Cutaneous and Hair Development

        230 Loss of nuclear membrane protein SUN2 leads to transient alopecia and hair follicle structure defects

        R Stewart,1A Zubek,2 K Rosowski,3 S Schreiner,1 M King1 and V Horsley3,21 Cell Biology, Yale School of Medicine, New Haven, CT, 2 Dermatology, Yale School of Medicine, New Haven, CT and 3 Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT
        The goal of this study is to investigate the role of nuclear membrane proteins of the SUN (Sad1p, Unc-84) family in hair follicle development. Cytoskeletal proteins, cell adhesion proteins, and nucleoskeletal proteins (e.g. keratins, cadherins, lamins, respectively) are known to play critical roles in cutaneous and hair follicle integrity, development and homeostasis. These molecules can regulate cell behavior by scaffolding signaling molecules and allowing changes in physical tension throughout the cell to guide organelle position and shape and modulate cell-cell adhesion and migration during skin development and wound healing. The role of the nuclear membrane proteins that link the cytoskeleton and nucleoskeleton (i.e. SUN proteins and nesprins) has not been previously reported in skin. Here we show that transgenic knockout (KO) of SUN2 leads to dramatically altered hair follicle structure and alopecia during murine anagen I. However, the follicles recover normal structure and the alopecia resolves during the second hair cycle. Ultrastructural study of the SUN2 KO hair follicle bulbs demonstrates widen intercellular spaces and altered number of desmosomes between the cells of the inner root sheath (IRS) and the outer root sheath (ORS). Microscopically, the SUN2 KO follicle morphology ranges from distortion to dramatic fragmentation in the suprabulbar region extending to the infundibulum of the follicle. Lineage tracing studies show delayed progenitor cell upward migration in follicles lacking SUN2 suggesting defective hair outgrowth. Upregulation of SUN1 expression during anagen II leads to functional hair growth recovery, suggesting compensation by SUN isoforms. This study demonstrates an essential role for SUN proteins in hair follicle structural integrity and growth, and suggests an important role of physical force transduction between cell surface and nucleus in hair structure and function.

        231 Apical cues induce asymmetric division of epidermal basal cells through PIP3-PDK1 pathway

        T Dainichi,1,2 MS Hayden,2,3 Y Miyachi,1 K Kabashima1 and S Ghosh21 Department of Dermatology, Kyoto University, Kyoto, Japan, 2 Department of Microbiology & Immunology, Columbia University College of Physicians & Surgeons, New York, NY and 3 Department of Dermatology, Columbia University College of Physicians & Surgeons, New York, NY
        Tissues with different cell types are generated from common progenitors through asymmetric cell division (ACD). The differentiation of the epidermis begins with the basal progenitor cells, and ACD in a perpendicular orientation relative to the basement membrane promotes cell differentiation and organizes the stratified epithelium. However, both the molecular cues that trigger organization of the apical complex during ACD and the signaling pathways that drive activation of apical complex components remain to be defined. Phosphoinositide dependent kinase 1 (PDK1) is a serine/threonine kinase, and its activity depends on phosphatidyl inositol 3-kinase (PI-3 kinase) signaling pathways from growth factor receptors and adhesion molecules. We generated mice lacking PDK1 in keratinocytes (PDK1CKO), which display severe defects in epithelial differentiation and stratification resulting in perinatal lethality. Thus, PDK1 is essential for the development of stratified epidermis. The aim of this study is to address the mechanism of PDK1-dependent development of epidermis and identify its cues. ACD in basal cells in E17.5 epidermis from PDK1CKO was significantly decreased compared to wild-type epidermis (P = 0.0003) while symmetric cell division was not affected. Cell-cell contact stimuli induce production of PIP3 at the apical side of basal cells. In wild-type epidermis PDK1 asymmetrically localizes to the apical side of dividing basal cells (P = 1.5E-13). PDK1 recruits and activates atypical protein kinase C (aPKC), and nucleates formation of the apical complex. PDK1CKO keratinocytes do not undergo calcium-induced activation of aPKC or differentiation in vitro or in vivo. Thus PDK1 regulates both activation and spatial organization of key signaling pathways in response to apical cues acting on basal progenitor cells in developing epidermis.

