Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms

Abstract

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 (PDGFalphaR) is fused to the cytosolic domain of Drosophila Torso (alpha(Tor)) or the mouse fibroblast growth factor receptor 1 (alpha(FR)). alpha(Tor) homozygous embryos exhibit significant rescue of neural crest and angiogenesis defects normally found in PDGFalphaR-null embryos yet fail to rescue skeletal or extraembryonic defects. This phenotype was associated with the ability of alpha(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 alpha(FR) chimeric receptor fails to rescue any aspect of the PDGFalphaR-null phenotype. Instead, alpha(FR) expression leads to a gain-of-function phenotype highlighted by ectopic bone development. The alpha(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.

FIG. 1.

Targeted knock-ins of α^GFP, α^FR, and α^Tor at the PDGFαR locus. (A) Schematic representation of the chimeric receptors and relevant binding sites for downstream effector molecules. (B) Schematic of the region of the wild-type PDGFαR genomic locus and the cDNA knock-in constructs. Hatched boxes represent the positions of exons. SA, splice acceptor; 3PA, triple polyadenylation sequence. Inset boxes are Southern blot analyses of wild-type (+/+) or targeted (α^FR/+ or α^GFP/+) ES clones with a probe corresponding to the shaded box and NheI-digested DNA. The probe hybridizes to a 4-kb fragment which indicates correct targeting (arrow) and an additional 8.7-kb fragment due to a duplication of this area in the α^FR and α^Tor cDNA. (C) Top left panel, analysis of GFP expression in unstained cells derived from an α^GFP/+ E9.5 embryo. Top right panel, cells from same embryo stained for PDGFαR expression with an antibody conjugated to PE. FACS analysis of cell lines expressing chimeric receptors (middle panel, α^Tor/α^Tor; lower panel, α^FR/−) by using an antibody that recognizes the extracellular domain of the PDGFαR conjugated to PE. The purple filled peak represents signal from αR^−/− cells, the solid green trace represents signal from cells expressing the chimeric receptors (α^Tor/α^Tor or α^FR/−), and the dotted red line is the trace of cells expressing the wild-type PDGFαR.

FIG. 2.

Expression patterns of α^GFP in embryonic and adult tissues. (A) Expression in polar trophectoderm of E4.5 blastocysts. Bottom panel, visible light; top panel, UV. (B) Expression in extraembryonic ectoderm of an E6.5 embryo. Top panel, UV; bottom panel, visible light. Arrows denote the boundary of embryonic and extraembryonic tissues, and Reichert's membrane was not removed. (C) Whole-mount E10.5 embryo α^GFP/+. (D) Magnification of the caudal somite. (E) E13.5 littermates. Left embryo, α^GFP/+; right embryo, α^GFP/α^GFP. Expression in whole-mount E18.5 organs, lung (F), stomach (G, top portion), and transverse section of adult stomach in the glandular region (G, lower portion). (H) Sagittal section of adult heart. (I) Whole-mount adult eye. (J) Sagittal section of ribs and spinal cord of an E16.5 α^GFP/+ embryo. (K) E18.5 α^GFP/+ transverse section through ribs. Whole-mount (L) and transverse section (M) of an E14.5 α^GFP/+ placenta visualized under fluorescence. ICM, inner cell mass; NT, neural tube; S, somites; V, ventricle; A, aorta; L, lens; R, ribs; P, perichondrium; La, labyrinth; ST, spongiotrophoblast layer; CA, chorioallantoic layer; U, umbilical cord.

FIG. 3.

Partial rescue of embryonic defects by α^Tor. (A to C) Wild-type (A) and PDGFαR^−/− (B and C) E14.5 whole-mount embryos. (D to F) Wild-type (D) and α^Tor/α^Tor (E and F) E17.5 embryos. (G to I) Cleft face phenotype of PDGFαR^−/− (G) and α^Tor/α^Tor (H) E14.5 embryos and E15.5 α^Tor/α^Tor embryos (I). (J and K) Transverse sections through E14.5 embryos at heart level for PDGFαR^−/− (J) and α^Tor/α^Tor (K) embryos. Arrows indicate incomplete septum formation.

FIG. 4.

Rescue of vascular development by α^Tor. Whole-mount PECAM staining in the mid- and forebrain region of E9.5 wild-type (A), PDGFαR ^−/− (B), and α^Tor/α^Tor (C) embryos is shown. Also shown is PECAM staining in the midbrain of E11.5 wild-type (D), PDGFαR ^−/− (E), and α^Tor/α^Tor (F) embryos.

FIG. 5.

Skeletal defects in α^Tor mice. Skeletons of E16.5 embryos. (A) Wild-type ventral view of ribs and sternum. (B) α^Tor/α^Tor ventral view of rib cage. (C) α^Tor/α^Tor dorsal view of spinal column. (D) Wild-type dorsal view of spinal column with limbs removed. Arrows denote the positions of sternum.

FIG. 6.

Placental defects in α^Tor mice. (A to C) α^GFP/+ (A), α^GFP/α^GFP (B), and α^GFP/α^Tor (C) E13.5 whole-mount placentas visualized under fluorescence. (D, F, and G) Hematoxylin and eosin staining of E13.5 transverse placental sections of α^GFP/+ (D), α^GFP/α^GFP (F), and α^GFP/α^Tor (G) embryos. (E) Transverse frozen section at E13.5 placenta. (H and I) α^GFP/α^Src (H) and α^GFP/α^PI3K (I) whole-mount placentas at E14.5. CA, chorioallantoic place; U, umbilical cord; L, labyrinth. Arrows denote positions of the epithelial layer between chorioallantoic layer and labyrinth, which is absent in mutants.

FIG. 7.

α^FR dominant phenotypes. (A to C) α^GFP/+ (A), α^GFP/α^GFP (B), and α^GFP/α^FR (C) E9.5 whole-mount embryos visualized under fluorescence. (D and E) E17.5 α^FR/+ embryo (D) and skeleton (E). Arrows denote extra digits and ectopic bone. (F and G) Alcian blue-stained transverse sections through nasal cartilage of E16.5 wild-type (F) and α^FR/+ (G) embryos. Arrows indicate cartilage growth in front of the eye. (H and I) ASMA staining of transverse sections of E16.5 wild-type (H) or α^FR/+ (I) placentas. Arrows denote positively stained chorioallantoic trophoblasts. L, labyrinth; CA, chorioallantoic plate; E, eye.

FIG. 8.

Activation of MAP kinase and PI3-kinase signaling pathways. (A) Western blot analysis of whole-cell lysates from wild-type (WT) or α^Tor/α^Tor (left panels) or α^FR/+ (right panels) primary embryonic fibroblasts stimulated with 30 ng of PDGF-AA/ml harvested at the time points indicated above the lanes (in minutes). Blots were probed with antibodies to phosphorylated MAP kinase (p-Erk), phosphorylated Akt (downstream of PI3-kinase), or Ras-GAP as a loading control. (B) Western blot analysis of cell lysates from Ph fibroblasts transfected with wild-type PDGFαR or α^FR stimulated with PDGF-AA for the indicated times. The expression levels of receptors were normalized by FACS sorting, and protein level loading controls were quantified by Ponceau S staining (not shown). Blots were probed with antibodies to phosphorylated Erk or phosphorylated Akt. Bottom right panel, Western blot analysis of FRS2 immunoprecipitation of lysates from indicated cell types. Blots were probed with antibodies to phosphorylated tyrosine.