dividing neurons were detected. These new data suggest that new neurons are recruited early from the pool of proliferating progenitor cells and lead to a lasting effect of adult neurogenesis.     

Regulation of parietal cell migration by gastrin in the mouse

Recent studies suggest that gastrin regulates parietal cell maturation. We asked whether it also regulates parietal cell life span and migration along the gland. Dividing cells were labeled with 5'-bromo-2'-deoxyuridine (BrdU), and parietal cells were identified by staining with Dolichos biflorus lectin. Cells positive for D. biflorus lectin and BrdU were reliably identified 10-30 days after BrdU injection in mice in which the gastrin gene had been deleted by homologous recombination (Gas-KO) and wild-type (C57BL/6) mice. The time course of labeling was similar in the two groups. The distribution of BrdU-labeled parietal cells in wild-type mice was consistent with migration to the base of the gland, but in Gas-KO mice, a higher proportion of BrdU-labeled cells was found more superficially 20 and 30 days after BrdU injection. Conversely, in transgenic mice overexpressing gastrin, BrdU-labeled parietal cells accounted for a higher proportion of the labeled pool in the base of the gland 10 days after BrdU injection. Gastrin, therefore, stimulates movement of parietal cells along the gland axis but does not influence their life span.     

Unusual morphological damage of Purkinje cells following postnatal BrdU administration in the cerebellar cortex of mouse

Postnatal development of the cerebellum lasts for weeks in rodents and can be disturbed by systemic 5-bromo-2'-deoxyuridine (BrdU) administration. This thymidine analogue incorporates into the DNA of proliferating cells, and result in more or less serious damage or death granule cells, the most actively dividing neuronal population in the developing cerebellar cortex. Further consequences of postnatal BrdU administration are the interrupted postnatal migration and integrations as well as partial loss of cerebellar Purkinje cells. In the present study, C57B16 mice were administered with 50 μg/g body weight BrdU, one sc. injection daily, between P0 and P11 postnatal days, respectively.Large "cavities" appeared in the cytoplasm of a subpopulation of Purkinje cells by P7 in about one-third of administered animals, their number are size of the cavities (and PCs exhibiting unusual morphology) decreased. EM studies revealed that the unusual Purkinje cells received numerous axonal inputs of unknown origin, first of all on their somatic and dendritic spines. The transitory appearance of a subpopulation of Purkinje cells possessing unusual morphology refers to the influence of other (neuronal, glial, or both) cells on their regular differentiation.     

Neuronal migration during the early development of the cerebral cortex

Fixed cerebral vesicles of mouse foetuses were fractured and examined with the scanning electron microscope. This method provides a study of the three dimensional developmental features of the pseudostratified columnar epithelium up to the formation of the early cortex plate. Matrix cells are a cell population of homogeneous shape, however, mitotic cells are easily identified by their spherical form. The external surface of the brain is formed by the closely packed end feet of these cells covered by a basal membrane. The formation of the cortical plate is the result of a continuous cell migration in columnar arrangement towards the pia. Glioependymal cells extend along the whole brain wall and most likely provide guidance for the migrating cell cords. The formation of the so-called migratory zone is a consequence of the growth of the basal and the horizontal prolongations of emigrating cells. The significance of the cell to cell contacts for the neuronal migration processes is discussed.     

Dual labeling with 5‐bromo‐2'‐deoxyuridine and 5‐ethynyl‐2'‐deoxyuridine for estimation of cell migration rate in the small intestinal epithelium