        232 β-catenin activation regulates epithelial growth non-cell autonomously within the hair follicle stem cell niche

        P Myung,1 E Deschene,2 P Rompolas,2 G Zito,2 T Yang,2 M Taketo,3 I Saotome2 and V Greco2,11 Dermatology, Yale University, New Haven, CT, 2 Genetics, Yale Stem Cell Center, Yale Cancer Center, Yale University, New Haven, CT and 3 Pharmacology, Kyoto University, Kyoto, Japan
        Wnt/β-catenin signaling is critical for tissue regeneration and cancer. However, it is unclear how Wnt/β-catenin controls stem cell behaviors to coordinate tissue growth. Using in vivo time lapse imaging, we show that genetic activation of β-catenin specifically within hair follicle stem cells generates new organized axes of hair growth through oriented cell divisions and coordinated cellular displacement. Additionally, β-catenin activation is sufficient to induce growth within the stem cell pool independently of mesenchymal niche signals previously shown to be required for hair follicle regeneration. Further, we demonstrate that wild type cells contribute to induced hair growths and that β-catenin mutant cells act non-cell autonomously to activate Wnt signaling within wild type cells via Wnt ligand upregulation. Collectively, this study demonstrates a novel mechanism by which Wnt/β-catenin signaling controls stem cell-dependent tissue growth non–cell autonomously and carries broader implications toward understanding mechanisms of tumor growth and regeneration.

        233 Association between Interleukin 18 polymorphisms and alopecia areata in Koreans

        Y Oh, B Lew and W Sim Dermatology, College of Medicine, Kyunghee University, Seoul, Republic of Korea
        Alopecia areata(AA) is a common, chronic and inflammatory disease. It is considered to be a tissue-specific autoimmune disease. The exact mechanism causing to the AA is not fully elucidated. But human leukocyte antigen(HLA) molecules and various inflammatory proteins, such as interleukin-1(IL1) and autoimmune regulator, are considered to be involved in the pathogenesis of AA. Interleukin 18(interferon-gamma-inducing factor) (IL18) is an important proinflammatory cytokine belonging to the IL-1 family that is produced by a wide range of immune cells, such as monocytes, activated macrophages and Kupffer cells. This study investigated whether IL18 single nucleotide polymorphisms(SNPs) are associated with the susceptibility to AA in Korean population. We tested 2 SNPs(one promoter SNP : rs187238, -137G/C and exonic SNP rs549908, Ser35Ser) associated with the development of AA, and also assessed the relationship between 2 SNPs of IL18 and the clinicopathologic features using a case-control approach. 233 AA patients and 243 healthy control subjects were recruited. Rs187238 and rs549908 in IL18 showed significant differences between AA and control subjects. In conclusion, our data suggest that the IL18 may be a risk factor for susceptibility to AA in Korean population. And, we assessed the relationship between IL18 SNPs and the clinicopathologic features (onset age, family history, type of AA, involvement of nail, and involvement of body hair) of AA. However, we did not observe significant association with IL18.

        234 Effect of miR-203 silencing in cultured human keratinocytes and reconstructed epidermis

        C Serre, C Capallere, F Labarrade, L Bergeron, C Plaza, V Busuttil, J Botto and N Domloge Ashland INC, Sophia Antipolis, France
        Epidermis undergoes a continuous renewal through the proliferation and differentiation of keratinocytes anchored to the dermo-epidermal junction. This multistep process requires a complex and coordinated program of gene expression and inhibition. Recent findings suggest that microRNAs, which are post-transcriptional regulators of gene expression, play an essential role in the renewal of epidermis, particularly by controling the shift between basal proliferation of keratinocytes and suprabasal differentiation. Long non-coding RNAs (LncRNAs) also participate in the structure of the epidermis. Bioinformatics is a powerful tool allowing the modelization of the interactions between expressed genes, microRNAs and LncRNAs. We established a bioinformatics model of keratinocyte differentiation in order to understand the interactions between the key players in the regulation of keratinocyte differentiation. MicroRNA-203 (miR-203) is a master regulator of the “exit from stemness” by controlling ΔNp63 expression. With the aim of establishing a reconstructed epidermal test model to study the role of particular microRNAs on epidermal differentiation, we used an anti-miR approach and down-regulated miR-203 expression in keratinocytes, and studied the effect of the silencing in cultured cells, and after epidermal reconstruction.

