Expression and function of WNT4 involved in larvae development and limb regeneration in Portunus trituberculatus

Abstract

The wingless-related integration site (WNT) proteins are a family of secreted glycoproteins that are evolutionarily conserved and are believed to be involved in evolution in vertebrates and invertebrates. WNT signaling pathways may be associated with limb regeneration and development in crustaceans. However, the detail mechanisms remain unclear. Therefore, the distribution of WNT4 in the hepatopancreasmuscle, hemocyte, ganglion, heart, eyestalk, gill tissue, and different larvae development stages of the swimming crab (Portunus trituberculatus) were characterized using immunofluorescence, real-time PCR, and Western blotting. Significant PtWNT4 expression was detected in heart and eyestalk. In addition-PtWNT4 was expressed in all larval stages of P. trituberculatus with a dynamic expression pattern, especially in the eyestalk and other organs in the carapace area. The injection of WNT4 dsRNA into regenerative limbs significantly decreased PtWNT4 mRNA levels in the eyestalk, heart, and muscle, resulting in 1.9-fold, 2.2-fold, and 2.7-fold decreases compared with those detected in the group injected with crab saline (P<0.05), respectively, indicating successful gene silencing. Overall, expression analysis on the WNT4 using RNAi provides an insight to its functional mechanism during limb regeneration in P. trituberculatus. The results not only demonstrated the requirement for WNT4 in limb regeneration of Crustaceans, but also suggested its ability to promote larval development at specific stages.

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Abbreviations

CXCR4:

C-X-C motif chemokine receptor 4

SHH:

sonic hedgehog

WNT:

wingless-related integration site

BCC:

basal cell carcinoma

WNT4:

wingless-type MMTV integration site family member-4

WNT3a:

wingless-type MMTV integration site family member-3a

WNT5a:

wingless-type MMTV integration site family member-5a

WNT7a:

wingless-type MMTV integration site family member-7a

GFP:

green fluorescent protein

References

  1. Agata K, Tanaka T, Kobayashi C, Kato K, Saitoh Y. 2003. Intercalary regeneration in planarians. Developmental Dynamics, 226(2): 308–316, https://doi.org/10.1002/dvdy.10249.

    Article  Google Scholar 

  2. Bely A E, Nyberg K G. 2010. Evolution of animal regeneration: re-emergence of a field. Trends in Ecology & Evolution, 25(3): 161–170, https://doi.org/10.1016/j.tree.2009.08.005.

    Article  Google Scholar 

  3. Caprioli A, Villasenor A, Wylie L A, Braitsch C, Marty-Santos L, Barry D, Karner C M, Fu S, Meadows S M, Carroll T J, Cleaver O. 2015. Wnt4 is essential to normal mammalian lung development. Developmental Biology, 406(2): 222–234, https://doi.org/10.1016/j.ydbio.2015.08.017.

    Article  Google Scholar 

  4. Chen Y, Alman B A. 2009. Wnt pathway, an essential role in bone regeneration. Journal of Cellular Biochemistry, 106(3): 353–362, https://doi.org/10.1002/jcb.22020.

    Article  Google Scholar 

  5. Chen Z M, Zhu J Y, Fu Y L, Richman A, Han Z. 2016. Wnt4 is required for Ostia development in the Drosophila heart. Developmental Biology, 413(2): 188–198, https://doi.org/10.1016/j.ydbio.2016.03.008.

    Article  Google Scholar 

  6. Cooper R L. 1998. Development of sensory processes during limb regeneration in adult crayfish. Journal of Experimental Biology, 201(Pt 11): 1 745–1 752.

    Google Scholar 

  7. Fleming P A, Muller D, Bateman P W. 2007. Leave it all behind: a taxonomic perspective of autotomy in invertebrates. Biological Reviews, 82(3): 481–510, https://doi.org/10.1111/j.1469-185X.2007.00020.x.

