Fate of the anterior neural ridge and the morphogenesis of the xenopus forebrain

The fate of the anterior neural ridge was studied by following the relative movements of simultaneous spot applications of DiI and DiO from stage 15 through stage 45. These dye movements were mapped onto the neuroepithelium of the developing brain whose shape was gleaned from whole-mount in situs to neural cell adhesion molecule and dissections of the developing nervous system. The result is a model of the cell movements that drive the morphogenesis of the forebrain. The midanterior ridge moves inside and drops down along the most anterior wall of the neural tube. It then pushes forward a bit, rotates ventrally during forebrain flexing, and gives rise to the chiasmatic ridge and anterior hypothalamus. The midanterior plate drops, forming the floor of the forebrain ventricle, and, keeping its place behind the ridge, it gives rise to the posterior hypothalamus or infundibulum. The midlateral anterior ridge slides into the lateral anterior wall of the neural tube and stretches laterally into the optic stalk and retina, and then rotates into a ventral position. The lateral anterior ridge converges to the most anterior part of the dorsal midline during neural tube closure, then rotates anteriorly, and gives rise to telencephalic structures. Whole-mount bromodeoxyuridine labeling at these stages showed that cell division is widespread and relatively uniform throughout the brain during the late neurula and early tailbud stages, but that during late tailbud stages cell division becomes restricted to specific proliferative zones. We conclude that the early morphogenesis of the brain is carried out largely by choreographed cell movements and that later morphogenesis depends on spatially restricted patterns of cell division. © 1995 John Wiley & Sons, Inc.     

Bromodeoxyuridine/DNA analysis of replication in CHO cells after exposure to UV light

The effects of ultraviolet light on cellular DNA replication were evaluated in an asynchronous Chinese hamster ovary cell population. BrdUrd incorporation was measured asa function of cell-cycle position, using an antibody against bromodeoxyuridine (BrdUrd) and dual parameter flow cytometric analysis. After exposure to UV light, there was an immediate reduction ( 50%) of BrdUrd incorporation in S phase cells, with most of the cells of the population being affected to a similar degree. At 5 h after UV, a population of cells with increased BrdUrd appeared as cells that were in G1 phase at the time of irradiation entered S phase with apparently increased rates of DNA synthesis. For 8 h after UV exposure, incorporation of BrdUrd by the original S phase cells remained constant, whereas a significant portion of original G1 cells possessed rates of BrdUrd incorporation surpassing even those of control cells. Maturation rates of DNA synthesized immediately before or after exposure by alkaline elution, were similar. Therefore, DNA synthesis measured in the short pulse by anti-BrdUrd fluorescence after exposure to UV light was representative of genomic replication. Anti-BrdUrd measurements after DNA damage provide quantitative and qualitative information of cellular rates of DNA synthesis especially in instances where perturbation of cell-cycle progression is a dominant feature of the damage. In this study, striking differences of subsequent DNA synthesis rates between cells in G1 or S phase at the time of exposure were revealed.     

Detection of S-Phase Cells in Smear Cytology Using in Vitro Bromodeoxyuridine Labeling

A technique Is described for rapid detection of S-pha?e cells of tumor tissues in smear specimens using bromodeoxyuridine (BrdU) immunostaining. Mouse NR-S1 tumors and human tumor specimens were prepared for smear cytology after incubation in RPMI 1640 culture medium containing 200 μM BrdU at 37 °C under 3 atm for 1 hr. Samples were fixed in 70% ethanol for 30 min and used immediately or air dried for 30 min. Samples were then denatured in either 4 N HC1 or 0.07 N NaOH to prepare partially single-stranded DMA. Fixation with air drying for 30 min followed by 30 min in 70% ethanol and 1 min denaturation with 0.07 N NaOH resulted in satisfactory staining quality. Cultured tumor specimens were processed for routine paraffin sections after smears were made for cytology. The labeling indices of the smear specimens and of the paraffin sections gave similar results. This technique should be useful in evaluating the cell proliferative potential of tumor tissue in smear cytology without processing paraffin sections.     

Progress in outgrowth culture from rabbit tracheal explants: Balance between proliferation and maintenance of differentiated state in epithelial cells

Summary Primary cultures of rabbit tracheal cells were obtained as outgrowths from explants of tracheal mucosa. A 30% collagen substratum containing serum and minimal essential medium was required for obtaining an outgrowth of epithelial cells keeping their differentiated characteristics. The tracheal epithelial cells obtained near the explant in the first days of culture presented morphologic similarities with normal tracheal epithelium. Cultures contained basal cells and epithelial polarized cells that exhibited apical tight junctions and desmosomes. Ciliated cells stayed functional during all time culture. Their number slightly increased at the beginning of the culture and then stayed constant when the total number of cells increased. Development of the outgrowth was rapid and significant inasmuch as the outgrowth surface reached 30 times that of the explant after less than 8 days. This was linked to cellular proliferation, as demonstrated by the incorporation of bromodeoxyuridine (BrdU) in phase-S nuclei and the revelation of BrdU using an immunofluorescence technique. The epithelial nature of the outgrowth cells and the absence of contamination with fibroblasts were established by positive staining with anti-keratin antibody and by negative staining with anti-vimentin antibody, respectively. This work was supported by DRET and by CIFRE grant awarded to S. R.     

