Regeneration of amputated mice digit tips by including Wnt signaling pathway with CHIR99021 and IWP-2 chemicals in limb organ culture system

Articles in Press

Document Type : Original Article

Authors

1 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, 1665659911, Iran

2 Department of Developmental Biology, University of Science and Culture, Tehran, Iran

10.22038/ijbms.2024.76957.16643

Abstract

Objective(s): Mammals have limited limb regeneration compared to amphibians. The role of Wnt signaling pathways in limb regeneration has rarely been studied. So, this study aimed to investigate the effect of Wnt-signaling using chemicals CHIR99021 and IWP-2 on amputated mice digit tips regeneration in an in vitro organ culture system.
Materials and Methods: The distal phalanx of paws from C57BL/6J mouse fetuses at E14.5, E16.5, and E18.5 was amputated. Then, the hands were cultured for 7 days. Subsequently, paws were treated with 1–50 µg/ml concentration of CHIR99021 and 5–10 µg/ml concentration of IWP-2. Finally, the new tissue regrowth was assessed by histological analysis, immunohistochemistry for BC, TCF1, CAN, K14, and P63 genes, and beta-catenin and Tcf1 genes were evaluated with RT-qPCR. 
Results: The paws of E14.5 and E16.5 days were shrinkaged and compressed after 7 days, so the paws of 18.5E that were alive were selected. As a result, newly-grown masses at digit tips were observed in 25 and 30 µl/ml concentrations of the CHR99021 group but not in the IWP2 treatment (*P<0.05; **P<0.01). qRT-PCR analysis confirmed the significant up-regulation of beta-catenin and Tcf1 genes in CHIR99021 group in comparison to the IWP-2 group (P<0.05). Moreover, Alcian-blue staining demonstrated the presence of cartilage-like tissue at regenerated mass in the CHIR group. In immunohistochemistry analysis beta-catenin, ACN, Keratin-14, and P63 protein expression were observed in digit tips in the CHIR-treated group.  
Conclusion: By activating the Wnt signaling pathway, cartilage-like tissue formed in the blastema-like mass in the mouse’s amputated digit tips. 

