BrdU Exposure Research Articles

Cell cycle analysis in the rat external granular layer evaluated by several bromodeoxyuridine immunoperoxidase staining protocols.

An important step in bromodeoxyuridine (BrdU) immunohistochemistry is the production of single-stranded DNA to make the incorporated BrdU accessible to the antibodies. This paper examines the effect of distinct DNA denaturation pretreatments (DNase I, sodium citrate buffer, endonuclease Eco RI and exonuclease III, and HCl hydrolysis) on detection of BrdU. We found that all the methods used in the partial denaturation of DNA combined good nuclear immunostaining with acceptable tissue integrity. We also observed that these immunohistochemical protocols revealed a spatial pattern in the distribution of DNA-synthesizing cells within the cerebellar external granular layer (EGL) of 10-day-old rats, allowing us to estimate the fraction of S-phase cells. Our results indicate that detection of BrdU-stained cells is affected by the distinct histological procedures used in such detection. Additionally, as the duration and phases of the cell cycle in EGL neuroblasts are estimated in accordance with BrdU detection, an effect on this detection can render the measurement of cell cycle inaccurate. The present work shows that DNase I and citrate buffer, at appropriate conditions, may be good alternatives for acid denaturation. However, they are less sensitive than autoradiographic techniques that use 3H-thymidine administration. Finally, current data reveal that short survival times after a single BrdU exposure do not seem to affect the cell cycle progression of the EGL neuroblasts.

Repeated exposure to 5-bromo-2′-deoxyuridine causes decreased proliferation and low-grade inflammation in the lungs of mice

Incorporation of 5-bromo-2′-deoxyuridine (BrdU) into proliferating cells has been used to label dividing cells in many tissues. Although BrdU has been shown to be genotoxic, teratogenic and mutagenic, such adverse effects have largely been ignored by researchers. We determined whether long-term BrdU exposure causes any histopathological changes in the lungs of mice. Eight-week-old male C57/BL6J mice were administered BrdU by intraperitoneal injection on 3 consecutive days of each week for 14 weeks. While no obvious structural changes such as tissue damage, fibrosis, emphysema, airway remodeling, vascular thickening or tumorigenesis were noted, a moderate degree of macrophage infiltration was observed in the airways and lung parenchyma in the lungs of the mice exposed repeatedly to BrdU (BrdU-exposed mice). The proliferative activities of the airway and alveolar epithelial and mesenchymal cells were reduced in the BrdU-exposed mice, although the numbers of these cells in the lungs were maintained. Double immunofluorescence study of the lungs of the BrdU-exposed mice showed overexpression of IL-6 in the airway epithelial and alveolar wall cells, some of which were also double-positive for BrdU. These results indicate that long-term exposure to BrdU inhibits cell proliferation and induces low-grade inflammation in the lungs of mice. Our findings underscore the need for caution in the interpretation of studies that involve long-term exposure to BrdU.

Valproic acid decreases cell proliferation and migration in the cerebellum of zebrafish larvae

Valproic acid (VPA) is used as an antiepileptic drug or mood stabilizer. Recent studies have shown that exposure to VPA during embryonic development alters neural progenitor cell proliferation. The main goal of this investigation was to elucidate the effects of VPA on cell proliferation in the cerebellum of zebrafish larvae using immunohistochemistry. 5-bromo-2'-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA)-labeled cells were mostly found in the ventral cerebellum proliferation zone and external granular layer of the cerebellum from 5 days postfertilization zebrafish immediately after treatment with BrdU for 12 h. Subsequently, BrdU-labeled cells were mostly found in the medial zone of the cerebellar plate 48 h after terminating BrdU treatment. Pretreatment with 2-mM VPA for 3 h prior to BrdU exposure reduced the number of BrdU-labeled cells both immediately and 48 h after terminating BrdU exposure. Posttreatment with VPA following BrdU exposure reduced the migration of BrdU-labeled cells, represented by a decreased number of BrdU-positive cells in the medial zone of the cerebellar plate. These results suggest that VPA may delay brain development by hindering cell proliferation and migration during the early developmental period.

