Profiling the Spatial Dynamics of Limb Patterning in Regeneration and Development Using Enhanced V3 HCR‐FISH Probe Design

Abstract

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