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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.

The hair follicle (HF) grows during the anagen phase from precursors in the matrix that give rise to each differentiated HF layer. Little is known about the lineal relationship between these layer-restricted precursors and HF stem cells. To understand how the HF stem cells regenerate the typical anagen organization, we conducted in vivo clonal analysis of key stages of the HF cycle in mice. Unexpectedly, we found that the pool of HF stem cells contains precursors with both multipotent and restricted contributions. This implies that the lineal relationships between HF stem cells (persisting during telogen) and layer-restricted precursors (in the germinative layer), responsible for HF elongation during anagen, are not stereotyped. Formation of the matrix at each cycle is accompanied by the transient expansion of an intermediary pool of precursors at the origin of the germinative layer and by the progressive restriction of cell dispersion. The regionalization of clonal patterns within the outer HF structure (the outer root sheath) suggests that the position of the precursors might be a crucial factor in determining their fate. The presence of HF stem cells with multipotent contribution and the progressive segregation of HF lineages upon anagen activation indicate that each HF renewal cycle constitutes an authentic morphogenetic process. A comprehensive model was constructed based on the different clonal patterns observed. In this model, the positions of the precursors relative to the dermal papilla together with the progressive restriction of cell dispersion are part of the mechanism that restricts their contribution to the different HF lineages.