    Article  Google Scholar 

  8. Fradkin L G, Dura J M, Noordermeer J N. 2010. Ryks: new partners for Wnts in the developing and regenerating nervous system. Trends in Neurosciences, 33(2): 84–92, https://doi.org/10.1016/j.tins.2009.11.005.

    Article  Google Scholar 

  9. Fu Y Y, Liu L, Wang C L, Zhu F, Liu X. 2019. Suppression of limb regeneration by RNA interference of WNT4 in the swimming crab Portunus trituberculatus. Comparative Biochemistry and PhysiologyPart D: Genomics and Proteomics, 234: 41–49, https://doi.org/10.1016/j.cbpb.2019.05.001.

    Article  Google Scholar 

  10. Fu Y Y, Zhu F, Liu L, Lu S K, Ren Z M, Mu C K, Li R H, Song W W, Shi C, Ye Y F, Wang C L. 2018. iTRAQ-based proteomic analysis identifies proteins involved in limb regeneration of swimming crab Portunus trituberculatus. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 26: 10–19, https://doi.org/10.1016/j.cbd.2018.02.003.

    Google Scholar 

  11. Grau S M, Cooke I M. 1992. Peptidergic neurons of the crab, Cardisoma carnifex, in defined culture maintain characteristic morphologies under a variety of conditions. Cell and Tissue Research, 270(2): 303–317, https://doi.org/10.1007/BF00328016.

    Article  Google Scholar 

  12. Guo X Z, Day T F, Jiang X Y, Garrett-Beal L, Topol L, Yang Y Z. 2004. Wnt/β-catenin signaling is sufficient and necessary for synovial joint formation. Genes & Development, 18(19): 2 404–2 417, https://doi.org/10.1101/gad.1230704.

    Article  Google Scholar 

  13. Haas B J, Whited J L. 2017. Advances in decoding Axolotl limb regeneration. Trends in Genetics, 33(8): 553–565, https://doi.org/10.1016/j.tig.2017.05.006.

    Article  Google Scholar 

  14. Hamilton P W, Sun Y, Henry J J. 2016. Lens regeneration from the cornea requires suppression of Wnt/β-catenin signaling. Experimental Eye Research, 145: 206–215, https://doi.org/10.1016/j.exer.2016.01.003.

    Article  Google Scholar 

  15. Hartmann C, Tabin C J. 2000. Dual roles of Wnt signaling during chondrogenesis in the chicken limb. Development, 127(14): 3 141–3 159.

    Google Scholar 

  16. Harzsch S, Vilpoux K, Blackburn D C, Platchetzki D, Brown N L, Melzer R, Kempler K E, Battelle B A. 2006. Evolution of arthropod visual systems: development of the eyes and central visual pathways in the horseshoe crab Limulus polyphemus Linnaeus, 1758 (Chelicerata, Xiphosura). Developmental Dynamics, 235(10): 2 641–2 655, https://doi.org/10.1002/dvdy.20866.

    Article  Google Scholar 

  17. He J, Gao Y, Wang W, Xie J J, Shi H, Wang G S, Xu W J. 2016. Limb autotomy patterns in the juvenile swimming crab (Portunus trituberculatus) in earth ponds. Aquaculture, 463. 189–192, https://doi.org/10.1016/j.aquaculture.2016.05.043.

    Article  Google Scholar 

  18. Hopkins P M. 1993. Regeneration of walking legs in the fiddler crab Uca pugilator. American Zoologist, 33(3): 348–356, https://doi.org/10.1093/icb/33.3.348.

    Article  Google Scholar 

  19. Hopkins P M. 2001. Limb regeneration in the fiddler crab, Uca pugilator: hormonal and growth factor control. American Zoologist, 41(3): 389–398, https://doi.org/10.1093/icb/41.3.389.

    Google Scholar 

  20. Hunger C, Ödemis V, Engele J. 2012. Expression and function of the SDF-1 chemokine receptors CXCR4 and CXCR7 during mouse limb muscle development and regeneration. Experimental Cell Research, 318(17): 2 178–2 190, https://doi.org/10.1016/j.yexcr.2012.06.020.