        235 Keratin 79 marks a novel population of migratory keratinocytes that mediates the formation and regeneration of the hair follicle canal / infundibulum

        NA Veniaminova,1 AN Vagnozzi,1 JF Reiter2 and S Wong11 Dermatology, University of Michigan, Ann Arbor, MI and 2 University of California San Francisco, San Francisco, CA
        The hair follicle is comprised of multiple layers of concentric epithelial cells surrounding a central hair shaft. The infundibulum (INF) is located at the distal-most aspect of the hair canal and is poorly characterized. Our studies reveal that the INF is multi-layered, biochemically distinct, and maintained by Lrig1-expressing stem cells in the isthmus, but not by bulge stem cells. In particular, we identify a novel keratin, Keratin 79 (K79), as a marker of early differentiating keratinocytes that line the suprabasal layers of the INF. During morphogenesis, K79+ cells are specified in hair germs and migrate distally out into the epidermis. This process is recapitulated during anagen I, when K79+ cells are specified within the reactivated secondary hair germ and stream along the anterior club hair bulge. MMP-9 is localized to migratory streams during development, suggesting that proteolysis may weaken cellular junctions prior to the formation of the hair follicle lumen. We further show that maintenance of the INF relies on Notch signaling, and that disruption of Notch in Lrig1+ stem cells induces cyst formation. These cysts resemble those observed in human acne comedones. In summary, we have identified a novel marker of a previously uncharacterized population of cells, whose outward movements likely mediate formation of the hair follicle canal.

        236 Interest of adenosine triphosphate in chronic hair loss treatment

        M Lévêque, N Castex-Rizzi and S Bessou-Touya Pierre Fabre Dermo-Cosmétique, Toulouse, France
        Chronic hair loss in men and in women is caused by shortening of anagen duration, the active growing phase of hair growth cycle. As anagen phase duration reduced, the frequency of the hair growth cycles accelerates and the capacity of hair follicles (HFs) to generate new hair shafts rapidly runs out. Adenosine triphosphate (ATP) is a universal endogenous molecule known to constitute an essential energy source for cells. The aim of this study was to evaluate the effect of ATP on hair follicles survival ex vivo and on anagen key factors expression in HFs. Microdissected human scalp HFs were incubated with ATP for 6 or 48 hours (gene expression analysis using qRT-PCR) and up to 28 days (morphometrically assessment of HFs apoptosis). Human dermal papilla cells (DPCs) were incubated with ATP for 24 hours and Keratinocyte Growth Factor (KGF) expression level was measured in cell culture supernatants using ELISA analysis. We found that ATP inhibited hair bulb degeneration with 25% of survival improvement after 28 days of culture. qRT-PCR analysis revealed that ATP increased KGF and fibronectin mRNA levels in HFs up to respectively 251% and 183% compared to control HFs. KGF production by cultured DPCs was also increased by ATP with a mean of 37% of stimulation. In conclusion, ATP delayed spontaneous HFs regression and therefore could prolonged anagen phase duration. KGF being a key growth factor for HFs development during anagen phase, one of the mechanisms that could explain this result is the enhancement of KGF production by DPCs after ATP incubation. Fibronectin, as a major glycoprotein of HFs dermal sheath, is required for HFs anchorage and enhances DPCs aggregation. The results of this study indicate that ATP is a good candidate for chronic hair loss treatment as it would help to maintain HFs in anagen phase and would also reinforce HFs anchorage.

        237 3D sebocyte spheroids induce terminal differentiation markers and lipogenesis offering an improved model for drug testing

        S Compton, RL Wolf and B Buehrer ZenBio Inc, Research Triangle Park, NC
        Acne and related skin disorders arise predominately as a result of dysfunction of the pilosebaceous unit and include abnormal sebum production and inflammation. Although there has been significant progress in defining the causative factors and molecular mechanisms involved in acne and related oily/dry skin disorders, there remains a need for safe and efficacious treatments for these diseases. This will require the development of well characterized and validated human cell based models for basic research that are compatible with high throughput systems for drug screening. Using adult human primary sebocytes and novel immortalized sebocyte cell lines we have developed a high throughput 3D human sebocyte model suitable for in vitro screening. Unlike traditional monolayer cultures human sebocytes grown in 3D have a higher expression of terminal differentiation markers (MC5R, MUC1/EMA,), genes involved in lipid synthesis (FASN, SREBP1, PPARG, LCN2) and lower levels of the early sebocyte marker KRT7. In addition increases in lipogenic genes were observed following treatment with the LXR agonist T0901317 and insulin indicating that 3D sebocytes are functionally responsive to known regulators of lipogenesis. Taken together these results suggest that culturing sebocytes in 3D platforms may provide a more biologically relevant model for sebocyte differentiation and for identifying novel compounds for acne and other skin related disorders.

