Epigenesis in developing avian scales. III. Stage-specific alterations of the developmental program caused by 5-bromodeoxyuridine

As an approach to the study of a developmental program, 5-bromodeoxyuridine (BrdU) was administered to chick embryos in ovo at various stages of avian scale formation. This brought about stage-specific alterations in morphogenesis in the anterior tarsometatarsus such as feathered scales, from Day 6 through Day 6 1/2; feathers only, from Day 6 3/4 through Day 7 1/4; scalelessness and rudimentary scales, from Day 7 7/8 through Day 8 1/8; and partial ridge scales, from Day 8 1/8 through Day 10. The effects of BrdU were completely nullified by an excess dose of thymidine which instantly suppressed BrdU incorporation into nuclear DNA. Effects of BrdU causing scalelessness were further examined. The percentage of BrdU labeled cells was immunohistochemically detected. It increased linearly in both the epidermis and dermis, reaching nearly 100% 24 hr following its injection on Day 8. However, scale forming potency, as assayed by the area of scale epidermis on Day 11, decreased with the duration of BrdU incorporation into the cells and disproportionately dropped at 15 hr when about 50% of the cells had incorporated BrdU. Scalelessness was also produced when the period of the incorporation of BrdU exceeded 15 hr. Time sequence observations demonstrated epidermal cell shape, polarity, alignment, and packing density to be remarkably disordered so that the placode and interplacode failed to develop on Day 9 1/4. Epidermal-dermal recombinations were carried out by exchanging normal tissues with those treated with BrdU in the anterior tarsometatarsus. The results clearly showed defects in the dermis at the time of reassociation, giving rise to scalelessness.     

Study of the Lineage and Cell Cycle of Small Micromeres in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

A study was made of 1st cell cycle of small micromeres, segregated at the 5th cleavage cycle, in the sea urchin embryos of Hemicentrotus pulcherrimus . For identification of small micromeres, the embryos were pulse labeled with 5-bromodeoxyuridine (BrdU) at the 1st cleavage. Using multiparametric microfluorometry equipped with a scanning stage (Tanaka, 1990), DNA content, extent of BrdU incorporation, protein content and the extent of ³H-thymidine labeling were measured on identical individual cells dissociated from an embryo. The findings of the present study are as follows. There is a short period of time between the telophase and onset of DNA replication. The period of DNA replication is 5 hr and after which, asynchronous mitosis takes place to produce 8 cells before hatching. The long S period is 83% the total 6 hr of the cell cycle. The rate of DNA accumulation is quite small during the initial one third of S but increases later in this phase. The degree of chromatin condensation remains high even during the S phase but it is low in large micromeres. The cell cycle may possibly be related causally to the development of small micromeres. The developmental significance of cell cycle duration, particularly that of DNA replication is discussed.     

Possible effect of the cell synchronization method on the pattern of 5-bromodeoxyuridine incorporation into early synthesized DNA

Synchronously and asynchronously growing chick embryo fibroblasts have been used to study the pattern of 5-bromodeoxyuridine (BrdU) incorporation into DNA. In the synchronous cell system, the density of unifilarly substituted DNA is about 0.010 g/ml higher during first half of S phase than during second half of S phase. The density of unifilarly substituted DNA isolated from asynchronously growing cells is similar to that of DNA synthesized during the second half of S phase of synchronously growing cells for a given concentration of analogue in culture medium. Reassociation kinetics experiments have shown the oversubstitution to occur at the level of early synthesized repeated and/or intermediate DNA sequences. It is then assumed that the oversubstitution is due to some metabolic changes caused by the synchronization procedure itself. As BrdU incorporation into early replicating DNA is known to induce alterations of the cell metabolism, the implication of this phenomenon is discussed at the level of the inhibition of transformation which takes place when chick embryo fibroblasts are infected with Rous sarcoma virus during G1 and subsequently treated with BrdU during early S phase.     

