Multiple receptor tyrosine kinases are expressed in adult rat retinal ganglion cells as revealed by single-cell degenerate primer polymerase chain reaction

Animals and surgical procedures

Female Sprague-Dawley rats (each weighing 225–250 g) were obtained from B&K Universal (Sollentuna, Sweden). For axotomy experiments female and male Dark Agouti rats each weighing 130–250 g were used (B&K Universal, Sollentuna, Sweden), sex and weight differing between groups and occasions of operation. For experimental manipulations, animals were anaesthetized with an intraperitoneal injection mixture of xylazine (10–15 mg/kg, Rompun; Bayer AG, Leverkusen, Germany) and ketamine (30–100 mg/kg, Ketalar; Parke-Davis, England). Rats were fed ad libitum, housed in standard cages in temperature-controlled rooms with a 12-hour light and 12-hour dark cycle, light intensity ranging from 8 to 24 lux. Experiments were carried out in accordance with European Community guidelines and Association for research in vision and ophthalmology for use of animals in ophthalmic and vision research. The local ethics committee for experimental animals (Uppsala Djurförsöksetiska nämnd) scrutinized the procedures. RGCs were retrogradely labelled from the superior colliculi using previously described techniques (6,19,20). In brief, after exposing the surface of the superior colliculi, a piece of gel foam soaked in 0.9% NaCl, 10% dimethylsulphoxide (Merck), and 3% FluoroGold (Fluorochrome Inc., Englewood, Colorado, USA) or True Blue (Molecular Probes, Leiden, Netherlands) was applied onto these structures. Rats were sacrificed 7 days after tracer application by an intraperitoneal injection of 3–4 mL pentobarbital (100 mg/mL).

For intra-orbital optic nerve transection an incision was made in the skin covering the superior orbital rim (6), the superior orbital contents were dissected, and superior and external muscles were sectioned. Following rotation of the eye, the dura mater sheath was longitudinally opened and the optic nerve was completely transected as close as possible to the eye. Care was taken to avoid damage to the retinal blood supply and the retinal perfusion after operation was confirmed using an ophthalmoscope. Animals were sacrificed 12 hours, 1, 3, 4, 7, or 14 days after the transection. Each group contained 4–8 animals. Optic nerve transection (ONT) was chosen since this type of injury affects mainly the RGCs and to a lesser extent the other cells in the retina.

Isolation of RGCs, Q RT PCR, and cloning

Total retina cDNA was synthesized from mRNA prepared from dissected retina. RGCs were isolated, and single-cell cDNA synthesis was performed as previously described (16). In brief, separation of the GCL from the rest of the retina was performed on a glass slide after enzymatic treatment of the retina. Individual, FluoroGold-labelled RGCs were identified in fluorescence microscopy, aspirated using a micropipette, washed, and transferred to reaction tubes for reverse transcription of mRNA. For the RGC-specific cDNA used for verification purposes, more than ten isolated RGCs were pooled, and the cDNA from those cells was used as a template. Negative controls were cell cDNA reactions with reverse transcriptase omitted. The medium containing the cells after layer separation was analysed for extracellular mRNA contaminants using Q RT PCR with Gapdh primers (16). Positive controls were performed on single cells by using primers to β-actin, Gapdh, Ret, Met, ErbB2, and TrkB (16). The conserved amino acid sequences, without gaps, HRDLA(A/T)RN and DVWS(F/Y/L)G(V/I) (Figure 1), were reverse-translated to nucleotide sequences with nucleotide sequence degeneracies of 6.5 × 10⁵ and 1.6 × 10⁴, respectively. This is a high degree of degeneracy for primer pairs (1010), but it has been shown to give effective amplification (21). The concentration of each specific primer with this degeneracy is very low, and inosines can be introduced to lower degeneracy, thus increasing the concentration of the specific primers in the ‘primer mix’. We used an empirical approach to find optimal primers and conditions (see below) for amplification which gave the following primer sequences that contain inosines: 5´-CA(C/T)(A/C)GIGA(C/T)(C/T)TIGCI(G/A)CI(C/A)GIAA-3´ and 5´-A(C/T)ICCI(A/T)(A/G)I(C/G)(A/T)CCAIAC(A/G)TC-3´. The degeneracy of the primers including the inosines is 64 for both primers. Inosine is a purine that can form base pairs with the other bases, cytidine, thymidine, and adenosine, but to a lesser degree with guanosine. The approach using multiple pools of primers in order to decrease primer degeneracy was disqualified by the limited amount of cDNA template for each reaction. The programs CODEHOP (22,23) and HYDEN (21) facilitate primer design. The typical PCR reaction contained 60 pmol of degenerate primers, 15 μL of single-cell cDNA reaction, buffer (15 mM Tris-HCl, 50 mM KCl), 0.2 mM of each of the deoxyG,A,T,CTP (dNTP), 2.5 mM MgCl₂, and 2.5 U Amplitaq Gold DNA polymerase (Perkin-Elmer, Boston, MA, USA). DNA polymerase was activated (95°C for 10 min) followed by 2 cycles (94°C for 1 min, 37°C for 5 min, 65°C for 3 min) and 48 cycles (94°C for 1 min, 53.4°C for 5 min, 65°C for 3 min) using slow ramping (1°C/sec) between annealing and extension steps. PCR reactions were analysed by 1.8%–2.2% agarose gel electrophoresis, and PCR fragments of correct size were excised under ultra violet (UV)-light and extracted using silica-based chemistry (Qiagen, Hilden, Germany). Extracted PCR fragments were cloned in pMOSBlue vector using pMOSBlue blunt end cloning kit (Amersham, Buckinghamshire, UK). Clones were sequenced, and homology searches were done using NCBI BLAST against translated GenBank/Swiss Prot databases, and ClustalW was used for alignments. A positive identification required an identical match to a rat protein sequence or a match on an orthologous sequence with an E-value <10–30. Partial sequences were disregarded. For specific PCR primers, see Table I.

