Screening of FGF target genes in Xenopus by microarray: temporal dissection of the signalling pathway using a chemical inhibitor

Abstract

Microarray is a powerful tool for analysing gene expression patterns in genome-wide view and has greatly contributed to our understanding of spatiotemporal embryonic development at the molecular level. Members of FGF (fibroblast growth factor) family play important roles in embryogenesis, e.g. in organogenesis, proliferation, differentiation, cell migration, angiogenesis, and wound healing. To dissect spatiotemporally the versatile roles of FGF during embryogenesis, we profiled gene expression in Xenopus embryo explants treated with SU5402, a chemical inhibitor specific to FGF receptor 1 ( FGFR1), by microarray. We identified 38 genes that were down-regulated and 5 that were up-regulated in response to SU5402 treatment from stage 10.5 -11.5 and confirmed their FGF-dependent transcription with RT-PCR analysis and whole-mount in situ hybridization (WISH ). Among the 43 genes, we identified 26 as encoding novel proteins and investigated their spatial expression pattern by WISH. Genes whose expression patterns were similar to FGFR1 were further analysed to test whether any of them represented functional FGF target molecules. Here, we report two interesting genes: one is a component of the canonical Ras-MAPK pathway, similar to mammalian mig6 (mitogen-inducible gene 6) acting in muscle differentiation; the other, similar to GPCR4 (G-protein coupled receptor 4), is a promising candidate for a gastrulation movement regulator. These results demonstrate that our approach is a promising strategy for scanning the genes that are essential for the regulation of a diverse array of developmental processes.

Genomic profiling of Mixer and Sox17β targets during Xenopus endoderm development

“…G protein-coupled receptors are known to transduce numerous extracellular signals into cells and regulate various aspects of cell proliferation (Marinissen and Gutkind, 2001). Recently, Gpr-4 was identified in a separate microarray experiment as a target gene for FGF signaling in Xenopus (Chung et al, 2004). In this study, Gpr-4 transcript was also observed in the endoderm and functional analysis of this gene revealed a potential role for Gpr-4 in regulating gastrulation movements (Chung et al, 2004).…”

“…Recently, Gpr-4 was identified in a separate microarray experiment as a target gene for FGF signaling in Xenopus (Chung et al, 2004). In this study, Gpr-4 transcript was also observed in the endoderm and functional analysis of this gene revealed a potential role for Gpr-4 in regulating gastrulation movements (Chung et al, 2004). Gpr-4 also has oncogenic properties and has been shown to regulate pH and ERK activity (Sin et al, 2004;Ludwig et al, 2003;Bektas et al, 2003).…”

“…Four of the six endodermally expressed genes are either known receptors (Cxcr4 and Gpr-4) or membrane-spanning proteins (March8 and Xtwik2) (Chavez et al, 1999;Moepps et al, 2000;Goto et al, 2003;Chung et al, 2004). Borg4 may be a member of a signal transduction cascade (Joberty et al, 1999).…”

The role of FGF signaling in the establishment and maintenance of mesodermal gene expression in Xenopus

“…The way in which maternal determinants and FGF and nodal pathways cooperate to induce mesoderm has been examined in several ways, including analysis on arrays Smith, 2000, 2002;Chung et al, 2004). However, while a limited number of up-and down-regulated targets have been identified, we do not have a good understanding of how these signals cooperate to produce the final pattern of the embryo.…”

Wnt Signaling in Vertebrate Axis Specification

“…Many Wntinducible genes, including Cdx4, Xpo, Meis, and Marginal coil (Xmc), have been previously identified as FGF-responsive genes (Table 1) (Amaya et al 1993;Frazzetto et al 2002;Aamar and Frank 2004;Chung et al 2004;Keenan et al 2006). In studies using dominant-negative FGF receptor constructs and specific morpholino oligonucleotides, FGF signaling has been reported to be essential for the Wnt pathway to activate its targets during anteroposterior axis development (McGrew et al 1997;Maegawa et al 2006;Burks et al 2009).…”