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Figure 2. Kermit2mut inhibits Xenopus gastrulation. (A) Control embryos injected with water close their blastopores and (D) assemble a dense FN matrix. (B) Embryos injected with kermit2 also gastrulate and (E) assemble FN matrix normally. (C) Embryos injected with kermit2mut mRNA fail to close the blastopore and (F) have a sparse FN matrix. (G) Xbra in situ hybridizations indicate normal mesodermal patterning in (G) control embryos, as well as (H) embryos expressing kermit2, and in embryos expressing (I) kermit2mut. Note that axial extension is inhibited in (I). (J) Western blot demonstrating equal expression of HA tagged kermit2 and kermit2 constructs. Embryos injected with water (cont) do not express the construct, while embryos injected with RNA encoding kermit2 (k2) and kermit2mut (k2mut) express equal amounts of either construct. Molecular mass markers are indicated on the right of the panel. (K) Kermit2mut expression does not inhibit FN protein accumulation. Western blots demonstrate that there is no substantial change in FN protein expression (FN) in water injected embryos (cont), embryos expressing kermit2 (k2), or embryos expressing kermit2mut (k2mut). Molecular mass markers are indicated on the right of the panel. (A) size marker = 200 mM. (D) size marker = 30 mM. (G) size marker 100 mM.
doi:10.1371/journal.pone.0010665.g002 |
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Figure 6. Inhibition of FN matrix assembly is not due to IGF signaling. (A, B) Control embryos close the blastopore by stage 12 and elaborate a dense FN matrix. (C, D) Embryos expressing a dominant negative IGFR-1 construct (dnIGFR-1) appear similar to control embryos and elaborate a dense FN matrix. (E) Xbra expression in control embryos. (F) Xbra patterning is not altered by blocking IGF signaling. There is a minor effect on axial extension that is clearly revealed in tadpoles (G) Control tadpoles (top) are longer than tadpoles resulting from embryos expressing dnIGFR-1 (middle). The dnIGFR-1 construct results in anterior defects including reduced or absent eyes (arrowhead). Tadpoles obtained from embryos that express kermit2mut show severe anterior truncations and mesodermal defects (bottom). (H) Western blots demonstrating inhibition of IGF signaling by the dnIGFR-1 construct. The phosphorylation of Akt (pAkt) seen in controls (cont) is not maintained in animal caps that express the dominant negative IGFR-1 construct (dnIGFR). Bottom panel shows total Akt expression in the same lysate. Molecular mass is indicated to the left of the panel.
doi:10.1371/journal.pone.0010665.g006 |
