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Platelet-derived growth factor receptor (PDGFR) signaling is required for normal gastrulation in Xenopus laevis. Embryos deprived of PDGFR signaling develop with a range of gastrulation-specific defects including spina bifida, shortened anteroposterior axis, and reduced anterior structures. These defects arise because the involuting mesoderm fails to move appropriately. In this study, we determine that inhibition of PDGFR signaling causes prospective head mesoderm cells to appear in the blastocoel cavity at the onset of gastrulation, stage 10. These aberrant cells undergo apoptosis via the caspase 3 pathway at an embryonic checkpoint called the early gastrula transition (EGT). They are TUNEL-positive and have increased levels of caspase 3 activity compared to control embryos. Apoptotic death of these mesoderm cells can be prevented by co-injection of mRNA encoding Bcl-2 or by injection of either a general caspase inhibitor or a caspase 3-specific inhibitor. Prevention of cell death, however, is not sufficient to rescue gastrulation defects in these embryos. Based on these data, we propose that PDGFR signaling is necessary for survival of prospective head mesoderm cells, and also plays an essential role in the control of their cell movement during gastrulation.
Fig. 1. Inhibition of PDGFR signaling causes mesoderm cells to accumulate in the blastocoel cavity at the beginning of gastrulation. Xenopus embryos co-injected at the 2- to 4-cell stage with β-gal-NLS mRNA and one of the following mRNAs: (A, E–H) PDGFR-FS, (B) XPDGFRα, (C, I–L) PDGFR-37, or (D) XPDGFRα/PDGFR-37 at a 10:1 ratio. Embryos are assessed at stages (A–D) 41, (E, I) 9, (F, J) 10, (G, K) 10+, and (H, L) 10.5. (A) PDGFR-FS- and (B) XPDGFRα-injected embryos develop normally. (C) PDGFR-37-injected embryos develop with gastrulation-specific defects. An example of a normal, mild, and severe phenotype is shown (C). (D) These defects are rescued by over-expression of wild-type XPDGFRα. (I–L) When assessed earlier in development, mesoderm cells with diffuse β-gal staining are found to appear in the blastocoel cavity of PDGFR-37-injected embryos beginning at stage 10. (E–H) At all stages, PDGFR-FS-injected embryos develop normally, and their mesoderm cells have nuclear β-gal staining. (M) The number of detached cells in the blastocoel cavity at stages 10, 10+, and 10.5 was assessed. Error bars represent standard error.
Fig. 2. Inhibition of PDGFR induces apoptosis via the caspase 3 pathway at gastrulation. Nicked DNA, an indicator of apoptosis, is visualized by terminal-UTP-nicked-end labeling (TUNEL). (A) Uninjected embryos treated with DNase I before TUNEL labeling have TUNEL-positive cells around the embryos periphery. (B) PDGFR-FS-injected embryos do not TUNEL-positive mesoderm cells. (C) PDGFR-37-injected embryos have TUNEL-positive cells in the blastocoel cavity. (D) An enlargement TUNEL positive cells within the square in C. Note in the blastocoel, small cells are TUNEL-positive (black arrowheads) and large cells are not (red arrowheads). (E) Caspase 3 activity was assessed at stages 10 and 11. Uninjected embryos (Con), embryos injected with PDGFR-37 (R-37) or PDGFR-FS (FS) mRNA at the 2- to 4-cell stage, embryos treated with 0.1 mg/ml cycloheximide (Chx) at the 32-cell stage. Note caspase 3 activity is increased in PDGFR-37-injected embryos compared to uninjected or PDGFR-FS-injected controls. Error bars represent standard error.
Fig. 3. Over-expression of Bcl-2 in PDGFR-37-injected embryos rescues the cell death but not gastrulation movements of mesoderm cells. Embryos that received mRNAs encoding β-gal-NLS and (A, D) PDGFR-FS, (G) Bcl-2, (B, E, H) PDGFR-37, or (C, F, I) Bcl-2/PDGFR-37 at a 10:1 ratio at the 2- to 4-cell stage were fixed and stained for β-gal at stages (A–C) 11 and (D–I) 26. Note the presence of cells with diffuse β-gal staining in the blastocoel of PDGFR-37-injected embryos (B, arrows; a mild phenotype is shown—see Fig. 4 for severe PDGFR-37 phenotypes) and their absence in Bcl-2/PDGFR-37-injected embryos (C) at stage 11. Also note the presence of lumps along the dorsal axis of Bcl-2/PDGFR-37-injected embryos at the tailbud stage (F, arrowheads). Gastrula-staged embryos are oriented animal up, dorsoanterior right.
Fig. 4. Inhibition of caspases rescues cell death but not gastrulation movements in PDGFR-37-injected embryos. Embryos were co-injected dorsoanteriorly with mRNAs encoding β-gal NLS and (A–F) PDGFR-FS or (G–L) PDGFR-37 at the 2- to 4-cell stage. At the blastula stage (stages 8–9), the blastocoel cavity was injected with 400 uM of (B, E, H, K) a general caspase inhibitor (z-VAD-fmk) or (C, F, I, L) a caspase 3 inhibitor (z-DEVD-fmk). The embryos were cultured until stage 11.5 or 28 before being fixed and stained for β-gal. Note, no β-gal-positive cells in the blastocoel of PDGFR-FS-injected embryos (A–C) and in the PDGFR-37-injected embryos, the rescue of cells with diffuse β-gal staining by caspase inhibitors (compare G with H, I). Caspase inhibitor-injected embryos, however, still exhibit gastrulation defects at stage 28 (compare J with K, L). Cells with diffuse β-gal staining are indicated (arrows).
Fig. 5. PDGFR signaling is not required for convergent extension of open-faced Keller explants. Keller explants were dissected at stage 10.25 from embryos co-injected with Gap43-GFP and either PDGFR-FS or PDGFR-37 mRNAs (see Material and methods). The explants were cultured until stage 20. GFP-positive explants were fixed in MEMFA to quench the GFP fluorescence, and subjected to TUNEL staining using dUTP conjugated to fluorescein. (A) PDGFR-FS-injected explants elongate to a similar extent as (D) PDGFR-37-injected explants. (B, C) PDGFR-FS-injected explants contain a similar number of TUNEL-positive, fluorescein-labeled, cells as (E, F) PDGFR-37-injected explants. Examples of TUNEL-positive cells are indicated (arrowheads). The explants from three independent experiments are shown in A and D.
