Fig 1.
Expression pattern of CUL4B embryos at E6.5 and E7.5.
(A) Immunostaining was performed for CUL4B (red), the embryonic ectoderm marker OCT3/4 (green), and the nuclear marker DAPI (blue) in E6.5 Cul4b WT (Cul4b+/Y, +/Y), knockout (Cul4bΔ/Y, Δ/Y), and heterozygous (Cul4bΔ/+, Δ/+) embryos. Staining of CUL4B is observed in the extraembryonic ectoderm (exe) and the embryonic ectoderm (ee) of WT embryos and in the ee of Cul4bΔ/+embryos with random inactivation of X chromosomes. The CUL4B signal was not detected in either the ee or exe of Cul4bΔ/Y embryos. Due to paternal X-inactivation in the extraembryonic tissue of Cul4bΔ/+embryos, the CUL4B signal was barely detectable. Scale bar: 50 μm. More than three embryos per genotype were analyzed for each experiment. (B) Immunostaining of E7.5 embryos was performed. Colors and symbols are the same as described in (A). Cul4bΔ/+ embryos exhibited random CUL4B signals in the ee and only slight expression of CUL4B in the chorionic ectoderm (ce) due to paternal X-inactivation in extraembryonic tissue. No CUL4B protein signal was detected in the ee or ce of Cul4bΔ/Y embryos, and this effect was accompanied by a reduction in embryo size. A trace amount of cells showing CUL4B staining in a fraction of CE cells might represent a few extraembryonic mesoderm cells that escape the random X-chromosome inactivation of Cul4b expression in Cul4bΔ/+ embryos. The upper (E6.5) and lower (E7.5) right panels show percentage of CUL4B-positive cells within the OCT4-positive cells in ee of Cul4b+/Y, Cul4bΔ/Y and Cul4bΔ/+ embryos, respectively. The red (CUL4B), green (OCT4), and blue (DAPI) fluorescence signals within the dotted areas were visually counted individually. All embryos were generated from Cul4b+/Δ females mated with Cul4b+/Y WT males or Cul4blox/Y;Prm1-Cre males. More than three embryos per genotype were analyzed for each experiment. Scale bar: 100 μm. Data are expressed as means + standard errors of the mean. ***: p<0.001 was compared with +/Y group. ###: p <0.001 was compared with Δ/+ group.
Table 1.
Genotype analysis of breeding of Cul4blox/+; Zp3-Cre females mated with WT males*.
Table 2.
Offspring of Cul4bΔ/+ heterozygous female mice generated from Cul4b+/Y males mated with Cul4b+/Δ females*.
Fig 2.
Analysis of extraembryonic tissues of E7.5 embryos.
(A) Immunostaining for the pluripotent markers CDX2 (red) and EOMES (green) was performed on sections of the chorionic ectoderm (ce) from Cul4b+/Y, Cul4bΔ/Y, and Cul4bΔ/+ embryos (E7.5). Yellow squares indicate the cells in the chorionic ectoderm that show positive staining. Nuclei are stained with DAPI (blue). Scale bar: 100 μm. Shown on the right panel is the visually counted CDX2 and EOMES positive cells normalized with respective DAPI (blue) positive cells in the ce of Cul4b+/Y, Cul4bΔ/Y and Cul4bΔ/+ embryos, respectively. (B) Immunostaining of the extraembryonic lineage marker, AP-2γ (green), in Cul4b+/Y, Cul4bΔ/Y, and Cul4bΔ/+ embryos (E7.5). Nuclei are stained with DAPI (blue). Cells expressing AP-2γ outside the yellow region are considered to be within a region comprising TGCs. The Cul4b genotype for embryonic (bottom) and extraembryonic (top) tissues are marked separately by (+) for WT and (Δ) for KO. The Cul4bΔ/Y embryos contain fewer TGCs, while the Cul4bΔ/+ embryos contain similar numbers of TGCs as the Cul4b+/Y embryos at E7.5. All embryos were generated from Cul4bΔ/+ females mated with Cul4b+/Y WT males or Cul4blox/Y;Prm1-Cre males. More than three embryos per genotype were analyzed for each experiment. Scale bar: 200 μm. Shown on the right panel is the visually counted AP-2γ positive cells normalized with the number of respective DAPI (blue) positive cells outside the yellow region of Cul4b+/Y, Cul4bΔ/Y and Cul4bΔ/+ embryos, respectively. Data are expressed as means + standard errors of the mean. *: p <0.05 and ***: p <0.001 were compared with +/Y group. #: p <0.05, ##: p <0.01 and ###: p <0.01 were compared with Δ/+ group.
Fig 3.
Expression of mesoderm and primitive streak markers in Cul4b-null embryos.
Transcripts of Brachyury (A-D), Eomes (E-H), and Fgf8 (I-L) were detected in Cul4b+/Y and Cul4bΔ/Y embryos by whole-mount RNA in situ hybridization experiments. (A, B) At E6.5, transcripts of the primitive streak marker, Brachyury, were detected in the nascent primitive streak of Cul4b+/Y embryos but not in the primitive streak of Cul4bΔ/Y embryos. (C, D) A slight decrease in Brachyury was detected at E7.5 in Cul4bΔ/Y embryos. (G, H) Expanded expression of Eomes was detected in the primitive streak of Cul4bΔ/Y embryos compared to that of Cul4b+/Y embryos. (I, J) Transcript levels of Fgf8 in Cul4b+/Y embryos at E6.5 were similar to those in Cul4b+/Y embryos. (K, L) Transcript levels of Fgf8 in Cul4b+/Y and Cul4bΔ/Y embryos at E7.5. Scale bar: 200 μm. (M, N) Sections of whole-mount RNA in situ hybridizations performed for E7.5 embryos. Expanded expression of Eomes was detected in Cul4bΔ/Y embryos (N1’-N3’) compared with that in their control littermates (Cul4b+/Δ) (N1-N3). Sections represent distances of 175 μm and 77 μm (M2 and M3), 126 μm and 49 μm (M2’ and M3’), 182 μm and 84 μm (N2 and N3), and 140 μm and 84 μm (N2’ and N3’), respectively, from the bottom of the embryos examined. Bracket: Ctl, Control; PS, primitive streak; arrow: ectoderm, double arrow: mesoderm, triple arrow: visceral endoderm. All embryos were generated from Cul4b+/Δ females mated with Cul4b+/Y WT males or Cul4blox/Y;Prm1-Cre males.
