AP-2? is Required for Maintenance of Multipotent Mammary Stem Cells

Mammary gland ductal morphogenesis depends on the differentiation of mammary stem cells (MaSCs) into basal and luminal lineages. The AP-2? transcription factor, encoded by Tfap2c, has a central role in mammary gland development but its effect in mammary lineages and specifically MaSCs is largely unknown. Herein, we utilized an inducible, conditional knockout of Tfap2c to elucidate the role of AP-2? in maintenance and differentiation of MaSCs. Loss of AP-2? in the basal epithelium profoundly altered the transcriptomes and decreased the number of cells within several clusters of mammary epithelial cells, including adult MaSCs and luminal progenitors. AP-2? regulated the expression of genes known to be required for mammary development including Cebpb, Nfkbia, and Rspo1. As a result, AP-2?-deficient mice exhibited repressed mammary gland ductal outgrowth and inhibition of regenerative capacity. The findings demonstrate that AP-2? can regulate development of mammary gland structures potentially regulating maintenance and differentiation of multipotent MaSCs. Overall design: Tfap2c fl/fl / Krt5-Cre mice were treated with either intraperitoneal corn oil or 4OHT for one week at four weeks postnatally determine effect of conditional knockout of Tfap2c on mammary stem cells. The fourth mammary glands of the mice were harvested at 9 weeks age. Two mice of each condition were submitted for sequencing using the manufacturer recommended Chromium (10x Genomics) and Illumina technologies. The amplified cDNA was constructed into 3' expression libraries and pooled together in separate lanes of a 150 base-pair, paired-end chemistry flow cell. The Illumina HiSeq 4000 was used with Agilent Bioanalyzer, and average library sizes ranged from 329 bp to 432 bp in length. The adapter sequence of the library was 124 bases in length. Basecalls were converted into FASTQ files by the University of Iowa Genomics Division using the Illumina bcl2fastq software. These samples captured an estimated 3,546 to 5,545 number of cells with mean reads per cell ranging from 63,842 to 94,776. Transcripts were mapped using Cell Ranger Version 3.0.1 and mm10 reference genome (GRCm38.91).