Development of a ZRS Reporter System for the Newt () During Terrestrial Limb Regeneration

Overview

Journal Biomedicines

Specialty Biomedical Engineering

Date 2024 Nov 27

PMID 39595071

Authors

Martin Miguel Casco-Robles

Ryosuke Ikeda

Fumiaki Maruo

Chikafumi Chiba

Affiliations

Soon will be listed here.

Abstract

Background: Newts, a type of urodele amphibian, offer remarkable insights into regenerative medicine due to their extraordinary tissue regeneration capabilities-a challenging feat in humans. During limb regeneration of adult newts, fascinating cellular and molecular processes are revealed, including scarless healing, de-differentiation of mature cells, and regeneration of limbs and digits. Sonic hedgehog (Shh), crucial for vertebrate limb development, is regulated by the zone of polarizing activity regulatory sequence (ZRS) in the limb bud zone of polarizing activity (ZPA). The metamorphosed (terrestrial) newt can reactivate Shh during regeneration, facilitating proper limb patterning. Cell types capable of regulating the ZRS in metamorphosed newts remain unknown. The identification of such cell types provides invaluable insight into novel regenerative mechanisms.

Objective: In this study, we developed the first newt ZRS reporter.

Methods: We isolated and characterized the newt ZRS enhancer (nZRS), identifying conserved DNA binding sites. Several binding sites with medical relevance were conserved in the newt ZRS. In functional analysis, we developed a system composed of a transgenic nZRS reporter newt and a new newt anti-Shh antibody, which allowed Shh monitoring during limb regeneration.

Results: We identified a group of Schwann cells capable of ZRS reporter and Shh protein expression during terrestrial limb regeneration.

Conclusions: This system provides a valuable in vivo approach for future genetic studies of patterning during limb regeneration.

References

Sandelin A, Alkema W, Engstrom P, Wasserman W, Lenhard B . JASPAR: an open-access database for eukaryotic transcription factor binding profiles. Nucleic Acids Res. 2003; 32(Database issue):D91-4. PMC: 308747. DOI: 10.1093/nar/gkh012. View

Notredame C, Higgins D, Heringa J . T-Coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol. 2000; 302(1):205-17. DOI: 10.1006/jmbi.2000.4042. View

Ekker S, Ungar A, Greenstein P, von Kessler D, Porter J, Moon R . Patterning activities of vertebrate hedgehog proteins in the developing eye and brain. Curr Biol. 1995; 5(8):944-55. DOI: 10.1016/s0960-9822(95)00185-0. View

Lettice L, Devenney P, De Angelis C, Hill R . The Conserved Sonic Hedgehog Limb Enhancer Consists of Discrete Functional Elements that Regulate Precise Spatial Expression. Cell Rep. 2017; 20(6):1396-1408. PMC: 5561167. DOI: 10.1016/j.celrep.2017.07.037. View

Casco-Robles R, Watanabe A, Eto K, Takeshima K, Obata S, Kinoshita T . Novel erythrocyte clumps revealed by an orphan gene Newtic1 in circulating blood and regenerating limbs of the adult newt. Sci Rep. 2018; 8(1):7455. PMC: 5945717. DOI: 10.1038/s41598-018-25867-x. View

Smith J, Timoshevskaya N, Timoshevskiy V, Keinath M, Hardy D, Voss S . A chromosome-scale assembly of the axolotl genome. Genome Res. 2019; 29(2):317-324. PMC: 6360810. DOI: 10.1101/gr.241901.118. View

Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey M, Baker M . The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. BMJ Open Sci. 2021; 4(1):e100115. PMC: 7610906. DOI: 10.1136/bmjos-2020-100115. View

Tickle C . An historical perspective on the pioneering experiments of John Saunders. Dev Biol. 2017; 429(2):374-381. DOI: 10.1016/j.ydbio.2017.05.028. View

Lettice L, Williamson I, Wiltshire J, Peluso S, Devenney P, Hill A . Opposing functions of the ETS factor family define Shh spatial expression in limb buds and underlie polydactyly. Dev Cell. 2012; 22(2):459-67. PMC: 3314984. DOI: 10.1016/j.devcel.2011.12.010. View

10.

Konno K, Yamasaki M, Miyazaki T, Watanabe M . Glyoxal fixation: An approach to solve immunohistochemical problem in neuroscience research. Sci Adv. 2023; 9(28):eadf7084. PMC: 10348680. DOI: 10.1126/sciadv.adf7084. View

11.

Liu Z, Jin Y, Chen L, Wang Y, Yang X, Cheng J . Specific marker expression and cell state of Schwann cells during culture in vitro. PLoS One. 2015; 10(4):e0123278. PMC: 4393255. DOI: 10.1371/journal.pone.0123278. View

12.

Kintner C, Brockes J . Monoclonal antibodies to the cells of a regenerating limb. J Embryol Exp Morphol. 1985; 89:37-55. View

13.

Kurosaka H, Takano-Yamamoto T, Yamashiro T, Agata K . Comparison of molecular and cellular events during lower jaw regeneration of newt (Cynops pyrrhogaster) and West African clawed frog (Xenopus tropicalis). Dev Dyn. 2007; 237(2):354-65. DOI: 10.1002/dvdy.21419. View

14.

Kumar A, Godwin J, Gates P, Garza-Garcia A, Brockes J . Molecular basis for the nerve dependence of limb regeneration in an adult vertebrate. Science. 2007; 318(5851):772-7. PMC: 2696928. DOI: 10.1126/science.1147710. View

15.

Eroglu E, Yen C, Tsoi Y, Witman N, Elewa A, Araus A . Epicardium-derived cells organize through tight junctions to replenish cardiac muscle in salamanders. Nat Cell Biol. 2022; 24(5):645-658. PMC: 9106584. DOI: 10.1038/s41556-022-00902-2. View

16.

Tanaka H, Ng N, Yu Z, Casco-Robles M, Maruo F, Tsonis P . A developmentally regulated switch from stem cells to dedifferentiation for limb muscle regeneration in newts. Nat Commun. 2016; 7:11069. PMC: 4820895. DOI: 10.1038/ncomms11069. View

17.

Bolivar S, Navarro X, Udina E . Schwann Cell Role in Selectivity of Nerve Regeneration. Cells. 2020; 9(9). PMC: 7563640. DOI: 10.3390/cells9092131. View

18.

Casco-Robles M, Yamada S, Miura T, Chiba C . Simple and efficient transgenesis with I-SceI meganuclease in the newt, Cynops pyrrhogaster. Dev Dyn. 2010; 239(12):3275-84. DOI: 10.1002/dvdy.22463. View

19.

Griffin J, Thompson W . Biology and pathology of nonmyelinating Schwann cells. Glia. 2008; 56(14):1518-1531. DOI: 10.1002/glia.20778. View

20.

Chen W, Tsissios G, Sallese A, Smucker B, Nguyen A, Chen J . In Vivo Imaging of Newt Lens Regeneration: Novel Insights Into the Regeneration Process. Transl Vis Sci Technol. 2021; 10(10):4. PMC: 8362625. DOI: 10.1167/tvst.10.10.4. View