RNA-seq of WT 23hpf zebra fish embryo heads treated with DMSO (control), Dasatinib 150uM, IWR1 25uM, YODA1 10uM, Rockout 100uM or CaliculinA 0.1uM.

The folding of the optic cup during embryonic development is a highly mechanic process. In our laboratory we want to demonstrate how the Retinal Pigmented Epithelium (RPE) cells are essential for the correct morphogenesis of the eye. Our experimental workflow has shown that the hippo and B-catenin pathways are involved in the acquisition of the particular geometry of the RPE cells and that this is what drives the proper folding of the optic cup. The aim of this RNA-seq is to analyse how transcription is affected in the zebra fish embryos after disturbing mechanosensitive pathways that alter RPE cells morphology and, therefore, optic cup folding. Dasatinib (150uM), IWR1 (25uM) and Rockout (100uM) are inhibitory drugs of the hippo pathway, Wnt pathway and tension respectively. Yoda1 (10uM) y an agonist of the mechanosensitive Piezo1 calcium channel, and CaliculynA (0.1uM) increase tension and was used as a positive control. WT zebra fish embryos were collected in 1 cell stage and incubated at 28ºC until 7hpf. Embryos were dechorionated with a pronase solution. Then, 50 embryos were added to each plate with E3 medium and the proper concentration of the corresponding drug. Embryos were incubated at 25ºC until the next morning. When the optic cups of the control condition (DMSO) were folded, heads of 10 embryos of each drug treatment were cut and added to a RNAse free Eppendorf and snap-freeze in liquid Nitrogen. We collected 3 biological replicates for each drug treatment. RNA extraction was done following the manufacturer instructions of the RNeasy Mini Kit from QIAGEN, Cat no. 74104. Extracted RNA eluted in 15uL Nuclease-free H2O was sent for library preparation and sequencing to Novogene. This enterprise creates RNA libraries through polyA capture and reverse transcription into cDNA, followed by quality assessment. 12G of sequencing was performed in 150 bp paired-end sequencing using Illumina PE150 technology.