A Regeneration Toolkit

Abstract

The ability of animals to replace injured body parts has been a subject of fascination for centuries. The emerging importance of regenerative medicine has reinvigorated investigations of innate tissue regeneration, and the development of powerful genetic tools has fueled discoveries into how tissue regeneration occurs. Here, we present an overview of the armamentarium employed to probe regeneration in vertebrates, highlighting areas where further methodology advancement will deepen mechanistic findings.

Keywords: gene editing; lineage tracing; mice; model systems; salamanders; stem cells; tissue regeneration; zebrafish.

Figure 1.. The regeneration toolkit.

Table includes major regeneration model systems and includes established, recent, and future tools employed in each system.

Figure 2.. Genetic ablation strategies in regeneration.

(A-C) Genetic strategy to inhibit cell proliferation in type A pericytes following spinal cord injury (A). Sagittal view of the lesion site shows diminished PDGFRβ-positive fibrotic scar in Tamoxifen-treated (C) animals relative to vehicle controls (B) at 2 weeks post-injury. (D) Cartoon indicating ablation of epicardial cells from the ventricular surface induced in explanted hearts of transgenic zebrafish using the metronidazole/nitroreductase system. Epicardium (green) regenerates in a wave from the base of the ventricle to the apex (red arrows indicate direction of regeneration). (E) Epicardial sheets growing ex vivo segregate into large, binucleated leader cells with indicators of high tension (white, pMLC), and smaller, mononucleate follower cells (above yellow dashed line). These cell behaviors mimic epicardial regeneration in vivo. (F) Schematic showing the use of dual recombinases for precise lineage tracing. In this example, a specific marker, c-Kit is expressed in two subpopulations (cardiomyocytes and non-cardiomyocytes). In Tnni3–Dre × Kit–Cre^ER × IR1 cardiomyocytes, Dre first removes one loxP site and ZsGreen from the interleaved reporter allele, precluding potential Kit-Cre–loxPmediated labeling within cardiomyocytes. (G)Simultaneous single-cell profiling of lineages and cell types. During development, CRISPR– Cas9 edits record cell lineage in mutated barcodes at multiple time points during development (Early T1 in blue and late T2 in yellow). Simultaneous recovery of transcriptomes and barcodes from the same cells can be used to generate cell lineage trees and also classify them into discrete cell types (c1–c6). Images in (A-C) are adapted from Dias et al., 2018. Images in (D) are adapted from Wang et al., 2015. Image in (E) is adapted from Cao et al., 2017. Image in (F) is adapted from He et al., 2017. Image in (G) is adapted from Raj et al., 2018.

Figure 3.. Gene regulation and manipulation.

(A) The dual inducible, fluorescent, and functional genetic mosaic (ifgMosaic) method was used to analyze the proliferative capacity of single cells with different combinations of Notch and VEGF signaling. Schematic representation of the genetic constructs of iMb-Vegfr2-Mosaic and iChr-Notch-Mosaic mice and representative confocal micrographs of retinal vessels expressing the different combinations of genes and fluorescent proteins (expression cassettes A-F). Higher-magnification pictures show dual fluorescent clones. Histogram shows frequency and clone size according to their dual nuclei and membrane color (Notch- and VEGF-signaling level). (B) A DNA element upstream of leptin b (LEN) directs regeneration-dependent gene expression in the zebrafish fin and cardiac ventricle. Top panels show transgene expression in regenerating fins at 2 days post-amputation (dpa) (Arrows, blastemal eGFP). Bottom panels indicate enhancer activation in resected ventricular region at 3 dpa (Arrows, endocardial eGFP). (C) Rib fracture (arrows) activates a distinct, adult-specific regulatory region that is inactive embryonically. A 17.8 kb genomic fragment (Ph7) activates lacZ expression in POD4 rib fractures in wholemount (left) and cross-sections (right). (D) In Situ Capture of Locus-Specific Chromatin Interactions by Biotinylated dCas9. In this system, dCas9 is biotinylated in vivo by the biotin ligase BirA together with sequence-specific sgRNAs, followed by isolation of macromolecules associated with the targeted sequence. The purified protein, RNA, and DNA complexes are identified and analyzed by proteomics, RNA-seq, and 3C-seq. Image in (A) is adapted from Pontes-Quero et al., 2017. Images in (B) are adapted from Kang et al., 2016. Images in (C) are adapted from Guenther et al., 2015. Image in (D) is adapted from Liu et al., 2017.