Specific ablation of Hippo signalling component Yap1 in retinal progenitors and Müller cells results in late onset retinal degeneration

2.1 Mouse models

All animal experiment protocols were approved by the Institutional Animal Care and Use Committee of Sichuan Provincial People's Hospital (Chengdu, Sichuan, China) and conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All animals were housed in a temperature-controlled room (24°C) with a 12-h light/dark cycle. Mice were maintained on a C57BL/6J background.

Mice with Yap1 deletion specifically in retinal in embryonic RPCs and in mature Müller glial cells were generated using the Cre-loxP system. Briefly, Yap1^flox/flox mice (Yap1^tm1.1Dupa/J, Jackson Laboratory, stock number: 027929) (N. Zhang et al., 2010) were crossed with the Chx10-Cre transgene (Jackson Laboratory, stock number: 005105) (Rowan & Cepko, 2004) transgenic mice to yield progeny with the genotype Yap1^loxP/+; Chx10-Cre. Then, the Yap1^loxP/+; Chx10-Cre mice were crossed with Yap1^flox/flox animals to generate Yap1^flox/flox; Chx10-Cre (Yap1^ChxKO) mice. Yap1^flox/flox littermates were used as controls. In addition, to monitor the expression pattern of the Cre enzyme, a tdTomato reporter gene was used (strain name: Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J; Jackson Laboratory, stock number: 007914). In the presence of the Cre enzyme, the stop codon before the tdTomato expression cassette was removed, which permits the expression of tdTomato (red fluorescence) in Cre-positive cells.

2.2 Genotyping

Mouse genomic DNA samples were extracted from mouse tails and genotyped using PCR to screen for the floxed Yap1 alleles using the primers Yap1-flox-F (5′-AGGACAGCCAGGACTACACAG-3′) and Yap1-flox-R (5′-CACCAGCCTTTAAATTGAGAAC-3′). Chx10-Cre was genotyped using the generic Cre primers Cre-F (5′-TGCCACGACCAAGTGACAGCAATG-3′) and Cre-R (5′-ACCAGAGACGCAAATCCATCGCTC-3′). The tdTomato mice were genotyped using the following primers provided by the JAX mouse service: oIMR9020, 5′-AAGGGAGCTGCAGTGGAGTA-3′; oIMR9021, 5’-CCGAAAATCTGTGGGAAGTC-3′; oIMR9103, 5′-GGCATTAAAGCAGCGTATCC-3′; and oIMR9105, 5′-CTGTTCCTGTACGGCATGG-3′. All amplification reactions were performed using a master mix (Invitrogen) according to the manufacturer's instructions. The first cycle of PCR consisted of holding at 95°C for 5 min, followed by 32 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 30 s. The PCR products were separated by DNA electrophoresis on a 3% agarose gel.

2.3 Electroretinography (ERG)

For electroretinographic evaluation, following overnight dark adaptation, mice were anesthetized with an intraperitoneal injection of xylazine (80 mg/kg) and ketamine (16 mg/kg) in normal saline. Additional anesthetic was given if akinesia was inadequate. The equipment and protocol used here have been described previously (Yang et al., 2019). Briefly, the responses of dark-adapted, rod-mediated ERGs to short-wavelength flashes generated by a photopic stimulator over 4.0-log units to the maximum intensity were recorded. Cone-mediated ERGs were recorded in response to white flashes after 10 min of complete light adaptation. The signals were sampled at 0.8 ms intervals and averaged.

2.4 Histological analysis

For hematoxylin and eosin staining (H&E), enucleated eyes from control and Yap1^ChxKO mice were fixed overnight in 1.22% glutaraldehyde and 0.8% paraformaldehyde in 0.08 M phosphate buffer, embedded in paraffin and then cut into 5 μm sections. To ensure that sections used for quantification came from the same eccentricity, the globe was embedded in the same orientation. Sections that encompassed the ON were selected for staining with H&E according to a standard protocol.

2.5 Semithin-section processing of the ON

ONs were dissected from eyes and treated with a solution containing 4% paraformaldehyde and 0.2% glutaraldehyde for 24 h. After rinsing three times in 0.1 M phosphate buffer (pH 7.4) for 15 min each, tissues were fix with 1% osmic acid and 0.1 M phosphate buffer (pH 7.4) at room temperature (20°C) for 2 h. After rinsing three times in 0.1 M phosphate buffer (pH 7.4) for 15 min each. Stained tissues were dehydrated in graded concentrations of ethanol, embedded in acetone: 812 embedding medium = 1:1 for 2–4 h, and in acetone: 812 embedding medium = 1:2 infiltration, overnight, and finally in 812 embedding medium for 5–8 h. Samples were inserted into an embedding plate containing 812 embedding medium, and placed overnight in an incubator at 37°C, followed by polymerization at 60°C for 72 h. Semithin Section (1 μm) were cut transversely from segments of ONs caudal to the eyeball and stained with 1% toluidine blue.

