Generation of a ror2-EGFP transgenic Xenopus line for tracking Ror2-expressing cells during limb and intestinal development.

During Xenopus metamorphosis, thyroid hormone (TH) induces extensive remodeling of the intestine including the development of adult epithelial stem cells (ASCs). Ror2 (receptor tyrosine kinase-like orphan receptor 2), a receptor involved in non-canonical Wnt signaling, is expressed in a limited population of larval epithelial cells that undergo dedifferentiation into ASCs. To examine when and how the fate of Ror2-expressing larval epithelial cells is specified toward the ASC lineage, we successfully generated a new transgenic Xenopus expressing EGFP under the control of the ror2 promoter (Ror2-EGFP Tg) to visualize Ror2-expressing cells. This was achieved using the recently developed NEXTrans method, a CRISPR-Cas9-based targeted transgenesis system for Xenopus laevis. We found that 19.4% (13/67) of F0 tadpoles expressed DsRed1, a transgenic marker, in one or both eyes. Efficient germline transmission (approximately 30%-70%) was achieved in F1 Tg tadpoles. Interestingly, strong EGFP signals were observed in the limb buds and were subsequently detected in Col2A1-positive chondrocytes at stage 54, when all five toes had developed in the hindlimb. Co-expression of endogenous ror2 and transgene-derived EGFP was confirmed in the hindlimb buds by hybridization chain reaction (HCR). This co-expression was also confirmed in the intestinal epithelium as early as premetamorphic stage 54. This new transgenic line provides a valuable tool for tracking Ror2-expressing cells during limb development and intestinal stem cell formation in Xenopus.