In situ hybridization histochemistry

Rat retinas were sectioned (12 μm) using a cryostat, and sections were mounted on poly-D-lysine-coated slides. Oligonucleotides complementary to the available rat receptor mRNAs identified in the PCR analysis were used as probes (Table II). Controls were performed by using a non-sense probe or by adding 100 times excess of unlabelled probe in order to compete out the labelled probe. Oligonucleotides were labelled with ³⁵S-ATP (NEN, Perkin Elmer, Boston MA) using terminal deoxynucleotidyl transferase (Amersham, Buckinghamshire, UK) and purified on ProbeQuant G-50 columns. ³⁵S-labelled oligonucleotides (10⁶ counts per minute/slide) in hybridization solution were hybridized to sections in a humidified chamber at 42°C for 14–16 hours. Slides were washed in sodium saline citrate (SSC) at 56°C, rinsed in water, dehydrated, dipped in NTB2 Kodak photoemulsion, and exposed for 4–6 weeks. Slides were developed in D19 developer (Amersham, Buckinghamshire, UK), counterstained with cresyl violet, and viewed and documented in a Zeiss Axioplan2 microscope.

Immunohistochemistry

Rats were anaesthetized and perfused with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). Eyes were postfixed, cryoprotected, sectioned (12 μm) using a cryostat, and mounted on Superfrost Plus slides. Sections were pre-incubated in 0.3% Triton X-100 in PBS (PBT) and incubated overnight with primary antibodies: rabbit α-mouse Csf-1R (1:200; #06-174, UpState), mouse or rabbit α-mouse Vegfr-2 (1:25, CH-11; 1:500, #07-158, UpState), rabbit α-mouse Pdgfrα,β (1:50, provided by L. Rönnstrand, Ludwig Institute for Cancer Research, Uppsala branch, the antibody recognizes both α and β types), goat α-mouse Ret (1:50; R&D Systems), mouse (1:40; 75133) or rabbit α-TrkB (1:200; 1494 R&D Systems), goat α-human Sky (1:50; Santa Cruz Biotech) in PBT with 5% of appropriate serum(s). Secondary antibodies were Alexa Fluor 488/594 conjugated donkey α-rabbit (1:50; Molecular Probes); Alexa Fluor 488/594 conjugated chicken α-goat (1:50; Molecular Probes), and Alexa Fluor 488/594 conjugated goat α-mouse (1:50; Molecular Probes). PDGF-α,βR was detected using biotinylated anti-rabbit (1:30; Vector) and a Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA, USA) with diaminobenzidine substrate. Negative controls were performed by omitting the primary antibody. Slides were mounted with Vecta-Shield (Vector Laboratories, Burlingame, CA, USA), and viewed and documented with a Zeiss Axioplan2 microscope. Cell counting in at least three retinas was performed in the central regions of the retina. Only cells with clear True Blue labelling were counted in ten visual fields (40× objective) from each retina. Percentages were calculated in relation to the total number of clearly True Blue-labelled cells in the ganglion cell layer.

