ECM-related gene expression by DRG neurons
We hypothesized that there would be gene expression differences between the IB4+ and IB4- cells given the observed growth responses in culture. Our results support the hypothesis to some extent, noting that there were intrinsic differences (eg., at t=0) in gene expression as assessed by qRT-PCR, with the IB4+ cells showing lower expression of AdamTs1, Fn, Icam1, Lamb1, Plat, but higher expression of Plaur. While several of these are clearly involved in axonal growth or regeneration in the peripheral nervous system (Fn1 (fibronectin) [30–34], Icam1, Lamb1, Plaur and Plat[13, 36, 37]), their expression in neurons has been less well described.
Discrepancies between the microarray and qRT-PCR results
As noted in the Results, the qRT-PCR data did not completely agree with the array results in terms of differences between the IB4- and IB4+ cells. This was particularly evident at t=24LN where there were no significant differences between the populations (Figure 3). The TaqManR assays also picked up differences within each group over time that that did not correspond with, nor were detectable in the array assays. There are a number of potential explanations for the discrepancies. A basic issue relates to the assay platforms and the sequences probed by the different methods. Specific primer sequences are not available for the TaqMan assays nor does the microarray platform (SABiosciences) provide sequence information with respect to the panels of oligonucleotides used to create the arrays, thus caution is required in interpreting or comparing results from these two very different approaches. Quantitative RT-PCR assays are more reliable and sensitive to changes in mRNA expression although such assays can also be subject to caution (http://miqe.gene-quantification.info). It is also possible that the discrepancies could be due to the different platforms probing different regions of the genes or that distinct isoforms are being expressed by the populations. For example, Spp1/OPN exists as three isoforms derived from alternative splicing and these have been reported to have differing effects on cellular behaviour including regulating expression of Plau, the ligand for Plaur . Several splice variants of Plaur/uPAR are also reported in cancer with differing expression and biological activity in malignant vs non-malignant cells .
The differences in protein expression detected by ICC did not agree with the quantitation of gene expression differences, although one would not necessarily expect a direct correlation [40, 41]. Post-transcriptional mechanisms including translation and post-translational modification as well protein turnover will modulate protein steady state levels . Post-transcriptional mechanisms have been suggested to contribute to discrepancies in plasminogen enzyme activities compared to mRNA levels in neonatal DRG neurons . The difference could also point to post-translational modifications or the presence of proteins translated from splice variants, since alternative-spliced isoforms have been described for several of these targets as noted above, albeit in cancer or metastasizing cells (eg., [39, 43]). In any case, the ICC provided confirmation of the genes, including those not previously described or detected at the protein level in the DRG (eg., Bsg, AdamTs1 ; MMP 14, MMP19, RT-Aw2, Ctgf).
Technical and biological variability between the different assays contribute to the observed discrepancies. Thus, while the microarray approach was useful for screening purposes, the quantitative changes in gene expression between the populations that we have presented are based upon the data obtained from qRT-PCR analyses.
The tissue plasminogen (uPAR/Plaur and tPA/Plat) system in sensory neurons
Many cells types use extracellular proteolysis to modify the ECM or cell-cell contacts in order to respond to environmental cues. Enzymes involved include MMPs and members of the plasminogen activator/plasmin system. Two types of plasminogen activators are tPA/Plat and uPA/Plau or urokinase; uPAR/Plaur is the receptor for uPA. Both PAs convert plasminogen into active plasmin, which is a protease with targets that include ECM components, growth factors and cellular receptors .
Our data show that Plat was expressed at higher levels in the IB4- cells at t=0, but was increased in the IB4+ cells by 24 hr. tPA plays a key role in growth and regeneration in the nervous system , with tPA enhancing sensory axon regrowth in the spinal cord in a model of spinal cord injury . LN has been shown to be degraded by tPA activation of plasminogen and to be necessary for PC12 cell neuritogenesis on LN . Plat may play a similar role here in the LN-induced growth of the IB4- neurons.
Our results showed that Plaur was expressed at higher levels in the IB4+ neurons compared to the IB4- cells at t=0; both were increased after 24 hr. The relative increases in Plaur with time in culture were several-fold higher than any of the other genes investigated by qRT-PCR; similar large increases have been observed in cultures of neonatal DRG neurons . The reason for this is unclear, particularly with the suggested role of Plaur in nerve regeneration, and the reported lack of growth of the IB4+ cells in vitro and in vivo[5, 8, 9]. However, considering that these cells are able to undergo neuritogenesis in culture when GDNF is added, it suggests that they do have the capacity to regenerate, provided the appropriate growth factors are also made available.
The Plaur gene encodes for a cell surface receptor (Plaur in rats, uPAR in mice and humans) that binds urokinase plasminogen activator (uPA), resulting in the conversion of plasminogen to plasmin to promote pericellular matrix degradation . In cancer, the plasminogen activator system has been extensively studied for its role in cell migration and invasion . Interestingly, uPAR has been reported to be necessary for nerve growth factor (NGF)-induced neurite extension in PC12 cells . Furthermore, an increase in Plaur/uPAR expression in sensory neurons after injury has been previously described. Hayden and Seeds  showed that uPAR and uPA mRNA levels significantly increased in neonatal mouse DRG neurons during the first day of culture. In subsequent in vivo nerve injury experiments, Siconolfi and Seeds  reported that DRG neuron uPAR mRNA expression increased by 8 hr post-injury, while uPA did not increase until day three. These studies suggested that the plasminogen activator system was likely to play a key role in peripheral nerve regeneration. The role of uPAR in peripheral nerve function and regeneration has only recently been more completely elucidated. In a recent report, using uPAR null mice, Previtali and colleagues have shown that lack of uPAR inhibits nerve regeneration following injury, which was attributed to a decrease in fibrinolytic activity in the damaged nerve and concomitant deposition of fibrin and vitronectin . This work suggests that uPAR/Plaur is key to the appropriate remodeling of the ECM necessary for nerve regeneration , although no differences in the neuronal subtypes were investigated.
