Retinoic Acid Signaling Sequentially Controls Visceral and Heart Laterality in Zebrafish
2011; Elsevier BV; Volume: 286; Issue: 32 Linguagem: Inglês
10.1074/jbc.m111.244327
ISSN1083-351X
AutoresSizhou Huang, Jun Ma, Xiaolin Liu, Yaoguang Zhang, Lingfei Luo,
Tópico(s)Hemispheric Asymmetry in Neuroscience
ResumoDuring 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: 1019-1031Crossref PubMed Google Scholar). One of the papers showed disturbed sideness of nodal and lefty in mouse embryos treated with RA antagonist (16Chazaud C. Chambon P. Dollé P. Development. 1999; 126: 2589-2596Crossref PubMed Google Scholar), whereas the second exhibited unaffected sideness of nodal and pitx2 in raldh2−/− mouse mutant (44Niederreither K. Vermot J. Messaddeq N. Schuhbaur B. Chambon P. Dollé P. Development. 2001; 128: 1019-1031Crossref PubMed Google Scholar). This discrepancy could also exist in zebrafish (45Kawakami Y. Raya A. Raya R.M. Rodríguez-Esteban C. Belmonte J.C. Nature. 2005; 435: 165-171Crossref PubMed Scopus (224) Google Scholar), and be explained by the following four possible reasons. First, other RA synthesis enzymes like Raldh1, Raldh3, and Raldh4 play redundant roles in the raldh2 mutant or morphant. But BMS453, a pan-retinoic acid receptor antagonist, was able to achieve a more complete inhibition of RA signaling. Second, maternal Raldh2 protein or mRNA plays an important role in setting up the left-sided Nodal signal. Actually, maternal transcripts of raldh2, raldh3, and raldh4 have all been reported in zebrafish (46Alexa K. Choe S.K. Hirsch N. Etheridge L. Laver E. Sagerström C.G. PLoS One. 2009; 4: e8261Crossref PubMed Scopus (28) Google Scholar, 47Liang D. Zhang M. Bao J. Zhang L. Xu X. Gao X. Zhao Q. Gene Expr. Patterns. 2008; 8: 248-253Crossref PubMed Scopus (30) Google Scholar). From this aspect, BMS453 or DEAB could also achieve a more complete inhibition of RA signaling. Third, RA antagonists result in stronger phenotypes than raldh2 mutation, which has been reported in two raldh2 mutant alleles in zebrafish (46Alexa K. Choe S.K. Hirsch N. Etheridge L. Laver E. Sagerström C.G. PLoS One. 2009; 4: e8261Crossref PubMed Scopus (28) Google Scholar). Fourth, raldh2 morpholino only represents a knockdown of Raldh2. A certain percentage of Raldh2 activity may still exist. Therefore, differences between chemical inhibitors and genetic inactivation have been sufficiently interpreted.Although RA signaling is required in more than one aspect of visceral organ development, such as pancreas differentiation and visceral laterality (29Jiang Z. Song J. Qi F. Xiao A. An X. Liu N.A. Zhu Z. Zhang B. Lin S. PLoS Biol. 2008; 6: e293Crossref PubMed Scopus (28) Google Scholar) (Fig. 1), our results demonstrate that LR effects of BMS453 treatment on heart and visceral organs are specific other than indirect. First, both predominantly right-sided distribution of spawMO and unilaterally left-sided distribution of exogenous spaw mRNA specifically rescued randomization of visceral laterality but not defective pancreas differentiation caused by BMS453 (Figs. 1, F and G, and 4, G, H, M, and N). Second, both BMS453 treatments before and after 2So caused no obvious early phenotype up to 25 hpf when laterality decisions have already been made (supplemental Fig. S6), ruling out the possibility that the LR phenotypes are indirect effects of earlier phenotypes. Third, defective laterality of heart and visceral organs caused by DEAB could be efficiently rescued by exogenous RA (Figs. 1K and 2O). Fourth, BMS453 or DEAB treatments led to differentiation phenotypes of cardiac progenitors before 2So (Fig. 2, C–E and I–K) and defective heart laterality mainly after 2So (Fig. 2, F–H and L–N). They are temporally separated.BMS453 and DEAB lead to similar laterality phenotypes at the same stage, which confirms that the phenotypes are due to blockage of RA signaling. However, BMS453 appears to be a stronger RA antagonist than DEAB. First, BMS453 treatment from 32-cell to 2So leads to a complete absence of exocrine pancreas as well as dramatic reduction of β-cells (Figs. 1, F and G, and 4, D–H, M, and N). In contrast, a residual of exocrine pancreas and mild reduction of β-cells were observed in the majority of embryos treated with DEAB (Fig. 1, I and J). Second, ratios of defective heart laterality were a little higher in embryos treated with BMS453 than DEAB (Fig. 2, C–O). These differences between BMS453 and DEAB can be explained by different mechanisms of how these chemicals work. BMS453, whose inhibitory effects cannot be rescued by exogenous RA, is a pan-retinoic acid receptor antagonist. DEAB is an inhibitor of RA synthase, and its effects can be efficiently rescued by exogenous RA.Both fgf8 knockdown and overexpression resulted in decreases in charon transcription around KV (5Hong S.K. Dawid I.B. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2230-2235Crossref PubMed Scopus (76) Google Scholar) (supplemental Fig. S4, A–C), indicating that a certain Fgf8 concentration is necessary for charon activation. This also explains why decreases in charon transcription after BMS453 treatment were exacerbated by a high concentration of fgf8MO, but partially rescued by a low concentration of fgf8MO (supplemental Fig. S4, D–F).Increases in heart size and expansion of cardiac bmp4 expression caused by BMS453 or DEAB treatment from 32-cell to 2So (Figs. 2, C–E and I–K, and 6B) should result from increases in cell number of cardiac progenitors (48Keegan B.R. Feldman J.L. Begemann G. Ingham P.W. Yelon D. Science. 