Blog
About

  • Record: found
  • Abstract: found
  • Article: found
Is Open Access

Lineage tracing of genome-edited alleles reveals high fidelity axolotl limb regeneration

Read this article at

Bookmark
      There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

      Abstract

      Salamanders are unparalleled among tetrapods in their ability to regenerate many structures, including entire limbs, and the study of this ability may provide insights into human regenerative therapies. The complex structure of the limb poses challenges to the investigation of the cellular and molecular basis of its regeneration. Using CRISPR/Cas, we genetically labelled unique cell lineages within the developing axolotl embryo and tracked the frequency of each lineage within amputated and fully regenerated limbs. This allowed us, for the first time, to assess the contributions of multiple low frequency cell lineages to the regenerating limb at once. Our comparisons reveal that regenerated limbs are high fidelity replicas of the originals even after repeated amputations.

      Related collections

      Most cited references 25

      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs

      The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5′ adenine were improved by rescuing 5′ end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.
        Bookmark
        • Record: found
        • Abstract: found
        • Article: not found

        Cells keep a memory of their tissue origin during axolotl limb regeneration.

        During limb regeneration adult tissue is converted into a zone of undifferentiated progenitors called the blastema that reforms the diverse tissues of the limb. Previous experiments have led to wide acceptance that limb tissues dedifferentiate to form pluripotent cells. Here we have reexamined this question using an integrated GFP transgene to track the major limb tissues during limb regeneration in the salamander Ambystoma mexicanum (the axolotl). Surprisingly, we find that each tissue produces progenitor cells with restricted potential. Therefore, the blastema is a heterogeneous collection of restricted progenitor cells. On the basis of these findings, we further demonstrate that positional identity is a cell-type-specific property of blastema cells, in which cartilage-derived blastema cells harbour positional identity but Schwann-derived cells do not. Our results show that the complex phenomenon of limb regeneration can be achieved without complete dedifferentiation to a pluripotent state, a conclusion with important implications for regenerative medicine.
          Bookmark
          • Record: found
          • Abstract: found
          • Article: not found

          Whole-organism lineage tracing by combinatorial and cumulative genome editing.

          Multicellular systems develop from single cells through distinct lineages. However, current lineage-tracing approaches scale poorly to whole, complex organisms. Here, we use genome editing to progressively introduce and accumulate diverse mutations in a DNA barcode over multiple rounds of cell division. The barcode, an array of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 target sites, marks cells and enables the elucidation of lineage relationships via the patterns of mutations shared between cells. In cell culture and zebrafish, we show that rates and patterns of editing are tunable and that thousands of lineage-informative barcode alleles can be generated. By sampling hundreds of thousands of cells from individual zebrafish, we find that most cells in adult organs derive from relatively few embryonic progenitors. In future analyses, genome editing of synthetic target arrays for lineage tracing (GESTALT) can be used to generate large-scale maps of cell lineage in multicellular systems for normal development and disease.
            Bookmark

            Author and article information

            Affiliations
            [1 ]deptDepartment of Molecular, Cellular and Developmental Biology Yale University New HavenUnited States
            [2 ]deptDepartment of Chemistry Yale University New HavenUnited States
            [3 ]deptDepartment of Pharmacology Yale University New HavenUnited States
            Stowers Institute for Medical Research United States
            Stowers Institute for Medical Research United States
            Contributors
            ORCID: http://orcid.org/0000-0001-7436-3531
            ORCID: http://orcid.org/0000-0002-8456-2005
            Role: Reviewing Editor,
            Stowers Institute for Medical Research United States
            Journal
            eLife
            Elife
            eLife
            eLife
            eLife Sciences Publications, Ltd
            2050-084X
            16 September 2017
            2017
            : 6
            28917058 5621835 25726 10.7554/eLife.25726
            © 2017, Flowers et al

            This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

            Product
            Funding
            Funded by: FundRef http://dx.doi.org/10.13039/100004829, Connecticut Innovations;
            Award ID: Seed Grant 15RMA-YALE-09
            Award Recipient :
            Funded by: FundRef http://dx.doi.org/10.13039/100009633, Eunice Kennedy Shriver National Institute of Child Health and Human Development;
            Award ID: Individual Postdoctoral Fellowship F32HD086942
            Award Recipient :
            Funded by: FundRef http://dx.doi.org/10.13039/100004829, Connecticut Innovations;
            Award ID: Established Investigator Award 15-RMB-YALE-01
            Award Recipient :
            Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
            Award ID: Predoctoral Training Fellowship T32GM007499
            Award Recipient :
            The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
            Categories
            Short Report
            Developmental Biology and Stem Cells
            Custom metadata
            CRISPR-based lineage tracing in the axolotl shows that regenerated limbs are composed of the same cell lineages in the same frequencies as those that gave rise to the original limb.

            Life sciences

            other, ambystoma mexicanum, crispr, regeneration, lineage tracing

            Comments

            Comment on this article