Evolutionary Divergence of Platelet-Derived Growth Factor Alpha Receptor Signaling Mechanisms

Receptor tyrosine kinases (RTKs) direct diverse cellular and developmental responses by stimulating a relatively small number of overlapping signaling pathways. Specificity may be determined by RTK expression patterns or by differential activation of individual signaling pathways. To address this issue we generated knock-in mice in which the extracellular domain of the mouse platelet-derived growth factor alpha receptor (PDGFαR) is fused to the cytosolic domain of Drosophila Torso (α(Tor)) or the mouse fibroblast growth factor receptor 1 (α(FR)). α(Tor) homozygous embryos exhibit significant rescue of neural crest and angiogenesis defects normally found in PDGFαR-null embryos yet fail to rescue skeletal or extraembryonic defects. This phenotype was associated with the ability of α(Tor) to stimulate the mitogen-activated protein (MAP) kinase pathway to near wild-type levels but failure to completely activate other pathways, such as phosphatidylinositol (PI) 3-kinase. The α(FR) chimeric receptor fails to rescue any aspect of the PDGFαR-null phenotype. Instead, α(FR) expression leads to a gain-of-function phenotype highlighted by ectopic bone development. The α(FR) phenotype was associated with a failure to limit MAP kinase signaling and to engage significant PI3-kinase response. These results suggest that precise regulation of divergent downstream signaling pathways is critical for specification of RTK function

Identification and validation of PDGF transcriptional targets by microarray-coupled gene-trap mutagenesis - Supplemental Materials.

We developed a versatile, high-throughput genetic screening strategy by coupling gene mutagenesis and expression profiling technologies. Using a retroviral gene-trap vector optimized for efficient mutagenesis and cloning, we randomly disrupted genes in mouse embryonic stem (ES) cells and amplified them to construct a cDNA microarray. With this gene-trap array, we show that transcriptional target genes of platelet-derived growth factor (PDGF) can be efficiently and reliably identified in physiologically relevant cells and are immediately accessible to genetic studies to determine their in vivo roles and relative contributions to PDGF-regulated developmental processes. The same platform can be used to search for genes of specific biological relevance in a broad array of experimental settings, providing a fast track from gene identification to functional validation