        238 ATP-dependent chromatin remodeler Brg1 regulates the establishment of the topological lineage-specific interactome for the Loricrin gene in keratinocytes during epidermal development

        A Mardaryev,1 V Rapisarda,1 K Poterlowicz,1 V Emelianov,2 I Malashchuk,1 J Yarker,1 J Rudolf,1 T Sharova,2 A Sharov,2 M Fessing1 and VA Botchkarev1,21 Centre for Skin Sciences, University of Bradford, Bradford, United Kingdom and 2 Dermatology, Boston University, Boston, MA
        During development, execution of distinct cell differentiation programs is accompanied by establishment of specific topological arrangements between the genes and their distal regulatory elements in the nucleus. Here, we show that during epidermal development, ATP-dependent chromatin remodeler Brg1, serving as a direct target for p63 transcription factor, regulates relocation of the Epidermal Differentiation Complex (EDC) locus and its constituent Loricrin gene towards the nuclear interior into a compartment enriched in SC35-positive speckles. ChIP-seq data show that Brg1 binds to distinct domains within the EDC locus and regulates expression of Loricrin gene in keratinocytes. Chromatin conformation capture (4C) analyses of epidermal keratinocytes reveal that nuclear neighbourhood of the Loricrin gene is enriched by actively transcribed genes located on chromosome 3 (cis-interactions), as well as on other chromosomes (trans-interactions). Loricrin is not expressed in thymocytes, and its 4C interactomes in thymocytes and keratinocytes showed only 8% of common genes, demonstrating a marked lineage-specificity in the nuclear neighbourhood for Loricrin gene underlying its active (keratinocytes) or inactive (thymocytes) status. ChIP-seq data revealed enrichment of the 4C Loricirin interactome in keratinocytes by the enhancer-specific histone modifications, as well as by the Satb1, Ctcf and cohesin-binding sites that partially overlap with Brg1-binding sites. Thus, Brg1 plays an essential role in remodelling of the higher-order chromatin structure of the EDC locus and, together with other chromatin regulators, contributes to the establishment of lineage-specific nuclear neighbourhood for Loricrin gene required for is efficient expression in epidermal keratinocytes during skin development and differentiation.

        239 Transcriptional modulation by a hair growth promoting formulation in inter-follicular- and hair follicle-associated human keratinocytes: Comparative analysis by genome-wide expression profiling

        T Hengl, K Riegel, S Krischok, K Schlinzig and HF AbtsBiopharmacy Dermatology, Merz Pharmaceuticals GmbH, Frankfurt a.M., Germany
        In vitro cultured human keratinocytes are an important tool to investigate general aspects of skin physiology. In the epidermal skin layer the normal human epidermal keratinocytes (NHEK) generate the multi-layered skin barrier by a distinct differentiation process. Within the hair follicle the hair follicle associated keratinocytes (HHFK) build up the major part of the follicle and form as result of another differentiation program the hair-shaft. While general aspects of keratinocyte physiology can be investigated in both cell-types the analyses of hair-specific processes require the use of HHFKs. We established cultivation of keratinocytes in a minimal growth medium (MGM) as an in vitro model for undersupplied keratinocytes, mimicking the situation of diminished hair growth for analyzing the effect of a hair growth promoting formulation, Panto(vi)gar. To investigate the genes that are modulated during Panto(vi)gar treatment we performed an Agilent whole genome array expression analysis using HHFK and NHEK cultivated either in a minimal growth medium (MGM) alone or supplemented with a Panto(vi)gar in vitro correlate (P-IC). From the approximately 1700 P-IC modulated sequences 479 sequences are detected in both cell types whereas 373 sequences appeared to be affected only in HHFK. In accordance with the P-IC induced cellular phenotype, genes that are involved in cell cycle, proliferation and metabolic processes are modulated by P-IC in NHEK and HHFK. Furthermore P-IC appears to regulate genes associated with cell death, extracellular matrix, stress responses and hair follicle physiology. Further analysis of the established pool of P-IC regulated genes in NHEK and HHFK allows us to differentiate in more detail between positive effects of Panto(vi)gar on keratinocytes in general and on hair follicle associated keratinocytes in particular.

