The authors have declared that no competing interests exist.
Conceived and designed the experiments: MRZ JAB. Performed the experiments: MRZ JAB. Analyzed the data: MRZ JAB. Contributed reagents/materials/analysis tools: MRZ JAB. Wrote the paper: MRZ JAB.
Pax3 has numerous integral functions in embryonic tissue morphogenesis and knowledge of its complex function in cells of adult tissue continues to unfold. Across a variety of adult tissue lineages, the role of Pax3 is principally linked to maintenance of the tissue’s resident stem/progenitor cell population. In adult peripheral nerves, Pax3 is reported to be expressed in nonmyelinating Schwann cells, however, little is known about the purpose of this expression. Based on the evidence of the role of Pax3 in other adult tissue stem and progenitor cells, it was hypothesised that the cells in adult peripheral nerve that express Pax3 may be peripheral glioblasts. Here, methods have been developed for identification and visualisation of Pax3 expressant cells in normal 60 day old mouse peripheral nerve that allowed morphological and phenotypic distinctions to be made between Pax3 expressing cells and other nonmyelinating Schwann cells. The distinctions described provide compelling support for a resident glioblast population in adult mouse peripheral nerve.
The
There are conflicting reports about the expression of
To delineate whether the production of additional mouse transcripts of
As such, specific primers were designed to amplify the mRNA of mouse
Gel electrophoresis of PCR amplification products of
In the adult peripheral nervous system, C-fibre neurons are not myelinated and are organised into a bundle in which many nerve fibres are ensheathed by one NMSC for conduction of peripheral afferent signals. NMSCs have a characteristic morphology that consists of long branching networks of cytoplasmic processes which coalesce in a plexiform manner with adjacent nonmyelinated bundles
In this study, mouse NMSCs were clearly visualised by fluorescence microscopy of teased sciatic nerve fibre specimens (data not shown); the morphology of the cell is stated as 2–4 µm in diameter across the cytoplasmic extensions and 4–5 µm in diameter across the nuclear region. The length of the cell is between 80–200 um and the nucleus is between 12–20 um in length. The nucleus of the NMSC is centrally located (as opposed to the peripheral location of the nucleus of a myelinating Schwann cell) and the nonmyelinated C-fibres that traverse longitudinally across the NMSC nucleus form it into a characteristic ‘cigar’ or spindle shape such as has been described for rat NMSCs
It is known that nonmyelinated bundles contain two classes of C-fibres, those dependent on nerve growth factor (NGF) that express low-affinity nerve growth factor receptor (p75Ngfr) and those dependent on glial-derived neurotrophic factor that express glial-derived neurotrophic factor family receptor-α1
A micrograph of a whole mount sciatic nerve fascicle preparation labeled with p75Ngfr (red) reveals the plexiform comingling and exchange of NGF-dependent C-fibres between adjacent nonmyelinated bundles. Cell nuclei are visualised using Hoechst DNA dye (blue). Images were acquired using scanning laser confocal microscopy.
RT-PCR results verified that
Development of the Pax3 immunofluorescent labeling method commenced using frozen sections of mouse sciatic nerve pre- and/or postfixed with 4% w/v paraformaldehyde (PFA) and a secondary indirect immunofluorescence procedure. In both tangental and longitudinal sections, a nuclear Pax3 label was undetectable. As indicated by the RT-PCR results, Pax3 expression levels were expected to be relatively low, thus, a tertiary (avidin/biotin) indirect immunofluorescence procedure was also performed on the frozen sections. When this method was analysed, levels of non-specific background staining were high and a nuclear Pax3 label remained undetectable (data not shown). Next, individual 2 mm lengths of fascicles from adult mouse sciatic nerve were pre- and/or postfixed in PFA and teased into individual Schwann cell/axons and indirect immunofluorescence methods were tested for Pax3 labeling. Various tissue permeabilisation protocols were also assessed as to their effects on cellular and extracellular integrity, nonspecific staining and intensity of immunofluorescent Pax3 label. Results showed that all methods trialed had a Pax3 label of low intensity and, in many specimens, Schwann cell structure was not optimal (data not shown). Positive control samples were processed during each of the teased fibre immunolabeling experiments using Krox24, a transcription factor reported expressed in Schwann cells of adult peripheral nerve
A sciatic nerve fascicle post-fixed for 2 hours in PFA was labeled with an antibody targeted at the Krox24 transcription factor. In this micrograph, a myelinating Schwann cell nucleus (indicated by the arrow) shows Krox24 positivity.
