First published online 5 May 2004
doi: 10.1242/dev.01151
Development 131, 2619-2630 (2004)
Published by The Company of Biologists 2004
Tissue-specific G1-phase cell-cycle arrest prior to terminal differentiation in Dictyostelium
Guokai Chen1,
Gad Shaulsky2 and
Adam Kuspa1,2,*
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
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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).
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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.
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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.
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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).
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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.
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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).
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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.
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© The Company of Biologists Ltd 2004
