Cloning and Characterizing Wnt11b Variants

Full-length cDNAs for wnt11b.L were amplified from wild-type or wnt11b^−/− mutant oocyte total RNA using a high-fidelity polymerase (Q5, New England Biolabs). PCR products were cloned into pCR8/GW/TOPO (Invitrogen) and individual clones were verified by sequencing. Primer sequences are presented in Table S1. Desired clones in the (correct) 5′L1-3′L2 orientation were inserted via recombination into a pCS2+ Gateway-converted vector (Custom Vector Conversion Kit; Invitrogen). Template DNAs for sense transcripts were prepared from wnt11b/pcs2+ plasmids by NotI digestion. Capped messenger RNA was synthesised using SP6 mMessage mMachine kits (Ambion). Eb3-gfp/pcs2+ RNA was similarly prepared as previously described (Olson et al., 2015 (link)). Mouse Lrp6 in pβ/RN3P was used as previously described (Kofron et al., 2007 (link)), prepared by SfiI/T3 digestion and transcription. RNAs for zebrafish dvl2-mcherry, rab5-gfp and rab11-gfp were gifts from D. Slusarski (The University of Iowa, IA, USA).