Organ LR asymmetry defect in embryos with aplnr ligands loss or gain of function. (A, G) Majority of control embryos expressed left-sided liver marker cp in day 3 (A. a1, G. column 1, 90.2%, n=51). Liver LR asymmetry was disturbed in apela MO injected embryos (A. a2-a3, G. column 2, 30.6%, n=121, p<0.01) and apela mRNA injected embryos (G. column 3, 34.9%, n=103, p<0.01). In transgenic line Tg(fabp10:GFP), the liver LR defect was also observed, displayed left-sided and right-sided liver in 76.5% and 23.5% of apela morphants, respectively (B. b2 and b3, n=51), but the GFP in liver region started to express in day 5 (B. b2 and b3). (C) Head marker otx5 was expressed in the middle telencephalon (C. c1, H. column 1, 100%, n=32), but many of apela morphants (C. c2, H. column 2, 65.4%, n=26) and aplnrb morphants (H. column 3 61.7%, n=34) showed both-sided otx5 in telencephalon. (D, I) Apln loss of function resulted heart LR asymmetry defect (D. d2-d4, I), displayed reversed loop (D. d4, I, 24.7%, n=117, p<0.05), normal loop (D. d2, I, 58.9%, n=117, p<0.01) and linear heart (D. d3, I, 16.2%, n=117, p<0.05). (E. e1-e3, G) In apln morphants, liver development was not delayed (E), but 25.4% liver LR asymmetry defect was found (E. e2-e3, G. column 4, n=185, p<0.001). Apela mRNA or apln mRNA gain of function also resulted in liver LR asymmetry defect, 34.9% (p<0.01) and 37.2% (p<0.01) of embryos displayed right-sided or both-sided expression of cp (G. column 5, n=97). When compared with the liver LR defect in apela morphants (G. column 2, 30.6%, n=121), high ratio of embryos co-injected with apela MO and aplnra MO (G. column 6, 37.1%, n=70), or embryos co-injected with apela MO and aplnrb MO (G. column 7, 40.0%, n=91) showed liver LR asymmetry defect. In embryos injected with apela/apln sgRNAs with Cas9 protein, the livers also showed left right asymmetry defect (G. column 8 and 9). L, left; R, right; V, ventral view; D3, day 3; D5, day 5.
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