Intracellular genetic tracing reveals that cardiac endothelium does not have hematopoietic potential during embryonic development

Cardiac resident macrophages play an essential role in the development, homeostasis, repair and regeneration of the heart. However, the developmental origins of cardiac macrophages remain controversial. During development, hematopoietic cells are generated at several sites, including the yolk sac, aorta, and the heart tube. Defining the origin of hematopoietic cells is made difficult due to their mobile nature. As a result, there has been controversy surrounding the origins of tissue-resident macrophages, as it remains unclear whether these cells arise in situ or whether they depend on an upstream hematopoietic stem cell precursor. Recently, Shigeta and colleagues1 documented that the endocardium of the developing heart represents the hemogenic endothelium site for transient definitive hematopoiesis that contributes to cardiac macrophages, which were essential for valvular remodeling in the developing heart. To define the origin of macrophages present in the heart tube during development, the authors used cardiac explants devoid of circulating cells, thus allowing examination of hematopoietic cells originating only from the cardiac tube. They then tested the ability of the cardiac endothelium to produce macrophages using the Nfatc1-Cre line, which specifically labels cells of the endocardium. Based on their findings, the authors concluded that tissue-resident macrophages in the heart tube arise directly from the endocardium, and that this new population is specifically required for valvular remodeling during development. Additionally, the same group has also reported that the Nkx2-5-labeled endocardium represents a transient definitive hematopoietic site for generating circulating blood cells during endocardium-to-mesenchyme transition.

Because the conclusion of the recent studies was not strongly supported by previous genetic tracing studies, given the non-specific nature of conventional Cre-loxP tracing tools, Bin Zhou and colleagues used an intracellular labelling system based on the advantage of the synthetic Notch (synNotch) system that permanently traces the cell fate of target cells after cell contact in vivo2. SynNotch receptors provide extraordinary flexibility in engineering cells with customized sensing/response behaviors to user-specified extracellular cues3. In this study, the synNotch ligand of sender cells was constructed by replacing the Notch ligand with a membrane-attached green fluorescent protein (mGFP). The synNotch receptor of receiver cells was generated by replacing the intracellular domains with the tetracycline (tet) transactivator (tTA), while retaining the transmembrane domain for recognition by gamma-secretase. To permanently trace the cell fate of receiver cells after contact, the authors combined the synNotch tool with the Cre-loxP system that further drive tetO-Cre and Rosa26tdTomato reporters. Thus, contact between sender and receiver cells causes the nuclear translocation of tTA to activate the tetO promoter which subsequently induces Cre-loxP recombination to label receiver cells with tdTomato. Thus, they used this system to specifically label the endocardium and re-investigate its hemogenic potential during development. Intercellular genetic tracing data show that the endocardium of the developing heart is not hemogenic and does not contribute any cardiac macrophages or circulating blood cells during embryogenesis. In conclusion, this cell contact-based system coupled with Cre-lox recombination enabled genetic tracing at a particular anatomical location, thus enriching the genetic tools that can be used for cell fate mapping.