Morpholino

For microinjection, we used injection buffer (24% glycerol, 20 mM HEPES pH 8.0 and 120 mM KCl). The morpholino (Gene Tools, Philomath, OR, USA) sequences are in the reagent table, and the in-needle concentration with injection buffer was 1.0 mM. Two nonoverlapping translation-blocking morpholinos for Opsin2 and ChAT were used to confirm the function specificity (S1 and S2 Figs). For negative control experiments, we injected a random MO or only injection buffer. The concentrations of MOs in the needles were as follows:
Opsin2-MO1 (0.4 mM), 5’-AGTTTGCCATCTTTGTGTTGCTTCG -3’
Opsin2-MO2 (0.4 mM), 5’-CGCCAATAACCACTGATCACAGTCG -3’
ChAT-MO1 (0.2 mM), 5’-ACGATTAGGCATGTGGTTCATGTAT -3’
ChAT-MO2 (1.0 mM), 5’-TGGAACGTCCAATAGTGGTATTGTA -3’
Gcm-MO, 5’-GCTTTGGACTAACCTTTTGCACCAT -3’ [34 (link)], and
Random-MO (1.0 mM).
Microinjections into fertilized eggs were performed as previously described [60 (link), 61 ]. After microinjection, the embryos were washed with FSW three times and stored with 50 μg/ml kanamycin until the desired stages were reached.

Microinjection was performed according to a previously described method [54 ] with injection buffer (24% glycerol, 20 mM HEPES pH 8.0 and 120 mM KCl). The morpholino (Gene Tools, Philomath, OR, USA) sequences and the in-needle concentrations in injection buffer were as follows:
Go-Opsin MO1 (0.8–1.0 mM): 5′-ATCTTCTTGAATATGCTTCCGCGCC-3′,
Go-Opsin MO2 (1.0–1.5 mM): 5′-ACGAATTCATTGTGGTCAAATCCGC-3′,
5HT₂ MO1 (0.5–1.0 mM): 5′-GGAAAGGAACATCTCAGATCGGCCT-3′,
5HT₂ MO2 (0.5 mM): 5′-GATGTCCTTATGGTATGTGCA-3′,
nNOS MO1 (1.0–1.5 mM): 5′-GGAAAGGAACATCTCAGATCGGCCT-3′ (previously characterized) [23 ], and
TPH MO (1.2 mM): 5′-ACAGAGTAGGACGTTGATGATCTAT-3′ (the specificity was checked by immunohistochemistry for serotonin (Additional file 1: Fig. S4D)).
Two non-overlapping translation-blocking morpholinos for Go-Opsin and 5HT₂ were used to confirm the specificity of their function (Additional file 1: Fig. S6b, 7c). For negative control experiments, we injected random MO (1.0–1.5 mM, Gene Tools, Additional file 1: Fig. S6a) or injection buffer only.
The DNA construct for the putative cis-regulatory element of 5HT₂ was prepared and injected as previously described [55 ]. Five thousand base-pairs of the genomic DNA of H. pulcherrimus were isolated and combined with a DNA sequence encoding Venus.

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)].

Microinjection was performed according to a previously described method (Yaguchi, 2019 (link)) with injection buffer (24% glycerol, 20 mM HEPES pH 8.0 and 120 mM KCl). The morpholino (Gene Tools, Philomath, OR, United States) sequences and the in-needle concentrations in injection buffer were as follows:
Rx-MO1 (1.9–3.8 mM): 5′- GGG​TGA​TGC​GCT​CCA​TCC​ATT​GTT​A -3′,
Rx-MO2 (1.0–1.9 mM): 5′- TTT​GTG​ACT​GAT​CGT​CTT​TCC​AAA​C -3′,
Msi1-MO1 (0.5–1.0 mM): 5′- AAC​CCT​CAA​CTA​AAA​AGG​CCC​AAT​A-3′,
Msi1-MO2 (1.9 mM): 5′- GAA​TTG​GCA​AAC​GGT​CCT​TCT​TAA​C-3′,
and Hbn-MO1 (0.7 mM): 5′- AAA​ATG​AAC​GGA​ACA​AGT​CCA​GTG​T -3’.
(previously characterized) (Yaguchi and Yaguchi, 2019 (link)).
Two non-overlapping translation-blocking morpholinos for Rx and Musashi were used to confirm the specificity of their function (Supplementary Figures S1, S2). For negative control experiments, we injected random MO (2.0 mM: Gene Tools, Supplementary Figure S1) or injection buffer only.