Aplnra/b Sequentially Regulate Organ Left-Right Patterning via Distinct Mechanisms

Abstract

The G protein-coupled receptor APJ/Aplnr has been widely reported to be involved in heart and vascular development and disease, but whether it contributes to organ left-right patterning is largely unknown. Here, we show that in zebrafish, aplnra/b coordinates organ LR patterning in an apela/apln ligand-dependent manner using distinct mechanisms at different stages. During gastrulation and early somitogenesis, aplnra/b loss of function results in heart and liver LR asymmetry defects, accompanied by disturbed KV/cilia morphogenesis and disrupted left-sided Nodal/spaw expression in the LPM. In this process, only aplnra loss of function results in KV/cilia morphogenesis defect. In addition, only apela works as the early endogenous ligand to regulate KV morphogenesis, which then contributes to left-sided Nodal/spaw expression and subsequent organ LR patterning. The aplnra-apela cascade regulates KV morphogenesis by enhancing the expression of foxj1a, but not fgf8 or dnh9, during KV development. At the late somite stage, both aplnra and aplnrb contribute to the expression of lft1 in the trunk midline but do not regulate KV formation, and this role is possibly mediated by both endogenous ligands, apela and apln. In conclusion, our study is the first to identify a role for aplnra/b and their endogenous ligands apela/apln in LR patterning, and it clarifies the distinct roles of aplnra-apela and aplnra/b-apela/apln in orchestrating organ LR patterning.

Keywords: apela/apln; aplnra/b; left right patterning; midline; spaw.

Figure 1

Organ left-right lateral defect in embryos treated with different ways. (A-D, O) The pattern of heart displayed in controls and aplnra/b morphants. Compared with controls (A, 98.6%, n=72), embryos injected with aplnra MO displayed normal-loop (B, 62.5%, n=112; p<0.01), reversed-loop (C, 17.8%, n=112; p<0.01) and no loop (D, 19.6%, n=112; p<0.01). (O, column 1 to 4) The cartogram of heart LR defect for embryos treated with different MOs or mRNA. Only more than 18.2% of aplnrb morphants displayed heart LR defect (O, column 4, n=115; p<0.05). Aplnra mRNA injection (O, column 3, 26.8%, n=71; p<0.05) partially rescued the heart LR defect in aplnra morphants (O, column 2, 37.5%, n=112). (E-J, P). While 97.3% of control embryos showed left-sided expression of cp (E, n=112), 18.2.0% (F, n=176, p<0.01), 9.1% (G, n=176, p<0.05) and 10.8% (P, n=176, p<0.05) of aplnra morphants showed both sided and right-sided expression of cp, or disappear. (H-J, P). Compared with that in aplnra morphants, aplnra mRNA partially rescued liver LR defect (24%, n=121, p<0.05). In aplnra/b double morphants, the expression of cp was greatly downregulated (100%, n=195, p<0.001), and more embryos displayed liver LR defect (H-J, P, 51.3%, n=195, p<0.05) than that in aplnra morphants (P, 38.1%, n=176). In embryos injected with aplnra sgRNAs and Cas9 protein, 15.8%; and 6.4% of them displayed right sided and both sided/middle liver (P, n=233, p<0.05). (K-N, Q). otx5 was expressed in the middle telencephalon. All the embryos injected with Cont MO displayed the expression of otx5 in the middle telencephalon (K, 100%, n=32, and Q, column 1).While 61.7% of embryos injected with aplnrb MO showed both-sided otx5 in telencephalon (M, Q, n=34), only 5.5% of aplnra morphants displayed both-sided otx5 (Q, 37). For observation, heart, ventral view; cp, spaw and otx5, dorsal view.

