Germline Transgenic Methods for Tracking Cells and Testing Gene Function during Regeneration in the Axolotl

Abstract

The salamander is the only tetrapod that regenerates complex body structures throughout life. Deciphering the underlying molecular processes of regeneration is fundamental for regenerative medicine and developmental biology, but the model organism had limited tools for molecular analysis. We describe a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma mexicanum (axolotl) that open up the cellular and molecular genetic dissection of regeneration. We demonstrate tissue-dependent control of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. Furthermore, we demonstrate the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. Finally, we inducibly overexpress the cell-cycle inhibitor p16INK4a, which negatively regulates spinal cord regeneration. These tissue-specific germline axolotl lines and tightly inducible Cre drivers and LoxP reporter lines render this classical regeneration model molecularly accessible.

Live Imaging of Axolotl Digit Regeneration Reveals Spatiotemporal Choreography of Diverse Connective Tissue Progenitor Pools

“…Upon Cre-mediated recombination, these transgenes generate combinatorial expression of fluorescent proteins, endowing cells with different, heritable color identities. We used a tamoxifen-inducible Cre recombinase driven by the ubiquitous CAGGS promoter that induced random patterns of ubiquitous, multicolored fluorescence (Khattak et al., 2013). To specifically label connective tissue within the limb, we implemented embryonic transplantation of LPM from the double transgenic animals into nontransgenic hosts (Figure S1B) (Kragl et al., 2009).…”

“…Transgenic founders were allowed to grow to sexual maturity and F1 progeny were screened for brightness, penetrance, and stability of transgene expression by the default nuclear hrGFPII expression and also after recombination. Double transgenic animals were created by breeding Brainbow animals to an already established CAGGs::ERT-Cre-ERT-T2A-GFPnls line (Khattak et al., 2013). Double transgenic animals from the initial breeding and subsequent F1 double transgenic animals were used as donors for embryonic transplantation and clonal analysis.…”

Mapping hematopoiesis in a fully regenerative vertebrate: the axolotl

“…This suggests that adult regeneration may be an acquired trait in axolotls that might be inducible in mammals if the required genetic pathways are mapped. Recent advances in production of transgenic axolotls, [6][7][8][9][10] complete mapping of the axolotl transcriptome, 11 and production of gene expression arrays [12][13][14][15][16] finally allow molecular mapping of regeneration pathways. Additionally, given the extensive conservation of synteny between axolotls and humans, this amphibian may be a powerful genetic model to study hematopoietic cell function in scar-free wound healing and tissue regeneration.…”

Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease

“…Recently, several transgenic axolotl lines have been created to track cell fates and visualize the activation of signaling pathways during regeneration (Khattak et al, 2013a;Sobkow et al, 2006). The study of gene function in regeneration now benefits from several improvements in the delivery of exogenous DNA constructs into adult axolotl tissues (Khattak et al, 2013b;Whited et al, 2013); however, to date, no studies describe the targeted induction of mutations to perturb the function of endogenous genes.…”