ERRATUM Development 137, 1775 (2010) doi:10.1242/dev.053116 Abnormal hair follicle development and altered cell fate of follicular keratinocytes in transgenic mice expressing Np63 Rose-Anne Romano, Kirsten Smalley, Song Liu and Satrajit Sinha There was a technical problem with the ePress version of Development 137, 1431-1439 published on 24 March 2010. A number of Greek symbols were missing from the text and figure headings in the Results section. A replacement ePress article was published on 31 March 2010. The online issue and print copy are correct. We apologise to authors and readers for this error. DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE 1431 Development 137, 1431-1439 (2010) doi:10.1242/dev.045427 © 2010. Published by The Company of Biologists Ltd Abnormal hair follicle development and altered cell fate of follicular keratinocytes in transgenic mice expressing DNp63a Rose-Anne Romano1, Kirsten Smalley1, Song Liu2,3 and Satrajit Sinha1,* SUMMARY The transcription factor p63 plays an essential role in epidermal morphogenesis. Animals lacking p63 fail to form many ectodermal organs, including the skin and hair follicles. Although the indispensable role of p63 in stratified epithelial skin development is well established, relatively little is known about this transcriptional regulator in directing hair follicle morphogenesis. Here, using specific antibodies, we have established the expression pattern of DNp63 in hair follicle development and cycling. DNp63 is expressed in the developing hair placode, whereas in mature hair its expression is restricted to the outer root sheath (ORS), matrix cells and to the stem cells of the hair follicle bulge. To investigate the role of DNp63 in hair follicle morphogenesis and cycling, we have utilized a Tet-inducible mouse model system with targeted expression of this isoform to the ORS of the hair follicle. DNp63 transgenic animals display dramatic defects in hair follicle development and cycling, eventually leading to severe hair loss. Strikingly, expression of DNp63 leads to a switch in cell fate of hair follicle keratinocytes, causing them to adopt an interfollicular epidermal (IFE) cell identity. Moreover, DNp63 transgenic animals exhibit a depleted hair follicle stem-cell niche, which further contributes to the overall cycling defects observed in the mutant animals. Finally, global transcriptome analysis of transgenic skin identified altered expression levels of crucial mediators of hair morphogenesis, including key members of the Wnt/b-catenin signaling pathway, which, in part, account for these effects. Our data provide evidence supporting a role for DNp63a in actively suppressing hair follicle differentiation and directing IFE cell lineage commitment. KEY WORDS: Differentiation, Hair follicle, Transcription, p63, Mouse INTRODUCTION continuous with the basal layer of the epidermis. After birth, by The multilayered stratified epithelium of the epidermis develops from postnatal day 16 (P16), the hair follicle reaches full maturation and a single layer of ectoderm progenitor cells through a tightly regulated enters a regressive state known as catagen. At this stage, the lower series of events during embryogenesis. In addition to forming the two thirds of the hair follicle undergoes rapid degeneration through epidermis, a subset of surface ectodermal cells develop into the a process involving apoptosis, where the dermal papilla comes to pilosebaceous units that include the hair follicle and sebaceous gland. rest just below the bulge region. The bulge is the permanent portion In mice, pelage hair follicle morphogenesis is initiated during of the hair follicle and serves as a reservoir for hair follicle stem cells embryonic development at E14.5 and is governed by a series of (Cotsarelis, 2006). It is during the resting telogen stage that the old inductive cues shared between keratinocytes committed to a hair hair shaft is shed from the hair canal. Following telogen, a new cycle follicle cell fate and mesenchymal cells of the underlying dermis of hair regeneration, anagen, is initiated. Not surprising, the complex (Fuchs, 2007). These signaling events result in the formation of local hair development program is governed by several conserved thickenings, or placodes, in the overlying epithelium (Hardy, 1992). signaling pathways including Wnt (Fuchs, 2007; Millar, 2002; Beneath the underlying epidermal thickenings of the placodes, Schneider et al., 2009). mesenchymal cells of the dermis localize, increase in density and Indeed, genetic studies have unequivocally demonstrated the form a cluster that later develops into specialized cells known as the Wnt/b-catenin signaling pathway to be crucial in regulating hair dermal papilla (DP), which are crucial for