Tang TL et al. (1995), The SH2-containing protein-tyrosine phosphatase...

XB-ART-20095

Cell 1995 Feb 10;803:473-83.

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The SH2-containing protein-tyrosine phosphatase SH-PTP2 is required upstream of MAP kinase for early Xenopus development.

Tang TL , Freeman RM , O'Reilly AM , Neel BG , Sokol SY .

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SH-PTP2, the vertebrate homolog of Drosophila corkscrew, associates with several activated growth factor receptors, but its biological function is unknown. We assayed the effects of injection of wild-type and mutant SH-PTP2 RNAs on Xenopus embryogenesis. An internal phosphatase domain deletion (delta P) acts as a dominant negative mutant, causing severe posterior truncations. This phenotype is rescued by SH-PTP2, but not by the closely related SH-PTP1. In ectodermal explants, delta P blocks fibroblast growth factor (FGF)- and activin-mediated induction of mesoderm and FGF-induced mitogen-activated protein (MAP) kinase activation. Our results indicate that SH-PTP2 is required for early vertebrate development, acting as a positive component in FGF signaling downstream of the FGF receptor and upstream of MAP kinase.

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Species referenced: Xenopus
Genes referenced: acta4 actc1 fgf2 fn1 inhba mapk1 ptpn11 ptpru tbxt

