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. 2018 Nov 20;25(8):1997–2007.e7. doi: 10.1016/j.celrep.2018.10.072

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© 2018 The Author(s)

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

lrrc10 Is Required for Heart Regeneration in Zebrafish

(A) RNAScope RNA expression analysis shows that lrrc10 is expressed specifically in all the MF20-positive myocardium at 7 dpa in surface fish and Pachón as well as in zebrafish at 7dpi. Arrows point to absent expression in non-myocardial tissues, such as the wound, valves, and bulbus arteriosus. Expression around the wound is higher in surface fish and zebrafish compared with Pachón (arrowheads).

(B) lrrc10 is especially higher expressed in the compact myocardium (arrowheads) compared with the trabecular myocardium close to the wound (demarcated with line) in surface fish and zebrafish. This difference is absent in Pachón.

(C) Zebrafish lrrc10 mutants 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-mount in situ hybridization using an lrrc10 antisense probe in 3 dpf (days post fertilization) lrrc10^−/− (n = 12) and wild-type embryos (n = 25) shows that lrrc10 expression is specifically present in the wild-type heart (arrow) but absent in the mutant, confirming knockout of lrrc10.

(F) AFOG staining showing large blue collagen scar in the lrrc10 mutant compared with the wild-type control at 60 dpi.

(G) Wound size at 60 dpi in lrrc10^−/− (n = 7) and wild-type controls (n = 7), unpaired two-tailed t test.

(H) PCNA/Mef2 staining on lrrc10^−/− and controls at 7dpi.

(I) No significant difference in the number of PCNA/Mef2-positive cells surrounding the wound in lrrc10^−/− (n = 5) compared with wild-type hearts (n = 6) at 7 dpi. Unpaired two-tailed t test, equal variance.

(J) PCNA/Mef2 staining on lrrc10^−/− 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 in lrrc10^−/− (n = 5) compared with wild-type hearts (n = 6) at 7 dpi. Unpaired two-tailed t test, equal variance.

All scale bars, 100 μm. BA, bulbus arteriosus; CW, compact wall; Lu, lumen; Va, valves. Results are presented as mean ± SEM.