title: Development and maturation of the immune system in zebrafish, Danio rerio: a gene expression profiling, in situ hybridization and immunological study.
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-affinity 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 42dpf before decreasing to less than the initial 1dpf expression level in adult fish (aged 105dpf). Recombination activating gene-1 (Rag-1) expression levels increased rapidly (by 10-fold) between 3 and 17dpf, reaching a maximum between 21 and 28dpf before decreasing gradually. However, in adult fish aged 105dpf, the expression level of Rag-1 had dropped markedly, and was equivalent to the expression level at 3dpf. 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 3dpf and their expression levels increased steadily to the adult range between 4 and 6 weeks post-fertilization (wpf). Using tissue-section ISH, Rag-1 expression was detected in head kidney by 2wpf while IgLC-1, 2 and 3 were detected in the head kidney and the thymus by 3wpf onwards. Secreted Ig was only detectable using immuno-affinity purification and Western blotting by 4wpf. Humoral response to T-independent antigen (formalin-killed Aeromonas hydrophila) and T-dependent antigen (human gamma globulin) was observed in zebrafish immunized at 4 and 6wpf, respectively, indicating that immunocompetence was achieved. The findings reveal that the zebrafish immune system is morphologically and functionally mature by 4-6wpf.
authors.affiliation: Department of Biological Sciences, The National University of Singapore, 10 Kent Ridge Crescent, 119260, Singapore.
authors.forename: S H
authors.initials: SH
authors.lastname: Lam
authors.forename: H L
authors.initials: HL
authors.lastname: Chua
authors.forename: Z
authors.initials: Z
authors.lastname: Gong
authors.forename: T J
authors.initials: TJ
authors.lastname: Lam
authors.forename: Y M
authors.initials: YM
authors.lastname: Sin
mesh_terms: D000818:Animals; D000917:Antibody Formation; D015153:Blotting, Western; D002454:Cell Differentiation; D004587:Electrophoresis, Agar Gel; D020869:Gene Expression Profiling; D018398:Homeodomain Proteins / Q000032:analysis / Q000378:metabolism; D007136:Immunoglobulins / Q000032:analysis / Q000276:immunology; D017403:In Situ Hybridization; D007668:Kidney / Q000166:cytology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013950:Thymus Gland / Q000166:cytology; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics / Q000276:immunology*
chemical_list: D018398:Homeodomain Proteins; D007136:Immunoglobulins; D012333:RNA, Messenger; C064658:RAG-1 protein