Quantitative Q RT PCR

Total RNA was prepared from whole retinas using Trizol (Invitrogen). The RNA was analysed for purity with Agilent RNA 6000 Assay (Agilent Technologies, USA). After treatment with RNase-free DNase (Promega, Madison, WI), 1 μg of total RNA was used for cDNA synthesis with TaqMan Reverse Transcriptase reagents using random hexamer primers (PE Applied Biosystems). Analysis of mRNA levels was performed using SYBR Green I assay in combination with ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). Sequence-specific primers were designed using Primer Express software (Applied Biosystems), for sequences see Table III. PCR reactions were carried out on duplicate cDNA samples in a 96-well plate using final concentrations of 1× SYBR Green PCR Master Mix (Applied Biosystems) and 300 nM of forward and reverse primers respectively (Table III). The PCR conditions were: 95ºC for 10 min, followed by 40 cycles of: 95ºC for 15 s, 60ºC for 60 s. Specific amplification was confirmed by generating dissociation curves and separation by agarose gel (3%) electrophoresis. For each sample, a C_T (threshold cycle) value corresponding to the PCR cycle at which the fluorescent signal reaches a threshold above the base-line emission was determined. Based on this, initial target mRNA levels were calculated, and relative differences between operated (subjected optic nerve transection) and unoperated animals were determined. As controls, the relative mRNA levels of the housekeeping genes β-actin and Gapdh were measured in each group of normal and axotomized tissues. The control genes did not show differences in the mRNA levels between the groups and thus were not affected by optic nerve transection. β-Actin was chosen as reference gene, and all collected data were normalized to β-actin expression. Thy-1, an RGC-specific gene, was analysed in order to monitor changes of expression in RGCs. Statistical analyses were performed using analysis of variance (ANOVA). P-values less than 0.05 were considered significant.

PCR controls

To identify RTKs expressed by RGCs we used degenerate primer PCR directed towards conserved sequences in the tyrosine kinase domain (Figure 1A). The PCR was applied on cDNA from acutely isolated adult RGCs. Both as controls and as part of the experiment, specific primers to several genes that are expressed by all cells (Gapdh, β-actin), in RGCs TrkB, Ret (24), as well as to genes not being expressed by RGCs (TrkA, rhodopsin), were included in the analysis (Table IV). Included were also Met and ErbB2 as candidate RTKs that we have previously found being expressed in RGCs (16). A pair of random primers was used as a negative control (non-sense). The specific primer PCRs were used to optimize and to control the procedure as also discussed elsewhere (16). In optimized standard reactions, primers spanning exon/intron boundaries in the actin gene did not produce any products. PCR using degenerate or specific primers on single-cell cDNA reactions without reverse transcription did not give any PCR products. Both controls confirm that the samples were free from genomic DNA contamination (Figure 2A). These kinds of controls were included in parallel to the analyses. The highly expressed housekeeping gene, Gapdh, could not be amplified from the medium in which cells were aspirated after the retinal layer separation (see Figure 1B and C), confirming that the surrounding solutions were free from mRNA contaminants.

Figure 2.

Agarose gel electrophoresis analysis of PCR products from single-cell degenerate primer PCR. A: Control analyses of the single-cell PCR showing negative and positive control reactions. (Lanes: neg. ctrl., PCR on cDNA reaction without reverse transcriptase; single retinal ganglion cell cDNA analysed using specific primers to Gapdh and TrkB; neg. ctrl. medium, control for extracellular RNA contamination; Actin cDNA and Actin genomic, controls for cDNA synthesis and genomic DNA contamination with primers that give a 138 bp fragment with the actin cDNA and 601 bp with genomic DNA as templates; M, 100 bp DNA size marker ladder). B: PCR on seven representative single cells using degenerate primers directed towards conserved sequences in the tyrosine kinase domain of receptor tyrosine kinases. (Lanes: negative controls and markers as in A; positive control using specific primers for Actin and Gapdh; 1–7, degenerate primer PCR on single retinal ganglion cell cDNA). Specific PCR products are approximately 220 bp. Cells 2, 3, 5, and 7 are considered positive, while 1, 4, and 6 are negative.

