Previously, we demonstrated i) that ergocalciferol (vitamin D 2 ) increases axon diameter and potentiates nerve regeneration in a rat model of transected peripheral nerve and ii) that cholecalciferol (vitamin D3) improves breathing and hyper-reflexia in a rat model of paraplegia. However, before bringing this molecule to the clinic, it was of prime importance i) to assess which form – ergocalciferol versus cholecalciferol – and which dose were the most efficient and ii) to identify the molecular pathways activated by this pleiotropic molecule. The rat left peroneal nerve was cut out on a length of 10 mm and autografted in an inverted position. Animals were treated with either cholecalciferol or ergocalciferol, at the dose of 100 or 500 IU/kg/day, or excipient (Vehicle), and compared to unlesioned rats (Control). Functional recovery of hindlimb was measured weekly, during 12 weeks, using the peroneal functional index. Ventilatory, motor and sensitive responses of the regenerated axons were recorded and histological analysis was performed. In parallel, to identify the genes regulated by vitamin D in dorsal root ganglia and/or Schwann cells, we performed an in vitro transcriptome study. We observed that cholecalciferol is more efficient than ergocalciferol and, when delivered at a high dose (500 IU/kg/day), cholecalciferol induces a significant locomotor and electrophysiological recovery. We also demonstrated that cholecalciferol increases i) the number of preserved or newly formed axons in the proximal end, ii) the mean axon diameter in the distal end, and iii) neurite myelination in both distal and proximal ends. Finally, we found a modified expression of several genes involved in axogenesis and myelination, after 24 hours of vitamin supplementation. Our study is the first to demonstrate that vitamin D acts on myelination via the activation of several myelin-associated genes. It paves the way for future randomised controlled clinical trials for peripheral nerve or spinal cord repair.

Funding: This work was financially supported by public grants from Aix-Marseille University, CNRS (Centre National de la Recherche Scientifique), DGA (Délégation Générale des Armées), and by private grants from various associations (Association Libre d’Aide à la Recherche sur la Moelle Epinière, Demain Debout, Combattre la Paralysie) and foundations (Avenir, Intermarché). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2013 Chabas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Calciferols are FDA-approved molecules used for preventing rickets or treating psoriasis. Nonetheless, there is currently no indication for neurological disorders or trauma. Therefore, in order to move closer to patients, we devised a pharmacological study based on the weekly delivery of an oral dose (low or high) of either ergocalciferol or cholecalciferol. For the low dose, we maintained our initial choice of 100 IU/kg/day (700 IU/kg/week) that potentiated some functional recovery [1] . For the high dose, we elected the concentration of 500 IU/kg/day (3,500 IU/kg/week) that has proven to be safe in humans. A study looking at graded doses of cholecalciferol, delivered daily to 38 healthy men during 8 weeks, found that the dose of 50,000 IU (the equivalent of a bolus of 500 IU/kg/day for a man weighing 100 kg) was safe, without jeopardizing the phosphocalcic homeostasis [19] . A similar outcome - no hypocalcaemia and no hypercalciuria - was reported by two further studies on escalating doses of cholecalciferol (up to 88,000 IU/day), administered during long periods (up to 6 years) to patients with multiple sclerosis [20] , [21] . In order to avoid repeated handling of the animals, we also opted for a weekly administration of calciferol, a choice that is supposed not to interfere with vitamin D efficiency since it takes 2 months to return to baseline level after administration of a single high dose of cholecalciferol [22] .

However, very little is known about the role of vitamin D during myelination. It is established that the VDR is present in both oligodendrocytes and Schwann cells. When added to cultured myelinating cells, calcitriol induces an upregulation of the transcripts coding for VDR and NGF [17] , [18] but has no effect on the mRNA level of Myelin Basic Protein (MBP) or ProteoLipid Protein (PLP) [17] . In order to better understand the putative role of vitamin D on myelination, we performed a comparative pangenomic transcriptome study, after a 24-hour incubation of dorsal root ganglion cells and/or Schwann cells with calcitriol.

