First published online 9 July 2008
doi: 10.1242/dev.024539
Development 135, 2729-2738 (2008)
Published by The Company of Biologists 2008
Maternal depletion of CTCF reveals multiple functions during oocyte and preimplantation embryo development
Le-Ben Wan1,
Hua Pan2,
Sridhar Hannenhalli3,
Yong Cheng4,
Jun Ma2,
Andrew Fedoriw1,*,
Victor Lobanenkov5,
Keith E. Latham4,
Richard M. Schultz2 and
Marisa S. Bartolomei1,
1 Department of Cell and Developmental Biology, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104, USA.
2 Department of Biology, University of Pennsylvania, Philadelphia, PA 19104,
USA.
3 Department of Genetics and Penn Center for Bioinformatics, University of
Pennsylvania, Philadelphia, PA 19104, USA.
4 The Fels Institute for Cancer Research and Molecular Biology, and Department
of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140,
USA.
5 Laboratory of Immunopathology, NIAID, NIH, Rockville, MD 20852, USA.
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Fig. 1. CTCF depletion in oocytes and embryos. (A) Quantification of
nuclear CTCF immunofluorescence staining in growing oocytes. Ntg and Tg
indicate oocytes derived from 10-day-old Ntg and Tg littermates, respectively.
Lines 1, 12 and 21 are shown. *P<0.0001;
n=number of oocytes. (B) Immunofluorescence images of
CTCF-stained oocytes and embryos. One confocal section of an egg or embryo is
shown per panel, with red indicating CTCF-stained nuclei. Line 1 is shown.
Scale bar: 40 µm. (C) Schematic of CTCF depletion in oocytes and
embryos. Ntg females were mated to Tg males and  50% of female progeny
inherited the transgene. All oocytes from Tg females were CTCF depleted.
CTCF-replete oocytes and embryos are depicted in brown, whereas CTCF-depleted
oocytes and embryos are depicted in white. Pink and blue circles represent
maternal and paternal pronuclei, respectively. GV, germinal vesicle; hr, hours
post-hCG.
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Fig. 3. Meiotic defects in CTCF-depleted oocytes. Eggs having undergone GVBD
by 2 hours of culture were processed for diakinesis spreads. Eggs that have
undergone PBE by 16 hours of culture were processed for M2 spreads. Line 1 is
shown. Seven percent (3/42) of Tg eggs that have undergone PBE contained
roughly 20 bivalents. n=number of oocytes. Scale bar: 0.1 µm.
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Fig. 4. Early mitotic defects leading to apoptosis. (A) Embryo
development in culture. One-cell embryos were cultured to 72 hours post-hCG
and categorized according to cell number. Line 1 is shown. n=number
of embryos. (B) Autoradiogram and (C) quantification of TRC
upregulation. Two-cell embryos from Line 1 were radiolabeled at various
timepoints post-cleavage. At each timepoint, TRC expression in 3-5 embryos
derived from Tg mice were normalized to TRC expression in an equivalent number
of embryos derived from Ntg mice (relative expression=1). Early, Mid and Late
indicate embryos radiolabeled at 6, 12 and 21 hours post-cleavage;
*P<0.05; n=number of experiments. + indicates
Spindlin, an abundant maternal protein. (D) Embryo development in vivo.
Cleavage-stage embryos were flushed at 72 hours post-hCG and categorized
according to cell number. Lines 1, 12 and 21 are shown. n=number of
embryos. (E) Immunofluorescence images of DAPI-stained embryos in D.
One confocal section of an embryo is shown per panel, with green indicating
DAPI-stained nuclei. Line 1 is shown. Tg embryos have ectopic nuclei. Arrow,
large ectopic nucleus; arrowhead, small ectopic nucleus. Scale bar: 20 µm.
(F) Percentages of embryos in D with ectopic nuclei. Embryos were
categorized according to whether large (white bars) or small (gray bars)
ectopic nuclei were observed. Lines 1, 12 and 21 are shown.
*P<0.05; **P<0.0001;
n=number of embryos. (G) Apoptosis at 120 hours post-hCG.
One-cell embryos were cultured to 120 hours post-hCG and TUNEL stained. One
embryo is shown per panel, with green indicating DAPI-stained nuclei and red
indicating TUNEL staining. Line 1 is shown. Only 7% (2/28) of embryos derived
from Tg mice versus 94% (29/31) of embryos derived from Ntg littermates formed
a blastoceol cavity by 120 hours post-hCG. Scale bar: 40 µm.
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Fig. 5. Maternal pronuclear transfer and Ctcf mRNA-injection experiments.
(A) Maternal pronuclear transfer experiments. Maternal pronuclei (mPNs)
were reconstructed with one-cell embryos and cultured until 72 hours post-hCG.
The average cell number per transfer-type at 72 hours post-hCG is indicated.
Ntg and Tg (Line 1) indicate mPNs or embryos derived from Ntg and Tg
littermates, respectively. Bars labeled A are significantly different from
bars labeled B (overall P<0.0001). n=number of embryos.
(B) Immunofluorescence images of CTCF-stained embryos after cytoplasmic
mRNA microinjection. One-cell embryos from Line 1 were microinjected with CTCF
or control GFP mRNA, and cultured until 72 hours post-hCG. One confocal
section of an embryo is shown per panel, with red indicating CTCF-stained
nuclei. Scale bar: 40 µm. (C) Embryo development after cytoplasmic
mRNA microinjection. The average cell number per microinjection-type at 72
hours post-hCG is indicated. Bars labeled A are significantly different from
bars labeled B (overall P<0.0001). n=number of
embryos.
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© The Company of Biologists Ltd 2008
P<10-5.(B) Percentages of
CTCF binding sites within 5 kb of all, upregulated and downregulated genes.
Among sites within 5 kb of downregulated genes, the percentage of upstream
sites was significantly higher than the percentage of downstream sites,
compared with background. *P<0.05.