The success rate in the PCR analysis, as defined by cells that gave amplification with the Gapdh primers and were blank in the controls, was on average 82% but varied from 100% to 48% between experiments (cell-picking occasions). Using specific primers for Gapdh, TrkB, and Ret, 56% of the cells were positive for Gapdh and TrkB mRNA, and 20% of the cells were positive for Gapdh and Ret mRNA.

Isolation and identification of cDNA fragments from RGCs encoding the tyrosine kinase domain

More than 40 cells were used for optimization of the degenerate primer PCR protocol, and typically 45% of these degenerate primer PCR amplifications gave products. This was lower than when using the specific primers. Optimized PCR was then carried out on 20 RGCs, and 10 cells gave clear PCR products of the predicted sizes. Seven such amplifications are shown in Figure 2B. The PCR fragments of the expected sizes (Figure 2B) were cloned and sequenced. Using the NCBI BLAST search against available databases with the PCR sequences as query, 25.4% of the sequences (110) were identical to 12 RTK and 2 cytoplasmic tyrosine kinases (E-values <10^-30, orthologues excluded); Axl (‘anexelekto’/UFO/Ark/Tyro7, Q8VI99, AB067527), Csf-1R (colony stimulating factor-1 receptor, Q00495), Eph A4 (highly similar to mouse Eph A4), Pdgfrβ (platelet-derived growth factor receptor β, Q05030), Ptk7 (protein tyrosine kinase 7, highly similar to mouse Ptk7, AAH27800), Ret (CAC10584), Ros (AAA40968), Sky (Tyro3/Etk2/Dtk/Brt/Rse/Tif, P55146), TrkB (NP 036863), TrkC (Q03351), Vegfr-2 (Flk1/Kdr, NP 037194), Vegfr-3 (Flt4, AAL13269), and Jak1, 2 (XP 342873, NP 113702), while the rest (74.6%) only displayed partial, low, or no similarity at all (E-values >10^-20) to the tyrosine kinase domain sequence. The number of tyrosine kinase sequences found using the degenerate PCR primer amplification from each individual cell varied substantially, ranging from only one tyrosine kinase sequence in several copies up to ten of the identified sequences among clones that were derived from a particular cell (Table IV, column 1). Furthermore, specific primers to TrkB gave PCR products in 68% of the cells that were positive for Gapdh, which is in agreement with histological analysis where a majority of the RGCs express TrkB (25). In contrast, only 2% of the cloned sequences corresponded to TrkB, and those came from just one cell out of the ten successful analyses.

Verification of RTK expression in retina and RGCs

The expression of the identified RTK as well as additional genes were checked in retina using specific primer Q RT PCR analysis of cDNA from whole retina as well as from picked (Figure 1B and C) and pooled RGCs (RGC-specific cDNA from >10 RGCs). Rhodopsin could be amplified from total retina but not from the RGC-specific cDNA. TrkA could not be amplified from RGC cDNA. The other RTKs (TrkB, Ret, Met, ErbB2) could be amplified from both total retina and RGC-specific cDNA. The random sequence primers did not produce any amplification (Table IV).

Among the 12 identified RTKs, rat sequences for Ptk7 and Eph A4 were not available, and their expression in retina was not verified. The expression and main function of Eph A4 in retina and RGCs are well documented (26,27), and this gene was not further analysed. The RTK Ros was identified using the Q RT PCR strategy as being expressed in RGCs, but its expression could neither be conclusively verified in retina using Q RT PCR nor using histological methods. Axl and Vegfr-3 mRNA expression could be verified using PCR but the histological methods did not give confirmation of these results (Table IV and Figure 3A).

Figure 3.