Small intestinal epithelium is a self‐renewing system in which the entire sequence of cell proliferation, differentiation, and removal is coupled to cell migration along the crypt‐villus axis. We examined whether dual labeling with different thymidine analogues, 5‐bromo‐2'‐deoxyuridine (BrdU) and 5‐ethynyl‐2'‐deoxyuridine (EdU), can be used to estimate cell migration rates on the villi of small intestines in rats. Rats received a single intraperitoneal injection of BrdU and EdU within a time interval, and signals in tissue sections were examined by immunohistochemistry and the “click” reaction, respectively. We successfully observed BrdU‐ and EdU‐positive cells on the epithelium with no cross‐reaction. In addition, we observed an almost complete overlapping of BrdU‐ and EdU‐positive cells in rats administered simultaneously with BrdU and EdU. By calculating the cell migration rate by dividing the distance between the median cell positions of the distribution of BrdU‐ and EdU‐positive cells by the time between the injection of BrdU and EdU, we estimated approximately 9 and 5 μm/h for the cell migration rates on the villi in the jejunum and ileum, respectively. We propose that dual labeling with BrdU and EdU within a time interval, followed by detecting with immunohistochemistry and the click reaction, respectively, is useful to estimate accurately the cell migration rate in the intestinal epithelium in a single animal.     

New GABAergic interneurons in the adult neocortex and striatum are generated from different precursors

Ongoing neurogenesis in the adult mammalian dentate gyrus and olfactory bulb is generally accepted, but its existence in other adult brain regions is highly controversial. We labeled newly born cells in adult rats with the S-phase marker bromodeoxyuridine (BrdU) and used neuronal markers to characterize new cells at different time points after cell division. In the neocortex and striatum, we found BrdU-labeled cells that expressed each of the eight neuronal markers. Their size as well as staining for gamma-aminobutyric acid (GABA), glutamic acid decarboxylase 67, calretinin and/or calbindin, suggest that new neurons in both regions are GABAergic interneurons. BrdU and doublecortin-immunoreactive (BrdU+/DCX+) cells were seen within the striatum, suggesting migration of immature neurons from the subventricular zone. Surprisingly, no DCX+ cells were found within the neocortex. NG2 immunoreactivity in some new neocortical neurons suggested that they may instead be generated from the NG2+ precursors that reside within the cortex itself.     

Newly proliferated cells in the adult male amygdala are affected by gonadal steroid hormones

Gonadal steroid hormones play an important role in the proliferation, survival, and activation of neurons. The present study was performed to examine the effects of testosterone and its metabolites on newly proliferated cells in the amygdala of adult male meadow voles (Microtus pennsylvanicus). Treatment with testosterone propionate (TP) in castrated males resulted in plasma testosterone levels similar to males following mating. TP-treated males displayed a significant increase in the density of cells labeled with a cell proliferation marker (BrdU) in the amygdala. Treatment with estradiol benzoate (EB) exerted a similar effect as TP on the density of BrdU-labeled cells, whereas 5alpha-dihydrotestosterone (DHT) was ineffective. A larger proportion (approximately 44%) of the BrdU-labeled cells in the amygdala displayed a neuronal phenotype, and a lesser percentage (approximately 35%) displayed a glial progenitor phenotype; however, treatment effects were not found in either population of cells. Hormonal effects appeared to be site-specific as no group differences were found in the dentate gyrus of the hippocampus or ventromedial hypothalamus. Finally, a time course study indicated that BrdU-labeled cells in the amygdala are present as early as 30 min following an acute injection of BrdU. Together, these data suggest that gonadal steroid hormones influence the number of newly proliferated cells in the amygdala, most likely by acting through an estrogenic mechanism, and these effects may be exerted on locally proliferating progenitors within the amygdala.     

