Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage.

Adam Balic,Adrian Sherman,Hazel Gilhooley,Pete Kaiser,Helen M Sang,Carla Garcia-Morales,David W Burt,David A Hume,Valerie Garceau,Lonneke Vervelde,Maria W Gutowska

doi:10.1242/dev.105593

Abstract

We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens.

Highlights

Macrophages participate in a wide range of processes during embryonic development and throughout life, including organogenesis and homeostasis, clearance of apoptotic cells, pathogen recognition, phagocytosis and destructions of pathogens, and antigen presentation (Pollard, 2009; Jones and Ricardo, 2013; Wynn et al, 2013)
Comparison of mammalian Fms intronic regulatory element (FIRE) with CNE3 in birds and reptiles identified several regions of ultra-conserved sequence (Fig. 1C). These ultra-conserved regions contain the precise binding sites of transcription factors AP1 and PU.1 that are occupied in the macrophage nucleus (Tagoh et al, 2002) and are required for macrophage lineage-specific transcription of Csf1r in mice (Fig. 1C,D) (Sauter et al, 2013)
To test the function of the candidate chicken FIRE sequence, we produced expression of green fluorescent protein (eGFP) reporter constructs containing the chicken colony stimulating factor 1 receptor (CSF1R) promoter region (Garceau et al, 2010) with or without the CNE3 region. eGFP expression was detected in stably transfected HD11 macrophage cells only when CNE3 was included, whereas no expression was detected in transfected DF-1 fibroblast cells

Summary

Introduction

Macrophages participate in a wide range of processes during embryonic development and throughout life, including organogenesis and homeostasis, clearance of apoptotic cells, pathogen recognition, phagocytosis and destructions of pathogens, and antigen presentation (Pollard, 2009; Jones and Ricardo, 2013; Wynn et al, 2013). The mononuclear phagocyte system in mammals is a family of cells derived from a shared progenitor, and includes blood monocytes, tissue macrophages and classical dendritic cells. These cells are found throughout the body and can be detected by immunocytochemical localisation of lineage-restricted surface markers (Hume, 2006). Chickens lack lymph nodes (McCorkle et al, 1979) and lymphoid tissues with equivalent function are difficult to visualise and isolate, which makes the isolation of cells and analysis of local immune responses challenging

Methods

Results

Conclusion