| CUL4belongs to Cullin protein family, which contains an evolutionarily conserved cullin homology domain consisting of about200amino acids at the C-terminus. Cullins are molecular scaffolds that organize the largest class of RING E3ligases, which is known as the CULLIN-RING ligase complexes (CRLs), and they have crucial roles in ubiquitination of cellular proteins. The Cul4gene is conserved during evolution from fission yeast to humans. While S. pombe, Drosophila melanogaster and A. thaliana encode a single Cul4gene, the mammal and zebrafish express two Cul4paralogs, Cul4A and Cul4B. Although encoded by two different genes, the mammalian CUL4A and CUL4B proteins share80%identity. Compared with CUL4A, CUL4B has an extended N terminal region. Recent studies indicate that CRL4complex participate in a broad variety of physiologically and developmentally controlled processes, such as cell cycle progression, transcription, signal transduction, apoptosis, and DNA repair and chromatin remodeling.Acting as the scaffold component, CUL4assembles with ROC1at its C terminus and with Damaged DNA Binding protein1(DDB1) at its N terminus to form CRL4complex. As CUL4A and CUL4B share a high degree of homology and utilize the same adapter protein DDB1in assembling CRL4complexes, they can potentially target the same substrates and function redundantly in some cellular functions. However, when mutated or disrupted, those two genes appeared very different consequences. Mutations of CUL4B in human causes an X-linked mental retardation (XLMR) syndrome. The present study found that Cul4b null mice are embryonic lethal, the mice with deletion of Cul4b in epiblast displayed hippocampus-related spatial learning and memory deficiencies. Cul4a knockout mice were viable and displayed no apparent developmental phenotype except that the knockout males were sterile due to impairment in spermatogenesis. Furthermore, the CRBN-mediated CRL4A E3ligase activity was critical for several important developmental processes, such as limb outgrowth and otic vescles development in zebrafish and chick embryos. Knockdown of Crbn or Cul4a caused specific defects in pectoral fin and otic vesicle development in zebrafish, which is similar with thalidomide-treated embryos. Thus, unlike in the mouse, cul4a may play a more important role during zebrafish development. In summary, these results suggest that the biological function of Cul4during embryonic development is very important and the two CUL4genes are not entirely redundant in mammals, but the early embryonic death obstacles the further research of its function, which need to establish a new model.Zebrafish(Danio rerio) is a good model organism for embryonic developement. Its genomic organization and embryonic development have been well characterized. Morpholino antisense oligos knockdown technology is widely used in zebrafish system. It prevents translation or splicing of the targeted gene by binding to RNA. Zebrafish have two cul4gene, cul4a and cul4b gene. Zebrafish cul4protein shares high identity to human CUL4and is highly conserved. Therefore in this study, we choose the zebrafish as a model for cul4a biological function research.Part1. Expression pattern of zebrafish cul4geneIn order to investigate the biological functions of cul4using zebrafish model, we examined the protein structure and expression pattern of zebrafish cul4. 1) As shown by sequence alignment, zebrafish cul4and human CUL4proteins are highly identical; zebrafish cul4a and cul4b have high sequence homology with human CUL4A and CUL4B respectively.2) We also searched zebrafish, mice and humans CUL4gene from Ensembl database, and then compared their location on chromosomes. We found that in all the three species, genes distributed around the location of CUL4A and CUL4B are similar. This showed that cul4a and cul4b are conserved among vertebrate organisms.3) The cul4a and cul4b mRNA levels of embryos at different developmental stages (0,3,6,12,24,36,48,72and96hpf) were determined by quantitative real-time PCR. cul4a and cul4b mRNA appeared immediately after fertilization in the zygote and express throughout the whole developmental stages.4) To determine the spatial and temporal expression pattern of cul4a during zebrafish development, whole-mount in situ hybridization (ISH) was carried out. At1-cell and8-cell stages, cul4a was highly expressed, consistent with the data from real-time RT PCR. During zebrafish embryonic development, cul4a was widely expressed in the whole embryos, and highly expressed in heart and pectoral fins at48hpf, which was similar to the data previously reported.Part2. Phenotype analysis of the cul4a knockdown zebrafishIn this part, we used the Morpholinos based knockdown technology to generate the zebrafish model and analyze the potential physiological function of cul4during early development:1) To analyze the potential physiological function of cul4a and cul4b