Developmental expression and organisation of fibrinogen genes in the zebrafish

Summary

The zebrafish is a model organism for studying vertebrate development and many human diseases. Orthologues of the majority of human coagulation factors are present in zebrafish, including fibrinogen. As a first step towards using zebrafish to model human fibrinogen disorders, we cloned the zebrafish fibrinogen cDNAs and made in situ hybridisations and quantitative reverse transcription-polymerase chain reactions (qRT-PCR) to detect zebrafish fibrinogen mRNAs. Prior to liver development or blood flow we detected zebrafish fibrinogen expression in the embryonic yolk syncytial layer and then in the early cells of the developing liver. While human fibrinogen is encoded by a three-gene, 50 kilobase (kb) cluster on chromosome 4 (FGB-FGA-FGG), recent genome assemblies showed that the zebrafish fgg gene appears distanced from fga and fgb, which we confirmed by in situ hybridisation. The zebrafish fibrinogen Bβ and γ protein chains are conserved at over 50% of amino acid positions, compared to the human polypeptides. The zebrafish Aα chain is less conserved and its C-terminal region is nearly 200 amino acids shorter than human Aα. We generated transgenic zebrafish which express a green fluorescent protein reporter gene under the control of a 1.6 kb regulatory region from zebrafish fgg. Transgenic embryos showed strong fluorescence in the developing liver, mimicking endogenous fibrinogen expression. This regulatory sequence can now be used for overexpression of transgenes in zebrafish hepatocytes. Our study is a proof-of-concept step towards using zebrafish to model human disease linked to fibrinogen gene mutations.

