A recent comparison of transcription between innervated and denervated limbs of the Mexican axolotl provided the first global, transcriptional description of the limb regeneration program (Monaghan et al., 2009
). That study used a small format microarray (~4500 probes with 3271 presumptive human orthologs) to detail gene expression of whole blastemas at 5 days and 14 days after limb amputation. However, a more comprehensive analysis of gene expression was needed in concert with an earlier and more precise tissue-sampling scheme to thoroughly investigate the transcriptomics of blastema formation. To this end, we investigated transcription within axolotl epithelium and subjacent cells during the first week of limb regeneration with the primary goal of identifying a core set of genes that are likely to be necessary for limb regeneration. To meet this goal, we devised a strategy that allowed us to subtract out genes common to all injury responses as well as to identify genes that are uniquely expressed in limbs. We further selected genes specific to limbs that regenerate (NL) rather than regress (DL) to identify genes associated with blastema formation. Our study is the most detailed molecular analysis of limb regeneration to date and is the first to identify genes specific to the limb regeneration process by comparing the general wound healing response outside a limb field. Overall, the genes identified here will be useful as tissue specific markers for regenerating limbs and candidates for regulating blastema formation.
At both histological and transcriptional levels, we show that the initial injury response is similar between NL, DL, and FW. The time to re-epithelialization was within 1 dpi and many of the same genes were differentially regulated in NL, DL, and FW. Interestingly, many of these injury-response genes are similarly regulated in mammals, suggesting conservation of some aspects of wound healing among tetrapods.
Previous studies have shown that there are fundamental differences between limb skin and flank skin. When forelimb skin is replaced with grafts of flank skin in newts and axolotls, limb regeneration is defective (Tank, 1984
; Tank, 1987
). We compared gene expression differences between NL and FW and identified transcripts with limb-specific expression patterns. For example, the homeobox-containing transcription factor, emx2
, was highly expressed in uninjured limb skin and was up-regulated after injury only in limb samples (supplementary material Table S1
). Mouse emx2
null mutants fail to form a scapula during development () (Capellini et al., 2010
; Pellegrini et al., 2001
) and newt emx2
is expressed in a graded proximodistal manner mainly in the epidermis of regenerating newt limbs (Beauchemin et al., 1998
and other limb-specific genes identified in our study (supplementary material Table S1
) may regulate limb-specific patterning events during regeneration.
Our analysis also identified a connection between salamander limb-enriched genes and orthologs that are associated with limb deformities in mammals (). For example, numerous genes involved in the Wnt/Planar cell polarity (PCP) signaling pathway were up-regulated in DL and NL, but remained at baseline levels in FW. Activation of PCP signaling by Wnt5 ligand through Vangl and Ror2 activation regulates limb bud elongation during mammalian development (Gao et al., 2011
) and Wnt5a activity is necessary for axolotl limb regeneration (Ghosh et al., 2008
). WNT/PCP signaling is thought to stabilize cellular polarity in epithelium of developing limbs, organize directional cell migration, and regulate directional cell proliferation (Wang et al., 2011
). Overall, it is clear that activation of Wnt signaling through Wnt5a is necessary for limb outgrowth, but the key problem is to identify the property of salamander limbs that allows this pathway to re-activate after injury while not being induced after a flank injury. It is possible that sustained expression of genes like emx2
into post-embryonic and larval stages allows accessibility of this important signaling pathway in adult axolotls.