2.6 Immunohistochemistry

For immunohistochemistry, eyes were removed from euthanized mice by intraperitoneal injection of pentobarbital (75 mg/kg) and by cervical dislocation and fixed in 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) for 1 h at 4°C, followed by cryoprotection in 30% sucrose for 2 h. The lenses were removed, and the eyes were embedded in optimal cutting temperature (OCT) solution and sectioned at a 10 μm thickness. After blocking and permeabilization with 10% normal donkey serum and 0.2% Triton X-100 in phosphate buffer for 1 h, the sections were labeled with the primary antibody overnight at 4°C. The primary antibodies used are shown in Table S1. The sections were rinsed in phosphate-buffered saline (PBS) three times, Alexa Fluor 594/488-conjugated goat anti-mouse/rabbit secondary antibody (Cat #A11005 and A11008, 1:500 dilution; Invitrogen) was applied, and nuclei were counterstained with DAPI (Cat #D8417; Sigma) for 1 h at room temperature. Images were captured on a Zeiss LSM 800 confocal scanning microscope. In addition, the fluorescence intensity was quantified using ZEN 2.3 imaging software.

2.7 Retinal flat mounts and cell count

Retinas were dissected from enucleated eyes and flattened. Retinas were immersed in 4% paraformaldehyde for 24 h at 4°C, then blocked in PBS containing 1% bovine serum albumin and 0.5% Triton X-100 and incubated with polyclonal rabbit anti-NeuN antibody (Cat #24307, 1:200 dilution; CST) for 12 h at 4°C. After several washes, the retinas were incubated with the secondary antibody, Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Cat #A11008, 1:250 dilution; Invitrogen) for 4 h at room temperature, and subsequently washed with PBS. To precisely evaluate the loss of RGCs, each retinal quadrant was divided into four zones—nasal, temporal, dorsal, and ventral—each located 1 mm from the ON head. Cells in GCL were counted in each unit area by two investigators in a blinded fashion, and the scores were averaged.

2.8 5-Ethynyl-2′-deoxyuridine (EdU) labeling assay

To detect cell proliferation in retinas, mice at P3, P30, 12-month old and pregnant females were first weighed and injected intraperitoneally with EdU (Cat #E10187; Thermo) (100 mg/gm of body weight) in PBS. The treated mice were killed after 3 h. EdU-positive cells were subsequently stained with the Click-iT EdU Alexa Fluor-488 Imaging Kit (Cat #C10337; Thermo). For cell cycle exit analysis, sectionswere double labeled with EdU (S-phase marker) and cell proliferation marker (Ki67).

2.9 Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End labeling (TUNEL) assay

Apoptotic cell death was detected by the TUNEL assay according to the manufacturer's protocol (Cat #11684795910; Roche Diagnostics) using prepared frozen sections. Images were captured on a Zeiss LSM 800 confocal scanning microscope.

2.10 Western blot

Tissues were lysed in radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail (Cat #11697498001; Roche) and a phosphatase inhibitor (Cat #4906845001; Roche). The protein concentration of the lysates was determined using a DC Protein Assay according to the manufacturer's instructions (Cat #500-0122; Bio-Rad). Equal amounts of protein were separated on SDS polyacrylamide gels and transferred onto NC membranes (Cat #HATF00010; Millipore). The blots were blocked with 8% nonfat dry milk in Tris-buffered saline solution with Tween® 20 detergent for 2 h at room temperature. Then, the membranes were incubated with the primary antibodies in blocking solution overnight at 4°C. The primary antibodies used for western blotting are shown in Table S1. The primary antibodies were detected with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:5000; Bio-Rad), and the signal was developed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Cat #34577; Thermo). The relative intensity of the immunoreactive bands was quantified using the gel analysis tool provided in ImageJ software. The intensity of the proteins of interest was normalized to that of glyceraldehyde 3-phosphate dehydrogenase. At least three independent western blots were conducted, and one typical blot is presented.