        240 Cell interaction and growth in a 3-D heterotypic hair-follicle spheroid model mimicking diffuse hair-loss

        T Hengl,1 S Krischok,1 K Riegel,1 N Ansari,2 E Stelzer2 and HF Abts11 Biopharmacy Dermatology, Merz Pharmaceuticals GmbH, Frankfurt a.M., Germany and 2 Buchman Institute for Molecular Life Science (BMLS), Goethe University, Frankfurt a.M., Germany
        Cells in a three-dimensional (3-D) environment receive characteristic biophysical and biochemical signals that are essential for obtaining organ specific cell functions. Associated processes like differentiation or morphogenesis can be simulated in vitro by using 3-D culture models. We were interested in a 3-D culture system mimicking more closely the in vivo situation of the human hair follicle. Such a model would help to understand the mode of action of hair-growth promoting formulations as well as to test drug candidates for their ability to improve hair growth. We used an in vitro heterotypic hair follicle spheroid model comprising of human dermal papilla cells (DP) and human hair-follicle associated keratinocytes (FK). Using automated live cell fluorescence microscopy we analyzed the specific migration pattern of DP and FK during the initial spheroid formation process. After spheroid formation the cell type specific distribution in the 3-D spheroid model was examined by light sheet-based fluorescence microscopy. To address the beneficial effect of the hair growth promoting formulation Panto(vi)gar, we mimicked the situation of diffuse hair loss in vitro by a specific minimal growth medium (MGM) allowing to analyze the positive properties of Panto(vi)gar ingredients by MGM supplementation. The formation process of the heterotypic spheroid was impaired in MGM whereas the MGM supplementation with a Panto(vi)gar in vitro correlate restored the spheroid formation process. Taken together, we investigated 3-D heterotypic spheroids as a simple hair follicle model system and analyzed the impact of the hair-growth formulation Panto(vi)gar on spheroid formation in real-time by conventional and light sheet-based fluorescence microscopy. Further studies will help to gain a better understanding of the mode of action of well-established hair growth promoting formulations like Panto(vi)gar and enable us to identify new compounds for hair growth promotion.

        241 Integration of microRNA-mRNAeExpression profiles reveals regulatory networks controlling hair cycle progression

        K Poterlowicz, M Alam, E Aunin, M Fessing and NV BotchkarevaCentre for Skin Sciences, University of Bradford, Bradford, United Kingdom
        Hair cycle-associated tissue remodeling is governed by tightly regulated gene expression programs. MicroRNAs (miRs) act as one of the essential components of the gene expression regulatory machinery. To define co-regulatory miR-mRNA networks, inversely correlated changes in the miRs (n=618) and mRNA (n=1895) expression profiles during hair cycle in mouse skin were integrated with sequence-based miR target prediction algorithm Targetscan. Overall, 2673 putative connections between 318 miRs and 871 mRNAs have been identified. Functional analysis of the network signatures highlighted enrichment of the genes involved in cell cycle, apoptosis, transcriptional regulation, chromatin remodeling, Wnt, EGF and FGF signaling pathways. Network analyses revealed that the majority of miRs showed higher expression levels in telogen versus anagen skin, while their mRNA targets were predominantly expressed in anagen. Investigation of the network topology revealed its scale-free structure, in which miR-mRNA interactomes were organized into subnetworks of highly connected miR and mRNA hubs. The miR-29 family was identified as a major component of the miR modular structure and showed the strongest associations with 150 predicted target genes during the hair cycle. qPCR confirmed that miR-29s are highly expressed in telogen versus anagen skin. Validation of miR-29 sub-networks using transfection of primary keratinocytes with miR-29a mimic confirmed that miR-29a significantly decreases expression of epigenetic regulators such as Cbx2, Bahd1, Dcp2, Dip2b, Tet2, and Tet3. Interestingly, Tet3 enzyme involved in DNA hydroxymethylation is highly expressed in anagen skin, and has also been identified as the most connected gene in the miR-mRNA hair cycle network, which can be targeted by 34 miRs. Thus, this study provides an important platform for further analyses of the miR-mRNA co-regulatory networks during the hair cycle, and identified the miR-29 family as one of the important regulators of epigenetic machinery contributing to the hair cycle progression.

        242 Age-associated changes in gene expression programs in human hair follicle

        A Patel,1 K Poterlowicz,1 G Westgate,1 N Farjo2 and NV Botchkareva11 Centre for Skin Sciences, University of Bradford, Bradford, United Kingdom and 2 Farjo Hair Institute, Manchester, United Kingdom
        Hair aging is manifested by the reduced rate of hair growth, hair thinning, and changes in the hair fibre structure and color. To investigate changes in the molecular signature associated with hair follicle aging, separate from pattern hair loss, RNA was obtained from hair follicles of the occipital area of healthy male patients of different age groups (30-35 year-old versus 60+ year-old) and processed for global microarray analysis. Microarray data validated by qPCR and immunohistochemistry revealed decrease in the expression of proliferation-associated genes (cyclins, cyclin-dependent kinases and PCNA) in the hair follicles obtained from 60+ year-old subjects. Substantial changes were also seen in the expression of cell differentiation-associated genes, including keratins and keratin-associated proteins, including decrease in expression of stem cell-associated keratins, K15 and K19 was detected in the hair follicles of older subjects. Over 190 transcription factors were differentially expressed in the hair follicles of young and aged subjects. qPCR confirmed the increase in expression of insulin responsive transcription factors FOXO1 and FOXO4, known to be implicated in aging. Also, hair follicle aging is associated with decrease in the SCF signalling, as expression of SCF and its receptor c-kit was down-regulated in older subjects. In addition, older subjects demonstrated decreased levels of Wnt antagonists, such as DKK1, DKK2, SOST and SOSTDC1 and increase in BMP2 expression. Furthermore, several interleukins and their receptors showed increased expression in older individuals, suggesting the association between chronological aging and changes in immune status of the hair follicles. Thus, this study suggests that human hair follicle aging is accompanied by altered programs of gene expression, including changes in cell proliferation- and differentiation-associated genes, as well as the genes that encode the components of selected signalling pathways and transcription factors controlling hair growth, pigmentation and immune response.

