Skip to main content

Advertisement

SpringerLink
Log in

Proteomic Analysis Reveals the Contribution of TGFβ/Smad4 Signaling Pathway to Cell Differentiation During Planarian Tail Regeneration

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

After planarian tail is cut off, posterior end of the remaining fragment will regenerate a new tail within about 1 week. However, many details of this process remain unclear up to date. For this reason, we performed the dynamic proteomic analysis of the regenerating tail fragments at 6, 12, 24, 72, 120, and 168 h post-amputation (hpa). Using two-dimensional electrophoresis (2-DE) in combination with MALDI-TOF-TOF/MS analysis, a total of 1088 peptides were identified as significantly changed between tail-cutting groups and 0-h group, 482 of which have identifiable protein names. Of these 482 proteins, there were 111 originating from the Turbellaria. Protein functional categorization showed that these 111 proteins are mainly related to differentiation and development, transcription and translation, cell signal transduction, and cell proliferation. The screening of key protein considered the transcription factor Smad4 as important protein for planarian tail regeneration. Cell signaling pathway analysis, combined with proteomic profiling of regenerating tail fragment, showed that TGFβ/Smad4 pathway was activated during planarian tail regeneration. Based on a comprehensive analysis of 2-DE MALDI-TOF-TOF/MS and bioinformatics analyses, it could be concluded that TGFβ/Smad4 pathway perhaps plays an important role in tail regeneration via promoting cell differentiation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

Hpa:

Hours post-amputation

CNS:

Central nervous system

CHAPS:

3-((3-Cholamido-propyl) dimethylammonio)-1-propanesulfonate

IEF:

Isoelectric focusing

CBB:

Coomassie brilliant blue

HCCA:

α-Cyano-4-hydroxycinnamic acid

TFA:

Trifluoroacetic acid

ACN:

Acetonitrile

qRT-PCR:

Quantitative real-time PCR

SNR:

Signal to noise ratio

RFSC:

Revised feature score criterion

BD:

Bhattacharyya distance

ED:

Euclidean distance

PCC:

Pearson correlation coefficient

FC:

Fold change

References

  1. Gentile, L., Cebrià, F., & Bartscherer, K. (2011). The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration. Disease Models & Mechanisms, 4, 12–19.

    Article  CAS  Google Scholar 

  2. Newmark, P. A., Wang, Y., & Chong, T. (2008). Germ cell specification and regeneration in planarians. Cold Spring Harbor Symposia on Quantitative Biology, 73, 573–581.

    Article  CAS  Google Scholar 

  3. Sikes, J. M., & Newmark, P. A. (2013). Restoration of anterior regeneration in a planarian with limited regenerative ability. Nature, 500, 77–80.

    Article  CAS  Google Scholar 

  4. Yazawa, S., Umesono, Y., Hayashi, T., Tarui, H., & Agata, K. (2009). Planarian Hedgehog/Patched establishes anterior-posterior polarity by regulating Wnt signaling. Proceedings of the National Academy of Sciences U S A, 106, 22329–22334.

    Article  CAS  Google Scholar 

  5. Adell, T., Cebrià, F., & Saló, E. (2010). Gradients in planarian regeneration and homeostasis. Cold Spring Harbor Perspectives in Biology, 2, a000505.

    Article  Google Scholar 

  6. Baguñà, J., Romero, R., Saló, E., Collet, J., Auladell, C., Ribas, M., Riutort, M., García-Fernàndez, J., Burgaya, F., & Bueno, D. (1990). Growth, degrowth and regeneration as developmental phenomena in adult freshwater planarians. In H.-J. Marthy (Ed.), Experimental embryology in aquatic plants and animals (pp. 129–162). New York: Plenum Press.

    Chapter  Google Scholar 

  7. Newmark, P. A., & Sánchez Alvarado, A. (2000). Bromodeoxyuridine specifically labels the regenerative stem cells of planarians. Developmental Biology, 220, 142–153.

