We performed three sets of RNAscope stainings as described in Method Details; Staining (1) Islr, Aqp4, Gdf10 (2) Mfge8, Aqp4, Agt (3) Slc6a9, Slc6a11, Agt. Full sagittal brain sections were scanned and stitched to a large image as described above. To quantify expression of the relevant markers (Figure 3F), we processed the images as follows. In order to allow overlap of spatial information from multiple images we aligned the three images using a set of 16 reference points. We manually registered these points using the DAPI channel as guide for the general anatomy. Images were transformed into the common coordinates using affine transform (MATLAB, “fitgeotrans” function) on the reference points and pixels outside the relevant domain were set to zero. The steps for image processing were as follows: (1) Enhance each channel between percentile 50 to 100. (2) Calculate background using “adapttresh” function MATLAB. (3) Subtract the background. (4) Re-enhance with gene specific parameter. (5) To obtain the RNAscope spots binary image, calculate extended maxima transform (“imextendemax” MATLAB), followed by fill holes (“imfill”) and remove all objects larger than 50