during early development, we designed six Morpholino antisense oligo nucleotides. For cul4a: cul4a MO, which targets the splice donor site of exon2to interfere with mRNA splicing, resulting in a frameshift and creating a stop codon, cul4a CoMO, a control morpholino antisense oligonucleotide, which is a modified sequence of cul4a MO2with five mismatches, and cul4a MOl, which targets the5’UTR of cul4a mRNA. We also esigned three morpholino antisense oligo nucleotides for cul4b:cul4b MO, which targets the splice donor site of exon2to interfere with mRNA splicing, cul4b MOl, which targets the5’UTR of cul4b mRNA, and a control morpholino antisense oligonucleotide, cul4b CoMO, which is a modified sequence of cul4b MOl with five mismatches. We microinjected these Morpholinos into embryos at1-4cell stage. To validate the efficacy of cul4a MO and cul4b MO, primers located in flanking exons were employed for RT-PCR of RNA samples extracted from24hour post fertilization (hpf) wild-type embryos and MO-injected embryos. We found that injection of4ng of cul4a MO or cul4b MO resulted in complete exclusion of exon2from the cul4a mRNA or cul4b mRNA, which was confirmed by cDNA sequencing.2) To test the requirement of cul4a and cul4b during development, we injected cul4a and cul4b MO or CoMO into single-cell stage embryos and observed developmental phenotypes from12hpf to72hpf. cul4a knockdown embryos showed abnormalites at the early stage of development. By48hpf-3dpf, cul4a knockdown embryos displayed an array of profound morphological defects, including tail curling, hindbrain edema, stunted or completely absent pectoral fins and pericardial edema accompanied with heart looping defects, while embryos injedted with CoMO did not show any significant defects, demonstrating that the phenotypes were not due to an injection artifact. Moreover, the phenotypes of cul4a knockdown embryos were dosage-dependent. But there was no obvious abnormality in cul4b knockdown embryos. We designed and synthesized zebrafish cul4a and cul4b mRNA (Zcul4a and Zcul4b), human CUL4A and CUL4B mRNA (HCUL4A and HCUL4B) o rescue cul4a knockdown phenotype. The phenotypes of cul4a knockdown embryos were partially rescued by co-injection of Zcul4a mRNA, suggesting that the phenotypes produced by cul4a MO were caused by the specific inhibition of cul4a. Interestingly, co-injection of cul4a MO and HCUL4A or HCUL4B mRNA partially rescued the morphant phenotypes, respectively, demonstrating that human CUL4A or CUL4B mRNA could compensate for the loss of the endogenous zebrafish cul4a mRNA. However, co-injection of cul4a MO and Zcul4b mRNA did not rescue the phenotypes of embryos caused by cul4a MO. Furthermore, we also analyzed the effect of cul4a and cul4b double knockdown on zebrafish development. The phenotypes of cul4a/cul4b double knockdown embryos were grossly similar to those of cul4a knockdown embryos, which again negates any notion of important function by cul4b during zebrafish development. Thus, only cul4a, but not cul4b, is indispensable for normal zebrafish development.3) Paraffin section H&E staining, BrdU incorporation experiment and acridine orange staining were carried to analysis head and trunk anomalies. We found that in cul4a kncokdown embryonic brain, the cell number was reduced, cell proliferation decreased, and apoptosis in head and trunk increased.4) To further define the cardiac abnormality in cul4a morphants, we injected CoMO or cul4a MO into zebrafish embryos of the transgenic line Tg (cmlc:gfp) in which enhanced green fluorescent protein was expressed in all cardiac cells. Although the initial heart tubes in cul4a morphants were indistinguishable from those in controls at24-33hpf, abnormalities were noted for subsequent cardiac development in cul4a MO-injected embryos. At48hpf, whereas the heart tubes appeared to loop to the right in control embryos, they remained relatively straight in cul4a MO morphants. At60hpf, while there was no obvious perturbation in cwic-driven GFP expression in cul4a MO morphants when compared to the controls,, the heart remained in the configuration of a cylinder. cul4a morphants also showed significantly slower heart rhythm than the controls. Together, these data revealed that the cardiac development in cul4a morphants was structurally and functionally impaired. To determine the developmental stage when cardiac development was first perturbed in cul4a morphants, we injected embryos with CoMO or cul4a-MO and processed them for in situ hybridization with various cardiac markers, cardiac myosin light chain2(cmlc2), a pan-cardiac marker. We found that the cardiac defect caused by cul4a deficiency was not evident until the cardiac looping stage. To examine whether the chamber identity was altered in cul4a morphants, we determined cardiac chamber specification using monoclonal antibodies against pan cardiac as well as chamber-specific myosin markers, MF20, S46and