    Article  CAS  Google Scholar 

  8. Agata, K., & Umesono, Y. (2008). Brain regeneration from pluripotent stem cells in planarian. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 363, 2071–2078.

    Article  CAS  Google Scholar 

  9. Sandmann, T., Vogg, M. C., Owlarn, S., Boutros, M., & Bartscherer, K. (2011). The head-regeneration transcriptome of the planarian Schmidtea mediterranea. Genome Biology, 12, R76.

    Article  CAS  Google Scholar 

  10. Liu, S. Y., Selck, C., Friedrich, B., Lutz, R., Vila-Farré, M., Dahl, A., Brandl, H., Lakshmanaperumal, N., Henry, I., & Rink, J. C. (2013). Reactivating head regrowth in a regeneration-deficient planarian species. Nature, 500, 81–84.

    Article  CAS  Google Scholar 

  11. Fraguas, S., Barberán, S., Iglesias, M., Rodríguez-Esteban, G., & Cebrià, F. (2014). Egr-4, a target of EGFR signaling, is required for the formation of the brain primordia and head regeneration in planarians. Development, 141, 1835–1847.

    Article  CAS  Google Scholar 

  12. Conte, M., Deri, P., Isolani, M. E., Mannini, L., & Batistoni, R. (2009). A mortalin-like gene is crucial for planarian stem cell viability. Development Biology, 334, 109–118.

    Article  CAS  Google Scholar 

  13. Rink, J. C., Vu, H. T., & Sánchez Alvarado, A. (2011). The maintenance and regeneration of the planarian excretory system are regulated by EGFR signaling. Development, 138, 3769–3780.

    Article  CAS  Google Scholar 

  14. Hubert, A., Henderson, J. M., Ross, K. G., Cowles, M. W., Torres, J., & Zayas, R. M. (2013). Epigenetic regulation of planarian stem cells by the SET1/MLL family of histone methyltransferases. Epigenetics, 8, 79–91.

    Article  CAS  Google Scholar 

  15. Kao, D., Felix, D., & Aboobaker, A. (2013). The planarian regeneration transcriptome reveals a shared but temporally shifted regulatory program between opposing head and tail scenarios. BMC Genomics, 14, 797.

    Article  Google Scholar 

  16. Kaur, B., Sharma, M., Soni, R., Oberoi, H. S., & Chadha, B. S. (2013). Proteome-based profiling of hypercellulase-producing strains developed through interspecific protoplast fusion between Aspergillus nidulans and Aspergillus tubingensis. Applied Biochemistry and Biotechnology, 169, 393–407.

    Article  CAS  Google Scholar 

  17. Tejada-Romero, B., Carter, J. M., Mihaylova, Y., Neumann, B., & Aboobaker, A. A. (2015). JNK signalling is necessary for a Wnt- and stem cell-dependent regeneration programme. Development, 142, 2413–2424.

    Article  CAS  Google Scholar 

  18. Gurley, K. A., Elliott, S. A., Simakov, O., Schmidt, H. A., Holstein, T. W., & Sánchez Alvarado, A. (2010). Expression of secreted Wnt pathway components reveals unexpected complexity of the planarian amputation response. Developmental Biology, 347, 24–39.

    Article  CAS  Google Scholar 

  19. Gurley, K. A., Rink, J. C., & Sánchez Alvarado, A. (2008). Beta-catenin defines head versus tail identity during planarian regeneration and homeostasis. Science, 319, 323–327.

    Article  CAS  Google Scholar 

  20. Long, A. B., Kaiser, W. J., Mocarski, E. S., & Caspary, T. (2013). Apaf1 apoptotic function critically limits Sonic hedgehog signaling during craniofacial development. Cell Death and Differentiation, 20, 1510–1520.

    Article  CAS  Google Scholar 

  21. Reuter, H., März, M., Vogg, M. C., Eccles, D., Grífol-Boldú, L., Wehner, D., Owlarn, S., Adell, T., Weidinger, G., & Bartscherer, K. (2015). β-Catenin-Dependent Control of Positional Information along the AP Body Axis in Planarians Involves a Teashirt Family Member. Cell Report, 10, 253–265.

