December 2012
Volume 53, Issue 13
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Biochemistry and Molecular Biology  |   December 2012
Changes in mRNA Expression of Class 3 Semaphorins and Their Receptors in the Adult Rat Retino-Collicular System after Unilateral Optic Nerve Injury
Author Affiliations & Notes
  • Anil Sharma
    From the School of Anatomy, Physiology and Human Biology, The University of Western Australia, Crawley, WA, Australia; and the
  • Margaret A. Pollett
    From the School of Anatomy, Physiology and Human Biology, The University of Western Australia, Crawley, WA, Australia; and the
  • Giles W. Plant
    From the School of Anatomy, Physiology and Human Biology, The University of Western Australia, Crawley, WA, Australia; and the
    Stanford Partnership for Spinal Cord Injury and Repair, Stanford Institute for Neuro-Innovation and Translational Neurosciences, School of Medicine, Stanford University, Stanford, California.
  • Alan R. Harvey
    From the School of Anatomy, Physiology and Human Biology, The University of Western Australia, Crawley, WA, Australia; and the
  • Corresponding author: Anil Sharma, School of Anatomy, Physiology and Human Biology, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia; anil@propriocept.com
Investigative Ophthalmology & Visual Science December 2012, Vol.53, 8367-8377. doi:10.1167/iovs.12-10799
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      Anil Sharma, Margaret A. Pollett, Giles W. Plant, Alan R. Harvey; Changes in mRNA Expression of Class 3 Semaphorins and Their Receptors in the Adult Rat Retino-Collicular System after Unilateral Optic Nerve Injury. Invest. Ophthalmol. Vis. Sci. 2012;53(13):8367-8377. doi: 10.1167/iovs.12-10799.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Increasing interest in the role of Class 3 Semaphorins (Sema3s) in plasticity and repair in the injured mammalian central nervous system prompted us to characterize changes in Sema3 expression after optic nerve (ON) injury.

Methods.: We used unilateral ON transection (ONT) and ON crush (ONC) models in conjunction with quantitative polymerase chain reaction (qPCR), and in situ hybridization (ISH) to characterize postinjury changes in the expression of the Sema3s and their receptors in the rat retina, optic nerve, and superior colliculus (SC).

Results.: We observed no changes in mRNA expression in axotomized retinas at 1 or 14 days after ONT, but there was a transient increase for Sema3b, Sema3f, L1cam, and Plxna3 at 3 days postinjury. There was no change in transcript expression in the deafferented contralateral SC 1 day following ONT, but there was a transient increase in Plxna2 at 3 days, and a decrease in Sema3e, L1cam, and Plxna4a mRNA levels by 14 days. There were also several changes in transcript expression in the unlesioned contralateral retina and ipsilateral SC that differed from those seen in axotomized retina and contralateral SC. At the injury site after ONC, there was a reduction in Sema3b and Sema3f mRNA at 6 hours, returning to control levels by 1 day, and a transient increase in SEMA3A immunoreactivity at 6 hours.

Conclusions.: These new data on Class 3 Semaphorins and their receptors provide more information about the complex reactive events that occur bilaterally in the retino-collicular system following unilateral adult ON injury.