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	Figure 1. Molecular Cloning and Expression of XSH-PTP2 (A) Deduced amino acid sequence for XSH-PTP2. SH2 domains are indicated by solid lines and the PTP domain by italics. (13) Comparison of the structure of XSH-PTP2 to HSH-PTP2 and csw, indicating a high degree of conservation. Percent identities are indicated. XSH-PTP2 and HSH-PTP2 lack an insert in the phosphatase domain. Two potential tyrosine phosphorylation sites are conserved from XSH-PTP2 to HSH-PTP2; one is also found in csw. All three homologs retain a proline-rich sequence within their C-termini. (C) Northern blot of SH-PTP2 expression in early Xenopus development. Numbers across the top indicate developmental stage. XSHPTP2 is expressed as an approximately 3 kb transcript, present in oocytes (M) and fertilized eggs (stage 1) through tadpole (stage 38). The blot was reprobed with Xenopus fibronectin to control for RNA integrity. Fibronectin RNA levels increase in the later stages of normal embryogenesis. The apparent decrease in XSH-PTP2 RNA expression in mid-embryonic stages is not observed reproducibly. (D) Maternal expression of XSH-PTP2 protein. Recombinant HSHPTP2 (50 and 100 ng) in total cell lysates from baculovirus-infected Sf9 cells (lanes A and B, respectively) and soluble protein from the equivalent of one-half (1/2) or one oocyte (lanes C and D, respectively) were detected by immunoblotting with anti-PTPID/SH-PTP2 monoclonal antibody.
	Figure 2. Microinjection Constructs Shown are schematics of human and Xenopus SH-PTP1 and SH-PTP2 including the N- (SH2-N) and C-terminal (SH2-C) SH2 domains, the PTP domain, and the C-terminal tail. HSH-PTP2 mutant constructs are also depicted including the PTP-inactive construct (AP), containing a 31 amino acid deletion in the critical catalytic core, and three C-terminal tyrosine to phenylalanine point mutants, consisting of two single point mutations (Y542F, Y580F) and the double point mutant (Y542,580F).
	Figure 3. AP RNA Injection Results in Embryos with Severe Tail Truncations Four- to eight-cell embryos were injected in the dorsal marginal zone with wild-type XSH-PTP2 or AP RNA and allowed to develop• (A-D) Morphology of injected embryos. Embryos were scored for developmental abnormalities at stage 18 (A and B) and stage 44 (C and D): embryos injected with XSH-PTP2 RNA (A and C); embryos injected with AP RNA (B and D). The phenotype of AP-injected embryos at stage 18 reflects failure of blastopore closure at the end of gastrulation. At stage 44, the extreme tail truncation of AP-injected embryos is evident• (E and F) Histological analysis. A transverse section at the back of the head of a normal, XSH-PTP2 RNA-injected tadpole is shown in (E). Brain (b), somites (s), and notochord (nc) are indicated. The scale bar in (E) represents 100 I~m, For comparison, in a more posterior section, a AP RNA-injected embryo is shown (F). Split notochord and neural tube (nt) are evident; somites are not duplicated• (G) Dose response of AP RNA injection. Increasing amounts of AP RNA were injected into 4- to 8-cell embryos, which were allowed to develop to stage 18 and scored for tail truncation. (H) Inhibition of Xbra expression by injection of AP RNA. Northern blot of RNA from stage 11 embryos, injected in the marginal zone of all four blastomeres at the 4-cell stage with water control (C), SH-PTP2 PTP domain mutant RNA (AP), wild-type XSH-PTP2 RNA (WT), dominant negative FGFR RNA (FRD), or wild-type FGFR RNA (FR), andprobed with the indicated early mesodermal markers. Injection of either AP or FRD inhibits Xbra expression. The same blot rehybridized with a fibronectin probe (FN) serves as a control for RNA loading and integrity.
	Figure 4. AP-Injected Embryos Are Rescued By Coinjection of Wild- Type XSH-PTP2 (A-C) Embryos were injected at the 4- to 8-cell stage with the indicated RNAs. (A) Embryos injected with wild-type XSH-PTP2. (B) Embryos coinjected with AP and wild-type XSH-PTP1 (5 ng AP:2.5 ng XSHPTP1); all of the embryos shown display the mutant phenotype. (C) Coinjection of AP with wild-type XSH-PTP2 RNA (5 ng AP:2.5 ng XSHPTP2) rescues almost all the embryos; in the top left corner is an embryo that was not rescued. (D) Immunoblot analysis of injected embryos. Embryos coinjected with AP RNA and wild-type HSH-PTP1 RNA (5 ng AP:2.5 ng HSH-PTP1) were harvested at stage 11 for protein analysis. (Left panel) Anti- SH-PTP1 immunoblot: no HSH-PTP1 is detectable in the uninjected lane (1), but full-length HSH-PTP1 is found in the coinjected lane (2). This blot was reprobed with anti-PTP1D/SH-PTP2 monoclonal antibody (right panel). AP protein is detected in the coinjected lane (2). Cross-reaction of this antibody with XSH-PTP2 (upper band seen in both lanes 1 and 2, right panel) acts as a control for protein loading and shows the relative levels of AP and endogenous XSH-PTP2.
	Figure 5. AP Injection Blocks FGF-Mediated Elongation of Animal Caps One- to two-cell embryos were injected with AP RNA, then allowed to develop to stage 8. Animal caps were treated for 3 hr with 100 ng/ml bFGF or 5 ng/ml human recombinant activin A. (a) Untreated controls; (b) uninjected animal caps treated with bFGF; (c) animal caps from embryos injected with AP FIN& treated with bFGF; (d) animal caps from embryos injected with wild-type XSH-PTP2 RNA, treated with bFGF; (e) untreated controls; (f) uninjected animal caps treated with activin; (g) animal caps from embryos injected with AP RNA, treated with activin; (h) animal caps from embryos injected with wildtype XSH-PTP2 RNA, treated with activin.
	Figure 6. AP Inhibits Expression of Early and Late Mesodermal Markers in Animal Caps (A) Inhibition of Xbra expression. One- to two-cell embryos were injected with AP or wild-type XSH-PTP2 RNA, then allowed to develop to stage 8. Animal caps were either left untreated (minus) or treated for 3 hr with 100 ng/ml bFGF (~olus F) or 5 ng/ml activin A (plus A), and RNA was prepared at stage 11. The resulting Northern blot was probed with the early marker Xbra and fibronectin (FN). Expression of these markers in total embryos (TE) is also shown. Injection of Ap RNA inhibits Xbra expression, whereas injection of wild-type XSHPTP2 (WT) has no effect. Xbra expression is not detected in ~Pinjected embryos even upon longer exposure of the blot. (B) Inhibition of muscle actin expression. One- to two-cell embryos were injected with AP or wild-type XSH-PTP2 RNA, and animal caps, prepared at stage 8 as in (A), were either left untreated (minus) or treated for 3 hr with 100 ng/ml bFGF or 5 ng/ml activin A, as indicated. RNA prepared at stage 11 was used for Northern analysis with an actin probe. In both the uninjected and XSH-PTP2 RNA-injected lanes, induction of muscle-specific actin (arrow) is evident upon bFGF treatment. Ap injection blocks muscle actin induction. An analogous result is seen for activin-induced muscle actin expression. The actin probe cross-hybridizes with cytoplasmic actin (the two upper bands seen in both blots), which is not induced by either bFGF or activin A treatment, thus providing a control for RNA loading.
	Figure 7. Ap RNA Injection Blocks MAPK Activation by FGF One- to two-cell embryos were injected in the animal pole with Ap RNA and allowed to develop to stage 8. Animal caps were dissociated in calcium-free, magnesium-free, normal amphibian media (see Experimental Procedures), treated with 100 nglml bFGF for 5 min, and pelleted and frozen for protein analysis. Total lysates from these cells were separated by SDS-PAGE and transferred to Immobilon. The blot was probed with anti-Xenopus MAPK rabbit polyclonal antibodies followed by anti-rabbit-horseradish peroxidase secondary antibodies and development by ECL. Duplicate experiments are shown. The decrease in electrophoretic mobility denotes MAPK activation. MAPK activation is blocked in animal cap cells from AP-injected embryos.