Knockdown experiments

We tested one antisense morpholino oligonucleotide (MO) each for prork1a and prokr1b (Supplementary Table ^S3). Both were splice-blocking MOs synthesised by Gene Tools LLC (Oregon, USA). Morpholinos were dissolved in Danieau’s solution (58 mMNaCl; 0,7 mMKCl; 0,4 mM MgSO₄. H₂O; 0,6 mMCa(NO₃)₂; 5 mMHepes pH 7.2) at 2 mM and stored at −80 °C. Embryos were microinjected at the 1–4 cell stage with rhodamine dextran (Molecular Probes) co-injected as a tracer. As a control for non-specific effects, a standard control morpholino (ctrl-MO) was injected, which targets the human β-globin gene. Morpholinos were tested for efficacy and toxicity by injecting different doses in tg(gnrh3:EGFP) embryos and evaluating them for morphological defects (Supplementary Fig. ^S1A-C). After injection, embryos were raised in fish water at 28 °C and observed until the developmental stage of interest. Embryos that were to be imaged after 24 hpf were treated with PTU. For imaging, embryos were anaesthetized using tricaine (ethyl 3-aminobenzoate methanesulfonate salt, Sigma; 25x stock = 0.08 g in 20 ml of distilled H₂O) in fish water. Injected embryos (morphants) were embedded at 48 hpf in UltraPure Low Melting Point Agarose (Thermo Fisher Scientific) and photographed using a confocal laser scanning microscope (Nikon C2) with a 20x objective.