Academic journal article Genetics

A Screen for Genes That Influence Fibroblast Growth Factor Signal Transduction in Drosophila

Academic journal article Genetics

A Screen for Genes That Influence Fibroblast Growth Factor Signal Transduction in Drosophila

Article excerpt

ABSTRACT

The misexpression of an activated form of the FGF receptor (FGFR) Breathless in conjunction with downstream-of-FGF-receptor (Dof), an essential signaling molecule of the FGF pathway, in the Drosophila eye imaginal discs impairs eye development and results in a rough eye phenotype. We used this phenotype in a gain-of-function screen to search for modifiers of FGF signaling. We identified 50 EP stocks with insertions defining at least 35 genes that affect the rough eye phenotype. Among these genes, 4 appear to be specific for FGFR signaling, but most of the genes also influence other signaling pathways, as assessed by their effects on rough eyes induced by other activated receptor tyrosine kinases (RTKs). Analysis of loss-of-function alleles of a number of these genes in embryos indicates that in many cases the products are provided maternally and are involved in germ cell development. At least two of the genes, sar1 and robo2, show a genetic interaction with a hypomorphic dof allele, suggesting that they participate in FGF-mediated morphogenetic events during embryogenesis.

FIBROBLAST growth factor (FGF) receptors are Iigand-activated transmembrane glycoproteins that transmit signals to a variety of intracellular targets. In Drosophila two FGF receptors, Breathless (Bd) and Heartless (HtI), lead to the phosphorylation of MAP kinase in the mesoderm and the trachea! system, as well as in a number of other cell types, and are required for the morphogenesis of these tissues (KLAMBT et al. 1992; BEIMAN et al. 1996; GISSELBRECHT et al. 1996; SUTHERLAND et al. 1996; GABAY et al. 1997). A cytoplasmic molécule, downstream-of-FGF-receptor (Dof), also known as Heartbroken or Stumps, is important for the proper transduction of signals from both FGF receptors (MiCHELSON et al. 1998; VINCENT et al. 1998; IMAM et al. 1999). In rfo/mutant embryos, mesoderm migration and trachéal branching are defective and phosphorylated MAPK fails to accumulate in these tissues. A constitutively active form of Ras can provide a partial rescue of the defects, suggesting that Dof acts upstream of Ras. Biochemical analysis and protein interaction experiments performed in yeast cells indicate that Dof binds directly to the FGF receptors via an essential protein domain, the Dof-BANK-BCAP domain, and becomes phosphorylated upon activation of the receptor (BATTERSBY et al. 2003; WILSON et al. 2004). Functional studies with mutant forms of Dof have suggested that one protein that could bind to Dof and participate in transmission of the FGF signal is the protein phosphatase Corkscrew, but the potential binding site is neither sufficient nor necessary for all aspects of signal transmission (PETIT et al 2004; WILSON et al 2004). It is therefore not presently fully understood how signals generated upon activation of the FGF receptors are transmitted to intracellular targets to promote cellular differentiation and morphogenesis.

Genetic screens for modifiers of mutant eye phenotypes have proved successful in identifying components of signaling pathways regulated by receptor tyrosine kinases (RTKs) (KARIM et al. 1996; HUANG and RUBIN 2000; REBAY et al 2000; THERRIEN et al 2000). These screens were based on the observation that the expression of a gain-of-function, ligand-independent form of the RTK Sevenless in the Drosophila eye causes all ommatidial precursors to develop as neurons and leads to a rough eye phenotype (BASLER et al. 1991). FGF signaling is not required for eye development (CASCi et al. 1999), but we found that the expression of a constitutively active FGF receptor in the eye together with Dof (also normally absent from the eye) caused a rough eye phenotype. On the basis of this observation, we performed a gain-of-function screen to identify genes whose mis- or overexpression might influence this effect. We screened a large panel of fly stocks (referred to here as "EP lines"), which carry P-element insertions that allow the tissue-specific expression of nearby genes (R0RTH 1996). …

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