Macrophage-specific gene functions in Spi1-directed innate immunity.
The Spi1/Pu.1 transcription factor plays a crucial role in myeloid cell development in vertebrates. Despite extensive studies of Spi1, the controlled gene group remains largely unknown. To identify genes dependent on Spi1, we used a microarray strategy using a knockdown approach in zebrafish embryos combined with fluorescence-activated cell sorting of myeloid cells from transgenic embryos. This approach of using knockdowns with specific green fluorescent protein-marked cell types was highly successful in identifying macrophagespecific genes in Spi1-directed innate immunity. We found a gene group downregulated on spi1 knockdown, which is also enriched in fluorescence-activated cell-sorted embryonic myeloid cells of a spi1:GFP transgenic line. This gene group, representing putative myeloidspecific Spi1 target genes, contained all 5 previously identified Spi1-dependent zebrafish genes as well as a large set of novel immune-related genes. Colocalization studies with neutrophil and macrophage markers revealed that genes cxcr3.2, mpeg1, ptpn6, and mfap4 were expressed specifically in early embryonic macrophages. In a functional approach, we demonstrated that gene cxcr3.2, coding for chemokine receptor 3.2, is involved in macrophage migration to the site of bacterial infection. Therefore, based on our combined transcriptome analyses, we discovered novel early macrophage-specific marker genes, including a signal transducer pivotal for macrophage migration in the innate immune response.
Zakrzewska
Anna
A
Institute of Biology, Leiden University, Leiden, The Netherlands.
Cui
Chao
C
Stockhammer
Oliver W
OW
Benard
Erica L
EL
Spaink
Herman P
HP
Meijer
Annemarie H
AH
D000818:Animals; D030801:Animals, Genetically Modified; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D006410:Hematopoiesis / Q000235:genetics / Q000276:immunology; D007113:Immunity, Innate / Q000235:genetics*; D017403:In Situ Hybridization; D008264:Macrophages / Q000276:immunology* / Q000502:physiology; D020411:Oligonucleotide Array Sequence Analysis; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000276:immunology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D054367:Receptors, CXCR3 / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012480:Salmonella Infections / Q000235:genetics / Q000276:immunology; D012486:Salmonella typhimurium; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000276:immunology*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics / Q000276:immunology; D029961:Zebrafish Proteins / Q000235:genetics* / Q000276:immunology*; D000099232:Proto-Oncogene Protein Spi-1
D049452:Green Fluorescent Proteins; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D054367:Receptors, CXCR3; D011994:Recombinant Proteins; D015534:Trans-Activators; D029961:Zebrafish Proteins; D000099232:Proto-Oncogene Protein Spi-1