Morpholino-Mediated Knockdown in Sea Urchin Embryos

The morpholino (Gene Tools, Philomath, OR, USA) sequences and the in-needle concentration with 24% glycerol were as follows:
Hbn-MO1 (0.7 mM): 5’- AAAATGAACGGAACAAGTCCAGTGT -3’,
Hbn-MO2 (2.0 mM): 5’- TAGGAGAACCAACGACCGCCGTCAT -3’,
Nodal-MO (0.2 mM): 5’- AGATCCGATGAACGATGCATGGTTA -3’,
Lefty-MO (0.4 mM): 5’- AGCACCGAGTGATAATTCCATATTG -3’,
FoxQ2-MO (0.2 mM): 5’- TCATGATGAAATGTTGGAACGAGAG -3’,
BMP2/4-MO (0.4 mM): 5’- GACCCCAATGTGAGGTGGTAACCAT -3’,
LRP6-MO1 (1.9 mM): 5’- GAAAGGTTTCAAGGCAGCCCATTTC -3’,
LRP6-MO2 (1.5 mM): 5’- TGCCGTTGACTAAATATCATCTACA -3’,
Wnt6-MO1 (3.8 mM): 5’- ACGTGTCCACTCCATCTTGTAATAC -3’,
Wnt6-MO2 (1.9 mM): 5’- TCGTCCAGCGATTTAATAAAGAGCT -3’,
Wnt7-MO1 (3.8 mM): 5’- ATAACCACACCAAgTTgggCCgCAT -3’, and
Wnt7-MO2 (1.9 mM): 5’- GCTCAGCGATGCCCGATGGATAAAA -3’.
Two non-overlapping morpholinos that blocked the translation of Hbn, LRP6, Wnt6 and Wnt7 were used to confirm the specificity of their function. For negative control experiments, we injected 24% glycerol into eggs.
mRNAs were synthesized from linearized plasmids using the mMessage mMachine kit (Thermo Fisher Scientific) and injected at the indicated concentrations in 24% glycerol in needles: hbn-mRNA (0.1 μg/μl), Δ-cadherin (0.3–0.6 μg/μl; [22 (link)]), and myc-mRNA (0.1 μg/μl). Microinjections into fertilized eggs and into one blastomere at the two-cell stage were performed as previously described [13 (link)].