Functional Study Of Activation-induced Cytidine Deaminase(Aicda) Gene In Zebrafish

The emergence of the lymphocyte based adaptive immunity in jawed vertebrates is a milestone in the evolutionary process of animal immune system. It is capable of recognizing specific antigen, distinguishing self-cells from non-self-cells, and maintaining immunological memory. B cells and immunoglobulins they express, are the essential components of adaptive immune system. AID (activation-induced cytidine deaminase), which can deaminate cytidine in DNA and transform cytidine into uracil, is a key factor involved in the expression of immunoglobulin. Through the repair of uracil, AID eventually triggers molecular events such as somatic hypermutation (SHM), gene conversion (GC) and classic switch recombination (CSR). These mechanisms can reshape the variable and constant regions of immunoglobulin and thus enrich the antibody repertoire. During the evolution of vertebrate’s immune system, these mechanisms, such as SHM and CSR, did not evolve simultaneously. In lower vertebrates such as fishes, SHM is only found at a very low level while CSR is absent. In order to explore these mechanisms in lower vertebrates, an Aicda knockout zebrafish model was generated and used to investigate the origin and evolution of SHM and CSR.The Aicda knockout zebrafish were generated by using TALEN technology and the commonly used Tubingen zebrafish line. In the mutant Aicda allele, a7bp deletion was found at the5’portion of the second exon, which would cause a frameshift in translation. Although4splicing variants of Aicda mRNA were identified in the Aicda mutated fish, none of them can be translated into a normal AID protein. Based on this model, the expressional manner of IgM and IgZ was analyzed. Compared with wild-type zebrafish, the IgZ expression level in Aicda knockout zebrafish was significantly higher in organs such as skin, gill and kidney. However, Aicda knockout did not have such influence on the IgM expression at the RNA level. The IgM and IgZ levels in serum were also detected by ELISA, revealing that both IgM and IgZ levels in Aicda knockout zebrafish were higher than those in wild-type zebrafish. Since the mutations in intron are thought to be less selected during the B cell development, intron sequences downstream of JH gene segments were amplified and sequenced for investigating the impact of Aicda knockout on SHM. To do so, a nested PCR was designed for the amplification of the JH intron downstream of recombined VDJ exons. From more than2000sequenced clones, we identified295unique sequences. By analyzing these clones, we found that the mutation rates in both wild-type and Aicda knockout zebrafish were remarkably lower than in mice, suggesting that the SHM level in zebrafish is much lower than that in mammals. In addition, we found that Aicda knockout had no obvious impact on the level of SHM in zebrafish. To analyze whether Aicda knockout could influence the antibody affinity to specific antigens, both wild-type and knockout fish were immunized by TNP-BSA. The variable segments of IgM in both fish groups were amplified using5’RACE, and a comparative analysis revealed that the TNP-BSA immunized Aicda knockout zebrafish had a limited usage of variable gene segments, while the diversity of recombined CDR3region was also reduced. Based on ELISA detection, we showed that the anti-TNP and anti-Vibrio anguillarum specific IgM level in wild-type and Aicda knockout zebrafish were almost at the same level. Additionaly, when the zebrafish vaccinated by a live attenuated vaccine against Vibrio anguillarum were challenged by pathogenic strain of Vibrio anguillarum, we found that Aicda knockout had no impact on the survival rates. In order to explore the origin of CSR, a nested PCR was used to amplify the potential recombined products of CSR in wild-type zebrafish. However, these efforts provided no evidence to the presence of CSR in zebrafish.In summary, by analyzing the Aicda knockout zebrafish model, we found that Aicda knockout affected the expression of immunoglobulin. However, owing to the extremely low SHM rate triggered by AID and the lack of germinal center in zebrafish, affinity maturation is not observed. As a result, Aicda gene knockout leads to no obvious immunodeficiency. Our data shed insights into the early evolution of SHM and CSR in lower vertebrates.