proliferation of hair follicle follicle morphogenesis. The importance of this pathway is illustrated matrix cells (Jahoda et al., 1984; Panteleyev et al., 2001; Paus et al., by experiments where the coordinate action of b-catenin or its 1999; Reynolds and Jahoda, 1992). nuclear mediators Tcf3 or Lef1 are blocked. Thus, in mice with Reciprocal mesenchymal-epithelial crosstalk continues during the conditional ablation of the Ctnnb1 gene (which encodes for b- early stage of development, stimulating rapid proliferation and catenin) or constitutive expression of the Wnt inhibitor Dkk1, hair down-growth of hair matrix cells to encase the DP. Subsequently, placode formation is severely compromised (Andl et al., 2002; matrix cells differentiate into specialized structures of the hair Huelsken et al., 2001). In agreement with these findings, Lef1-null follicle, which include the inner root sheath (IRS) and hair shaft animals lack hair, whereas transgenic animals that express a compartments of the hair follicle (Panteleyev et al., 2001). The IRS transcriptionally inactive form of Lef1 display altered differentiation is completely surrounded by an outer root sheath (ORS), which is and the reprogramming of hair follicle cell fate (Merrill et al., 2001; Niemann et al., 2002; van Genderen et al., 1994; Zhou et al., 1995). These studies have established a fundamental role for the Wnt/b- 1 2 Department of Biochemistry, and Department of Biostatistics, Center for Excellence catenin signaling pathway in the early epithelial-mesenchymal in Bioinformatics and Life Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA. 3Department of Biostatistics, Roswell Park Cancer Institute, Buffalo, events that specify hair follicle cell fate, initiate hair patterning and NY 14263, USA. direct hair follicle morphogenesis. As with most developmental programs, the signaling events tied to the maturation of hair follicles *Author for correspondence ([email protected]) are intimately associated with a gene expression program dictated Accepted 25 February 2010 by transcription factors. DEVELOPMENT 1432 RESEARCH ARTICLE Development 137 (9) The transcription factor p63, a member of the p53 family, plays by Dox chow withdrawal from the lactating mother. For triple transgenic an important role in the development of stratified epithelium of the experiments, DNp63aBG mice were mated to TOPGAL.lacZ mice, in the skin and its appendages. Mice with deletion of the Trp63 gene absence of Dox, to generate TOPGALDNp63aBG animals. exhibit severe developmental abnormalities including limb Western blot truncations and defects in skin epidermal stratification and Western blot analysis was performed as previously described (Romano et differentiation (Mills et al., 1999; Yang et al., 1999). Moreover, these al., 2009). Primary antibodies were used at the following dilutions: HA animals lack ectodermal organs such as teeth, hair follicles and (Roche, 1:5000), b-tubulin (Chemicon, 1:10000), Lef1 (Upstate, 1:2000), glandular structures. Although the p63 knockouts have provided b-catenin (Sigma 1:2000), K15 (Thermo Scientific, 1:2000), Gata3 (Santa valuable insights into understanding epidermal development, thus Cruz, 1:2000), RR-14 (1:5000) and Sox9 (Santa Cruz, 1:2000). far it has not been an ideal model system to study hair follicle morphogenesis. This is primarily owing to the severe developmental Immunostaining Stainings using paraffin embedded sections were performed as previously arrest observed in these animals, including a complete block in described (Romano et al., 2009). Primary antibodies used at the indicated placode formation. Another issue that has complicated studies of dilutions were DNp63 (RR-14; 1:50), p63 (1:50; Santa Cruz, 4A4), K5, K10, p63 is the existence of multiple p63 isoforms, each with potentially K6, loricrin and filaggrin (1:200; gift from Dr Julie Segre, NHGRI, distinct molecular properties. The Trp63 gene encodes for multiple Bethesda, USA), Sox9 (1:50; Santa Cruz), K15 (1:50; Thermo Scientific), functionally distinct protein isoforms, including TAp63 and DNp63, K17 (1:200; gift from Dr Pierre Coulombe, John Hopkins University, New which contain unique N-terminal segments that harbor independent York, USA), AE13/AE15 (1:10; gift from Dr T. T. Sun, New York activation properties (Helton et al., 2008; Yang et al., 1998). In University, New York, USA), Elf5 (1:50, Santa Cruz), Gata3 (1:50, Santa addition, both TA and DN transcripts are differentially spliced at the Cruz), b-catenin (1:100, Santa Cruz), Lef1 (1:100, Upstate), Ki67 (1:100, 3Ј end, generating proteins with unique C-termini designated as a, Novacastra), PCNA (1:50, Dako Cytomation) and S100A6 (1:100, Thermo b and g isoforms. The fact that all isoforms of p63 are absent in the