| Mutations of CUL4B in human, are one of the major causes of X-linked mental retardation (XLMR) syndrome. To date, at least 12 families of XLMR syndrome have been reported to be attributable to base substitutions or deletions in CUL4B. In addition to mental retardation, those patients also manifest short stature, abnormal gait, impaired speech, seizure, ataxia, tremor and other abnormalities.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 E3 ligase complexes, which recognize and target various substrates for proteosomal degradation. Human cullin family consists of eight members, CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, CUL7 and PARC. Among them, CUL4A and CUL4B have the highest degree of homo logy, with 83% identity in protein sequences. There is only one ortholog, Cul4, in lower organisms.So far only a few substrates of CRL4B complexes have been identified, including estrogen receptor a (ERa), cyclin E, topoisomerase I (Topo Ⅰ), Peroxiredoxin Ⅲ (PrxⅢ), WDR5, TSC2, Jab1/CSN5 and β-catenin. These substrates involve in a broad variety of physiologically and developmentally controlled processes such as cell cycle progression, replication, and DNA damage response.To illuminate the mechanism of mental retardation which caused by CUL4B mutation, we generated the mouse model of CUL4B-mediated XLMR by using Cre/loxp strategy. This study includes four parts.Part 1. The expression pattern of Cul4b in mouse tissuesAs the first step to characterize Cul4b function, we systematically analyzed the expression pattern of Cul4b and its subcellular location in mouse tissues.1) Western blot analyses of proteins extracted from different organs of mice at different development stages showed that Cul4b expressed in many tissues of mice during embryonic and adult stages. The highest level was detected in brain, while the lowest level was detected in adult heart. Furthermore, the immunohistochemistry of the tisssues was consisit with the Western blot analyses. Cul4b was also located mainly in the nucleus in brain and other tissues.2) To understand the role of Cul4b in brain, Western blot and immunohistochemistry were used to analyze the expression pattern of Cul4b in the brain of the mice at different developmental stages including E14.5, E17.5, P0, P14, and adult etc. We found that Cul4b was highly expressed in brain during all the stages detected, especially in the cortex and hippocampus. Furthermore, Cul4b was highly expressed in the postmitonic cells, but not in the proliferating neural progenitor cells in the subventricular zone.3) The immunofluorescence analysis indicated that Cul4b was detected in NeuN positive cells, suggesting that Cul4b is highly expressed in neurons.Part 2. Generation of Cul4b floxed miceBecause CUL4B-deficient cells are strongly selected against, we envisaged that it might be difficult to generate Cul4b deficient embryonic stem (ES) cells via conventional knockout technology. Therefore we used the Cre/loxP strategy to generate Cul4b floxed mice. First, a Cul4b floxed targeting vector was constructed. In this vector, exons 3-5 were floxed by two loxP sites. A neo gene flanked by two FRT sites and a TK gene were also inserted into intron 5 and vector backbone respectively for ES cell seletion. And then the targeting vector was linearized and eletroporated into 129 male ES cells for homologous recombination. Ninety six clones were screened by long-range PCR and Southern blot. Two correctly targeted clones were identified. Targeted ES cells were injected into C57BL/6J blastocysts to produce chimaeric mice, which were then used for germline transmission to produce Cul4b floxed mice.Nither male hemizygous (Cul4bflox/Y) nor female heterozygous/homozygous (Cul4b+(flox/Cul4flox/flox) for the Cul4b floxed allele showed any apparent phenotype, suggesting that the floxed allele did not disturb the normal function of CuUb gene.To verify that Cul4bflox can be rendered nonfunctional by the expression of Cre, due to the removal of exons 3-5, we generated nervous system-specific knockout mice (Cul4bflox/Y;Nestin-Cre mice) and sequenced the cDNA of Cul4b prepared from the brain. We observed that exon 2 was indeed spliced onto exon 6, as a result of removal of exons 3-5. The deletion would also result in a frameshift, generating 8 missense condons followed by a stop condon. The mutant allele is thus predicated to generate a peptide of merely 28 amino acids, if it can be expressed. Thus, deletion of exons 3-5 created a null mutation. Furthermore, the Cul4b mRNA level in brains of the conditional knockout mice is much lower than that of littermate wild-type control, suggesting that the truncation mutation also resulted in nonsense-mediated decay, as observed in patients with CUL4B nonsense mutation. Western blot analyses showed that Cul4b protein was nearly absent in brain tissues of conditional knockout mice, indicating that deletion of exons 3-5 in Cul4b results in a null mutation.Part 3. Characterization of constitutive Cul4b null miceTo investigate the function of Cul4b during mouse development, Cul4b floxed mice were crossed to Ella-Cre transgenic mice to generate constitutive Cul4b null mice. The following results were obtained.1) While Cul4b+/null females were recovered, no Cul4null/Y mice were present in the weaned pups, suggesting that Cul4bmll/Y comceptuses can not develop to term or die before weaning.2) To determine the exact time