Polysialic acid regulates the clustering, migration, and neuronal differentiation of progenitor cells in the adult hippocampus

Newborn cells of the adult dentate gyrus in the hippocampus are characterized by their abundant expression of polysialic acid (PSA), a carbohydrate attached to the neural cell adhesion molecule (NCAM). PSA+ newborn cells of the dentate gyrus form clusters with proliferating neural progenitor cells, migrate away from these clusters, and terminally differentiate. To identify the roles of PSA in the development of adult progenitors of the dentate gyrus, we injected endoneuraminidase N (endoN) into the hippocampus of adult rats to specifically cleave PSA from NCAM. Two days later, we administered the mitotic marker, 5-bromo-2'-deoxyuridine (BrdU). Three days after BrdU injection, BrdU+ cells were found inside and outside the clusters of newborn cells. In endoN-treated animals, the total number of BrdU+ cells was not changed but significantly more BrdU+ cells were present within clusters, suggesting that PSA normally facilitates the migration of progenitors away from the clusters. Seven days post-BrdU injection, endoN-treated animals had significantly more BrdU+ cells which were also positive for the mature neuronal nuclear marker NeuN compared with controls, indicating that the loss of PSA from progenitor cells increases neuronal differentiation. This report is the first demonstration that PSA is involved in controlling the spatio-temporal neuronal maturation of adult hippocampal progenitors in the normal brain. In vitro, the removal of PSA from adult-derived neural progenitors significantly enhanced neuronal differentiation, strengthening our in vivo findings and indicating that PSA removal on isolated progenitor cells, apart from a complex in vivo environment, induces neuronal maturation.     

Identification of putative bovine mammary epithelial stem cells by their retention of labeled DNA strands

Stem cells appear to retain labeled DNA for extended periods because of their selective segregation of template DNA strands during mitosis. In this study, proliferating cells in the prepubertal bovine mammary gland were labeled using five daily injections of 5-bromo-2-deoxyuridine (BrdU). Five weeks later, BrdU-labeled mammary epithelial cells were still evident. The percentage of BrdU-labeled epithelial cells was greatest in the lower region of the mammary gland, near the gland cistern, and was decreased toward the periphery of the parenchymal region, where the ducts were invading the mammary fat pad. Increased numbers of BrdU-labeled epithelial cells in basal regions of the gland are likely a consequence of decreased proliferation rates and increased cell cycle arrest in this area. In peripheral regions of mammary parenchyma, the percentage of heavily labeled epithelial cells averaged 0.24%, a number that is consistent with estimates of the frequency of stem cells in the mouse mammary gland. Epithelial label-retaining cells seemingly represent a slowly proliferating population of cells, as 5.4% of heavily labeled cells were positive for the nuclear proliferation antigen Ki67. Because epithelial label-retaining cells contain estrogen receptor (ER)-negative and ER-positive cells, they apparently comprise a mixed population, which I suggest is composed of ER-negative stem cells and ER-positive progenitors. Continuing studies will address the usefulness of this technique to identify bovine mammary stem cells and to facilitate studies of stem cell biology.     

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.     

The effects of social environment on adult neurogenesis in the female prairie vole

In the mammalian brain, adult neurogenesis has been found to occur primarily in the subventricular zone (SVZ) and dentate gyrus of the hippocampus (DG) and to be influenced by both exogenous and endogenous factors. In the present study, we examined the effects of male exposure or social isolation on neurogenesis in adult female prairie voles (Microtus ochrogaster). Newly proliferated cells labeled by a cell proliferation marker, 5-bromo-2'-deoxyuridine (BrdU), were found in the SVZ and DG, as well as in other brain areas, such as the amygdala, hypothalamus, neocortex, and caudate/putamen. Two days of male exposure significantly increased the number of BrdU-labeled cells in the amygdala and hypothalamus in comparison to social isolation. Three weeks later, group differences in BrdU labeling generally persisted in the amygdala, whereas in the hypothalamus, the male-exposed animals had more BrdU-labeled cells than did the female-exposed animals. In the SVZ, 2 days of social isolation increased the number of BrdU-labeled cells compared to female exposure, but this difference was no longer present 3 weeks later. We have also found that the vast majority of the BrdU-labeled cells contained a neuronal marker, indicating neuronal phenotypes. Finally, group differences in the number of cells undergoing apoptosis were subtle and did not seem to account for the observed differences in BrdU labeling. Together, our data indicate that social environment affects neuron proliferation in a stimulus- and site-specific manner in adult female prairie voles.     