Introduction
This study aimed to further our understanding of the role of the Class 3 Semaphorins (Sema3s) in the complex events that occur after injury to the adult mammalian central nervous system (CNS). Greater knowledge about the cellular and molecular changes that occur after CNS trauma will aid in the development of effective interventions to overcome intrinsic restrictions on regenerative growth. Members of the secreted Sema3 family are involved in the CNS injury response, 13 in most cases restricting plasticity and regrowth. Characterization of changes in the expression of the Sema3s after injury to the mammalian CNS is incomplete, however, thus we set out to quantify changes in expression of the Sema3s and their receptors in the days after injury to the rat optic nerve, an established model for neurotrauma and regeneration research. 46  
The relevance of using the rat visual system is supported by observations that Sema3s can be repulsive to retinal ganglion cells (RGCs), 712 and rat RGCs express at least some of the requisite receptor components. 13 It is therefore likely that the Sema3s contribute to abortive regenerative responses after visual system injury. For example, in the rat there are reports of short-term increases in SEMA3A, but not NRP1 in the retina 2 to 3 days after intraorbital optic nerve transection (ONT). 14,15 In addition, inhibition of SEMA3A increases survival of RGCs following ONT. 15 Sema3a has also been implicated as a factor mediating RGC survival in the goldfish, where after optic nerve (ON) injury, exogenous SEMA3A significantly reduces RGC survival. 16 Interestingly in the goldfish, which unlike higher vertebrates is able to functionally regenerate, SEMA3A expression after ON injury is reduced in the ganglion cell layer 3 days postinjury, returning to control levels by 7 days. 16  
In this study, both intraorbital ONT and ON crush (ONC) models were used. These models are commonly used to study postlesion molecular and cellular changes in the retina, the retinofugal pathway, and central retino-recipient targets. 6,1719 After ONT, cellular responses in the rat retina can be divided into three general time periods: early, delayed, and late. These periods approximate 0 to 4 days, 4 to 14 days, and more than 14 days after the injury; up to approximately 4 days after ONT there is little overt change in the cellular composition of the affected retina, followed by a rapid decline in the number of RGCs that continues for approximately 2 weeks. 2024  
Molecular changes in the retina following ONT can also be roughly divided into early, delayed, and late time periods, 25 but follow a slightly different time profile to cellular changes. 2528 mRNAs for many transcription factors are upregulated in the axotomized retina as early as 4 hours following ONT, but expression drops to approximately control levels by 3 days. 25 Such early changes in gene expression may reflect a commitment to apoptosis by RGCs in response to axotomy. 2931 Longer-term molecular changes in axotomized retinal tissue include upregulation of ciliary neurotrophic factor (CNTF), 32 basic fibroblast growth factor (bFGF), 33 EphA, 34 Caspase-8 activity, 35 and Netrin-1 receptors. 36 More recently, a large-scale analysis of protein expression after ONT has revealed changes in expression of a diverse range of genes in the retina at early, delayed, and late time periods. 37  
After ONT, there are also changes in the deafferented superficial layers of the contralateral SC, including reactionary synaptogenesis from remaining projections 3846 and changes in glial cell reactivity. 4750 Deafferented SC also reexpresses at least some axon guidance molecules that can influence growth of embryonic and adult RGC axons. 34,51 ONC also results in damage to RGC axons, the extent related to the severity of the crush. 52 Six hours after ONC injured RGC axons retract from the injury site and after 1 week, injured axons have formed sprouts into the lesion site 53 ; however, this regeneration is generally abortive 6,54 due to a significant extent to the inhibitory milieu of the injury site. 1,4,55  
At the time of this study, there were few validated antibodies against the Sema3s and their ligands, indeed for some of those proteins there were no available antibodies. Because of this we chose to quantitatively analyze mRNA changes in the retina and SC at 1, 3, and 14 days after ONT using quantitative reverse-transcriptase PCR (qPCR). Additionally, we began to characterize early changes in Sema3 expression at the injury site itself using ONC 6 combined with in situ hybridization and immunohistochemistry. 
Methods
Experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, National Health and Medical Research Council (Australia) guidelines, and were approved by the Animal Ethics Committee of The University of Western Australia. Animals were sourced from the Animal Resources Centre of Western Australia. Immediately after surgery all animals received a subcutaneous injection of analgesic (0.02 mg/kg buprenorphine; Temgesic; Reckitt and Colman, Hull, UK), and intramuscular (IM) injection of antibiotics (0.1 mL penicillin; Benacillin; Ilium, Troy Laboratories, Glendenning, Australia). 
Optic Nerve Transection
Twenty adult (8- to 10-week-old) female Wistar rats were anesthetized by intraperitoneal (IP) injection of ketamine (10 mg/kg; Ilium) and xylazine (80 mg/kg; Ilium). An incision was made above the left eye, superior rectus muscle cut, and the ON exposed by blunt dissection. A suture (6-0 Ethicon; Johnson & Johnson, North Ryde, Australia) was tied through the ON sheath and nerve approximately 5 mm distal from the eye and the ON was then transected as described by Solomon and colleagues. 56 Complete transection was confirmed by retraction of the unanchored ON and examination of the eye confirmed no damage to the ophthalmic artery. In this part of the study, 6 to 8 animals were used for qPCR analysis per time point. 
Rats were euthanized by an overdose of pentobarbitone sodium (IP, 500 ng/g body weight; Virbac, Milperra, Australia) at 1, 3, or 14 days after ONT. Immediately after euthanasia, the following tissues were dissected and immediately placed into RNAlater (Ambion, Austin, TX): injured retina, uninjured contralateral retina, proximal (to the eye) injured ON, distal injured ON, uninjured contralateral ON, ipsilateral and contralateral (to the injured ON) SC. Tissue was stored overnight at 4°C before longer storage at −20°C. 
Optic Nerve Crush
Four adult (8- to 10-week-old) female Wistar rats were anesthetized as described above. The ON was crushed approximately 3 mm from its exit from the eye with a pair of No. 3 micro-forceps (2000 microforceps, Medicon Tuttlingen, Germany) for exactly 5 seconds. Confirmation of integrity of the ophthalmic artery was obtained by examination of the eye. All nerve crushes were performed on the same day and by the same surgeon. Two rats were used for each time point (6 hours or 1 day postinjury) and compared with 2 uninjured rats. 
At the appropriate postinjury time points, rats were anesthetized by IP injection of pentobarbitone sodium (500 ng/g body weight; Virbac), before transcardial perfusion with 0.05% (wt/vol) heparin (David Bull Laboratories, Lidcombe, Australia) in PBS and then 4% paraformaldehyde (wt/vol; Sigma-Aldrich, Castle Hill, Australia) in 0.1 M Sorenson's Buffer (4% PFA; pH 7.4). ONs were dissected out and postfixed for a further 30 minutes in 4% PFA before being placed in diethylpyrocarbonate (DEPC)-treated PBS containing 30% sucrose (wt/vol; Sigma-Aldrich) overnight at 4°C for cryoprotection. 
Over the following days, the PBS/sucrose solution was gradually replaced with Jung tissue freezing medium (Leica Microsystems, Mt. Waverley, Australia). Infused with tissue freezing medium, tissue was snap frozen in isopropanol (2-propanol; Sigma-Aldrich). Sections were then cut on a cryostat, placed on SuperFrostPlus slides (Menzel-Gläser, Braunschweig, Germany), and then stored at −80°C until processing by in situ hybridization (ISH). 
qPCR Analysis
Retina and SC were homogenized in 1 mL Tri Reagent (Molecular Research Center, Cincinnati, OH) using a Kinematica PT-2100 (Kinematica A.G., Lucerne, Switzerland) for approximately 10 to 20 seconds before RNA extraction according to the manufacturer's instructions. RNA concentration was measured by spectrophotometry in a Nanodrop ND-1000 (Nanodrop; Thermo Fisher Scientific, North Ryde, Australia) and 1.5 μg RNA treated with recombinant DNase I (rDNase I; DNA-free; Ambion). 
First strand cDNA was synthesized using Omniscript (Qiagen, Doncaster, Australia). All temperature steps were carried out in an MJ Research PTC-1000 thermocycler (MJ Research, Waltham, MA). One modification was the inclusion of two extra incubation steps, added to increase primer annealing (25°C) and decrease secondary and tertiary RNA structures (55°C). The full incubation protocol was thus 25°C 30 minutes, 37°C 60 minutes, 55°C 30 minutes. Control cDNA samples (n = 4–5) were uninjured adult retina and SC from a previous study. cDNA was purified using the AxyPrep PCR Cleanup Kit (Axygen Biosciences, Union City, CA) and a Qiavac 24 vacuum manifold (Qiagen). 
All comparable tissue samples were processed at the same time. That is, all retina in one run, all ON segments in another run, and all SC in another run. mRNA transcript expression of Sema3a, Sema3b, Sema3c, Sema3e, Sema3f, Plxna1, Plxna2, Plxna3, Plxna4a, Nrp1, Nrp2, and L1cam was quantified, along with mRNA of two reference genes, Rnr1 (18s ribosomal RNA) and Ppia. Primer pairs for each assay were designed using Primer3 57 and NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and are listed in Table 1. Specificity was ensured by single peak melting curves, and single bands after gel electrophoresis. qPCR runs were performed on a Rotor-Gene 3000 or 6000 (Qiagen), using Bio-Rad iQ SYBR 2X Mastermix (Bio-Rad, Gladesville, Australia) in the formula: 5 μL master mix, 1 μL each 5 μM forward and reverse primers, 1 μL cDNA template, 2 μL double deionized water (DDW). 
Table 1. 
 
