Absence of Vsx1 expression in the normal and damaged mouse cornea

Animals

All experiments on wild type mice were performed on 129S1 inbred mice (The Jackson Laboratory, Bar Harbor, ME). Vsx1:τLacZ knock-in mice [4] were maintained on a 129S1 genetic background. All mouse work was done with approval from the University of Victoria Animal Care Committee, in accordance with the Canadian Council for Animal Care.

RNA extraction

Mice were euthanized by cervical dislocation and tissues dissected in chilled phosphate buffered saline (PBS), transferred to 2 ml microcentrifuge tubes containing 1 ml Trizol (Invitrogen, Carlsbad, CA) and Precellys CK28 ceramic beads (Precellys, Montigny-le-Bretonneux, France) and then frozen on dry ice. Tissue homogenization was performed on a Precellys-24 homogenizer using two 10-s pulses. Homogenized tissue was spun for 10 min at 12,000× g at 2–8 °C, the supernatant transferred into fresh tubes and then kept at room temperature for 5 min. Next, 0.2 ml chloroform was added per ml of Trizol in the initial tubes, the tubes shaken, left to sit for 3 min at room temperature and then samples were centrifuged at 12,000× g for 15 min at 2–8 °C. The upper aqueous phase was then transferred into a fresh tube, 0.5ml isopropyl alcohol was added and samples were mixed by shaking. Following 10 min at room temperature and a 10-min centrifuge at 2–8 °C, 12,000× g, the supernatant was removed and resulting RNA pellet was washed using 75% ethanol before being stored in distilled RNase/DNase-free water (Gibco, Invitrogen,  Carlsbad, CA).

qRT–PCR

Primers for Vsx1 were designed using Primer 3Plus software while those for housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh), myofibroblast marker alpha-smooth muscle actin (Acta2) and rhodopsin (Rho) were taken from the literature (Table 1) [20-22]. Specific amplification was verified through comparison of PCR product sequences against published cDNA sequences. Cloning for sequencing was preformed using pGEMT Easy Vector protocol (Promega, Madison, WI) and QIAprep Miniprep kit (Qiagen Inc., Mississauga, Ontario). RT reactions were preformed as per Quantitect Reverse Transcription kit (Qiagen) protocol and reactions run as per Quantitect SYBR Green PCR kit (Qiagen): 15 min 95 °C, 35× (15 s 94 °C, 30 s 60 °C, 30 s 72 °C) using a Stratagene Mx300p (Agilent Technologies, Inc., Santa Clara, CA). Relative expression between samples was calculated using the 2^-ΔΔCt method [23] using the housekeeping gene GAPDH to normalize.

Corneal wounding procedure

Our alkali-burn corneal wounding protocol was based on previously published protocols [24,25]. Briefly, 4 μl of 0.2 M NaOH was applied to the corneas of anesthetized mice for 30 s followed by flushing with 10 ml of saline solution. Mice were then euthanized using cervical dislocation at desired time points post-wounding. Mice undergoing the wounding procedure were injected subcutaneously with buprenorphine (0.1 mg/kg) pre-wounding, and every 12 h for up to 3 days after the procedure to provide analgesia. One drop of Proparacaine was also applied to eyes immediately before NaOH application as a topical anesthesia. All mice were monitored throughout the experiments in accordance with University of Victoria animal care guidelines.

Immunolabeling

Eyes removed from euthanized mice were transferred to chilled PBS and bisected longitudinally using scissors to expose inner ocular chambers. Bisected eyes were then fixed for 1 h in 4% paraformaldehyde/PBS on ice followed by washing in PBS and overnight cyroprotection in 30% sucrose/PBS at 4 °C. Tissue was frozen in Tissue-Tek OCT (Sakura Finetek, Torrance, CA) and sectioned at a thickness of 14 μm and stored at −20 °C. Sections were washed with PBS and cleared in 1% Triton X-100/PBS for 30 min. Blocking was performed in 10% horse serum/PBS for 1 h before incubating with primary rabbit anti-Vsx1 antibody [3] used at a 1:100 dilution in 1% horse serum/PBS. Secondary labeling was performed using an Alexa-Fluor-488 conjugated donkey anti-rabbit (1:100 in PBS). Nuclei were stained with Draq-5 (Biostatus, Leicestershire, UK). Slides were imaged using a Nikon confocal microscope (Nikon, Mississauga, Ontario). Post-imaging adjustment of levels in order to observe non-specific labeling was done in Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA) by changing the maximum levels value from 256 to 26.