An alternate method, consisting of a short post-fixation of dried whole mount sciatic nerve fascicles with 4°C acetone was subsequently found to preserve both tissue morphology and Pax3 antigenicity, therefore, a Pax3 labeling procedure was developed using this method of fixation and analysed using scanning laser confocal microscopy. Results showed strong Pax3 immunoreactivity in cell nuclei randomly distributed along the length of the 60 day old sciatic nerve trunk. In the whole mount mouse sciatic nerve fascicle specimens analysed, relatively 2% of cell nuclei were positive for Pax3 when compared to the total number of Hoechst stained nuclei visible along the length of the nerve (
The cell indicated by the arrow has the characteristic morphology of the NMSCs associated with NGF-dependent C-fibres. Note the spindle shaped nucleus (blue) and p75+ cytoplasmic extensions (red).
It was hypothesised that cells of adult nerve that express Pax3 would co-express peripheral glioblast markers, therefore co-localisation studies were performed using antibodies against Pax3 and a marker of neural crest cells, transcription factor SRY-related high-mobility group box-2 (Sox2). To date, Sox2 expression has been thought limited to embryonic glioblasts
Whole mount sciatic nerve fascicles co-immunolabeled with Pax3 (green), Sox2 (red) and Hoechst DNA dye (blue) revealed that Pax3 expressant nuclei co-express stem cell marker Sox2 (indicated by the arrows). Images were acquired using scanning laser confocal microscopy.
This study was primarily concerned with the expression of a developmental transcription factor, Pax3, in adult mouse peripheral nerve. No studies have discussed Pax3 protein expression in normal adult mouse NMSCs and thus it was compelling to investigate and contemplate the implications of
The Pax3 expression pattern seen in adult peripheral nerve is similar to that seen in adult skeletal muscle where Pax3/7 expressing stem (satellite) cells account for up to 4% of the total myonuclei
Based on the evidence of the role of Pax3 in other adult tissue stem and progenitor cells, and taken together with evidence that a population of cells exist in adult peripheral nerve that express Pax3
Labeling procedures allowed several distinctions to be made between Pax3 expressing cells and other NMSCs. Firstly, it was seen that the Pax3+ cells co-expressed p75Ngfr in the nucleus but lacked p75Ngfr+ bipolar cytoplasmic extensions. While it is possible that Pax3 expressant cells may associate with glial-derived neurotrophic factor dependent C-fibres (future studies should address this question), the p75Ngfr nuclear expression pattern would be curious; on the other hand, the p75Ngfr/Pax3+ nuclear expression is consistent with that of a peripheral glioblast
The morphologic and phenotypic differences of the Pax3 expressing cells described here lend credence to the theory that peripheral glioblasts may be retained in peripheral nerve after birth
Finally, in the mouse after birth, a subset of Schwann cells that associate with C-fibres differentiate toward a nonmyelinating phenotype, re-establish dependency on paracrine signaling for survival
Peripheral nerve injuries, in which the nerve trunk is severed, result in separation of the axon from the nucleus and a subsequent inflammatory response called Wallerian degeneration. A fundamental characteristic of Wallerian degeneration is the reported plasticity of adult myelinating Schwann cells to revert from the myelinogenic transcriptional program (or differentiated state) into the cell cycle and back
Here, the question arises as to why the peripheral nerve trunk would harbour resident glioblasts when myelinating Schwann cells have the capability to de-differentiate. Several suppositions come to mind. Resident glioblasts would proliferate (initially) at a greater rate than a lost myelinating cell, which must extrude and degrade myelin debris before it undergoes proliferation and thus resident glioblasts could be an early source of regenerative Schwann cells. In support of this notion is a study by Griffin et al
Furthermore, there are very few studies outlining the mechanisms of nonmyelinated nerve regeneration and it is possible that resident glioblasts may have a role along these lines. Support for this comes from two studies. Neurofibromatosis Type 1 (NF1) is characterised by loss of the
Finally, a recent study has demonstrated a population of Pax3+ immature Schwann cells in the cutaneous nerve plexus of the dermis
In 2008, Griffin and Thompson stated that “the possibility of a population of Schwann cell precursors in adult nerves is largely unexplored”
Methods were also developed and described that allowed the visualisation and further characterisation of NMSCs that associate with NGF-dependent C-fibres of normal 60 day old mouse peripheral nerve. To date, neurological studies of this kind have been performed on larger animals such as frog, rat, cat and dog. The intricate and complex morphological characteristics of mouse NMSCs described here, along with the procedures for imaging these cells, provides a foundation for further studies of NMSCs in mouse and may be particularly useful for studies using transgenic animals.
