Retinoic Acid Signaling Sequentially Controls Visceral and Heart Laterality in Zebrafish

Abstract

During zebrafish development, the left-right (LR) asymmetric signals are first established around the Kupffer vesicle (KV), a ciliated organ generating directional fluid flow. Then, LR asymmetry is conveyed and stabilized in the lateral plate mesoderm. Although numerous molecules and signaling pathways are involved in controlling LR asymmetry, mechanistic difference and concordance between different organs during LR patterning are poorly understood. Here we show that RA signaling regulates laterality decisions at two stages in zebrafish. Before the 2-somite stage (2So), inhibition of RA signaling leads to randomized visceral laterality through bilateral expression of nodal/spaw in the lateral plate mesoderm, which is mediated by increases in cilia length and defective directional fluid flow in KV. Fgf8 is required for the regulation of cilia length by RA signaling. Blockage of RA signaling before 2So also leads to mild defects of heart laterality, which become much more severe through perturbation of cardiac bmp4 asymmetry when RA signaling is blocked after 2So. At this stage, visceral laterality and the left-sided Nodal remain unaffected. These findings suggest that RA signaling controls visceral laterality through the left-sided Nodal signal before 2So, and regulates heart laterality through cardiac bmp4 mainly after 2So, first identifying sequential control and concordance of visceral and heart laterality. During zebrafish development, the left-right (LR) asymmetric signals are first established around the Kupffer vesicle (KV), a ciliated organ generating directional fluid flow. Then, LR asymmetry is conveyed and stabilized in the lateral plate mesoderm. Although numerous molecules and signaling pathways are involved in controlling LR asymmetry, mechanistic difference and concordance between different organs during LR patterning are poorly understood. Here we show that RA signaling regulates laterality decisions at two stages in zebrafish. Before the 2-somite stage (2So), inhibition of RA signaling leads to randomized visceral laterality through bilateral expression of nodal/spaw in the lateral plate mesoderm, which is mediated by increases in cilia length and defective directional fluid flow in KV. Fgf8 is required for the regulation of cilia length by RA signaling. Blockage of RA signaling before 2So also leads to mild defects of heart laterality, which become much more severe through perturbation of cardiac bmp4 asymmetry when RA signaling is blocked after 2So. At this stage, visceral laterality and the left-sided Nodal remain unaffected. These findings suggest that RA signaling controls visceral laterality through the left-sided Nodal signal before 2So, and regulates heart laterality through cardiac bmp4 mainly after 2So, first identifying sequential control and concordance of visceral and heart laterality. IntroductionDuring vertebrate development, left-right (LR) 2The abbreviations used are: LRleft-rightLPMlateral plate mesodermDEAB4-diethylaminobenzaldehydeMOmorpholino oligoRAretinoic acidSosomite stageDMSOdimethyl sulfoxidehpfhours post-fertilizationEepiboly stage. asymmetry is evident both in heart and visceral organs such as liver, pancreas, and gut. Situs inversus totalis, in which there is complete right to left reversal of the thoracic and abdominal organs, occurs once in about 8,000 persons. Although situs inversus is usually of no medical consequence, heterotaxia, incomplete reversal of organs, occurs with higher frequency and causes significant medical problems. These problems are observed not only in humans but also in nearly all vertebrates in whom LR asymmetry needs to be established during development. In zebrafish, LR asymmetric signals are first established around KV, a ciliated organ that generates directional fluid flow (1Essner J.J. Vogan K.J. Wagner M.K. Tabin C.J. Yost H.J. Brueckner M. Nature. 