2005; 307: 247-249Crossref PubMed Scopus (170) Google Scholar, 49Waxman J.S. Keegan B.R. Roberts R.W. Poss K.D. Yelon D. Dev. Cell. 2008; 15: 923-934Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Because cardiac looping is not only controlled by the laterality decisions but also affected by the differentiation of cardiomyocyte, the mild role of RA signaling before 2So in regulating heart laterality could be a result of increases in cardiac progenitors. It is also possible that RA signaling before 2So mildly regulates heart laterality through the left-sided Nodal signal or other unknown mechanisms. At 22So, the asymmetric, bilateral cardiac primordia have fused in zebrafish. In embryos treated with BMS453, although there is no delay in embryonic development (supplemental Fig. S5), the bilateral cardiac primoridia have not yet fused (Fig. 6, B–E). This delay in fusion of cardiac primordia could be a result of perturbed heart laterality, or a result of increases in cardiac progenitors, or an independent effect.Knockdown of Bmp4 in the dorsal forerunner cells or ectopic expression of noggin3 lead 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, except for unaffected formation of KV in bmp4 morphant, mechanisms underlying regulation of the left-sided Nodal signal by bmp4 remain unknown. Expression patterns during gastrulation (only in the ventral region and prechordal plate) precludes Bmp4 as an upstream factor of RA and FGF signaling in regulating ciliogenesis. Expression of bmp4 around KV at 10So remained unaffected in embryos treated with BMS453 from 32-cell to 2So (supplemental Fig. S7), excluding bmp4 as a downstream factor of RA signaling. Therefore, unlike RA and FGF signaling that regulate cilia length, bmp4 most probably plays parts in other aspects of ciliogenesis or even independent of ciliogenesis.Both blockage of RA signaling and exogenous RA represent disturbance of LR asymmetry under the control of RA signaling, which could explain why both BMS453 and RA treatments do lead to similar effects on the left-sided Nodal signal (34Tsukui T. Capdevila J. Tamura K. Ruiz-Lozano P. Rodriguez-Esteban C. Yonei-Tamura S. Magallón J. Chandraratna R.A. Chien K. Blumberg B. Evans R.M. Belmonte J.C. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 11376-11381Crossref PubMed Scopus (234) Google Scholar) (Fig. 3). It has been reported that spaw morphant displays disturbed cardiac bmp4 asymmetry as well as defective heart laterality (11Long S. Ahmad N. Rebagliati M. Development. 2003; 130: 2303-2316Crossref PubMed Scopus (298) Google Scholar, 21Chocron S. Verhoeven M.C. Rentzsch F. Hammerschmidt M. Bakkers J. Dev. Biol. 2007; 305: 577-588Crossref PubMed Scopus (124) Google Scholar), and reversed asymmetry of lefty2 is accompanied by reversed heart laterality (50Baker K. Holtzman N.G. Burdine R.D. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13924-13929Crossref PubMed Scopus (55) Google Scholar). However, it is likely that if the left-sided Nodal is present, repression of the Nodal signal in the right LPM is less significant for cardiac bmp4 asymmetry and heart laterality (Figs. 2C, 3, B and H, and 6B). In other words, the Nodal asymmetry could be inconsistent with cardiac bmp4 asymmetry and heart laterality. This inconsistency has been previously reported both in mice and zebrafish (44Niederreither K. Vermot J. Messaddeq N. Schuhbaur B. Chambon P. Dollé P. Development. 2001; 128: 1019-1031Crossref PubMed Google Scholar, 51Lopes S.S. Lourenço R. Pacheco L. Moreno N. Kreiling J. Saúde L. Development. 2010; 137: 3625-3632Crossref PubMed Scopus (89) Google Scholar). One reference showed normal sideness of Nodal signal in the LPM of raldh2−/− mouse, but its heart laterality was defective (44Niederreither K. Vermot J. Messaddeq N. Schuhbaur B. Chambon P. Dollé P. Development. 2001; 128: 1019-1031Crossref PubMed Google Scholar). The other showed bilateral expression of spaw in aei−/− zebrafish mutant, but its heart laterality remains unaffected (51Lopes S.S. Lourenço R. Pacheco L. Moreno N. Kreiling J. Saúde L. Development. 2010; 137: 3625-3632Crossref PubMed Scopus (89) Google Scholar). The concomitance of asymmetrically normal spaw and lefty2, correct visceral laterality, disturbed cardiac bmp4, and aberrant heart laterality (Figs. 1H, 2, F–H, 3, C and I, and 6E) indicates that cardiac bmp4 asymmetry and heart laterality are not under the direct control of the left-sided Nodal signal, and cardiac bmp4 is insignificant to the determination of visceral laterality. Furthermore, from our results, although LR asymmetry is phenotypically evident first in the heart, its laterality determination even takes place later than that of visceral organs.Taken together, our data suggest that RA signaling regulates LR patterning of organs at two stages, shedding new light on how concordance of visceral and heart laterality is achieved. Before 2So, RA signaling regulates cilia length and directional fluid flow in KV by the mediation of fgf8. This is critical for setting up the left-sided Nodal signal that dictates visceral laterality. From 2So on, because a possible new RA resource appears in the anterior LPM, the mild regulatory role of RA signaling before 2So in controlling heart laterality becomes much more significant after 2So through asymmetric cardiac bmp4. 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.
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