In addition, plasminogen-independent roles for Plaur have been explored. For example, Plaur has also been shown to interact with cell surface molecules such as integrins [51–55] and mediate MEK/ERK signaling through FAK or Src [56–58]. Plaur also regulates the activity of MMPs after sciatic nerve crush . Thus, the increase in Plaur mRNA and protein in the absence of uPA expression may point to a uPA-independent role(s) in these cultured DRG neurons. Our data, combined with the previous literature, suggest that Plaur expression may be involved in essential interactions with the ECM in these neurons. However, further experiments are necessary to investigate whether the increased expression of Plaur in the IB4+ cells plays any part in the reported diminished ability of these neurons to regenerate compared to other classes of DRG neurons [2, 6, 8–10].
Integrin expression in DRG neurons
The microarrays detected that integrins β1, β4 and α5 were consistently expressed by these neurons. We have previously detected expression of α1, α5, and α3 integrins along with β1 by ICC, and have shown that β1 is required for LN-induced neurite growth . While Integrin β1 was found to be decreased in the IB4+ cells by both array and ICC analysis, the qRT-PCR did not detect significant differences at t=0 between the 2 groups. However, after 24 hr culture on LN, increased gene expression in both the IB4+ and IB4- cells was detected by qRT-PCR. While our initial hypothesis was that differences in integrin expression might contribute to the relatively rapid initial growth from IB4- cells compared to IB4+, these data indicate that this is not the only factor in the growth response.
Other studies have pointed to an important contribution of integrin α7β1 expression to the regeneration promoting effects of preconditioning lesions [60, 61]. However, α7 is not expressed by IB4+ neurons, which have a reduced regenerative capacity, nor does forced expression of α7 lead to any rescue of this lack of growth [8, 9, 17]. Integrin α7 was not detected by the arrays, although standard RT-PCR showed that the mRNA was expressed in both populations (data not shown). However, as we have previously been unable to detect α7 by ICC in our cultures using several different antibodies, it is possible that while the mRNA is present, there is a low level of translation not detectable by our methods.
Other genes of interest detected on the microarray screen
Our initial approach was to use the arrays as a platform to screen for genes that were differentially expressed between two populations of DRG neurons and might conceivably contribute to the differences in neurite growth induced by LN. This approach identified the genes that were then further assessed by qRT-PCR as noted above. A number of other genes were identified that displayed relatively high levels of expression (compared to the housekeeping genes) in both sets of neurons, but were not detectably changed over time in culture. Several of these (Bsg, Sparc, Adamts1, Mmp14) have only recently been described in embryonic DRG neurons using a similar microarray approach, but neither Bsg nor AdamTs1 was detected at the protein level . For others (Mmp19, Ctgf, RT1-Aw2), to our knowledge it is the first time that they have been reported to be expressed in adult DRG neurons, although Ctgf was picked up in a large scale screening of genes associated with outgrowth in embryonic DRG, using RNA isolated from whole ganglia . IHC to detect a number of these proteins showed that they are expressed in the ganglia both in neurons and in associated non-neuronal cells DRGs (Figure 9; also Additional files 7-8: Figures S3-4).
Several of these genes have been studied for their role in ECM remodeling in other cell types. Remodeling of ECM components clears a path for cell migration and plays a role in peripheral nerve regeneration [13, 62]. Of interest, MMP14 (also known as MT1-MMP) is a membrane-bound MMP that regulates the turnover and integrin-mediated endocytosis of fibronectin , and our results show neuronal and nuclear expression in the DRG sections. BSG (also known as EMMPRIN) is a type 1 integral membrane receptor that has the ability to complex with integrin α3β1  and induce MMP expression [65, 66]. It has also been shown to mediate neuron-glial interactions important in the development of the Drosophila eye . IHC results demonstrate expression in DRG neurons, with an apparent cytoplasmic and cell surface localization. As these genes were robustly expressed in DRG neurons, they could be important of the response of DRG neurons to their extracellular environment in vivo.
Another gene not previously reported as being expressed in DRG neurons is RT1-Aw2. RT1-Aw2 is an MHC class I molecule , and MHC class I molecules have recently been implicated in the immunoregulatory functions of synaptic plasticity after nerve injury  and in neuroinflammation . This could suggest that RT1-Aw2 plays a key role in antigen presentation and neuro-immune interactions in these cultured adult neurons. RT1 is expressed in DRG neurons and appears to be enriched on the cell surface (Figure 9K).
The genes (Bsg, Cst3, Ctsb, Ctsd, Ctsl, Mmp14, Mnp19, Sparc) detected by the microarray screen as being relatively highly expressed are involved in the degradation of extracellular and intercellular proteins and could play a role in the response of the neurons to ECM cues in axonal growth . Because there were no differences observed between the populations on the arrays, these genes were not subjected to further analyses by qRT-PCR. However, given the discrepancies observed between the two assays, we will investigate this further. Future studies will focus on these genes to understand what role they play in ECM remodeling that could promote axonal growth in in vitro and in vivo systems.
In summary we have investigated whether the observed growth differences between IB4+ and IB4- neurons were associated with any differences in the expression of genes associated with the ECM. Several differences were observed, which may play a role in the growth response or represent a general response to cellular injury.