    Article  CAS  Google Scholar 

  22. Fraguas, S., Barberán, S., & Cebrià, F. (2011). EGFR signaling regulates cell proliferation, differentiation and morphogenesis during planarian regeneration and homeostasis. Developmental Biology, 354, 87–101.

    Article  CAS  Google Scholar 

  23. Adler, C. E., Seidel, C. W., McKinney, S. A., & Sánchez Alvarado, A. (2014). Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria. eLife, 3, e02238.

    Article  Google Scholar 

  24. Salo, E., Pineda, D., Marsal, M., Gonzalez, J., Gremigni, V., & Batistoni, R. (2002). Genetic network of the eye in Platyhelminthes: expression and functional analysis of some players during planarian regeneration. Gene, 287, 67–74.

    Article  CAS  Google Scholar 

  25. Irvine, S. Q., & Martindale, M. Q. (2000). Expression patterns of anterior Hox genes in the polychaete Chaetopterus: correlation with morphological boundaries. Developmental Biology, 217, 333–351.

    Article  CAS  Google Scholar 

  26. Isolani, M. E., Abril, J. F., Saló, E., Deri, P., Bianucci, A. M., & Batistoni, R. (2013). Planarians as a model to assess in vivo the role of matrix metalloproteinase genes during homeostasis and regeneration. PLoS One, 8, 55649.

    Article  Google Scholar 

  27. Cebrià, F., & Newmark, P. A. (2007). Morphogenesis defects are associated with abnormal nervous system regeneration after roboA RNAi in planarians. Development, 134, 833–837.

    Article  Google Scholar 

  28. Saló, E., Tauler, J., Jimenez, E., Bayascas, J. R., Gonzalez, J., Garcia Fernandez, J., & Baguña, J. (2001). Hox and parahox genes in flatworms: characterization and expression. American Zoologist, 41, 652–663.

    Google Scholar 

  29. Callaerts, P., Muñoz-Marmol, A. M., Glardon, S., Castillo, E., Sun, H., Li, W. H., Gehring, W. J., & Saló, E. (1999). Isolation and expression of a Pax-6 gene in the regenerating and intact Planarian Dugesia(G)tigrina. Proceedings of the National Academy of Sciences U S A, 96, 558–563.

    Article  CAS  Google Scholar 

  30. Reddien, P. W., Bermange, A. L., Murfitt, K. J., Jennings, J. R., & Sánchez Alvarado, A. (2005). Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria. Developmental Cell, 8, 635–649.

    Article  CAS  Google Scholar 

  31. Weimer, J. M., Stanco, A., Cheng, J. G., Vargo, A. C., Voora, S., & Anton, E. S. (2008). A BAC Transgenic Mouse Model to Analyze the Function of Astroglial SPARCL1 (SC1) in the Central Nervous System. Glia, 56, 935–941.

    Article  Google Scholar 

  32. Rouhana, L., Shibata, N., Nishimura, O., & Agata, K. (2010). Different requirements for conserved posttranscriptional regulators in planarian regeneration and stem cell maintenance. Developmental Biology, 341, 429–443.

    Article  CAS  Google Scholar 

  33. Salvetti, A., Batistoni, R., Deri, P., Rossi, L., & Sommerville, J. (1998). Expression of DjY1, a protein containing a cold shock domain and RG repeat motifs, is targeted to sites of regeneration in planarians. Developmental Biology, 201, 217–229.

    Article  CAS  Google Scholar 

  34. Di Bernardini, E., Campagnolo, P., Margariti, A., Zampetaki, A., Karamariti, E., Hu, Y., & Xu, Q. (2014). Endothelial lineage differentiation from induced pluripotent stem cells is regulated by microRNA-21 and transforming growth factor β2 (TGF-β2) pathways. Journal of Biological Chemistry, 289, 3383–3393.

    Article  Google Scholar 

  35. Gonzalez-Sastre, A., Molina, M. D., & Salo, E. (2012). Inhibitory Smads and bone morphogenetic protein (BMP) modulate anterior photoreceptor cell number during planarian eye regeneration. International Journal of Developmental Biology, 56, 155–163.