Primer Pairs for Each qPCR Assay
Table 1. 
 
Primer Pairs for Each qPCR Assay
Gene 5′ Primer 3′ Primer
L1cam GCC CTG AGC TTG AAG ACA TC GCC CTT CCA CCA GTA CGT TA
Nrp1 GGA GCT ACT GGG CTG TGA AG ATG TCG GGA ACT CTG ATT GG
Nrp2 GGC ATT TGT ACG CAA GTT CA GGG CTT TGA GTC TGT CCA GTC
Plxna1 CCA ACT CCT CTA CCT TCA CGA GTG TTC CCT TAG TAG CCA GCA G
Plxna2 TGG GGA CTA AGA GTG GCA AG CAC AAT GAG GAT CCC CTG AG
Plxna3 CAC CCA GAT TCA CCC ACT AAC GAT TCC TCC ATC TCA CAT ACG A
Plxna4a GCA TTA AGG ACC GCC TAC AG AGA CTG GAA TGC CAC GTA CC
Ppia AGC ATA CAG GTC CTG GCA TC TTC ACC TTC CCA AAG ACC AC
Rnr1 GAT CCA TTG GAG GGC AAG TCT CCA AGA TCC AAC TAC GAG CTT TTT
Rpl19 CTG AAG GTC AAA GGG AAT GTG CCT CCT TGC ACA GAG TCT TGA
Sema3a ATA TGC AAG AAT GAC TTT GGA GGA C AAG GAA CAC CCT TCT TAC ATC ACT C
Sema3b GCT GTC TTC TCC ACC TCC AG ACA TGC CAG GTC TTG GGT AG
Sema3c AGA CGT GAG ACA CGG GAA TC TTC ATT CAG TTT AAC CCT CCT TCC
Sema3e GCG TCA GTG ATG GCT ACA GA CAA AAC CCG GAC ATA ATT GG
Sema3f CTC TTC CAA GAG GCA ACA ACT G TTT GCA TTG GAA TTG AAA CCA C
Efficiencies for each individual qPCR reaction were calculated using LinRegPCR, 58,59 the means calculated for each primer pair in each tissue type (Table 1). Cq (quantification cycle) values were obtained from Rotor-Gene 6.0 software using the inbuilt second derivative maximum (SDM) equation. Mean efficiencies were used in the delta Cq equation to calculate relative expression levels. 60 Initial fluorescence levels were calculated, thus being directly proportional to initial template levels, 61 similar to the method described by Schefe and colleagues. 62 Expression levels were then normalized to the geometric mean of the reference genes. 
Statistical Analysis of qPCR Data
To ensure correct statistical handling of data, they were tested for conformity to normal distribution (Shapiro-Wilk W test), and homogeneity of variance (Levene's test) between groups. When normality and equal variance of populations could be assumed, one-way ANOVA was used to test for changes between groups, with Dunnet's post hoc test to analyze changes between postoperative groups and the control uninjured group. If either normality or equivalence of variance was not confirmed, then data were Log10 transformed and the tests run again. In case where transformation did not make variance of populations equal, Welch ANOVA was run on untransformed data to test for changes between groups, with a Games-Howell post hoc test to analyze changes between postoperative groups and the control uninjured group. Finally, if transformed data did not have a normal distribution, changes between groups was assessed by Kruskal-Wallis, and differences between uninjured and injured groups assessed by Mann-Whitney U tests. All statistical tests were performed in SPSS 19.0 (IBM, Armonk, NY). 
Riboprobe Production
Plasmids for riboprobes were kindly provided by Professor Joost Verhaagen (Netherlands Institute for Brain Research), then cloned and purified using plasmid purification column (Qiagen Maxiprep Kit; Qiagen, Australia). All plasmids used have been described previously. 13 Antisense probes were produced by EcoR I restriction endonuclease (RE; New England Biolabs, Ipswitch, MA) digest followed by in vitro RNA transcription (IVT) with digoxigenin (dig)-labeled RNA (Roche, Castle Hill, Australia) and T7 RNA polymerase (Roche). Riboprobes were then hydrolyzed for 1 hour at 60°C, precipitated with LiCl and 100% ethanol, resuspended in DEPC-treated DDW, and stored at −80°C before use. 
In Situ Hybridization
Sections on slides were processed for ISH with the above riboprobes using methods described previously, 13 with α-dig-AP fragments (Roche) and NBT/BCIP (Roche). Sections were then differentiated in 70% ethanol if necessary, and stored in PBS before proceeding to ISH. 
Immunofluorescence
After ISH processing, sections were processed for immunohistochemistry (IHC). Antibody diluent was 0.1% (wt/vol) Triton-X100, 10% (vol/vol) normal goat serum (NGS; Chemicon, Temecula, CA), in PBS. Primary antibody incubation was 4°C overnight in a humidified chamber, followed by secondary fluorescent antibody incubation for 2 hours in a dark humidified chamber at room temperature. Slides were coverslipped using fluorescent mounting media (DAKO, Campbellfield, Australia). Primary antibodies were α-βIII-tubulin (Tubb3; TUJ1 clone), rabbit polyclonal, 1:2,000 dilution (Covance, Vienna, VA); α-ED1 (Cd68), mouse monoclonal, 1:200 dilution (Serotec, Oxford, UK); α-GFAP (glial acidic fibrillary protein), rabbit polyclonal, 1:1,000 dilution (DAKO); α-OLIG2, rabbit polyclonal, 1:250 dilution (Chemicon). Dilutions and sources of secondary antibodies were: α-rabbit Cy3, goat raised, 1:400 dilution (Jackson ImmunoResearch, West Grove, PA); α-mouse FITC, goat raised, 1:400 dilution (ICN Cappel, Aurora, OH). 
Immunoperoxidase
Two polyclonal antibodies, C17 and N15 (Santa-Cruz Biotechnology, Santa-Cruz, CA), which bind to two separate epitopes of the human SEMA3A protein (c-terminus, C17; n-terminus, N15) were used for anti-SEMA3A immunostaining (generous gifts from Professor Verhaagen, Netherlands Institute for Neuroscience). These antibodies have been shown to have specific and overlapping profiles with each other, and another anti-SEMA3A antibody in both IHC and Western blots. 63,64  
Sections were immunostained according to standard protocols. Antibody diluents were 1% (wt/vol) BSA (Sigma-Aldrich), 0.2% (vol/vol) Triton X-100 in PBS. Primary antibody (1:50 dilution) incubation was overnight at 4°C in a humidified chamber, followed by secondary antibody (biotinylated α-goat; 1:800 dilution; Vector Laboratories, Burlingame, CA) incubation for 2 hours at room temperature, and then ABC reagent (1:200 dilution in PBS) treatment (Vectastain Elite ABC kit, Vector, Switzerland). Color reaction used Pierce diaminobenzidine (DAB) substrate (Thermo Fisher Scientific) at room temperature. Slides were coverslipped using Aquamount (BDH, London, UK). 
Results
For qPCR data, overall statistics across time points and within tissues are given in Table 2, and presented graphically in Figures 1 and 2. In axotomized retinas, out of all the transcripts tested there was a significant increase in expression of the following mRNAs: Sema3b (P < 0.01), Sema3f (P < 0.05), L1cam (P < 0.05), and Plxna3 (P < 0.01) (Fig. 1). All four gene mRNA levels followed the same time-course of expression, with a transient increase above control levels at 3 days after injury, but no significant change seen at either 1 or 14 days. 
Figure 1. 
 
