DEV009290 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. SHP-2 is expressed during early Xenopus development. (A) Western blot of in vitro translated Xenopus SHP-2 in the sense orientation or anti-sense orientation (Neg.) probed with an anti-SHP-2 antibody. (B) Western blot analysis of early Xenopus embryogenesis at stages 10.5 (gastrula) through stage 40 (late tadpole) with a SHP-2-specific antibody. Anti-EF-2 was used as a loading control. (C) Western blot analysis of SHP-2 and Tmy in whole embryos and isolated heart tissue from stage 37 embryos with α-tubulin as the loading control.

• Supplemental Figure S2 -

  Fig. S2. Shp-1 expression in Xenopus. (A) Whole-mount in situ hybridization of uninjected embryos at stages 22-37 with a Shp-1-specific probe. Anterior, left; posterior, right. We observe that similar to studies of Shp-1 expression in mice, Xenopus Shp-1 expression is observed in the developing hematopoetic lineage (stage 29, 33 and 37 embryos, arrowheads). (B) In situ hybridization of transverse serial sections through a stage 29 Xenopus heart with probes against Tbx20, Shp-2 and Shp-1, with Shp-1 being the most-anterior section followed by Tbx20 and Shp-2. The arrows point to corresponding Tbx20-positive tissue in adjacent sections. Scale bars: 1 mm in A; 100 μm in B.

• Supplemental Figure S3 -

  Fig. S3. Shp-2 rescues expression of cardiac tissue in a dose-dependent manner. Whole-mount antibody staining of cardiac explants derived from either uninjected embryos or embryos injected with 1 ng of FL-Shp2 or Shp-2 N308D. Embryos shown at stage 37 stained with an antibody against myosin heavy chain (MHC, red). Anterior, left; dorsal, top. Scale bar: 0.5 mm.

• Supplemental Figure S4 -

  Fig. S4. Shp-2 N308D does not block cardiac commitment, migration or fusion. Whole-mount in situ hybridization of control, full-length Shp-2 injected, or Shp-2 N308D injected embryos at stage 26 with cardiac-specific probes Nkx2.5, Tbx5 or Tbx20.

• Supplemental Figure S5 -

  Fig. S5. Gata4, Gata5 and Gata6 expression in SHP-2-inhibited explants. Whole-embryo controls (whole) and cardiac explants were isolated at stage 22 and cultured until stage 37 in DMSO or NSC-87877. In situ hybridization was performed on explants with Gata4, Gata5 and Gata6. Scale bars: 0.5 mm.

• Supplemental Figure S6 -

  Fig. S6. Dose response of the SHP-2 inhibitor NSC-87877. (A) Whole-mount in situ analysis of whole embryo and cardiac explants with a Nkx2.5-specific probe. All explants were removed and treated at stage 22 and cultured in either carrier (DMSO) with the respective dose of NSC-87877 until stage 37. Upper left-hand panel shows a representative Nkx2.5 in situ of a stage 37 embryo. Scale bars: 0.5 mm for whole embryo; 1 mm for explants. (B) Western blot analysis of active and total ERK in cardiac explants cultured in DMSO or increasing doses of NSC-87877 until stage 37, with α-tubulin as a loading control.