2.11 Statistical analysis

Statistical analysis was performed using GraphPad Prism 6 software. The datasets were tested for normal distribution using Shapiro–Wilk test. For normally distributed data, statistical significance was determined by Student's t test or analysis of variance (ANOVA). If the data was not normally distributed, nonparametric statistic was used. All p values were calculated by Student's t test or ANOVA followed by a, Tukey, Dunnett or Sidak's multiple comparisons test as appropriate. A p < 0.05 was considered statistically significant.

3.1 Generation of Yap1 conditional knockout mice using Chx10-Cre lines

Previous studies have demonstrated that Yap1 is widely expressed in developing eyes as well as in RPE, CB, cornea and retinal Müller cells in adult mouse (Hamon et al., 2017; Williamson et al., 2014). To confirm the expression pattern of Yap1 in both embryonic and adult retinas, cryosections of E14.5 mouse embryos and retinas from postnatal 30 (P30) mouse were prepared and immunostained with the primary antibodies anti-YAP. In wildtype embryonic E14.5 retinae, YAP exhibit ubiquitous expression in the NR and in particular were enriched in neuroblastic layers (NBL) (Figure 1a), where mainly retinal progenitors reside. In contrast, post-mitotic GCL cells presented little YAP expression (Figure 1a), suggesting specific localization of YAP in retinal progenitors. In adult mouse retina, the signal intensity of YAP was restricted in the ON, CB, as well as INL (Figure 1b, upper panels), and their position and morphology in INL strongly suggests that YAP could be specifically expressed in Müller glial cells (Figure 1b, lower panels). Additionally, we analysed the protein expression profiles of YAP in mouse retina at different ages. As shown in Figure 1c, yap was steady expressed during embryonic (E14.5) and early postnatal life (P3), while it showed approximately 1.67-fold increase in maturated retinal neurons (2-month old) (Figure 1c,d). Interestingly, YAP expression level was decreased in aged retinas (12-month old) compared to that in 2-month-old mice, while it's still slightly higher than that at embryonic stage (Figure 1c,d).

To investigate the underlying role of YAP specifically in embryonic and adult mouse retina, we developed Yap1 conditional knockout mice by crossing Yap1 exon 1–2 floxed mice (Yap1^flox/flox) with BAC-Tg Chx10-Cre mice (Figure S1a), which droves Cre expression in the majority of E9-P2 RPCs, as well as in a subset of retinal Müller cells at adult stage (Kowalchuk et al., 2018; Lu et al., 2013; Ueki et al., 2009). Their progenies were intercrossed to yield littermate Yap1^flox/flox; Chx10-Cre (hereafter Yap1^ChxKO), Yap1^flox/+; Chx10-Cre (hereafter Yap1^Het) and Yap1^flox/flox (hereafter Yap1^Ctrl, served as control) mice (Figure S1b). Moreover, a strong Cre reporter line, ROSA26-tdTomato, was introduced to monitor the specific expression of Chx10-Cre. We crossed ROSA26-tdTomato reporter mice with Yap1^flox/flox; Chx10-Cre mice to generate Yap1^flox/flox; Chx10-Cre; Rosa mice (Figure S2a). A loxP-flanked STOP sequence before the tdTomato expression cassette is deleted in Cre-expressing cells, and subsequently, tdTomato is expressed.

Immunostaing data of tdTomato-expressing retinas from E14.5 and P30 mice suggested that the tdTomato was expressed distinctly in the NR as well as Müller glial cells in E14.5 and P30 mice retina respectively (Figure S2b,c), verifying the specific expression of this Cre line. Besides, we found that YAP distinctly expressed in the ROSA⁺ Müller glial cells in adult Chx10-Cre; ROSA mice (Figure S2d), confirming the specific YAP expression in mature retinas. To further evaluate the knockout efficiency of YAP in these cells, we prepared cerebral and ocular/retinal lysates from E14.5/P30 Yap1^ChxKO mice and subjected to immunoblotting analysis. The expression level of YAP in E14.5 Yap1^ChxKO eyes was reduced to approximately 36% of that in controls (Figure 1e and 1g), and reduced to approximately 27% in P30 Yap1^ChxKO retina (Figure 1f,g), while the YAP expression in both Yap1^ChxKO cerebral cortex did not change (Figure 1e,f). Considering that YAP is also expressed in other retinal cells, the excision efficiency was fairly good. The immunostaining results further verified this. The YAP signals were robust in the tdTomato expressed NR in control retinas but were almost absent in the Yap1^ChxKO NR (Figure 1h). Besides, the sections of P30 mice retinas were co-immunostained with YAP and PKCα, a marker for RBCs. Although still expressed in PKCα marked by RBCs, YAP staining disappeared in areas where Müller cells are mainly occupied (Figure 1i). Together, these results suggested the precise excision of YAP in embryonic RPCs and retinal Müller cells.

