Eased CD86 and MHC class II expression, indicating that these DC were capable of maturation (data not shown). Langerin expression by cultured EpCAM+ cells was low as in comparison with freshly isolated epidermal LC. QRTPCR revealed a rise in Langerin mRNA expression by cultured LC-like cells over the first 72 hours. Accordingly, flow cytometry revealed a peak in intracellular Langerin protein expression following 96 hours (Figure 1c). The number of LC-like cells per properly decreased after 120 hours. in vitro effects of Wnt signaling modulators on LC-like cells To investigate the involvement of Wnt signaling in LC development, we initially characterized effects of Wnt protein and also the Wnt antagonist Dkk1 on the improvement ofJ Invest Dermatol. Author manuscript; readily available in PMC 2012 March 01.Becker et al.Pagemurine LC-like DC in C57BL/6 bone marrow cultures. Initial dose response studies revealed maximal effects of Wnt3A and Dkk1 at 100 ng/ml and 1000 ng/ml respectively (information not shown). Addition of Wnt3A (100 ng/ml), that is recognized to activate the Wnt/-catenin signaling pathway (Kishida et al., 1999), into bone marrow cultures resulted in modest increases within the numbers of LC-like DC that were recovered following 72 hours ( 33 ; p0.05, Figure 2a). In contrast, the potent Wnt inhibitor Dkk1 (1000 ng/ml) decreased the number of LC-like cells accumulating in cultures that were not supplemented with Wnt3A protein ( 21 , p0.05, Figure 2a). Total leukocyte numbers, determined at 72 hours, did not adjust drastically in the presence of Wnt3A or Dkk1 (Figure 2b). These final results indicate that Wnt3A includes a modest selective impact on the development of LC-like cells in vitro, and suggest that small amounts of endogenous Wnt proteins may possibly be present and active in bone marrow cultures. Influence of intraepidermal Wnt signaling on LC in vivo To assess probable effects of Wnt signaling on LC improvement in situ, we initially characterized LC within the epidermis of K5-rtTA; tetO-Dkk1 DT mice (Supplemental Figure 1). Keratinocytes in these mice produce the Wnt inhibitor Dkk1 after exposure to doxycycline (Chu et al., 2004). Dkk1 was induced within the skin of young mice by feeding doxycycline to nursing mothers beginning on postnatal day 0 (P0). This approach avoids the limb and dental defects that would result from earlier exposure of creating mice to Dkk1 (Chu et al., 2004). Due to a lack of availability from the DT mice, we performed subsequent research with K14-KRM1; K5-rtTA; tetO-Dkk1 TT mice. In TT mice, the Wnt inhibiting impact of Dkk1 is potentiated in keratinocytes by the extra expression of KRM1 in K14 expressing cells. Direct effects of Dkk1 on LC or LC precursors are expected to become identical in DT and TT mice. LC precursors enter murine skin soon soon after epidermal differentiation is completed and undergo a massive burst of proliferation amongst postnatal days 2 (P2) and 7 (P7), reaching “adult” numbers inside the first two weeks following birth. (Chang-Rodriguez et al., 2004; Chang-Rodriguez et al., 2005; Chorro et al., 2009; Elbe-Burger and Schuster, 2010; Toll-like Receptor 6 Proteins web Kobayashi et al., 1987; Tripp et al., 2004). Thus, it was anticipated that an effect of Wnt inhibition by Dkk1 would be evident before P14 if Wnt proteins were involved in LC improvement. Dkk1 induction resulted in an apparent Nuclear Receptor Subfamily 4 Group A Member 2 Proteins Source physique size and hair phenotype. DT and TT mice have been smaller and had much less terminal hair than their littermate controls. This confirms that administration of doxycycline to nursing mothers.