Fish lines and embryo manipulation

Zebrafish embryos were generated by natural spawning, maintained under standard conditions, and staged according to Kimmel et al., 1995. The following lines were used: Tg(cldnb:lyn-egfp) RRID:ZFIN_ZDB-ALT-060919-2 (Haas and Gilmour, 2006), TgBAC(cxcr4b:lifeact-citrine) RRID:ZFIN ID: ZDB-ALT-160901–3 (Fuentes et al., 2016), TgBAC(cxcr4b:h2a-mcherry) RRID:ZFIN ID: ZDB-ALT-180131–1 (Colak-Champollion et al., 2019), Tg(cldnb:lyn-mscarlet), Tg(krt4:dsred) RRID:ZFIN ID: ZDB-ALT-120127–5 (O'Brien et al., 2012). For generation of Tg(cldnb:lyn-mscarlet) transgenic fish, the lyn-mscarlet (Bindels et al., 2017) DNA sequence was codon optimized for zebrafish expression (Horstick et al., 2015) and commercially synthesized. This fragment was cloned downstream of the 4.2 kb claudinb promoter fragment (Gerety et al., 2013), which drives expression in the lateral line primordium and periderm, among other tissues. This construct was cloned between sites for the Tol1 transposon (Koga et al., 2008) and 20 ng of plasmid DNA was injected with 80 ng of tol1 mRNA into one-cell stage zebrafish embryos. Founders were screened by fluorescence for high expression in the lateral line primordium.

For generation of chimeric PLLp, embryos were dechorionated at ~2 hpf and placed in embryo media with 100 U/mL penicillin and 0.1 mg/mL streptomycin (Roche). When the embryos had reached high-sphere stage (~3.5-4hpf), they were placed in individual wells made in agarose by a custom-printed mold. An Eppendorf CellTram Vario connected to a glass capillary needle with the tip removed at approximately the diameter of an embryonic cell was used to gently aspirate cells from the donor embryo and place them in the host embryo. After transplantation, embryos were placed in individual chambers of a 48-well plate in embryo medium with Penicillin and Streptomycin and grown overnight at 28°C. Embryos were screened at 24hpf for expression of the donor transgene in the PLLp.