2002; 418: 37-38Crossref PubMed Scopus (294) Google Scholar, 2Essner J.J. Amack J.D. Nyholm M.K. Harris E.B. Yost H.J. Development. 2005; 132: 1247-1260Crossref PubMed Scopus (474) Google Scholar, 3Yost H.J. Curr. Biol. 2003; 13: R808-R809Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Ciliogenesis, key to the initiation of LR asymmetry, is dependent on fibroblast growth factor (FGF) signaling (4Neugebauer J.M. Amack J.D. Peterson A.G. Bisgrove B.W. Yost H.J. Nature. 2009; 458: 651-654Crossref PubMed Scopus (188) Google Scholar, 5Hong S.K. Dawid I.B. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2230-2235Crossref PubMed Scopus (76) Google Scholar). Then, LR asymmetry is conveyed and stabilized in the lateral plate mesoderm (LPM), patterning left and right sides of the embryo (6Raya A. Izpisúa Belmonte J.C. Nat. Rev. Genet. 2006; 7: 283-293Crossref PubMed Scopus (185) Google Scholar). A critical event of LR patterning at this stage is the left-sided expression of nodal/southpaw (spaw) in the LPM (7Levin M. Johnson R.L. Stern C.D. Kuehn M. Tabin C. Cell. 1995; 82: 803-814Abstract Full Text PDF PubMed Scopus (651) Google Scholar, 8Collignon J. Varlet I. Robertson E.J. Nature. 1996; 381: 155-158Crossref PubMed Scopus (481) Google Scholar, 9Lowe L.A. Supp D.M. Sampath K. Yokoyama T. Wright C.V. Potter S.S. Overbeek P. Kuehn M.R. Nature. 1996; 381: 158-161Crossref PubMed Scopus (414) Google Scholar, 10Lustig K.D. Kroll K. Sun E. Ramos R. Elmendorf H. Kirschner M.W. Development. 1996; 122: 3275-3282Crossref PubMed Google Scholar, 11Long S. Ahmad N. Rebagliati M. Development. 2003; 130: 2303-2316Crossref PubMed Scopus (298) Google Scholar). The left-sided Nodal/Spaw then activates lefty1 in the ventral neural tube, lefty2 and pitx2 in the LPM, all exclusively on the left side (12Meno C. Ito Y. Saijoh Y. Matsuda Y. Tashiro K. Kuhara S. Hamada H. Genes Cells. 1997; 2: 513-524Crossref PubMed Scopus (229) Google Scholar, 13Meno C. Saijoh Y. Fujii H. Ikeda M. Yokoyama T. Yokoyama M. Toyoda Y. Hamada H. Nature. 1996; 381: 151-155Crossref PubMed Scopus (371) Google Scholar, 14Piedra M.E. Icardo J.M. Albajar M. Rodriguez-Rey J.C. Ros M.A. Cell. 1998; 94: 319-324Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 15Ryan A.K. Blumberg B. Rodriguez-Esteban C. Yonei-Tamura S. Tamura K. Tsukui T. de la Peña J. Sabbagh W. Greenwald J. Choe S. Norris D.P. Robertson E.J. Evans R.M. Rosenfeld M.G. Izpisúa Belmonte J.C. Nature. 1998; 394: 545-551Crossref PubMed Scopus (436) Google Scholar). Lefty1 and Lefty2 antagonize Nodal activity, thus generating a negative feedback loop to restrict the extent and duration of Nodal signaling. The left-sided expression of nodal/spaw, lefty2, and pitx2 are highly conserved in vertebrates to ensure proper LR patterning (6Raya A. Izpisúa Belmonte J.C. Nat. Rev. Genet. 2006; 7: 283-293Crossref PubMed Scopus (185) Google Scholar).Retinoic acid (RA) and bone morphogenetic protein signals have been reported to regulate several aspects of LR asymmetry. Treatment of RA antagonist leads to randomization of heart looping and perturbed sideness of nodal in mouse (16Chazaud C. Chambon P. Dollé P. Development. 1999; 126: 2589-2596Crossref PubMed Google Scholar), but roles of RA signaling in the determination of heart and visceral laterality in zebrafish remain unknown. In Xenopus, chick, and mouse, bone morphogenetic protein signaling is required to repress Nodal activity in the right LPM, thus ensuring right-sided laterality (17Ramsdell A.F. Yost H.J. Development. 1999; 126: 5195-5205Crossref PubMed Google Scholar, 18Rodríguez Esteban C. Capdevila J. Economides A.N. Pascual J. Ortiz A. Izpisúa Belmonte J.C. Nature. 