Keywords

Main Subjects

References

1. Choi Y, Meng F, Cox CS, Lally KP, Huard J, Li Y. Regeneration and regrowth potentials of digit tips in amphibians and mammals. Int J Cell Biol 2017; 2017:5312951.
2. Eming SA, Martin P, Tomic-Canic M. Wound repair and regeneration: Mechanisms, signaling, and translation. Sci Transl Med 2014; 6:265sr266.
3. Southerland KW, Xu Y, Peters DT, Lin X, Wei X, Xiang Y, et al. Skeletal muscle regeneration failure in ischemic-damaged limbs is associated with pro-inflammatory macrophages and premature differentiation of satellite cells. Genome Med 2023; 15:95-114.
4. Yang Y. Wnts and wing: Wnt signaling in vertebrate limb development and musculoskeletal morphogenesis. Birth Defects Res C Embryo Today 2003; 69:305-317.
5. Marigo V, Johnson RL, Vortkamp A, Tabin CJ. Sonic hedgehog differentially regulates expression of GLI and GLI3 during limb development. Dev Biol 1996; 180:273-283.
6. Eloy-Trinquet S, Wang H, Edom-Vovard F, Duprez D. Fgf signaling components are associated with muscles and tendons during limb development. Dev Dyn 2009; 238:1195-1206.
7. Fernandez-Teran M, Ros MA. The Apical Ectodermal Ridge: morphological aspects and signaling pathways. Int J Dev Biol 2008; 52:857-871.
8. Lorda-Diez CI, Duarte-Olivenza C, Hurle JM, Montero JA. Transforming growth factor beta signaling: The master sculptor of fingers. Dev Dyn 2022; 251:125-136.
9. Zuniga A, Zeller R. Dynamic and self-regulatory interactions among gene regulatory networks control vertebrate limb bud morphogenesis. Curr Top Dev Biol 2020; 139:61-88.
10. Steinhart Z, Angers S. Wnt signaling in development and tissue homeostasis. Development 2018; 145:dev146589.
11. Li X, Han Y, Li G, Zhang Y, Wang J, Feng C. Role of Wnt signaling pathway in joint development and cartilage degeneration. Front Cell Dev Biol 2023; 11:1181619.
12. Oichi T, Otsuru S, Usami Y, Enomoto-Iwamoto M, Iwamoto M. Wnt signaling in chondroprogenitors during long bone development and growth. Bone 2020; 137:115368.
13. Lin YC, Haas A, Bufe A, Parbin S, Hennecke M, Voloshanenko O, et al. Wnt10b-GSK3beta-dependent Wnt/STOP signaling prevents aneuploidy in human somatic cells. Life Sci Alliance 2021; 4:e202000855.
14. Gao Y, Chen N, Fu Z, Zhang Q. Progress of Wnt Signaling Pathway in Osteoporosis. Biomolecules 2023; 13:483-505.
15. Li C, Furth EE, Rustgi AK, Klein PS. When you come to a fork in the road, take it: Wnt signaling activates multiple pathways through the APC/Axin/GSK-3 complex. Cells 2023; 12:2256-2272.
16. Holzem M, Boutros M, Holstein TW. The origin and evolution of Wnt signalling. Nat Rev Genet 2024.
17. Albrecht LV, Tejeda-Munoz N, Bui MH, Cicchetto AC, Di Biagio D, Colozza G, et al. GSK3 inhibits macropinocytosis and lysosomal activity through the Wnt destruction complex machinery. Cell Rep 2020; 32:107973.
18. Raymond MJ, Jr., McCusker CD. Making a new limb out of old cells: Exploring endogenous cell reprogramming and its role during limb regeneration. Am J Physiol Cell Physiol 2024; 326:C505-C512.
19. Walczynska KS, Zhu L, Liang Y. Insights into the role of the Wnt signaling pathway in the regeneration of animal model systems. Int J Dev Biol 2023; 67:65-78.
20. Kawakami Y, Rodriguez Esteban C, Raya M, Kawakami H, Marti M, Dubova I, et al. Wnt/beta-catenin signaling regulates vertebrate limb regeneration. Genes Dev 2006; 20:3232-3237.
21. Lovely AM, Duerr TJ, Qiu Q, Galvan S, Voss SR, Monaghan JR. Wnt signaling coordinates the expression of limb patterning genes during axolotl forelimb development and regeneration. Front Cell Dev Biol 2022; 10:814250.