3.2 Yap1 depletion in RPCs has no detectable effect on retinal structure in embryonic and neonatal mice

Previous studies have implicated YAP in retinal development by modulate retinal cell proliferation (Kim et al., 2016; H. Zhang et al., 2012). In present study, Cre-mediated recombination in Yap1^ChxKO mice led to YAP ablation in the majority of retinal progenitors in E9-P2 mice, therefore we set out to assess the consequences of the specific excision of YAP in developing retinas. We first compared histologic sections from E14.5 control and Yap1^ChxKO retinas at the early stages of retinal development (Figure S3a). In control retinas, the NBL zone was occupied by proliferating progenitor cells and the inner retina distributed postmitotic neurons. Regrettably, the Yap1^ChxKO retina presented no visible defects on the gross retinal mmorphology compared with controls (Figure S3a, upper panels). To examine the differentiation of retinal progenitors, position-matched sections of the H&E staining were immunostained with Brn3a, which marks the postmitotic neurons in embryonic retina (Wang et al., 2002). However, in line with histologic result, E14.5 Yap1 deficient and control retinas contained comparable proportions of Brn3a positive postmitotic neurons (Figure S3a, lower panels), indicated that there was no detectable abnormality in retinal development of Yap1 mouse in this stage.

We next examine the P9 retina to evaluate the role of Yap1 at the late periods of retinal development after birth. Likewise, the Yap1^ChxKO retina appeared to resemble the control retinas in morphology, exhibiting comparable thicknesses of outer and INLs (Figure S3b). Surprisingly, no obvious alterations was observed neither in the thickness of ONL/INL nor in cell number of GCL in Yap1^ChxKO mouse retina at 4-mouths age, compared to those in control littermates (Figure S4). Overall, these observations suggested that Yap1 depletion in RPCs has no detectable effect on retinal structure in embryonic and neonatal mice.

3.3 YAP is essential for the normal visual function and retinal maintenance in adult retinas

Since YAP is also expressed in Müller cells which maintain retinal function and homeostasis at the adult period of mice, we next evaluate the physiological status of the retina at the adult period using photopic and scotopic electroretinograms (ERGs) of control and Yap1^ChxKO mice at 10 months of age. Scotopic ERG recordings, which detect the responses of rod-driven circuits, revealed reduced amplitudes of both a-waves and b-waves compared with those of the controls at each flash intensity (Figure 2a). The mean amplitudes of the a- and b-waves of Yap1^ChxKO mice were decreased by approximately 56.7% and 52.5%, respectively (Figure 2c,d). The dark-adapted amplitude reductions for both a- and b-waves suggested that Yap1^ChxKO retinas not only have photoreceptor dysfunction, but also deficits in visual signal transmission from photoreceptors to inner neurons. Likewise, the photopic b-wave and flicker amplitudes, which mainly reflect the status of cone cells, were also decreased in Yap1^ChxKO mice (Figure 2b and 2e).

Moreover, the total amplitude of oscillatory potentials (OPs), which serves as a sensitive functional indicator of inner retina (Heynen et al., 1985), was reduced to 43%, 49%, 57% of that of controls in 0.3, 3, and 20 cd·s/m², respectively, in Yap1^ChxKO mice (Figure S5). The reduction of OPs amplitude provided additional evidence for the impaired physiological status within the Yap1^ChxKO inner retina. Together, ERG data suggested that the visual function of Yap1^ChxKO mice was significantly compromised by the loss of YAP.

To determine the pathological changes underlying the abnormal ERG results, histologic analysis was performed to examine the retinal morphology in Yap1^ChxKO retinas at 10 months of age. In accordance with the ERG results, the Yap1^ChxKO mice showed a remarkable thinning of outer and INLs that worsened toward the central retina, accompanied by a shorter outer segment (OS) (Figure 3a,b). Besides, a 53.2% decrease in GCL cell number was observed compared to that in control retinas (Figure 3c), indicating significant RGC loss. Moreover, this retinal degeneration worsened in Yap1^ChxKO mice at 14 months of age (Figure S6). We, however, did not detect any alterations neither in thickness of ONL/INL nor in cell number of GCL in Yap^Het mouse retina, compared to those in control littermates (Figure S6).