Targeted disruption of Mcm10 causes defective embryonic cell proliferation and early embryo lethality

Minichromosome maintenance 10 (MCM10) is a conserved, abundant nuclear protein, which plays a key role in the initiation of eukaryotic chromosomal DNA replication and elongation. To elucidate the physiological importance of MCM10 in vivo, we generated conventional knockout mice. No MCM10-null embryos were recovered after E8.5, and the mutation was found to be lethal before the implantation stage. Mutant embryos showed apparently normal growth until the morula stage, but growth defects after this stage. The dramatic reduction of 5-bromo-2-deoxyuridine (BrdU) incorporation in the mutant embryo, followed by cell death, suggests that defective cell proliferation may underlie this developmental failure. Taken together, these findings provide the first unequivocal genetic evidence for an essential and non-redundant physiological role of MCM10 during murine peri-implantation development.     

Chromosome banding and DNA replication patterns in bird karyotypes

The karyotypes of the domestic chicken (Gallus domesticus), Japanese quail (Coturnix coturnix), and griffon vulture (Gyps fulvus) were studied with a variety of banding techniques. The DNA replication patterns of bird chromosomes, analyzed by incorporation of 5-bromodeoxyuridine (BrdU) and deoxythymidine (dT), are presented here for the first time. In particular, the time sequence of replication of the ZZ/ZW sex chromosomes throughout the S-phase was meticulously analyzed. BrdU and dT incorporation are very useful methods to identify homoeologies between karyotypes, as well as rearrangements that occurred in the macroautosomes during speciation. The Z chromosomes of the three birds displayed the same replication patterns, indicating a high degree of evolutionary conservation. In the homogametic male, BrdU and dT incorporation revealed no evidence of asynchronous replication between euchromatic bands in the ZZ pair. The same was true of the three Z chromosomes in a triploid-diploid chimeric chicken embryo. Minor replication asynchronies between the homologous ZZ or ZZZ chromosomes were restricted to heterochromatic C-bands. These results confirm that, in the ZZ male/ZW female sex-determining system of birds, dosage compensation for Z-linked genes does not occur by inactivation of one of the two Z chromosomes in the homogametic male. The heterochromatic W chromosomes of the three species showed bright labeling with distamycin A/mithramycin counterstain-enhanced fluorescence and exhibited significantly delayed DNA replication. The nucleolus organizers of birds, frequently located in microchromosomes, were also distinguished by bright distamycin A/mithramycin fluorescence.     

Decrease of DNA synthesis in amniotic fluid cells during the middle part of S-phase revealed by differential chromosome staining after incorporation of BrdU

The time sequence of DNA replication in partially synchronized human amniotic fluid cells has been analysed, employing BrdU incorporation techniques. —Regardless of the interval between removal of the methotrexate/uridine block and addition of BrdU during S-phase, the treatment results in an R-type replication pattern. Conversely, replacement of BrdU containing medium by another one with thymidine yields G-type replication patterns. A thymidine pulse during the first 4 h of S-phase results in R-type replication patterns; from 7–10 h after block removal it produces G-type pattern. In between, only faint red staining dots can be found indicating a marked decrease of replicational activity during the middle part of the S-phase.     

Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S,and G2 Phases of the Cell Cycle in Trypanosomatids

Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU‐ and EdU‐incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and “click” chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re‐estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring.     

In vivo BrdU-33258 Hoechst analysis of DNA replication kinetics and sister chromatid exchange formation in mouse somatic and meiotic cells

BrdU (5-bromodeoxyuridine)-33258 Hoechst methods have been adapted for in vivo analyses of replication kinetics, sister chromatid differentiation and sister chromatid exchange (SCE) formation in mice. Sufficient in vivo BrdU substitution for cytological detection was effected with multiple intraperitoneal injections of the analogue. The combination of centromere staining asymmetry and sister chromatid differentiation at metaphase permits unambiguous determination of the number of replications in BrdU and dT (deoxythymidine) undergone by individual cells. Late-replicating regions in marrow and spermatogonial chromosomes are highlighted by bright fluorescence after sequential incorporation of BrdU followed by dT during a single DNA synthesis period. SCEs are analyzed in marrow and spermatogonial metaphases after successive complete cycles of BrdU and dT incorporation. Significant induction of SCE was observed with both mitomycin C and cyclophosphamide; the latter drug requires host-mediated activation to be effective. In meiotic metaphase cells harvested two weeks after BrdU incorporation, satellite DNA asymmetry, sister chromatid differentiation and SCE could be detected in a few chromosomes, most frequently the X and the Y.     

