QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Burns, J. L. Articles by Hassan, A. B. Search for Related Content PubMed PubMed Citation Articles by Burns, J. L. Articles by Hassan, A. B. Social Bookmarking                         What's this?

Cell survival and proliferation are modified by insulin-like growth factor 2 between days 9 and 10 of mouse gestation

Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK

View larger version (60K): [in a new window]   Fig. 1. In situ analysis of cell death E9-E9.5. (A) Acridine Orange staining of pyknotic cells in unfixed embryos (19 somites). Conventional fluorescence microscopy (inserts) at low power reveal similar sized and stained embryos, despite differences in folding and angles of view (left panel, wild type=Igf2+m/+p; right panel Igf2+m/-p). Confocal microscopy and maximum projection of the same embryos (18x5µm sections at 20 µm intervals, large images) reveal the distribution of multiple areas of focal labelling above background (see text). (B) TUNEL assay of 7 µm cryosection (wild type=Igf2+m/+p embryo (upper) and littermate Igf2+m/-p (lower)). A complex distribution of labelling was observed in both sections. Scale bars: 500 µm in A; 100 µm in B.

View larger version (41K): [in a new window]   Fig. 2. Analysis of droplet spreads of embryo cell suspensions. (A,B) Droplet spreads of suspensions from whole wild-type (A) and Igf2+m/-p (B) embryos stained with DAPI and visualised with fluorescence microscopy. Mitotic figures (arrow in A) and pyknotic cells with less than 2C DNA content (arrows in B) can be identified. (C) Similarity in the distribution of fluorescence emitted per nuclei from litter-matched whole embryo spreads, (wild type (blue) n=436 nuclei, Igf2+m/-p (red) n=499 nuclei). (D) Significantly increased pyknotic cell number in Igf2+m/-p cell spreads were observed (**P=0.012 Student’s t-test, wild type (blue) n=2341 nuclei from seven embryos, Igf2+m/-p (red) n=1277 nuclei from seven embryos). No significant differences (NS) were detected for mitotic cell counts (P=0.47). Scale bars: 10 µm in A,B.

View larger version (23K): [in a new window]   Fig. 3. Flow cytometric analysis of whole embryo cell suspensions. (A,B) Flow cytometric quantification of total cell number (unbroken line) and 95% confidence interval (broken lines) from pooled wild-type (A, n=65) and Igf2+m/-p (B, n=84) embryos dated by seminal plugs and using a quadratic line of best fit (Minitab). Inserts show linear plots of loge cell number versus age (days). (C) Box plots of normalised (100%) cell number (subtracting sub-G1) of each embryo relative to the mean cell number of the respective genotype in each litter (box=interquartile range, vertical line=95% confidence interval, horizontal line=median). Insert shows pooled values. (D) Ratios of mean cell numbers per litter for Igf2+m/-p (-p) over wild-type (+p) embryos matched per litter and aged by the mean cell number of wild-type embryos in each litter (blue diamonds, n=15 litters, 140 embryos, P=0.005 at E11, Student’s t-test). Line shows linear regression of all data points. (E) Somite number plotted against loge cell number for wild type (blue, n=65) and Igf2+m/-p (red, n=84). (F) Ratios of mean somite numbers and line of best fit for Igf2+m/-p (-p) over wild type (+p) embryos matched per litter and aged by the mean cell number of wild-type embryos in each litter (n=10 litters, 124 embryos). Litters with at least two embryos of each genotype were used.

