The Molecular Differentiation of Anatomically Paired Left and Right Mantles of the Pacific Oyster Crassostrea gigas

  • Lei Wei
  • Fei Xu
  • Yuzhi Wang
  • Zhongqiang Cai
  • Wenchao Yu
  • Cheng He
  • Qiuyun Jiang
  • Xiqiang Xu
  • Wen Guo
  • Xiaotong Wang
Original Article

Abstract

Left-right (L-R) asymmetry is controlled by gene regulation pathways for the L-R axis, and in vertebrates, the gene Pitx2 in TGF−β signaling pathway plays important roles in the asymmetrical formation of organs. However, less is known about the asymmetries of anatomically identical paired organs, as well as the transcriptional regulation mechanism of the gene Pitx in invertebrates. Here, we report the molecular biological differences between the left and right mantles of an invertebrate, the Pacific oyster Crassostrea gigas, and propose one possible mechanism underlying those differences. RNA sequencing (RNA-seq) analysis indicated that the paired organs showed different gene expression patterns, suggesting possible functional differences in shell formation, pheromone signaling, nerve conduction, the stress response, and other physiological processes. RNA-seq and real-time qPCR analysis indicated high right-side expression of the Pitx homolog (cgPitx) in oyster mantle, supporting a conserved role for Pitx in controlling asymmetry. Methylation-dependent restriction-site associated DNA sequencing (MethylRAD) identified a methylation site in the promoter region of cgPitx and showed significantly different methylation levels between the left and right mantles. This is the first report, to our knowledge, of such a difference in methylation in spiralians, and it was further confirmed in 18 other individuals by using a pyrosequencing assay. The miRNome analysis and the TGF-β receptor/Smad inhibition experiment further supported that several genes in TGF−β signaling pathway may be related with the L/R asymmetry of oyster mantles. These results suggested that the molecular differentiation of the oyster’s paired left and right mantles is significant, TGF−β signaling pathway could be involved in establishing or maintaining the asymmetry, and the cgPitx gene as one of genes in this pathway; the different methylation levels in its promoter regions between L/R mantles was the one of possible mechanisms regulating the left-right functional differentiation.

Keywords

Pacific oyster Asymmetry of paired organs Mantle The gene Pitx Methylation Promoter region 

Notes

Acknowledgements

We thank Peter W H Holland and Jordi Paps for helpful suggestions upon critical reading of the manuscript, as well as Guofan Zhang for helpful discussion regarding the experimental design.

Compliance with Ethical Standards

Competing Interests

The authors declare that they have no conflict of interest.

Supplementary material

10126_2018_9806_MOESM1_ESM.xlsx (37 kb)
ESM 1 (XLSX 37 kb)
10126_2018_9806_MOESM2_ESM.docx (66 kb)
ESM 2 (DOCX 66 kb)
10126_2018_9806_MOESM3_ESM.xlsx (63 kb)
ESM 3 (XLSX 62 kb)
10126_2018_9806_MOESM4_ESM.xlsx (87 kb)
ESM 4 (XLSX 87 kb)
10126_2018_9806_MOESM5_ESM.xlsx (13 kb)
ESM 5 (XLSX 12 kb)
10126_2018_9806_MOESM6_ESM.xlsx (10 kb)
Table S7 Different expressed miRNA related with TGF−β pathway. (XLSX 9 kb)