Targeted disruption of Mcm10 causes defective embryonic cell proliferation and early embryo lethality

Minichromosome maintenance 10 (MCM10) is a conserved, abundant nuclear protein, which plays a key role in the initiation of eukaryotic chromosomal DNA replication and elongation. To elucidate the physiological importance of MCM10 in vivo, we generated conventional knockout mice. No MCM10-null embryos were recovered after E8.5, and the mutation was found to be lethal before the implantation stage. Mutant embryos showed apparently normal growth until the morula stage, but growth defects after this stage. The dramatic reduction of 5-bromo-2-deoxyuridine (BrdU) incorporation in the mutant embryo, followed by cell death, suggests that defective cell proliferation may underlie this developmental failure. Taken together, these findings provide the first unequivocal genetic evidence for an essential and non-redundant physiological role of MCM10 during murine peri-implantation development.     

Anomalous neural differentiation induced by 5-bromo-2'-deoxyuridine during organogenesis in the rat

The influence of 5-bromo-2'-deoxyuridine (BrdU) on rat embryo development and neurogenesis was investigated using a rat conceptus culture system during organogenesis (pregnancy days 10-13). The embryos and visceral yolk sacs of conceptuses cultured with BrdU were examined for overall growth, morphological anomalies, incorporation of radiolabeled BrdU into DNA, and neurotransmitter enzyme activities in embryos. In addition, neural tubes from cultured whole embryos were isolated and mechanically dissociated into fragments and cultured again to assess neural cell differentiation into neuron-like cells. BrdU was found to incorporate differentially into embryonic and visceral yolk sac DNA with simultaneous stage-specific retardation and anomalous organogenesis in proportion to the increasing concentrations used. Neural tube differentiation of cultured embryos was markedly altered, and there were morphologically distinct neural anomalies. The neurite outgrowth from neuroblast cells (type 1) of explanted spinal neural tube fragments from BrdU-treated embryos was markedly reduced in length and number compared to those from similar areas of embryos grown without BrdU. In contrast, BrdU at the same doses did not affect differentiation of a number of neural tissue-related enzymes. These results indicate that BrdU incorporation into DNA of primordial embryonic cells significantly affects neurogenesis and differentiation of neurites from neuroblasts, which is a specific neural cytodifferentiation characteristic of neuronal cells.     

Double labeling with non-isotopic in situ hybridization and BrdU immunohistochemistry: calmodulin (CaM) mRNA expression in post-mitotic neurons of the olfactory system.

We describe a novel histochemical procedure for simultaneous detection of mRNA expression by in situ hybridization (ISH) and DNA synthesis on cells that are pulse-labeled with bromodeoxyuridine (BrdU) by immunohistochemistry (ICC). Pregnant rats were injected with BrdU at embryonic Day 20 and the olfactory bulbs of their pups were collected daily. The expression of calmodulin (CaM) mRNA was analyzed by ISH with an anti-sense digoxigenin-labeled riboprobe and BrdU incorporation by indirect ICC. Starting 5 days after BrdU injection, a few tufted and granular neurons of the olfactory bulb were observed to be double labeled for CaM mRNA and BrdU. To study the olfactory neuroepithelium, adult animals were injected with BrdU, sacrificed after 30 days, and the nasal mucosa dissected and decalcified. The co-expression of CaM mRNA and BrdU incorporation was then analyzed in the olfactory neuroepithelium: BrdU-positive primary olfactory neurons were also CaM mRNA positive. The combination of ISH and ICC on the same section resulted in improved BrdU staining with respect to both increased intensity and reduced background levels. The procedure described here can be applied to a variety of problems in developmental biology and is of potential value for correlating the timing of specific mRNA expression with the birth date of a cell type of interest.     

