The patterning of limbs in development utilizes spatial and temporal signaling dynamics which guide the formation of a complete limb from undefined tissue. Although they may play different roles, a group of key factors guide the development of most species. This process is recapitulated in the limb regeneration of the Mexican axolotl, and the possibility of regeneration in humans will require a precise understanding of how these factors guide this patterning. Previously, observation of limb patterning was restricted by in situ hybridization imaging techniques which had limited resolution and could not reliably compare the collection of essential genes on a single tissue. Now, we have applied V3 Fluorescent in situ Hybridization by Hybridization Chain Reaction (HCR‐FISH) to the model to generate clear, bright, multiplexed images of the expression of these factors. These multiplexed images are the first of their kind in the field, and allow for the overlapping of key factors in both axolotl salamander and murine models to draw essential conclusions regarding each gene’s role in limb patterning within and between species. Development of an efficient probe‐design pipeline and oligonucleotide amplification parameters has improved signal strength, selectivity, and cost effectiveness. Furthermore, we have designed novel V3 HCR‐FISH probes targeting the introns of actively transcribed sequences allows for the identification of actively transcribed RNA via nuclear‐specific signal. Our techniques have not only been applied to confirm predictions derived from single cell RNA‐seq data regarding the roles of Sonic Hedghog (Shh), Fibroblast Growth Factor 8 (Fgf8), and Gremlin 1 (Grem1), in axolotl limb patterning, but also showcased significant differences between the axolotl and murine limb models. These results indicate that application of V3 HCR‐FISH allows for the robust visualization of multi‐gene transcriptional dynamics with a clarity previously impossible in both regenerative and developmental models. Support or Funding Information This project was supported by NSF grants 1558017 and 1656429
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. Support or Funding Information This project was funded by grants NSF 1656429, NSF 1558017, and NIH OD024909
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