Experimental procedures were carried out in accordance with the provisions of the National Health and Medical Research Council Australian Code for Responsible Conduct of Research (2007), the Australian code of practice for the care and use of animals for scientific purposes (2004) and the Animal Welfare Act (2002). Experimentation was approved by the Edith Cowan University Animal Ethics Committee (project approval code 06-A7 ZIMAN). The age of the animals was chosen to reflect the cellular makeup of adult or mature tissue. All the investigations described were undertaken using 60 day old male mice that were provided by the Animal Resources Centre (Canning Vale, Western Australia).
Mice were sacrificed by CO2 narcosis at 20%/min v/v and the sciatic nerves were rapidly excised in an aseptic field. Nerves were dissected and ligated under a Leica Zoom 2000 dissecting microscope, with care taken to remove connective fascia from the epineurium. Freshly removed nerve tissue was immediately frozen by immersion in liquid nitrogen and stored at −80°C until further use. Total RNA was isolated from single sciatic nerves using TriReagent (Molecular Research Center, Inc.) and homogenisation with a glass-col mortar and pestle. For each extraction, RNA purity and concentration were assessed using a Bioanalyzer (Agilent).
First strand cDNA was synthesised from 2 µg of isolated RNA using an OmniScript system (Qiagen) and an oligo(dT)18 primer (10 µM) (Qiagen). Reverse transcription was carried out at 37°C for 1 hour in a total volume of 20 µl. Negative controls included reactions without Omniscript reverse transcriptase. PCR amplifications were performed using a TaqDNA Polymerase Kit (Qiagen) and a negative control (without template DNA) was included in every experiment. The PCR reaction was conducted with the following oligonucleotides, designed using OligoAnalyser 3.1 (Integrated DNA Technologies) and Primer-BLAST (NCBI):
(R)
(R)
(R)
(R)
Internal controls for cDNA were performed using PCR amplification of mouse housekeeping gene
(R)
Positive controls for primers were performed using total RNA isolated from embryonic day 11 mice and PCR negative controls eliminated cDNA as primer template from each PCR reaction. PCR products were resolved on 1.5% w/v agarose gels and visualised under UV light using a Geldoc system. PCR products were sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems) and an ABI Prism 3730 48 capillary sequencer. Sequences were aligned with known sequences in GenBank using the multiAlign tool in Angis, available on GenBank.
To prepare fresh frozen sections of sciatic nerve, animals were sacrificed by cervical disslocation. The sciatic nerves were surgically excised, immersed in Tissue Tek O.C.T. (Sakura Finetek Europe) and frozen in liquid nitrogen cooled N-methyl butane (Sigma). Tissue blocks were cryosectioned at 9 µm onto SuperFrost slides (Menzel-Gläser), dried and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PFA) for 30 minutes. Sections were washed in phosphate buffered saline (PBS) 3 times for 5 minutes prior to subsequent processing or storage at −80°C. To prepare pre-fixed frozen sections, animals were anaesthetised with Nembutal (Abbott) and transcardially perfused through the left ventricle; a constant flow (10 mL/min) of PBS (10 mL) followed by ice cold PFA in 0.1 M phosphate buffer at pH 7.4 (50 mL) was established using a peristaltic pump. Sciatic nerves were surgically excised, post-fixed in PFA for 6 hours before immersion in 30% sucrose for 48 hours. Tissues were rinsed in PBS, immersed in Tissue Tek O.C.T. and frozen in liquid nitrogen cooled N-methyl butane prior to cryosectioning at 9 µm onto SuperFrost slides (Menzel-Gläser). Slides were dried prior to processing or storage at −80°C.
