Toshiteru Morita

Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus

In mammalian cells, it has been shown that adjacent multiple DNA replicons, termed a replicon cluster or a replicon domain, are replicated coordinately in a defined temporal order during the DNA synthetic (S) phase. However, no intranuclear structure of this replicon domain has been revealed in the nucleus labelled with [3H]thymidine at the limited resolution level of autoradiography. By immunofluorescent staining with antibody against 5-bromodeoxyuridine (BrdU), we succeeded in detecting novel, intranuclear ring-like structures of replicating replicon domains that were organized temporarily during the S phase of mammalian cells with incorporated BrdU.

Intracellular localization and metabolism of DNA polymerase α in human cells visualized with monoclonal antibody

Indirect immunofluorescence microscopy with monoclonal antibody against DNA polymerase alpha revealed the intranuclear localization of DNA polymerase alpha in G1, S, and G2 phases of transformed human cells, and dispersed cytoplasmic distribution during mitosis. In the quiescent, G0 phase of normal human skin fibroblasts or lymphocytes, the alpha-enzyme was barely detectable by either immunofluorescence or enzyme activity. By exposing cells to proliferation stimuli, however, DNA polymerase alpha appeared in the nuclei just prior to onset of DNA synthesis, increased rapidly during S phase, reached the maximum level at late S and G2 phases, and was then redistributed to the daughter cells through mitosis. It was also found that the increase in the amount of DNA polymerase alpha by proliferation stimuli was not affected by inhibition of DNA synthesis with aphidicolin or hydroxyurea.

Immunolocalization of Ku-proteins (p80/p70): Localization of p70 to nucleoli and periphery of both interphase nuclei and metaphase chromosomes☆

Distribution on both nuclei and metaphase chromosomes of Ku-proteins, recognized by autoantibodies from a patient with systemic lupus erythematosus, has been studied using a specific monoclonal antibody (mAbH6) that recognizes p70, one Ku-protein. Observation with either a conventional fluorescent microscope or a confocal laser scanning microscope revealed mAbH6-stained p70 antigen localized on both nuclear periphery and nucleoli of human interphase cells. The specific staining of nucleoli with mAbH6 has been confirmed using isolated nucleoli from rat liver in which the staining was seen as fine granules surrounding nucleolar DNA. During mitosis p70 antigen moved away from association with the nuclear envelope region to localization on the periphery of condensed chromosomes with no apparent staining of chromosome interior. The p70 antigen was copurified with DNA fragments by immunoaffinity column chromatography using mAbH6. The mAbH6 staining of both nuclear periphery and nucleoli was lost upon digestion with DNase I at low concentrations. These results suggest that p70 antigen is connected with these nuclear structures through DNA.

MOUSE EMBRYONIC SUBMANDIBULAR GLAND EPITHELIUM LOSES ITS TISSUE INTEGRITY DURING EARLY BRANCHING MORPHOGENESIS

During the development of the mouse submandibular gland, the epithelium undergoes not only shape changes to produce extensively branched lobules and stalk, but also changes in cell arrangement from a cell mass to a cavitated cell sheet. The present study examined the organization in the developing epithelium of intercellular adhesion systems and of actin‐containing microfilaments. E‐cadherin and β‐catenin, which are components of cell‐to‐cell adherens junctions in epithelial cells, were distributed along the cell periphery of almost the entire epithelium of the submandibular gland at all stages examined and were mainly localized at the apical region of the oral epithelium. Actin‐containing microfilaments, which are associated with cell‐to‐cell adherens junctions, showed a distribution similar to that of those molecules. In contrast, although the distributions of desmoplakins I/II, major desmosomal proteins; and ZO‐1 (a tight junction protein) were seen in the oral epithelium and proximal stalk of the submandibular gland epithelium, signals representing these molecules were absent from or much reduced in the submandibular gland epithelium of the cell mass at the 12‐ and 13‐day stages. In the 14‐day gland, they strongly appeared in the cells facing the appearing lumens, whereas they were weakly scattered within the terminal lobules that were still a part of the cell mass. These findings suggest that cell‐to‐cell adhesion systems are differentially regulated during the epithelial morphogenesis of the submandibular gland and that the integrity of the submandibular gland epithelium is lost during the early stages of development, indicating the tissue to be a rather plastic structure.