Induction of chromosome shattering by ultraviolet irradiation and caffeine: comparison of whole cell and partial-cell irradiation

Synchronized and asynchronously growing cells of a V79 sub-line of the Chinese hamster were either partial-cell irradiation (λ, 254 nm) or laser-UV-microirradiated (λ, 257 nm). Post-incubation with caffeine (1–2 mM) often resulted in chromosome shattering, which was a rare event in the absence of this compound. In experiments with caffeine, the following results were obtained.

Shattering of all the chromosomes of a cell (generalized chromosome shattering, GCS) was induced by partial-cell irradiation at the first post-irradiation mitosis when the UV fluence exceeded and “threshold” valued in the sensitive phases of the cell cycle (G1 and S). GCS was also induced by laser-UV-microirradiation of a small part of the nucleus in G1 of S whereas microirradiation of cytoplasm beside the nucleus was not effective. An upper limit of the UV fluence in the non-irradiated nuclear part due to scattering of the microbeam was experimentally obtained. This UV fluence was significantly below the threshold fluence necessary to induce GCS in whole-cell irradiation experiments. In other cells, partial nuclear irradiation resulted in shattering of a few chromosomes only, while the majority remained intact (partial chromosomes shattering, PCS). G1/early S was the most sensitive phase for induction of GCS by whole-cell and partial nuclear irradiation. The frequency of PCS was observed to increase when partial nuclear irradiation was performed either at lower incident doses or at later stages of S. We suggest that PCS and GCS indicate 2 levels of chromosome damage which can be produced by the synergistic action of UV irradiation and caffeine. PCS may be restricted to microirradiated chromatin whereas GCS involves both irradiated and unirradiated chromosomes in the microirradiated nucleus.     

The measurement of chemically-induced DNA repair synthesis in human cells by BND-cellulose chromatography.

Repair synthesis in human cells in tissue culture can be readily separated from semi-conservative DNA synthesis with the aid of a benzoylated naphthoylated DEAE cellulose (BND-cellulose) column. Cells are incubated with a radioactive DNA precursor during treatment with a repair-inducing agent. An inhibitor of semi-conservative DNA synthesis (hydroxyurea) is added to slow the progression of the DNA growing point. The cells are lysed and after treatment with ribonuclease and pronase the lysates are sheared and passed through a BND-cellulose column. Native DNA is eluted with I M NaCl. Any increase in radioactivity in the native DNA is due to repair synthesis and the specific repair activity (nucleotides inserted per mug of DNA) can be determined from radioactivity and absorbancy measurements. Repair can also be measured in the region of the DNA growing point by fractionation of the material eluted from BND-cellulose with 50% formamide. Repair was not detected in N-acetoxy-2-acetylaminofluorene (AAAF)-treated lymphoblasts derived from an individual with xeroderma pigmentosum although methyl methanesulfonate (MMS)-induced repair was observed in these cells.     

Cell Kinetic Evidence Suggests Elevated Oxidative Stress in Cultured Cells of Bloom's Syndrome

Bromodeoxyuridine/Hoechst flow cytometry was used to analyse disturbed cell proliferation of fibroblasts and lymphoblastoid cells from Bloom's syndrome (BS). Fibroblasts show poor activation, arrest in the G2 phase of the cell cycle along with a prolongation of the Gl phase. This pattern of perturbed cells proliferation is akin to that elicited in normal fibroblasts by 4-hydroxy-nonenal, a breakdown product of lipid peroxides. Treatment with vitamin E improved growth of BS fibroblasts more strongly than growth of normal fibroblasts. Lymphoblastoid cells from BS, to the contrary, experience only a minor arrest in the G2 phase after one round of bromodeoxyuridine incorporation, but are strongly inhibited during and after the second S phase. Thus, their cell cycle arrest is dependent upon BrdU incorporation, as has been found previously in normal cells exposed to elevated concentrations of oxygen or paraquat, a superoxide generating compound. These results suggest that BS cells may suffer from an elevated, endogenous generation of oxygen free radicals.     

Myoblast proliferation and syncytial fusion both depend on connexin43 function in transfected skeletal muscle primary cultures

Muscles are formed by fusion of individual postmitotic myoblasts to form multinucleated syncytial myotubes. The process requires a well-coordinated transition from proliferation, through migratory alignment and cycle exit, to breakdown of apposed membranes. Connexin43 protein and cell-cycle inhibitor levels are correlated, and gap junction blockers can delay muscle regeneration, so a coordinating role for gap junctions has been proposed. Here, wild-type and dominant-negative connexin43 variants (wtCx43, dnCx43) were introduced into rat myoblasts in primary culture through pIRES-eGFP constructs that made transfected cells fluoresce. GFP-positive cells and vitally-stained nuclei were counted on successive days to reveal differences in proliferation, and myotubes were counted to reveal differences in fusion. Individual transfected cells were injected with Cascade Blue, which permeates gap junctions, mixed with FITC-dextran, which requires cytoplasmic continuity to enter neighbouring cells. Myoblasts transfected with wtCx43 showed more gap-junctional coupling than GFP-only controls, began fusion sooner as judged by the incidence of cytoplasmic coupling, and formed more myotubes. Myoblasts transfected with dnCx43 remained proliferative for longer than either GFP-only or wtCx43 myoblasts, showed less coupling, and underwent little fusion into myotubes. These results highlight the critical role of gap-junctional coupling in myotube formation.