Changes in mRNA in ipsilateral axotomized (grey bars) and contralateral uninjured (white bars) retinas at 1, 3, and 14 days following unilateral ONT. Statistical tests are against control retina mRNA (black bar) from uninjured adult rats. Individual graphs are shaded where expression changed significantly during at least one time point in either tissue. In the ipsilateral retina Sema3b, Sema3f, L1cam, and Plxna3 changed transcript expression compared with controls, and all increased at 3 days postinjury. Within the contralateral retina, Sema3b, Sema3f, and L1cam showed increased transcript expression. For Sema3b the increase was observed only after 14 days. Sema3f increased expression at 3 days after posttransection, but this increase was not statistically significant by 14 days. L1cam mRNA increased from 3 days and into at least 14 days postinjury. U, uninjured control; error bars: ± 1 SEM; *P < 0.05; **P < 0.01. n = 4–8 per group.
Figure 1. 
 
Changes in mRNA in ipsilateral axotomized (grey bars) and contralateral uninjured (white bars) retinas at 1, 3, and 14 days following unilateral ONT. Statistical tests are against control retina mRNA (black bar) from uninjured adult rats. Individual graphs are shaded where expression changed significantly during at least one time point in either tissue. In the ipsilateral retina Sema3b, Sema3f, L1cam, and Plxna3 changed transcript expression compared with controls, and all increased at 3 days postinjury. Within the contralateral retina, Sema3b, Sema3f, and L1cam showed increased transcript expression. For Sema3b the increase was observed only after 14 days. Sema3f increased expression at 3 days after posttransection, but this increase was not statistically significant by 14 days. L1cam mRNA increased from 3 days and into at least 14 days postinjury. U, uninjured control; error bars: ± 1 SEM; *P < 0.05; **P < 0.01. n = 4–8 per group.
Figure 2. 
 
Changes in mRNA in ipsilateral (grey bars) and deafferented contralateral (white bars) SC hemispheres at 1, 3, and 14 days following unilateral ONT. Statistical tests are against control superior colliculus mRNA (black bar) from uninjured adult rats. Individual graphs are shaded where expression changed significantly during at least one time point in either tissue. In the contralateral SC, both L1cam and Plxna4a had reduced mRNA expression compared with controls at 14 days following injury. In contrast, Plxna2 transcript expression was higher than controls at both 3 and 14 days after injury. Within the ipsilateral SC, Sema3e, L1cam, and Plxna4a had lower levels of transcript expression compared with controls 14 days postinjury. Plxna2 had a temporary increase in transcript expression at 3 days after injury, with expression falling to control levels at 14 days postinjury. U, uninjured control; error bars: ± 1 SEM; *P < 0.05; **P < 0.01. n = 5–6 per group.
Figure 2. 
 
Changes in mRNA in ipsilateral (grey bars) and deafferented contralateral (white bars) SC hemispheres at 1, 3, and 14 days following unilateral ONT. Statistical tests are against control superior colliculus mRNA (black bar) from uninjured adult rats. Individual graphs are shaded where expression changed significantly during at least one time point in either tissue. In the contralateral SC, both L1cam and Plxna4a had reduced mRNA expression compared with controls at 14 days following injury. In contrast, Plxna2 transcript expression was higher than controls at both 3 and 14 days after injury. Within the ipsilateral SC, Sema3e, L1cam, and Plxna4a had lower levels of transcript expression compared with controls 14 days postinjury. Plxna2 had a temporary increase in transcript expression at 3 days after injury, with expression falling to control levels at 14 days postinjury. U, uninjured control; error bars: ± 1 SEM; *P < 0.05; **P < 0.01. n = 5–6 per group.
Table 2. 
 
Statistical Results (P) for Changes in mRNA Expression after ONT
Table 2. 
 
