To investigate how loss of cdon affects the patterning of the eye we have generated cdon mutant lines using the CRISPR/Cas9 technology. The general morphology of the mutants appeared normal and no evident eye defects were found. However, cdon-/- mutants showed a reduction in the expression levels of pax2, a gene essential for patterning of the eye. Despite this decrease, the expression domain of the optic stalk was expanded, indicating that Cdon is required to limit the distal region of the eye, well in line with previous work from the host lab. Furthermore, the mutant embryos were more sensitive to Hh signaling inhibitors and showed changes in the expression levels of gli genes, Hh downstream targets, suggesting that the absence of Cdon impairs Hh signaling. To test if compensation by related genes was responsible for the mild phenotype observed in the generated cdon-/- mutants, we carried out qPCR and ISH analysis of boc and gas1 in cdon-/- embryos and injected guide RNAs against both genes in cdon-/- background. Crossing cdon-/- and boc+/- (available at the host lab) mutant lines was also performed. There was no up-regulation or changes in the expression patterns of boc in cdon-/- mutants and boc inactivation had no consequences on the cdon-/- phenotype. In contrast, the expression of gas1 was up-regulated and with a wider expression domain but we did not succeeded in proving that this up-regulation could compensate cdon function because the selected guide RNAs against gas1 were rather inefficient and did not lead to gene inactivation. To visualize Shh protein in vivo and determine the role that Cdon has on the dispersion of Hh protein we planned to generate a zebrafish shh:kaede knock-in line. Despite the use of different strategies and tools, we did not succeeded in generating the corresponding zebrafish line. To circumvent this problem, we turned to the medaka fish. We attempted to generate a shh:gfp knock-in using the reported biotin 5’ ends donor vector modification strategy. We are currently genotyping the injected generation to identify founders. In parallel, we are also generating gas1 and cdon mutants in medaka fish to pursue our aim. To visualize Cdon-Hh interaction, we planned to use BiFC assay and to outcross shh:kaede line to cdon-/- mutant. We have generated Venus-Shh and Venus-Cdon fusion proteins and evaluated VN-Shh – VN-Cdon, VN-Shh – VC-Cdon, VC-Shh – VN-Cdon and VC-Shh – VC-cdon interactions. Unfortunately, we could not establish the best combination of Shh/Venus and Cdon/Venus fusion proteins nor generate the shh:kaede knock-in in zebrafish, as already mentioned. We hope that the use of the medaka fish lines will provide the right tools to address this question.