1999; 401: 243-251Crossref PubMed Scopus (181) Google Scholar, 19Yokouchi Y. Vogan K.J. Pearse 2nd, R.V. Tabin C.J. Cell. 1999; 98: 573-583Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 20Chang H. Zwijsen A. Vogel H. Huylebroeck D. Matzuk M.M. Dev. Biol. 2000; 219: 71-78Crossref PubMed Scopus (120) Google Scholar). In zebrafish, bone morphogenetic protein signaling regulates LR asymmetry at two distinct developmental time points. Shortly after KV has been formed during early segmentation, bone morphogenetic protein signaling is necessary to repress spaw in the right LPM, similar to other vertebrates (21Chocron S. Verhoeven M.C. Rentzsch F. Hammerschmidt M. Bakkers J. Dev. Biol. 2007; 305: 577-588Crossref PubMed Scopus (124) Google Scholar). In later segmentation at around the 22-somite (22So) stage prior to the initiation of cardiac jogging, bmp4 is asymmetrically distributed in the cardiac field with stronger expression on the left side (22Chen J.N. van Eeden F.J. Warren K.S. Chin A. Nüsslein-Volhard C. Haffter P. Fishman M.C. Development. 1997; 124: 4373-4382Crossref PubMed Google Scholar). This asymmetric bmp4 is required for the determination of heart laterality (21Chocron S. Verhoeven M.C. Rentzsch F. Hammerschmidt M. Bakkers J. Dev. Biol. 2007; 305: 577-588Crossref PubMed Scopus (124) Google Scholar, 22Chen J.N. van Eeden F.J. Warren K.S. Chin A. Nüsslein-Volhard C. Haffter P. Fishman M.C. Development. 1997; 124: 4373-4382Crossref PubMed Google Scholar, 23Smith K.A. Chocron S. von der Hardt S. de Pater E. Soufan A. Bussmann J. Schulte-Merker S. Hammerschmidt M. Bakkers J. Dev. Cell. 2008; 14: 287-297Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar).Ectopic expression of bmp4 on the right side results in reversal of heart laterality, but visceral organs are unaffected (24Schilling T.F. Concordet J.P. Ingham P.W. Dev. Biol. 1999; 210: 277-287Crossref PubMed Scopus (101) Google Scholar). Induction of exogenous noggin3 at the 16-somite stage causes perturbation of heart laterality, but visceral laterality remains normal (21Chocron S. Verhoeven M.C. Rentzsch F. Hammerschmidt M. Bakkers J. Dev. Biol. 2007; 305: 577-588Crossref PubMed Scopus (124) Google Scholar). High occurrence of heterotaxia in nature proposes that asymmetries of heart and visceral organs may involve different regulatory mechanisms and need to be coordinated. However, key mechanistic differences and concordance between visceral and heart laterality during LR patterning remain to be elucidated.Here we demonstrate that inhibition of RA signaling before 2So leads to randomized laterality of visceral organs through bilateral expression of spaw, which resulted from increases in cilia length and defective directional fluid flow in KV. Fgf8 is required to mediate the control of cilia length by RA signaling. Inhibition of RA signaling before 2So also results in mild defects of heart laterality, which become much more severe through disturbance of cardiac bmp4 asymmetry when RA signaling is blocked after 2So. However, visceral laterality and left-sided spaw and lefty2 remain unaffected at this stage. Our study first identifies RA signaling as the key signaling pathway that sequentially controls visceral and heart laterality, so that RA signaling achieves their concordance during LR patterning.DISCUSSIONIn summary, our results suggest that RA signaling regulates LR patterning of organs at two stages in zebrafish. Before 2So, RA signaling is critical for setting up the left-sided Nodal signal through regulation of cilia length and KV fluid flow. fgf8 is required to mediate this regulation. The left-sided Nodal signal is the key dictator of visceral laterality so that RA signaling controls visceral laterality before 2So. Roles of RA signaling in the determination of heart laterality are mild before 2So, and become much more significant after 2So through regulation of cardiac bmp4 asymmetry. Although previous studies have reported that visceral and heart laterality is sometimes uncoordinated so that their asymmetries may need