Single-Cell Analysis Reveals a Hair Follicle Dermal Niche Molecular Differentiation Trajectory that Begins Prior to Morphogenesis

Abstract

Summary: Delineating molecular and cellular events that precede appendage morphogenesis has been challenging due to the inability to distinguish quantitative molecular differences between cells that lack histological distinction. The hair follicle (HF) dermal condensate (DC) is a cluster of cells critical for HF development and regeneration. Events that presage emergence of this distinctive population are poorly understood. Using unbiased single-cell RNA sequencing and in vivo methods, we infer a sequence of transcriptional states through which DC cells pass that begins prior to HF morphogenesis. Our data indicate that Wnt/β-catenin signaling is required to progress into an intermediate stage that precedes quiescence and differentiation. Further, we provide evidence that quiescent DC cells are recent progeny of selectively proliferating cells present prior to morphogenesis and that are later identified in the peri-DC zone during DC expansion. Together, these findings provide an inferred path of molecular states that lead to DC cell differentiation.

An updated classification of hair follicle morphogenesis

“…Critically, single‐cell RNA sequencing has allowed for a more dynamic understanding of developmentally associated transcriptional dynamics, through the use of computational modelling, as well as identification of cellular heterogeneity. Since pseudotemporal ordering of cell fates in the dermis allowed for revealing a putative differentiation trajectory from upper dermal fibroblasts to pre‐DC to Stage 3 and 4 DC, it is conceivable that cognate analyses of epidermal differentiation will add in the future increased temporal resolution and, in combination with dermal differentiation analyses, will shed light on critical signalling crosstalk. Through observations of in vivo phenomena in genetic abrogation studies, substantial insights into the staging of early HF morphogenesis have been made, to drive understanding of signalling necessity.…”

“…Knockout of the epidermal transcription factor ΔNp63 prevents expression of Wnt target genes in early epidermal progenitors and subsequent HF formation, further confirming the important role of epidermis‐derived Wnts and broad dermal Wnt signalling activity in pre‐Pc fate specification and HF induction. Interestingly, recent single‐cell RNA sequencing analyses suggest that upper dermal fibroblasts are transitioning towards DC fate specification at this early induction stage prior to morphogenesis …”

“…In contrast to the other fibroblast‐type cells in the mesenchyme, including fibroblasts that will transition into pre‐DC, pre‐DC cells are already post‐mitotic, indicating that acquisition of DC fate is concomitant with the shutdown of the cell cycle machinery . Like the pre‐Pc, there are high levels of Wnt signalling activity in pre‐DC, when compared to other dermal fibroblasts; in fact, Wnt activation is necessary for acquisition of DC fate and DC formation . Pre‐DC cells, ranging from 1 to ~15‐20 cells, are randomly dispersed among dermal fibroblasts and located right below pre‐Pc cells (separated by the basement membrane), which by contrast form a contiguous unit (Figure ).…”

“…Pre‐DC cells, ranging from 1 to ~15‐20 cells, are randomly dispersed among dermal fibroblasts and located right below pre‐Pc cells (separated by the basement membrane), which by contrast form a contiguous unit (Figure ). Pre‐DC can be discerned by de novo expression of Foxd1 and Sox2, as well as by upregulation of Tbx18 and highest expression of pandermal fibroblast marker Twist2 , which begins ramping up expression prior to acquisition of DC fate among upper dermal fibroblasts (Figure ). Foxd1, Sox2, Tbx18, Bmp4, Bmp7, Hhip and Fgf10 have been previously identified as highly expressed signature genes in the aggregated DC (Figure 5).…”

“…Such advances have permitted the definition of molecular signatures of Pc and DC, and neighbouring cell types, as well as identified migration as the main cellular mechanism of Pc and DC formation . They have also allowed for the discovery of precursors to the Pc (pre‐Pc), multipotent fibroblasts that give rise to the DC and the fated precursors of DC (pre‐DC), by their molecular properties and prior to identifiable changes in cell morphologies and arrangement. Finally, they have enabled identification of suprabasal SOX9 + precursors to HF stem cells after placode formation …”