title: CRISPR-mediated genomic deletion of Sox2 in the axolotl shows a requirement in spinal cord neural stem cell amplification during tail regeneration.
abstract: The salamander is the only tetrapod that functionally regenerates all cell types of the limb and spinal cord (SC) and thus represents an important regeneration model, but the lack of gene-knockout technology has limited molecular analysis. We compared transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) in the knockout of three loci in the axolotl and find that CRISPRs show highly penetrant knockout with less toxic effects compared to TALENs. Deletion of Sox2 in up to 100% of cells yielded viable F0 larvae with normal SC organization and ependymoglial cell marker expression such as GFAP and ZO-1. However, upon tail amputation, neural stem cell proliferation was inhibited, resulting in spinal-cord-specific regeneration failure. In contrast, the mesodermal blastema formed normally. Sox3 expression during development, but not regeneration, most likely allowed embryonic survival and the regeneration-specific phenotype. This analysis represents the first tissue-specific regeneration phenotype from the genomic deletion of a gene in the axolotl.
authors.affiliation: DFG Center for Regenerative Therapies, Dresden (CRTD), Technische Universität Dresden, Dresden 01307, Germany.
authors.forename: Ji-Feng
authors.initials: JF
authors.lastname: Fei
authors.affiliation: DFG Center for Regenerative Therapies, Dresden (CRTD), Technische Universität Dresden, Dresden 01307, Germany.
authors.forename: Maritta
authors.initials: M
authors.lastname: Schuez
authors.affiliation: DFG Center for Regenerative Therapies, Dresden (CRTD), Technische Universität Dresden, Dresden 01307, Germany.
authors.forename: Akira
authors.initials: A
authors.lastname: Tazaki
authors.affiliation: DFG Center for Regenerative Therapies, Dresden (CRTD), Technische Universität Dresden, Dresden 01307, Germany.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Taniguchi
authors.affiliation: DFG Center for Regenerative Therapies, Dresden (CRTD), Technische Universität Dresden, Dresden 01307, Germany.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Roensch
authors.affiliation: DFG Center for Regenerative Therapies, Dresden (CRTD), Technische Universität Dresden, Dresden 01307, Germany. Electronic address: elly.tanaka@crt-dresden.de.
authors.forename: Elly M
authors.initials: EM
authors.lastname: Tanaka
mesh_terms: D000558:Ambystoma mexicanum / Q000196:embryology / Q000235:genetics / Q000502:physiology*; D029845:Amphibian Proteins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D049109:Cell Proliferation; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D017353:Gene Deletion; D018507:Gene Expression Regulation, Developmental; D055786:Gene Knockout Techniques; D008969:Molecular Sequence Data; D058953:Neural Stem Cells / Q000166:cytology*; D012038:Regeneration; D055748:SOXB1 Transcription Factors / Q000235:genetics*; D058630:Spinal Cord Regeneration
chemical_list: D029845:Amphibian Proteins; D055748:SOXB1 Transcription Factors