Identifying the genes necessary to regenerate an injured brain - insights from a basal chordate, Ciona intestinalis

Methods

Summary

Stem Cell Markers. 

We will use alkaline phosphatase (AP), which has been previously employed as a stem cell marker in Ciona and other animals, and pulse-chase labeling with EdU, which is effective in following the migration of progenitor cells.

AP is a universal marker for germ line and adult stem cells, including vertebrate neural stem cells.  It is also known that AP knockdown impairs neural stem cell proliferation and differentiation in mammals. AP will be detected using the BCIP-AP histochemical assay.

To trace migrating stem cells from their origin to the regenerating brain we will use pulse-chase labeling with the cell proliferation marker EdU. EdU is an indicator of current and past DNA replication, and was used previously in Ciona. The logic is that cells labeled during a short EdU "pulse" following brain removal represent the progeny of stem cells involved in brain regeneration, and their specific contribution to the brain should be established by their accumulation in the regenerating organ during a "chase".

In 2019, a stem-cell-specific antibody (PIWI) and a second method involving transgenic animals and chimeras will be used to confirm the EdU results.

Spatial cues for the regenerating brain.

To test the possibility that the severed nerve tracts, which were connected to the original brain prior to its removal provide the spatial cues for organizing the development of brain precursor cells into differentiated brain cells, we will remove the residual neural tracts by manual dissection and/or by chemical means after brain ablation, and determine the effects on all aspects of brain regeneration, including neuron and glia differentation, using specific antibodies.

If negative results are obtained we will entertain alternatives, such as the nearby pharynx wall or the dorsal blood vessels serving as spatial cues.

Genetic regulation of brain regeneration.

We will identify the regulatory genes responsible for Ciona brain regeneration by sequecing isolated branchial sac and brain transcriptomes. We will isolate and sequence the total RNA present in intact Ciona brains and branchial sac before brain ablation, and at periodic intervals within the next few weeks during brain regeneration. The identity of the genes will be confirmed by BLAST to the existing databases. We will be most interested in genes encoding transcription factors or components of signaling pathways, which would be likely regulators of brain regeneration.

In 2019, the expression levels of up-regulated genes will be validated by quantitative PCR, and the expression location of transcripts (e.g. branchial sac stem cell niche, specific brain parts, or both) will be determined by in situ hybridization. Finally, we will address the role(s) of these genes in brain regeneration by RNAi mediated gene knockdown, which we have developed for Ciona , and/or by CRISPR-Cas mediated gene editing, which is also available for Ciona.