Spatial and temporal changes in chondroitin sulfate distribution in the sclerotome play an essential role in the formation of migration patterns of mouse neural crest cells

We have examined the roles of pertinent extracellular matrix molecules in the formation of the neural crest cell migration patterns in the sclerotome of the mouse embryo. The present data indicate that permissiveness for migration is inversely correlated with chondroitin sulfate content. Experimental removal of chondroitin sulfate proteoglycans in the embryo causes neural crest cells to migrate even within the posterior half of the somite, which they do not invade ordinarily. Moreover, three different sclerotomal regions defined by the presence or absence of the ventromedial and/or ventrolateral pathways are present along the anteroposterior axis and undergo systematic temporal changes that affect migration patterns. The most anterior portion of the sclerotome is conducive to both ventromedial and ventrolateral migration (Anterior Region). The intermediate portion is conducive to ventromedial migration only (Intermediate Region). No neural crest cells are seen within the posterior portion of the sclerotome (Posterior Region). At this level, they are observed exclusively in the dorsolateral space adjacent to the roof of the neural tube. With advancing embryonic development, the rostrocaudal length of the Anterior Region decreases and is accompanied by a corresponding enlargement of the Intermediate Region. These results suggest that temporal and regional differences in the sclerotome contribute to the neural crest cell migration patterns in the mouse. To refine our understanding of the underlying mechanisms, regional differences and temporal changes in the distribution of extracellular matrix molecules have been examined during migration. In the sclerotome, chondroitin sulfate displays distinct distribution patterns that are closely correlated with the migration patterns of mouse neural crest cells. Furthermore, their migration patterns are altered in embryos treated with the inhibitors of chondroitin sulfate proteoglycan biosynthesis, sodium chlorate, and β‐d‐xyloside. In inhibitor‐treated embryos, neural crest cell migration occurs even in the posterior portion of the sclerotome. The metameric organization of dorsal root ganglia is disturbed in these embryos. Our observations provide novel evidence for the importance of sclerotomal chondroitin sulfate distribution patterns in mouse crest cell migration patterns. We conclude that systematic spatiotemporal changes in the distribution of chondroitin sulfate proteoglycans are a key requisite for the formation of migration patterns of mouse neural crest cells in the sclerotome. Dev Dyn 1999;214:55–65.

New monoclonal antibody (4E9R) identifies mouse neural crest cells.

In order to analyze migration patterns of mouse neural crest cells, we produced a rat anti‐mouse monoclonal antibody (4E9R) which identifies these cells. The distribution of 4E9R‐immunoreactive cells was examined in migratory stages of mouse neural crest cells, ranging from embryonic day (Ed) 8.5 to 10.5. In the cephalic region of Ed 8.5 embryos, some mesencephalic mesenchymal cells close to neural folds were 4E9R‐positive. In Ed 9.0–9.5 embryos, streams of 4E9R‐immunoreactive cells extending from cranial neural tubes to maxillary processes and to first visceral arches were found in the lateral region and some of these cells were localized in presumptive trigeminal ganglia. In the same embryonic stages, 4E9R‐positive cells were present in mesenchymal cells around the optic and otic vesicles. In the trunk region of Ed 9.5–10.5 embryos, 4E9R‐positive cell masses were observed in dorsolateral spaces adjacent to neural tubes. The presence of 4E9R‐immunoreactive cells in somitic segments was restricted within the anterior halves and these cells were seen under the dermomyotome and/or in the medial portion of the sclerotome. These cells colonized in presumptive dorsal root ganglia and in the surroundings of the dorsal aorta, the embryonic area in which sympathetic ganglia are formed. 4E9R‐positive cells were also found just under the epidermis. These observations indicate that the distribution of 4E9R‐positive cells is similar to that of amniote neural crest cells reported previously. Furthermore, the data suggest that the migratory behavior of mouse neural crest cells at trunk levels may be different between rostral and caudal levels within an anterior half of the sclerotome, and that sympathetic ganglia may be formed by neural crest cells migrating along “ventromedial pathways” as well as “ventrolateral pathways” at hindlimb‐bud levels of mouse embryos. In primary cultures of mouse neural crest cells, approximately 80% of the cells were 4E9R‐positive on culture day 2. Further, we have shown in cultures treated with colchicine or cytochalasin B that 4E9R antigens are present in intermediate filaments. When image analysis with a confocal laser scanning microscope was performed on primary cultures of mouse neural crest cells, the intracellular localization of 4E9R antigens in these cells was comparable to that of vimentin, which is a typical intermediate filament in most mesenchymal cells in embryonic stages examined. Since the distribution of 4E9R‐positive cells and anti‐vimentin‐immunoreactive cells was different in mouse embryos, it is suggested that 4E9R antigens are vimentin‐related and specifically expressed in a few cell types including mouse neural crest cells. These results indicate that a rat anti‐mouse monoclonal antibody 4E9R is useful for the identification of mouse neural crest cells during the migratory stages.

