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. 2012;7(5):e37070.
doi: 10.1371/journal.pone.0037070. Epub 2012 May 14.

Lack of Cul4b, an E3 ubiquitin ligase component, leads to embryonic lethality and abnormal placental development

Baichun Jiang  1 , Wei Zhao, Jupeng Yuan, Yanyan Qian, Wenjie Sun, Yongxin Zou, Chenhong Guo, Bingxi Chen, Changshun Shao, Yaoqin Gong
Affiliations

Lack of Cul4b, an E3 ubiquitin ligase component, leads to embryonic lethality and abnormal placental development

Baichun Jiang et al. PLoS One. 2012.

Abstract

Cullin-RING ligases (CRLs) complexes participate in the regulation of diverse cellular processes, including cell cycle progression, transcription, signal transduction and development. Serving as the scaffold protein, cullins are crucial for the assembly of ligase complexes, which recognize and target various substrates for proteosomal degradation. Mutations in human CUL4B, one of the eight members in cullin family, are one of the major causes of X-linked mental retardation. We here report the generation and characterization of Cul4b knockout mice, in which exons 3 to 5 were deleted. In contrast to the survival to adulthood of human hemizygous males with CUL4B null mutation, Cul4b null mouse embryos show severe developmental arrest and usually die before embryonic day 9.5 (E9.5). Accumulation of cyclin E, a CRL (CUL4B) substrate, was observed in Cul4b null embryos. Cul4b heterozygotes were recovered at a reduced ratio and exhibited a severe developmental delay. The placentas in Cul4b heterozygotes were disorganized and were impaired in vascularization, which may contribute to the developmental delay. As in human CUL4B heterozygotes, Cul4b null cells were selected against in Cul4b heterozygotes, leading to various degrees of skewed X-inactivation in different tissues. Together, our results showed that CUL4B is indispensable for embryonic development in the mouse.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of Cul4b flox mice.
(A) Strategy for generation of Cul4b floxed targeting vector. On the top was shown the wild-type allele of Cul4b gene. The targeting vector and targeted allele were shown in the middle and at the bottom, respectively. BamHI (labeled B) and XbaI (labeled X) sites are indicated. Black bars indicating the positions of the probes used in Southern blots are also indicated. (B) Southern blot analysis of genomic DNA isolated from wild-type ES cells and selected ES cell clones. BamHI digested DNA was hybridized with 5′ probe and XbaI digested DNA was hybridized with 3′ probe. WT, wild-type allele; F, flox allele. (C) cDNA sequencing of Cul4b gene from the brain tissue of brain-specific knockout mice (Cul4b flox/Y;Nesin-Cre). As predicted, exon 2 was spliced onto exon 6 after the excision of exons 3–5. (D) Real-time RT-PCR analysis of Cul4b mRNA isolated from brain tissues of wild-type males (Cul4b +/Y;Nestin-Cre +/−), wild-type females (Cul4b+/+;Nestin-Cre +/−), heterozygous females (Cul4b +/flox;Nestin-Cre +/−), and conditional knock-out males (Cul4b flox/Y;Nestin-Cre +/−). (E) Western blot analysis of Cul4b protein isolated from brain tissues of wild-type, heterozygous and conditional knockout mice using an anti-Cul4b antibody. Gapdh was used as a loading control.
Figure 2
Figure 2. Morphology and histology of Cul4b null embryos.
(A) Uterus excised from pregnant female at 12.5 dpc. Arrows indicate absorbed embryos. (B) Photomicrographs of E7.5 embryos dissected from surrounding deciduas tissue of the same uterus. The two embryos on the left appear normal in size and morphology and the two on the right were much smaller and were partially deteriorated. The bar represents 200 µm. (C) H&E staining of paraffin sections of wild-type and Cul4b null embryos at 7.5 dpc. Littermate embryos at 7.5 dpc were paraffin embedded and cross sectioned together with their surrounding deciduas. The genotype of each embryo was determined by immunohistochemistry using an anti-Cul4b antibody, as shown below. (D) Immunohistochemistry of paraffin sections of wild-type and Cul4b null embryos at 7.5 dpc with an anti-Cul4b antibody. (E–F) Photomicrographs with higher magnification of the stained section shown in (D).
Figure 3
Figure 3. Decreased proliferation and increased apoptosis in Cul4b null embryos.
(A) Paraffin sections of wild-type and Cul4b null embryos at 7.5 dpc were stained with an antibody against Ki67, a proliferation marker. Sections were counterstained with haematoxylin. Lower panels are the higher magnification of the upper panels. (B) Paraffin sections of wild-type and Cul4b null embryos at 7.5 dpc were analyzed by immunostaining against BrdU, and counterstained with DAPI. Lower panels are the higher magnification of the upper panels. (C) Paraffin sections of wild-type and Cul4b null embryos at 7.5 dpc were analysed by TUNEL assay for labeling apoptotic cells, and counterstained with DAPI. Lower panels are the higher magnification of the upper panels. (D) Paraffin sections of wild-type and Cul4b null embryos at 7.5 dpc were stained with an antibody against cyclin E. Sections were counterstained with haematoxylin. Lower panels are the higher magnification of the upper panels.
Figure 4
Figure 4. Growth retardation of Cul4b heterozygous mice during embryonic development.
(A) Bodyweights of Cul4b heterozygous mice and littermate wild-type females after birth. Data were presented as mean±SD. N = 8, *: p<0.05; **: p<0.01; ***: p<0.001. (B–E) Representative photographs of Cul4b heterozygous embryos and littermate wild-type controls at 9.5 (B), 10.5 (C), 12.5 (D) and 14.5 (E) dpc. The bar represents 1 mm in (B–C) and 2 mm in (D) and (E), respectively.
Figure 5
Figure 5. Morphology and histology of placentas of wild-type, Cul4b heterozygous and absorbed embryos at 14.5 dpc.
(A) Representative photographs of placentas of wild-type, Cul4b heterozygous and absorbed embryos at 14.5 dpc. (B) H&E staining of radial sections of placentas. sp, spongiotrophoblast layer; la, labyrinthine layer. Lower panels are the higher magnification of the upper panels. (C) Immunohistochemisty of radial sections of placentas with an antibody to PECAM, an angiogenesis marker. Middle panels are the higher magnification of the upper panels, and lower panels are the higher magnification of the middle panels.
Figure 6
Figure 6. Characterization of X chromosome inactivation by Cul4b expression in heterozygous mice.
(A–C) Percentages of cells positive for Cul4b of Cul4b heterozygous mice and littermate wild-type female controls at 4 months (A), 3 weeks (B) and newborn (C). More than 2,000 cells of each tissue were scored. Hi, hippocampus; Ki, kidney; Li, liver; Lu, lung. Data were presented as mean±SD. *: p<0.05; **: p<0.01; ***: p<0.001. (D–E) Representative images of liver (D) and hippocampus (E) at 3 weeks stained with an antibody against Cul4b. Sections were counterstained with haematoxylin. Lower panels are the higher magnification of the upper panels. (F) Immunohistochemistry of paraffin sections of Cul4b heterozygous embryos at 7.5 dpc with an anti-Cul4b antibody. Embryos at 7.5 dpc were paraffin embedded and cross sectioned together with their surrounding deciduas. Middle and right panels are the higher magnification of the left panel.
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