Most tissue-resident macrophage (RTM) populations are seeded by waves of embryonic hematopoiesis and are self-maintained independently of a bone marrow contribution during adulthood. A proportion of RTMs, however, is constantly replaced by blood monocytes, and their functions compared to embryonic RTMs remain unclear. The kinetics and extent of the contribution of circulating monocytes to RTM replacement during homeostasis, inflammation, and disease are highly debated. Here, we identified Ms4a3 as a specific gene expressed by granulocyte-monocyte progenitors (GMPs) and subsequently generated Ms4a3 TdT reporter, Ms4a3 Cre , and Ms4a3 CreERT2 fate-mapping models. These models traced efficiently monocytes and granulocytes, but no lymphocytes or tissue dendritic cells. Using these models, we precisely quantified the contribution of monocytes to the RTM pool during homeostasis and inflammation. The unambiguous identification of monocyte-derived cells will permit future studies of their function under any condition.

Most tissue-resident macrophage (RTM) populations are seeded by waves of embryonic hematopoiesis and are self-maintained independently of a bone-marrow contribution during adulthood. A proportion of RTMs, however, is constantly replaced by blood monocytes and their functions compared to embryonic RTM remains unclear. The kinetics and extent of the contribution of circulating monocytes to RTM replacement during homeostasis, inflammation and disease is highly debated. Here, we identified Ms4a3 as a specific marker expressed by granulocyte-monocyte progenitors (GMPs) and subsequently generated Ms4a3 TdT reporter and Ms4a3 Cre -Rosa TdT fate mapper models to follow monocytes and their progenies. Our Ms4a3 Cre -Rosa TdT model traced efficiently blood monocytes (97%) and granulocytes (100%), but no lymphocytes or tissue dendritic cells. Using this model, we precisely quantified the contribution of monocytes to the RTM pool during homeostasis and inflammation. The unambiguous identification of monocyte-derived cells will permit future studies of their function under any condition.

Summary Myeloid cells, including dendritic cells (DCs), granulocytes, monocytes, monocyte-derived cells and macrophages, are important players in the immune response, but their identification is not as clear as lymphocytes, especially in tissues. This protocol details the step-by-step procedure for the analysis of myeloid populations in various mouse tissues by flow cytometry. For complete details on the use and execution of this protocol, please refer to Liu et al. (2019) .

In this study, the mechanism by which GSK-3β regulates protein synthesis and lipid deposition was investigated in zebrafish (Danio rerio). The vector of pEGFP-N1-GSK-3β was constructed and injected into the muscle of zebrafish. It was found that the mRNA and protein expression of tuberous sclerosis complex 2 (TSC2) was significantly increased. However, the mRNA and protein expression of mammalian target of rapamycin (mTOR), p70 ribosomal S6 kinase 1 (S6K1), and 4E-binding protein 1 (4EBP1) was significantly decreased by the pEGFP-N1-GSK-3β vector in the muscle of zebrafish. In addition, the mRNA and protein expression of β-catenin, CCAAT/enhancer binding protein α (C/EBPα), and peroxisome proliferators-activated receptor γ (PPARγ) was significantly decreased, but the mRNA expression of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), ATP-citrate lyase (ACL), and HMG-CoA reductase (HMGCR) was significantly increased by the pEGFP-N1-GSK-3β vector. The activity of FAS, ACC, ACL, and HMGCR as well as the content of triglyceride (TG), total cholesterol (TC), and nonesterified fatty acids (NEFA) were significantly increased by the pEGFP-N1-GSK-3β vector in the muscle of zebrafish. The content of free amino acids Arg, Lys, His, Phe, Leu, Ile, Val, and Thr was significantly decreased by the pEGFP-N1-GSK-3β vector. The results indicate that GSK-3β may participate in regulating protein synthesis via TSC2/mTOR signaling and regulating lipid deposition via β-catenin in the muscle of zebrafish (Danio rerio).

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Apoptosis, also named programmed cell death, is a highly conserved physiological mechanism. Apoptosis plays crucial roles in many life processes, such as tissue development, organ formation, homeostasis maintenance, resistance against external aggression, and immune responses. Apoptosis is regulated by many genes, among which Apoptosis Inhibitor-5 (API5) is an effective inhibitor, though the structure of API5 is completely different from the other known Inhibitors Of Apoptosis Proteins (IAPs). Due to its high expression in many types of tumors, API5 has received extensive attention, and may be an effective target for cancer treatment. In order to comprehensively and systematically understand the biological roles of API5, we summarized the evolution and structure of API5 and its roles in anti-apoptosis in this review.