References

  1. Aguilera F, Mcdougall C, Degnan BM (2014) Evolution of the tyrosinase gene family in bivalve molluscs: independent expansion of the mantle gene repertoire. Acta Biomater 10:3855–3865CrossRefPubMedGoogle Scholar
  2. Anders S, Huber W (2012) Differential expression of RNA-Seq data at the gene level—the DESeq package. Embl. http://www.bioconductor.org/packages/release/bioc/vignettes/DESeq/inst/doc/DESeq.pdf. Version: 1.30.0
  3. Anders S, Pyl PT, Huber W (2015) HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31:166–169CrossRefPubMedGoogle Scholar
  4. Béven L, Adenier H, Kichenama R, Homand J, Redeker V, Le CJ, Ladant D, Chopineau J (2001) Ca2+-myristoyl switch and membrane binding of chemically acylated neurocalcins. Biochemistry 40:8152–8160CrossRefPubMedGoogle Scholar
  5. Barbagallo B, Prescott HA, Boyle P, Climer J, Francis MM (2010) A dominant mutation in a neuronal acetylcholine receptor subunit leads to motor neuron degeneration in Caenorhabditis elegans. J Neurosci 30:13932–13942CrossRefPubMedPubMedCentralGoogle Scholar
  6. Blank S, Arnoldi M, Khoshnavaz S, Treccani L, Kuntz M, Mann K, Grathwohl G, Fritz M (2003) The nacre protein perlucin nucleates growth of calcium carbonate crystals. J Microsc 212:280–291CrossRefPubMedGoogle Scholar
  7. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622CrossRefPubMedGoogle Scholar
  8. Cock PJ, Fields CJ, Goto N, Heuer ML, Rice PM (2009) The Sanger FASTQ file format for sequences with quality scores, and the Solexa/Illumina FASTQ variants. Nucleic Acids Res 38:1767–1771CrossRefPubMedPubMedCentralGoogle Scholar
  9. Cummins SF, Xie F, De Vries MR, Annangudi SP, Misra M, Degnan BM, Sweedler JV, Nagle GT, Schein CH (2007) Aplysia temptin—the ‘glue’ in the water-borne attractin pheromone complex. FEBS J 274:5425–5437CrossRefPubMedGoogle Scholar
  10. Dodenhof T, Dietz F, Franken S, Grunwald I, Kelm S (2014) Splice variants of perlucin from Haliotis laevigata modulate the crystallisation of CaCO3. PLoS One 9:e97126CrossRefPubMedPubMedCentralGoogle Scholar
  11. Duboc V, Röttinger E, Lapraz F, Besnardeau L, Lepage T (2005) Left-right asymmetry in the sea urchin embryo is regulated by nodal signaling on the right side. Dev Cell 9:147–158CrossRefPubMedGoogle Scholar
  12. Grande C (2010) Left-right asymmetries in Spiralia. Integr Comp Biol 50:744–755CrossRefPubMedGoogle Scholar
  13. Grande C, Martin-Duran JM, Kenny NJ, Truchado-Garcia M, Hejnol A (2014) Evolution, divergence and loss of the Nodal signalling pathway: new data and a synthesis across the Bilateria. Int J Dev Biol 58:521–532CrossRefPubMedGoogle Scholar
  14. Grande C, Patel NH (2009) Nodal signalling is involved in left-right asymmetry in snails. Nature 457:1007–1011CrossRefPubMedGoogle Scholar
  15. Harvey RP (1998) Links in the left/right axial pathway. Cell 94:273–276CrossRefPubMedGoogle Scholar
  16. His E (1996) Embryogenesis and larval development in Crassostrea gigas, experimental data and field observations on the effect of tributyltin compounds. In: Champ MA, Seligman PF (eds) Organotin. Chapman & Hall, LondonGoogle Scholar
  17. Hudson C, Yasuo H (2005) Patterning across the ascidian neural plate by lateral Nodal signalling sources. Development 132:1199–1210CrossRefPubMedGoogle Scholar
  18. Ihle JN (1996) STATs: signal transducers and activators of transcription. Cell 84:331–334CrossRefPubMedGoogle Scholar
  19. Jezkova E, Kajo K, Zubor P, Grendar M, Malicherova B, Mendelova A, Dokus K, Lasabova Z, Plank L, Danko J (2016) Methylation in promoter regions of PITX2 and RASSF1A genes in association with clinicopathological features in breast cancer patients. Tumor Biol 37:15707–15718CrossRefGoogle Scholar
  20. Kaartinen V, Warburton D (2003) Fibrillin controls TGF-β activation. Nat Genet 33:331–332CrossRefPubMedGoogle Scholar
  21. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL (2013) TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14:R36CrossRefPubMedPubMedCentralGoogle Scholar
  22. Kirchhof P, Kahr PC, Kaese S, Piccini I, Vokshi I, Scheld HH, Rotering H, Fortmueller L, Laakmann S, Verheule S (2011) PITX2c is expressed in the adult left atrium, and reducing Pitx2c expression promotes atrial fibrillation inducibility and complex changes in gene expression. Circ Cardiovasc Genet 4:123–133CrossRefPubMedGoogle Scholar