Birth, survival and differentiation of neurons in an adult crustacean brain

Life-long neurogenesis is a characteristic feature of many vertebrate and invertebrate species. In decapod crustaceans, new neurons are added throughout life to two cell clusters containing local (cluster 9) and projection (cluster 10) interneurons in the olfactory pathway. Adult-born neurons in clusters 9 and 10 in crayfish have the anatomical properties and chemistry of mature neurons by 6 months after birth. Here we use 5-bromo-2'-deoxyuridine (BrdU) incorporation to pulse label mitotically active cells in these cell clusters, followed by a survival time of up to 8 months, during which crayfish (Cherax destructor) were sacrificed at intervals and the numbers of BrdU-labeled cells quantified. We find a decrease in the numbers of BrdU-labeled cells in cell cluster 10 between the first and second weeks following BrdU exposure, suggesting a period of cell death shortly after proliferation. Additional delayed cell divisions in both cell clusters are indicated by increases in labeled cells long after the BrdU clearing time. The differentiation time of these cells into neurons was defined by detection of the first immunoreactivity for the transmitter SIFamide in cluster 10 BrdU-labeled cells, which begins at 4 weeks after BrdU labeling; the numbers of SIFamide-labeled cells continues to increase over the following month. Experiments testing whether proliferation and survival of Cluster 10 cells are influenced by locomotor activity provided no evidence of a correlation between activity levels and cell proliferation, but suggest a strong influence of locomotor activity on cell survival.

P53 Mutation suppresses adult neurogenesis in medaka fish (Oryzias latipes)

Tumor suppressor p53 negatively regulates self-renewal of neural stem cells in the adult murine brain. Here, we report that the p53 null mutation in medaka fish (Oryzias latipes) suppressed neurogenesis in the telencephalon, independent of cell death. By using 5-bromo-29-deoxyuridine (BrdU) immunohistochemistry, we identified 18 proliferation zones in the brains of young medaka fish; in situ hybridization showed that p53 was expressed selectively in at least 12 proliferation zones. We also compared the number of BrdU-positive cells present in the whole telencephalon of wild-type (WT) and p53 mutant fish. Immediately after BrdU exposure, the number of BrdU-positive cells did not differ significantly between them. One week after BrdU-exposure, the BrdU-positive cells migrated from the proliferation zone, which was accompanied by an increased number in the WT brain. In contrast, no significant increase was observed in the p53 mutant brain. Terminal deoxynucleotidyl transferase (dUTP) nick end-labeling revealed that there was no significant difference in the number of apoptotic cells in the telencephalon of p53 mutant and WT medaka, suggesting that the decreased number of BrdU-positive cells in the mutant may be due to the suppression of proliferation rather than the enhancement of neural cell death. These results suggest that p53 positively regulates neurogenesis via cell proliferation.

Hyperactivity induced by prenatal BrdU exposure across several experimental conditions

Behavioral results are sometimes not reproducible even in the positive controls of developmental neurotoxicity (DNT) tests. Effects of several factors on the results should be considered. In the present paper, we examined the effects of strain-, gender-, and test-condition differences on BrdU-induced hyperactivity. The results showed that BrdU-induced hyperactivity was reproducible in two rat strains (SD and F344 rats), rodent species (rat and mouse), and both sexes. When the level of background sound in a test room was increased, the hyperactivity was persistent, resulting in no effect of background sound on BrdU-induced hyperactivity. Thus, we have demonstrated that the BrdU-animal model is a useful positive control via prenatal exposure to validate the entire DNT test process.

The Endothelial Antigen ESAM Monitors Reversible Conversion of Hematopoietic Stem Cells Between Dormancy and Self-Renewal

