Beschreibung:
<jats:sec><jats:label /><jats:p>Unlike humans, axolotl salamanders are capable of restoring sensory and motor function after spinal cord injury. Upon tail amputation or spinal cord transection, endogenous neural stem cells (NSCs) increase proliferation and differentiate into multiple neural and glial cell types of the central and peripheral nervous system. How NSCs transition from a resting state to a regenerative state and coordinate proper neural differentiation is poorly understood. Here, we study the global changes that occur in NSCs after spinal cord injury as well as specific changes in gene transcription as the NSC regenerative program unfolds. We first studied global changes in NSCs after injury by measuring global RNA synthesis temporally and spatially through incorporation of 5‐ethynyl‐uridine into nascent RNA. Substantial upward shifts in overall transcriptional output occurred in specific NSC populations during regeneration, which was correlated with loss of facultative repressive chromatin marks, and was dependent on Hippo‐YAP signaling. We also found an evolutionarily conserved upregulation of nascent RNA production in active neural progenitors in axolotls and mice. In order to dig deeper into neural stem cell dynamics during regeneration, we adopted multiplexed fluorescence in situ hybridization (FISH) in tissue section and whole mount tissues. We produced a user‐friendly, sharable, Docker Container for automated high‐throughput design of Third Generation Hybridization Chain Reaction (HCR) FISH probe sets. An advantage of HCR‐FISH is that it utilizes non‐enzymatic signal amplification, while maintaining low background signal, leading to high signal‐to‐noise detection of mRNAs. We demonstrate that the HCR‐FISH method is amenable to multiplexing through five sequential rounds of hybridization (SeqFISH). Using our probe design pipeline and HCR‐FISH, we imaged NSC activation and differentiation in tissue sections collected after injury using 15 simultaneously‐detected probe sets indicative to CNS and PNS cell types. Whole mount HCR‐FISH for three neural markers were also imaged simultaneously to assess NSC and neuron responses after injury. Overall, the genetic neuroanatomical mapping performed here using molecular classification of NSCs provides novel information on how NSCs respond to injury to repair a CNS injury.</jats:p></jats:sec><jats:sec><jats:title>Support or Funding Information</jats:title><jats:p>This project was funded by grants NSF 1656429, NSF 1558017, and NIH OD024909</jats:p></jats:sec>