Inducible Rbpms-CreERT2 Mouse Line for Studying Gene Function in Retinal Ganglion Cell Physiology and Disease

Abstract

Retinal ganglion cells (RGCs) are the sole output neurons conveying visual stimuli from the retina to the brain, and dysfunction or loss of RGCs is the primary determinant of visual loss in traumatic and degenerative ocular conditions. Currently, there is a lack of RGC-specific Cre mouse lines that serve as invaluable tools for manipulating genes in RGCs and studying the genetic basis of RGC diseases. The RNA-binding protein with multiple splicing (RBPMS) is identified as the specific marker of all RGCs. Here, we report the generation and characterization of a knock-in mouse line in which a P2A-CreER^T2 coding sequence is fused in-frame to the C-terminus of endogenous RBPMS, allowing for the co-expression of RBPMS and CreER^T2. The inducible Rbpms-CreER^T2 mice exhibited a high recombination efficiency in activating the expression of the tdTomato reporter gene in nearly all adult RGCs as well as in differentiated RGCs starting at E13.5. Additionally, both heterozygous and homozygous Rbpms-CreER^T2 knock-in mice showed no detectable defect in the retinal structure, visual function, and transcriptome. Together, these results demonstrated that the Rbpms-CreER^T2 knock-in mouse can serve as a powerful and highly desired genetic tool for lineage tracing, genetic manipulation, retinal physiology study, and ocular disease modeling in RGCs.

Keywords: Cre recombinase; RGC and axonal degeneration; RGC development; glaucoma; optic neuropathy.

Figure 1

Generation and confirmation of Rbpms-CreER^T2 mice. (A) Rbpms-CreER^T2 targeting strategy by fusing P2A-CreER^T2 sequence in-frame to the C-terminus of Rbpms. (B) Representative long-range PCR amplification results show the 742 bp external fragment at 5′ and the 2072 bp amplicon at 3′ of knock-in sequence, confirming the correct allele using 5′ and 3′ external primer sets, respectively. (C) Representative PCR genotyping of a litter from crossing of Rbpms^CreERT2/+ mice identify a 335 bp Cre fragment and a 347 bp wild type fragment, confirming the correct targets using Cre and Rbpms wt primers, respectively. (D) Co-labeling with anti-RBPMS and anti-CRE antibodies in 6 weeks retinas show the overlapping expression of CreER^T2 and endogenous RBPMS. (E) Immunostaining with antibodies against RBPMS and POU4F1 of wild type, Rbpms^CreERT2/+ and Rbpms^{CreERT2/CreERT2} retinal whole-mounts show the similar expression of RBPMS and POU4F1 in the ganglion cell layer (GCL). (F,G) Quantification of the RBPMS⁺ (F) and POU4F1⁺ (G) cells reveals no significantly difference among wild type, Rbpms^CreERT2/+ and Rbpms^{CreERT2/CreERT2} retinas. n = 3. Statistical significance was assessed by one-way ANOVA followed by Tukey’s multiple comparisons test. ONL and INL denote outer nuclear layer and inner nuclear layer, respectively. Scale bars equal 50 μm.

Figure 2

Rbpms-CreER^T2 knock-in does not alter retinal structure and transcriptome. (A) Representative images of H&E stained retinal cryosections reveal comparable retinal cellular structure among the wild type, Rbpms^CreERT2/+ and Rbpms^{CreERT2/CreERT2} mice. (B) Quantification of cell number in the ganglion cell layer (GCL) within 250 µm × 250 µm region shows no significant changes among the wild type, Rbpms^CreERT2/+ and Rbpms^{CreERT2/CreERT2} mice. (C,D) Volcano plots display the RNA-Seq results. Note that using the criteria of adjusted p < 0.05 and fold change > 2, no differentially expressed gene (DEG) with significant change is identified (C) between wild type and Rbpms^CreERT2/+ retinas and (D) between wild type and Rbpms^{CreERT2/CreERT2} retinas. (E–J) Heatmaps show the expression level of selected genes expressed in individual retinal cell types. Note that there is no significant difference among the control, Rbpms^CreERT2/+ and Rbpms^{CreERT2/CreERT2} retinas of in the expression of marker genes specific for (E) retinal ganglion cell (RGC), (F) photoreceptor cell (PC), (G) horizontal cell (HC), (H) bipolar cell (BC), (I) amacrine cell (AC), and (J) Müller glial cell (MC). The cutoff criteria are set as adjusted p < 0.05 and fold change > 2. n = 3. Statistical significance was assessed by one-way ANOVA followed by Tukey’s multiple comparisons test. ONL indicates outer nuclear layer, INL and GCL denote inner nuclear layer and ganglion cell layer, respectively. Scale bar equals 50 μm.

