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An early step in establishing left-right (LR) symmetry in zebrafish is the generation of asymmetric fluid flow by Kupffer's vesicle (KV). As a result of fluid flow, a signal is generated and propagated from the KV to the leftlateral plate mesoderm, activating a transcriptional response of Nodal expression in the leftlateral plate mesoderm (LPM). The mechanisms and molecules that aid in this transfer of information from the KV to the leftLPM are still not clear. Here we provide several lines of evidence demonstrating a role for a member of the TGFβ family member, Dvr1, a zebrafish Vg1 ortholog. Dvr1 is expressed bilaterally between the KV and the LPM. Knockdown of Dvr1 by morpholino causes dramatically reduced or absent expression of southpaw (spaw, a Nodal homolog), in LPM, and corresponding loss of downstream Lefty (lft1 and lft) expression, and aberrant brain and heart LR patterning. Dvr1 morphant embryos have normal KV morphology and function, normal expression of southpaw (spaw) and charon (cha) in the peri-KV region and normal expression of a variety of LPM markers in LPM. Additionally, Dvr1 knockdown does not alter the capability of LPM to respond to signals that initiate and propagate spaw expression. Co-injection experiments in Xenopus and zebrafish indicate that Dvr1 and Spaw can enhance each other's ability to activate the Nodal response pathway and co-immunoprecipitation experiments reveal differential relationships among activators and inhibitors in this pathway. These results indicate that Dvr1 is responsible for enabling the transfer of a left-right signal from KV to the LPM.
Fig. 5. Dvr1 and Spaw mutually enhance the activation of the Nodal response pathway in Xenopus. (A) Method of injection of mRNAs for Xenopus animal cap assay. D is dorsal and V is ventral. Embryos were evaluated for ectopic xbra expression at stage 10.5. The black arrow on the diagram indicates ectopic xbra expression and the red arrowhead indicates endogenous xbra expression. (B)E) Evaluation of ectopic xbra expression in embryos injected with (B) control GFP mRNA, (C) dvr1 mRNA, (D) spaw mRNA and (E) dvr1 and spaw mRNA. (F) Quantification of the area of ectopic xbra expression in dvr1 (n=53; 17/53 had no ectopic expression), spaw (n=51; 19/51 had no ectopic expression) and dvr1+spaw (n=76) mRNA injected embryos. Results are averages from 4 different batches of embryos, and include embryos that exhibited no ectopic xbra expressionfor. Error bars: s.e.m.
Fig. 6. Spaw and Dvr1 do not Co-Immunoprecipitate. (A) Scheme of protein co-precipitation assay. Xenopus embryos were injected with mRNAs at 2 cell stage and collected at stage 10. Cleared lysates were loaded directly onto gels (nput or in parallel were used for immunoprecipitation by anti-myc or anti-Flag epitopes, which were then loaded on gels and processed for Western Blot analysis. (B) Western result with anti-HA antibody shows that there is a strong interaction between Spaw and Charon, but not Spaw and Dvr1. Anti-FLAG IP was used as a control for IP.
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