Asymmetrical growth, differential cell proliferation, and dynamic cell rearrangement underlie epithelial morphogenesis in mouse molar development

During molar development from the cap to bell stage, the morphology of the enamel knots, inner dental epithelium, and epithelial-mesenchymal junction dynamically changes, leading to the formation of multiple cusps. To study the basic histological features of this morphogenetic change, we have investigated the cell arrangement, mitosis, and apoptosis simultaneously in the developing first lower molar of the mouse by means of BrdU injection and immunostaining for P-cadherin, BrdU, and single-stranded DNA. At the typical cap stage, the primary enamel knot shows a characteristic cell arrangement, absence of mitosis, and abundant apoptosis, but also actively dividing cells at its lateral margins. Two secondary enamel knots then appear in the anterior part of the tooth germ. One is completely non-proliferating, whereas the other contains dividing cells, indicating asymmetrical growth of the inner dental epithelium. From this transitional stage to the early bell stage, additional minor BrdU-negative domains appear, and at the same time, the cell arrangement in the inner dental epithelium rapidly changes to show regional differences. Comparisons between the histology and the distribution of BrdU-positive cells have revealed that both the regionally different cell rearrangement and the differential cell proliferation in the enamel knots and inner dental epithelium probably play a significant role in multiple cusp formation.     

Proliferation and migration of glial precursor cells in the developing rat spinal cord

The mechanisms that control the production and differentiation of glial cells during development are difficult to unravel because of displacement of precursor cells from their sites of origin to their permanent location. The two main neuroglial cells in the rat spinal cord are oligodendrocytes and astrocytes. Considerable evidence supports the view that oligodendrocytes in the spinal cord are derived from a region of the ventral ventricular zone (VZ). Some astrocytes, at least, may arise from radial glia. In this study a 5-Bromo-2′-deoxyuridine (BrdU) incorporation assay was used to identify proliferating cells and examine the location of proliferating glial precursor cells in the embryonic spinal cord at different times post BrdU incorporation. In this way the migration of proliferating cells into spinal cord white matter could be followed. At E14, most of the proliferating cells in the periventricular region were located dorsally and these cells were probably proliferating neuronal precursors. At E16 and E18, the majority of the proliferating cells in the periventricular region were located ventrally. In the white matter the number of proliferating cells increased as the animals increased in age and much of this proliferation occurred locally. BrdU labelling showed that glial precursor cells migrate from their ventral and dorsal VZ birth sites to peripheral regions of the cord. Furthermore although the majority of proliferating cells in the spinal cord at E16 and E18 were located in the ventral periventricular region, some proliferating cells remained in the dorsal VZ region of the cord.     

Morphological evidence of basal keratinocyte migration during the re-epithelialization process

The regeneration of wounded stratified epithelium is accomplished via the migration of keratinocytes from the margins of the wound. However, the process of keratinocyte migration on the wound surface and the role of epithelial stem cells during re-epithelialization remain to be elucidated. Therefore, we administered BrdU to embryonic mice and generated epithelial defects on the buccal mucosa of these mice at two weeks after birth, using CO₂ laser irradiation, with which we removed the entire thickness of the epithelium. In the unwounded epithelium, cytokeratin 14, p63, and BrdU were localized within the basal layer of the epithelium, but the majority of cells within the regenerated epithelium were immunopositive for these proteins. PCNA-negative and BrdU-positive basal keratinocytes, which evidence a slow cell cycle, were localized solely within the basal layer of the unwound epithelium facing the tips of dermal papillae. After laser irradiation, these basal keratinocytes facing the tips of the papillae evidenced positive immunoreactivity for PCNA, in addition to BrdU. These results indicate that epithelial stem cells of oral mucosa may be localized in the basal layer of the epithelium facing the tips of dermal papillae, and may migrate laterally with other basal keratinocytes in response to external stimuli. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.