Animals and tissue collection

Experiments were performed at 19- to 21-day-old mice (C57BL/6) Mtr-knock-out (Mtr-cKO) in retina tissue, achieved by Cre/Lox system using Thy-1.2 as a promoter of Cre recombinase. Animals have been treated following the National Institute of Health Guide for the Care and Use of Laboratory Animals, in an accredited establishment (Institut National de la Santé et de la Recherche Médicale, U1256) according to the UE guidelines 2010–63-UE and to French governmental decree 2013–118 and the authorization number Apafis #12,851. Mice were euthanized at D21 with an overdose of isoflurane. Tissues were rapidly harvested and kept frozen in liquid nitrogen and then stored at − 80 °C until biochemical/molecular and LC–MS/MS analyses or otherwise used directly for experiments that need fresh tissues.

Behavioral testingAssessment of bilateral eyelids opening and visual acuity test

We followed the pups on days 12, 13, 14, 15, and 16 of their lives, three times a day until the full eyes are open, to register any eyes development delay. The visual acuity performances were evaluated using a behavioral test adapted from Prusky et al. [35]. Behavioral observations of 12 wild-type and 12 Mtr-cKO mice (including 6 females and 6 males per group) were conducted using a video-tracking system (Viewpoint) allowing a high standardization between runs.

A rectangular swimming pool of 65 cm long and 33 cm wide, at the end of which are two corridors, was used and adapted for carrying out the visual acuity test. The pool was filled with water (25 °C) to a height of 3 cm allowing a walking swim. A platform hidden in the water and/or a visual stimulus, corresponding to a checkerboard whose squares are 15 mm in length, could be added.

The visual acuity test involves the vision, the dorso-median striatum, and the dorsolateral striatum [36]. This test involves stimulus–response association memory, that is, the memory that prompts the animal to go in a direction based on an understanding of an association between the visual stimulus and the platform. To carry out this test, each lane of the swimming pool was deposited or not a platform and a visual stimulus. This test requires a preliminary training stage, carried out at D20, in which the animal will try to assimilate that the platform is in the same place as the visual stimulus, regardless of the location. For the training stage, the visual stimulus and the platform are placed in the two lanes of the swimming pool, and then, the mouse is placed in front of the platforms. When the mouse reaches the platform, it is moved to the location of the reward (a heated cage). During this training step, each mouse undergoes 10 passages. After twenty-four hours allowing memory consolidation, the visual acuity test is performed (D21). During this test, the platform and the visual stimulus are placed in only one corridor, and their location is alternated between each pass following a pseudorandom pattern. Thus, the mouse is placed in front of the corridors (release spot) where it makes its choice between the left or right corridor based on the visual recognition of the stimulus. During this step, each mouse undergoes 19 passages, and 2 parameters are considered: the ability to find the platform directly (success) or not (failure) and the latency for the mice to get to the platform from the release spot.

Eye fundus

The observation of the fundus is done externally on the animal anesthetized under isoflurane. It is a noninvasive method of exploration and imaging allowing the observation of the retina. The examination is performed under general anesthesia, and an endoscopic probe is used to view the retina after the pupil is dilated with Tropamide solution (Mydriaticum 2 mg/0.4 mL). The probe is positioned and guided by an experienced operator for a comprehensive examination of the retina. The endoscope is a mobile device, AIDA Compact II system (Karl Storz) adapted to the study of small rodents [37]. The endoscope is equipped with a light source, an optical system utilizing optical fibers, and various channels for operator control. The images (videos or photographs) are digitized and then analyzed by an ophthalmologist.

Methylome analysis

For methylome studies, four retina samples were used per group. Genomic DNA was extracted using QIAamp DNA Kits (Qiagen) following the manufacturer’s instructions. Tissue lysis was performed overnight at 56 °C, and the DNA was eluate in 50 µl elution buffer. DNA concentration was measured using the Quant-iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen), and DNA quality was analyzed using TapeStation instrument (4150 Agilent) and genomic DNA ScreenTape Assay kit (Agilent).