  23. Kishore U, Gaboriaud C, Waters P, Shrive AK, Greenhough TJ, Reid KBM, Sim RB, Arlaud GJ (2004) C1q and tumor necrosis factor superfamily: modularity and versatility. Trends Immunol 25:551–561CrossRefPubMedGoogle Scholar
  24. Kumari A, Srinivasan R, Vasishta RK, Wig JD (2009) Positive regulation of human telomerase reverse transcriptase gene expression and telomerase activity by DNA methylation in pancreatic cancer. Ann Surg Oncol 16:1051–1059CrossRefPubMedGoogle Scholar
  25. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359CrossRefPubMedPubMedCentralGoogle Scholar
  26. Levin M (1997) Left-right asymmetry in vertebrate embryogenesis. BioEssays 19:287–296CrossRefPubMedGoogle Scholar
  27. Levin M (2005) Left-right asymmetry in embryonic development: a comprehensive review. Mech Dev 122:3–25CrossRefPubMedGoogle Scholar
  28. Liang X, Su J, Zheng G, Jian L, Zhang G, Wang H, Xie L, Zhang R (2013) Correction: patterns of expression in the matrix proteins responsible for nucleation and growth of aragonite crystals in flat pearls of Pinctada fucata. PLoS One 8:e66564CrossRefPubMedPubMedCentralGoogle Scholar
  29. Lin J, Patel SR, Cheng X, Cho EA, Levitan I, Ullenbruch M, Phan SH, Park JM, Dressler GR (2005) Kielin/chordin-like protein, a novel enhancer of BMP signaling, attenuates renal fibrotic disease. Nat Med 11:387–393CrossRefPubMedGoogle Scholar
  30. Livak KJ, Schmittgen TD (2012) Analysis of relative gene expression data using real-time quantitative PCR and the 2–△△CT method. Methods 25:402–408CrossRefGoogle Scholar
  31. Lowe CJ (2008) Molecular genetic insights into deuterostome evolution from the direct-developing hemichordate Saccoglossus kowalevskii. Philos Trans R Soc Lond B Biol Sci 363:1569–1578CrossRefPubMedPubMedCentralGoogle Scholar
  32. Lowe CJ, Terasaki M, Wu M, Jr RMF, Runft L, Kwan K, Haigo S, Aronowicz J, Lander E, Gruber C (2006) Dorsoventral patterning in hemichordates: insights into early chordate evolution. PLoS Biol 4:e291CrossRefPubMedPubMedCentralGoogle Scholar
  33. Márquez-Aliaga A, Jiménez-Jiménez AP, Checa AG, Hagdorn H (2005) Early oysters and their supposed Permian ancestors. Palaeogeogr Palaeoclimatol Palaeoecol 229:127–136CrossRefGoogle Scholar
  34. Martin-Duran JM, Vellutini BC, Hejnol A (2016) Embryonic chirality and the evolution of spiralian left-right asymmetries. Philos Trans R Soc Lond B Biol Sci 371(1710):20150411CrossRefPubMedPubMedCentralGoogle Scholar
  35. Meehan RR, Stancheva I (2001) DNA methylation and control of gene expression in vertebrate development. Essays Biochem 37:59–70CrossRefPubMedGoogle Scholar
  36. Mercer DK, Iqbal M, Miller P, Mccarthy AJ (1996) Screening actinomycetes for extracellular peroxidase activity. Appl Environ Microbiol 62:2186–2190PubMedPubMedCentralGoogle Scholar
  37. Mercola M (2003) Left-right asymmetry: nodal points. J Cell Sci 116:3251–3257CrossRefPubMedGoogle Scholar
  38. Mercola M, Levin M (2001) Left-right asymmetry determination in vertebrates. Annu Rev Cell Dev Biol 17:779–805CrossRefPubMedGoogle Scholar
  39. Mika A, Minibayeva F, Beckett R, Lüthje S (2004) Possible functions of extracellular peroxidases in stress-induced generation and detoxification of active oxygen species. Phytochem Rev 3:173–193CrossRefGoogle Scholar
  40. Miyamoto H, Kajihara K (2005) Conserved ribosomal protein sequences S5, S18, S27, S30 in the Pacific oyster Crassostrea gigas and Crassostrea virginica. Memoirs of the School of Biology-Oriented Science and Technology of Kinki University, vol 16, pp 1–5Google Scholar
  41. Namigai EK, Kenny NJ, Shimeld SM (2014) Right across the tree of life: the evolution of left–right asymmetry in the Bilateria. Genesis 52:458–470CrossRefPubMedGoogle Scholar
  42. Pai VP, Vandenberg LN, Blackiston D, Levin M (2012) Neurally derived tissues in Xenopus laevis embryos exhibit a consistent bioelectrical left-right asymmetry. Stem Cells Int 2012:353491CrossRefPubMedPubMedCentralGoogle Scholar
  43. Palmer AR (2009) Animal asymmetry. Curr Biol 19:R473–R477CrossRefPubMedGoogle Scholar
