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

Jiang B, Zhao W, Yuan J, Qian Y, Sun W, Zou Y, Guo C, Chen B, Shao C, Gong Y - PLoS ONE (2012)

Bottom Line: 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 cells were selected against in Cul4b heterozygotes, leading to various degrees of skewed X-inactivation in different tissues.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Experimental Teratology, Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong, China.

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 mutation, Cul4b 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 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 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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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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pone-0037070-g006: 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.

Mentions: Because Cul4b is X-linked, Cul4b heterozygous females are functional mosaics in terms of the expression of Cul4b. The somatic cells are either Cul4b functional or depending on the choice of the X-chromosome that becomes inactivated. In the XLMR family with CUL4B mutation, X-chromosome inactivation (XCI) was extremely skewed in peripheral blood cells of carriers, due to the selection that favors the cells in which the mutant CUL4B allele is inactivated [17], [20]. We first determined the XCI pattern in 4-month-old adult Cul4b heterozygous females by immunohistochemical analysis for Cul4b expression. If XCI were balanced in Cul4b heterozygous females, the proportion of cells expressing Cul4b should be ∼50% of that in wild-type females. On the other hand, if XCI is skewed toward Cul4b allele, as in human CUL4B heterozygous carriers, the percentage of cells expressing Cul4b should be closer to that in wild-type females. As shown in Fig. 6A, the percentages of cells expressing Cul4b in kidney, liver and lung were identical between Cul4b heterozygous females and wild-type females, suggesting that XCI is extremely skewed in those organs. XCI in hippocampus was also skewed, but to a lesser extent, since the percentage of Cul4b positive cells in heterozygotes remained lower than that in wild-type. Western blot assay showed a similar trend (Figure S2). While the expression levels of Cul4b in liver and lung in Cul4b heterozygous mice were comparable to those in wild type mice, the expression levels in cortex and hippocampus were decreased in Cul4b heterozygous mice compared to those in wild type mice.


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

Jiang B, Zhao W, Yuan J, Qian Y, Sun W, Zou Y, Guo C, Chen B, Shao C, Gong Y - PLoS ONE (2012)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351389&req=5

pone-0037070-g006: 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.
Mentions: Because Cul4b is X-linked, Cul4b heterozygous females are functional mosaics in terms of the expression of Cul4b. The somatic cells are either Cul4b functional or depending on the choice of the X-chromosome that becomes inactivated. In the XLMR family with CUL4B mutation, X-chromosome inactivation (XCI) was extremely skewed in peripheral blood cells of carriers, due to the selection that favors the cells in which the mutant CUL4B allele is inactivated [17], [20]. We first determined the XCI pattern in 4-month-old adult Cul4b heterozygous females by immunohistochemical analysis for Cul4b expression. If XCI were balanced in Cul4b heterozygous females, the proportion of cells expressing Cul4b should be ∼50% of that in wild-type females. On the other hand, if XCI is skewed toward Cul4b allele, as in human CUL4B heterozygous carriers, the percentage of cells expressing Cul4b should be closer to that in wild-type females. As shown in Fig. 6A, the percentages of cells expressing Cul4b in kidney, liver and lung were identical between Cul4b heterozygous females and wild-type females, suggesting that XCI is extremely skewed in those organs. XCI in hippocampus was also skewed, but to a lesser extent, since the percentage of Cul4b positive cells in heterozygotes remained lower than that in wild-type. Western blot assay showed a similar trend (Figure S2). While the expression levels of Cul4b in liver and lung in Cul4b heterozygous mice were comparable to those in wild type mice, the expression levels in cortex and hippocampus were decreased in Cul4b heterozygous mice compared to those in wild type mice.

Bottom Line: 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 cells were selected against in Cul4b heterozygotes, leading to various degrees of skewed X-inactivation in different tissues.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Experimental Teratology, Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong, China.

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 mutation, Cul4b 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 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 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.

Show MeSH
Related in: MedlinePlus