Hm560

For tissue sections, embryos/larvae were hand‐dechorionated when necessary and fixed in 4% paraformaldehyde in 0.1 M pH 7.4 phosphate buffer saline overnight at 4°C. After three washes in PBS, embryos were embedded in a solution containing 10% sucrose and 1.5% agarose. Blocks were cryoprotected in 30% sucrose in PBS overnight at 4°C. A total of 12 μm coronal sections were obtained in a cryostat (Thermo Scientific HM560).
For life imaging, embryos at the proper stage were anesthetized as usual and embedded in 1.5% low melting agarose (ThermoFisher Scientific R0801) in E3 medium.

Eye size was measured as described previously (Lind et al., 2014 ; Mitkus et al., 2018 (link)). The eye or head of a freshly dead bird was frozen at −80°C and sectioned horizontally in a cryotome (HM-560, Thermo Fisher Scientific, Waltham, MA, USA). Photographs (Canon EOS 500D camera with a Canon Ultrasonic 100 mm macro objective) of the head or eye block were taken every 100 or 150 μm, with a ruler at the same distance from the camera serving as a scale. We measured eye dimensions from the photograph featuring the largest pupil and longest lens path length, using ImageJ (Schneider, 2012 (link)). Eye size was measured as the axial length from the cornea apex to the back of the sclera. Corneal and lens thickness were measured along this line. For additional species, we used axial length measurements from Ritland (1982) .

A donor cornea was preserved and transported in Optisol-GS at 4°C, and was used within 11 days from preservation. The age of donor was 27 years old.
Fresh corneal tissues were embedded in optimal cutting temperature (OCT) compound and frozen sections were cut using a microtome-cryostat (HM560, Thermo Fisher Scientific Inc., Walldorf, Germany) in 10 μm. After drying for 30 minutes at room temperature, tissue sections were washed with Tris-buffered saline (TBS; Takara) 3 times, and incubated with TBS containing 5% donkey serum and 0.3% Triton X-100 for 1 hour to block non-specific reactions. Each section was then incubated with primary antibodies listed in Table 3 at 4°C overnight. Subsequently, slides were again washed with TBS 3 times, and incubated with a 1: 200 dilution of their respective Alexa Fluor 488-conjugated secondary antibodies (Life Technologies) and 2μg/mL Hoechst 33342 (#B2261, Sigma-Aldrich) for 2 hours at room temperature. The slides were mounted with a drop of Permafluor mountant (Thermo Scientific) to reduce photobleaching and observed by fluorescent microscopy (Axio Observer D1; Carl Zeiss Jena Gmbh, Jena Germany). For each of the primary antibodies, isotype specific rabbit IgG and goat IgG (#AB-105-C, #AB-108-C; R&D Systems, Minneapolis, MN) were used as negative controls at the same dilution as the primary antibodies.

Six-week-old C57BL/6JRj (Janvier, Genest Saint Isle, France) mice were euthanized by CO₂ administration and cervical dislocation. Eyes were removed and prepared as follows: a hole just behind the ora serrata was made, and the eyeballs were placed in 4% (w/v) PFA in 0.12 M phosphate buffer, pH 7.2 for 1 h at RT. The lens was removed, and the eyecups were fixed again for 20 min in 4% PFA and cryoprotected with increasing concentrations of ice-cold sucrose in 0.12 M phosphate buffer, pH 7.2 (10% for 1 h and 30% overnight). Subsequently, the eyecups were embedded in 7.5% gelatin, 10% sucrose 1 × PBS and frozen in a dry ice-cooled isopentane bath. Subsequently, 10-μm retinal sections were made with a cryostat (model HM560; Thermo Fisher; Microm, Walldorf, Germany) [18 (link)], mounted with a mounting medium (Mowiol preparation, Sigma) on glass slides (Super-Frost, Thermo Fisher Scientific, Waltham, MA, USA) and kept at −80 °C.