EKLF/Klf1 expression specifies a unique macrophage subset during mouse erythropoiesis

ABSTRACT: Purpose: We characterized the role of EKLF in erythroblast island macrophages in E13.5 mouse fetal liver by analysing the transcriptomes of F4/80+ macrophages from WT and EKLF-/- by RNA-Seq. In addition we analyzed the transcriptomes of F4/80+ fetal liver macrophages from an EKLF/GFP mouse, where GFP acts as a surrogate for EKLF expression, to determine the genes enriched in EKLF/GFP+ macrophages compared to EKLF/GFP- macrophages where EKLF is not expressed. Finally, we used single cell RNA sequencing to resolve the heterogeneous population of F4/80+ macrophages from WT. Methods: For RNA-Seq from WT and EKLF-/- F4/80+ macrophages were FACS sorted from primary E13.5 mouse fetal livers and RNA was isolated using the Trizol method. F4/80+ macrophages were also FACS sorted from the EKLF/GFP mouse and the GFP+ and GFP- populations were collected. The GFP+ population had low cell numbers and therefore RNA was isolated using an Agilent RNA Nanoprep kit. For single cell sequencing, E13.5 fetal livers were stained with F4/80-PE antibody and the F4/80-PE+ cells were purified using an EZ-Sep PE selection kit (mouse). 25,000 cells were submitted for single cell sequencing for the Chromium V3 platform. Library preparation was done using the standard Illumina Truseq workflow, and libraries were sequenced in a Hiseq 2500 for WT and EKLF-/-, or Novaseq for EKLF/GFP and single cell sequencing. Results: We found that 1954 genes are differentially expressed in EKLF-/- F4/80+ macrophages vs WT and 2330 genes are differentially enriched in EKLF/GFP+ F4/80+ macrophages using DESeq2. Of these, 504 are common and constitute the EKLF-dependent gene expression program in F4/80+ fetal liver macrophages. We resolved the F4/80+ WT macrophages into 13 clusters based on gene expression and find that 23% of the total F4/80+ cells express EKLF. Conclusions: We find that the F4/80+ fetal liver macrophage are a unique cell type. We identify the EKLF-dependent gene expression program in these macrophages and determine an important transcription circuit governed by EKLF that constitute cell cycle and other Klf and Sp family transcription factors. Single cell sequencing showed a highly heterogeneous population of macrophages with activated macrophage as well as erythro-myeloid characteristics. Based on the expression of EKLF, we identified specific cell surface markers for EKLF+ F4/80+ macrophages.

ORGANISM(S): Mus musculus

PROVIDER: GSE156153 | GEO | 2021/03/16

REPOSITORIES: GEO