Abstract
Tyrosinase, encoded by Tyr, is a key rate-limiting enzyme in melanin biosynthesis. Knockout of Tyr results in a distinct albino phenotype, making it a widely used target for evaluating gene-editing efficiency. Here, we found that the tyrosinase-deficient skin melanophore lineage of Xenopus tropicalis (X. tropicalis) tadpoles shows strong autofluorescence under the GFP filter, which may interfere with in vivo fluorescence imaging. Through spectral scanning analysis, we characterized the emission spectrum of the autofluorescence under commonly used excitation wavelengths for fluorescent proteins. Based on this, we established a reference protocol for identifying and excluding such interference in Tyr-targeted knockin studies. Furthermore, knockout of the GTP cyclohydrolase 2 gene (Gch2) using CRISPR-Cas9 significantly reduced the fluorescence intensity induced by tyrosinase deficiency, indicating an essential role of the enzyme and its mediated pterine biosynthesis in the generation of the autofluorescence. This study systematically characterized these fluorescent mutant melanophores in X. tropicalis tadpoles, providing a practical basis for avoiding fluorescent interference in experimental science and a new perspective on pigment cell development and evolution.