Like other neurosteroids, the genomic action of calcitriol is mediated by a nuclear receptor, the VDR, a member of the steroid/thyroid hormone super-family of transcription regulation factors. After hetero-dimerisation with nuclear receptors of the retinoic X receptor (RXR) family, the VDR and its ligand bind to vitamin D responsive elements (VDRE), located in the promoter regions of hundreds of target genes [9] . For example, a VDRE has been found upstream from genes coding for Brain Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF) and Neurotrophin 3 (NT3) [9] . As a result, vitamin D regulates the expression of NGF [10] , [11] , [12] , NT3 and NT4 [13] , and Glial cell line-Derived Neurotrophic Factor (GDNF) [14] . When added to cultured hippocampal cells, calcitriol increases neurite outgrowth [12] . Conversely, when vitamin D is removed from the diet of pregnant rat females, decreased expression of NGF is observed in the brains of neonate [15] and adult offspring [16] .

Vitamin D is a group of seco-steroid hormones, including the fungi-derived form of vitamin D, named vitamin D 2 or ergocalciferol, and the animal-derived form of vitamin D, named vitamin D 3 or cholecalciferol. After two separate hydroxylations, performed by two P450 enzymes (25-hydroxylase and 1-alpha-hydroxylase, respectively), both calciferols give rise to the active form (1,25(OH)2D), referred to as calcitriol [2] . Initially, it was thought that liver and kidneys were the only organs responsible for the production of calcitriol. However, it is now clearly established that many tissues, including the brain [3] , express vitamin D 1-alpha-hydroxylase. Moreover, vitamin D receptors (VDRs) are widely distributed throughout the brain, in rats [4] , [5] , [6] , [7] , [8] as well as in humans [3] .

In a previous study, using a rat model of nerve trauma, we demonstrated that vitamin D 2 is a potent compound that promoted axon sparing/regeneration and improved physiological maturation [1] . We also observed that vitamin D 2 supplementation induced an increase in axon diameter, suggesting that myelination was probably enhanced [1] . However, we had no direct evidence that vitamin D is a true myelinating agent.

Materials and Methods

Animals Six-day-old (for DRG cultures) and 5-week-old (for Schwann cells) Sprague Dawley (Charles River®, Les Oncins, France) male rats were used for the in vitro study and 8-week-old male Sprague Dawley rats, weighting 250–300 g (Charles River®, Les Oncins, France) were used for the pharmacological study. All animals were housed in smooth-bottomed plastic cages at 22°C with a 12-hour light/dark cycle. Food (Purina®, rat chow) and water were available ad libitum. Anaesthesia and surgical procedures were performed according to the French law on animal welfare and the Animal Care Committees of Aix-Marseille University and the? CNRS (Centre National de la Recherche Scientifique) approved our protocols. Furthermore, experiments were performed following the recommendations provided in the Guide for Care and Use of Laboratory Animals (U.S. Department of Health and Human Services, National Institutes of Health) and in accordance with the European Community council directive of 24 November 1986 (86/609/EEC).

Experimental Design In a first set of experiments, the rats (n = 36) were randomised into six groups. A Control (C) group (n = 6) included animals on which no surgery was performed. Animals were deeply anaesthetised (Pentobarbital Sodique® Sanofi Santé Animale, 60 mg/kg). Surgical procedures were performed aseptically under binoculars. The peroneal nerve from the left limb was dissected free from the surrounding tissues on a length of 3–4 cm and a 1 cm segment was removed. The nerve segment was immediately replaced in inverted position, and sutured at the two free nerve stumps by three epineurial stitches (Ethilon® 10–0, Ethicon Inc., Johnson & Johnson Company, Auneau, France). Muscles and skin were stitched (Vicryl® 3–0, Ethicon Inc., Johnson & Johnson Company, Auneau, France). Immediately after lesioning, rats were orally fed weekly with a 500 μl bolus of either ergocalciferol, at the dose of 100 IU/kg/day (D2–100 group, n = 6) or 500 IU/kg/day (D2–500 group, n = 6), or cholecalciferol, at the dose of 100 IU/kg/day (D3–100 group, n = 6) or 500 IU/kg/day (D3–500 group, n = 6), or the excipient (triglycerides) (Vehicle group, n = 6). Rats were weighted every week and the delivered vitamin D doses were adjusted accordingly. For example, a 300 g rat from the D-100 groups received a weekly dose of 210 IU. All compounds were purchased from Crinex Laboratories (Montrouge, France). In a second set of experiments, we increased the statistical power of our study by replicating experiments with 18 rats randomised into 3 groups, as follows: Control group (n = 6); Vehicle group (n = 6) and D3–500 group (n = 6).

