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Figure 3—figure supplement 1.

Segmentation of the extracellular spaces with Ilastik to study corneal stroma organization.1. An enucleated eye was placed with the cornea facing down on a glass-bottom microwell dish.2. A z-stack of confocal optical sections was acquired between the corneal epithelium and the lens epithelium, and then deconvoluted with AutoQuant X3. 3. z-sections corresponding to the area of interest (Anterior or Posterior) were selected. 4. After uploading the z-sections in Ilastik, a pixel classification was done to train the classifier to recognize pixels belonging to the cells or to the extracellular spaces. For this, a trained user manually labeled pixels with a different color for each class: cells were labeled in yellow and extracellular spaces in red. Boxed region indicates the area shown at higher magnification below. Various examples of each class were given to the classifier to optimize the segmentation. Quality of the segmentation was checked with the prediction by a trained user. 5. Segmented masks were then exported and the quantification of the amount and the average size of extracellular spaces were performed using Fiji.Figure 7—figure supplement 1.

Live imaging of the cornea boundary area.Enucleated eye was placed facing down in a glass-bottom microwell dish. The observed field was centered on the major arterial circle (yellow line). A z-stack was acquired from the corneal epithelium to the presumptive iris by confocal microscopy (49 to 109 μm per stack). and then deconvoluted with AutoQuant X3. z-sections corresponding to the area of interest were selected. The major arterial circle (yellow dotted line) is the reference to define the boundary between the cornea and the sclera. Quantifications of the corneal area covered by blood vessels and the percentage of corneal and stromal thickness with blood vessels were done with Fiji on the maximum intensity projections. MAC, major arterial circle.