        243 Prostaglandin I2 (prostacyclin) is playing a role in regulating rat hair follicle growth

        KG KhidhirDepartment of Biology, University of Sulaimani, Al-Sulaimaniyah, Iraq
        Hair growth disorders cause serious psychological distress, but they are poorly controlled. The prostaglandin (PG) and prostamide (PM) F2α analogues used for the treatment for glaucoma, ocular hypertension, stimulates eyelash growth as a side effect. The mechanism of action by which these drugs stimulate eyelash hair growth is poorly understood, but we have recently shown that receptors for PGF2α (FP) and prostamide F2α are expressed in scalp hair follicles; and prostaglandin F2α stimulates the growth of rodent back follicles in vivo and human scalp follicles in organ culture. Therefore, to determine whether another common prostaglandin, PGI2 (prostacyclin) may also have role in hair growth regulation, the expression of genes for PGI2 receptors (IP) in rat hair follicle were investigated using Reverse Transcription Polymerase Chain Reaction (RT-PCR). Lower hair follicles were individually microdissected for RNA extraction from neck region of 5 adult male Wistar rats with appropriate ethical approval. RT-PCR analysis showed expression of the IP gene in the isolated lower hair follicles (n=5). The identity of PCR products were confirmed by sequence analysis. Therefore, hair follicle express the genes for PGI2 receptor (IP) suggesting that PGI2 may also play a role in regulating hair growth through acting on the hair follicle bulb; and further analysis of the actual roles of PGI2 in hair follicle may lead to a novel treatment for hair disorders.

        244 Impact of Dlx3 deficiency in isthmus/infundibulum for hair shaft development

        J Kim, O Duverger, J Hwang and M MorassoLaboratory of Skin Biology, NIH, NIAMS, Bethesda, MD
        Dlx3 is a homeodomain transcription factor essential in epidermal, osteogenic, and placental development. Autosomal dominant mutations in DLX3 are responsible for Tricho-Dento-Osseous syndrome, characterized by defects in hair, tooth, and bone development. We have previously demonstrated that epidermal-specific deletion of Dlx3 during embryogenesis prevents the formation of the hair shaft and inner root sheath (IRS) during hair morphogenesis. With characterization of Dlx3 expression during postnatal hair growth, we showed high levels of the protein in hair follicle stem cells in the bulge as well as in epidermal stem cells in the isthmus/infundibulum area. The latter has been shown to be involved in sebaceous gland development and in epidermal differentiation upon injury. Using K14creERT inducible mice with suboptimal doses of tamoxifen, we were able to delete Dlx3 specifically in the isthmus/infundibulum area without affecting the bulge. Consistent with these observations, full anagen hair induced by depilation after tamoxifen induction showed absence of Dlx3 in the epidermis and isthmus/infundibulum area, while Dlx3 was still expressed in the bulb and all layers of the developing shaft. Deletion of Dlx3 in the isthmus/infundibulum stem cell niche did not affect the progression of hair growth and the expression pattern of hair-related keratins. However, ultrastructural analysis of the hair revealed defects in the structure of the shaft with different degrees of cuticle anomalies. These results indicate that (1) the epidermal stem cells in the isthmus/infundibulum area influence hair shaft formation, even though they do not directly contribute to hair development; (2) Dlx3 expression in these cells is critical for normal hair cuticle formation.

