The first objective of this proposal was to identify the contribution of the RPE in optic cup folding both in zebrafish and eye organoids.
First, I established the culture of several cell lines and got trained in different techniques for transgenesis and mutageneisis in cells with the organization of a workshop in our institute. This will allow me the generation of eye organoids from mouse ESCs in the near future.
Second, by using super resolution microscopy and cell tracking and shape pipelines, I defined cell trajectories and morphology changes of prospective retinal and pigmented cells in the wild type zebrafish eye. Now I am comparing features with YAP mutant eyes to identify mechanical defects that might explain the lack of optic cup folding in these mutants. I generated dominant-negative (dn) YAP constructs, both in a plasmid valid for direct expression experiments and transgenic fish carrying dn-YAP downstream of the UAS promoter that will provide me the higher penetrance of the phenotype required for these analysis.
Moreover I developed force maps of the zebrafish optic cup folding using the Beta-actin_Myosin:GFP transgenic line. I used these force maps as a reference to conduct laser ablation experiments to measure the relevance of rigidity of the cells from different compartments. I will use this information to compare relevance of the RPE cells in YAP mutants, and confirm the YAP-mediated cell tension contribution during optic cup folding.
The second objective of this proposal was to study the convergence of Wnt and YAP signaling
First I analyzed the spatiotemporal dynamics of Wnt and YAP signaling using reporter lines. Due to temporal incongruences with the results obtained using YAP reporter lines provided by a different lab, I decided to generate my own line and designed and alternative strategy using RNA-seq data from our lab. The earliest TF expressed during RPE differentiation belong to the YAP and Wnt pathways, which are tead3b and tcf12, respectively. Using double fluorescent in situ hybridization, I confirmed that both YAP and Wnt signaling pathways are active in the same cells very early during RPE differentiation, and I will use them as reliable readouts.
Second, we set up the use of the Wnt inhibitor IWR-1, the Wnt activator BIO and the YAP inhibitor Verteporfin in the context of optic cup folding. I am using them to manipulate Wnt and YAP signaling and confirm whether Wnt signaling is required for YAP activation and viceversa.
Third, I analyzed the consequences of blocking the LINC complex during optic cup folding by injecting dn-Nesprins forms in zebrafish embryos. Interestingly, this phenocopies the lack of YAP activity in the mutants, supporting the mechanical-dependent activation of YAP in the eye. To circumvent the limitation of the different ranges of phenotypic intensity obtained after injection of dn-Nesprins, I developed transgenic fish lines carrying egfp-nesprin1/2-kash downstream of UAS. Crossing these fish with bhlhe40:gal4ff fish, will allow me to carry out tissue-specific and uniform inhibition of Nesprins, valid for the RNA- and ATAC-seq experiments planned.
Finally, I establish a new tool to interfere with tension in a localized manner, by transferring single beads soaked in the the mechanical cytoskeleton inhibitor Rockout.
