Primitive Myeloid Leukocytes Formation and Their Roles in Epimorphic Regeneration in Zebrafish

In vertebrates, macrophages and neutrophils are major myeloid lineages essential for host defense, embryo- and organo-genesis, tissue homeostasis and regeneration. Yet, the molecular basis that dictates the cell fate of macrophage and neutrophil during ontogeny and their precise actions during tissue regeneration remain undefined.;In the first part of the thesis, the role of interferon regulatory factor (IRF) 8, a transcription factor of IRF family, in zebrafish primitive myelopoiesis was investigated. Whole-mount in situ hybridization (WISH) revealed that during zebrafish embryogenesis irf8 was expressed specifically in macrophages but not neutrophils. Suppression of Irf8 function in zebrafish by antisense morpholino oligonucleotides (MO) caused a depletion of macrophages and an enhanced output of neutrophils, without affecting the overall number, proliferation and survival of primitive myeloid cells. On the other hand, overexpression of Irf8 in zebrafish promoted macrophage formation at the expense of neutrophil development. These findings demonstrate that Irf8 is a critical determinant for neutrophil versus macrophage fate choice during zebrafish primitive myelopoiesis.;In the second part of the thesis, the in vivo actions of macrophages and neutrophils during zebrafish tail fin regeneration were analyzed in Tg(coro1a:eGFP/lyz:Dsred) transgenic fish, in which macrophages and neutrophils were marked by distinct fluorescent colors. Upon tail fin amputation, macrophages and neutrophils responded immediately and mobilized toward the lesion, but neutrophils with a faster motility. As a result, within the first 6 hours post amputation (hpa), the inflammation stage, neutrophils were the primary cells that scavenged the dead cell debris. The activated neutrophils had a limited phagocytosis activity and quickly underwent cell death. In the resolution stage, from 6-14 hpa, macrophages became the dominant scavengers, removing the cell debris and apoptotic cells with a remarkable efficiency and capacity, to resolve the local inflammation. Finally, in the regeneration stage, the filtrated macrophages evolved into elongating appearance and continuously invigilated the lesion to facilitate the regrowth of the damaged tissue. In accordance with their behaviors, ablation of macrophages severely impaired the inflammation resolution and the subsequent regeneration, resulting in the formation of large vacuoles in the regenerated fins. Our study documents the distinctive behaviors and functions of macrophages and neutrophils during tissue regeneration.