Visualizing and Quantifying the Spatiotemporal Expression of Proximodistal Pattern-Establishing Genes in the Regenerating Axolotl Forelimb

Abstract Body : How a regenerating axolotl limb establishes positional identity is unknown. One potential determinate of positional identity is retinoic acid (RA). However, how the spatiotemporal expression patterns of genes that regulate RA synthesis, transportation, and breakdown differs between proximal and distal blastemas has not been studied. Further, how the expression patterns of these genes are related to classic patterning genes also remains elusive. We performed bulk RNAseq on distal and proximal blastemas followed by differential expression analysis with DESeq2. To provide spatial context to the genes we identified, we performed V3 HCR-FISH targeting genes of interest. We utilized a pipeline developed in-house to quantify FISH dots with the rsFISH FIJI plugin. We quantified the expression of the genes identified in our RNAseq in both the epithelium and mesenchyme of regenerating blastemas to understand the spatiotemporal dynamics of pattern-establishing and RA-responsive genes. Our RNAseq analysis identified Cyp26b1, which breaks down RA, as differentially expressed in proximal and distal blastemas. Using our V3 HCR-FISH pipeline, we show that Cyp26b1 is more highly expressed specifically in the distal blastema mesenchyme than that in proximal blastemas. In addition, we find that members of the RA synthesis pathway are more highly expressed proximally and localize to the epithelium. RA-response genes, such as Crabp2 and RA receptors, localize to the mesenchyme, and are also more highly expressed proximally. Our results indicate that an RA gradient could be generated by RA synthesis and breakdown in the limb epithelium, with higher RA synthesis and responsiveness seen in proximal blastemas. We conclude that dynamic gene expression, in accord with relative RA levels controlled by Cyp26b1, potentially establishes the regenerating limb’s proximodistal axis.