We have developed the means to follow axolotls as they hunt artificial prey so that we can quantitate their behavior. We have shown that injury to the visual portion of the brain eliminates this behavior, and that after three months, the behavior is restored. We have also constructed a 3D behavioral tracking arena that uses four parallel cameras and infrared lighting to film the spontaneous and visually-evoked behaviors of axolotl over many hours and days. The resolution is sufficient to accurately reconstruct the tail and limb movements using automated analyses. We are now beginning to film baseline behaviors in control animals to ultimately compare to regenerating animals.
We have generated a single-nucleus transcriptomics atlas of all regions of the axolotl brain using both droplet microfluidic-based and combinatorial barcoding-based approaches. We have developed the computational pipeline to process and integrate the data and to perform clustering to identify distinct neuronal, glial and non-neural cell types. We have computationally compared the cell clusters in the axolotl brain atlas to cell subclasses defined in the mouse brain cell atlas, and have found homologous cell types across all brain regions. We have identified a diversity of Ependymoglia stem cell states in regionally distinct niches, largely defined by differential morphogen signaling signatures. We have further generated a single-nucleus atlas of chromatin accessibility of the axolotl brain and are currently analyzing this data to learn the gene regulatory basis of cell type identity in the axolotl brain. To complement the single-nucleus data, we have generated a spatial transcriptomics atlas using the MERFISH technology targeting 500 genes on both coronal and sagittal slices across the axolotl brain. We have developed the computational pipeline to process and analyze the data. We are in the process of integrating single-nucleus and spatial transcriptomics data. This comprehensive Axolotl brain atlas will serve as a foundation to later follow how the different cell types are restored after injury.
We have developed several methodologies to follow neural circuits in the axolotl brain. One of the main goals of the project is an anatomical reconstruction of a complete synaptic connectivity of a control and regenerated axolotl optic tectum to compare wiring changes following regeneration. For this, a reproducible preparation method was required to preserve the fine ultrastructure across the axolotl brain. We optimized many parameters in the processing protocols to fix and stain the brain to ensure uniform data quality across the optic tectum and now have a reproducible protocol in place and are in the process of collecting our first volume electron microscopy datasets. In parallel, we optimized a previously published protocol for axolotl that combines the labeling of cell-type specific proteins using immunohistochemistry to correlate fluorescent imaging with electron microscopy. We demonstrated that this also works well in axolotl using several fluorescent antibodies that are known to label cells in the optic tectum.
In order to functionally characterize the activity of tectal neurons in response to visual stimuli, we procured and constructed a three-photon laser scanning microscope to be able to imaged throughout the depth of the optic tectum. We are in the process of measuring the achievable depth at which we can imaged non-invasively.
Finally, we have established cultures of primary ependymoglia derived from different regions of the axolotl brain and have screened different conditions to grow these neural stem cells in 3-dimensional organoid cultures and to differentiate them into neurons. We have generated single-cell transcriptomics data of the cultures to molecularly define the cells and compare them to their primary counterparts. We find brain regional signatures maintained in vitro as well as an injury response signature that is induced during passaging. We are now exploring delivery methods to introduce the CRISPR-Cas9 system into the cells for future genome editing.
