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First published online 5 May 2004
doi: 10.1242/dev.01151

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Tissue-specific G1-phase cell-cycle arrest prior to terminal differentiation in Dictyostelium

¹ Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
² Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

View larger version (29K): [in a new window]   Fig. 2. Proportions of cellular DNAs during development. Changes in the relative proportions of the chromosomal DNA, mitochondrial DNA (mtDNA) and the extrachromosomal ribosomal DNA (rDNA) were estimated by quantification on pulsed field gels. (A) High molecular weight DNAs were separated by pulsed field gel electrophoresis and stained with ethidium bromide. The three DNA species were quantified by digital image analysis using several different exposures. Phosphoimager analyses of Southern hybridizations were used to correct for the proportion of each DNA trapped in the wells. The signal obtained for mtDNA is shown as an example (B). (C) Changes in the proportions of mtDNA, rDNA and chromosomal DNA are shown for a representative experiment. Note that their proportions do not change during the time cells undergo mitosis (indicated by the bracket). (D) The measured peak values for flow cytometry DNA profiles of mononucleated cells are plotted for the same population of cells described in C (filled circles). The expected peak values were calculated from the proportions of the different DNA species in C and assuming that all cells at 6 hours are in G2 and that all cells that divided later remained in G1 (open circles), or progressed through an S phase and arrested in G2 (open squares).

View larger version (27K): [in a new window]   Fig. 1. Mitosis and cellular DNA content during development. (A) Wild-type AX4 cells were plated for development on filters, and counted at different times. (B) Mitosis was monitored in the same samples by counting nuclei in propidium iodide-stained cells. For cellular and nuclear counts, eight determinations were made for each sample of developing cells and the means (±s.e.m.) are reported. (C) Flow cytometry profiles of the DNA content in whole cells at various times of development. The profiles corresponding to the mononucleated cells are shown. Spores and stalk cells were purified from fruiting bodies after 36 hours of development as described in Materials and methods. (D) The cellular DNA content for each time of development is summarized by the peak values (±s.e.m.) of the mono-nucleated cell flow cytometry profiles. This graph summarizes multiple profile determinations from one experiment that is representative of four separate experiments.

View larger version (69K): [in a new window]   Fig. 3. DNA synthesis during development. DNA synthesis during development was monitored by BrdU incorporation. (A) High molecular DNA was separated on standard pulsed-field gels and BrdU was detected by immunostaining after Southern transfer of the DNA. The amount of BrdU incorporation into chromosomes was compared to level of labeling observed in growing cells during one cell cycle and normalized to the amount of chromosomal DNA on the blot, as detected with a chromosomal probe (B). See text for details.

View larger version (21K): [in a new window]   Fig. 4. DNA Synthesis during spore germination and recovery to growth phase. Spores were heat-shocked, and allowed to recover in liquid growth media. (A) The increase in nuclei and the peak level of the DNA content profile of mononucleated cells is plotted over the time of the first cell doubling. (B) DNA synthesis was measured by BrdU incorporation as in Fig. 3, using growing cells that had been labeled for one cell cycle as a control. (C) The percentage of chromosomal DNA synthesis in germinating spores relative to one cell cycle of growing cells was estimated from the BrdU incorporation in B. The values are normalized to the amount of chromosomal DNA on the blot, determined by Southern hybridization as described in Fig. 3. At the 30-hour time point, there was 52% the BrdU incorporation observed in growing cells after only a 10% increase in the number of nuclei. At 21-hours, some BrdU incorporation into chromosomal DNA was detectable upon longer exposures of the blot (arrow).

View larger version (64K): [in a new window]   Fig. 5. Visualization of chromosome content by FISH. Fluorescence in situ hybridization was carried out with a single copy probe corresponding to the tagB/tagC/tagD locus on chromosome 4. The representative images are two-dimensional projections of the three-dimensional images used to score the presence of the locus. The percentage of nuclei with a single observable locus is shown. (A) Amoebae that had been developing for six hours on filters showing 4 of 6 nuclei that clearly contain two loci and two nuclei that were counted as having a single locus. Arrows point to signals within these two nuclei considered too weak to be scored as a locus. (B) Amoebae that had emerged from spores that were germinating for 12 hours showing eight nuclei that contain a single tag/C/D locus.

View larger version (29K): [in a new window]   Fig. 6. DNA content in prespore and prestalk cells of migrating slugs. (A) Wild-type cells (AX4) marked with a prestalk reporter gene (ecmA/gfp) were developed on agar to the slug stage and dissected into two fractions enriched in anterior or posterior cells. (B) Cells were dissociated, fixed and stained with propidium iodide to obtain population profiles of their DNA content. (C) The cells were divided into GFP-negative (window A) and GFP-positive (window B) populations that were confirmed by direct visual inspection of sorted cells. The percentage of the total cell population in each window is shown. (D) The DNA content profiles of the GFP-negative cells in window A of the slug posteriors (upper panel) and the GFP-positive cells in window B of the anterior cells (lower panel).

View larger version (39K): [in a new window]   Fig. 7. Prespore and prestalk cell DNA content during development. Wild-type cells (AX4) marked with a prespore reporter gene (cotB/GFP), or a prestalk reporter gene (ecmA/GFP), were allowed to develop on filters (A). The ecmA/GFP slug is the same image as shown in Fig. 6. (B) Flow cytometry profiles of cells at 18 hours of development show the distribution of cells expressing GFP. (C) GFP-positive cells were collected by fluorescence activated cell sorting as illustrated in (B), stained with propidium iodide, and their DNA content was determined by flow cytometry. Peak values of the DNA content profiles for mononucleated cells are plotted.