An aliquot of 100 ng of DNA was utilized for library preparation, bisulfite conversion, and amplification using the Diagenode Premium RRBS Kit (Liege, Belgium) as per the manufacturer’s guidelines. A total of 8 samples were mixed before the bisulfite conversion step. We used the methylated and unmethylated spike-in controls included in RRBS Diagenode kit to estimate the bisulfite conversion efficiency. The results indicated no over-conversion as the conversion rate of the methylated spike-in was below 2% and no under-conversion as the conversion rate of the unmethylated spike-in was above 98% as shown in Additional file 1: Table S1. Every library pool was quantified using Quant-iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen), and the average size of DNA fragments was estimated with a 2100 Bioanalyzer instrument (Agilent) using High Sensitivity DNA Kit (Agilent). Every pool was then denatured in NaOH and diluted at 1.8 pm with 20% of PhiX Control v3 (Illumina). Sequencing was carried out on a NextSeq 550 machine (Illumina) using the NextSeq 500/550 High Output v2 Kit (Illumina), 75 cycles in single-end mode. After demultiplexing, Fastq files containing all the sequencing reads per sample were generated and used in downstream bioinformatics pipelines. Quality control was performed with FastQC v0.11.5. Adapters, 5’ and 3’ adjustments for possible end-repair bases and low-quality bases were removed using Trim Galore! v0.6.6 (FastQC) and Trim Galore! was downloaded from https://www.bioinformatics.babraham.ac.uk/. Bisulfite-treated reads were then aligned (using Bowtie v2.4.4) to the GRCm38/mm10 mouse reference genome followed by methylation calling using Bismark v0.22.3 22 (parameters for the mapping step: –non_bs_mm –bam –nucleotide_coverage; parameters for the methylation calls: –cytosine_report –comprehensive –merge_non_CpG).

All differential methylation analyses were performed between Mtr-cKO and WT groups under R 4.1 (RStudio v1.4.1106) with the MethylKit v1.16.1 package 23. The dataset was first filtered for low coverage (CpGs with coverage below 10x) and for extremely high coverage to exclude reads with PCR bias (CpGs with coverage more than the 99.9th percentile of coverage), then normalized following the median depth method, and finally merged by allowing one maximum missing position per group. After evaluation of the correlation structure between samples, differential CpGs were identified with the “calculateMethDiff” function, with false discovery rate (FDR) correction following the Benjamini–Hochberg procedure. The differentially methylated regions (DMRs) were identified within a window size of 2000 bp. DMRs were annotated using the mouse GRCm38/mm10 genome as a reference, with RefSeq curated (NCBI) and GENCODE VM22 (Ensembl) databases queried and prepared with the UCSC table browser (https://genome.ucsc.edu/).

Bioinformatics analysis

We performed enrichment analyses on CpGs exhibiting increased or decreased methylation (methylation fold change > 25 and a cumulative q-value < 0.01) and on DMRs with a methylation fold change > 15, and a cumulative q-value < 0.01 using “Gene Ontology Biological Process” and/or “Reactome Pathway Database” to summarize statistically top ten significant terms based on their calculated FDR. Differentially methylated CpGs and DMRs are summarized on a volcano plot using GraphPad.

Immunofluorescence cell imaging on flat-mounted retina

The eyes of 21-day-old mice, enucleated, are fixed with 4% PFA for 1 h at room temperature. After fixation, eyes were sectioned at the limbus, and then, the cornea and the lens were removed. The retinas were carefully separated from the choroid, and sclera. They were then washed in PBS and incubated in a 1 ml tube with a solution of primary antibodies (RBPMS #GTX118619, GENETEX; PNA #10,134,522 Fisher Scientific) diluted 1:100 in BSA-Triton overnight at 4 °C with gentle agitation. After incubation, the retinas were washed 3 times for 5 min with 1% PBS—Tween, at 4 °C and under slow agitation, and then incubated with 200μL of secondary antibody (Anti-Rabbit Abcam) diluted to 1/1000 and DAPI (Sigma) diluted to 1/3000 in BSA–Triton for 2 h at room temperature with stirring. The retinas were mounted flat between two coverslips with mounting fluid (Dako Fluorescent Mounting Medium) and observed under a confocal microscope (Nikon C2). For each retina, an image was recorded in 3 distinct zones: top, bottom, and right. The cell counting was performed using a blind manual technique by an external experimenter who was not aware of the phenotype to eliminate possible bias, using the Icy v2.4 software. For the retinal ganglion cells (RGCs), all the cells were counted and the number of RGCs was related to the number of total cells, while, for the cones, a rectangle with an area of 2 µm2 was used for counting. This rectangle was placed in the center-top, center, and center-bottom of each image, and then, the number of cones/mm2 was related to the number of nuclei.

