Sonic hedgehog (Shh) signaling plays a critical role in hair follicle development and skin cancer, but how it controls these processes remains unclear. Of the three Gli transcription factors involved in transducing Shh signals in vertebrates, we demonstrate here that Gli2 is the key mediator of Shh responses in skin. Similar to Shh −/− mice, Gli2 −/− mutants exhibit an arrest in hair follicle development with reduced cell proliferation and Shh-responsive gene expression, but grossly normal epidermal differentiation. By transgenic rescue experiments, we show that epidermal Gli2 function alone is sufficient to restore hair follicle development in Gli2 −/− skin. Furthermore, only a constitutively active form of Gli2, but not wild-type Gli2, can activate Shh-responsive gene expression and promote cell proliferation in Shh −/− skin. These observations indicate that Shh-dependent Gli2 activator function in the epidermis is essential for hair follicle development. Our data also reveal that Gli2 mediates the mitogenic effects of Shh by transcriptional activation of cyclin D1 and cyclin D2 in the developing hair follicles. Together, our results suggest that Shh-dependent Gli2 activation plays a critical role in epithelial homeostasis by promoting proliferation through the transcriptional control of cell cycle regulators.[Keywords: Shh; Gli2; hair follicle; mitogen; D-type cyclins] Supplemental material is available at http://www.genesdev.org.

Natural extracellular matrix (ECM) can regulate the interactions between cells and ligands by exhibiting heterogeneous nano-sequences periodically displaying adhesive ligands, such as RGD ligand in vivo. [1,2] Cell-adhesive ECM proteins, such as fibronectin, vitronectin, and collagen, were shown to form periodically sequenced RGD ligand-bearing nanostructures (67-100 nm). [1] Periodic structure in reflectance was also observed from native tissues. [2] The ligation of integrin with adhesive ligand mediates the assembly of cytoskeletal actin filaments and focal adhesion (FA) complexes to activate mechanosensing signaling pathways that can regulate cellular differentiation. [3,4] Strategically developing materials with heterogeneously sequenced ligand nanostructures can emulate ECM [5] microenvironment to help elucidate the interactions between cells and nano-ligands with tunable frequency and sequences. This can effectively regulate diverse cellular adhesion and functionality in vivo, such as FA, mechanosensing, and differentiation of stem cells. [6] The native extracellular matrix (ECM) can exhibit heterogeneous nanosequences periodically displaying ligands to regulate complex cell-material interactions in vivo. Herein, an ECM-emulating heterogeneous barcoding system, including ligand-bearing Au and ligand-free Fe nano-segments, is developed to independently present tunable frequency and sequences in nano-segments of cell-adhesive RGD ligand. Specifically, similar exposed surface areas of total Fe and Au nano-segments are designed. Fe segments are used for substrate coupling of nanobarcodes and as ligand-free nanosegments and Au segments for ligand coating while maintaining both nanoscale (local) and macroscale (total) ligand density constant in all groups. Low nano-ligand frequency in the same sequences and terminally sequenced nano-ligands at the same frequency independently facilitate focal adhesion and mechanosensing of stem cells, which are collectively effective both in vitro and in vivo, thereby inducing stem cell differentiation. The Fe/RGD-Au nanobarcode implants exhibit high stability and no local and systemic toxicity in various tissues and organs in vivo. This work sheds novel insight into designing biomaterials with heterogeneous nano-ligand sequences at terminal sides and/or low frequency to facilitate cellular adhesion. Tuning the electrodeposition conditions can allow synthesis of unlimited combinations of ligand nano-sequences and frequencies, magnetic elements, and bioactive ligands to remotely regulate numerous host cells in vivo.

In the crystallisation of nanomaterials, an assembly-based mechanism termed ‘oriented attachment’ (OA) has recently been recognised as an alternative mechanism of crystal growth that cannot be explained by the classical theory. However, attachment alignment during OA is not currently tuneable because its mechanism is poorly understood. Here, we identify the crystallographic disorder-order transitions in the OA of magnetite (Fe3O4) mesocrystals depending on the types of organic surface ligands on the building blocks, which produce different grain structures. We find that alignment variations induced by different surface ligands are guided by surface energy anisotropy reduction and surface deformation. Further, we determine the effects of alignment-dependent magnetic interactions between building blocks on the global magnetic properties of mesocrystals and their chains. These results revisit the driving force of OA and provide an approach for chemically controlling the crystallographic order in colloidal nanocrystalline materials directly related to grain engineering.

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Micro/nanotopographies (MNTs) have been reported to enhance the osseointegration of biomaterials and modulate cell functions, but the underlying mechanisms are incompletely understood. We hypothesized that transient receptor potential vanilloid 4 (TRPV4) may mediate the topographically induced osteoblastic differentiation of bone marrow stem cells (BMSCs) by regulating the NFATc1 and Wnt/β-catenin signaling. To test this hypothesis, murine BMSCs were cultured on polished titanium (Ti) discs (PT) and Ti discs carrying titania nanotubes (i.e., MNTs) with diameters of ∼30 and ∼100 nm (termed TNT-30 and TNT-100, respectively). It was found that the MNTs (in particular TNT-100) promoted the expression and activation of TRPV4. Inhibition of TRPV4 in BMSCs cultured on TNT-100 reduced the expression of osteoblastic genes and the gene expression and protein levels of NFATc1 and Wnt3a/β-catenin and also decreased nuclear translocation of NFATc1 and β-catenin (all vs uninhibited BMSCs). Conversely, activation of TRPV4 in BMSCs cultured on PT increased the expression of the osteoblastic gene and the gene expression and protein level of NFATc1 and Wnt3a/β-catenin and also enhanced the nuclear translocation of NFATc1 and β-catenin (all vs unactivated BMSCs). These differences suggest that the MNTs promoted TRPV4 expression and activation to enhance intracellular Ca2+, which further increased the nuclear translocation of NFATc1 and stimulated the Wnt/β-catenin signaling, thus leading to upregulated expression of osteoblastic genes. These results indicate TRPV4 to be a mediator in MNT-induced osteoblastic differentiation of BMSCs.