Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Lorea Iturri; Javier Saenz Coronilla; Yvan Lallemand; Elisa Gomez Perdiguero

doi:10.3791/55305

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Method Article

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

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DOI:

10.3791/55305

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July 17th, 2017

Lorea Iturri¹^,² , Javier Saenz Coronilla¹ , Yvan Lallemand¹ , Elisa Gomez Perdiguero¹

¹Department of Developmental and Stem Cell Biology, CNRS UMR3738, Department of Immunology, Institut Pasteur, ²Cellule Pasteur UPMC, University Pierre et Marie Curie

Summary

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Abstract

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

Introduction

There are several successive but overlapping waves of hematopoietic progenitors during development whose myeloid progeny remain into adulthood. First, unipotent "primitive" progenitors emerge in the mouse yolk sac ¹^,² between E7.5-E8.25 and give rise to embryonic macrophages without any monocytic intermediate. Whether macrophages derived from primitive progenitors persist in the adult brain as microglia remains a subject of active investigation. Second, Erythro-Myeloid Precursors (EMPs) arise in the yolk sac at E8.5, enter the bloodstream and colonize the embryo. EMPs emerge from the yolk sac hemogenic....