    Article  Google Scholar 

  21. Iglesias M, Almuedo-Castillo M, Aboobaker A A, Saló E. 2011. Early planarian brain regeneration is independent of blastema polarity mediated by the wnt/β-catenin pathway. Developmental Biology, 358(1): 68–78, https://doi.org/10.1016/j.ydbio.2011.07.013.

    Article  Google Scholar 

  22. Kobayashi T, Tanaka H, Kuwana H, Inoshita S, Teraoka H, Sasaki S, Terada Y. 2005. Wnt4-transformed mouse embryonic stem cells differentiate into renal tubular cells. Biochemical and Biophysical Research Communications, 336(2): 585–595, https://doi.org/10.1016/j.bbrc.2005.08.136.

    Article  Google Scholar 

  23. Kragl M, Knapp D, Nacu E, Khattak S, Maden M, Epperlein H H, Tanaka E M. 2009. Cells keep a memory of their tissue origin during axolotl limb regeneration. Nature, 460(7251): 60–65, https://doi.org/10.1038/nature08152.

    Article  Google Scholar 

  24. Liu L, Fu Y Y, Zhu F, Mu C K, Li R H, Song W W, Shi C, Ye Y F, Wang C L. 2018. Transcriptomic analysis of Portunus trituberculatus reveals a critical role for WNT4 and WNT signalling in limb regeneration. Gene, 658: 113–122, https://doi.org/10.1016/j.gene.2018.03.015.

    Article  Google Scholar 

  25. Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods, 25(4): 402–408, https://doi.org/10.1006/meth.2001.1262.

    Article  Google Scholar 

  26. Maginnis T L. 2006. The costs of autotomy and regeneration in animals: a review and framework for future research. Behavioral Ecology, 17(5): 857–872, https://doi.org/10.1093/beheco/arl010.

    Article  Google Scholar 

  27. Majidinia M, Aghazadeh J, Jahanban-Esfahlani R, Yousefi B. 2018. The roles of Wnt/β-catenin pathway in tissue development and regenerative medicine. Journal of Cellular Physiology, 233(8): 5 598–5 612, https://doi.org/10.1002/jcp.26265.

    Article  Google Scholar 

  28. Nicol B, Guerin A, Fostier A, Guiguen Y 2012. Ovary-predominant wnt4 expression during gonadal differentiation is not conserved in the rainbow trout (Oncorhynchus mykiss). Molecular Reproduction & Development, 79(1): 51–63, https://doi.org/10.1002/mrd.21404.

    Article  Google Scholar 

  29. Roy R, Gardiner D M. 2002. Cyclopamine induces digit loss in regenerating axolotl limbs. Journal of Experimental Zoology, 293(2): 186–190, https://doi.org/10.1002/jez.10110.

    Article  Google Scholar 

  30. Sagi A, Manor R, Ventura T. 2013. Gene silencing in crustaceans: from basic research to biotechnologies. Genes, 4(4): 620–645, https://doi.org/10.3390/genes4040620.

    Article  Google Scholar 

  31. Satoh A, Suzuki M, Amano T, Tamura K, Ide H. 2005. Joint development in Xenopus laevis and induction of segmentations in regenerating froglet limb (spike). Developmental Dynamics, 233(4): 1 444–1 453, https://doi.org/10.1002/dvdy.20484.

    Article  Google Scholar 

  32. Schubert M, Holland L Z, Holland N D. 2000. Characterization of two amphioxus Wnt genes (AmphiWnt4 and AmphiWnt7b) with early expression in the developing central nervous system. Developmental Dynamics, 217(2): 205–215, https://doi.org/10.1002/(SICI)1097-0177(200002)217:2<205::AID-DVDY7>3.0.CO;2-F.

    Article  Google Scholar 

  33. Singh B N, Doyle M J, Weaver C V, Koyano-Nakagawa N, Garry D J. 2012. Hedgehog and Wnt coordinate signaling in myogenic progenitors and regulate limb regeneration. Developmental Biology, 371(1): 23–34, https://doi.org/10.1016/j.ydbio.2012.07.033.

