In vivo detection of programmed cell death during mouse heart development

Kristel Martínez-Lagunas,Yoshifumi Yamaguchi,Marco C Deruiter,Caroline Geisen,Bernd K Fleischmann,Masayuki Miura,Michael Hesse,Cora Becker

doi:10.1038/s41418-019-0426-2

Abstract

Despite the great progress on the cell biology of programmed cell death (PCD), its incidence and exact time course during embryonic and particular heart development are still unclear. This is also due to the lack of models enabling to directly identify and monitor PCD cells at different time points in vivo. Herein we report generation of transgenic murine embryonic stem cell and mouse models expressing secreted Annexin V-YFP under control of the CAG promoter. This enables to visualize and quantify PCD in vitro and in vivo during embryonic development. At early embryonic stages we found Annexin V-YFP+ fluorescent cells in known areas of PCD, such as the otic ring and at the site of neural tube closing, underscoring its specificity for detection of PCD. We have focused our detailed analysis primarily on PCD in the embryonic heart for a better understanding of its role during development. Our findings reveal that PCD peaks at early stages of cardiogenesis (E9.5–E13.5) and strongly decreases thereafter. Moreover, the PCD cells in the heart are predominantly cardiomyocytes, and an unexpected area of prominent cardiac PCD are the ventricular trabeculae (E9.5–E14.5). Thus, the sA5-YFP mouse line provides novel insight into the incidence and relevance of cardiac PCD during embryonic development ex- and in vivo.

Highlights

The reporter consists of a CAG-sA5-yellow fluorescent protein (YFP) expression cassette [9] (Supplementary Fig. 1a), in which the ubiquitous CAG promoter drives the expression of a fusion protein consisting of a secretion signal peptide(s), the human Annexin V (A5) and YFP
A major challenge in the field of programmed cell death (PCD) is its accurate detection, quantification, and observation over time ex- and in vivo, in the mammalian heart [38, 39]. It is still not clear, where and to which extent PCD occurs during embryonic development. We met this challenge by establishing a genetic PCD detection system in transgenic mice consisting of ubiquitously expressed sA5 fused to YFP
Since cleaved caspase 3 (cCasp3) is a key marker of early–intermediate [27, 28], and terminal dUTP nick end labeling (TUNEL) of late PCD our data suggest that based on its overlap with these two markers sA5-YFP labels early–intermediate PCD stages

Summary

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The Annexin V-based assay is one of the most sensitive techniques to detect exposure of phosphatidylserine (PS) residues in the outer membrane of cells undergoing PCD in vitro, ex vivo, and in vivo [8, 9] This method detects cells in various phases of the PCD cascade ranging from the early phase prior to morphological nuclear changes until the late phase [10]. This technique faces limitations, when used in vivo, strongly limiting this tool for its in vivo usage [5]. We have validated the functionality of the CAG-sA5-YFP system as a PCD marker in vitro and in vivo, and used it to provide detailed insight into extent and localization of PCD in the developing mouse heart

Methods

Results

Discussion

Compliance with ethical standards