Statistical Results (P) for Changes in mRNA Expression after ONT
Ipsilateral Retina Contralateral Retina Ipsilateral Superior Colliculus Contralateral Superior Colliculus
Sema3a 0.850 0.871 N/A N/A
Sema3b 0.009 0.005 0.074 0.035
Sema3c 0.257 0.647 0.386 0.080
Sema3e 0.227 0.924 < 0.001 0.083
Sema3f 0.011 0.003 0.596 0.181
Nrp1 0.148 0.354 0.564 0.242
Nrp2 0.121 0.394 0.459 0.242
L1cam 0.023 0.045 0.001 0.008
Plxna1 0.004 0.396 0.418 0.430
Plxna2 0.138 0.655 0.005 0.024
Plxna3 0.003 0.008 0.920 0.820
Plxna4a 0.024 0.120 0.001 0.006
In the contralateral retina that was not directly affected by ONT, the following genes showed increased transcript levels: Sema3b (P < 0.01), Sema3f (P < 0.01), and L1cam (P < 0.05) (Fig. 1). mRNA levels for Sema3b appeared to rise as soon as 3 days after injury, but this increase was only statistically significant at 14 days. In contrast, Sema3f transcript expression increased at 3 days after injury, although the elevated expression at 14 days did not reach statistical significance. Finally, L1cam mRNA levels were statistically higher at both 3 and 14 days after ONT. 
In the deafferented SC contralateral to the ONT, there were changes in mRNA levels for the following genes: L1cam (P < 0.01), Plxna2 (P < 0.05), and Plxna4a (P < 0.01) (Fig. 2). Both L1cam and Plxna4a showed reduced mRNA expression at 14 days postinjury. In contrast, Plxna2 transcript levels increased at 3 days after ON injury and remained significantly higher at 14 days. Sema3c mRNA expression may also change after injury, judging from the close statistical test (P = 0.08), and around 6-fold increase at 14 days. 
In the SC ipsilateral to ONT (Fig. 2) there were somewhat similar changes in gene expression after injury. Significantly altered transcript levels were seen for Sema3e (P < 0.001), L1cam (P < 0.01), Plxna2 (P < 0.01), and Plxna4a (P < 0.01) (Fig. 2). Sema3e, L1cam, and Plxna4a all had reduced transcript expression levels by 14 days postinjury. Conversely, Plxna2 mRNA expression, as in the contralateral SC, was elevated at 3 days after injury, but values did not differ from control levels at either 1 or 14 days. It is possible that Sema3b transcript expression may also change after injury, given the 4-fold increase at 3 days and close to alpha significance (P = 0.07). 
Surprisingly, no transcript for Sema3a could be detected in either hemisphere of the SC following injury. The expression of Sema3a in the naïve SC is low, and borders on the edge of detection. It is unclear whether the expression of Sema3a in the SC after ONT was lost, or was beyond the sensitivity of this qPCR assay; control samples in the same runs were positive and ran as normal. In addition, mRNA was extracted in sufficient quantity and quality from ON stumps; however, no transcripts could be detected in the resultant cDNA, including the abundantly expressed Rnr1. The source of this technical difficulty was unclear. 
After ONC, the injury site in 6-hour and 1-day tissue was identifiable by gross morphology and/or by increased GFAP immunoreactivity, and the presence of macrophages (ED1 immunolabeling) 6 hours but not necessarily at 1-day postcrush (Fig. 3A). βIII-tubulin immunostaining was also used to assist in identifying the crush site (data not shown). 
Figure 3. 
 
Identification and characterization of the optic nerve crush site. Adult rat ONs were crushed by forceps, removed at 6 hours and 1 day after injury, and processed by ISH and immunohistochemistry. C, F and I show uninjured ON. The injury site (arrows) was identifiable by GFAP immunostaining, and increased expression of ED1 (Cd68; activated macrophages) (A). There was no discernible staining by ISH for Sema3a in injured ON (B). The relatively strong expression of Sema3b in the uninjured ON (C) appears to be downregulated 6 hours after injury, especially at the injury site (D); however, expression returns to normal levels after 1 day (E). There is no appreciable change in Sema3c expression in the ON after injury (F–H). Sema3f expression immediately surrounding the injury site appears to fall considerably at 6 hours after injury (compare I and J); however, Sema3f-positive cells are still seen adjacent to the injury (small arrows). Sema3f expression increases back to around control levels from 1 day after injury, and transcript expression does not appear to colocalize with OLIG2 expression (L). Large arrows: crush site; small arrows: Sema3-positive cells; rostral: right; scale bar: 100 μM. n = 2 per group.
Figure 3. 
 
Identification and characterization of the optic nerve crush site. Adult rat ONs were crushed by forceps, removed at 6 hours and 1 day after injury, and processed by ISH and immunohistochemistry. C, F and I show uninjured ON. The injury site (arrows) was identifiable by GFAP immunostaining, and increased expression of ED1 (Cd68; activated macrophages) (A). There was no discernible staining by ISH for Sema3a in injured ON (B). The relatively strong expression of Sema3b in the uninjured ON (C) appears to be downregulated 6 hours after injury, especially at the injury site (D); however, expression returns to normal levels after 1 day (E). There is no appreciable change in Sema3c expression in the ON after injury (F–H). Sema3f expression immediately surrounding the injury site appears to fall considerably at 6 hours after injury (compare I and J); however, Sema3f-positive cells are still seen adjacent to the injury (small arrows). Sema3f expression increases back to around control levels from 1 day after injury, and transcript expression does not appear to colocalize with OLIG2 expression (L). Large arrows: crush site; small arrows: Sema3-positive cells; rostral: right; scale bar: 100 μM. n = 2 per group.
Sema3a mRNA was not detectable by ISH in either the uninjured or injured ON (Fig. 3B); however, immunoperoxidase staining did show SEMA3A immunopositive cells at 6 hours and 1 day, but not in the naïve nerve (Fig. 4). SEMA3A immunoreactivity was similar between both antibodies at 6 hours (Figs. 4D, 4E), with increased staining within the injury site as well as adjacent vascular areas. One day postinjury, tissue stained with anti-SEMA3A C17 antibody revealed a low level of SEMA3A immunoreactivity restricted to the peripheral margins of the ON (Fig. 4F). 
Figure 4. 
 
Immunoperoxidase staining of Sema3a expression in the injured rat ON using antibodies against both the n-terminus (N15) and c-terminus (C17) of SEMA3A. (A) No staining was observed in naïve ON stained without the presence of primary antibody (no primary control). There was no specific cellular staining for SEMA3A inside the ON with either antibody, but there were some SEMA3A-positive cells in the surrounding sheath (B, C). At 6 hours after injury, there was an induction of SEMA3A expression within the injury site and adjacent areas that appeared to be vasculature, although the number of positive cells was small (D, E). At 1 day after ON crush, the expression of SEMA3A at the injury site is indistinguishable from the uninjured nerve (F). Small arrows, immunopositive cells; large arrows, crush site. Scale bar: 100 μM.
Figure 4. 
 