Keywords

Fibrinogen - zebrafish - gene cluster - transgenic - liver

• References

• 1 Doolittle RF. Fibrinogen and fibrin. Annu Rev Biochem 1984; 53: 195-229.
• 2 Mosesson MW. Fibrinogen and fibrin structure and functions. J Thromb Haemost 2005; 03: 1894-1904.
• 3 Fuller GM, Zhang Z. Transcriptional control mechanism of fibrinogen gene expression. Ann NY Acad Sci 2001; 936: 469-479.
• 4 Kant JA, Fornace Jr. AJ, Saxe D. et al. Evolution and organization of the fibrinogen locus on chromosome 4: gene duplication accompanied by transposition and inversion. Proc Natl Acad Sci USA 1985; 82: 2344-2348.
• 5 de Moerloose P, Neerman-Arbez M. Congenital fibrinogen disorders. Semin Thromb Hemost 2009; 35: 356-366.
• 6 Danesh J, Lewington S, Thompson SG. et al. Plasma fibrinogen level and the risk of major cardiovascular diseases and nonvascular mortality: an individual participant meta-analysis. J Am Med Assoc 2005; 294: 1799-1809.
• 7 Vorjohann S, Fish RJ, Biron-Andreani C. et al. Hypodysfibrinogenaemia due to production of mutant fibrinogen alpha-chains lacking fibrinopeptide A and polymerisation knob 'A'. Thromb Haemost 2010; 104: 990-997.
• 8 Suh TT, Holmback K, Jensen NJ. et al. Resolution of spontaneous bleeding events but failure of pregnancy in fibrinogen-deficient mice. Genes Dev 1995; 09: 2020-2033.
• 9 Gulledge AA, Rezaee F, Verheijen JH. et al. A novel transgenic mouse model of hyperfibrinogenemia. Thromb Haemost 2001; 86: 511-516.
• 10 Hogan KA, Merenbloom BK, Kim HS. et al. Neonatal bleeding and decreased plasma fibrinogen levels in mice modeled after the dysfibrinogen Vlissingen/ FrankfurtIV. J Thromb Haemost 2004; 02: 1484-1487.
• 11 Lieschke GJ, Currie PD. Animal models of human disease: zebrafish swim into view. Nat Rev Genet 2007; 08: 353-367.
• 12 Jagadeeswaran P, Kulkarni V, Carrillo M, Kim S. Zebrafish: from hematology to hydrology. J Thromb Haemost 2007; 05 (01) 300-304.
• 13 Doolittle RF. Step-by-step evolution of vertebrate blood coagulation. Cold Spring Harb Symp Quant Biol 2009; 74: 35-40.
• 14 Doolittle RF. Coagulation in vertebrates with a focus on evolution and inflammation. J Innate Immun 2011; 03: 9-16.
• 15 Lin HF, Traver D, Zhu H. et al. Analysis of thrombocyte development in CD41-GFP transgenic zebrafish. Blood 2005; 106: 3803-3810.
• 16 Thattaliyath B, Cykowski M, Jagadeeswaran P. Young thrombocytes initiate the formation of arterial thrombi in zebrafish. Blood 2005; 106: 118-124.
• 17 O'Connor MN, Salles II, Cvejic A. et al. Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins. Blood 2009; 113: 4754-4762.
• 18 Kimmel CB, Ballard WW, Kimmel SR. et al. Stages of embryonic development of the zebrafish. Dev Dyn 1995; 203: 253-310.
• 19 Paw BH, Zon LI. Primary fibroblast cell culture. Methods Cell Biol 1999; 59: 39-43.
• 20 Pinkel D, Landegent J, Collins C. et al. Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and trans-locations of chromosome 4. Proc Natl Acad Sci USA 1988; 85: 9138-9142.
• 21 Rice P, Longden I, Bleasby A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 2000; 16: 276-277.
• 22 Urasaki A, Morvan G, Kawakami K. Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics 2006; 174: 639-649.
• 23 Thisse C, Thisse B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc 2008; 03: 59-69.
• 24 Kawakami K. Transposon tools and methods in zebrafish. Dev Dyn 2005; 234: 244-254.
• 25 Fisher S, Grice EA, Vinton RM. et al. Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish. Nat Protoc 2006; 01: 1297-1305.
• 26 The Danio rerio Sequencing Project. Wellcome Trust Sanger Institute. Available at: http://www.sanger.ac.uk/Projects/D_rerio/
• 27 Thisse B, Thisse C.. Fast Release Clones: A High Throughput Expression Analysis. ZFIN Direct Data Submission. ( http://zfinorg ) 2004
• 28 Ober EA, Field HA, Stainier DY. From endoderm formation to liver and pancreas development in zebrafish. Mech Dev 2003; 120: 5-18.
• 29 Fort A, Borel C, Migliavacca E, Antonarakis SE, Fish RJ, Neerman-Arbez M. Regulation of fibrinogen production by microRNAs. Blood 2010; 116: 2608-2615.
• 30 Fort A, Fish RJ, Attanasio C, Dosch R, Visel A, Neerman-Arbez M. A liver enhancer in the fibrinogen gene cluster. Blood 2011; 117: 276-282.
• 31 Fujita PA, Rhead B, Zweig AS. et al. The UCSC Genome Browser database: update 2011. Nucleic Acids Res 2011; 39 (Database issue) D876-882.
• 32 Jagadeeswaran P, Cykowski M, Thattaliyath B. Vascular occlusion and thrombosis in zebrafish. Methods Cell Biol 2004; 76: 489-500.
• 33 Jagadeeswaran P, Paris R, Rao P. Laser-induced thrombosis in zebrafish larvae: a novel genetic screening method for thrombosis. Methods Mol Med 2006; 129: 187-195.
• 34 Sheehan J, Templer M, Gregory M. et al. Demonstration of the extrinsic coagulation pathway in teleostei: identification of zebrafish coagulation factor VII. Proc Natl Acad Sci USA 2001; 98: 8768-8773.
• 35 Hoeprich Jr. PD, Doolittle RF. Dimeric half-molecules of human fibrinogen are joined through disulfide bonds in an antiparallel orientation. Biochemistry 1983; 22: 2049-2055.
• 36 Weissbach L, Grieninger G. Bipartite mRNA for chicken alpha-fibrinogen potentially encodes an amino acid sequence homologous to beta- and gamma-fibrinogens. Proc Natl Acad Sci USA 1990; 87: 5198-5202.
• 37 Doolittle RF, Kollman JM. Natively unfolded regions of the vertebrate fibrinogen molecule. Proteins 2006; 63: 391-397.
• 38 Ahituv N, Prabhakar S, Poulin F. et al. Mapping cis-regulatory domains in the human genome using multi-species conservation of synteny. Hum Mol Genet 2005; 14: 3057-3063.
• 39 Sigrist CJ, Cerutti L, de Castro E. et al. PROSITE, a protein domain database for functional characterization and annotation. Nucleic Acids Res 2010; 38 (Database issue) D161-166.