Genes involved in other important signaling pathways were also up-regulated specifically in limb samples. For example, genes associated with retinoic acid (RA) signaling were dynamically expressed in limbs after injury. Retinoic acid is an important signaling molecule involved in the development and regeneration of limbs; disruption of this pathway disrupts limb formation (Blum and Begemann, 2012
; Kikuchi et al., 2011
; Maden, 1998
; Maden, 2007
). We found that crabp1
was only up-regulated in NL and was expressed exclusively in the limb mesenchyme. In contrast, we found that crabp2
was up-regulated in NL, DL, and FW at 7 dpi. Our findings are in accordance with previous studies showing that CRABP protein is up-regulated during regeneration, although it is unclear whether these studies were detecting CRABP1 or CRABP2 (Maden et al., 1989
; McCormick et al., 1988
). CRABPs are intracellular RA binding proteins that are thought to shuttle RA to the nucleus to regulate RA-mediated transcription, which may explain why we observe their expression during limb regeneration (Noy, 2000
). We also found that aldh1a3
, enzymes involved in the synthesis of RA during development, were up-regulated in NL and DL at 1 dpi and aldh1a3
was expressed specifically in cells resembling perineural fibroblasts in peripheral nerve bundles. Altogether, our data suggest that RA signaling is a dynamic process during limb regeneration and identifies the genes that may mediate the necessity of RA during epimorphic regeneration (Blum and Begemann, 2012
; Kikuchi et al., 2011
Beyond signaling pathways, structural proteins showed very specific transcriptional profiles in regenerating tissues. Numerous keratins (krt5
, and krt13
) and keratin-associated molecules (eppk1
) were up-regulated after injury and were enriched in limbs. Furthermore, some genes like krt8
were highly nerve-dependent. Keratins are components of intermediate filaments that protect the structural integrity of cells, but have recently been implicated in other cellular processes including cell motility, cell signaling, cell growth, and cancer metastasis (Karantza, 2011
; Windoffer et al., 2011
). Although previous studies in newts have identified keratins NvKII, krt8
, and krt18
in mesenchymal and WE cells during limb regeneration (Ferretti et al., 1991
; Ferretti and Ghosh, 1997
) and knockdown of krt8
in newt blastemal cells in vitro
decreased DNA synthesis (Corcoran and Ferretti, 1997
), our understanding of these proteins during regeneration remains poor. Functional testing is necessary to determine if keratin proteins play solely a supportive, structural role during regeneration or whether they are mediating cell signaling to promote growth or patterning. Together, the highly limb-specific and nerve-dependent expression patterns of the keratin genes strongly suggest that they are integral to the formation of the blastema.
Other limb-enriched genes were more quantitatively different than FW rather than being expressed exclusively in the limb. For example, two possible salamander-specific genes, sodefrin-like
(axo22108-r) and methyltransferase-like
(axo23458-r), were up-regulated in NL, DL, and FW, exclusively in the epidermis (; data not shown), but expression was only maintained in NL and DL. This suggests that these molecules are not limb-specific, although sustained expression in the limb WE may impose some necessary function to the WE during limb regeneration. Regardless, the fact that these genes seem to be unique to salamanders (Campbell et al., 2011
) and show strong and specific expression in the WE warrants further functional studies.
A surprising result was the observation that myelin-associated genes were up-regulated and both limb-specific and nerve-dependent. Myelinated peripheral nerves permeate throughout the uninjured limb, but only naked sensory nerve fibers are found in uninjured epidermis of animals (Boulais and Misery, 2008
). Hence, our tissue collection scheme did not sample myelinated nerve fibers in uninjured samples, yet injured NL and DL samples contained transected nerve bundles located just proximal to the WE. This likely explains why mRNA levels of myelin-associated genes increased above baseline in NL at 1 dpi. The fact that myelin-associated gene mRNA did not increase in DL suggests that expression of these genes was lost following denervation. A similar phenomenon takes place in mammals, where peripheral nerve fiber transection down-regulates expression of myelin-associated genes in distal Schwann cells (Hall, 2005
). This result is interesting because it suggests that Schwann cells are affected early after denervation, which may have detrimental effects on downstream blastema formation. In newts, the protein Anterior Gradient 2 is expressed in Schwann cells after limb amputation and supplemental Anterior Gradient 2 can partially rescue regeneration in the denervated state (Kumar et al., 2007
). Others have shown that denervation in axolotls induces peripheral nerves to become inhibitory to limb regeneration, suggesting that they may secrete inhibitory factors (Irvin and Tassava, 1998
; Tassava and Olsen-Winner, 2003
). It will be critical in future experiments to determine if the response of Schwann cells to denervation is the cause of a loss of blastema formation.
The proliferation of blastema cells is known to be a target of the nerve during limb regeneration (Stocum, 2011
). In order to increase our sensitivity for identifying proliferation-associated genes during regeneration, we directly compared NL to DL without comparing samples to baseline or FW. This analysis showed that by 7 dpi, approximately 50% of the genes that were higher in NL versus DL were associated with the cell cycle, supporting the notion that the cell cycle is the primary target of denervation. Most of these genes were only different at 7 dpi, suggesting that our study identified the genes likely upstream of the cell proliferation effect of denervation. This result highlights that the limb-enriched and nerve-enriched genes we identified in our study are excellent candidates for regulating the increase in cell proliferation that is characteristic to limb regeneration. Overall, our study used a focused approach to identify the genes that are likely necessary for limb regeneration and showed that many of these genes are expressed in specific tissues and before considerable outgrowth takes place in the limb. The identification of these genes is an important advance in our ability to tease apart the cellular and molecular mechanisms that drive regeneration and will be a useful resource for regeneration researchers that may be looking for specific genes to analyze during early blastema formation.