Serumal Vitamin D Assessment At 3 months post-surgery and one week after the last vitamin D delivery, blood was collected from the Vehicle, D2–500 and D3–500 groups (n = 12 for Vehicle and D3–500 groups; n = 6 for D2–500 group). Serumal calcidiol (25(OH)D2+25(OH)D3) levels were quantified with a standard commercial radioimmuno-assay (La Timone Hospital, Marseille, France).

Functional Assessment of Hind Limb Recovery The peroneal functional index (PFI) indicating the functional alteration of the experimental nerve when compared with the opposite side was calculated by the method of Bain et al. [23] with the following formula: PFI = 174.9[(ePL-nPL)/nPL]+80.3[(eTS-nTS)/nTS]-13.4. The PFI recovery rate was defined with a score from −100 to −13.4, where −13.4 represents normal function and −100 a total absence of contraction. Hindlimb paws were marked with black ink and footprints were recorded each week on paper track, copied in a high-resolution scanner, and digitalised images were analysed. Data concerning each animal were individually identified. The parameters measured for both normal (n) and operated (e) feet were footprint length (PL, or longitudinal distance between the tip of the longest toe and the heel) and total toe spread (TS, or cross-sectional distance between the first and fifth toes). Footprints were obtained and analysed on a weekly basis, from the second to the eleventh week after surgery, by an investigator blinded to the treatment groups. All animals had been conditioned to walk homogeneously into the recording apparatus three times per day over five days during the week before surgery.