Fig 4.
Histology of the embryos at E6.5, E7.0 and E7.5.
(A) Representative embryos at E6.5, and Cul4bΔ/Y (KO1, KO2) embryos displayed a slightly smaller size than WT embryos. Arrowheads indicate the boundary of ee and exe. ee, embryonic ectoderm; exe, extraembryonic ectoderm; epc, ectoplacental cone. Scale bar: 50 μm. (B) Representative embryos at E7.0, WT embryos underwent the formation of the posterior amniotic fold (paf). Cul4bΔ/Y (KO3, KO4) embryos displayed a phenotype of delayed morphology or had abnormal paf (*). Arrowheads indicate the boundary of ee and exe. Scale bar: 100 μm. (C) Representative embryos at E7.5. WT embryos underwent the formation of three cavities, including the amniotic cavity (ac), the exocoelomic cavity (eec), and the chorionic cavity (cc). Cul4bΔ/Y (KO5, KO6) embryos displayed a single proamniotic cavity or formation of a posterior amniotic fold (paf) with accumulation of epiblasts (*). All embryos were generated from Cul4b+/Δ females mated with Cul4b+/Y WT males or Cul4blox/Y;Prm1-Cre males. Arrowheads indicate the boundary of ch, chorionic ectoderm; al, allantois. Scale bar: 100 μm.
Fig 5.
Loss of Cul4b in the ICM formed from blastocysts cultured in vitro causes proliferation defects.
(A) Images of cultured Cul4b WT, Het, and KO blastocysts at day 7 are shown. (B) Growth areas in the ICM of Cul4b KO blastocysts were severely decreased compared to the WT and Het blastocysts. (C) A comparison of ICM growth rates among the Cul4b WT, Het, and KO blastocysts suggests that deficiencies in Cul4b lead to proliferation defects. All embryos were generated from Cul4b+/Δ females mated with Cul4b+/Y WT males or Cul4blox/Y;Prm1-Cre males. More than three independent experiments were performed. WT (Cul4b+/Y and Cul4b+/+); Het, heterozygous (Cul4bΔ/+ and Cul4b+/Δ); KO (Cul4bΔ/Y and Cul4bΔ/Δ); TGCs, trophoblast giant cells (arrowheads). Scale bar: 100 μm.
Fig 6.
Expression of extraembryonic ectoderm and ectoplacental cone markers in Cul4b-null embryos.
Transcripts of Bmp4 (A-D), Hand1 (E-H), and Mash2 (I-L) were detected in Cul4b+/Y and Cul4bΔ/Y embryos in whole-mount RNA in situ hybridization experiments. At E6.5 and E7.5, the transcript levels of the extraembryonic ectoderm marker Bmp4 and the ectoplacental cone marker Hand1 cannot be discriminated between Cul4b+/Y and Cul4bΔ/Y embryos (A-H). The expression levels of the extraembryonic ectoderm and ectoplacental cone marker Mash2 were not distinct between Cul4b+/Y and Cul4bΔ/Y embryos at E6.5 (I, J). A severe reduction in the chorionic ectoderm in Cul4bΔ/Y embryos was observed compared to that in Cul4b+/Y embryos at E7.5 (K, L). All embryos were generated from Cul4b+/Δ females mated with Cul4b+/Y WT males or Cul4blox/Y;Prm1-Cre males. Scale bar: 200 μm.
Fig 7.
The parietal endoderm and visceral endoderm were not affected in Cul4b-null embryos.
Cul4b+/Y and Cul4bΔ/Y embryos at E7.5 were sectioned and immunostained for GATA-4 (red), OCT-4 (green), and DAPI (blue). Dashed yellow lines indicate the cells with GATA-4 expression that are localized to regions of the parietal endoderm and visceral endoderm. All embryos were generated from Cul4b+/Δ females mated with Cul4b+/Y WT males or Cul4blox/Y;Prm1-Cre males. Scale bar: 100 μm.
Fig 8.
Immunoblotting of G1/S-phase cyclins in the embryo proper of Cul4b-null embryos at E9.5.
(A) Lower expression levels of cyclins D1, D2, and E were detected in Cul4b KO embryos (Cul4bΔ/Y and Cul4bΔ/Δ) compared to that in Cul4b WT (Cul4b+/Y) and Cul4b Het (Cul4b+/Δ) embryos. The detection of β-actin was included as a loading control. Cyclin D3 showed comparable protein levels in all three embryo types. (B) Densitometry values for the protein levels of cyclins, (C) The β-catenin and phosphor-β-catenin signals detected in each of the three embryo types were normalized to the average signal for the Cul4b WT embryo extracts, which was set to 1, with the data obtained from three independent experiments. Each Cul4b KO embryo sample represents a collection of samples from three identical genotypes of E9.5 Cul4b KO embryos. All embryos were generated from Cul4b+/Δ females mated with Cul4b+/Y WT males or Cul4blox/Y;Prm1-Cre males. ***P < 0.001.