Fitc labeling mix

WISH, FISH, and two color FISH were performed as previously described (Forbes-Osborne et al., 2013 (link), Pillai-Kastoori et al., 2014 (link)). Antisense RNA probes were prepared by in vitro transcription of linearized plasmids containing a portion of the coding sequence of the gene of interest using T7 polymerase and digoxigenin (DIG) or fluorescein (FITC) labeling mix (Roche Applied Science, Indianapolis, IN). The her4 and kaede containing plasmids were prepared by cloning PCR products into the pGEM-T-easy vector (Promega, Madison, WI). The sequences of all PCR primers used in this study are presented in Table S1. Images were obtained on an inverted fluorescent microscope (Eclipse Ti-U; Nikon Instruments), and were exported into Adobe Photoshop for figure preparation.

Xenopus embryos were obtained by in vitro fertilization and cultured in standard solution (MMR). RNAs and MOs were injected on the dorsal side (bilaterally) or animal side (both ventral blastomeres for BMP transcription, all 4 cells for anti-phospho-Smad1 Western blot). For rescue of anterior structures, apcdd1 MO (20ng) were injected alone or together with 1 ng Bmp2, Bmp4, Bmp7, admp MO in the marginal zone of both dorsal blastomeres at the 4-cell stage and collected at stage 34. At the specified stages, according to Nieuwkoop and Faber, embryos were fixed for in situ hybridization, or extracts prepared for transcription or protein gels. RNA for injection and in situ hybridization was prepared with mMessage Machine with SP6, T7 or T3 RNA polymerases (Ambion), and T3 or T7 polymerase (Roche), for in situ probes with digoxigenin or FITC labeling mix (Roche), respectively. Morpholino oligonucleotides against Xenopus BMPs were: admp MO, 5’ –GGTCCATCTCATCAAGCTGCAGCTC-3’(Reversade and De Robertis, 2005 (link)), Bmp2 MO, 5′-GATCCCAGCGACC ATTGTCAACCTG-3′; Bmp4 MO, 5′-CAGCATTCGGTTACCAGGAATCATG-3′; Bmp7 MO, 5′-TTACTGTCAAAGCATTCATTTTGTC-3′(Reversade et al., 2005 (link)), and were co-injected with apcdd1 MO at 1 ng /dorsal blastomere.