Abstract 2337Although hematopoietic stem cells (HSC) are characterized with self-renewal and pluri-potential, their cell-cycle status and differentiating behavior do fluctuate according to the physiological requirement. In the homeostatic state of adult bone marrow (BM), HSC are likely to be quiescent so that they can evade exhaustion or mutation. However, when BM is injured by irradiation and/or anti-cancer drugs, HSC need to proliferate to restore normal hematopoiesis. Then, after re-establishment of homeostasis, activated HSC return to be quiescent. Molecular crosstalk between HSC and BM microenvironment is thought to elaborately control the status of HSC, but precise mechanisms remain unknown. If the conversion of HSC between dormancy and self-renewal could be accurately monitored, the method should be useful to understand how the HSC status is regulated.Our previous study demonstrated that endothelial cell-selective adhesion molecule (ESAM) is a useful marker for murine HSC throughout life. In the present study, we examined if the ESAM level reflects the HSC status between dormancy and activation. Firstly we monitored ESAM levels of the Lin− Sca1+ c-kit+ (LSK) HSC-enriched fraction in BM after a single 5-FU injection (150 mg/kg) by flow cytometry. From 2 to 9 days after the 5-FU injection, ESAM levels on the LSK fraction remarkably increased. Indeed, the mean fluorescence intensity of ESAM expression on HSC increased by 9.6-fold in 5 days after 5-FU injection. The increase of ESAM expression was more drastic than that of other endothelial-related markers such as CD34 (1.6-fold). After reaching to the maximum peak around day 5–6, ESAM level gradually decreased and returned to the homeostatic level by 12 days after 5-FU. Interestingly, the ESAM up-regulation on HSC was abrogated when inhibitory drugs for NF-kB and topoisomerase-II were given after 5-FU injection. Furthermore, short-term BrdU exposure proved that the ESAMhi cells after 5-FU treatment are actually active in the cell cycle status. Then, the immuno-histochemical analyses were performed to locate the activated HSC in 5-FU treated BM. Since more than 80% of the Lin− ESAMhi Sca1+ cells were found within 20 μm from vascular endothelium, the activated HSC seemed to be intimate with endothelial cells and/or vascular-related cells.Next, we performed functional assessments of the ESAMlow LSK and ESAMhi LSK fractions sorted from 5-FU-treated BM. In methylcellulose cultures, while both fractions contained a number of hematopoietic progenitors, CFU-Mix, primitive multipotent progenitors, were significantly enriched in the ESAMhi fraction (10±0 vs. 48.5±2.1 per 200 ESAMlow or ESAMhi LSK cells, respectively). In the in vivo long-term reconstitution assays, we transplanted 2,000 CD45.1+ ESAMlow or ESAMhi LSK cells with 2 × 105 CD45.2+ competitor BM cells into lethally irradiated CD45.2+ mice. Sixteen weeks after transplantation, the mice transplanted with ESAMhi LSK cells showed significantly higher chimerisms of CD45.1+ cells than those transplanted with ESAMlow LSK, suggesting that long-term HSC are enriched in the ESAMhi fraction. It is noteworthy that the ESAMhi CD45.1+ LSK fraction re-constituted a CD45.1+ LSK population in the CD45.2+ recipient BM, whose ESAM expression levels lowered to the homeostatic level.The results above suggested that ESAM expression level mirrors the activation status of HSC after BM injury. However, it remains unclear if ESAM plays an important role in the hematopoietic recovery. Although we did not observe significant phenotypes except slight anemia in homeostatic ESAM KO mice, we presumed that substantial BM stress might reveal physiological importance of the ESAM expression. At day 5 after injecting 200mg/kg 5-FU, we found that leukocytes and platelet were remarkably decreased in KO mice. Furthermore, the KO mice showed severe anemia (Hb; WT 10.4±1.1 g/dl vs. KO 6.0±1.7 g/dl at day 10), and two of five mice died at day 12. In addition, we observed LSK Flt3− HSC as well as total mononuclear cells more significantly decreased in the KO mice.In summary, our data have shown that ESAM serves as a strong tool to monitor the conversion between dormancy and proliferation of adult BM HSC. In addition, the data from ESAM KO mice have suggested that ESAM is indispensable for normal hematopoietic recovery after BM injury. Further studies should address physiological meanings of the high ESAM level on active HSC. Disclosures:No relevant conflicts of interest to declare.

