SHP-2 is required for the maintenance of cardiac progenitors

Abstract

The isolation and culturing of cardiac progenitor cells has demonstrated that growth factor signaling is required to maintain cardiac cell survival and proliferation. In this study, we demonstrate in Xenopus that SHP-2 activity is required for the maintenance of cardiac precursors in vivo. In the absence of SHP-2 signaling, cardiac progenitor cells downregulate genes associated with early heart development and fail to initiate cardiac differentiation. We further show that this requirement for SHP-2 is restricted to cardiac precursor cells undergoing active proliferation. By demonstrating that SHP-2 is phosphorylated on Y542/Y580 and that it binds to FRS-2, we place SHP-2 in the FGF pathway during early embryonic heart development. Furthermore, we demonstrate that inhibition of FGF signaling mimics the cellular and biochemical effects of SHP-2 inhibition and that these effects can be rescued by constitutively active/Noonan-syndrome-associated forms of SHP-2. Collectively, these results show that SHP-2 functions within the FGF/MAPK pathway to maintain survival of proliferating populations of cardiac progenitor cells.

Fig. 1. Inhibition of SHP-2 activity results in loss of MHC expression

Tissue explants show identical cardiac expression profiles as intact Xenopus embryos. (A) Whole-mount antibody staining of cardiac differentiation with tropomyosin (Tmy; red) or myosin heavy chain (MHC; red) antibodies as indicated, in whole embryos and cardiac explants at stages 22 (neurula), 29 (tailbud) and 37 (tadpole). (B) Whole-mount in situ hybridization for early heart markers Tbx5, Tbx20, Nkx2.5 in whole embryos and cardiac explants at stage 22, 29 and 37, as indicated. (C) MHC expression is dependent on SHP-2 activity. Whole-mount antibody staining for MHC (red) in cardiac explants taken from uninjected (control) embryos or embryos injected with Shp-2 N308D and treated with either buffer or DMSO carrier as controls or with NSC-87877 as indicated. BF, bright field.

Fig. 2. SHP-2 activity is required for the maintenance of expression of cardiac markers

(A–D) Cardiac explants isolated and cultured in DMSO or NSC-87877 beginning at stage 22. In situ hybridization performed on explants with probes specific for Nkx2.5 (A), Tbx5 (B), Tbx20-(C) and MLC1v′ (D) at stages 22, 26, 29 and 33, as indicated. Black arrows denote Tbx5 expression at the leading edge of the cardiac ridge at stage 33. (E) In situ hybridization of Nkx2.5 in uninjected (control) or Shp-2 N308D-injected explants treated with DMSO or NSC-87877 beginning at stage 22, and assessed at stage 37. (F) In situ with Nkx2.5 of anterior region of whole Xenopus embryos cultured in DMSO (control) or NSC-87877. H, heart; P, pharyngeal arches.

Fig. 3. SHP-2 activity is required for pharyngeal mesoderm

(A) Schematic of tissues and rudimentary organ structures in Xenopus tissue explants. (B) Cardiac explants isolated and cultured in DMSO or NSC-87877 (NSC) beginning at stage 22. In situ hybridization performed on explants with Tbx1, Fgf8, at stages 22, 26, 29 and 33. (C) Whole-mount in situ hybridization of Endocut, Endodermin, Sox2, Ami and Xmsr at stage 37; whole embryos and cardiac explants treated with DMSO (control) or NSC-87877, as indicated.

Fig. 4. SHP-2 is required for cardiac cell survival

TUNEL staining of control cardiac explants (DMSO) and explants treated with NSC-87877. Cell death examined in explants at stages 22, 26, 29 and 33, as indicated. Red arrows denote cardiac cells, yellow arrows endodermal cells. Double in situ (red)/TUNEL (dark blue) using a Tbx5-specific probe on stage 26 explants that were cultures in DMSO or NSC-87877 from stage 22–26. Black arrow points to Tbx5-expressing cells in NSC-87877-treated explants.

