AN EXAMINATION OF NOTCH SIGNALLING IN THE REGENERATING SPINAL CORD OF THE AXOLOTL, Ambystoma mexicanum: ESTABLISHMENT AND CHARACTERIZATION OF AN AXOLOTL OPTOGENETIC CONSTRUCT FOR UPREGULATION OF NOTCH1 SIGNALLING IN VIVO.

Abstract

The Notch signalling pathway is a juxtacrine signalling pathway conserved across vertebrate and invertebrate species and is known to be a potent regulator of progenitor cell fate decisions during nervous system development. The dysregulation of the Notch pathway has been implicated in the establishment of an anti-neurogenic environment following spinal cord injury in mammals that ultimately prevents functional recovery. In regeneration-competent species, where both neurons and glia are produced by resident progenitor cells in response to trauma, Notch appears to be regulated differently. In the regeneration-competent axolotl, very little is known regarding the role of the Notch signalling pathway in the establishment of a regeneration permissive environment. Here I report that the axolotl possesses a homolog of the Notch1 receptor and qPCR data indicate that its expression decreases significantly at 7 days post injury in caudal spinal cord tissue. I further report that the Notch downstream target gene, Hes1, is expressed in the spinal cord 3 days following injury and that bath application of the indirect Notch inhibitor, tert-Butyl (S)-{(2S)-2-[2-(3,5 difluorophenyl)acetamido]propanamido} phenylacetate (DAPT), impairs spinal cord regeneration. Finally, I have modified an existing optogenetic plasmid construct that allows for light-dependent temporal and spatial Notch receptor-independent signalling through the axolotl Notch intracellular domain (NICD). In vitro characterization has included the determination of an effective membrane anchor for this construct and the production of an appropriate light-insensitive negative control plasmid. This research has provided evidence for a role of Notch during spinal cord regeneration in the axolotl and provided a unique optogenetic tool to facilitate the determination of the in vivo role that Notch signalling plays during spinal cord regeneration in the axolotl.