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molecular states and instructive signals occurring at the single cell level be examined to fully grasp how
individual cells traverse the differentiation cascade as well as discern how specific cell states can re-emerge
in breast cancer and contribute to metastasis.

A NEW VIEW OF THE MAMMARY HIERARCHY
Single-cell sequencing has emerged as a powerful tool capable of uncovering the transcriptional heterogeneity
of diverse cell types within a tissue . Although transcriptome profiling of single cells was reported over
[47]
20 years ago [48,49] , only recently has it become a widely utilized technique. Technological advances in areas
such as microfluidics and in situ barcoding have allowed for high throughput, cost effective sequencing of
hundreds to thousands of cells within a given sample . Furthermore, the ability to analyze large numbers
[50]
of cells at high resolution has mandated the development of innovative approaches for downstream data
analysis. Software tools have recently become available to infer evolutionary history and lineage relationships
within heterogeneous cell populations .
[51]
Recently, several independent laboratories have utilized scRNA-seq to identify epithelial subpopulations
and their differentiation trajectory within the mammary gland [11-14] . Together these studies have examined
the complete mammary developmental cycle encompassing embryonic through adult stages and pregnancy
through post-weaning. The transcriptional landscape generated from these studies has provided critical
insight into epithelial differentiation, transforming the way in which we view the mammary epithelial
hierarchy. The new model emerging from these studies emphasizes the fluid and gradual progression of
epithelial differentiation and reveals a greater degree of cellular heterogeneity than had previously been
acknowledged.

SINGLE-CELL SEQUENCING OF THE MAMMARY EPITHELIUM
Prior to birth, sc-RNA sequencing has shown that the epithelium consists of a continuum of primitive
cellular states in which unique subpopulations could not be detected . This supports previous findings
[12]
generated from gene expression analysis of bulk fMaSCs, demonstrating that fMaSCs are comprised of
a single CD24+CD49f+ population . During embryogenesis, the majority of mammary epithelial cells
[32]
exhibit dual expression of both luminal and myoepithelial signature genes [12,32,52] . This hybrid gene expression
signature is associated with multipotent potential and a loss of dual lineage expression as cells become
restricted to unipotent potential .
[52]
At birth, many mammary epithelial cells maintain a hybrid gene expression profile . This dual lineage
[12]
signature may include rare bipotent progenitor populations previously observed by lineage tracing
studies [28,38] . However, the majority of this population likely represents cells spanning various transitional
stages of lineage restriction. Cells become increasingly more lineage restricted throughout puberty; however,
the precise timing of the major bifurcation event between luminal and basal cells remains unclear. In work
by Pal et al. , scRNA-sequencing of mammary epithelium from 2 (pre-puberty), 5 (puberty), and 10 (adult)
[53]
week-old mice demonstrated that before puberty, the mammary epithelial cell populations are largely
homogeneous, expressing a basal (myoepithelial)-like gene signature. Upon puberty, a fraction of cells
upregulate expression of luminal genes such as Epcam, and Keratins 8, 18, and 19, suggesting that bifurcation
initiates at this time . However, Giraddi et al. , specifically assessed changes in the early postnatal
[12]
[53]
epithelium by examining glands from mice at day zero (PN0) and day 4 (PN4) after birth. In contrast to the
report by Pal and colleagues, they found that two distinct cellular populations, one containing myoepithelial
cells and the other containing luminal precursors, begin to emerge as early as PN4. While these studies
confirm that the mammary epithelium remains in a primitive state until postnatal stages during which
lineage restriction commences, the specific timing of those commitments remains unclear.