Immunoperoxidase staining of Sema3a expression in the injured rat ON using antibodies against both the n-terminus (N15) and c-terminus (C17) of SEMA3A. (A) No staining was observed in naïve ON stained without the presence of primary antibody (no primary control). There was no specific cellular staining for SEMA3A inside the ON with either antibody, but there were some SEMA3A-positive cells in the surrounding sheath (B, C). At 6 hours after injury, there was an induction of SEMA3A expression within the injury site and adjacent areas that appeared to be vasculature, although the number of positive cells was small (D, E). At 1 day after ON crush, the expression of SEMA3A at the injury site is indistinguishable from the uninjured nerve (F). Small arrows, immunopositive cells; large arrows, crush site. Scale bar: 100 μM.
Sema3b was readily detectable in both the naïve and injured rat ON by ISH (Figs. 3C–E). Sema3b-positive cells were arranged longitudinally along the ON. Around the injury site, Sema3b transcript levels were reduced at 6 hours, but not at 1 day after injury. Sema3c-expressing cells were also arranged longitudinally along the ON (Figs. 3F–H). There was little change evident in Sema3c transcript expression after ON crush, save for the immediate proximal stump at 6 hours where there appeared to be reduced expression (Fig. 3G). 
Sema3f-positive cells also appeared in lineal strips along the ON (Figs. 3I, 3J). At 6 hours after ONC there was a reduction in Sema3f-positive cells in, and immediately surrounding, the crush site, but Sema3f-positive cells were seen nearby (small arrows, Figs. 3I, 3J). By 1 day postinjury, expression of Sema3f appeared similar to naïve ON (data not shown). Sema3f transcript expression did not colocalize with OLIG2 expression (Fig. 3K). 
Additionally, Sema3b-, Sema3c-, and Sema3f-positive cells were evident in the sheath surrounding the ON, but there was no obvious colocalization of Sema3a, Sema3b, Sema3c, or Sema3f revealed by ISH staining with either GFAP or ED1 immunoreactivity (data not shown). 
Discussion
This study provides a quantitative assessment of Sema3 ligand and receptor mRNA expression changes in the rat retino-collicular system following unilateral ON injury. Changes in mRNA expression levels differed between the tissues investigated, both in terms of which Sema3-related transcripts were altered, and the time-course of these changes. Because the vast majority of rat RGCs project to the contralateral SC, 6567 we expected to see most changes in the axotomized retina and contralateral SC, with perhaps less obvious changes in the contralateral (uninjured) retina and ipsilateral SC. 
In axotomized retinas there were no observed differences in transcript expression at the early time point, 1 day after ONT. Other investigators have found changes in mRNA expression as soon as 4 hours following ON injury, 25,26,30,31 thus the lack of observed changes is not due to an intrinsic stasis in reaction to the injury. In contrast, a delayed (3 days postinjury) response was observed in axotomized retinas, with a transient increase in mRNA levels for Sema3b, Sema3f, L1cam, and Plxna3
A previous study found that SEMA3A protein levels were increased both at 3 and 14 days after ONT, measured by Western blot using a bespoke antibody against SEMA3A. 14 The discrepancy may be due to posttranslational changes in SEMA3A protein. It is also possible that the antibody used also recognized epitopes shared by one or more Sema3s, as the reported changes in expression are similar to the combined expression of Sema3b and Sema3f transcripts in this study. 
Although these data deal only with mRNAs, and biological implications of changes in their expression remain unclear, we think it important to discuss possible explanations for these changes. These reasoned speculations based on previously discovered changes following ONT, as well as what is currently known about the function of the Sema3s, are presented below. 
It is possible that the increase in mRNA for Sema3f, its receptor Plxna3, as well as the receptors for Sema3a, L1cam, and Plxna3, 68 may be involved in the apoptosis of RGCs following ONT. Increased expression was seen 3 days after injury, just before the onset of RGC loss, 2024 and when RGCs appear to have committed to an apoptotic phenotype. 22,29 Significantly, some Sema3s, including Sema3f, whose transcript levels were elevated, have been shown to be neurotoxic to RGCs and can induce apoptosis. 15,6973 Interestingly, Plxna3 mRNA is significantly elevated in the axotomized retina following injury, and is a receptor for Sema5a, which has also been implicated in abortive regeneration of RGC axons. 74 Furthermore, at 14 days after ONT, mRNA expression levels of Sema3f, Plxna3, and L1cam transcripts were no longer increased in axotomized retina, a time when the level of apoptosis is much lower. 
Note here that other retinal cells change their gene expression profiles at various times after ON injury. For example, bFGF expression is increased in photoreceptors, 33 and CNTF is increased in Müller cells. 75 Therefore, changes in transcript expression in the retina observed in this study may also reflect reactive changes in a variety of retinal cells, not just the primary axotomized cells, the RGCs. More detailed analysis will require the use of techniques such as laser micro-dissection and single-cell qPCR. 
Interestingly, there were also changes in mRNA expression levels of several Sema3 related transcripts in retina contralateral to the ONT. Cellular and gene expression changes in contralateral retina following unilateral ON injuries have previously been reported 7681 and theorized to be due to gliosis found in this tissue. 76,78 Changes in the eye contralateral to rodent experimental intraocular hypertension 82,83 and retinal ischemia 84 have also been reported. In rat, the contralateral retina has also been reported to contain a small number of retino-retinal projecting RGCs, 85 and these would be axotomized by the ONT. Further, RGC axons from contralateral retina are in direct contact with the injured axons from the ipsilateral retina at the optic chiasm. Transduction of injury signals to the contralateral retina may also progress by an autoimmune reaction similar to the human condition of sympathetic ophthalmia, 86 especially considering the longer-term changes in expression described here. Together, this reinforces the view already stated by others 77,82,83 that the contralateral eye is not an appropriate control tissue for injuries to the eye and optic nerve. The mechanism of effect, and possible implications are worthy of further study. For example, could therapeutic agents be administered to the “unaffected” eye to treat neuropathologies in the contralateral retinofugal pathway? 
The changes in L1cam, Plxna2, and Plxna4a in the contralateral SC at 3 and 14 days ONT might reflect a change in synaptic plastic potential/remodeling. The Sema3s are known to be important in synapse formation and plasticity, and may help pattern the maturing SC, 2 and the observed changes are seen at approximately the time that there is vacation of synaptic sites by degenerate retinal axons. 38,48,50  
There were no significant changes in Sema3 ligand expression in the contralateral SC, in disagreement with their previously speculated importance for guidance of RGCs in the SC. 9 Alternatively, because it has been established that after ONT the SC either reexpresses or increases expression of guidance molecules for RGCs, 34,51 the lack of such changes in Sema3 expression might represent a barrier to anatomically correct regeneration of axotomized RGC axons. 
Interestingly, the SC ipsilateral to ONT, which is only partially deafferented, also showed changes in mRNA expression. L1cam, Plxna2, and Plxna4a expression changes were generally similar to those seen in contralateral SC. In addition to the small RGC projection to the ipsilateral SC, 67 there are also bidirectional commissural projections, 87 and both pathways, albeit small, potentially may influence gene expression in the SC ipsilateral to the injury. Bilateral changes in gene expression in the SC following ONT have been observed previously, 34 and may additionally indicate a reactive change in other brain regions projecting to both SC hemispheres, or global activation of inflammatory signaling due to CNS insult. 
GFAP and ED1 immunostaining aided identification of the crush site and was in general agreement with previous studies. 8890 A lack of Sema3a mRNA expression in uninjured ON is consistent with previous results, 14 and is perhaps not surprising because Sema3a transcripts are not found in oligodendrocytes or astrocytes. 74 In contrast to the ISH data, immunoperoxidase staining did reveal evidence of Sema3a-positive cells in the injured ON, as described previously, 14 possibly because it is a more sensitive technique. Although the immunostaining results in this study were not identical between both anti-SEMA3A antibodies, they were in broad agreement. The distribution of Sema3a-positive signal is similar to the distribution of macrophages in this and other studies. 88,89 The SEMA3A-positive staining found in the meninges surrounding the ON also supports previous research reporting SEMA3A-positive meningeal fibroblasts in the spinal cord. 9193 Sema3b-, Sema3c-, and Sema3f-ISH positive cells were observed in the sheath surrounding the ON, possibly indicating their expression in fibroblasts, as described for the spinal cord. 92 Sema3f-expressing cells in the ON did not obviously colocalize with OLIG2-positive profiles and thus are unlikely to be oligodendrocytes. 
Further characterization of which cells express the Sema3s and their receptors in the naïve and injured retino-collicular system is clearly required, and will become easier to perform as validated antibodies against the Sema3s become commercially available. Nevertheless, our quantitative analysis of transcripts provides useful new information on altered expression of some Sema3s and their coreceptors after unilateral visual system trauma, and provides clues for future research aimed at understanding the role of these molecules in degenerative and regenerative events. 
Acknowledgments
We thank Ying Hu for performing the optic nerve crush procedures. 
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Footnotes
 Supported by grants from the Western Australia Neurotrauma Research Program.
Footnotes
 Disclosure: A. Sharma, None; M.A. Pollett, None; G.W. Plant, None; A.R Harvey, None
Figure 1. 
 
