RNA Sequencing Analysis and Multiplexed Imaging of Secreted Growth Factors in Dorsal Root Ganglia Neurons During Axolotl Limb Regeneration

Abstract Body : Introduction
Unlike humans, axolotl salamanders can regenerate complex tissues such as their limbs throughout their lives. Upon amputation, the axolotl limb regenerates via a collection of lineage restricted progenitor cells known as the blastema. One important question which remains to be comprehensively answered in this model is the role played by nerves during regeneration.

The primary neuronal components of the blastema consist of afferent sensory connections and efferent motor neuron inputs derived from the dorsal root ganglia (DRG) and spinal cord, respectively. Neuronal innervation is required for blastemal cell mitogenesis, and denervation of the limb’s peripheral nerves results in a failure to replace the lost tissue. It is widely acknowledged that this dependency on neural innervation is the result of peptidergic nerve-derived factors (NDFs) that originate in the somas and are then secreted to promote blastema cell mitogenesis. While several studies have implicated certain peptides as candidate NDFs, no study has yet used modern transcriptomic approaches to identify neuronally expressed growth factors capable of influencing blastema cell proliferation.

Methods
We first generated bulk RNA sequencing data from DRGs innervating either regenerating or uninjured limbs. Differentially expressed transcripts were then identified via DESeq2 analysis. We combined these expression data with those from a previously published RNA sequencing dataset generated from blastema tissue to select for mRNAs expressed 1.5-fold higher in DRGs than in blastema tissue. Subsequently, these expression data were compared to an annotated ligand-receptor database to identify which candidates were secreted growth factors whose cognate receptors were also expressed in the blastema. Finally, V3 HCR fluorescence in-situ hybridization was used to confirm neuron-specific expression of these candidates in DRG tissues.

Results
In total, 559 genes were found to have significantly increased expression in DRGs which innervated regenerating limbs. Of those, 18 were identified as ligands which are: 1) expressed 1.5-fold higher in DRG tissue compared to blastema tissue; 2) known to act as secreted growth factors; and 3) have cognate receptors expressed in blastema cells. Notably, 3 of these candidates – Ptn, Chgb, and Adcyap1 – show specific co-localization with the neuronal marker β3-tubulin.

Conclusions
Altogether we have generated a dataset of 18 neuronally expressed growth factors with cognate receptors expressed within the blastema. Of these, 3 have never been studied in the context of limb regeneration and show neuron-specific expression. These candidates should serve as promising targets for future investigations into the molecular mechanisms underlying the neural regulation of limb regeneration.

Significance
Natural examples of regeneration provide critical information that could improve regenerative therapies, and the axolotl limb is a favorable model given the genetic and morphological similarities they share with humans. Human wound healing is also a nerve dependent process, such that neuropathy and aberrant innervation are leading causes of chronic wounds. Similarly, diabetic neuropathy and the consequential formation of chronic foot ulcers is the leading cause of non-traumatic limb amputations. Thus, a better understanding of how competent neural tissue facilitates proper tissue repair serves as a promising avenue for future regenerative therapies.