    Article  Google Scholar 

  36. Leonardo, R., Alessandra, S., Francesco, M. M., Annalisa, L., Paolo, D., Linda, M., Renata, B., Ena, W., & Vittorio, G. (2007). Deciphering the molecular machinery of stem cells: a look at the neoblast gene expression profile. Genome Biology, 8, R64.

    Article  Google Scholar 

  37. Shibata, N., Hayashi, T., Fukumura, R., Fujii, J., Kudome-Takamatsu, T., Nishimura, O., Sano, S., Son, F. Y., Suzuki, N., Araki, R., Abe, M., & Agata, K. (2012). Comprehensive gene expression analyses in pluripotent stem cells of a planarian, Dugesia japonica. International Journal of Developmental Biology, 56, 93–102.

    Article  CAS  Google Scholar 

  38. Cebrià, F., & Newmark, P. A. (2007). Morphogenesis defects are associated with abnormal nervous system regeneration after roboA RNAi in planarians. Development, 134, 833–837.

    Article  Google Scholar 

  39. Koinuma, S., Umesono, Y., Watanabe, K., & Agata, K. (2003). The expression of planarian brain factor homologs, DjFoxG and DjFoxD. Gene Expression Patterns, 3, 21–27.

    Article  CAS  Google Scholar 

  40. Huang, Y., Li, Z., & Ye, Q. (2016). Transcriptional regulation of genes involved in 3-hydroxypropionic acid production in response to aeration of recombinant Klebsiella pneumoniae. Applied Biochemistry and Biotechnology, 178, 1129–1140.

    Article  CAS  Google Scholar 

  41. Rao, N., Jhamb, D., Milner, D. J., Li, B., Song, F., Wang, M., Voss, S. R., Palakal, M., King, M. W., Saranjami, B., Nye, H. L., Cameron, J. A., & Stocum, D. L. (2009). Proteomic analysis of blastema formation in regenerating axolotl limbs. BMC Biology, 7, 83.

    Article  Google Scholar 

  42. King, M. W., Neff, A. W., & Mescher, A. L. (2009). Proteomics analysis of regenerating amphibian limbs: changes during the onset of regeneration. The International Journal of Developmental Biology, 53, 955–969.

    Article  CAS  Google Scholar 

  43. Padhi, B. K., Joly, L., Tellis, P., Smith, A., Nanjappa, P., Chevrette, M., Ekker, M., & Akimenko, M. A. (2004). Screen for genes differentially expressed during regeneration of the zebrafish caudal fin. Developmental Dynamics, 231, 527–541.

    Article  CAS  Google Scholar 

  44. Nishidate, M., Nakatani, Y., Kudo, A., & Kawakami, A. (2007). Identification of novel markers expressed during fin regeneration by microarray analysis in medaka fish. Developmental Dynamics, 236, 2685–2693.

    Article  CAS  Google Scholar 

  45. Umesono, Y., & Agata, K. (2009). Evolution and regeneration of the planarian central nervous system. Development, Growth & Differentiation, 51, 185–195.

    Article  CAS  Google Scholar 

  46. Eguizabal, C., Montserrat, N., Veiga, A., & Izpisua Belmonte, J. C. (2013). Dedifferentiation, transdifferentiation, and reprogramming: future directions in regenerative medicine. Seminars in Reproductive Medicine, 31, 82–94.

    Article  Google Scholar 

  47. Stoltz, J. F., Bensoussan, D., De Isla, N., Zhang, L., Han, Z., Magdalou, J., Huselstein, C., Ye, J. S., Li, N., Decot, V. & Reppel, L. (2016). Stem cells and vascular regenerative medicine. Clinical hemorheology and microcirculation.

  48. Tasaki, J., Shibata, N., Nishimura, O., Itomi, K., Tabata, Y., Son, F., Suzuki, N., Araki, R., Abe, M., Agata, K., & Umesono, Y. (2011). ERK signaling controls blastema cell differentiation during planarian regeneration. Development, 138, 2417–2427.