3.4 Degeneration of rod and cone photoreceptors in Yap1^ChxKO retinas

Retinal ONL consists of the nuclei of photoreceptors, which are classified into two types according to differences in morphology and function: rods for dim light vision and cones for daylight and high-acuity vision. To characterize the specific cell types affected in ONL of Yap1^ChxKO retina, immunostaining was performed against antibodies of Rhodopsin (rod OS marker) and NaK pump (rod IS marker) to analyze the subcellular changes in rods using retinal sections from 10-month-old mice. Though the rhodopsin marked OS was well organized and the signal was strictly restricted to the rod OS, the Yap1^ChxKO mice showed a decrease in the length of the OS in the central retina, with a nearly 50% reduction (9.73 ± 0.6 vs. 17.36 ± 0.9 mm) in OS length near the ON (Figure 4a,b). This result was further confirmed by the immunoblotting: a approximately 43% reduction rhodopsin content was observed in Yap1^ChxKO retinas (Figure 4c), suggesting degeneration of rod cells.

To determine the effect of YAP deficiency on cone photoreceptors, we immunolabeled retinal cryosections from 10-month-old control and Yap1^ChxKO mice with cone arrestin (cArr), which revealed a reduction in cone number throughout the Yap1^ChxKO retina (Figure 4d,e), compared to that of controls. Moreover, the retinal sections were coimmunostained with an L/M-opsin antibody to mark the cone OS and Alexa Fluor-594-conjugated PNA, which mainly labels the cone matrix sheaths associated with all cones. As expected, a mildly decrease in the number of M-cone cells was observed in the Yap1^ChxKO retina, with a shortened, disorganized, and misshapen cone OS compared to that of controls (Figure 4f,g), suggesting that YAP depletion also results in the degeneration of cone photoreceptors.

3.5 Yap maintains the survival of multiple cell types in INL

INL, the second-order neurons layer in retinal system, plays essential roles in transmitting and integrating visual information from photoreceptors to third-order retinal neurons, such as the ganglion cells. ERG recordings revealed the reduced amplitudes of scotopic b-wave and OPs, suggesting dysfunction of inner retina. We, therefore, used cell type specific antibody markers to label and quantify RBCs, HCs, and ACs, which constitutes the major neuronal circuits in INL. Immunostaining using RBC specific marker PKCα revealed the malformation and loss of RBCs in Yap1^ChxKO retina compare to that of controls (Figure 5a). The mean numbers of PKCα-positive cells in the control and Yap1^ChxKO mice were 19.8 ± 0.8 and 12.9 ± 1.3 cells/200 μm of retina length, respectively (Figure 5a,b), suggesting a nearly 35% reduction in PKCα-positive RBC number.

To detect the status of HCs and ACs, we performed immunostaining on retina sections from 10-month-old mice for anti-Calbindin, which marks HC and AC somata and dendrites in retina (Soto et al., 2013). In Yap1^Ctrl retinas, intensely stained Calbindin-positive HC somata and dendrites are positioned at the border between INL and OPL, while lightly-stained Calbindin-positive AC somata are located in the inner INL and IPL (Figure 5c). Compared to the controls, Yap1^ChxKO retinas had fewer and fainter Calbindin-positive HCs (Figure 5c,d, yellow arrowheads) and ACs (Figure 5c,d, white triangles). Together, these observations revealed the death of multiple neuronal cell types in Yap1^ChxKO INL, which led to the significantly thinner INL and compromised inner circuits in aged Yap1^ChxKO retinas.

3.6 RGC death and ON degeneration in Yap1^ChxKO retinas

Given impaired OPs amplitude and remarkably cell loss in GCL were observed in Yap1^ChxKO mice, we next set out to investigate the GCL pathologies. To directly visualize the cell content in GCL of control and Yap1^ChxKO mice, wholemount retinas from 10-month-old mice were immunostained with a specific neuronal marker NeuN, which labels cells resided predominantly in the GCL (Tan et al., 2020). To this end, we imaged four equivalent prescribed areas each located 1 mm from the ON head (nasal, temporal, ventral, and dorsal) per retina and quantified the cell density of GCL by determining NeuN-positive cells per unit area. In Yap1^ChxKO retinas, a significant decrease in NeuN-positive cells in all four quadrants was observed (Figure 6a). Quantitative analysis revealed a 28.6%; 26.7%; 25.4%, and 26.1% reduction of NeuN-positive cells compare to that of WT in nasal, temporal, ventral, and dorsal area, respectively (Figure 6b). To observe RGCs in the GCL, immunohistochemical analysis of Brn-3a (RGC marker) was performed in 10 months Yap1^Ctrl and Yap1^ChxKO retinas. The Brn3a immunofluorescence was precisely restricted within the GCL in the two groups (Figure 6c). Consistent with the results of NeuN immunofluorescence, the Brn3a staining revealed that the mean numbers of Brn-3a-positive cells were 19.7 ± 1.2 and 13.8 ± 0.8 cells/200 μm of retina length in the Yap1^Ctrl and Yap1^ChxKO groups, respectively, indicating a significant reduction of RGCs in Yap1^ChxKO retinas (Figure 6d).

