Van Stry M et al. (2005), Distinct effectors of platelet-derived growth f...

XB-ART-1839

Proc Natl Acad Sci U S A 2005 Jun 07;10223:8233-8. doi: 10.1073/pnas.0502885102.

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Distinct effectors of platelet-derived growth factor receptor-alpha signaling are required for cell survival during embryogenesis.

Van Stry M , Kazlauskas A , Schreiber SL , Symes K .

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Platelet-derived growth factor receptor (PDGFR) signaling is essential for normal embryonic development in many organisms, including frog, mouse, zebrafish, and sea urchin. The mode of action of PDGFR signaling during early development is poorly understood, however, mostly because inhibition of signaling through either the PDGFRalpha or PDGFRbeta is embryonic lethal. In Xenopus embryos, disruption of PDGFRalpha signaling causes migrating anterior mesoderm cells to lose direction and undergo apoptosis through the mitochondrial pathway. To understand the mechanism of PDGFRalpha function in this process, we have analyzed all known effector-binding sites in vivo. By using a chemical inducer of dimerization to activate chimera PDGFRalphas, we have identified a role for phospholipase Cgamma (PLCgamma) in protecting cells from death. PDGFRalpha-mediated cell survival requires PLCgamma and phosphatidylinositol 3-kinase signaling, and that PDGFRalpha with binding sites for these two signaling factors is sufficient for this activity. Other effectors of PDGFRalpha signaling, Shf, SHP-2, and Crk, are not required for this process. Thus, our findings show that PDGFRalpha signaling through PLCgamma and phosphatidylinositol 3-kinase has a protective role in preventing apoptosis in early development. Furthermore, we demonstrate that small molecule inducers of dimerization provide a powerful system to manipulate receptor function in developing embryos.

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Species referenced: Xenopus
Genes referenced: crk fkbp1a nr0b2 pdgfra pik3ca ppp3ca shf

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	Fig. 1. Schematic of iPDGFRα mutants. To dissect PDGFRα signaling, tyrosines that when phosphorylated (P) bind and activate specific downstream effectors were replaced by phenylalanine (black squares) by site-directed mutagenesis. (A) Subtraction mutants contain mutations that allow binding and activation of all but one downstream effector. (B) Add-back mutants contain mutations to allow binding and activation of one or more downstream effector. (C) iPDGFRα is a fusion protein of the myristoylation signal from v-Src, three tandem repeats of FKBP12 containing point mutations G89P and I90K to block calcineurin binding, and the cytoplasmic domain of the PDGFRα with or without specific Y→F mutations. The addition of the dimerizer, AP1510, activates the receptor kinase through the induced dimerization of two of the receptor fusion proteins.
	Fig. 2. PDGFR signaling through PLCγ and PI3K, but not through SHP-2, Shf, and Crk, is required for mesoderm cell survival. (A and B) Embryos were coinjected with mRNA encoding β-gal and iPDGFRα or β-gal, PDGFR-37 (R37), and the following iPDGFRαs. (C and D) wt. (E and F) F572/74. (G and H) F720. (I and J) F731/42. (K and L) F762. (M and N) F988. (O and P) F1018. At the beginning of gastrulation (stage 10), AP1510 or DMSO was injected into the blastocoel. At the midgastrula stage (stage 11), β-gal expression was visualized (shown in blue). The stained embryos were dissected and scored for the presence or absence of nonnuclear β-gal-stained cells in the blastocoel cavity, within the vitelline membrane, or in the process of being excluded from the embryo (see red arrowhead in K), indicating the presence of apoptotic cells. The percentage of embryos containing apoptotic cells was calculated. Representative saggital sections of these embryos are shown. (G–P) Note that when signaling through PLCγ and PI3K is prevented (I, J, and M–P), activation of the receptor with AP1510 did not restore cell survival, whereas cell survival is restored when signaling through SHP-2, Shf, and Crk is prevented (G, H, K, and L). Arrowheads indicate apoptotic mesoderm cells outside the blastocoel cavity. Note that overexpression of iPDGFRα-wt mRNA alone does not cause apoptosis in mesoderm cells.
	Fig. 3. PDGFR signaling through a single downstream effector is not sufficient for mesoderm cell survival. (A and B) Embryos were coinjected with mRNA encoding β-gal and iPDGFRα or β-gal, PDGFR-37 (R37), and the following iPDGFRαs. (C and D) wt. (E and F) Y572/74. (G and H) Y720. (I and J) Y731/42. (K and L) Y762. (M and N) Y988. (O and P) Y1018. At the beginning of gastrulation (stage 10), AP1510 or DMSO was injected into the blastocoel. At the midgastrula stage (stage 11), β-gal expression was visualized (shown in blue). Representative saggital sections of these embryos are shown. (E–P) Note that single effectors do not restore cell survival. Arrowheads indicate apoptotic mesoderm cells outside the blastocoel cavity.
	Fig. 4. PDGFR signaling through PLCγ and PI3K is required for mesoderm cell survival. (A and B) Embryos were coinjected with mRNA encoding β-gal and iPDGFRα or β-gal, PDGFR-37 (R37), and the following iPDGFRαs. (C and D) wt. (E and F) F720/762. (G and H) F4. At the beginning of gastrulation (stage 10), AP1510 or DMSO was injected into the blastocoel. At the midgastrula stage (stage 11), β-gal expression was visualized (shown in blue). Representative saggital sections of these embryos are shown. As shown in E and F, only the presence of PLCγ, PI3K, and Src pYBs are required to restore mesoderm cell survival.
	Fig. 5. PDGFR signaling through PLCγ and PI3K is required for mesoderm cell survival. The data presented in Figs. 2, 3, 4 are shown in graph form. The percentage of embryos that do not contain apoptotic cells (i.e., cells with nonnuclear β-gal staining) is presented for embryos injected with mRNA as described in Figs. 2, 3, 4 and with DMSO (gray bars) or AP1510 (black bars). This percentage is low for some mutants compared with wt because there may be some basal activity of the receptor construct without the addition of dimerizer. Error bars represent standard error and were calculated from a minimum of three separate experiments. The data used to construct this graph is available in Table 1, which is published as supporting information on the PNAS web site.

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