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Figure 2. Sizzled Is a Feedback Inhibitor of BMP Signaling that Requires Xlr Activity(A) Endogenous Smad1 phosphorylation at early gastrula is increased by Szl MO.(B) An autoregulatory negative feedback loop is interrupted in Szl MO embryos, resulting in ventro-posterior accumulation of Szl transcripts.(C) Szl upregulation is mediated by BMP4 signaling.(D) Chd protein, but not Chd mRNA levels, are decreased by Szl knockdown (compare lanes 1 and 2). BMP2/4/7 MO injection serves as negative control.(E and F) Xlr mRNA overexpression or Chd knockdown increase ventral Szl transcripts, indicating increased BMP signaling.(G) Szl-Fc protein injection into the blastula cavity suppresses the pro-BMP effects of Xlr mRNA.(H) Szl-Fc protein has no effect in Chd-depleted embryos. Inset shows phenotype of Szl-Fc injection.(I) The role of Xlr in the regulation of Chordin anti-BMP activity. Tsg (Twisted gastrulation) is a cofactor of Chordin.(J) Microinjection of DN-Xlr mRNA expands Otx2 expression, a sign of dorsalization.(K) Szl MO feedback loop requires endogenous Xlr function since it is inhibited by DN-Xlr mRNA; compare with inset showing sibling embryo injected with Szl MO alone.All microinjection results were performed at least three times and minimum of 15 embryos were analyzed by hybridization in each sample.(L) Schematic diagram of the proposed BMP negative feedback loop via Sizzled, in which Szl inhibits Xlr activity. |
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Figure 4. The SzlD92N Point Mutation Mimicking Zebrafish ogon Inhibits Xlr Binding and Biological Activity(A) Diagram of Szl-Fc and SzlD92N/Ogon-Fc constructs.(B) BIAcore measurements of binding of 10 μg/ml Xlr in the flow to wild-type Szl-Fc or SzlD92N/Ogon-Fc immobilized on the chip. Note the ten-fold decrease in binding affinity to Xlr.(C–H) Microinjection of 40 nl of 25 μM wild-type Szl-Fc protein into the blastula cavity dorsalizes the Xenopus embryo (E) and rescues the Szl knockdown phenotype (F). Injection of same concentration of SzlD92N/Ogon-Fc protein had no obvious phenotypic effect on wild-type (G) or Szl MO (H) embryos. |