Injury to RGCs, central nervous system neurons that relay visual information to the brain, often leads to RGC axon degeneration. This prompted us to further evaluate the ON status in degraded Yap1^ChxKO retinas. Histological analysis revealed the thinning of the NFL near ON in Yap1^ChxKO mice at 12 months of age (Figure 6e,f). To determine details of axonopathy, toluidine blue-stained semithin sections of the ON from 12-month-old animals were prepared and the number of axons were quantified. Compared with control mice, the axons density was clearly lower in Yap1^ChxKO mice and accompanied with axonal swelling and extensive myelin debris (Figure 6g,h), indicating apparent axonal damage.

3.7 Müller glial malformation and reactive gliosis in Yap1^ChxKO retinas

Given the Yap1 was specifically removed in the mature retinal Müller cells, we next sought to investigate the pathologic changes of Müller glia cells in 10-months-old Yap1^ChxKO retinas, through using antibodies directed against glutamine synthetase (GS) to visualize cell bodies and processes of Müller glia cells. In control retinas, Müller glia cell bodies were orderly aligned in the INL, and their cell processes span across the retina and form the outer and inner limiting membranes (Figure 7a). By contrast, uneven distribution of GS-stained cell bodies was indicated in the Yap1^ChxKO retina, accompanied by the fewer and disorganized Müller glial cells (Figure 7a). The mean numbers of GS-positive Müller cells were 49.8 ± 1.1 and 40.9 ± 0.7 cells/250 μm of retina length in the Yap1^Ctrl and Yap1^ChxKO retinas, respectively (Figure 7b), indicated dysfunction and loss of Müller cells.

Meanwhile, immunostaining of retinal sections also revealed that the expression of glial fibrillary acidic protein (GFAP) was dramatically upregulated in degenerating Yap1^ChxKO retinas compared to control retinas (Figure 7c). Western blot analysis further confirmed this result, as revealed by the increased GFAP protein level in the Yap1^ChxKO retina (Figure 7d). These results suggested reactive gliosis and that Müller cells were activated as an indicator of retinal damage.

3.8 Yap1^ChxKO retinas showed normal cell proliferation during early retinogenesis but increased apoptosis at late stage

We further assessed the proliferation potential of RPCs using an in vivo EdU labeling assay. We pulsed E14.5 pregnant females with EdU followed by embryo harvest after 3 h. Retinal sections from E14.5 embryos and P3 mice were subjected to immunostaining with Ki67, along with EdU detection (Figure 8a and 8d). The numbers of Ki67⁺ cells exhibited no difference between the Yap1^Ctrl and Yap1^ChxKO samples at both ages, indicating that RPCs maintain the normal ability to proliferate in Yap1^ChxKO NBL, although with a slightly reduced rate (Figure 8a,b and 8d,e). Moreover, we quantified EdU⁺ (S-phase) RPCs as a percentage of total Ki67⁺ RPCs, which also showed no difference in S-phase RPCs in Yap1^ChxKO retinas compared to Yap1^Ctrl (Figure 8a–d and 8f). In addition, P30 and 8-month-old littermates were also accessed, while no proliferating cells was detected in both Yap1^ChxKO and Yap1^Ctrl retinas (Figure S7).

To investigate whether the YAP deficiency-induced retinal degeneration observed in aged mice could be linked to an increased cell death of the retinal cells, we investigated the extent of apoptosis in retina by TUNEL staining. Although no significant difference between Yap1^ChxKO and Yap1^Ctrl mice in E14.5, P9 and 2-month old, our TUNEL assay data showed that apoptotic cells were dramatically increased in 8-month-old Yap1^ChxKO mice retinas, especially in the ONL and INL, compared to that of Yap1^Ctrl mice (Figure 8g,h). These data indicated that the broad retina degeneration in aged Yap1^ChxKO mice should be mainly caused by increased cell apoptosis at late stage, rather than abnormal retinal cell proliferation during embryonic and early postnatal life.