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Additional Supporting Information may be found in the online version of this article.

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DVG_20825_sm_suppinfofig1.tif1533KSupplement Figure S1: ABP is disrupted in embryos injected with DNPar6. As expected from previous studies of DNaPKC (Chalmers et al., 2005), expression of DNPar6 decreased expression of the apical marker cytokeratin and increased expression of the basolateral marker β1-integrin. In all panels, arrows indicate normal and arrowheads abnormal expression.
DVG_20825_sm_suppinfofig2.tif24550KSupplement Figure S2: Expression of molecular constructs that disrupt either ABP or PCP alter LR patterning, regardless of which blastomere is targeted. A) Constructs were injected into one blastomere at the 4-cell stage. This localization was verified by expression of a lineage marker (β-gal) at the GRP, or absent from the GRP. Panels of st. 16-17 embryos show β-gal localization on the exterior neural folds (left panels) and specifically at the GRP on dissected dorsal explants (right panel). For all injections detailed in Table 2, β-gal localization was verified at st. 45 (examples shown). B) Injections into the left dorsal blastomere (red) target the GRP and contribute to LR-relevant flow, and injections into the right ventral blastomere (green) do not contribute to the GRP or LR-relevant flow. Blastomeres indicated by gray may make minor contributions to the GRP and/or LR-relevant flow. Regardless of which blastomere is targeted by these molecular constructs, significant levels of heterotaxia were observed. For all constructs, all blastomeres are significantly different from control embryos injected with β-gal alone (p<0.01). Significant differences were not observed when comparing the individual blastomeres pair-wise for each molecular treatment. Experiments for each blastomere and each construct include at least 50 embryos.
DVG_20825_sm_suppinfofig3.tif4291KSupplement Figure S3: Verification of molecular construct targeting in the conjoined twin model. A) In the first set of twin experiments, molecular constructs were injected at the 1-cell stage, and a secondary organizer was induced via injection of XSiamois at the 16-cell stage. Here, localization of the FITC-tagged Vangl2MO demonstrates that this molecular reagent was expressed throughout the developing twin. B) In the second set of twin experiments, molecular constructs were co-injected with XSiamois at the 16-cell stage. Here, localization of the FITC-tagged Vangl2MO demonstrates that this reagent was limited to a small number of cells in the induced twin. In all figures, yellow arrows indicate the original twin and blue arrows indicate the induced twin. For clarity, we have outlined the FITC signal in each twin with a white line.

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