Standard mo

In vitro-transcribed RNA of wild type Tnfa and DN Tnfr2 [15 (link)] was obtained following manufacturer’s instructions (mMESSAGE mMACHINE kit, Ambion). Morpholinos were diluted in DEPC-treated water at a concentration of 0.3 mM (Standard-mo, Gene Tools) 0.5 mM (Tnfa-MO, 5’-GCAGGATTTTCACCTTATGGAGCGT-3’ [42 (link)], 0.65 mM (Tnfr1-mo, 5’-ctgcattgtgacttacttatcgcac-3’ [15 (link)], 0.3 mM (Tnfr2-mo, 5’-ggaatctgtgaacacaaagggacaa-3’ [15 (link)]. Morpholinos and RNA were mixed in microinjection buffer and microinjected into the yolk sac of one-cell-stage embryos using a microinjector (Narishige) (0.5–1 nl per embryo). The same amount of MOs and/or RNA were used in all experimental groups. The efficiency of the MOs was checked by RT-PCR [15 (link),42 (link)].

To suppress Hr-Brachyury (Bra) function, we injected about 1000 pg of a 25-mer Bra morpholino oligonucleotide (MO; Gene Tools), covering the first methionine, into fertilized eggs. The nucleotide sequence was 5′-TTGTAATTGACATAATTCCTTGTAC-3′[7] (link), [19] (link). As a control, a standard MO (Gene Tools) was injected.
Hr-Tbx6[14] (link) and Hr-CKI-b ORFs were cloned into the RN3EX vector (RN3 vector [20] (link) with an extra XhoI site). mRNAs flanked by Xenopus globin UTRs were synthesized from each of the plasmids using the mMessage mMachine kit and poly (A) tailing kit (Ambion). 0.1 µg/µl of Hr-Tbx6 and 0.3 µg/µl of Hr-CKI-b mRNA solutions were injected into fertilized eggs. mCherry:Tbx6 mRNA was synthesized from the RN3EX vector with an insert in which the mCherry coding sequence was inserted in-frame into the 5′ end of the Tbx6 ORF. The synthesized mRNA had the original Tbx6 UTRs; 0.1 µg/µl of the mRNA solutions was injected. To visualize the descendant cells of the mesenchyme precursors, mRNA encoding histone H2B protein (from the appendicularian Oikopleura dioica) fused with mCherry fluorescent protein was synthesized from the plasmid pSD64TF carrying the ORF (kindly provided by Dr. A. Nishino, Hirosaki University).