LC–MS/MS analyses

Briefly, each retina is placed in a 2 mL tube from the Lysing kit CK28 (Precellys, Cat. No.03961-1-002) along with ceramic beads. This combination facilitates efficient homogenization and lysis of the retinas. To initiate the process, 200 µL of PBS is added to the retinas. The tube is then inserted into a Precellys24 homogenizer (Bertin Instruments) operating at 5000 rpm for 15 s, followed by 5 s of rest. This cycle is repeated twice to ensure thorough processing. Once the retinas are homogenized, 16 µL of the resulting sample is used for protein quantification using BCA kit (Interchim). A standard curve is prepared, comprising a mixture of retinol, retinal, retinoic acid. Subsequently, 100 µL of acetonitrile (Dasit Group) is added to facilitate retinoid extraction. The tubes are vigorously vortexed to promote thorough mixing, followed by the addition of methyl-tert-butyl ether (MTBE) (Acros organics). Further vortexing is performed, and the tubes are then centrifuged for 10 min at 13,000 rpm followed by adding a solution of 75% methanol and 25% of formic acid. Experiments were carried out using Shimadzu LCMS 8045 ESI Triple quadrupoles (Kyoto, Japan) and analyzed using Insight software v3.1 (Shimadzu, Kyoto, Japan).

RNA extraction and quantitative RT-PCR analysis

Five hundred nanograms of total RNA extracted for retina tissue using Nucleospin RNA plus kit (Macherey-Nagel) was subjected to 2-step RT-qPCR using PrimeScript™ RT Master Mix and SYBR® Premix Ex Taq™ (Takara, Saint-Germain-en-Laye, France) following the manufacturer’s instructions. Primers (Additional file 1: Table S2) were ordered from Eurogentec (Angers, France). Melting temperature was determined for each sample, and the expression of the genes of interest was normalized to those of Tbp and Hprt genes using BioRad CFX Maestro software.

Protein analysesProtein extraction and Western Blot/Wes Simple Protein analyses

Nitrogen-frozen retina samples were homogenized in RIPA lysis buffer complemented by phenylmethanesulfonyl fluoride 1% (Sigma), Sodium Orthovanadate 1% (Sigma), and Phosphatase Inhibitor Cocktail 0,5% (Roche). The protein concentration was determined using the BCA Protein Assay kit (Interchim) following the provider’s instructions. Considering the weak amount of total protein from the retina, analysis was performed using the Protein Simple Wes system (ProteinSimple, USA) or otherwise using classic western-blot experiments (in the case of failed compatibility between the used antibody and Wes). Western blot experiments were performed as described [38]. The membranes were incubated with Rhodopsin (#MAB5356, Merck Millipore) and RBPMS (#GTX118619, GENETEX) antibodies at 4 °C overnight. Peroxidase-labeled anti-rabbit (711-035-152, Interchim) or anti-mouse (715-035-152, Interchim) secondary antibodies were used at a 1:5000 dilution. The total amount of proteins per lane was normalized using α-tubulin (ab185067, Abcam), and densitometry analysis of the band intensity was achieved using ImageJ v1.53. The Wes automated capillary-based size sorting system (ProteinSimple, USA) was used for the analysis of protein expression. 0,4ug/uL of total protein was loaded to WES 25-well plates for separation (Protein Simple, USA) following provider instructions. Primary antibodies of MTR (#25,896-1-AP, Protein Tech), OPSIN (#AB5405, Millipore), and PAX6 (#ab5790, Abcam) were diluted at 1:75. The total amount of proteins per lane was normalized using α-tubulin (#2144, Cell Signaling). HRP-labeled anti-rabbit (ProteinSimple, USA) secondary antibodies were used at the provided concentration. The relative amount of each protein was analyzed through the areas under peaks from the chemiluminescence chromatograms by the Compass for SW software v6.0.0 (ProteinSimple, USA) with a virtual blot presentation.

Statistical analyses

Statistical analyses were performed under GraphPad Prism V10.0.2. Continuous variables and densitometry analyses were reported as means ± SEM. To analyze the data, the Mtr-cKO groups were compared using Student’s unpaired t test after confirming that the data followed Gaussian distributions and exhibited equal variances. Statistical significance was determined with a threshold of p value below 0.05, indicating significant differences between the compared groups.