Identification of Genomic Regions Required for DNA Replication during Drosophila Embryogenesis

A collection of Drosophila deficiency stocks was examined by bromodeoxyuridine (BrdU) labeling of embryos to analyze the DNA replication patterns in late embryogenesis. This permitted us to screen 34% of the genome for genes that when absent in homozygous deficiencies affect the cell cycle or DNA replication. We found three genomic intervals that when deleted result in cessation of DNA replication in the embryo, 39D2-3;E2-F1, 51E and 75C5-7;F1. Embryos deleted for the 75C5-7;F1 region stop DNA replication at the time in embryogenesis when a G(1) phase is added to the mitotic cell cycle and the larval tissues begin to become polytene. Thus, this interval may contain a gene controlling these cell cycle transitions. DNA replication arrests earlier in embryos homozygous for deletions for the other two regions. Analysis of the effects of deletions in the 39D2-3;E2-F1 region on DNA replication showed that the block to DNA replication correlates with deletion of the histone genes. We were able to identify a single, lethal complementation group in 51E, l(2)51Ec, that is responsible for the cessation of replication observed in this interval. Deficiencies that removed one of the Drosophila cdc2 genes and the cyclin A gene had no effect on replication during embryogenesis. Additionally, our analysis identified a gene, pimples, that is required for the proper completion of mitosis in the post-blastoderm divisions of the embryo.     

Host DNA replication is induced by geminivirus infection of differentiated plant cells

The geminivirus Tomato golden mosaic virus (TGMV) replicates in differentiated plant cells using host DNA synthesis machinery. We used 5-bromo-2-deoxyuridine (BrdU) incorporation to examine DNA synthesis directly in infected Nicotiana benthamiana plants to determine if viral reprogramming of host replication controls had an impact on host DNA replication. Immunoblot analysis revealed that up to 17-fold more BrdU was incorporated into chromosomal DNA of TGMV-infected versus mock-infected, similarly treated healthy leaves. Colocalization studies of viral DNA and BrdU demonstrated that BrdU incorporation was specific to infected cells and was associated with both host and viral DNA. TGMV and host DNA synthesis were inhibited differentially by aphidicolin but were equally sensitive to hydroxyurea. Short BrdU labeling times resulted in some infected cells showing punctate foci associated with host DNA. Longer periods showed BrdU label uniformly throughout host DNA, some of which showed condensed chromatin, only in infected nuclei. By contrast, BrdU associated with viral DNA was centralized and showed uniform, compartmentalized labeling. Our results demonstrate that chromosomal DNA is replicated in TGMV-infected cells.     

PAR-4/LKB1 regulates DNA replication during asynchronous division of the early C. elegans embryo

Regulation of cell cycle duration is critical during development, yet the underlying molecular mechanisms are still poorly understood. The two-cell stage Caenorhabditis elegans embryo divides asynchronously and thus provides a powerful context in which to study regulation of cell cycle timing during development. Using genetic analysis and high-resolution imaging, we found that deoxyribonucleic acid (DNA) replication is asymmetrically regulated in the two-cell stage embryo and that the PAR-4 and PAR-1 polarity proteins dampen DNA replication dynamics specifically in the posterior blastomere, independently of regulators previously implicated in the control of cell cycle timing. Our results demonstrate that accurate control of DNA replication is crucial during C. elegans early embryonic development and further provide a novel mechanism by which PAR proteins control cell cycle progression during asynchronous cell division.     

