Redefining the structure of the hair follicle by 3D clonal analysis.

The hair follicle (HF) is a multi-tissue mini-organ that self-renews periodically. However, the cellular organisation of this much-studied model is not fully understood. The structures of the outer layer and of the bulb, which ensures HF growth, have not been completely established. To clarify these points, we have conducted in vivo clonal analyses with 3D imaging in mice. The upper two-thirds of the HF outer layer consists of two clonally unrelated groups of cells that exhibit different modes of growth. They correspond to the basal outer root sheath (ORS) and the companion layer (Cp). The basal ORS has an unusual anisotropic mode of growth from a suprabulbar zone, which we named the privileged proliferation zone. The Cp has a stem/transient-amplifying mode of growth and is shown to be an HF internal structure. Furthermore, we describe an additional element, the bulb outer layer, which is contiguous and shares markers (e.g. Lgr5) with the basal ORS but is formed by a separate lineage that belongs neither to the ORS nor Cp lineage. It represents a novel element with proximal cells that are contiguous with the germinative layer in the bulb. In reference to its shape and position we named it the lower proximal cup (LPC). These clonal hierarchies reveal a novel model of HF organisation and growth based on two major entities: the basal ORS and the LPC plus the seven internal layers.