Electrophysiological Recordings Twelve weeks after surgery, rats were re-anaesthetised by an intra-peritoneal injection of solution containing sodium pentobarbital (Pentobarbital Sodique®, Sanofi Santé Animale, 60 mg.kg−1). A tracheotomy was performed and rats were artificially ventilated (Harvard® volumetric pump: rate 40–60 min−1, tidal volume 2–4 ml, Southmatick, MA, USA). The left peroneal nerve was dissected free from surrounding tissues on a length of 3–4 cm. Maximum relaxation rate. The twitch contraction of the tibialis anterior by nerve stimulation was induced with a neurostimulator (Grass S88K®, Grass Technologies, Astro-med Inc., Rhode Island, USA) rectangular delivering, through an isolation unit, a single shock (duration: 0.1 ms, frequency 0.5 Hz) and measured with a strength gauge (Micromanometer 7001®, Ugo Basile Srl., Biological Research Apparatus, Comerio VA, Italy). Twitches were analysed in terms of peak amplitude (A) and maximum relaxation rate (MRR), defined as the slope of a tangent drawn to the steepest portion of the relaxation curve. MRR was normalised to the total twitch amplitude (MRR/A = mean relaxation rate constant, ms−1), as suggested by Esau et al. [24] who showed that MRR values are linearly related to A. Twitches were recorded with a Biopac MP150® system (sampled at 2000 Hz, low-pass filtered at 150 Hz) and analysed with the AcqKnowledge® 3.7.3 software. Tetanus threshold. Muscle stimulation was induced by a pair of steel stimulating electrodes (inter-electrode distance: 4–5 mm) placed on the surface of the tibialis anterior muscle. Contractions were produced by the neurostimulator (Grass S88K®) delivering trains of rectangular pulses. After determining a threshold able to elicit a twitch, pulse train intensity was set to a supramaximal level. Tetanic threshold was obtained by increasing frequency by 5 Hz steps. The voltage was 20% higher than the voltage evoking a maximal twitch. The duration of stimulus trains was 500 ms, and trains were repeated each second to produce a series of contractions. Pulse duration was 2 ms and five single stimulations were delivered during each 500 ms train (10 Hz). Ventilatory response. According to one of our previous studies [25], changes in ventilation were recorded after tibialis anterior stimulation. The experiments were performed after regional circulatory occlusion which isolated and maintained the neural drive and abolished humoural communication. Repetitive muscle stimulation induces fatigue, which activates the muscle metabosensitive afferent fibres projecting to the bulbar respiratory centre and subsequently increases ventilation. To elicit electrically-induced muscle fatigue (EIF), rhythmic muscle contractions were produced by the neurostimulator (Grass® S88, Quincy, MA) which delivered pulse trains to the muscle surface electrode (pulse duration: 0.1 ms; frequency: 10 Hz, i.e., 5 shocks in each 500 ms train; duty cycle: 500/1500 ms, voltage range: 5 to 8 volts). The voltage was supramaximal, i.e. 20% higher than that used to elicit a maximal twitch. Fatigue was assessed from the decay of force throughout the 3-min EIF period. The strength of muscle contraction (the decay of force) was measured from the beginning to the end of this 3-min muscle electrical stimulation. We chose to stimulate the muscle directly because we previously showed that muscle low frequency stimulation is a strong activator of metabosensitive afferent fibres [26]. Ventilatory activity was recorded using a thermocouple inserted into the tracheal canula. Measurement was performed two minutes before EIF (rest condition) and 5 minutes after, and expressed in cycles/min. Changes in ventilatory activity after EIF was expressed in percent [Δcycle/min (%)] of the mean cycles recorded two minutes before muscle stimulation. Afferent activity. The proximal portion of the peroneal nerve was cut. In order to record the afferent activity from the tibialis anterior muscle, a few millimetres of the epineurial tissue were removed from the free end of the distal nerve using an operating microscope (x40, MZ75®, Leica, Heerbrugg, Switzerland). Then, the distal nerve was positioned on a monopolar tungsten electrode and immersed in paraffin oil. Nerve activity was recorded in reference to a nearby ground electrode implanted in a close muscle, amplified (50 to 100 K) and filtered (30 Hz to 10 kHz) by a differential amplifier (P2MP® SARL, Marseille, France). The afferent discharge was recorded (Biopac MP150® and AcqKnowledge® software, BIOPAC Systems, Inc., Goleta, USA) and fed into pulse window discriminators (P2MP® SARL, Marseille, France), which simultaneously analysed afferent populations. The output of these discriminators provided noise-free tracings (discriminated units) that were counted by a data analysis system (Biopac AcqKnowledge® software BIOPAC Systems, Inc., Goleta, USA) at 1 s intervals (in Hz) and then displayed on a computer. The discriminated units were counted and recorded on separate tracings. As previously described [1], [2], we recorded the response of muscle afferents after 1) a 3 min low frequency (10 Hz) electrical stimulation of the tibialis anterior muscle [simulated fatigue - Electrically-Induced Fatigue (EIF)], 2) an intra-muscular injection of capsaicin solution (655 μM in 50 µL of saline). The discharge rate of nerve afferents was averaged for a 1-min period preceding EIF or capsaicin injection (baseline discharge), and its maximal change was measured following activation. The increase in average afferent discharge rate after EIF or capsaicin injection was expressed as a percentage of average afferent discharge rate before activation. A 20-minute recovery period was allowed between EIF and capsaicin injection.

Muscular Atrophy Measurement After the electrophysiological study, rats were sacrificed by an intra-arterial overdose (1 ml) of sodium pentobarbital solution (Pentobarbital Sodique®, Sanofi Santé Animale, 60 mg.ml−1). The left tibialis anterior muscle was harvested and immediately weighted on a precision scale (Navigator™, N30330 model, OHAUS Corp., New Jersey, USA). Comparisons of muscle mass atrophy were performed using a muscle weight/body weight ratio.