  44. Paps J, Xu F, Zhang G, Holland PWH (2015) Reinforcing the egg-timer: recruitment of novel Lophotrochozoa homeobox genes to early and late development in the Pacific oyster. Genome Biol Evol 7:677–688CrossRefPubMedPubMedCentralGoogle Scholar
  45. Piedra ME, Icardo JM, Albajar M, Rodriguez-Rey JC, Ros MA (1998) Pitx2 participates in the late phase of the pathway controlling left-right asymmetry. Cell 94:319–324CrossRefPubMedGoogle Scholar
  46. Polansky JK, Kretschmer K, Freyer J, Floess S, Garbe A, Baron U, Olek S, Hamann A, Von BH, Huehn J (2008) DNA methylation controls Foxp3 gene expression. Eur J Immunol 38:1654–1663CrossRefPubMedGoogle Scholar
  47. Ponder WF, Lindberg DR (2010) Towards a phylogeny of gastropod molluscs: an analysis using morphological characters. Zool J Linnean Soc 119:83–265CrossRefGoogle Scholar
  48. Razin A, Kantor B (2005) DNA methylation in epigenetic control of gene expression. Prog Mol Subcell Biol 38:151–167Google Scholar
  49. Robinson MD, Mccarthy DJ, Smyth GK (2010) edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140CrossRefPubMedGoogle Scholar
  50. Ryan AK, Blumberg B, Rodriguezesteban C, Yoneitamura S, Tamura K, Tsukui T, Peña JDL, Sabbagh W, Greenwald J, Choe S (1998) Pitx2 determines left-right asymmetry of internal organs in vertebrates. Nature 394:545–551CrossRefPubMedGoogle Scholar
  51. Tang HK, Chao LY, Saunders GF (1997) Functional analysis of paired box missense mutations in the PAX6 gene. Hum Mol Genet 6:381–386CrossRefPubMedGoogle Scholar
  52. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, Van Baren MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:511–515CrossRefPubMedPubMedCentralGoogle Scholar
  53. Wan J, Oliver VF, Wang G, Zhu H, Zack DJ, Merbs SL, Qian J (2015) Characterization of tissue-specific differential DNA methylation suggests distinct modes of positive and negative gene expression regulation. BMC Genomics 16:49CrossRefPubMedPubMedCentralGoogle Scholar
  54. Wang J, Wu C, Xu C, Yu WC, Li Z, Li Y, Guo T, Wang X (2015a) Voltage-gated potassium ion channel may play a major role in the settlement of Pacific oyster ( Crassostrea gigas ) larvae. Aquaculture 442:48–50CrossRefGoogle Scholar
  55. Wang S, Lv J, Zhang L, Dou J, Sun Y, Li X, Fu X, Dou H, Mao J, Hu X, Bao Z (2015b) MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes. Open Biol 5.  https://doi.org/10.1098/rsob.150130
  56. Wang X, Li L, Zhu Y, Du Y, Song X, Chen Y, Huang R, Que H, Fang X, Zhang G (2013) Oyster shell proteins originate from multiple organs and their probable transport pathway to the Shell formation front. PLoS One 8:e66522CrossRefPubMedPubMedCentralGoogle Scholar
  57. Weiss G, Cottrell S, Distler J, Schatz P, Kristiansen G, Ittmann M, Haefliger C, Lesche R, Hartmann A, Corman J, Wheeler T (2009) DNA methylation of the PITX2 gene promoter region is a strong independent prognostic marker of biochemical recurrence in patients with prostate cancer after radical prostatectomy. J Urol 181:1678–1685CrossRefPubMedGoogle Scholar
  58. Wlizla, M. (2011). Evolution of nodal signaling in deuterostomes: insights from Saccoglossus kowalevskii. Dissertations & Theses - GradworksGoogle Scholar
  59. Yano M, Nagai K, Morimoto K, Miyamoto H (2006) Shematrin: a family of glycine-rich structural proteins in the shell of the pearl oyster Pinctada fucata. Comp Biochem Physiol B Biochem Mol Biol 144:254–262CrossRefPubMedGoogle Scholar
  60. Zhang G, Fang X, Guo X, Li L, Luo R, Xu F, Yang P, Zhang L, Wang X, Qi H (2012) The oyster genome reveals stress adaptation and complexity of shell formation. Nature 490:49–54CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Lei Wei
    • 1
  • Fei Xu
    • 2
  • Yuzhi Wang
    • 3
  • Zhongqiang Cai
    • 4
  • Wenchao Yu
    • 1
  • Cheng He
    • 1
  • Qiuyun Jiang
    • 1
  • Xiqiang Xu
    • 5
  • Wen Guo
    • 6
  • Xiaotong Wang
    • 1
  1. 1.School of AgricultureLudong UniversityYantaiChina
  2. 2.Institute of OceanologyChinese Academy of SciencesQingdaoChina
  3. 3.School of Life SciencesShandong UniversityJinanChina
  4. 4.Changdao Enhancement and Experiment StationChinese Academy of Fishery SciencesChangdaoChina
  5. 5.Qingdao OE Biotechnology Company LimitedQingdaoChina
  6. 6.Marine Biology Institute of Shandong ProvinceQingdaoChina

Personalised recommendations