Changes in mRNA in ipsilateral axotomized (grey bars) and contralateral uninjured (white bars) retinas at 1, 3, and 14 days following unilateral ONT. Statistical tests are against control retina mRNA (black bar) from uninjured adult rats. Individual graphs are shaded where expression changed significantly during at least one time point in either tissue. In the ipsilateral retina Sema3b, Sema3f, L1cam, and Plxna3 changed transcript expression compared with controls, and all increased at 3 days postinjury. Within the contralateral retina, Sema3b, Sema3f, and L1cam showed increased transcript expression. For Sema3b the increase was observed only after 14 days. Sema3f increased expression at 3 days after posttransection, but this increase was not statistically significant by 14 days. L1cam mRNA increased from 3 days and into at least 14 days postinjury. U, uninjured control; error bars: ± 1 SEM; *P < 0.05; **P < 0.01. n = 4–8 per group.
Figure 1. 
 
Changes in mRNA in ipsilateral axotomized (grey bars) and contralateral uninjured (white bars) retinas at 1, 3, and 14 days following unilateral ONT. Statistical tests are against control retina mRNA (black bar) from uninjured adult rats. Individual graphs are shaded where expression changed significantly during at least one time point in either tissue. In the ipsilateral retina Sema3b, Sema3f, L1cam, and Plxna3 changed transcript expression compared with controls, and all increased at 3 days postinjury. Within the contralateral retina, Sema3b, Sema3f, and L1cam showed increased transcript expression. For Sema3b the increase was observed only after 14 days. Sema3f increased expression at 3 days after posttransection, but this increase was not statistically significant by 14 days. L1cam mRNA increased from 3 days and into at least 14 days postinjury. U, uninjured control; error bars: ± 1 SEM; *P < 0.05; **P < 0.01. n = 4–8 per group.
Figure 2. 
 
Changes in mRNA in ipsilateral (grey bars) and deafferented contralateral (white bars) SC hemispheres at 1, 3, and 14 days following unilateral ONT. Statistical tests are against control superior colliculus mRNA (black bar) from uninjured adult rats. Individual graphs are shaded where expression changed significantly during at least one time point in either tissue. In the contralateral SC, both L1cam and Plxna4a had reduced mRNA expression compared with controls at 14 days following injury. In contrast, Plxna2 transcript expression was higher than controls at both 3 and 14 days after injury. Within the ipsilateral SC, Sema3e, L1cam, and Plxna4a had lower levels of transcript expression compared with controls 14 days postinjury. Plxna2 had a temporary increase in transcript expression at 3 days after injury, with expression falling to control levels at 14 days postinjury. U, uninjured control; error bars: ± 1 SEM; *P < 0.05; **P < 0.01. n = 5–6 per group.
Figure 2. 
 
Changes in mRNA in ipsilateral (grey bars) and deafferented contralateral (white bars) SC hemispheres at 1, 3, and 14 days following unilateral ONT. Statistical tests are against control superior colliculus mRNA (black bar) from uninjured adult rats. Individual graphs are shaded where expression changed significantly during at least one time point in either tissue. In the contralateral SC, both L1cam and Plxna4a had reduced mRNA expression compared with controls at 14 days following injury. In contrast, Plxna2 transcript expression was higher than controls at both 3 and 14 days after injury. Within the ipsilateral SC, Sema3e, L1cam, and Plxna4a had lower levels of transcript expression compared with controls 14 days postinjury. Plxna2 had a temporary increase in transcript expression at 3 days after injury, with expression falling to control levels at 14 days postinjury. U, uninjured control; error bars: ± 1 SEM; *P < 0.05; **P < 0.01. n = 5–6 per group.
Figure 3. 
 