concordance (21Chocron S. Verhoeven M.C. Rentzsch F. Hammerschmidt M. Bakkers J. Dev. Biol. 2007; 305: 577-588Crossref PubMed Scopus (124) Google Scholar, 24Schilling T.F. Concordet J.P. Ingham P.W. Dev. Biol. 1999; 210: 277-287Crossref PubMed Scopus (101) Google Scholar), our study first identifies RA signaling as the key signaling pathway that sequentially controls and coordinates visceral and heart laterality.Titrations for the cut-off time at the shield, 80%E, bud, 2So, and 4So have identified the significance of 2So (data not shown). Theoretically, on one hand, formation of KV and cilia are initially observed at 3–4So (2Essner J.J. Amack J.D. Nyholm M.K. Harris E.B. Yost H.J. Development. 2005; 132: 1247-1260Crossref PubMed Scopus (474) Google Scholar) (data not shown). Thus, RA signaling should be functional to regulate ciliogenesis before that. This point was proven by the unaffected sideness of spaw and lefty2 after RA treatment at 4So (Fig. 3, E and K). On the other hand, asymmetric expression of raldh2 in the anterior LPM initiates at 1–2So (Fig. 7H). Therefore, control of cardiac bmp4 asymmetry by RA signaling is enabled only after that. These two aspects fit our titration results and supply possible explanations why 2So becomes a critical cut-off time.Discrepancy between treatments of RA antagonist and raldh2 mutation have been previously reported in mice and discussed in two papers published by the same laboratory (16Chazaud C. Chambon P. Dollé P. Development. 1999; 126: 2589-2596Crossref PubMed Google Scholar, 44Niederreither K. Vermot J. Messaddeq N. Schuhbaur B. Chambon P. Dollé P. Development. 2001; 128: PubMed Google Scholar). of the papers sideness of nodal and in mouse with RA antagonist (16Chazaud C. Chambon P. Dollé P. Development. 1999; 126: 2589-2596Crossref PubMed Google Scholar), the unaffected sideness of nodal and pitx2 in mouse K. Vermot J. Messaddeq N. Schuhbaur B. Chambon P. Dollé P. Development. 2001; 128: PubMed Google Scholar). This also in zebrafish Y. A. R.M. C. Belmonte J.C. Nature. 2005; PubMed Scopus Google Scholar), and be by the possible other RA and roles in the raldh2 a acid was to a more complete inhibition of RA signaling. protein in setting up the left-sided Nodal of and have all been reported in zebrafish K. Choe S.K. N. E. 2009; PubMed Scopus Google Scholar, D. M. J. 2008; PubMed Scopus Google Scholar). this also a more complete inhibition of RA signaling. RA in stronger raldh2 which has been reported in two raldh2 in zebrafish K. Choe S.K. N. E. 2009; PubMed Scopus Google Scholar). raldh2 only a of A of activity may Therefore, differences between and have been RA signaling is required in more one of visceral organ development, such as and visceral laterality J. F. A. B. S. Biol. 2008; PubMed Scopus Google Scholar) (Fig. our results demonstrate that LR of treatment on heart and visceral organs are other both right-sided of and left-sided of exogenous spaw randomization of visceral laterality but not defective by and and and both treatments before and after 2So no early up to when laterality decisions have been the that the LR are of defective laterality of heart and visceral organs by be by exogenous RA and treatments to of cardiac before 2So (Fig. and and defective heart laterality mainly after 2So (Fig. and are and to similar laterality at the same stage, which that the are to of RA signaling. However, to be a stronger RA antagonist treatment from to 2So leads to a complete of as as of and and and In a of and mild of observed in the of with (Fig. and of defective heart laterality a higher in with (Fig. These differences between and be by different mechanisms of be by exogenous is a acid is of RA and be by exogenous fgf8 and resulted in in around KV S.K. Dawid I.B. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2230-2235Crossref PubMed Scopus (76) Google Scholar) that a Fgf8 is necessary for This also why in after treatment by a of but by a of in heart and of cardiac bmp4 expression by treatment from to 2So and and should from increases in of cardiac Ingham P.W. D. 2005; PubMed Scopus Google Scholar, K.D. D. Dev. Cell. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). cardiac looping is not only by the laterality decisions but also by the of the mild of RA signaling before 2So in heart laterality be a of increases in cardiac is also possible that RA signaling before 2So regulates heart laterality through the left-sided Nodal signal other At the bilateral cardiac have in zebrafish. In with there is no in the bilateral cardiac have not (Fig. This in of cardiac be a of perturbed heart laterality, a of increases in cardiac of in the expression of noggin3 to bilateral expression of spaw (21Chocron S. Verhoeven M.C. Rentzsch F. Hammerschmidt M. Bakkers J. Dev. Biol. 2007; 305: 577-588Crossref PubMed Scopus (124) Google Scholar). However, for unaffected formation of KV in bmp4 mechanisms regulation of the left-sided Nodal signal by bmp4 remain unknown. during in the ventral and as factor of RA and signaling in of bmp4 around KV at unaffected in with from to 2So bmp4 as a factor of RA signaling. Therefore, RA and signaling that regulate cilia bmp4 in other aspects of ciliogenesis of of RA signaling and exogenous RA disturbance of LR asymmetry the control of RA which why both and RA treatments to similar on the left-sided Nodal signal T. Capdevila J. Tamura K. P. Rodriguez-Esteban C. Yonei-Tamura S. J. K. Blumberg B. Evans R.M. Belmonte J.C. Proc. Natl. Acad. Sci. U.S.A. 1999; PubMed Scopus Google Scholar) (Fig. has been reported that spaw cardiac bmp4 asymmetry as as defective heart laterality S. Ahmad N. Rebagliati M. Development. 2003; 130: 2303-2316Crossref PubMed Scopus (298) Google Scholar, S. Verhoeven M.C. Rentzsch F. Hammerschmidt M. Bakkers J. Dev. Biol. 2007; 305: 577-588Crossref PubMed Scopus (124) Google Scholar), and asymmetry of lefty2 is by heart laterality K. Proc. Natl. Acad. Sci. U.S.A. 2008; PubMed Scopus Google Scholar). However, is that the left-sided Nodal is of the Nodal signal in the right LPM is significant for cardiac bmp4 asymmetry and heart laterality 3, and and In other the Nodal asymmetry be with cardiac bmp4 asymmetry and heart laterality. This has been previously reported both in mice and zebrafish K. Vermot J. Messaddeq N. Schuhbaur B. Chambon P. Dollé P. Development. 2001; 128: PubMed Google Scholar, S.S. R. N. J. Development. PubMed Scopus Google Scholar). normal sideness of Nodal signal in the LPM of mouse, but heart laterality was defective K. Vermot J. Messaddeq N. Schuhbaur B. Chambon P. Dollé P. Development. 2001; 128: PubMed Google Scholar). The other bilateral expression of spaw in zebrafish but heart laterality remains unaffected S.S. R. N. J. Development. PubMed Scopus Google Scholar). The of asymmetrically normal spaw and lefty2, visceral laterality, cardiac and heart laterality 3, and and that cardiac bmp4 asymmetry and heart laterality are not the control of the left-sided Nodal and cardiac bmp4 is to the determination of visceral laterality. from our LR asymmetry is evident first in the laterality determination later that of visceral our suggest that RA signaling regulates LR patterning of organs at two on concordance of visceral and heart laterality is Before 2So, RA signaling regulates cilia length and directional fluid flow in KV by the of This is critical for setting up the left-sided Nodal signal that visceral laterality. 