Purinergic Signals Regulate Daily S-Phase Cell Activity in the Ciliary Marginal Zone of the Zebrafish Retina

Regeneration and growth that occur in the adult teleost retina have been helpful in identifying molecular and cellular mechanisms underlying cell proliferation and differentiation. Here, it is reported that S-phase cell number, in the ciliary marginal zone (CMZ) of the adult zebrafish retina, exhibits day-night variations with a mid-light phase peak. Oscillations persist for 24 h in constant darkness (DD), suggesting control by a circadian component. However, variations in the S-phase nuclei number were rapidly dampened and not present during and after a second day in DD. An ADPβS treatment significantly enhanced S-phase activity at night to mid-light levels, as assessed by in vivo BrdU incorporation in a 2-h interval. Moreover, daylight increase in S-phase cell number was completely abolished when extracellular nucleotide levels or their extracellular hydrolysis by ectonucleoside triphosphate diphosphohydrolases (NTPDases) were significantly disrupted or when a selective antagonist of purinergic P2Y1 receptors was intraocularly injected before BrdU exposure. Extracellular nucleotides and NTPDase action were also important for maintaining nocturnal low levels of S-phase activity in the CMZ. Finally, we showed that mRNAs of NTPDases 1, 2 (3 isoforms), and 3 as well as of P2Y1 receptor are present in the neural retina of zebrafish. NTPDase mRNA expression exhibited a 2-fold increment in light versus dark conditions as assessed by quantitative RT-PCR, whereas P2Y1 receptor mRNA levels did not show significant day-night variations. This study demonstrates a key role for nucleotides, principally ADP as a paracrine signal, as well as for NTPDases, the plasma membrane-bound enzymes that control extracellular nucleotide concentration, for inducing S-phase cell entry in the CMZ-normally associated with retinal growth-throughout the light-dark cycle.

The effects of neonatal forebrain cholinergic lesion on adult hippocampal neurogenesis

Previous work in our laboratory indicated that cholinergic denervation by intraventricular infusion of 192-IgG-saporin on postnatal day 7 (N192S) reduced the number of cells in the dentate gyrus expressing doublecortin, a marker for immature neuroblasts. In addition, there was a suggestion that N192S impaired the neurogenic response to environmental enrichment (EE). The purpose of the present study was to further characterize the impact of N192S on the proliferation, differentiation and survival of newborn cells in the dentate gyrus. After 42days in EE or standard housing, all rats received injections of 5-bromo-2-deoxyuridine (BrdU) to label dividing cells. They were sacrificed either one day (to assess cell proliferation) or 28days later (to assess survival and differentiation of BrdU-labelled cells). EE failed to increase neurogenesis, thereby preventing determination of the effects of N192S on EE-induced neurogenesis. However, N192S by itself reduced the number of BrdU+ cells 1day after BrdU exposure, but did not alter the number of cells expressing the cell cycle marker Ki-67. The number of BrdU+ cells 28days after BrdU exposure was not affected by N192S. Confocal analysis of BrdU+ cells double-immunofluorescently stained to detect NeuN or S100B indicated that N192S did not alter the proportion of new cells that adopted a neuronal or glial identity. The most plausible explanation for these results is that N192S accelerates the death of newborn cells, but does not change their overall survival rate or phenotypic differentiation.

Stem and progenitor cell compartments within adult mouse taste buds

Adult taste buds are maintained by the lifelong proliferation of epithelial stem and progenitor cells, the identities of which have remained elusive. It has been proposed that these cells reside either within the taste bud (intragemmal) or in the surrounding epithelium (perigemmal). Here, we apply three different in vivo approaches enabling single-cell resolution of proliferative history to identify putative stem and progenitor cells associated with adult mouse taste buds. Experiments were performed across the circadian peak in oral epithelial proliferation (04:00 h), a time period in which mitotic activity in taste buds has not yet been detailed. Using double label pulse-chase experiments, we show that defined intragemmal cells (taste and basal) and perigemmal cells undergo rapid, sequential cell divisions and thus represent potential progenitor cells. Strikingly, mitotic activity was observed in taste cells previously thought to be postmitotic (labelled cells occur in 30% of palatal taste buds after 1 h of BrdU exposure). Basal cells showed expression of the transcription factor p63, required for maintaining the self-renewal potential of various epithelial stem cell types. Candidate taste stem cells were identified almost exclusively as basal cells using the label-retaining cell approach to localize slow-cycling cells (0.06 +/- 0.01 cells per taste bud; n = 436 taste buds). Together, these results indicate that both stem- and progenitor-like cells reside within the mammalian taste bud.