Identification and characterization of the optic nerve crush site. Adult rat ONs were crushed by forceps, removed at 6 hours and 1 day after injury, and processed by ISH and immunohistochemistry. C, F and I show uninjured ON. The injury site (arrows) was identifiable by GFAP immunostaining, and increased expression of ED1 (Cd68; activated macrophages) (A). There was no discernible staining by ISH for Sema3a in injured ON (B). The relatively strong expression of Sema3b in the uninjured ON (C) appears to be downregulated 6 hours after injury, especially at the injury site (D); however, expression returns to normal levels after 1 day (E). There is no appreciable change in Sema3c expression in the ON after injury (F–H). Sema3f expression immediately surrounding the injury site appears to fall considerably at 6 hours after injury (compare I and J); however, Sema3f-positive cells are still seen adjacent to the injury (small arrows). Sema3f expression increases back to around control levels from 1 day after injury, and transcript expression does not appear to colocalize with OLIG2 expression (L). Large arrows: crush site; small arrows: Sema3-positive cells; rostral: right; scale bar: 100 μM. n = 2 per group.
Figure 3. 
 
Identification and characterization of the optic nerve crush site. Adult rat ONs were crushed by forceps, removed at 6 hours and 1 day after injury, and processed by ISH and immunohistochemistry. C, F and I show uninjured ON. The injury site (arrows) was identifiable by GFAP immunostaining, and increased expression of ED1 (Cd68; activated macrophages) (A). There was no discernible staining by ISH for Sema3a in injured ON (B). The relatively strong expression of Sema3b in the uninjured ON (C) appears to be downregulated 6 hours after injury, especially at the injury site (D); however, expression returns to normal levels after 1 day (E). There is no appreciable change in Sema3c expression in the ON after injury (F–H). Sema3f expression immediately surrounding the injury site appears to fall considerably at 6 hours after injury (compare I and J); however, Sema3f-positive cells are still seen adjacent to the injury (small arrows). Sema3f expression increases back to around control levels from 1 day after injury, and transcript expression does not appear to colocalize with OLIG2 expression (L). Large arrows: crush site; small arrows: Sema3-positive cells; rostral: right; scale bar: 100 μM. n = 2 per group.
Figure 4. 
 
Immunoperoxidase staining of Sema3a expression in the injured rat ON using antibodies against both the n-terminus (N15) and c-terminus (C17) of SEMA3A. (A) No staining was observed in naïve ON stained without the presence of primary antibody (no primary control). There was no specific cellular staining for SEMA3A inside the ON with either antibody, but there were some SEMA3A-positive cells in the surrounding sheath (B, C). At 6 hours after injury, there was an induction of SEMA3A expression within the injury site and adjacent areas that appeared to be vasculature, although the number of positive cells was small (D, E). At 1 day after ON crush, the expression of SEMA3A at the injury site is indistinguishable from the uninjured nerve (F). Small arrows, immunopositive cells; large arrows, crush site. Scale bar: 100 μM.
Figure 4. 
 
Immunoperoxidase staining of Sema3a expression in the injured rat ON using antibodies against both the n-terminus (N15) and c-terminus (C17) of SEMA3A. (A) No staining was observed in naïve ON stained without the presence of primary antibody (no primary control). There was no specific cellular staining for SEMA3A inside the ON with either antibody, but there were some SEMA3A-positive cells in the surrounding sheath (B, C). At 6 hours after injury, there was an induction of SEMA3A expression within the injury site and adjacent areas that appeared to be vasculature, although the number of positive cells was small (D, E). At 1 day after ON crush, the expression of SEMA3A at the injury site is indistinguishable from the uninjured nerve (F). Small arrows, immunopositive cells; large arrows, crush site. Scale bar: 100 μM.
Table 1. 
 
Primer Pairs for Each qPCR Assay
Table 1. 
 
Primer Pairs for Each qPCR Assay
Gene 5′ Primer 3′ Primer
L1cam GCC CTG AGC TTG AAG ACA TC GCC CTT CCA CCA GTA CGT TA
Nrp1 GGA GCT ACT GGG CTG TGA AG ATG TCG GGA ACT CTG ATT GG
Nrp2 GGC ATT TGT ACG CAA GTT CA GGG CTT TGA GTC TGT CCA GTC
Plxna1 CCA ACT CCT CTA CCT TCA CGA GTG TTC CCT TAG TAG CCA GCA G
Plxna2 TGG GGA CTA AGA GTG GCA AG CAC AAT GAG GAT CCC CTG AG
Plxna3 CAC CCA GAT TCA CCC ACT AAC GAT TCC TCC ATC TCA CAT ACG A
Plxna4a GCA TTA AGG ACC GCC TAC AG AGA CTG GAA TGC CAC GTA CC
Ppia AGC ATA CAG GTC CTG GCA TC TTC ACC TTC CCA AAG ACC AC
Rnr1 GAT CCA TTG GAG GGC AAG TCT CCA AGA TCC AAC TAC GAG CTT TTT
Rpl19 CTG AAG GTC AAA GGG AAT GTG CCT CCT TGC ACA GAG TCT TGA
Sema3a ATA TGC AAG AAT GAC TTT GGA GGA C AAG GAA CAC CCT TCT TAC ATC ACT C
Sema3b GCT GTC TTC TCC ACC TCC AG ACA TGC CAG GTC TTG GGT AG
Sema3c AGA CGT GAG ACA CGG GAA TC TTC ATT CAG TTT AAC CCT CCT TCC
Sema3e GCG TCA GTG ATG GCT ACA GA CAA AAC CCG GAC ATA ATT GG
Sema3f CTC TTC CAA GAG GCA ACA ACT G TTT GCA TTG GAA TTG AAA CCA C
Table 2. 
 
Statistical Results (P) for Changes in mRNA Expression after ONT
Table 2. 
 
Statistical Results (P) for Changes in mRNA Expression after ONT
Ipsilateral Retina Contralateral Retina Ipsilateral Superior Colliculus Contralateral Superior Colliculus
Sema3a 0.850 0.871 N/A N/A
Sema3b 0.009 0.005 0.074 0.035
Sema3c 0.257 0.647 0.386 0.080
Sema3e 0.227 0.924 < 0.001 0.083
Sema3f 0.011 0.003 0.596 0.181
Nrp1 0.148 0.354 0.564 0.242
Nrp2 0.121 0.394 0.459 0.242
L1cam 0.023 0.045 0.001 0.008
Plxna1 0.004 0.396 0.418 0.430
Plxna2 0.138 0.655 0.005 0.024
Plxna3 0.003 0.008 0.920 0.820
Plxna4a 0.024 0.120 0.001 0.006
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