MEL-47, a novel protein required for early cell divisions in the nematode <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

In the early Caenorhabditis elegans embryo, a rapid succession of cell divisions, many of them asymmetric, form blastomeres that differ in size, cell cycle duration and developmental potential. These early cell cycles are highly regulated and controlled by maternally contributed products. We describe here a novel gene, mel-47, that is required maternally for the proper execution of the early cell cycles. mel-47(yt2) mutants arrest as completely disorganized embryos with 50–80 cells of variable size. The earliest defects we found are changes in the absolute and relative duration of the very early embryonic cell cycles. In particular, the posterior cell of the two-cell embryo divides late compared with its anterior sister. Frequently the daughter cells remain connected through chromatin bridges after the early cleavage divisions indicating that the chromosomes do not segregate properly. The cell cycle delay can be suppressed by knocking down a DNA replication check point. Therefore we propose that mel-47 is required for proper DNA replication in the early embryo. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Characterization of deoxyribonucleic acid synthesis and the transition from maternal to embryonic control in the 4-cell porcine embryo.

These studies were conducted to identify the point during the 4-cell stage at which the porcine embryo begins to control development. Reproductive tracts of gilts were flushed 48 h after the onset of estrus to obtain 1- and 2-cell embryos. To determine the duration of the 4-cell stage in vitro, development of 29 embryos was timed from cleavage to the 4-cell stage and from cleavage to the 8-cell stage. The average duration of the 4-cell stage was 50.5 h. The duration of the 4-cell stage was positively correlated (p < 0.01) with culture time in vitro before cleavage to the 4-cell stage. DNA content was determined by using the Feulgen's reaction and quantified with micro-densitometry. Staining units (SU; density x area) were calculated at 0, 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, and 36 h post-cleavage to the 4-cell stage (P4C). Results revealed a possible G1 phase (< 2 h) with DNA synthesis starting within 2 h P4C. DNA synthesis was completed by 16 h P4C, and was followed by an extended G2 phase. Embryos were evaluated for uptake and incorporation of [35S]methionine and for qualitative changes in protein profiles specific to time points during the 4-cell stage (2, 10, 14, 16, 18, 24, 30, and 40 h P4C). Methionine uptake and incorporation into protein followed similar patterns, both decreasing until 16-18 h P4C, followed by a steady increase through the 4-cell stage. Protein profiles revealed qualitative changes beginning at 14 and 16 h P4C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Strand breaks arising from the repair of the 5-bromodeoxyuridine-substituted template and methyl methanesulphonate-induced lesions can explain the formation of sister chromatid exchanges

5-Bromodeoxyuridine (BrdU)-induced sister chromatid exchanges (SCEs) are mainly determined during replication on a BrdU-substituted template. The BrdU, once incorporated, is rapidly excised as uracil (U), and the gap is repaired with the incorporation of BrdU from the medium, which leads to further repair. During the second S period in BrdU medium, this process continues as the strand acts as template. Experiments suggest that 3-amino-benzamide (3AB) delays the ligation of the gaps formed after U excision, resulting in enhanced SCE levels during the second cycle of BrdU incorporation. When normal templates of G1 cells are treated before BrdU introduction with methyl methanesulphonate (MMS), 3AB in the first cycle doubles the MMS-induced SCEs but has no effect on them during the second cycle. When the BrdU-substituted template is treated with MMS in G1 of the second cycle, 3AB again doubles the SCEs due to MMS and also enhances the SCEs resulting from delays in ligation of the gaps following U excision in the BrdU-substituted template. The repair processes of MMS lesions that are sensitive to 3AB and lead to SCEs take place rapidly, while the repair process of late repairing lesions that lead to SCEs appear to be insensitive to 3AB. A model for SCE induction is proposed involving a single-strand break or gap as the initial requirement for SCE initiation at the replicating fork. Subsequent events represent natural stages in the repair process of a lesion, ensuring replication without loss of genetic information. G1 cells treated with methylnitrosourea (MNU) and grown immediately in BrdU medium rapidly lose the O⁶-methylguanine from their DNA and the rate of loss is BrdU-dose dependent. The rapid excision of the U lesions can explain the effect of BrdU concentration on SCE reduction following both MNU or MMS treatment.