    Article  CAS  Google Scholar 

  49. Penheiter, S. G., Mitchell, H., Garamszegi, N., Edens, M., Doré Jr., J. J., & Leof, E. B. (2002). Internalization-dependent and -independent requirements for transforming growth factor beta receptor signaling via the Smad pathway. Molecular and Cellular Biology, 22, 4750–4759.

    Article  CAS  Google Scholar 

  50. Friedman, M., & Ståhl, S. (2009). Engineered affinity proteins for tumour-targeting applications. Applied Biochemistry and Biotechnology, 53, 1–29.

    Article  CAS  Google Scholar 

  51. Aoki, M., Yamashita, T., & Tohyama, M. (2004). EphA receptors direct the differentiation of mammalian neural precursor cells through a mitogen-activated protein kinase-dependent pathway. Journal of Biological Chemistry, 279, 32643–32650.

    Article  CAS  Google Scholar 

  52. Sánchez Alvarado, A., & Tsonis, P. A. (2006). Bridging the regeneration gap: genetic insights from diverse animal models. Nature Reviews Genetics, 7, 873–884.

    Article  Google Scholar 

  53. Hueston, J. L., & Suprenant, K. A. (2009). Loss of dystrophin and the microtubule-binding protein ELP-1 causes progressive paralysis and death of adult C. elegans. Developmental Dynamics, 238, 1878–1886.

    Article  CAS  Google Scholar 

  54. Runser, S., & Cerletti, N. (1995). Transforming growth factors beta: conformational stability and features of the denaturation of recombinant human transforming growth factors beta 2 and beta 3. Applied Biochemistry and Biotechnology, 22, 39–53.

    CAS  Google Scholar 

  55. Owens, P., Bazzi, H., Engelking, E., Han, G., Christiano, A. M., & Wang, X. J. (2008). Smad4-dependent desmoglein-4 expression contributes to hair follicle integrity. Developmental Biology, 322, 156–166.

    Article  CAS  Google Scholar 

  56. Song, B., Estrada, K. D., & Lyons, K. M. (2009). Smad signaling in skeletal development and regeneration. Cytokine Growth Factor Review, 20, 379–388.

    Article  CAS  Google Scholar 

  57. Orii, H., & Watanabe, K. (2007). Bone morphogenetic protein is required for dorso-ventral patterning in the planarian Dugesia Japonica. Development, Growth & Differentiation, 49, 345–349.

    Article  CAS  Google Scholar 

  58. Reddien, P. W., Bermange, A. L., Kicza, A. M., & Sánchez Alvarado, A. (2007). BMP signaling regulates the dorsal planarian midline and is needed for asymmetric regeneration. Development, 134, 4043–4051.

    Article  CAS  Google Scholar 

  59. Molina, M. D., Salò, E., & Cebrià, F. (2007). The BMP pathway is essential for re-specification and maintenance of the dorsoventral axis in regenerating and intact planarians. Developmental Biology, 311, 79–94.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Our work has been financially supported by the National Natural Science Foundation of China (No. 31401209).

Author information

Authors and Affiliations

  1. Animal Science and Technology School, Henan University of Science and Technology, 263, Kaiyuan Avenue, Luoyang, Henan Province, 471023, China

    Xiaoguang Chen

  2. College of Life Science, Henan Normal University, Xinxiang, 453007, China

    Cunshuan Xu

  3. Co-constructing Key Laboratory for Cell Differentiation Regulation, Xinxiang, 453007, China

    Cunshuan Xu

Authors
  1. Xiaoguang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cunshuan Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xiaoguang Chen.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, X., Xu, C. Proteomic Analysis Reveals the Contribution of TGFβ/Smad4 Signaling Pathway to Cell Differentiation During Planarian Tail Regeneration. Appl Biochem Biotechnol 182, 529–545 (2017). https://doi.org/10.1007/s12010-016-2342-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-016-2342-y

Keywords

Search

Navigation