        245 SULT1A1 activity assay predicts minoxidil response in the treatment of androgenetic alopecia

        A Goren,1,2J McCoy,1 J Roberts1 and T Lotti21 Applied Biology, Inc., Irvine, CA and 2 University of Rome (“G.Marconi”), Rome, Italy
        Topical minoxidil is the most common drug used for the treatment of androgenetic alopecia (AGA) in men and women. While topical minoxidil exhibits a good safety profile, the efficacy in the overall population remains relatively low at 30-40%. To observe significant improvement in hair growth, minoxidil is typically used daily for a period of at least 3-4 months. Due to the significant time commitment and low response rate, a biomarker for predicting patient response prior to therapy would be advantageous. Minoxidil is converted in the scalp to its active form, minoxidil sulfate, by the sulfotransferase enzyme SULT1A1. We hypothesized that SULT1A1 enzyme activity in the hair follicle correlates with minoxidil response for the treatment of AGA. Our retrospective study of a SULT1A1 activity assay demonstrates 95% sensitivity and 73% specificity in predicting minoxidil treatment response for AGA.

        246 Hedgehog signaling creates a perineural stem cell niche essential for maintaining sensory touch domes in skin

        Y Xiao, JS Williams and I Brownell Dermatology Branch, National Cancer Institute, Bethesda, MD
        Mammalian skin contains touch domes (TD) composed of keratinocytes in juxtaposition with neuroendocrine Merkel cells (MC) that are involved in slow adapting touch sensation. There is ongoing cellular turnover in TDs, however, the regulation of the progenitors that maintain the TD and its MCs remains unclear. In many adult tissues, Hedgehog (Hh) signaling regulates stem cell populations. Using reporter mice with LacZ knocked into the locus coding for the Hh target genes Gli1 and Gli2, we determined that the basal cell layer of the specialized TD epithelium is composed of Hh-responding cells. This is in contrast to the remainder of the IFE that was negative for Gli1 and Gli2. Next we used Gli1CreER/+ ; R26RLacZ/LacZ mice to fate map the Gli1-expressing cells in TDs. Labeled cells from the TD basal layer contributed to the overlying epithelium and to MCs indicating that Hh-responding progenitors provide both the epidermal and sensory cells of the TDs. When these mice were aged, the labeled cells were retained in the TDs for up to one year and did not migrate out into the surrounding IFE, indicating that self-renewing Gli1-expressing stem cells maintain the TDs as a unique lineage. Importantly, we showed that Sonic Hedgehog (Shh) expressing sensory nerves are the source of Hh signaling to TDs, as cutaneous denervation eliminated Gli1 expression from TDs, followed by eventual loss of TDs themselves. Furthermore, in adult mice with Shh genetically knocked out in peripheral nerves, TDs are strikingly smaller and have fewer MCs than control mice. Therefore the TD with its MCs is a unique epithelial lineage within the skin that is maintained by Gli1-expressing stem cells that are dependent on a perineural stem cell niche where nerves provide Shh signaling. These results demonstrate that neurons can trophically maintain the specialized sensory epithelia they are associated with by signaling directly to lineage specific epithelial stem cells.

        247 Dietary vitamin A dose dependently regulates bone morphogenetic protein 4 and wingless-related MMTV integration site signaling to alter the hair cycle

        L Suo,1 JP Sundberg2,3 and HB Everts11 The Ohio State University, Columbus, OH, 2 The Jackson Laboratory, Bar Harbor, ME and 3 Vanderbilt University Medical Center, Nashville, TN
        Previously we found that dietary vitamin A dose dependently altered the hair cycle, with high levels (12 & 28 IU/g diet) increasing anagen but excess levels (56 IU/g diet) reducing anagen. Our purpose here is to determine how dietary vitamin A regulates the hair cycle. Female C57BL/6J (B6) mice (n=8-10) were fed 4, 28, or 56 IU vitamin A/g diet for 16 weeks, while C3H/HeJ mice (n=3-4) were fed 0, 4, 12 or 28 IU vitamin A/g diet for 17 weeks, starting two weeks before these mice received a sham graft surgery. We found that retinoic acid (active metabolite of vitamin A) synthetic components retinal dehydrogenase 2 and cellular retinoic acid binding protein 2, as well as retinoic acid degradation enzyme cytochrome p450 26b1 co-localized with bulge stem cell marker CD34 during refractory telogen by immunofluorescence in B6 mice. We next compared the expression of important components in both wingless-related MMTV integration site (WNT) and bone morphogenetic protein 4 (BMP4) signaling pathways in all groups by immunohistochemistry. All hair follicles in B6 mice fed 56 IU vitamin A/g diet were in telogen, with significantly more BMP4 positive cells in the dermis (p<0.05), as well as significantly lower levels of WNT7A (p<0.05) and nuclear localized beta-catenin (CTNNB1; a marker of conical WNT signaling, p<0.05) within the hair follicle bulge compared with the telogen hair follicles in the other two diet groups. In contrast, moderately increasing dietary vitamin A increased WNT7A, which was only significant in the C3H/HeJ mice (p<0.01, 0 & 4 vs 12 & 28 IU groups), yet nuclear localized CTNNB1 was significantly increased in mice fed 4-28 IU vitamin A/g diet compared to mice fed no vitamin A (p<0.001). These findings suggests that feeding mice high levels of dietary vitamin A increases WNT signaling in the bulge to increase anagen, but excess dietary vitamin A also increases BMP in the dermis to arrest hair follicles in refractory telogen.

