Sense RNAs for microinjection were synthesized using the mMessage Machine kit (Ambion). cDNA plasmids were linearized and transcribed as follows: pCS105-Dse (BstX1, Sp6), pCS2-Dse* (NotI, Sp6), pCS2-nLacZ, (NotI, Sp6), pCS2-Flag-Sdc4 (NotI, Sp6; Muñoz et al., 2006) and pCMT-eGFP (NotI, Sp6; a kind gift from Eric Bellefroid, Université Libre de Bruxelles, Belgium). The Dse-MO (5′-GCTCCCCGAGTGTGAGTCCTCATTG-3′), Dse-5MM-MO (5′-GCTaCCCcAGTcTGAGTaCTaATTG-3′; lowercase letters represent mismatches), Dsel-MO (5′-ATGGTCCATTAGGAGAATAGTCAGT-3′) and standard control-MO (5′-CCTCTTACCTCAGTTACAATTTATA-3′) were obtained from Gene Tools LLC. Unless otherwise stated, mRNAs, DNAs and MOs were injected into the animal pole of all blastomeres at the two- or four-cell stage. A total of 45 ng MOs was injected per embryo. For single injections, a quarter of the MO amount with 125 pg nlacZ mRNA as a lineage tracer was used.