Identification of a stem cell niche in the zone of Ranvier within the knee joint

A superficial lesion of the articular cartilage does not spontaneously self-repair and has been suggested to be partly due to lack of progenitor cells within the joint that can reach the site of injury. To study whether progenitor cells are present within the joint, 3-month-old New Zealand white rabbits were exposed to bromodeoxyuridine (BrdU) for 12 consecutive days and were then sacrificed 4, 6, 10, 14, 28 and 56 days after the first BrdU administration. Presence of BrdU and localization of progenitor markers were detected using immunohistochemistry. After 10 days of BrdU exposure, BrdU-positive cells, i.e. proliferating cells, were abundantly detected in the epiphyseal plate, the perichondrial groove of Ranvier, and in all zones of the articular cartilage. After a wash-out period, BrdU-positive cells were still present, i.e. those considered to be progenitor cells, in these regions of the knee except for the proliferative zone of the epiphyseal plate. Cells in the perichondrial groove of Ranvier were further positive for several markers associated with progenitor cells and stem cell niches, including Stro-1, Jagged1, and BMPr1a. Our results demonstrate that a small population of progenitor cells is present in the perichondrial groove of Ranvier as well as within the articular cartilage in the knee. The perichondrial groove of Ranvier also demonstrates the properties of a stem cell niche.

Reduced HSPA9B Expression, a 5q31.2 Candidate Gene, in Primary Human CD34+ Cells Recapitulates Features of Ineffective Hematopoiesis Observed in MDS.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis. Deletions spanning chromosome 5q31.2 are among the most common karyotypic abnormalities in MDS, and evidence suggests that del(5q) maybe an early genetic event. We hypothesize that haploinsufficiency of one or more of the 28 candidate genes in the 5q31.2 commonly deleted segment (CDS) are important for MDS pathogenesis. Many genes in the CDS, including HSPA9B, have dose reduced mRNA levels in CD34+ cells from MDS patients with del(5q), and zebrafish carrying a heterozygous mutation in the orthologue of the human HSPA9B gene display increased apoptosis in blood cells. Therefore, we asked whether reduced HSPA9B expression in primary human hematopoietic cells can recapitulate key features of ineffective hematopoiesis (i.e. abnormal proliferation, apoptosis, and differentiation). To address this question, we purified human CD34+ hematopoietic progenitors from cord blood samples (>90% purity) and infected them with a shRNA expressing lentivirus that also carries the puromycin resistance gene. Cell culture densities were normalized after 4 days in selection medium, and 3 days later apoptosis (AnnexinV/7-AAD), cell cycle status (BrdU/7-AAD), and erythroid or myeloid differentiation was measured using flow cytometry. We performed 4–5 independent experiments using 5 individual shRNAs targeting HSPA9B (30% to >90% knockdown) and 2 control shRNAs. Cells transduced with control shRNAs expand 18.2 fold from days 4–7 in RBC unilineage differentiation culture medium (25ng/ml SCF, 10ng/ml IL-6, 10ng/ml IL-3 and 0.5U/ml Epo), vs. only a 0.8–5.2 fold expansion when HSPA9B is knocked down (N=5 for each shRNA, p≤0.007). The reduced cell numbers observed in HSPA9B knockdown cultures is associated with an increase in apoptosis and a decrease in the number of cells entering S-phase compared to control shRNA expressing cells. 8.2% of cells in control cultures were AnnexinV+/7AAD+ vs. 20–62% of cells in HSPA9B knockdown cultures (N=5 for each shRNA, p≤0.0004). Following 1 hour of BrdU exposure, 60% of cells in control cultures were in S-phase vs. only 22–48% of HSPA9B knockdown cultures (N=5 for each shRNA, p≤0.008). In addition, after 7 days in RBC unilineage differentiation culture medium, 58% of control shRNA expressing cells were CD71+/Gycophorin A+ vs. 7.1–32% of cells in the HSPA9B knockdown cultures (N=4 for each shRNA, p≤0.004). The decreased number of GPA+ cells in HSPA9B knockdown cultures was concomitant with retention of CD34+ expression on cells. Similar results were observed using myeloid unilineage culture conditions (10ng/ml SCF, 100ng/ml G-CSF, 20% FCS), where the number of cells expressing CD15+ was reduced from 47% in control cultures to 28–37% in HSPA9B knockdown cultures. A delay in erythroid and myeloid differentiation was confirmed by cell morphology. Lentiviral knockdown of the murine orthologue of HSPA9B (Hspa9a) in 2 independent transduction/transplantation experiments resulted in a loss of transduced cells over 2 months (2 shRNAs each) compared to a control shRNA, implicating a cell intrinsic defect when Hspa9a levels are reduced in vivo. Collectively, these results implicate that reduced HSPA9B expression in human CD34+ progenitor cells results in abnormal proliferation, increased apoptosis, and altered differentiation, key features of ineffective hematopoiesis, in a dose dependent manner.