Expression of receptor tyrosine kinase (RTK) mRNA in the retinal ganglion cell layer. Bright-field micrographs of oligonucleotide probe in situ hybridization analysis of the retinal ganglion cell layer in adult rat. Autoradiography for Axl (A), Sky (C), Vegfr-2 (E), Csf-1R (G), Pdgfrβ (I), Pdgfrα (K), TrkB (M), Ret (N), and Met (O) mRNA expression. B, D, F, H, J, and L: negative controls using 100 times molar excess of unlabelled oligonucleotides (GCL = ganglion cell layer). Scale bars 20 μm. Bar in B also valid for A, bar in D also valid for C–L, and bar in O also valid for M–N.

Axl and Sky/Tyro-3 belong to the same subfamily of RTKs. In contrast to Axl, labelling for the Sky was clear in retina and retinal ganglion cell layer using the different methods (Table IV, Figure 3C and 5B). Displaced amacrine cells constitute up to 50% of the cells in the ganglion cell layer, and to confirm the RGC localization of the identified receptors we studied whether Sky immunoreactivity was present on RGCs retrogradely traced with True Blue. As shown in Figure 5A–C, Sky is expressed by RGCs as its immunoreactivity co-localizes with True Blue-positive RGCs (Figure 5A–C).

Labelling for Vegfr-2 mRNA was found in the GCL (Figure 3E, F) and in the inner nuclear layer (data not shown). Vegfr-2 immunoreactivity was seen on True Blue-labelled RGCs in the GCL (Figure 5E–H). Weak labelling for Csf-1R mRNA was found in the GCL (Figure 3G, H) and Csf-1R-immunoreactivity was seen on True Blue-labelled RGCs (Figure 6I–K). Pdgfrβ was identified using the PCR strategy, and labelling for Pdgfrβ mRNA was found over cells in the GCL (Figure 3I, J). Labelling for Pdgfrα mRNA could also be detected (Figure 3K, L), and using an antibody that detects both Pdgfr α and β forms immunore activity was detected on cells in the GCL. Whole-mount Pdgfrαβ immunohistochemistry labelled RGCs in the GCL as well as axons in the optic fibre layer (Figure 4A, B). This result was confirmed by Pdgfr immunoreactivity in the GCL on FluoroGold-labelled RGCs (Figure 4C–E).

Figure 4.

Immunohistochemical localization of Pdgf receptors in the retinal ganglion cell layer. Immunohistochemistry using an antibody against both α and β forms of the Pdgf receptor. A, B, and D: Bright-field micrographs of adult rat retina with immunoreactivity visualized using diaminobenzidine precipitates. A, B: Micrographs of the vitreal side of whole-mount retina preparation with the focus plane in the ganglion cell layer. Pdgfrα,β immunoreactive cell (arrow). Immunoreactivity could also be seen in the optic fibre layer (out of focus in panels A and B but visible in cross-section shown in D). C–E: Cross-sections of retina with FluoroGold-labelled RGCs. C: Fluorescence micrograph showing FluoroGold-labelled RGCs (arrow-head). D: Pdgfrα,β immunoreactivity in the same section as shown in C. Note the displaced RGC in C, which is also labelled for Pdgfrα,β. E: Negative control (GCL = ganglion cell layer; OFL = nerve fibre layer; RGCs = retinal ganglion cells). Scale bar in A, B, and E, 20 μm, also valid for C and D.

RGCs express multiple RTKs

It was clear from the PCR and histochemical analyses that several of the analysed RTKs were expressed in RGCs. Not all RGCs were labelled, suggesting that there are subpopulations of RGCs that express the receptors. Counting RTK-immunoreactive True Blue-labelled cells in relation to the total number of True Blue-labelled cells, in the GCL of the central part of the retina, allowed the percentage of RGCs to be calculated for Sky, Vegfr-2, Csf-1R, Pdgfrαβ, TrkB, and Ret. The results are shown in Table IV (rightmost column) and Figure 5. The calculation is based on the fact that by applying FluoroGold to both superior colliculi 98.4% of the RGCs present in the adult rat retina are traced (6).

Figure 5.