Histology and Microscopy Histology assessments were performed with the three most interesting groups: Control, Vehicle and D3–500. For axon number counting, peroneal nerves (n = 6 per group) were harvested, washed in phosphate-buffered saline (PBS), immersed in a 4% paraformaldehyde-containing PBS solution during 24 hours and sectioned in three parts (proximal end, middle of the segment and distal end) before being immunostained with an anti-neurofilament antibody. For the Control group, a unique section, located at the middle of the virtually-sectioned segment, was collected. In each group, the samples were included in paraffin. After embedding, sections (5 μm) were cut on a microtome (RM2155, Leica®, Solms, Germany) and collected on SuperFrost Plus® slides (Gerhard Menzel-Glaser, GmbH, Germany). Then sections were immunostained with a mouse monoclonal antibody to the light chain of the neurofilament protein (NF-L 70 kDa, Dako MO762, dilution: 1/100) using a robot (Benchmark® XT, Ventana Medical Systems, Inc., Arizona, USA). After washing, an appropriate biotinylated-conjugated secondary antibody was applied to the sections. The final staining step was performed using diaminobenzidine (Ventana® iVIEW DAB 760 091, Ventana Medical Systems, Inc., Arizona, USA). For myelination assessment, peroneal nerves (n = 6 per group) were harvested free from surrounding tissues, washed in PBS (Gibco®, Life Technologies Corp., Saint Aubin, France) and immersed in a 4% glutaraldehyde-containing PBS solution during 24 hours. Samples were stained with p-phenylenediamine (PPD). After inclusion, semithin sections (0.8 µm) were cut using an ultramicrotome (Ultracut® R, Leica, Solms, Germany) and collected on SuperFrost Plus® slides. After being dried for 12 hours on a hot plate, sections were stained with a p-phenylenediamine-ethanol (70°) solution, washed in distilled water, dried for 5 hours on a hot plate and mounted with glycerol-containing medium (Glycergel®, DakoCytomation, Glostrup, Denmark). The slides were examined using an optical microscope (Eclipse® E800, Nikon, Champigny-sur-Marne, France) that was associated with a high-resolution colour digital camera (DXM 1200, Nikon). The slides were digitised and analysed with the Histolab software (Alphelys®, Plaisir, France). The following parameters were measured: axon number and axonal area. The counting was performed by a robot and therefore observer-associated biases were avoided. To assess G-ratio (i.e. the ratio between the diameter of the axon and the outer diameter of the myelinated fibre), slides were coded, 5 regions of interest in each section were randomly chosen and data analysis was performed blindly.

Cultures of Schwann Cells and Dorsal Root Ganglia Nerves from three rats were collected and the connective tissue surrounding nerve bundles was carefully discarded. Nerves were then cut into small pieces with a McIlwain chopper and enzymatically dissociated during 10 minutes with a solution of tryspin-EDTA (0.25% Tryspin, 1 mM EDTA, Invitrogen®, Life Technologies Corp., Saint Aubin, France) diluted in Hank's Balanced Salt Solution (HBSS) and then mechanically dissociated with a flamed Pasteur pipette. The enzyme activity was blocked with 9 ml of DMEM/Ham-F12 supplemented with foetal calf serum (10%) (DH/FCS), penicillin (50 U.ml−1) and streptomycin (50 μg.ml−1) (Invitrogen®), the cell suspension was centrifuged for 5 min at 300 g and the cell pellet was resuspended in DH/FCS before being plated onto poly-L-lysine-coated (2 μg.cm−2) flasks. When the required number of cells was obtained, Schwann cells were detached using trypsin (0.25%), centrifuged and re-seeded, at the density of 5,000 cells per cm2 in DMEM/Ham-F12 supplemented with insulin, transferrin and selenium (ITS, Invitrogen®) and TGFα (25 ng.ml−1, Sigma-Aldrich Co., Missouri, USA). Thoracolumbar dorsal root ganglia (DRG) were removed from young (Postnatal day 6) Sprague Dawley rats, anaesthetised with a lethal dose of inhaled isoflurane. Ganglia were transferred into Dulbecco’s modified Eagle medium (DMEM, Invitrogen®) supplemented with penicillin and streptomycin (Invitrogen®). The connective tissue was removed and ganglia from 10 rats were collected and seeded in a 6-well plate, precoated with poly-D-lysine (1 mg.ml−1, Sigma-Aldrich Co.) and laminin (10 μg.ml−1, Sigma-Aldrich Co.,) and cultured in DMEM/Ham-F12 supplemented with serum (10%), penicillin, streptomycin, NGF (50 ng.ml−1, Alomone Labs®, Jerusalem, Israel). Two days later, the culture medium was removed and replaced by serum-free Neurobasal medium (NB, Invitrogen®) supplemented with penicillin/streptomycin (40,000 IU/l, Invitrogen®) and B-27 supplement (Invitrogen®). Neurons and Schwann cells grew out from the whole ganglia and the culture was maintained as a mixed culture of the two cell types.