point when Cul4bnull/Y conceptuses die, timed mating was performed and embryos at different developmental stages were dissected. Genotyping of E9.5,10.5,12.5 and 14.5 embryos showed that no Cul4null/Y conceptuses were recovered on E9.5 and beyond, suggesting that these embryos implanted but died by E9.5. Although much smaller, Cul4b null embryos were recovered at expected ratio at E7.5 and 8.5. H&E staining of paraffin-embedded sections of E7.5 embryos confirmed that Cul4b null embryos are developmental retarded.3) To investigate what causes the developmental delay of the Cul4b null embryos, we analyzed the proliferation activity and apoptosis in E7.5 embryos. We found that proliferative cells were less abundant in Cul4b null embryos when compared to stagematched wild-type embryos. Furthermoe, the number of apoptotic cells was remarkably increased in the Cul4b null embryos. As observed in in vitro study, cyclin E was accumulated in Cul4b null embryos compared to littermate wild-type embryos. Taken together, these results suggested that the developmental delay in Cul4b null embryos is attibutalbe to a reduction in proliferation and an increase in apoptosis.4) Cul4b heterozygous mice were also recovered at a lower than expected ratio at weaning. Furthermore, the average body weight of heterozygous newborns was much smaller than that of wild-type controls. However, Cul4b heterozygous mice were able to gradually catch up after birth. Further analysis indicated that the growth retardation during prenatal development of Cul4b heterozygous mice was probably caused by the disorganization and impaired vascularization in placenta.Part 4. Characterization of nervous system-specific Cul4b knockout miceBecause of severe developmental arrest of Cul4b null embryos, we generated nervous system-specific Cul4b knockout mice to investigate the pathogenic mechanism leading to mental retardation. Cul4b floxed mice were crossed to Nestin-Cre transgenic mice, in which Cre recombinase was under the control of the rat nestin promoter and enhancer, and was expressed in nervous system during embryo development. Lack of Cul4b protein in Cul4bNestin-Cre mouse brains was confirmed by Western blot analysis and immunostaining.Cul4bNestion-Cre mouse did not show any apparent phenotype till 2 years old, with normal body weights and brain sizes. Morris water maze was used to evaluate the spatial learning and memory abilities of Cul4bNestin-Cre mouse. Open field test and Elevated plus maze were used to evaluate the anxiety level. Cul4bNestin-Cre mouse exhibited impaired spatial learning and memory, which is similar to the phenotypes of XLMR patients with CUL4B mutation. Therefore, Cul4bNestin-Cre mouse could be used as a good model for human XLMR.We next examined the histological organization of the brains of Cul4bNestin-Cre mouse by H&E staining. However, no significant difference was detected in Cul4bNestin-Cre mouse.To investigate the proliferation potentiality of neuronal progenitor cells in neuroepithelium of Cul4bNeslm-Cre mouse, Ki67 was immunostained to label proliferating cells. No difference in the number of Ki67-positive proliferating cells was detected in the Cul4bNestin-Cre mice at PO compared to littermate controls. TUNEL assay was used to detect the apoptotic cells in Cul4bNestin-Cre mouse brain. Few apoptotic cells were detected in cortex and hippocampous of Cul4bNestin-Cre mouse, which is consistent to that in littermate control mouse, suggesting that deletion of Cul4b does not affect programmed cell death in neural development.Because we found out that Cul4b was mainly expressed in postmitonic neurons, we examined the expression of NeuN in the brain sections by immunohistochemistry. There was no obvious change in Cul4bNestin-Cre mouse.The birthdating experiments were used to monitor the neurocytes migrated to superficial cortical layers, and we could not detect any difference in neurocytes migration between nervous system-specific knockout mice and controls.Primary neuron culture was used to examine dendrites and axon development, and no significant change was observed in Cul4bNestin-Cre mouse.To date, several CRL4B specific substrates have been identified. We therefore used Western blot to analyse the expression levels of cyclin E, WDR5, and (3-catenin. No significant changes in the levels of the protein was observed in Cul4bNestin-Cre mouse tissues when compared to the controls.In summary, we showed that Cul4b was expressed in all tissues examined, with highest level in brain, and lowest level in heart, in developmental embryos and adult. As in human cells, mouse Cul4b is mainly in the nucleus. However, in brain Cul4b was highly expressed in postmitonic neurons, instead of proliferative cells. Cul4b null mice were embryonic lethal at around 8.5 dpc. Cul4b null embryos showed a reduction in proliferative capacity and an increase in apoptosis, accompanied by an increased accumulation of cyclin E. Cul4b heterozygous mice exhibited prenatal growth retardation due to defects in placenta. Cul4bNestin-Cre mouse exhibited impaired spatial learning and memory, which is similar to the phenotypes of XLMR patients with CUL4B mutation. Therefore, we successfully generated a mouse model for X-linked mental retardation, which will be helpful for understaning the mechanism of mental retardation and identifying direct targets for mental retardation treatments. |
PDF Full Text Request