Maternal depletion of CTCF reveals multiple functions during oocyte and preimplantation embryo development - PubMed (original) (raw)

Maternal depletion of CTCF reveals multiple functions during oocyte and preimplantation embryo development

Le-Ben Wan et al. Development. 2008 Aug.

Abstract

CTCF is a multifunctional nuclear factor involved in epigenetic regulation. Despite recent advances that include the systematic discovery of CTCF-binding sites throughout the mammalian genome, the in vivo roles of CTCF in adult tissues and during embryonic development are largely unknown. Using transgenic RNAi, we depleted maternal stores of CTCF from growing mouse oocytes, and identified hundreds of misregulated genes. Moreover, our analysis suggests that CTCF predominantly activates or derepresses transcription in oocytes. CTCF depletion causes meiotic defects in the egg, and mitotic defects in the embryo that are accompanied by defects in zygotic gene expression, and culminate in apoptosis. Maternal pronuclear transfer and CTCF mRNA microinjection experiments indicate that CTCF is a mammalian maternal effect gene, and that persistent transcriptional defects rather than persistent chromosomal defects perturb early embryonic development. This is the first study detailing a global and essential role for CTCF in mouse oocytes and preimplantation embryos.

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Figures

Fig. 1

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.

Fig. 2

Fig. 2. Transcriptional misregulation in CTCF-depleted oocytes

(A) Percentages of genes within 0 (overlapping), 5, 10 and 50 kb of a CTCF-binding site. The proportions of nearby upregulated and especially downregulated genes were significantly higher than background (all genes) within every distance threshold. *P<0.05; **P<10−8, †P<10−7, ‡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.

Fig. 3

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.

Fig. 4

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.

Fig. 5

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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