Differentiation of lens and neural cells in chicken embryo is accompanied by simultaneous decay of DNA replication machinery

DNA polymerase alpha was detected in cells of developing chicken embryos by an immunofluorescent method using a monoclonal antibody specific for the high molecular weight polypeptide of chicken DNA polymerase alpha, and DNA polymerase beta was detected using a rabbit anti-chicken DNA polymerase beta antibody. In lens tissue of the 3- to 4-day chicken embryo, fluorescence with anti-DNA polymerase alpha antibody was detected in nuclei of lens epithelial cells but not in nuclei of lens fiber cells which had differentiated from epithelial cells. The localization of cells containing DNA polymerase alpha coincided with the distribution of cells capable of DNA replication as detected by [3H]thymidine autoradiography. Similar results were obtained during the differentiation of neural matrix cells to neuroblasts in the developing neural tube. In contrast to DNA polymerase alpha, DNA polymerase beta was detected in nuclei of both undifferentiated and differentiated cells of these tissues. Since the disappearance of DNA polymerase alpha was very rapid after the onset of differentiation, the DNA replication machinery in which DNA polymerase alpha plays a central role is thought to decay almost simultaneously with the onset of cellular differentiation in these tissues.

Branching Morphogenesis of Mouse Embryonic Submandibular Epithelia Cultured under Three Different Conditions

To investigate how the mesenchyme interacts with the epithelium, we employed three different culture systems: System A, in which intact submandibular gland rudiments at the mid 13‐day stage were cultured on Millipore filters; System B, in which the 13‐day epithelium and mesenchyme were separated once with dispase, recombined again, and cultured on the filter; System C, in which the separated 13‐day epithelium was clotted with Matrigel and cultured with the mesenchyme across the filter or in the presence of EGF instead of the mesenchyme. In Systems A and B, 13‐day epithelia expanded and produced similar lobules with narrow clefts and stalk. When the 13‐day epithelium was cultured in System C under the influence of the mesenchyme, it formed rather oval lobules with stalk that were superficially similar to those in System A, but narrow clefts, as seen in the intact early 13‐day gland, were rarely found in System C. Furthermore, no long stalk formation was observed when EGF was introduced in place of the mesenchyme. A bacterial collagenase from Clostridium histolyticum gave a considerable inhibition of branching of the 13‐day epithelium in Systems A and B, but no significant inhibition was observed in System C when the mesenchyme or EGF was employed as the source of diffusible factor(s). In contrast, although the 13‐day epithelium was significantly resistant to the action of heparitinase I from Flavobacterium heparinum in Systems A and B, the enzyme almost completely inhibited the expansion and branching of the epithelium in System C. Judging from these observations, we conclude that the mechanisms of lobular formation in Systems A and B are not the same as those in System C, where the epithelium is clotted with basement membrane matrix components during tissue culture.

Polyclonal Antibodies to DNA Modified with 4‐Nitroquinoline 1‐Oxide: Application for the Detection of 4‐Nitroquinoline 1‐Oxide‐DNA Adducts in vivo

Antibodies against 4‐nitroquinoline 1‐oxide (4NQO) adducts were elicited in rabbits immunized with 4NQO‐modified DNA complexed with methylated bovine serum albumin. In enzymelinked immunosorbent assay (ELISA), the antibodies could recognize either denatured or native 4NQO‐modified DNA, but not unmodified DNA, DNA modified with other carcinogens or free 4NQO derivative. Modification levels as low as 5 μmol of adduct per one mole DNA nucleotide (5 adducts/106 nucleotides) can be easily detected by the competitive ELISA. Indirect immunofluorescence staining by anti 4NQO‐DNA antibody indicated that the antibodies bound specifically to the nuclei of normal human skin fibroblast cells treated with 4NQO. The intensity of fluorescence was proportional to the dose of 4NQO used to treat the cells, and the fluorescence positive cells could be detected after treatment with 0.25μM 4NQO (which resulted in the formation of 104 adducts per cell). Applying the competitive ELISA to the quantitation of DNA‐adducts in rats treated with 4NQO, it was confirmed that the sensitivity of immunochemical assays was equivalent to that of isotopic assays. These methods should be helpful in studies on the formation of adducts and their removal in cells and tissues.

Dexamethasone-induced changes in morphology and keratin organization of rat thymic epithelial cells in primary culture

Dexamethasone (DM)-induced changes in morphology and keratin organization of rat thymic epithelial cells (TECs) in primary culture were studied. The morphology and keratin organization of TECs were greatly altered by the addition of more than 10 nM DM. In control cultures, small slender cells were predominant and grew faster than cells of other types. Most of them had keratin bundles on day 3, but lost the bundles on day 7. In experimental cultures containing more than 10 nM DM, the morphology of TECs altered to a polygonal form and the TECs formed colonies. They had fine interlacing bundles of keratin filaments all over the cytoplasm, and the bundles were maintained or increased in number with the day of culture. Keratin proteins in TECs treated with 100 nM DM for 7 days were 2-fold for 46 500 D, 4-fold for 49 000 D, 1.7-fold for 52 000 D and 3.5-fold for 55 500 D keratins to those in control TECs, respectively. Growth of TECs was inhibited by the addition of more than 10 nM DM. The effects of DM were reversible to some extent, for the morphology and keratin organization of TECs gradually changed into the control type by the removal of DM. These results suggested that DM greatly involved the growth and differentiation of rat TECs.