Fig. 5. Blocking the cardiac cell cycle results in loss of early, but not late, cardiac markers

(A) Cardiac explants isolated and cultured in DMSO or aphidicolin to block cells in S phase beginning at stage 22 and fixed at stage 37. Whole-mount immunostaining of explants with phosphohistone H3-specific antibody (red). In situ hybridization on explants with the early cardiac markers (B) Tbx5, Tbx20, Nkx2.5, (C) Gata4, Gata5 and Gata6, (D) with the cardiac differentiation markers Hsp27 and MLC1v′, and (E) by whole-mount immunostaining with the cardiac differentiation markers Tmy and MHC. (F) Explants were treated with DMSO or colchicine (Colch) to block cells in M phase of the cell cycle. In situ hybridization was performed to examine expression of Tbx5, Nkx2.5 and MLC1V′, as indicated. Aph, aphidicolin; BF, bright field; pH3, phosphohistone H3-specific antibody.

Fig. 6. SHP-2 is required for the survival of proliferating cardiac cells

(A) Xenopus cardiac explants were treated with the SHP-2 inhibitor NSC-87877 beginning at stage 22, 24, 26 or 29 as indicated, cultured to stage 37 and stained with an MHC antibody (red). (B) Representative transverse sections through stage 33, 35, 42 and adult Xenopus hearts with antibodies against MHC (green) or Tmy (green), phosphohistone H3 (red) and DAPI (blue). (C) Representative transverse sections through the cardiac tissue of a DMSO-treated and an explant treated with NSC-87877 beginning at stage 29, and stained with antibodies against phosphohistone H3 (red) and Tmy (green), as assessed at stage 37. (D) Cardiac mitotic index of explants treated beginning at stage 29 with DMSO (blue bar) or NSC-87877 (magenta bar) and assessed at stage 37. Bars represent the mean mitotic index of four explants per condition. Error bars denote the standard deviation. *, a statistically significant reduction in mitotic index of NSC-87877-treated explants (P=0.0021). BF, bright field; pH3, phosphohistone H3.

Fig. 7. SHP-2 is phosphorylated and interacts with FRS in vivo

(A) Transverse cryosections through the heart of control Xenopus embryos stained with tropomyosin to mark cardiac tissue (Tmy; green), DAPI to mark cell nuclei (blue), and anti-total SHP-2, anti-phospho-542 SHP-2 or anti-phospho-580 SHP-2 (all shown in red). Arrows denote endocardial cells that are negative for SHP-2. All samples from stage 37. (B) SHP-2 interacts with FRS in vivo. Hearts from FL-HA-SHP-2-derived embryos were dissected at stage 35 and immunoprecipitated with an anti-SHP-2 antibody (+) or beads with no antibody (−). Western analysis was then performed using antibodies specific for total SHP-2, phospho-542 SHP-2 and FRS-2. Note that the level of SHP-2 phopho-542 in the input was below levels of detection.

Fig. 8. FGF functions through SHP-2 to maintain the cardiac lineage

(A) Whole-mount in situ hybridization for Tbx5, Tbx20 and Nkx2.5 or whole-mount immunostaining for MHC (red) performed on explants treated with DMSO or the FGFR1 inhibitor SU5402. (B) Explants isolated from uninjected (control) or SHP-2 N308D-injected Xenopus embryos cultured in DMSO or SU5402 and analyzed by in situ hybridization for the cardiac markers Nkx2.5 and Tbx5. (C,D) Western blot analysis of DMSO-, NSC-87877- (C) or SU5402- (D) treated explants for phosphorylated and total ERK; α-tubulin was used as a loading control. (E) Explants were cut at stage 22 and then incubated in either modified Barth’s solution (MBS) or SU5402 until stage 35. Either endogenous SHP-2 was immunoprecipitated (IP) or explants were lysed (IB) and western analysis performed as in Fig. 7B.