Defining the Biomechanics of the Developing Heart through High-Speed Dynamic Fluorescent Imaging in Transgenic Quail Embryos

Final Report Summary - DYNIMHEART (Defining the Biomechanics of the Developing Heart through High-Speed Dynamic Fluorescent Imaging in Transgenic Quail Embryos)

The objectives of this proposal are organized in three main milestones:
1. To learn how to keep avian embryos (quail) alive under incubated microscopes for up to 48h, image them and reconstruct 3D images. It is also intended to find and image, molecular partners involved during heart morphogenesis, specifically Eph-ephrin receptors.
2. To generate molecular tools fluorescently tagged involved in the formation of endothelial and myocardial layers.
3. To generate specific software that help us track cells automatically during the developmental window we are imaging.

Description of the work performed since the beginning of the project
The main milestones achieved during the project are:
• In vivo imaging of quail embryos: Tg(tie1:H2B-YFP);Tg(pgk:H2B-chFP)) double Tg quail embryos permit all cells to be tracked and the ECs and endocardial cells to be separated and distinguished from other cells based on their spectral signatures using multispectral imaging approaches.
• Eph activation imaging through clustering: Eph ephrin are interesting candidates to study the separation of tissues. We have cloned different versions of this constructs, including a green version, EphB2-GFP; A yellow version, EphB2-YFP; A blue version, EphB2-CFP; and a red monomeric version EphB2-mRuby
• Eph activation imaging through tyrosine kinase indicators: novel indicators that specifically recognize the phosphorylated receptor and have a shift in the intensity of their signal have been used to report dynamically the activation of the Eph-ephrin pathway.
• Lentivirus production: we have generated several viruses such as pLenti:CAG::EphB2-GFP, pLenti:CAG::EphB2-m Ruby to generate transgenic animals with the proteins of interest fused to fluorescent reporters.

The main milestones achieved during the last year of the project are:
• Developing a new imaging tool for the study of protein dynamics and protein aggregation.
• Generation of stable cell lines expressing truncated or mutated versions of fluorescent EphB2
• Developing a new protocol for presentation of ligands and live cell imaging using TIRF microscopy.

Description of the main results achieved so far
• Dynamic imaging of Eph-ephrin activation. We have developed 2 different protocols to image the dynamic activation of one of the main important pathways involved in tissue boundary formation. We can capture with high level of resolution the aggregation of receptors and use that as a hallmark to detect in real time, not only the expression of the protein, but the moment when it gets activated.
• Production of lentiviruses suitable for trangenesis. Based on the previous result we have inserted the same constructs into lentiviral vectors that will allow us to transition from imaging cells in culture towards direct imaging of live embryos.

Last year update
• Enhanced Number & Brightness (eN&B): a new tool for the live imaging of cells. We have modified the N&B technique and give it higher temporal resolution and deeper pixel depth. By detrending photobleaching we can acquire time lapse series for more than one hour. Also, by resampling the acquisitions, we can compute distributions –rather than simply average values- of molecular species contained in a single pixel.
• A nucleated polymerization model for the activation of the Eph receptor. We propose a model where the big cluster formation of the EphB2 receptor is done through two mechanisms, namely: 1. Nucleation+chain polymerization is the process by which the ligand brings together 2 Eph molecules (initial nucleation) and monomers bind the nucleus making the complex grow. 2. Condensation is the mechanism by which two cluster merge and give rise a higher order cluster. Activation of the receptor takes place only during the step 1.

The expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).
Tissues form during development due to a highly organized and precise spatio-temporal regulation. Crucial players for the formation and maintenance of the integrity of tissues are the Eph receptors and their counterpart Ephrin. In the last few years a lot of progress has been done in imaging florescent constructs to determine, mainly, they expression in time and space. We have taken that approach one step forward and image the dynamics of the Eph receptor and develop a technique that can tell us, in living samples, when the pathway is getting activated. By doing so, we can take live imaging to a new, functional dimension. Imaging receptor activation will tell us, not only where the proteins are expressed, but at which moment during development they exert their function.

During this last year, we have developed a novel technique which we call enhanced Number and Brightness, building upon previous N&B protocol. The technique allows to image at an unprecedented level of resolution the aggregation of the EphB2 receptor in living cells. Receptor clustering is a direct consequence of ligand-induced activation. Implementing our technique in living embryos, which is not limited to EphB2 but to any protein that can be tagged fluorescently, will allow us to understand the key players involved in tissue separation and morphogenesis.

In conclusion, we provide to the scientific community with a novel tool to study morphogenesis. This was one of the main objectives of the implementation of this fellowship. One of our main goals was to learn from the vast know how at Fraser laboratory to image living embryos and cells, and bring exclusive technology to our European host lab.

Final Report Summary - DYNIMHEART (Defining the Biomechanics of the Developing Heart through High-Speed Dynamic Fluorescent Imaging in Transgenic Quail Embryos)

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