22. Yokoyama H, Maruoka T, Ochi H, Aruga A, Ohgo S, Ogino H, et al. Different requirement for Wnt/beta-catenin signaling in limb regeneration of larval and adult Xenopus. PLoS One 2011; 6:e21721.
23. Liu L, Fu Y, Zhu F, Mu C, Li R, Song W, et al. Transcriptomic analysis of Portunus trituberculatus reveals a critical role for WNT4 and WNT signalling in limb regeneration. Gene 2018; 658:113-122.
24. Sensiate LA, Marques-Souza H. Bone growth as the main determinant of mouse digit tip regeneration after amputation. Sci Rep 2019; 9:9720-9727.
25. Aztekin C, Storer MA. To regenerate or not to regenerate: Vertebrate model organisms of regeneration-competency and -incompetency. Wound Repair Regen 2022; 30:623-635.
26. Singh BN, Doyle MJ, Weaver CV, Koyano-Nakagawa N, Garry DJ. Hedgehog and Wnt coordinate signaling in myogenic progenitors and regulate limb regeneration. Dev Biol 2012; 371:23-34.
27. Yokoyama H, Ogino H, Stoick-Cooper CL, Grainger RM, Moon RT. Wnt/beta-catenin signaling has an essential role in the initiation of limb regeneration. Dev Biol 2007; 306:170-178.
28. Zhou C, Li Z, Lu K, Liu Y, Xuan L, Mao H, et al. Advances in human organs-on-chips and applications for drug screening and personalized medicine. Fundamental Research 2024.
29. Wang H, Brown PC, Chow ECY, Ewart L, Ferguson SS, Fitzpatrick S, et al. 3D cell culture models: Drug pharmacokinetics, safety assessment, and regulatory consideration. Clin Transl Sci 2021; 14:1659-1680.
30. Paloschi V, Sabater-Lleal M, Middelkamp H, Vivas A, Johansson S, van der Meer A, et al. Organ-on-a-chip technology: A novel approach to investigate cardiovascular diseases. Cardiovasc Res 2021; 117:2742-2754.
31. Staines KA, Brown G, Farquharson C. The ex vivo organ culture of bone. Methods Mol Biol 2019; 1914:199-215.
32. Aydelotte MB, Kochhar DM. Development of mouse limb buds in organ culture: Chondrogenesis in the presence of a proline analog, L-azetidine-2-carboxylic acid. Dev Biol 1972; 28:191-201.
33. Paradis FH, Yan H, Huang C, Hales BF. The murine limb bud in culture as an in vitro teratogenicity test system. Methods Mol Biol 2019; 1965:73-91.
34. Garber JC, Barbee RW, Bielitzki JT, Clayton LA, Donovan JC, Hendriksen C, et al. Guide for the care and use of laboratory animals. The National Academic Press, Washington DC 2011; 8:220.
35. Bijarchian F, Taghiyar L, Azhdari Z, Baghaban Eslaminejad M. M2c Macrophages enhance phalange regeneration of amputated mice digits in an organ co-culture system. Iran J Basic Med Sci 2021; 24:1602-1612.
36. Boivin GP, Bottomley MA, Schiml PA, Goss L, Grobe N. Physiologic, behavioral, and histologic responses to various euthanasia methods in C57BL/6NTac male mice. J Am Assoc Lab Anim Sci 2017; 56:69-78.
37. Narcisi R, Arikan OH, Lehmann J, Ten Berge D, van Osch GJ. Differential effects of small molecule WNT agonists on the multilineage differentiation capacity of human mesenchymal stem cells. Tissue Eng Part A 2016; 22:1264-1273.
38. Kim C, Jin J, Weyand CM, Goronzy JJ. The transcription factor TCF1 in T cell differentiation and aging. Int J Mol Sci 2020; 21:6497.
39. Steltzer SS, Abraham AC, Killian ML. Interfacial Tissue Regeneration with Bone. Curr Osteoporos Rep 2024; 22:290-298.
40. Kodytkova A, Dusatkova P, Amaratunga SA, Plachy L, Pruhova S, Lebl J. Integrative role of the SALL4 gene: From thalidomide embryopathy to genetic defects of the upper limb, internal organs, cerebral midline, and pituitary. Horm Res Paediatr 2024; 97:106-112.
41. Petersen CP. Wnt signaling in whole-body regeneration. Curr Top Dev Biol 2023; 153:347-380.
42. Zhong J, Jing A, Zheng S, Li S, Zhang X, Ren C. Physiological and molecular mechanisms of insect appendage regeneration. Cell Regen 2023; 12:9-20.
43. Daponte V, Tylzanowski P, Forlino A. Appendage Regeneration in Vertebrates: What Makes This Possible? Cells 2021; 10.
44. Ghosh S, Roy S, Seguin C, Bryant SV, Gardiner DM. Analysis of the expression and function of Wnt-5a and Wnt-5b in developing and regenerating axolotl (Ambystoma mexicanum) limbs. Dev Growth Differ 2008; 50:289-297.
45. Singh BN, Weaver CV, Garry MG, Garry DJ. Hedgehog and Wnt Signaling Pathways Regulate Tail Regeneration. Stem Cells Dev 2018; 27:1426-1437.
46. Yu L, Dawson LA, Yan M, Zimmel K, Lin YL, Dolan CP, et al. BMP9 stimulates joint regeneration at digit amputation wounds in mice. Nat Commun 2019; 10:424.
47. Takeo M, Chou WC, Sun Q, Lee W, Rabbani P, Loomis C, et al. Wnt activation in nail epithelium couples nail growth to digit regeneration. Nature 2013; 499:228-232.
48. Tang SN, Bonilla AF, Chahine NO, Colbath AC, Easley JT, Grad S, et al. Controversies in spine research: Organ culture versus in vivo models for studies of the intervertebral disc. JOR Spine 2022; 5:e1235.
49. Goldrick C, Guri I, Herrera-Oropeza G, O’Brien-Gore C, Roy E, Wojtynska M, et al. 3D multicellular systems in disease modelling: From organoids to organ-on-chip. Front Cell Dev Biol 2023; 11:1083175.
50. Giuliani S, Perin L, Sedrakyan S, Kokorowski P, Jin D, De Filippo R. Ex vivo whole embryonic kidney culture: a novel method for research in development, regeneration and transplantation. J Urol 2008; 179:365-370.
51. Yuta T, Tian T, Chiba Y, Miyazaki K, Funada K, Mizuta K, et al. Development of a novel ex vivo organ culture system to improve preservation methods of regenerative tissues. Sci Rep 2023; 13:3354.
52. Han X, Yoshizaki K, Tian T, Miyazaki K, Takahashi I, Fukumoto S. Mouse Embryonic Tooth Germ Dissection and Ex vivo Culture Protocol. Bio Protoc 2020; 10:e3515.
53. Monnickendam MA, Balls M. Amphibian organ culture. Experientia 1973; 29:1-17.
54. Urzi O, Gasparro R, Costanzo E, De Luca A, Giavaresi G, Fontana S, et al. Three-dimensional cell cultures: The bridge between in vitro and in vivo models. Int J Mol Sci 2023; 24:1-44.
55. Arostegui M, Underhill TM. Murine limb bud organ cultures for studying musculoskeletal development. Methods Mol Biol 2021; 2230:115-137.
56. Al-Lamki RS, Bradley JR, Pober JS. Human organ culture: Updating the approach to bridge the gap from in vitro to in vivo in inflammation, cancer, and stem cell biology. Front Med (Lausanne) 2017; 4:148.
57. Liu Y, Qi X, Donnelly L, Elghobashi-Meinhardt N, Long T, Zhou RW, et al. Mechanisms and inhibition of porcupine-mediated Wnt acylation. Nature 2022; 607:816-822.
58. Matsuura K, Hayashi N, Kuroda Y, Takiue C, Hirata R, Takenami M, et al. Improved development of mouse and human embryos using a tilting embryo culture system. Reprod Biomed Online 2010; 20:358-364.
59. Wang B, Khan S, Wang P, Wang X, Liu Y, Chen J, et al. A highly selective GSK-3beta inhibitor CHIR99021 promotes osteogenesis by activating canonical and autophagy-mediated Wnt signaling. Front Endocrinol (Lausanne) 2022; 13:926622.
60. Luo Y, Liu Y, Wang B, Tu X. CHIR99021-treated osteocytes with wnt activation in 3d-printed module form an osteogenic microenvironment for enhanced osteogenesis and vasculogenesis. Int J Mol Sci 2023; 24:6008.
61. Lee HJ, Wolosin JM, Chung SH. Divergent effects of Wnt/beta-catenin signaling modifiers on the preservation of human limbal epithelial progenitors according to culture condition. Sci Rep 2017; 7:15241.
Iranian Journal of Basic Medical Sciences

Articles in Press, Accepted Manuscript
Available Online from 29 June 2024

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