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Fig. 4 lrrc10Is Required for Heart Regeneration in Zebrafish(A) RNAScope RNA expression analysis shows thatlrrc10is expressed specifically in all the MF20-positive myocardium at 7 dpa in surface fish and Pacho ́naswell as in zebrafish at 7dpi. Arrows point to absent expression in non-myocardial tissues, such as the wound, valves, and bulbus arteriosus. Expression aroundthe wound is higher in surface fish and zebrafish compared with Pacho ́n (arrowheads).(B)lrrc10is especially higher expressed in the compact myocardium (arrowheads) compared with the trabecular myocardium close to the wound (demarcatedwith line) in surface fish and zebrafish. This difference is absent in Pacho ́n.(C) Zebrafishlrrc10mutants were generated by removing 601 nucleotides, including the ATG start site using CRISPR/Cas9.(D) Genotyping with primers just outside the deleted region amplifies a product of 833 bp in wild-types and a product of 232 bp in mutants.(E) Whole-mountin situhybridization using anlrrc10antisense probe in 3 dpf (days post fertilization)lrrc10/(n = 12) and wild-type embryos (n = 25) shows thatlrrc10expression is specifically present in the wild-type heart (arrow) but absent in the mutant, confirming knockout oflrrc10.(F) AFOG staining showing large blue collagen scar in thelrrc10mutant compared with the wild-type control at 60 dpi.(G) Wound size at 60 dpi inlrrc10/(n = 7) and wild-type controls (n = 7), unpaired two-tailed t test.(H) PCNA/Mef2 staining onlrrc10/and controls at 7dpi.(I) No significant difference in the number of PCNA/Mef2-positive cells surrounding the wound inlrrc10/(n = 5) compared with wild-type hearts (n = 6) at 7 dpi.Unpaired two-tailed t test, equal variance.(J) PCNA/Mef2 staining onlrrc10/and controls at 7dpi at the base of the ventricle.(K) Increased number of PCNA/Mef2-positive cells on the other side of the ventricle inlrrc10/(n = 5) compared with wild-type hearts (n = 6) at 7 dpi. Unpairedtwo-tailed t test, equal variance.All scale bars, 100mm. BA, bulbus arteriosus; CW, compact wall; Lu, lumen; Va, valves. Results are presented as mean±SEM.