In Vivo Lineage Tracing Defines the Role of Acinar-to-Ductal Transdifferentiation in Inflammatory Ductal Metaplasia

Chronic injury results in regeneration of normal pancreatic tissue and formation of a metaplasia of ductal phenotype. Metaplastic ductal lesions are seen in pancreatitis as well as in specimens of pancreatic cancer and are thought to represent a condition with increased risk of neoplasia. Acinar-to-ductal transdifferentiation is thought to be the source of this metaplasia. This has been suggested for flat duct-like lesions called tubular complexes and for lesions exhibiting a mucinous metaplasia. However, available studies are based on interpretation of static data rather than on direct evidence. Transdifferentiation from acinar to ductal cells has never been confirmed in the adult pancreas. Here, we use Cre-loxP-based genetic lineage tracing in vivo to investigate whether transdifferentiation of acinar cells contributes to regeneration and metaplasia in pancreatitis. The results show that transdifferentiation does not play a role in regeneration of normal tissue. Acinar cells are regenerated by preexisting acinar cells and not from other cell types. Three different types of metaplastic ductal lesions are observed and analyzed. Whereas the majority of metaplastic lesions are not of acinar origin, acinar-to-ductal transdifferentiation is identified in a minority of mucinous metaplastic lesions. Here, we provide direct evidence that acinar-to-ductal transdifferentiation occurs in the adult pancreas in vivo. However, it accounts for only a minority of metaplastic lesions.

Exposure to 5‐Bromo‐2′‐deoxyuridine induces oxidative stress and activator protein‐1 DNA binding activity in the embryo

During organogenesis the embryo is highly sensitive to oxidative stress. We hypothesize that oxidative stress and activation of a redox-sensitive transcription factor, activator protein-1 (AP-1), are early indicators of embryonic stress in response to a teratogenic insult. 5-Bromo-2'-deoxyuridine (BrdU) was chosen as a model teratogen to test this hypothesis; BrdU is a thymidine analog that is incorporated into replicating DNA. Timed pregnant CD1 mice were given vehicle or BrdU (400, 600, 800, or 1000 mg of BrdU/kg of body weight) on gestation day 9 (GD 9). Oxidative stress, assessed as the ratio of glutathione disulfide (GSSG) to reduced glutathione (GSH), and AP-1 DNA binding activity (c-Fos- and c-Jun-dependent DNA binding) were measured in the maternal livers and embryos 0.5, 3, and 6 hr after treatment. External and skeletal malformations were assessed on GD 18. N-acetylcysteine, a glutathione precursor, was coadministered with BrdU to further explore the relationship between teratogenicity and redox homeostasis. BrdU exposure produced a dose-dependent increase in skeletal malformations, which included polydactyly, and delayed ossification of the sternebrae and vertebrae. Exposure to teratogenic doses of BrdU depleted GSH concentrations and increased oxidative stress, as assessed by the GSSG:GSH ratio, in both maternal livers and embryos. While c-Jun DNA binding activity in embryos was not affected, c-Fos DNA binding activity was elevated significantly 3 hr after BrdU exposure. Coadministration of N-acetylcysteine decreased the skeletal malformations and AP-1 DNA binding activity induced by BrdU. BrdU exposure induced an embryonic stress response manifested as an increase in oxidative stress and AP-1 DNA binding activity; these data support the hypothesis that disturbances in redox homeostasis mediate the response of the conceptus to a teratogenic insult.