Figure 3

Analysis of Rbpms-CreER^T2 knock-in effects on visual function and retinal fundus features. (A,B) Optomotor response measurements show no significant changes in (A) visual acuity and (B) contract sensitivity among wild type, Rbpms^CreERT2/+ and Rbpms^{CreERT2/CreERT2} mice. (C) Pattern electroretinography (PERG) was performed on the same mice with the 50 cd.s/m² “natural” stimulus, averaged amplitude of P1 and N2 components from representative wild type, Rbpms^CreERT2/+ and Rbpms^{CreERT2/CreERT2} mice does not exhibit overt differences between each of the two groups. (D) Quantification of amplitudes of P1 and N2 in PERG. (E,F) Representative optical coherence tomography (OCT) images (E) and measurement of each layer thickness evaluated by OCT (F) show no significant difference among wild type, Rbpms^CreERT2/+ and Rbpms^{CreERT2/CreERT2} mice. (G) Fluorescein angiography (FA) examination reveals comparable retinal vessel features among wild type, Rbpms^CreERT2/+ and Rbpms^{CreERT2/CreERT2} mice. n = 4. Each bar represents the mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by Tukey’s multiple comparisons test (D) or two-way ANOVA (F). Scale bars equal 300 μm.

Figure 4

Assessment of Rbpms-CreER^T2 knock-in effects on retinal function. (A) Representative scotopic ERG waveforms from the wild type, Rbpms^CreERT2/+, and Rbpms^{CreERT2/CreERT2} mice at P42. (B) Representative photopic ERG waveforms from the same mice tested after scotopic ERG. (C,D) Summary of luminance-response functions for the dark-adapted ERG (C) a-wave and (D) b-wave. Two-way ANOVA test revealed that heterozygous and homozygous Rbpms-CreER^T2 knock-in has no significant effect on either a-wave (F_{10, 54} = 0.36, p = 0.96) or b-wave (F_{10, 54} = 0.11, p = 1.0). (E,F) Summary of luminance-response functions for the light-adapted ERG (E) a-wave and (F) b-wave. No significant difference is seen among the wild type, Rbpms^CreERT2/+, and Rbpms^{CreERT2/CreERT2} mice based on two-way ANOVA analysis of a-wave (F_{10, 54} = 0.53, p = 0.86) and b-wave (F_{10, 54} = 0.15, p = 1.0). n = 4. Data points represent the mean ± SEM.

Figure 5

Assessment of Cre recombinase activity in RGCs of Rbpms-CreER^T2 knock-in mice. (A) Representative images of flat-mount retinas of 8-week-old Rbpms^CreERT2/+; Rosa26^tdT/+ mice. One to three dosages of tamoxifen were administered starting at P50 and retinas were harvested 7 days later for immunolabeling with antibodies against RBPMS (green) and RFP (red) to detect tdTomato. (B) Representative enlarged views of flat-mount retinas in (A). (C) Representative images of immunolabeled retinal cryosections of 8-week-old Rbpms^CreERT2/+; Rosa26^tdT/+ mice with one to three dosages of tamoxifen. (D) Quantification of Rbpms-CreER^T2 recombinase efficiency and (E) Quantification of Rbpms-CreER^T2 recombinase specificity. The CreER^T2-mediated recombination efficiency was calculated as the percentage of tdTomato⁺-RBPMS⁺/total RGCs positive for RBPMS, the CreER^T2-mediated recombination specificity was calculated as the percentage of tdTomato⁺-RBPMS⁺/total tdTomato positive RGCs driven by RBPMS. n = 3. ONL indicates outer nuclear layer; INL and GCL denote inner nuclear layer and ganglion cell layer, respectively. The scale bars in (A) equal 1 mm while those in (B,C) equal 50 μm.

Figure 6

Characterization of Rbpms-CreER^T2 recombinase activity during early retinal development. (A,B) Determination of the onset of RBPMS expression in RGCs. Cryosections of wild type developing retinas at (A) E11.5 and (B) E12.5 were immunolabeled with anti-RBPMS (green) and anti-POU4F1 (red) and nuclear counterstained with DAPI (blue). Arrowheads indicate RBPMS expression and RBPMS-POU4F1 co-expression in the central retina ((B), left and central panels, respectively). Far right panels show the enlarged views of the boxed regions. (C–E) Representative images of retinal cryosections immunolabeled for RBPMS, tdTomato (RFP) and POU4F2 after induction by a single dosage of tamoxifen. IP injection of tamoxifen was administered at (C) E11.5, (D) E12.5, and (E) E13.5, and retinas were collected for immunolabeling 2 days after tamoxifen induction at E13.5, E14.5, and E15.5, respectively. DAPI (blue) was used to stain the nuclei. Far right panels show the enlarged views of the boxed regions. Scale bars equal 250 μm.

Figure 7

Cre recombinase activity in differentiated RGCs of embryonic Rbpms^CreERT2/+; Rosa26^tdT/+ mice. Representative images of retinal cryosections of Rbpms^CreERT2/+; Rosa26^tdT/+ mice immunolabeled with anti-RBPMS (green), anti-RFP (red), and anti-CDKN1B (grey) and nuclear counterstained with DAPI (blue). Tamoxifen was administered at (A) E11.5, (B) E12.5, and (C) E13.5 and retinas were collected for immunolabeling 2 days after tamoxifen induction at E13.5, E14.5, and E15.5, respectively. Far right panels show the enlarged views of the boxed regions. n = 3. Scale bars equal 250 μm.