Salamanders are capable of regenerating amputated limbs by generating a mass of lineage-restricted cells called a blastema. Blastemas only generate structures distal to their origin unless treated with retinoic acid (RA), which results in proximodistal (PD) limb duplications. Little is known about the transcriptional network that regulates PD duplication. In this study, we target specific retinoic acid receptors (RARs) to either PD duplicate (RA treatment or RARγ agonist) or truncate (RARβ antagonist) regenerating limbs. RARE-EGFP reporter axolotls showed divergent reporter activity in limbs undergoing PD duplication versus truncation, suggesting differences in patterning and skeletal regeneration. Transcriptomics identified expression patterns that explain PD duplication, including upregulation of proximal homeobox gene expression and silencing of distal-associated genes, whereas limb truncation was associated with disrupted skeletal differentiation. RARβ antagonism in uninjured limbs induced a loss of skeletal integrity leading to long bone regression and loss of skeletal turnover. Overall, mechanisms were identified that regulate the multifaceted roles of RARs in the salamander limb including regulation of skeletal patterning during epimorphic regeneration, skeletal tissue differentiation during regeneration, and homeostatic regeneration of intact limbs.
The authors declare no competing or financial interests.
M.N., P.S. and J.R.M. contributed to experimental design, experimentation, analysis of results and writing of the manuscript. J.P. and S.J.S contributed to experimentation. M.M. and S.R.V. contributed to experimental design, analysis of results and writing of the manuscript. All authors read and approved the manuscript.
This work was funded by Northeastern University (Start-up funds to J.R.M.), a National Science Foundation grant (1558017 to J.R.M. and M.M.), and the US Army Research Office (56157-LS-MUR to S.R.V.).
Microarray data have been deposited in NCBI GEO under accession number GSE93303.
Supplementary information available online at http://dev.biologists.org/lookup/doi/10.1242/dev.139873.supplemental
- Received May 16, 2016.
- Accepted December 30, 2016.