Transcriptome Study Calcitriol (1,25(OH)D3) (#D1530, Sigma-Aldrich Co.) was added at the concentration of 500 nM, during 24 hours, to serum-free DMEM/Ham-F12 supplemented with insulin, transferrin and selenium. Eight 25-cm2 flasks, containing either Schwann cells (n = 4), treated (n = 2) or not (n = 2) with calcitriol or a mixed culture of DRG and Schwann cells (n = 4), treated (n = 2) or not (n = 2) with calcitriol, were used. At the end of the incubation period, cultures of Schwann cells and cultures of DRGs and Schwann cells were trypsinised and centrifuged before being treated with RNeasy lipid minikit (#74804, Qiagen, Life Technologies Corp. California, USA). Unwanted genomic DNA was removed using DNase set kit (#79254, Qiagen). Purified total RNAs, from three pooled replicate cultures, were kept at −80°C and processed for hybridisation on genome-wide DNA microarrays within one month. All RNAs were checked for integrity using the 2100 BioAnalyzer (Agilent Technologies®, California, USA) and quantified using a ND-1000 spectrophotometer (NanoDrop, Thermo Fisher Scientific Inc., Massachusetts, USA). Cyanine-3-labeled cRNA was generated from 0.3 mg of RNA using the One-Color Low RNA Input Linear Amplification kit (Agilent Technologies) according to the manufacturer’s instructions, followed by purification on RNeasy column (Qiagen). All amplified cRNAs were checked for dye incorporation, cRNA yield and amplification profile. Only those fitting all quality criteria were fragmented for further hybridisation onto microarrays. Samples were then carefully hybridised onto Agilent Whole Rat Genome (4644K) Oligo Microarrays (G4131F). Microarrays were scanned using an Agilent DNA microarray scanner G2505B. Data are available on the ArrayExpress database (accession number E-MEXP-3491).

Microarray Data Analysis Individual microarray quality was evaluated based on QC report, pair-wise MA-plots, and box plots. Intra-array normalisation of raw signals from the 8 microarrays (corresponding to the four above-mentioned conditions in duplicate) was performed using Feature Extraction software 9.1.3.1 (Agilent Technologies®). Microarray normalised data were further exported into the Limma package, for inter-array normalisation using the quantile method. Statistical analysis was performed using the TIGR MeV (MultiExperiment Viewer) v4.4 software (http://www.tm4.org/mev.html) and the GeneANOVA program. Multi-way ANOVA model was implemented: first, to identify differentially regulated genes when accounting for the multiple sources of variation in the microarray experiment; second, to evaluate the effect of the main variable, the addition of calcitriol during 24 hours. Multiple test correction was further carried out using the false discovery rate (FDR) method. Cluster and Tree View softwares were used for unsupervised hierarchical clustering.

Overview of Functional Patterns Altered by Vitamin D Supplementation As a primary analysis, genes identified to be differentially-expressed were analysed for significant gene ontology clusters using DAVID Bioinformatics Resources (http://david.abcc.ncifcrf.gov/). Gene functional classification was used to rank gene ontology clusters by statistical over-representation of individual genes in specific categories relative to all genes in the same category on the filtered list [27]. As a secondary analysis, biological interpretation of the data from the DNA microarrays was performed using Ingenuity Systems Pathway Analysis (http://www.ingenuity.com/). This database builds networks on candidate genes/proteins and putatively associated genes/proteins according to the data collected in previous publications.

Quantitative PCR Validation of Under- and Over-expressed Genes The samples used for the microarray experiment were reverse transcribed. Synthesis of cDNA was performed with oligo dT, RNase Out and M-MLV RT enzyme (Invitrogen®). Four genes (Igf1, Prx, Spp1 and Tspan2) involved in axogenesis and myelination and one housekeeping gene (Gapdh) were selected for validation using the quantitative PCR technique. Before selecting Gapdh as a housekeeping gene, we checked that its expression remained steady in the gene array database. The PCR was performed using the TaqMan Gene Expression Master Mix (Applied Biosystems® #4369016, Life Technologies Corp., California, USA) with the following conditions: 40 cycles, 15 s at 95°C and 1 min at 60°C. Probes were purchased from Applied Biosystems®. The reference numbers for each gene were: Rn01775763 (Gapdh); Rn00710306_m1* (Igf1); Rn00576815_m1* (Prx); Rn00563571_m1* (Spp1) and Rn00574907_m1* (Tspan2).