Establishment of a transgenic system for in vivo detection of apoptosis in the developing heart

Abstract

All living organisms face death since the beginning of their lives. Cells commit suicide to sculpt correctly all organs during animal development. Currently, there exist at least 13 cell death modalities through which a cell is programmed to die. From these, the most studied is apoptosis. Despite the huge progress made in the field of apoptosis, little is known about its role in mouse heart development and it is still not clear if its upregulation or downregulation can lead to heart disease. One of the main reasons for this uncertainty is the lack of techniques that mark apoptotic cells at different time points in vivo and in fixed tissue. With a specific and broad detection method that overcomes the rapidity in which apoptotic cells die and disappear from tissues, it should be possible to confirm if apoptosis takes place during mouse heart development, in which areas and at which specific rates occurs during different embryonic stages and which are the cells that undergo apoptosis during heart morphogenesis. <br /> In this work, I addressed these issues by establishing a genetically encoded reporter tool used to visualize and quantify apoptosis during mouse heart development in vivo. This system is based on the detection of phosphatidylserine (PS) residues—exposed in the outer membrane of apoptotic cells—by Annexin V (A5). Commercial probes of A5 linked to fluorescent probes have been developed. These probes provide excellent results in cells, but have some limitations such as high cost and not-specific for fixed tissue, when used in fixed tissue or in living organisms by systemic injection. Therefore, this system was aimed to overcome these drawbacks by ubiquitously expressing a secreted human Annexin V (sA5) fused to the yellow fluorescent protein (YFP). The ubiquitous expression was ensured by the chicken β-actin promoter with CMV enhancer element (CAG). As cells undergo apoptosis, sA5-YFP binds with high affinity to its PS residues resulting in bright fluorescent signals. In this way, it was possible to better characterize, quantify and monitor apoptotic signals in the developing mouse heart over time.<br /> In summary, this study consisted of two major goals. The first one was to validate the sA5-YFP system, in vitro and in vivo, to specifically label apoptotic cells. The second goal was to use this system to describe the apoptotic patterns and rates during all stages of embryonic heart development in more detail. For the first part, I could show that the labeled sA5-YFP signals in transgenic mESCs, in EBs, and in sA5-YFP mice were apoptotic cells after inducing apoptosis ectopically or in entities (EBs and embryonic tissue such as neural tube, <br /> otic placode and yolk sac) in which apoptosis is a physiological process. These cells presented the typical morphological and biochemical changes of apoptotic cells. They rounded up, detached from the culture plate or tissue compound, formed membrane blebs or fragmented, and displayed condensed chromatin. Moreover, the sA5-YFP protein co-localized with active forms of pro-caspases (casp-8), effector caspases (active casp-3 and 7) and with TUNEL staining. Thus, I proved that the sA5-YFP system allows the visualization of cells and blebs (fragments of apoptotic cells) in different stages (early-intermediate-late) of apoptosis. <br /> For the second goal, I used the transgenic CAG-sA5-YFP mouse line to provide detailed information about apoptosis rates, distribution of apoptotic events and cells undergoing apoptosis during mouse heart development. I could show that apoptosis takes place at all stages of the mouse heart development starting from E9.5. I observed than less than 1.5% of the cells in the embryonic heart undergo physiological apoptosis and the apoptosis rates decrease as the heart ages. In contrast to earlier studies, apoptotic cells were observed very clearly in the primitive ventricle at E9.5 and in the lining of the trabeculae of both ventricles from E10.5 - E14.5 in sA5-YFP hearts. These events coincide with episodes of trabeculae formation, differentiation, remodeling, and compaction, suggesting that apoptosis might be necessary for chamber maturation. In addition, I noticed apoptotic events in the outflow tract, the interventricular septum, and atria, coinciding with previous reports. Lastly, I demonstrated that the labeled sA5-YFP apoptotic cells, found in the trabeculae, co-localize mainly with cardiomyocytes around days E13.5 - E14.5 of mouse heart development. These findings imply a role of cardiomyocyte apoptosis in ventricular differentiation. However, more experiments are needed to undercover the biological role of apoptosis in trabeculae formation. <br /> Last but not least, I provided evidence that the CAG-sA5-YFP mouse model enables the real-time imaging of induced apoptosis and physiological occurring apoptosis in the developing mouse embryo at E8.75 and in the embryonic mouse heart at E9. Apoptotic signals can be identified by their roundish morphology and bright sA5-YFP⁺ signals. Fluorescence intensity of sA5-YFP can be used as quantitative parameter to evaluate changes in apoptosis in vivo. This means that this reporter line can be used to monitor the distribution and morphological changes of apoptotic cells within specific tissues over time in living organisms. In conclusion, the sA5-YFP mouse has the potential to unravel the physiological role of apoptosis in the developing heart, its role in the formation of other mammalian organs, and in diseases displaying aberrant patterns of apoptosis.