Immunohistochemical localization of receptor tyrosine kinases (RTKs) in retrogradely filled retinal ganglion cells. Adult rat retinal ganglion cells (RGCs) were retrogradely filled with True Blue from the superior colliculi, and cross-sections of the retinas were subjected to epifluorescence immunohistochemistry. A, E, I, M, and Q: Micrographs showing RGCs with True Blue fluorescence (blue). B: Sky; F, N, and R: Vegfr-2; V: Ret immunoreactivity (green). J, O, and U: Csf-1R; and S: TrkB immunoreactivity (red). C, G, K, P, T, and X: Merge of panels to the left on same row. Arrows indicate labelled (A–K) and double-labelled (M–X) True Blue-identified RGCs. Arrow-heads indicate single-labelled RGCs. D, H, and L: Negative controls (GCL = ganglion cell layer). Scale bars in L and T are 25 μm and are also valid for A–T. Scale bar in X is 40 μm, also valid for U and V.

Co-labelling for RTKs in RGCs was analysed using double immunohistochemistry. Retinas with True Blue-labelled RGCs were used, and we found that Vegfr-2 and Csf-1R immunoreactivity (Figure 5M–P) overlapped in 49% ± 24% of the True Blue-labelled cells; 14% ± 12% of the cells were positive for Csf-1R and not for Vegfr-2 (Figure 5O, P). Very few cells that were labelled for Vegfr-2 and not for Csf-1R could be seen. Vegfr-2 immunoreactivity overlapped with that of TrkB in 46% ± 20% of the True Blue-labelled cells (Figure 5Q–T); 12% ± 11% of the cells were positive for TrkB and not for Vegfr-2. Just a few cells were labelled for Vegfr-2 and not for TrkB. The clear majority of Sky-labelled cells were also labelled for Vegfr-2 (data not shown). The analysis of the overlap of TrkB and Csf-1R was not conclusive due to uncertain staining but indicated that the patterns overlap (data not shown). The patterns of Csf-1R and Ret overlapped in the GCL (Figure 5U–X), and the TrkB labelling overlapped with those of Sky and Ret (data not shown) (24). A potential overlap between Ret and Sky patterns could not be analysed because both antisera were raised in goat, and Pdgfr immunoreactivity could not be analysed because the double labelling epifluorescence analysis was not conclusive.

Expression in retina after optic nerve transection (ONT)

Little is known about the roles for several of the above genes in the retina. So next we studied whether ONT induces changes on their mRNA expression level at 12 h, 1, 3, 4, 7, and 14 days after ONT. This analysis was done by using Q RT-PCR. Basal levels of Pdgfrβ, Csf-1R, Vegfr-2, Sky, and Axl, as well as two ligands for Axl/Sky, Gas6 and ProteinS, in normal and injured adult rat retina were analysed (Figure 6A). Levels of mRNA vary between the genes in normal retina, with high levels in retina of Vegfr-2, Gas6, and Pdgfrβ mRNA compared to Axl and Sky. The levels shown in Figure 6A should not be considered as absolute relative mRNA levels due to possible variances in amplification efficiency between primer pairs. However, our experience is that differences in amplification levels reflect the differences in mRNA levels. The expression levels were normalized using β-actin mRNA levels. β-Actin and Gapdh mRNA levels were 22 and 117 times higher, respectively, than that of Pdgfrβ in retina (data not shown). Thy-1 mRNA levels were analysed to visualize RGC-specific expression in relation to RGC loss. There was a marked decrease of Thy-1 mRNA levels that parallels the loss of RGCs after ONT (Figure 6B). The levels of Axl (Figure 6D) and ProteinS (Figure 6F) mRNA increased after ONT, while the mRNA levels of the other analysed genes remained in general unaltered compared to normal levels (Figure 6C–I).

Figure 6.

Relative mRNA levels in normal retina and retina from rats 12 hours, 1, 3, 4, 7, and 14 days after optic nerve transection. Relative mRNA levels were analysed using quantitative real-time PCR analysis. A: Relative amplification levels in normal retina. Relative mRNA levels for Thy-1 (B), Axl (C), Sky (D), Gas6 (E), ProteinS (F), Pdgfrβ (G), Csf-1R (H), and Vegfr-2 (I). Time point 0 represents normal levels and was assigned the value of 1. n = 6. Error bars show SEM. * indicates P <0.05 ANOVA.