dev maturation immune system lam et al

Development and maturation of the immune system in zebrafish, Danio rerio: a gene expression profiling, in situ hybridization and immunological study S.H. Lam a , H.L. Chua a , Z. Gong a , T.J. Lam a , Y.M. Sin a,b, * a Department of Biological Sciences, The National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260 b Singapore Fish Breeding and Immunization Centre, Teo Way Yong and Sons Pte. Ltd, Singapore Received 12 December 2002; revised 12 May 2003; accepted 13 May 2003 Abstract The development and maturation of the immune system in zebrafish was investigated using immune-related gene expression profiling by quantitative real-time polymerase chain reaction, in situ hybridization (ISH), immunoglobulin (Ig) detection by immuno-affi nity purification and Western blotting as well as immersion immunization experiments. Ikaros expression was first detected at 1 day post-fertilization (dpf) and thereafter increased gradually to more than two-fold between 28 and 42 dpf before decreasing to less than the initial 1 dpf expression level in adult fish (aged 105 dpf). Recombination activating gene-1 ( Rag-1) expression levels increased rapidly (by 10-fold) between 3 and 17 dpf, reaching a maximum between 21 and 28 dpf before decreasing gradually. However, in adult fish aged 105 dpf, the expression level of Rag-1 had dropped markedly, and was equivalent to the expression level at 3 dpf. T-cell receptor alpha constant region and immunoglobulin light chain constant region (IgLC) isotype-1, 2 and 3 mRNAs were detected at low levels by 3 dpf and their expression levels increased steadily to the adult range between 4 and 6 weeks post-fertilization (wpf). Using tissue-sec tion ISH, Rag-1 expression was detected in head kidney by 2 wpf while IgLC-1, 2 and 3 were det ected in the head kidney and the thymus by 3 wpf onwa rds. Secreted Ig was only detectable using immuno-affinity purification and Western blotting by 4 wpf. Humoral response to T-independent antigen (formalin-killed Aeromona s hydroph ila ) and T-depe nde nt ant ige n (human gamma glo bul in) was obs erved in zeb rafi sh immun ized at 4 and 6 wpf, respecti vely, indicat ing that immunoco mpete nce was achieved . The findings reveal that the zebra fish immune system is morpho logic ally and funct ional ly mature by 4– 6 wpf. q 2003 Published by Elsevier Ltd. Keywords: Zebr afish ; Immun e syst em matu rati on; Ikaros; Reco mbin ati on acti vati ng gene -1; T-ce ll rece ptor alph a cons tant regi on; Immunoglobulin light chain constant region isotypes; Humoral immunity 1. Introduction The zebra fish, Danio rerio, of fer s an att ractive model for the study of ontogenetic development of the immune syst em [1–7]. The relative ease wit h which zebrafish embryos and larvae can be studied, the powerful genetics which can be applied for the generation of mutants and transgenic animals, the vast genomic resources including the availability of many 0145-305X/04/$ - see front matter q 2003 Published by Elsevier Ltd. doi:10.1016/S0145-305X(03)00103-4 Developmental and Comparative Immunology 28 (2004) 9–28 www.elsevier.com/locate/devcompimm * Corre spond ing author. Addr ess: Depar tment of Biolo gical Sciences, The National University of Singapore, 10 Kent Ridge Cres cent , Sing apor e 119260. Tel.: þ65-6469161 9; fax: þ65- 67792486. E-mail address: info@teowa yyong.com. sg (Y.M. Sin). Abbreviations: dpf, day( s) post- ferti liza tion; HGG, huma n gam ma gl o bu li n; Ig, i mm un ogl obul i n; IgLC1, 2,3, immunoglobulin light chain constant region isotype 1,2,3; ISH, in situ hybr idiz ation; PCR, poly merase chai n reac tion; Rag-1, recombina tion activating gene-1; TCRAC, T-cell receptor alpha constant region; wpf, week(s) post-fert ilization.

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