2So a possible RA in the anterior LPM, the mild regulatory of RA signaling before 2So in controlling heart laterality becomes much more significant after 2So through asymmetric cardiac IntroductionDuring vertebrate development, left-right (LR) 2The abbreviations used are: LRleft-rightLPMlateral plate mesodermDEAB4-diethylaminobenzaldehydeMOmorpholino oligoRAretinoic acidSosomite stageDMSOdimethyl sulfoxidehpfhours post-fertilizationEepiboly stage. asymmetry is evident both in heart and visceral organs such as liver, pancreas, and gut. Situs inversus totalis, in which there is complete right to left reversal of the thoracic and abdominal organs, occurs once in about 8,000 persons. Although situs inversus is usually of no medical consequence, heterotaxia, incomplete reversal of organs, occurs with higher frequency and causes significant medical problems. These problems are observed not only in humans but also in nearly all vertebrates in whom LR asymmetry needs to be established during development. In zebrafish, LR asymmetric signals are first established around KV, a ciliated organ that generates directional fluid flow (1Essner J.J. Vogan K.J. Wagner M.K. Tabin C.J. Yost H.J. Brueckner M. Nature. 2002; 418: 37-38Crossref PubMed Scopus (294) Google Scholar, 2Essner J.J. Amack J.D. Nyholm M.K. Harris E.B. Yost H.J. Development. 2005; 132: 1247-1260Crossref PubMed Scopus (474) Google Scholar, 3Yost H.J. Curr. Biol. 2003; 13: R808-R809Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Ciliogenesis, key to the initiation of LR asymmetry, is dependent on fibroblast growth factor (FGF) signaling (4Neugebauer J.M. Amack J.D. Peterson A.G. Bisgrove B.W. Yost H.J. Nature. 2009; 458: 651-654Crossref PubMed Scopus (188) Google Scholar, 5Hong S.K. Dawid I.B. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2230-2235Crossref PubMed Scopus (76) Google Scholar). Then, LR asymmetry is conveyed and stabilized in the lateral plate mesoderm (LPM), patterning left and right sides of the embryo (6Raya A. Izpisúa Belmonte J.C. Nat. Rev. Genet. 2006; 7: 283-293Crossref PubMed Scopus (185) Google Scholar). A critical event of LR patterning at this stage is the left-sided expression of nodal/southpaw (spaw) in the LPM (7Levin M. Johnson R.L. Stern C.D. Kuehn M. Tabin C. Cell. 1995; 82: 803-814Abstract Full Text PDF PubMed Scopus (651) Google Scholar, 8Collignon J. Varlet I. Robertson E.J. Nature. 1996; 381: 155-158Crossref PubMed Scopus (481) Google Scholar, 9Lowe L.A. Supp D.M. Sampath K. Yokoyama T. Wright C.V. Potter S.S. Overbeek P. Kuehn M.R. Nature. 1996; 381: 158-161Crossref PubMed Scopus (414) Google Scholar, 10Lustig K.D. Kroll K. Sun E. Ramos R. Elmendorf H. Kirschner M.W. Development. 1996; 122: 3275-3282Crossref PubMed Google Scholar, 11Long S. Ahmad N. Rebagliati M. Development. 2003; 130: 2303-2316Crossref PubMed Scopus (298) Google Scholar). The left-sided Nodal/Spaw then activates lefty1 in the ventral neural tube, lefty2 and pitx2 in the LPM, all exclusively on the left side (12Meno C. Ito Y. Saijoh Y. Matsuda Y. Tashiro K. Kuhara S. Hamada H. Genes Cells. 1997; 2: 513-524Crossref PubMed Scopus (229) Google Scholar, 13Meno C. Saijoh Y. Fujii H. Ikeda M. Yokoyama T. Yokoyama M. Toyoda Y. Hamada H. Nature. 1996; 381: 151-155Crossref PubMed Scopus (371) Google Scholar, 14Piedra M.E. Icardo J.M. Albajar M. Rodriguez-Rey J.C. Ros M.A. Cell. 1998; 94: 319-324Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 15Ryan A.K. Blumberg B. Rodriguez-Esteban C. Yonei-Tamura S. Tamura K. Tsukui T. de la Peña J. Sabbagh W. Greenwald J. Choe S. Norris D.P. Robertson E.J. Evans R.M. Rosenfeld M.G. Izpisúa Belmonte J.C. Nature. 1998; 394: 545-551Crossref PubMed Scopus (436) Google Scholar). Lefty1 and Lefty2 antagonize Nodal activity, thus generating a negative feedback loop to restrict the extent and duration of Nodal signaling. The left-sided expression of nodal/spaw, lefty2, and pitx2 are highly conserved in vertebrates to ensure proper LR patterning (6Raya A. Izpisúa Belmonte J.C. Nat. Rev. Genet. 2006; 7: 283-293Crossref PubMed Scopus (185) Google Scholar).Retinoic acid (RA) and bone morphogenetic protein signals have been reported to regulate several aspects of LR asymmetry. Treatment of RA antagonist leads to randomization of heart looping and perturbed sideness of nodal in mouse (16Chazaud C. Chambon P. Dollé P. Development. 