    Article  Google Scholar 

  34. Stueckle T A, Likens J, Foran C M. 2008. Limb regeneration and molting processes under chronic methoprene exposure in the mud fiddler crab, Uca pugnax. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 147(3): 366–377, https://doi.org/10.1016/j.cbpc.2008.01.004.

    Google Scholar 

  35. Sun Y M, Yan Y, Zhang J J. 1984. The larval development of Portunus trituberculatus. Journal of Fisheries of China, 8(3): 219–226. (in Chinese with English abstract)

    Google Scholar 

  36. Tauc H M, Mann T, Werner K, Pandur P. 2012. A role for Drosophila Wnt-4 in heart development. Genesis, 50(6): 466–481, https://doi.org/10.1002/dvg.22021.

    Article  Google Scholar 

  37. Technau U, von Laue C C, Rentzsch F, Luft S, Hobmayer B, Bode H R, Holstein T W. 2000. Parameters of self-organization in Hydra aggregates. Proceedings of the National Academy of Sciences of the United States of America, 97(22): 12 127–12 131, https://doi.org/10.1073/pnas.97.22.12127.

    Article  Google Scholar 

  38. von Maltzahn J V, Chang N C, Bentzinger C F, Rudnicki M A. 2012. Wnt signaling in myogenesis. Trends in Cell Biology, 22(11): 602–609, https://doi.org/10.1016/j.tcb.2012.07.008.

    Article  Google Scholar 

  39. Zhang S, Li C Z, Yang Q H, Dong X H, Chi S Y, Liu H Y, Shi L L, Tan B P. 2016. Molecular cloning, characterization and expression analysis of Wnt4, Wnt5, Wnt6, Wnt7, Wnt10 and Wnt16 from Litopenaeus vannamei. Fish & Shellfish Immunology, 54: 445–455, https://doi.org/10.1016/j.fsi.2016.04.028.

    Article  Google Scholar 

  40. Zhu F, Fu Y Y, Mu C K, Liu Lei, Li R H, Song W W, Shi C, Ye Y F, Wang C L. 2018. Molecular cloning, characterization and effects of catechol-o-methyltransferase (comt) mRNA and protein on aggressive behavior in the swimming crab Portunus trituberculatus. Aquaculture, 495: 693–702, https://doi.org/10.1016/j.aquaculture.2018.06.055.

    Article  Google Scholar 

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Acknowledgment

The authors are grateful to all the laboratory members for their technical advice and helpful discussion.

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Corresponding authors

Correspondence to Lei Liu or Chunlin Wang.

Additional information

Supported by the National Natural Science Foundation of China (No. 31602152), the Major Agriculture Program of Ningbo (No. 2017C110007), and the K. C. Wong Magana Fund in Ningbo University. The funding body had no role in the study design, experimental implementation, interpretation of data, or writing of the manuscript

Data Availability Statement

All data generated and/or analyzed during this study are available from the corresponding author, LIU Lei, upon reasonable request.

Authors’ Contribution

REN Zhiming, FU Yuanyuan, and LIU Lei carried out the immunofluorescence, real-time PCR, and Western blotting experiments, LIU Xiao performed the RNAi experiments and data analysis. Experiments were planned and analyzed by LIU Lei and WANG Chunlin. Manuscript preparation was done by REN Zhiming, FU Yuanyuan, and LIU Lei.

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Ren, Z., Fu, Y., Liu, L. et al. Expression and function of WNT4 involved in larvae development and limb regeneration in Portunus trituberculatus. J. Ocean. Limnol. 39, 306–316 (2021). https://doi.org/10.1007/s00343-020-9291-6

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Keyword

  • wingless-type MMTV integration site family member-4 (WNT4)
  • limb regeneration
  • larvae development
  • Portunus trituberculatus
  • expression pattern
  • RNAi