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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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Decreased proliferation and increased apoptosis in Cul4b  embryos.(A) Paraffin sections of wild-type and Cul4b  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  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  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  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.
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pone-0037070-g003: Decreased proliferation and increased apoptosis in Cul4b embryos.(A) Paraffin sections of wild-type and Cul4b 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 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 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 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.

Mentions: To investigate what causes the developmental delay of the Cul4b embryos, we analyzed the proliferative activity and apoptosis in E7.5 embryos. The proliferation was evaluated by immunohistochemical staining of paraffin sections of E7.5 embryos for Ki67, a marker of proliferating cells. While there was a high level of proliferation in the wild-type embryos, proliferative cells were less abundant in Cul4b embryos (Fig. 3A). BrdU incorporation assay confirmed the decrease in proliferation in Cul4b embryos (Fig. 3B), suggesting that the proliferative activity was greatly compromised in Cul4b embryos.


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)

Decreased proliferation and increased apoptosis in Cul4b  embryos.(A) Paraffin sections of wild-type and Cul4b  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  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  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  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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351389&req=5

pone-0037070-g003: Decreased proliferation and increased apoptosis in Cul4b embryos.(A) Paraffin sections of wild-type and Cul4b 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 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 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 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.
Mentions: To investigate what causes the developmental delay of the Cul4b embryos, we analyzed the proliferative activity and apoptosis in E7.5 embryos. The proliferation was evaluated by immunohistochemical staining of paraffin sections of E7.5 embryos for Ki67, a marker of proliferating cells. While there was a high level of proliferation in the wild-type embryos, proliferative cells were less abundant in Cul4b embryos (Fig. 3A). BrdU incorporation assay confirmed the decrease in proliferation in Cul4b embryos (Fig. 3B), suggesting that the proliferative activity was greatly compromised in Cul4b embryos.

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