Live WT zebrafish was anesthetized and staged following the adult normal table.21 (link) Seven stages were chosen to capture major processes during the entire transition from larva to juvenile (see Table S2), and fish were staged by the SSL convention.21 (link) Samples were prepared following McMenamin et al.12 (link); briefly, staged fish were pooled, euthanized by an overdose of tricaine, blotted dry, and weighed in microcentrifuge tubes. Tissue was suspended in cold methanol with 1 mM 6-propyl-2-thiouracil (PTU; Sigma) and homogenized by sonication; homogenized samples were stored at –80°C until analysis. Samples were centrifuged, re-extracted with additional PTU-containing methanol. Supernatants were pooled and dried with a CentriVap Concentrator (Labconco) for ~5 hours, then resuspended in PBS. Samples were run on T4 and T3 ELISA kits (Diagnostic Automation Inc.) with two technical replicates and with three biological replicates for each stage. Plates were analyzed on a PerkinElmer Victor X5; mean absorbance value from three readings was used for subsequent analysis. Standard curves were generated with four-parameter logistic regression via online analysis software (www.elisaanalysis.com).