Whole-mount in situ hybridization for zrsr2

To generate a template for zrsr2 riboprobe synthesis for WISH, we performed RT-PCR using RNA from 5 dpf WT embryos and primers from the region shared by both isoforms (Supplementary Table S1). The RT-PCR product was cloned into a pCR4-TOPO vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequence-verified by Sanger sequencing. The plasmid DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen, Germantown, MD, USA), digested with SpeI, and labeled with a DIG labeling kit (Sigma-Aldrich, St. Louis, MO, USA) using T7 polymerase. Antisense riboprobes for all other WISH markers used in this study were also generated using a DIG labeling kit (Sigma-Aldrich, St. Louis, MO, USA). Zrsr2^hg129/+ fish were in-crossed, and embryos were collected at various developmental stages. Collected embryos were euthanized and fixed overnight in 4% PFA at 4 °C. WISH was performed as described previously [44 (link)]. For o-dianisidine staining, euthanized embryos were incubated in o-dianisidine (MP Biomedicals, Costa Mesa, CA, USA) solution for 15 min in the dark [45 (link)], followed by fixation in 4% PFA prior to imaging. Embryos used for WISH and o-dianisidine staining were genotyped via fluorescent PCR, as described previously [41 (link)], using REDExtract-N-Amp PCR ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) for product amplification.