To prepare pre-fixed teased nerves, animals were anaesthetised with 75 µg/g Nembutal (Abbott) and transcardially perfused through the left ventricle; a constant flow (10 mL/min) of PBS (10 mL) followed by ice cold paraformaldehyde (PFA) in 0.1 M phosphate buffer at pH 7.4 (50 mL) was established using a peristaltic pump. Sciatic nerves were immediately excised and postfixed in 4% w/v PFA for 18 hours at 4°C. Nerves were rinsed in PBS and prepared onto chilled polylysine slides (Menzel-Gläser) where fascicles were cut into 2 mm lengths and individual fibres were teased apart along the length by 0.2 mm entomology pins. Preparations were dried for 18 hours before immunohistochemical processing or storage at −80°C.
Whole mount preparations were prepared using freshly excised nerves which were obtained from animals sacrificed using CO2 narcosis. Sciatic nerves were excised and prepared on chilled polylysine slides where fascicles were separated and cut into 2 mm segments and mounted by the epineurium. Slides were dried overnight, post-fixed in acetone for 10 minutes at −20°C and rinsed in PBS at pH 7.4, before immunohistochemical processing or storage at −80°C.
Primary antibodies used were mouse monoclonal IgG2a anti-quail Pax3 (1∶10; Developmental Studies Hybridoma Bank); rabbit polyclonal anti-mouse Krox24 (1∶250; Aviva Systems Biology); rabbit polyclonal anti-mouse Sox2 (1∶200; Sapphire Bioscience) and rabbit polyclonal anti-mouse p75 nerve growth factor receptor (1∶500; Chemicon). Species specific secondary antibodies used were AlexaFluor488-conjugated to goat anti-mouse IgG2a (1∶500; Molecular Probes) and AlexaFluor546-conjugated to goat anti-rabbit IgG (1∶500; Molecular Probes).
Slides were rehydrated in Tris buffered saline (TBS) and permeabilised in 0.01% v/v Triton X100 (for 45 minutes at 25°C. Slides were washed in TBS 3 times for 10 minutes each prior to incubation in 10% v/v normal goat serum for 6 hours at 25°C. Primary antibodies were individually or simultaneously incubated with 0.2% v/v TritonX100 for 18 hours at 4°C. Specimens were washed in 0.05% v/v TBS/Tween20, 6 times for 30 minutes each, using gentle agitation. Secondary antibody incubation was done thereafter at 25°C for 20 minutes. Specimens were washed in TBS/Tween20, 6 times for 30 minutes using gentle agitation where the last wash contained Hoechst DNA dye 33342 (1 ng/ml). Coverslips were mounted with FluorSave medium (Calbiochem). Negative controls were processed at the same time but were not incubated with primary or secondary antibody.
Fluorescently labeled tissues were viewed with an Olympus BX51 microscope connected to an Olympus DP71 digital camera and digital images were collected in the Olympus analySIS FIVE program and transferred to the IrfanView (4.27) program for montage construction. The contrast and brightness of these images were unaltered. Whole mount specimens were imaged with a BioRad MRC 1000/1024 UV laser scanning confocal microscope on a Nikon Diaphot 300 with either a 40X objective (with zoom) or 60X immersion objective (without zoom) using a 351- and 488-nanometer argon laser and a 543-nanometer helium/neon laser. Gain and black level adjustments were performed to improve analogue to digital signal conversion and background noise was eliminated using a KALMAN filter. Z-stacks were collected using various step-sizes and KALMAN averaging was performed manually for each step. Digital images were collected and compiled in greyscale and subsequently pseudocoloured with hues approximate to the fluorescence emission spectra of the respective fluorophores using the Confocal Assistant™ (4.02) program. Images were transferred to Adobe Photoshop (7.0) and IrfanView (4.27) programs for montage construction. The images were unaltered.
The author would like to gratefully acknowledge Dr Paul Rigby for expert guidance and instruction using the scanning laser confocal microscope, Tammy Esmaili and Andrew Wilson for assistance with animal work, Dr Mark Brown and Rebecca Slattery for assistance with RT-PCR investigations, Dr Jennifer Thompson for insightful immunohistochemical advice and Dr Angus Stewart for instruments necessary for quality nerve fibre preparations. The Pax3 monoclonal antibody, developed by C.P. Ordahl, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA, USA. BioRad microscopy was carried out using facilities at the Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, which is supported by University, State and Federal Government funding.