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Figure 6. An Essential Role of Daam1 in Gastrulation and in the Wnt/Rho Signaling Pathway(A) N-Daam1 or XDaam1 MO inhibits gastrulation. RNA (2 ng) for N-Daam1 or Xdd1 (for comparison) or Xdaam1 MO (40 ng) was injected dorsally at 4-cell stage (st). XDaam1 MO effect was rescued by coinjection with human Daam1 (2 ng), but not β-gal (2 ng) RNA.(B) Embryos injected dorsally with N-Daam1 or Xdd1 RNA. N-Daam1 injected embryos show normal expression of mesodermal Xbra, and dorsal Gsc and Otx2 at st 10.5, but exhibit an Xbra expression surrounding a large-sized blastopore and missing from the involuting notochord at st 12. Gsc expression in control embryos at st 13 is observed in anterior mesoendoderm far from the closed blastopore (arrowheads), but remains in a dorsal region close to the open blastopore in N-Daam1 injected embryo. Otx-2 is expressed anteriorly in both mesodermal and overlying neural tissues in control embyos at st 13. In N-Daam1 injected embryos, two separate Otx-2 expression domains are obvious. A bisection shows that the dorsal margin expression near the blastopore reflects the anterior mesoderm that fails to involute, and the other is in neuroectoderm. Sox-2 is expressed in the neural plate at st 14. In N-Daam1 injected embryos, Sox-2 expression is seen in a broad dorsal region that surrounds the open blastopore and lacks neural plate morphology. Finally, mAb 12/101, which is muscle-specific, detects normal amount of muscle formation along a severe shortened/bent body axis in N-Daam1 injected embryos. N-Daam1 phenocopies Xdd1 in all cases.(C) A model for the Wnt/Rho signaling pathway (see Discussion). |
