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Mutating Fibroblast Growth Factor Receptor 1 (Fgfr1) in Zebrafish to Create a New Model of Craniosynostosis
Joanna P. Tomaszewski, M.S.1, Michael S. Gart, M.D.2, Ramy A. Shoela, M.D.1, Jacek Topczewski, Ph.D.1, Jolanta M. Topczewska, Ph.D.1, Arun K. Gosain, M.D.3.
1Ann & Robert H. Lurie Children's Hospital of Chicago Research Center, Chicago, IL, USA, 2Northwestern University Feinberg School of Medicine, Chicago, IL, USA, 3Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA.
Human genomic studies and research on animal models suggest that various loci and genetic mechanisms, notably mutations in fibroblast growth factor receptor 1 (FGFR1), are involved in the development of craniosynostosis (CS), or premature suture obliteration. The goal of this project is to create a zebrafish model of CS, which would present advantages over previously established rodent models. Homologous genes and similar genetic networks are involved in morphogenesis of craniofacial skeletal elements in both zebrafish and humans (Knight and Schilling, 2006). Furthermore, the ease of complex genetic manipulations makes the zebrafish an attractive model for elucidating the etiology of CS. We aim to mimic the mutation observed in Pfeiffer syndrome by generating a stable transgenic zebrafish line that allows for inducible expression of the mutated fgfr1(Pro252Arg) gene.
Two FGFR1 zebrafish paralogs, fgfr1a and fgfr1b, were detected in total RNA isolated from calvaria at 8-10mm standard length using RT-PCR. fgfr1b was cloned and mutagenized by a single base pair substitution to create the Pro252Arg mutation. Gateway Recombineering and the Tol2 system were used to create the transgenic construct, which includes an hsp70 promoter for inducible expression of the mutated gene and an IRES-driven GFP reporter for monitoring of ectopic expression. To assess the impact of the mutation on embryonic development, single cell stage embryos were injected with mutated fgfr1b mRNA and tested for expression of target genes of FGF signaling using whole mount in situ hybridization. Once the final form of transgenic cassette is confirmed, it will be injected into embryos to create a stable transgenic line.
Preliminary results of RNA in situ hybridization revealed that the expression domain of krox20 within the presumptive rombomeres 3rd and 5th is expanded in injected embryos, supporting the hypothesis that Pro252Arg mutation has an activating character in zebrafish (See Figure 1A,B). Furthermore, embryos injected with fgfr1b(Pro252Arg) mRNA phenotypically reveal decreased head size, consistent with fgfr1 upregulation (See Figure 1C).
An expanded krox20 expression domain at the embryonic stage of fgfr1b(Pro252Arg) mRNA injected embryos suggests that this mutation has activating character in zebrafish, indicating a conserved role of fgfr1 in cranial suture development among vertebrates. The effects of genetic manipulation on adult suture phenotype will be assessed through histological analysis, skeletal morphology, and cell proliferation will be studied in our transgenic fgfr1b(Pro252Arg) embryos.
This zebrafish model of CS will advance our understanding of the role of Fgfr1b in cranial suture morphogenesis and the etiology of the disorder. In the future, it can be used for genetic and chemical screens to search for genetic modifiers and therapeutic agents that alter CS.
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