The evaluation of early embryonic neurogenesis after exposure to the genotoxic agent 5-bromo-2′-deoxyuridine in mice

Developmental neurotoxicity (DNT) is an important issue in children's health. Neurogenesis occurs throughout the early fetal to the postnatal period. The proliferation of embryonic stem cells can be a target for toxicants, especially genotoxic compounds. 5-Bromo-2′-deoxyuridine (BrdU), a thymidine analogue, has been used as a marker for proliferating cells. However, we reported that prenatal BrdU exposure induced behavioral abnormalities such as hyperactivity in rat and mouse offspring. In this study, to further clarify the toxic effect of BrdU on the early neurogenesis and to examine the usefulness of the evaluation of this process in DNT, C57BL/6 mice were exposed to 100 mg/kg of BrdU once on gestational day (GD) 9 or 11, and serial sections from a wide variety of areas of the embryonic brains 24 h after the exposure were examined. BrdU exposure on GD11 induced cell death in some specific areas, such as the neocortex and striatum, but not in the substantia nigra, raphe and pons, even though BrdU was incorporated into those cells. BrdU decreased the number of cells positive for phosphorylated histone 3 (phospho-histone 3), a marker for proliferating cells at metaphase of mitosis, in the cortex, mammillary body and cerebellum, suggesting that BrdU affected the proliferation of neural stem cells. Exposure on GD9 did not induce cell death in the fetal brain. These results indicate that BrdU actually impaired the early neurogenesis, supporting the postnatal results, and demonstrated that embryonic neurogenesis has heterogeneous sensitivity to the genotoxic agents BrdU that differs according to the area and developmental stage. The evaluation of events in early neurogenesis such as the proliferation of neural stem cells shortly after chemical exposure will be one of the valuable endpoints for studying postnatal neurodevelopmental disorders.

Immunogold-labeled S-phase neoblasts, total neoblast number, their distribution, and evidence for arrested neoblasts in Macrostomum lignano (Platyhelminthes, Rhabditophora)

Neoblasts in Platyhelminthes are the only cells to proliferate and differentiate into all cell types. In Macrostomum lignano, the incorporation of 5'-bromo-2'-deoxyuridine (BrdU) in neoblasts confirmed the distribution of S-phase cells in two lateral bands. BrdU labeling for light and for transmission electron microscopy (TEM) identified three populations of proliferating cells: somatic neoblasts located between the epidermis and gastrodermis (mesodermal neoblasts), neoblasts located within the gastrodermis (gastrodermal neoblasts), and gonadal S-phase cells. In adults, three stages of mesodermal neoblasts (2, 2-3, and 3) defined by their ultrastructure were found. Stage 1 neoblasts where only seen in hatchlings. These stages either were phases within the S-phase of one neoblast pool or were subsequent stages of differentiating neoblasts, each with its own cell cycle. Regular TEM and immunogold labeling provided the basis for calculating the total number of neoblasts and the ratio of labeled to non-labeled neoblasts. Somatic neoblasts represented 6.5% of the total number of cells. Of these, 27% were labeled in S-phase. Of this fraction, 33% were in stage 2, 46% in stage 2-3, and 21% in stage 3. Immunogold labeling substantiated results concerning the differentiation of neoblasts into somatic cells. Non-labeled stage 2 neoblasts were present, even after a 2-week BrdU exposure. Double labeling of mitoses and FMRF-amide revealed a close spatial relationship of mesodermal neoblasts with the nervous system. Immunogold-labeled sections showed that nearly 70% of S-phase cells were in direct contact or within 5 microm from nerve cords.

The transcription factor MEF/ELF4 regulates the quiescence of primitive hematopoietic cells

The transcriptional circuitry that regulates the quiescence of hematopoietic stem cells is largely unknown. We report that the transcription factor known as MEF (or ELF4), which is targeted by the t(X;21)(q26;q22) in acute myelogenous leukemia, regulates the proliferation of primitive hematopoietic progenitor cells at steady state, controlling their quiescence. Mef null HSCs display increased residence in G0 with reduced 5-bromodeoxyuridine incorporation in vivo and impaired cytokine-driven proliferation in vitro. Due to their increased HSC quiescence, Mef null mice are relatively resistant to the myelosuppressive effects of chemotherapy and radiation. Thus, MEF plays an important role in the decision of stem/primitive progenitor cells to divide or remain quiescent by regulating their entry to the cell cycle.

