Understanding how to induce skin regeneration instead of scarring will have broad implications clinically and cosmetically (Walmsley et al., 2015b). One of the main characteristics of scars is the absence of hair follicles, indicating that their regeneration in a wound may be a critical step in achieving scar-less skin repair (Yang and Cotsarelis, 2010). Interestingly, human embryonic skin has the capacity to regenerate without scars (Lo et al., 2012). Similarly, neonatal and adult mouse skin has the capacity to regenerate small non-functional hair follicles under specific conditions (Figure 1c–d; Ito et al., 2007; Rognoni et al., 2016; Telerman et al., 2017). These insights have prompted efforts in the field to define the molecular triggers that promote hair development in skin, with the ultimate goal of devising a way to regenerate fully functional hairs in adult skin wounds as a therapeutic modality (Yang and Cotsarelis, 2010).
Human and mouse skin are similar in their overall structural complexity, indicating that mouse skin can be a useful model to study skin development and wound repair (Chen et al., 2013). Murine hair follicle and skin development primarily occurs between embryonic day 12.5 (E12.5) to post-natal day 21 (P21) (Müller-Röver et al., 2001). During this time, different fibroblast lineages are established that respond to the changes in the environment to support hair follicle and skin development (Driskell et al., 2013; Jiang et al., 2018; Rinkevich et al., 2015; Rognoni et al., 2016). Fibroblasts that support hair follicle development differentiate from the papillary fibroblast lineage, into dermal papilla (DP), dermal sheath (DS), and arrector pili (AP) cells (Driskell et al., 2013; Plikus et al., 2017). Reticular fibroblasts, which cannot differentiate into papillary fibroblast lineages, secrete extra-cellular-matrix (ECM) and form adipocytes (Driskell et al., 2013; Schmidt and Horsley, 2013). By post-natal day 2 (P2) fibroblast heterogeneity is fully established with the presence of the defined layers of the dermis (Figure 1a; Driskell et al., 2013). Skin maturation occurs after P2 with the formation of the AP and the completion of the first hair follicle cycle, which results in the loss of a defined papillary fibroblast layer (Figure 1b; Driskell et al., 2013; Rognoni et al., 2016; Salzer et al., 2018). We have previously shown that papillary fibroblasts are the primary source of de-novo dermal papilla during skin development, which are required for hair formation (Driskell et al., 2013). Furthermore, it has been suggested that adult murine skin form scars due to the lack of a defined papillary layer (Driskell et al., 2013; Driskell and Watt, 2015). Consequently, expanding this fibroblast layer in adult skin might support skin regeneration in adult mice.
The use of scRNA-seq in the murine skin has established useful cell atlases of the skin during development and homeostasis (Guerrero-Juarez et al., 2019; Haensel et al., 2020; Joost et al., 2020; Joost et al., 2018; Joost et al., 2016; Mok et al., 2019). In addition, scRNA-seq studies investigating wound healing have so far focused on comparing scarring, non-scarring, or regenerating conditions (Guerrero-Juarez et al., 2019; Haensel et al., 2020; Joost et al., 2018). These studies have helped to identified key markers of the newly discovered skin fascia, including Gpx3, Plac8, and Mest, which recently has been shown to contribute to scar formation (Correa-Gallegos et al., 2019; Grachtchouk et al., 2000; Joost et al., 2020). These, scRNA-seq studies have revealed that transgenic activation of the Shh pathway in the alpha-smooth-actin cells in scarring wounds, which includes pericytes, blood vessels, and myofibroblasts, can support small non-functional hair regeneration (Lim et al., 2018). However, activation of Shh pathway in dermal fibroblasts is associated with malignant phenotypes and may perturb development and homeostasis such that it may not be a safe target to support human skin regeneration clinically (Fan et al., 1997; Grachtchouk et al., 2000; Oro et al., 1997; Sun et al., 2020). Altogether, these findings suggest that an overall comparison of developing, homeostatic, scarring, and regenerating skin conditions will yield important discoveries for molecular factors that can safely support skin regeneration without harmful side effects.
The Wnt signaling pathway is involved in regulating development, wound healing, disease and cancer (Nusse and Clevers, 2017). Studies that activated Wnt and beta-catenin in skin have led to important discoveries for wound healing but have produced contrasting results in the context of fibroblast biology and hair follicle formation (Chen et al., 2012; Enshell-Seijffers et al., 2010; Hamburg-Shields et al., 2015; Lim et al., 2018; Rognoni et al., 2016). Wnts are a group of secreted protein that activates a cascade of events that stabilizes nuclear beta-catenin, which operates as a powerful co-transcription factor of gene expression. Importantly, beta-catenin alone cannot activate the expression of Wnt target genes without co-transcription factors. There are four Wnt co-transcription factors Tcf7 (Tcf1), Lef1, Tcf3 (Tcf7l1), and Tcf4 (Tcf7l2). These co-transcription factors modulate the functional outcome of Wnt signaling by binding to different target genes (Adam et al., 2018; Nguyen et al., 2009; Yu et al., 2012). In the context of wound healing and regeneration, it is not known how differential expression of Tcf/Lef can modulate fibroblast activity via Wnt signaling.
Since it has been shown that embryonic and neonatal skin have the potential to regenerate hair follicles upon wounding (Hu et al., 2018; Rognoni et al., 2016), we set out to identify the cell types and molecular factors that define this ability in order to transfer this regenerative potential to adult tissue. Our work has identified Lef1, as the factor in fibroblast of developing skin, that can transform adult tissue to regenerate.