1999; 126: 2589-2596Crossref PubMed Google Scholar), but roles of RA signaling in the determination of heart and visceral laterality in zebrafish remain unknown. In Xenopus, chick, and mouse, bone morphogenetic protein signaling is required to repress Nodal activity in the right LPM, thus ensuring right-sided laterality (17Ramsdell A.F. Yost H.J. Development. 1999; 126: 5195-5205Crossref PubMed Google Scholar, 18Rodríguez Esteban C. Capdevila J. Economides A.N. Pascual J. Ortiz A. Izpisúa Belmonte J.C. Nature. 1999; 401: 243-251Crossref PubMed Scopus (181) Google Scholar, 19Yokouchi Y. Vogan K.J. Pearse 2nd, R.V. Tabin C.J. Cell. 1999; 98: 573-583Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 20Chang H. Zwijsen A. Vogel H. Huylebroeck D. Matzuk M.M. Dev. Biol. 2000; 219: 71-78Crossref PubMed Scopus (120) Google Scholar). In zebrafish, bone morphogenetic protein signaling regulates LR asymmetry at two distinct developmental time points. Shortly after KV has been formed during early segmentation, bone morphogenetic protein signaling is necessary to repress spaw in the right LPM, similar to other vertebrates (21Chocron S. Verhoeven M.C. Rentzsch F. Hammerschmidt M. Bakkers J. Dev. Biol. 2007; 305: 577-588Crossref PubMed Scopus (124) Google Scholar). In later segmentation at around the 22-somite (22So) stage prior to the initiation of cardiac jogging, bmp4 is asymmetrically distributed in the cardiac field with stronger expression on the left side (22Chen J.N. van Eeden F.J. Warren K.S. Chin A. Nüsslein-Volhard C. Haffter P. Fishman M.C. Development. 1997; 124: 4373-4382Crossref PubMed Google Scholar). This asymmetric bmp4 is required for the determination of heart laterality (21Chocron S. Verhoeven M.C. Rentzsch F. Hammerschmidt M. Bakkers J. Dev. Biol. 2007; 305: 577-588Crossref PubMed Scopus (124) Google Scholar, 22Chen J.N. van Eeden F.J. Warren K.S. Chin A. Nüsslein-Volhard C. Haffter P. Fishman M.C. Development. 1997; 124: 4373-4382Crossref PubMed Google Scholar, 23Smith K.A. Chocron S. von der Hardt S. de Pater E. Soufan A. Bussmann J. Schulte-Merker S. Hammerschmidt M. Bakkers J. Dev. Cell. 2008; 14: 287-297Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar).Ectopic expression of bmp4 on the right side results in reversal of heart laterality, but visceral organs are unaffected (24Schilling T.F. Concordet J.P. Ingham P.W. Dev. Biol. 1999; 210: 277-287Crossref PubMed Scopus (101) Google Scholar). Induction of exogenous noggin3 at the 16-somite stage causes perturbation of heart laterality, but visceral laterality remains normal (21Chocron S. Verhoeven M.C. Rentzsch F. Hammerschmidt M. Bakkers J. Dev. Biol. 2007; 305: 577-588Crossref PubMed Scopus (124) Google Scholar). High occurrence of heterotaxia in nature proposes that asymmetries of heart and visceral organs may involve different regulatory mechanisms and need to be coordinated. However, key mechanistic differences and concordance between visceral and heart laterality during LR patterning remain to be elucidated.Here we demonstrate that inhibition of RA signaling before 2So leads to randomized laterality of visceral organs through bilateral expression of spaw, which resulted from increases in cilia length and defective directional fluid flow in KV. Fgf8 is required to mediate the control of cilia length by RA signaling. Inhibition of RA signaling before 2So also results in mild defects of heart laterality, which become much more severe through disturbance of cardiac bmp4 asymmetry when RA signaling is blocked after 2So. However, visceral laterality and left-sided spaw and lefty2 remain unaffected at this stage. Our study first identifies RA signaling as the key signaling pathway that sequentially controls visceral and heart laterality, so that RA signaling achieves their concordance during LR