View larger version (17K): [in a new window]   Fig. 4. DNA content and cell cycle parameters of embryo suspensions. (A,B) Representative distribution of DNA content at E9.25 (aged by cell number) of wild-type (A) and Igf2+m/-p (B) litter-matched embryos. Gates: Sub-G1=a, G1=2xb, S=c-(b+d) and G2=2xd. (C-F) Quantification of Sub G1(C), G1(D), S (E) and G2 (F) from DNA profiles of litter-matched embryos, as in A,B aged by wild-type embryos at E8.4±0.2 (n=10), E8.8±0.2 (n=26), E9.25±0.25 (n=18), E9.75±0.25 (n=24), E10.15±0.15 (n=22), E10.75±0.25 (n=6) and E11.52±0.25 (n=18). The differences were significant (*) only at E9.25 for sub-G1 (C, P=0.003). E9.75 S-phase (E, P=0.001) and E9.75 G2 (F, P=0.008) (Student’s t-test, 95% confidence interval). For D-F, percentages were of total number of viable cells (excluding subG1). Error bars indicate s.e.m.

View larger version (22K): [in a new window]   Fig. 5. Cytometric analysis of phosphorylated histone H3 in whole embryo cell suspensions. Right panel shows antibody labelling of mitotic chromatin coinciding with 4C DNA content compared with control (left panel) without primary antibody to serine 10 -phosphorylation of histone H3.

View larger version (37K): [in a new window]   Fig. 6. Analysis of caspase-mediated cell death in unfixed embryo cell suspensions. (A,B) Representative dual colour cytometry from wild type and Igf2+m/-p embryos at E9.25 (determined by wild-type embryo cell number) labelled with FAM-VAD-FMK and propidium iodide (PI). FAM-VAD-FMK labels cells with active caspases (1-9) and propidium iodide labels cells with disrupted membranes. FAM-VAD-FMK and PI-positive cells are undergoing apoptosis (area iii), PI-positive cells have disrupted membranes and may be in the late stages of cell death (area iv), FAM-VAD-FMK-positive but PI-negative cells maybe in the earlier commitment phase of cell death (ii) and cells with little caspase activity and intact membranes are viable (i). Numbers indicate mean percentage of whole embryos suspension in each quadrant as in C. (C) Summary of mean±s.e.m. for profiles as in A,B for E8.5± 0.25 (n=11), E9.25 (n=22) and E10.5± 0.25 (n=13) embryos. Significant differences occurred around E9.25, with increased caspase activity and cell permeability in cells from Igf2+m/-p embryos (P=0.044).

View larger version (29K): [in a new window]   Fig. 7. Cytometric analysis of proliferation determined by BrdU incorporation. (A) Pulse BrdU followed by cytometry of whole embryo suspensions after 1 hour. Typical profiles for wild-type (left panel) and Igf2+m/-p (right panel) litter-matched embryos aged at E9.75 by cell number. Numbers indicate mean cell number above the horizontal line as a percentage of whole embryos (n=16). (B) Pulse-chase BrdU followed by cytometry of whole embryo suspensions after 24 hours in vivo. Typical profiles for wild-type (left panel) and Igf2+m/-p (right panel) of litter-matched embryos injected at E8.5 and dissected at E9.5 (plugging). The cells above the horizontal line were labelled as after a single BrdU pulse. Numbers indicate mean cell number above the horizontal line as a percentage of whole embryos (n=12). (C) Mean±s.e.m. of percentage of pulse BrdU incorporation as in A at E8.5 (n=8), E9.75 (n=16) and E10.5 (n=13). The only significant differences occurred at E9.75 (P=0.006). (D) Means±s.e.m. of percentage of pulse (E8.5) chase (to E9.5) BrdU incorporation as in B. Fewer cells divided in Igf2+m/-p, as significantly more retained label as if pulsed at 1 hour (n=12, litter-matched samples).

View larger version (32K): [in a new window]   Fig. 8. Cell size analysis of unfixed cells in suspension. (A) Red blood cell (mouse) size distribution profiles following incubation in buffers of different osmolarity (insert) using a Coulter multi-sizer. (B) Red blood cell changes in cell size with osmolarity, from A. (C) Multiple cell diameter profiles from unfixed whole wild-type (blue, n=5) and Igf2+m/-p (red, n=6) litter-matched embryos, aged E9.5 using cell number of a fixed wild-type embryo.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter