Salamander‐Eci: An optical clearing protocol for the three‐dimensional exploration of regeneration

Abstract

Background: Salamander limb regeneration is a complex biological process that entails the orchestration of multiple cellular and molecular mechanisms in a three-dimensional space. Hence, a comprehensive understanding of this process requires whole-structure level explorations. Recent advances in imaging and optical clearing methods have transformed the study of regenerative phenomena, allowing the three-dimensional visualization of structures and entire organisms.Results: Here we introduce Salamander-Eci, a rapid and robust optical clearing protocol optimized for the widely used axolotl model, which allows simultaneous immunohistochemistry and Click-chemistry detection with minimal volume disruption. We provide examples of its application, from whole larva to adult limbs and organs, and complement it with an image analysis pipeline for volumetric cell quantification. Further, we offer a detailed 3D quantitation of cell proliferation throughout axolotl limb regeneration.Conclusions: Salamander-Eci enables the comprehensive volumetric analysis of regenerative phenomena at both local and systemic levels.

Toward whole tissue imaging of axolotl regeneration

“…Methods such as 2Eci and Salamander-Eci were found to efficiently clear axolotl tissues, preserve endogenous fluorescence and are compatible with dye labeling strategies such as Sytox Green. 46,47 We have already taken advantage of this approach and used it to study the complex cellular processes and clonal dynamics during axolotl limb regeneration. 36 This approach is particularly powerful when dealing with either fluorescent protein expression or antibody labeled tissues.…”

“…More recently we have seen the development and implementation of several clearing strategies on axolotl. Methods such as 2Eci and Salamander‐Eci were found to efficiently clear axolotl tissues, preserve endogenous fluorescence and are compatible with dye labeling strategies such as Sytox Green 46,47 . We have already taken advantage of this approach and used it to study the complex cellular processes and clonal dynamics during axolotl limb regeneration 36 .…”

Salamander Insights Into Ageing and Rejuvenation

“…As the evolutionarily closest organisms to humans capable of complex regeneration, salamanders constitute valuable models for regenerative biology studies. In particular, the axolotl— Ambystoma mexicanum— and the Iberian ribbed newt— Pleurodeles waltl— are two laboratory-tractable systems whose adoption has exponentially grown in recent years due to the ease of captive breeding and rearing ( Khattak et al, 2014 ; Joven et al, 2015 ), efficient transgenesis and genome editing methods ( Khattak et al, 2013b ; Hayashi et al, 2014 ; Fei et al, 2018 ; Cai et al, 2019 ), availability of genomic and transcriptomic information ( Elewa et al, 2017 ; Nowoshilow et al, 2018 ; Smith et al, 2019 ), and advanced imaging techniques ( Masselink and Tanaka, 2020 ; Subiran Adrados et al, 2020 ; Box 1 ). Newts present a conventional salamander life cycle, undergoing metamorphosis and becoming fully developed adults with reduced to imperceptible continuous growth.…”

Salamander models for elucidating mechanisms of developmental biology, evolution, and regeneration: Part one

“…A new innovation in tissue clearing of axolotls is also presented in this issue by Adrados et al using a method called Salamander-Eci. 14 This method builds upon previous clearing approaches, 15,16 but modified parameters to reduce tissue shrinkage that often occurs with previous Eci clearing approaches. They also show that Salamander-Eci is compatible with immunohistochemistry to stain specific cell types and click chemistry to image EdU-based cell proliferation.…”

“…They also predicted new approaches for reducing bottlenecks in workflows for imaging whole tissues and even entire appendages. A new innovation in tissue clearing of axolotls is also presented in this issue by Adrados et al using a method called Salamander‐Eci 14 . This method builds upon previous clearing approaches, 15,16 but modified parameters to reduce tissue shrinkage that often occurs with previous Eci clearing approaches.…”