Figure 2

Expression of left-sided Nodal signaling in LPM. (A. a1-a4, B) Expression of left-sided spaw in embryos. In control embryos, near all the embryos expressed left-sided spaw (A. a1, 91.4%, n=93). While in aplnra and aplnrb morphants, left-sided spaw expression pattern was changed (A. a2-a4, B), 13.6% (p>0.05), 7.8% and 47.6% (p<0.001) of aplnra morphants expressed right-sided, both-sided spaw or spaw disappeared (B, middle-left column, n=103). 10.2 % (p<0.05) and 6.1% (p>0.05) of aplnrb morphants showed right-sided and both sided spaw (B, middle-right column, n=98), majority of aplnrb morphants showed no staining for spaw (B, middle-right column, n=98, p<0.001). (C. c1-c4, D) The expression of left-sided lft2 in the heart progenitor field. In the control embryos, lft2 was expressed in the left side of heart progenitor field (C.c1, 94.2%, n=52). Lft2 was down-regulated in aplnra morphants (C. c2-c4), the left-sided expression pattern was also perturbed (C. c2-c4, D), with 9.0%, 6.3% and 52.5% of embryos showing right-sided, both-sided lft2 and lft2 disappeared, respectively (C. c2-c4; D, middle column, n=74). The lft2 expression pattern in aplnrb morphants (D, right column, n=93) was similar to that in aplnra morphants (D, middle column, n=74), but more embryos had no staining for lft2 in aplnrb morphants (D, right column, n=93) than in aplnra morphants (D, middle column, n=74). All the embryos were observed from dorsal view. L, Left; R, Right.

Figure 3

KV morphogenesis and Ciliogenesis in treated embryos. (A-D) In control, 91.3% of embryos showed normal KV (A, n=181), but 35.6% (p<0.001), 54.2% (p<0.001) and 10.2% (p<0.001) of embryos injected with aplnra MO showed normal KV, smaller KV and no KV, respectively (B, C and D. column2, n=175), this kind of KV phenotype also can be found in aplnra+b morphants (D. column 4, n=213, p<0.001) or apela morphants (D. column 7, n=208, p<0.001), embryos injected with apela mRNA (D. column 8, n=153, p<0.001) or embryos injected with apln mRNA (D. column 6, n=135, p<0.001) . In aplnrb or apln morphants, no distinct phenotype about KV was discovered (D. column 3(n=147) and column 5, n=193). Injection of aplnra mRNA partially restored the KV phenotype in aplnra morphants (D. column 9, n=116, p<0.05). (E-I) In the KV, compared with that in control morphants (E and H, n=25), the cilia number is decreased in aplnra morphants (F and H, n=28, p<0.01), difference was also found about the length of cilia (F, I, n=12, p<0.05). In aplnrb morphants, only mild difference about cilia number and cilia length was observed (G, H, I, n=9, p>0.05). Co-injection of aplnra mRNA with aplnra MO in the embryos resulted that the number (G, H, n=26, p<0.05) or length of cilia (G, I, n=14, p<0.05) was closed to that in control. *, p<0.05; **, p<0.01; NS, not significant.

Figure 4

Expression of sox17 and foxj1a. (A-E) Expression of sox17 in DFCs at 90% epiboly. In control embryos, 89.5% of embryos showed normal expression (A, n=88). In aplnra morphants, three kinds of expression pattern were discovered: mild decreased (B, 52.4%, n=89, p<0.001), scattered (C, 21.3%, n=89, p<0.001) and decreased (D, 19.6%, n=89, p<0.01). The area of sox17 expression was measured and in control embryos, the average level was 3.68x10³ uM² (E. e1 group, n=19), the average area in aplnra morphants was 2.55x10³ uM² (E. e2 group, n=22, p<0.05). Sox17 expression areas were also measured for aplnrb morphants, aplnra+b morphants, apela morphants, embryos injected with apln mRNA and embryos injected with apln mRNA, respectively (E, e3-e7 group). The average areas were 3.63x10³ uM² (E, e3 group, aplnrb morphants, n=15, p>0.05), 2.53x10³ uM² (E. e7 group, aplnra+b morphants, n=15), 3.28x10³ uM² (E, e4 group, apela morphants, n=15, p<0.05), 2.46x10³ uM² (E, e5 group, apln mRNA, n=15, p<0.01) and 2.64x10³ uM² (E, e6 group, apela mRNA, n=15, p<0.001), these data indicated sox17 expression was down reglated in all these treated embryos, excepted for aplnrb morphants. (F-J) Analysis of foxj1a expression. Foxj1a was expressed in DFCs at 90% epiboly in control (F) and treated embryos (G, H). Compared with control embryos (F, 80.5%, n=82), foxj1a was greatly down regulated in aplnra morphants (H-I, 68.9%, n=87, p<0.001) as well in aplnra+b morphants (I, 65.7%, n=76, p<0.001), but not in aplnrb morphants (I, 18.9%, n=74, p>0.05). Foxj1a expression area in control embryos, aplnra morphants, aplnrb morphants and aplnra+b morphants were average 3.75 x10³ uM². 2.56 x10³ uM², 3.57 x10³ uM² and 2.39 x10³ uM² respectively (I). Compared with that in control embryos, q-PCR experiment showed the quantity of foxj1a expression in aplnra morphants and aplnra+b morphants were down-regulated with 0.48 folds and 0.46 folds respectively, while the expression of foxj1a in aplnrb morphants was not affected (J). *, p<0.05; **, p<0.01; ***, p<0.001; NS, not significant.

