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Vertebrate gastrulation requires coordination of mesoderm specification with morphogenetic movements. While both of these processes require FGF signaling, it is not known how mesoderm specification and cell movements are coordinated during gastrulation. The related Sprouty and Spred protein families are recently discovered regulators of receptor tyrosine kinase signaling. We identified two genes for each family in Xenopus tropicalis: Xtsprouty1, Xtsprouty2, Xtspred1, and Xtspred2. In gain- and loss-of-function experiments we show that XtSprouty and XtSpred proteins modulate different signaling pathways downstream of the FGF receptor (FGFR), and consequently different developmental processes. Notably, XtSproutys inhibit morphogenesis and Ca(2+) and PKCdelta signaling, leaving MAPK activation and mesoderm specification intact. In contrast, XtSpreds inhibit MAPK activation and mesoderm specification, with little effect on Ca(2+) or PKCdelta signaling. These differences, combined with the timing of their developmental expression, suggest a mechanism to switch FGFR signal interpretation to coordinate mesoderm formation and cell movements during gastrulation.
Figure 1. Morphogenesis Requires FGFR Signaling Independent of MAPK(A) Western blots against embryo lysates from various stages probed for active MAPK (dp-MAPK) and pan-MAPK. MAPK activity peaked at stage 11 and decreased by stage 14, and then returned at stage 19.(B) Blots for dp-MAPK and pan-MAPK against DMZ lysates at corresponding stages. Again, MAPK activity peaked at stage 11 and decreased sharply at stage 12, remaining low until stage 18.(C) DMZ extension (μm) plotted against the corresponding stage. The majority of extension occurred between stages 12–16. Error bars represent standard deviation.(D) dp-MAPK blots from animal cap lysates incubated for 15 min with and without FGF2 in the presence of increasing concentrations of FGFR1 inhibitor (SU5402) or MEK1 inhibitor (U0126). Both drugs abolished FGF-induced MAPK activation in a dosage-dependent manner.(E) Embryos injected with 10 nl of 2 mM SU5402, U0126, or DMSO into the blastocell and cultured with the same inhibitor at stage 8 or 12.5. Stage 8 treated embryos were fixed at stage 11.5 and 29–30 for in situ hybridization for Xbra and cardiac actin, respectively. Both inhibitors blocked mesoderm specification and gastrulation movements when treated at stage 8. Embryos treated at stage 12.5 with SU5402 exhibited morphogenetic defects, but U0126-treated were unaffected.
Figure 2. Xtsproutys and Xtspreds Are Expressed in Similar Patterns, but at Slightly Different Times(A) Cartoon depiction of domain structures for each protein. Spry, conserved Sprouty domain; EVH-1, VASP homology domain; KBD, c-Kit binding domain.(B) In situ hybridizations for each gene. At gastrula stages (stage 10.5), Xtsproutys showed more extensive staining around the blastopore than Xtspreds (arrow). Neurula stages showed increased staining of Xtspreds and decreased Xtsproutys. Tail bud stages showed similar staining in branchial arches (br), otic vesicle (ov), and tail bud (tb), but Xtspred staining was absent in mid-brain/hind-brainisthmus (mhb) and forebrain (fb).(C) Quantitative real-time RT-PCR analysis of each gene’s expression over time. Relative expression levels showed Xtsproutys peak before stage 12.5 and then diminish, while Xtspreds levels increased after stage 12.5.
Figure 5. Loss of Xtsprouty and Xtspred Functions Causes Distinct Phenotypes(A) Cartoon depicting splice MO designs at either end of splice junctions.(B) RT-PCRs of MO-injected embryos. MO-injected lanes show bands either absent or shifted (arrowheads).(C and E) Embryos injected with MOs before first cleavage assayed for cardiac actin and FK506bp expression at stage 35 by in situ hybridization (purple). Embryos injected with 4-mis control MOs appeared identical to uninjected embryos (UI). Injection of combined Spry1 and Spry2 MOs (Spry 1+2) produced a truncated phenotype, while injection of Sprd1 and Sprd2 MOs (Sprd 1+2) produced a severe ventro-posteriorized phenotype.(D and F) Corresponding crosssections through trunks of embryos injected as above. Spry MO embryos appeared relatively normal in crosssection, but Sprd MO embryos showed disrupted somites and absence of a notochord (arrowhead).
