Hybridization-Chain Reaction (HCR) Fluorescent In Situ Hybridization (FISH).

For each gene, an HCR-FISH probe set targeting the coding sequence was custom-designed by Molecular Instruments with the number of probes varying between different sets. For each gene, the accession number, probe set size, amplifier/AlexaFluor dye used, and probe concentration are summarized in Supplementary Table 5. Because the sea lamprey embryo is highly opaque and auto-fluorescent, this HCR-FISH protocol needed to be adapted from the zebrafish HCRv3 FISH protocol⁶⁰ , with additional steps of bleaching and clearing of the tissue. First, sea lamprey embryos were gradually rehydrated at room temperature from 100% MeOH into 100% PBST (1X PBS + 0.1% Tween) in series of 5-minutes-long washes. Following this, embryos were then bleached using a freshly made solution (ingredients to be mixed in the following order: 5% Formamide, 0.5% SSC, 3% H₂O₂) then exposed under direct LED light (>10K Lumen) for 2h. This is followed by a few washes in 100% PBST. Embryos were kept at minimal density (about 5 embryos per tube) and were treated in 1ml of proteinase K solution (20μg/ml) for 8’, followed by a few washes in 100% PBST. Embryos were then re-fixed in 4% PFA for 20' (shaking). The detection and amplification stage follows the zebrafish HCRv3 protocol⁶⁰. The optimal probe input needs to be determined empirically for each probe set and target tissue and is further used to determine the volume of probe to be added to prepare a 2pmol probe/hybridization buffer solution (Supplementary Table 5).