Intrinsic and spontaneous neurogenesis in the postnatal slice culture of rat hippocampus

Organotypic slice culture preserves the morphological and physiological features of the hippocampus of live animals for a certain time. The hippocampus is one of exceptional regions where neurons are generated intrinsically and spontaneously throughout postnatal life. We investigated the possibility that neurons are generated continuously at the dentate granule cell layer (GCL) in slice culture of the rat hippocampus. Using 5-bromodeoxyuridine (BrdU) labelling and retrovirus vector transduction methods, the phenotypes of the newly generated cells were identified immunohistochemically. At 4 weeks after BrdU exposure, BrdU-labelled cells were found in the GCL and were immunoreactive with a neuronal marker, anti-NeuN. There were fibrils immunoreactive with anti-glial fibrillary acidic protein (GFAP), an astrocyte marker, in the layer covering the GCL and occasionally encapsulated BrdU-labelled nuclei. When the newly divided cells were marked with the enhanced green fluorescent protein (EGFP) using a retrovirus vector, these cells had proliferative abilities throughout the following 4-week cultivation period. Four weeks after the inoculation, the EGFP-expressing cells consisted of various phenotypes of both early and late stages of differentiation; some were NeuN-positive cells with appearances of neurons in the GCL and some were immunoreactive with anti-Tuj1, a marker of immature neurons. Some EGFP-expressing cells were immunoreactive with anti-GFAP or anti-nestin, a marker of neural progenitors. The present study suggests that slice cultures intrinsically retain spontaneous neurogenic abilities for their cultivation period. The combination of slice culture and retrovirus transduction methods enable the newly divided cells to be followed up for a long period.

Studies on BrdU labeling of hematopoietic cells: stem cells and cell lines.

Studies using chronic in vivo BrdU exposure, isolating primitive stem cells, and determining BrdU labeling, indicate that stem cells cycle. BrdU is also incorporated into DNA during damage/repair. DNA, which has incorporated BrdU due to cycle transit is heavier than normal, while the density of DNA with damage/repair incorporation is intermediate. DNA density of purified lineage-rhodamine low (rho(low)) Hoechst low (Ho(low)) stem cells or FDC-P1 cell line cells-was assessed in vitro, after exposure to cytokines and BrdU (cycling model) or cytokines and BrdU with bleomycin to induce strand breaks and hydroxyurea to halt cycle progression (damage/repair model). We determined DNA density using cesium chloride (CsCl) gradients and either fluorometry or dot blot chemiluminesence. DNA from BrdU labeled cycling Lin-rho(lo)Ho(lo) or FDC-P1 cells was heavier than normal DNA, while damage repair DNA had an intermediate density. We then assessed BrdU labeling of Lin-rho(lo)Ho(lo) cells in vivo. We found that 70.9% of lin-rho(lo)Ho(lo) cells labeled at 5 weeks. DNA density of these cells was low, in the damage/repair range, but similar results were obtained with stem cells, which had proliferated in vivo. Dilution of BrdU in in vitro culture of proliferating FDC-P1 cells also resulted in damage/repair density. We conclude that in vitro BrdU labeling models can distinguish between proliferation and damage/repair, but that we cannot obtain high enough in vivo levels to address this issue. All together, while we cannot absolutely exclude damage/repair as contributing to stem cell BrdU labeling, the data indicate that primitive bone marrow stem cells are probably a cycling population.

Identification of newborn cells by BrdU labeling and immunocytochemistry in vivo.

5-Bromodeoxyuridine, variously abbreviated as BrdU, BUdR, and BrdUrd, is a halogenated thymidine analog that is permanently integrated into the DNA of dividing cells during DNA synthesis in S-phase. BrdU can be immunocytochemically detected in vitro and in vivo, allowing the identification of cells that were dividing during the period of BrdU exposure. In vivo, it has been used to identify the“birthdate” of cells during development, to examine the fate of postnatally generated cells, and to label cells prior to transplantation for subsequent identification.