Fgf17b And Its Feedback Inhibitor, Mkp3, Function In Early Patterning Of Zebrafish Embryo

Fibroblast growth factors (FGFs) represent a large family of growth factors that haveimportant roles in cell division, growth and differentiation. During embryonic development invertebrates, they are major players in mesoderm induction, dorsoventral patterning,anteroposterior patterning of the neuroectoderm, and formation of many tissues and organs.More than twenty Fgf factors have been identified in vertebrates, with restricted expressionpatterns. Fgf factors bind to and activate the receptors Fgfrs of the receptor tyrosine kinases(RTK), which in turn activate several downstream signaling cascades, including phospholipaseC gamma (PLC-γ), phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase(MAPK) pathway. Because of diverse functions and involvement in many downstreamsignaling pathways, Fgf signaling level has to be tightly regulated via positive and negativefeedback loops. Fgf signaling pathway crosstalks with other signaling pathways, e.g., Nodal,Wnt and Hedgehog pathways, and these collaboratively control embryonic development.As a new vertebrate model, zebrafish is preferred by more and more developmentalbiologists since it has advantage over other vertebrate models in bringing the genetics anddevelopment biology together. However, the functions of Fgfs are not well studied in zebrafish.To date, only 7 members of Fgf family in zebrafish have been identified and 5 of them wellstudied. Considering redundant functions of different Fgf members, identification of moremembers will help elucidate regulatory mechanisms of Fgf signaling pathway in embryonicpatterning in zebrafish.In this study, a novel zebrafish Fgf gene, fgf17b, is identified during an in situ hybridizationscreen for tissue-specific genes. Sequence analysis and chromosome mapping reveal that it isan ortholog of human FGF17. RT-PCR and in situ hybridization identify two distinct phases offgf17b expression. During the first phase, from late blastulation to early gastrulation, fgf17b isexpressed in the blastoderm margin, with a gradient which is the highest on the dorsal and thelowest on the ventral. The second phase starts after the onset of segmentation, when fgf17b isexpressed in the forebrain, presomitic mesoderm and newly formed somites. The earlyexpression of fgf17b is positively regulated by Nodal signal.Impacts of Fgf17b on embryonic development are first studied by overexpression. Injectionwith fgf17b mRNA induces ectopic expression of the mesodermal marker ntl, indicating amesoderm induction activity. Overexpression of fgf17b results in dorsalization of embryos, withexpansion of the dorsal marker chordin and reduction of the ventral markers evel and gata2,suggesting that Fgf17b has dorsalizing activity. Furthermore, fgf17b overexpression promotesexpression of the posterior neuroectodermal marker hoxb1b while the the anteriorneuroectodermal marker otx1 is inhibited, demonstrating a positive role in neuroectodermalposteriorization. The fact that simultaneous knockdown of endogenous Fgf17b and Fgf8weakens expression of ntl and chordin and knockdown of Fgf17b compromises inhibitory effectof fgf3 overexpression on anterior neuroectoderm suggests that Fgf17 has redundant functionswith other Fgf members. Comparing overexpression effects of fgf3, fgf8 and fgf17b, we havefound that Fgf8 possesses stronger activities in mesoderm induction and dorsalization, Fgf3 hasstronger activity in neuroectodermal posteriorization, and Fgf17 has the strongest activities in allof the three processes.This study has identified mkp3 as an Fgf-responsive gene using cDNA microarray. Inzebrafish embryos its expression domains overlap those of fgf17b. In situ hybridization indicatesthat mkp3 expression is positively regulated by Fgf signal. Overexpression of mkp3 inhibitsformation of the dorsal mesoderm and the neuroectoderm, while knockdown of mkp3 promotesthe formation of mesoderm, doraslizes the gastrulae and posteriorizes the neuroectoderm. Wealso demonstrate that Mkp3 negatively regulates Fgf function in mesoderm induction anddorsoventral patterning.