Analysis of Vsx1 mRNA expression in the postnatal and mature mouse cornea

To examine Vsx1 mRNA expression, 4 primer pairs spanning Vsx1 exons 3–5 were designed. Only 3 of these primer pairs (Figure 1A) robustly amplified products from adult (2–8 months old) retinal cDNA of the predicted size (Figure 1B), with similar efficiencies (Figure 2A) and correct sequence (data not shown). In contrast to the retina, no qRT–PCR product was observed for any of the 3 Vsx1 primer pairs when amplifying from adult corneal cDNA (Figure 2 B, n=3 mice). No specific qRT-PCT product was observed after up to 40 cycles (data not shown).

We next examined retinal and corneal cDNA from wild type mice over the course of several postnatal time points (postnatal days 0, 3, 7, 14, and 21; n=3 mice for all time points; Figure 3). Consistent with the previously described onset of Vsx1 expression in mouse at postnatal day 5, retinal expression of Vsx1 was detected in our samples at post-natal day 7 onward. In contrast, no Vsx1 expression was detected in any of the corneal samples. Likewise, Rho (which served as a control for retinal contamination) was absent from the cornea.

Analysis of Vsx1 mRNA expression following corneal wounding

Corneal wounding experiments were performed on 8 wild type mice with one eye receiving an alkali burn and the other serving as an untreated control (4 mice with treated left eye, 4 with treated right). Wounded corneas appeared cloudy from 1 day post-wounding. Retinal and corneal tissue was taken 3 days after the corneal wounding with NaOH and the wounded morphology of treated corneas was confirmed through examination during dissection (Figure 4A,B). Alkali treated corneas were no longer transparent, appeared thicker and had considerably pitted surfaces compared to the unwounded control eyes from the same mouse. qRT–PCR results found no significant change in Vsx1 expression post-wounding in the retina samples. Vsx1 expression was not observed in untreated or alkali-wounded corneal samples but Acta2, a gene that is known to be upregulated following corneal alkali burn [25] was significantly unregulated by an average of 16.3 times in treated relative to untreated control eyes for both wild type and Vsx1^{τLacZ/τLacZ} mice (Figure 4C). A time course experiment set-up identically to the 3 day wounding found no Vsx1 expression in the corneas of wild type mice 6 h, 1 day, 3 days, 7 days or 14 days post-wounding although Acta2 was upregulated in each case (data not shown).

Histological examination of Vsx1 expression following corneal wounding

Immunohistological analysis aimed to confirm the qRT–PCR results in the cornea. No specific Vsx1 immunolabeling was observed in the corneas of alkali-burn wounded or untreated mice as compared to Vsx1^{τLacZ/τLacZ} mice, which do not have Vsx1-immunolabeling [4] and therefore serve as a negative control (Figure 5). It was noted that when the post-acquisition imaging pixel intensity level was elevated by an order of magnitude higher than the optimal near-saturation levels used for imaging retinal Vsx1, non-specific labeling was observed in both wild type and Vsx1^{τLacZ/τLacZ} alkali-burn treated corneas but not in untreated corneas (Figure 6). We next examined β-galactosidase knock-in reporter expression in normal and wounded corneas of Vsx1^{τLacZ/τLacZ} mice. The Vsx1 τLacZ knock-in reporter recapitulates the normal Vsx1 expression pattern in retinal bipolar cells [4] and, although it is present in the developing ventral hindbrain and spinal cord, it is not detected from the embryonic retina and cornea (Figure 7 and data not shown). Similar to the non-specific Vsx1 immunolabeling observed in the wounded cornea, a high degree of non-specific β-galactosidase immunolabeling was also observed in wild type and wounded cornea (data not shown). We therefore examined β-galactosidase activity using the X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside) chromogenic substrate. In contrast to robust β-galactosidase activity in the Vsx1^{τLacZ/τLacZ} retina, no activity was observed in wounded or untreated corneas (Figure 8).