Periodic Reporting for period 1 - MechaPattern (Coordination between cell identity, tissue mechanics and proportionate patterning during vertebrate eye formation)
Período documentado: 2019-04-01 hasta 2021-03-31
The generation of organs depends on the integration of different sources of information. The most relevant ones are cell identity information given by signaling genetic pathways and the inherent mechanical information of individual cells and tissues. In addition, these different inputs must be coordinated in a way that the organ is scaled and patterned proportionally to their final size. Although some of these aspects have been extensively studied independently, a comprehensive analysis about how they are integrated is missing.
The first aim of this proposal is to understand the coordination between these interdependent sources of morphogenetic cues using as a paradigm the development of the vertebrate eye.
Eye formation is described as a two-step process; first, the evagination of the eye progenitors to form the optic vesicles, and second, the infolding of the tissue into bi-layered optic cups. Two main territories are specified within the vertebrate optic cup: the inner layer, called neuroretina (NR) and the surrounding outer layer, the retinal pigmented epithelium (RPE). Decades of research have demonstrated that the development of a functional eye requires the interplay of different genetic programs, which are mostly driven by signaling pathways, together with physical forces and extracellular matrix mechanics. One of the biggest challenges in the field is to find coordination hubs between signaling genes that control cell fate decisions and mechanical properties of the tissue that drive cell collectives movements and ultimately control organ 3D shape. YAP, the Hippo nuclear transducer, is the perfect candidate to mediate this coordination, since it is able to sense and infer tissue mechanical inputs and it interacts with multiple signaling pathways.
These mechano-patterning interplay and scaling-invariant properties are needed to grant plasticity and adaptability to organs and tissues, essential attributes for processes such as homeostasis or regeneration. Therefore, this understanding about organ formation from a comprehensive morphogenetic and proportionate patterning perspective is required to establish the biological groundwork for next generation regenerative medicine. To contribute to this innovative field, I plan to compare properties of in vivo models of eye formation with in vitro 3D eye organoids generated from mouse Embryonic Stem Cells (mESCs). The second aim of the proposal is to analyze the maintenance of this morphogenetic and mechanical coordination in these organoids grown up outside the organism.
The first aim of this proposal is to understand the coordination between these interdependent sources of morphogenetic cues using as a paradigm the development of the vertebrate eye.
Eye formation is described as a two-step process; first, the evagination of the eye progenitors to form the optic vesicles, and second, the infolding of the tissue into bi-layered optic cups. Two main territories are specified within the vertebrate optic cup: the inner layer, called neuroretina (NR) and the surrounding outer layer, the retinal pigmented epithelium (RPE). Decades of research have demonstrated that the development of a functional eye requires the interplay of different genetic programs, which are mostly driven by signaling pathways, together with physical forces and extracellular matrix mechanics. One of the biggest challenges in the field is to find coordination hubs between signaling genes that control cell fate decisions and mechanical properties of the tissue that drive cell collectives movements and ultimately control organ 3D shape. YAP, the Hippo nuclear transducer, is the perfect candidate to mediate this coordination, since it is able to sense and infer tissue mechanical inputs and it interacts with multiple signaling pathways.
These mechano-patterning interplay and scaling-invariant properties are needed to grant plasticity and adaptability to organs and tissues, essential attributes for processes such as homeostasis or regeneration. Therefore, this understanding about organ formation from a comprehensive morphogenetic and proportionate patterning perspective is required to establish the biological groundwork for next generation regenerative medicine. To contribute to this innovative field, I plan to compare properties of in vivo models of eye formation with in vitro 3D eye organoids generated from mouse Embryonic Stem Cells (mESCs). The second aim of the proposal is to analyze the maintenance of this morphogenetic and mechanical coordination in these organoids grown up outside the organism.
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.
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.
The pillar of the greatest advances in biomedicine is to understand fundamental biological processes. This project will significantly advance the understanding in organ formation from new perspectives and aims to complement the available knowledge on stem cell differentiation, to ultimately establish the foundation for next generation regenerative medicine based on differentiating embryonic stem cells for tissue replacement.
The development of this research line will come with numerous socio-economic benefits. In terms of economical growth, it will generate many work opportunities for scientific students and experts with the promotion of a Unit specialized on Organoids and Biophysics in our institute. Moreover, I believe it has a great potential in the development of new biological devices for the use in regenerative medicine, which will incorporate the participation of public and private Biotech companies, and possibly, the generation of profitable patents.
Finally, the benefits for Society of this proposal are without question. The creation of knowledge and the improvement of the generation of organs in vitro will ultimately make possible the treatment of patients who can not be treated by standard organ transplantation due to the unavoidable shortage of donors and organ incompatibilities.
The development of this research line will come with numerous socio-economic benefits. In terms of economical growth, it will generate many work opportunities for scientific students and experts with the promotion of a Unit specialized on Organoids and Biophysics in our institute. Moreover, I believe it has a great potential in the development of new biological devices for the use in regenerative medicine, which will incorporate the participation of public and private Biotech companies, and possibly, the generation of profitable patents.
Finally, the benefits for Society of this proposal are without question. The creation of knowledge and the improvement of the generation of organs in vitro will ultimately make possible the treatment of patients who can not be treated by standard organ transplantation due to the unavoidable shortage of donors and organ incompatibilities.