ventricular myosin heavy chain (vmhc). We found that the chamber specification proceeded normally in cul4a morphant embryos. Cell proliferation, cellular organization and cell shape changes have been suggested to contribute to cardiac looping. To investigate the mechanism underlying the failure in heart looping in cul4a morphants, we investigated whether the number of cardiomyocytes was decreased in cul4a morphants. Examination of histological sections of cul4a MO injected embryos at3dpf showed that the walls of ventricle and atrium were markedly thinner than those in age-matched controls, and the cardiomyocytes were more scarcely distributed along the ventricular and atrial walls in cul4a morphants. Using TUNEL assay, immunostaining of PCNA and BrdU incorporation, We found that cardiac cell apoptosis increase and proliferation decrease in the heart of cul4a MO injected embryos, compared to that in control embryos. In addition the cardiac cells in the cul4a morphants also appeared to be disorganized compared to the controls, and they were irregularly shaped.5) Injection of cul4a MO also led to the truncation or absence of pectoral fins in zebrafish. Alcian Blue staining, which marks cartilage and bone structures in the developing embryos, also revealed shortened or absent pectoral fins in cul4a morphants. One-hour pulse of BrdU at33hpf revealed fewer proliferating (BrdU-positive) cells in the buds of pectoral fins in cul4a morphants than in control embryos. These results suggest that lack of proliferating cells is also responsible for the failure in the pectoral fin development.Part3. Cul4a regulates heart and pectoral fins development through regulating tbx5a gene expressionWe noted that the phenotypes displayed by cul4a morphants were reminiscent of those in hst mutant or tbx5a knockdown morphants. Both tbx5a and cul4a morphants exhibited structural defects in heart and pectoral fin. The common phenotypes shared between cul4a and tbx5a loss-of-function morphants prompted us to test whether the two lie in the same pathway. 1) We determined the levels of tbx5a mRNA and protein in control and cul4a MO-treated zebrafish embryos at32hpf by quantitative RT-PCR and found that tbx5expression was remarkably reduced in cul4a-MO-injected embryos compared to that in embryos injected with CoMO. Because tbx5is a transcription factor, we also measured the expression levels of its targets gene, fgf10and hand.2, we found that they were also significantly downregulated. Whole mount in situ hybridization using tbx5a probes also indicated tbx5a expression reduced in the heart and the buds of pectoral fins in cul4a morphants. We found that co-injection of tbx5a mRNA significantly rescued the developmental defects in cul4a morphants, with the percentage of embryos with malformed heart and pectoral fin significantly decreased. These results indicated that the phenotypes of cul4a morphants were mediated by downregulation of tbx5a.2) Because Cul4a can control tbx5a mRNA expression, the next focus of our research is the relationship between Cul4a and tbx5a gene. Due to lacking suitable antibodies against zebrafish, we choose H9C2cells, a rat myocardial cell line, for research. Consistent to the data in zebrafish, Tbx5mRNA and protein were also decreased in Cul4a knockdown H9C2cells.3) We detected Cul4a subcellular localization in H9C2cells by Western blot and immunofluorescence. We found that Cul4a was located in the nucleus in H9C2cells. To further confirm subcellular localization of Cul4a in vivo, we analyzed heart of fetal mice and adult mice by Western blotting and immunostaining. We found that Cul4a located in the cytoplasm and nucleus in E18.5fetal mice heart cells, but located mostly in the cytoplasm in adult mice heart cells.4) Four pairs of primers were designed corresponding to the sequence within-1～-1000bp of rat Tbx5gene. ChIP assay demonstrated that Cul4a directly binds to-908～-511bp upstream of Tbx5gene. H3K4trimethylation is a prevalent marker associated with transcriptional activation. ChIP assays showed that Cul4a knockdown resulted of H3K4trimethylation on Tbx5promoter locus decrease. These data demonstrated that Cul4a directly binds to the promoter of Tbx5gene and catalyzes for H3K4trimethylation on the Tbx5promoter locus, thus transcriptional activates Tbx5expression.In this study, we found that cul4a knockdown caused multiple organs and systems disorders, including head, trunk, heart, and pectoral fins abnormalities, then we found that tbx5a mRNA level was remarkable decreased in cul4a knockdown embryos compared to embryos injected with CoMO. Through in vitro experiments we found that Cul4a bound to-908～-511bp upstream of Tbx5gene and knockdown of Cul4a resulted H3K4trimethylation on Tbx5promoter locus decrease. Our investigation is important for understanding cul4a biological functions in development. |
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