Figure 5

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.

Figure 6

KV morphogenesis and left-sided Nodal signaling in apela morphants. (A-B) In control, 91.2% of embryos showed normal KV (A, n=136), but 37.6%, 53.3% and 9.1% of embryos injected with apela MO showed normal KV, mild smaller KV and no KV, respectively (B, C and D. column2, n=178). (C. c1-c5, D) Majority of control morphants expressed left-sided spaw in the LPM (C. c1, D left column, 91.4%, n=35). In apela MO morphants, 31.3%, 13.9% and 16.3% of embryos showed left-sided spaw, right-sided spaw and both-sided spaw (C. c2-c4, D middle column, n=30), while the spaw expression in most of apela morphants couldn't be found (C. c5, D middle column, 38.9%, n=86). (E-F) The Nodal downstream gene lft2 was checked. Compared with that in control embryos (E. e1, F right column, 94.2%, n=52), lft2 was down regulated and the left-sided expression pattern was disturbed in apela morphants (E. e2-e4, F middle column, n=39), displaying left-sided (17.9%), right-sided (10.25%), both-sided (5.12%) expression and the expression disappeared (66.7%). The disturbed KV morphogenesis and the perturbed spaw or lft2 expression were also observed in apln morphants (D, D, F, right column).

Figure 7

KV/cilia related signaling pathway analysis in apela morphants. (A-C) Compared with the control embryos (A. a1, B. b1 and C. c1), the expression of fgf8 and the downstream gene dnh9 were not changed in apela morphants (A. a2, 84.4%, n=32; C. c2, 65.6%, n=32), but the expression of erm was up regulated in apela morphants (B. b2, 85%, n=20). (E-D) Foxj1a was expressed in DFCs. Compared with that in control embryos (E. e1, 82.8%, n=35), foxj1a was down regulated in apela morphants (E. e3, 63.5%, n=104). The average area of foxj1a expression in control embryos and apela morphants were 3.87 x10³ uM² (n=21) and 2.75 x10³ uM², respectively (D, n=21, p<0.01). Q-PCR experiment indicated that foxj1a expression was down regulated with 0.61 folds compared with that in control embryos (F). *, p<0.05; **, p<0.01.

Figure 8

Expression of midline related genes in different treated embryos. (A. a1-a6) Shh expression at 24SS. Compared with that in control embryos (A. a1, 100%, n=31), no clearly decreased or increased expression of shh in midline was found in apela morphants (A. a2, 82.7%, n=29), aplnra+b double morphants (A. a4, 78.2%, n=23), aplnra morphants (A. a5, 85.1%, n=27) and aplnrb morphants (A. a6, 86.4%, n=22). In apln morphants, the expression of shh was slightly increased (A. a3, 67.9%, n=28). (B. b1-b6, C) Expression of lft1 at 20SS. In wild type embryos lft1 was expressed in 4 domains, including left telencephalon, left heart field, trunk midline and tail midline (B. b1, black arrow head, n=45), and in dorsal view, lft1 was found to be expressed in left telencephalon, left heart field, trunk midline (B. b2, black arrow head). In apln morphants, lft1 expression was found to be decreased with different phenotypes (B. b3-b6, n=85). Among all the alpn morphants, lft1 in midline was disappeared or decreased greatly in more than half of embryos (B. b4-b6, class 3 to class 5; C, the second column, 64.7%, n=85, p<0.01). The expression pattern of lft1 in apln morphants was also found in apela morphants (C, column 3, 58.2%, n=79, p<0.01), aplnra morphants (C, column 4, 60%, n=75, p<0.01) and aplnrb morphants (C, column 5, 56.2%, n=89, p<0.01), near or more than half of embryos showed greatly decreased lft1 in the trunk midline.