        248 Screening for autoantigen epitopes involved in the development of alopecia areata

        EH Wang,1 T Breitkopf,1 N Akhoundsadegh,1 JP Dutz,1,2 J Shapiro2 and KJ McElwee11 Dermatology and Skin Science, University of British Columbia, Vancouver, BC, Canada and 2 Dermatology and Skin Science, Vancouver General Hospital, Vancouver, BC, Canada
        The development of alopecia areata (AA) is believed to involve an autoimmune mechanism. AA presents as a non-scarring, inflammatory hair loss disease where CD4 and CD8 (CTL) T cells are required for the onset and progression of AA. Hair follicle (HF) antigens derived from keratinocytes (Kc) and/or melanocytes (Mc) have been suggested to be able to trigger auto-reactive CTL responses in AA subjects, but the exact epitope targets are not yet identified. We investigated the potential for a panel of epitopes expressed by human HF Kcs and Mcs to activate CTLs from AA subjects. Peripheral blood mononuclear cell (PBMC) populations were isolated from AA and healthy subjects with HLA-A2 serotypes. Synthesized HLA-A2 restricted peptides with specific sequences for trichohyalin (trichoh), mel1, MART1, tyrosinase, tyrosinase related protein-2 (TRP2) and GP100 were cultured with PBMCs. The frequency of activated IFNg secreting CTLs in PBMCs was measured using enzyme-linked immunosorbent spot (ELISpot) assays. Separate peptide epitope cocktails induced significantly higher CTL responses in AA subjects; trichoh (5 & 7-fold, p=0.001 & 0.02), tyrosinase (11-fold, p=0.02) and TRP2 (6-fold, p=0.03) respectively. CTL activation using single trichoh epitopes showed highly variable results, suggesting patients with different presentations of AA may have different primary epitope targets. Apoptosis assays revealed that culture media conditioned by AA PBMCs stimulated with both groups of trichoh peptides could significantly elevate the expression of apoptosis markers (5% & 6%) in primary HF Kcs in comparison to healthy subjects (both 2%). The data indicate that AA affected subjects present with an increased frequency of CTLs responsive to specific antigen epitopes originating from Kcs and Mcs. Activated CTLs can also induce apoptosis in HF Kcs via secretory factors. Potentially, trichohyalin could be a specific target for CTLs that cause AA.

        249 Microanatomy of psoriasis: Sebaceous gland atrophy contributes to downregulation of lipid metabolism genes

        L Rittié,1 T Tejasvi,1 LC Tsoi,2 WR Swindell,1 RP Nair1 and JT Elder11 Dermatology, Univ. Michigan, Ann Arbor, MI and 2 Biostatistics, Univ. Michigan, Ann Arbor, MI
        Lipid metabolism genes show decreased expression in psoriasis and several other skin diseases. Consistent with this, our recent RNA-seq study of lesional psoriatic (PP) vs normal (NN) skin revealed repression of a co-expression module containing genes enriched for lipid biosynthetic genes (enrichment p=1.5x10-11), suggestive of sebaceous gland (SG) atrophy as has previously described in psoriatic scalp. In addition, we noted decreased expression of modules associated with dermis and hair follicle (HF) structures (90% of HF transcripts decreased >2-fold). Hair loss was also detected from biopsy site photographs, which showed visible hairs in only 19/71 (26.8%) of PP lesions, compared to 50/71 (70.4%) of perilesional (PN) skin and 38/52 (73.1%) of NN skin (p < 10-6). Based on these findings, we carried out morphometric analysis of HF, SG, and eccrine glands (EGs) by 3D reconstruction of horizontally sectioned paired PP and PN biopsies. As expected, epidermal volume was higher in PP vs PN skin (5.5±0.6-fold, n=2 pairs). However, in contrast to our clinical observations, HF volume was increased in PP vs. PN skin (3.1±0.6-fold, p=0.052), due to increases in both length and diameter. EG ducts were also increased in volume (1.5±0.3-fold, p=0.22), mainly due to increased length. Interestingly, SG were markedly atrophic in PP vs PN skin (volumetric ratio = 0.18±0.13-fold), which was confirmed by Oil Red O staining of an independent set of biopsies. Moreover, SG count was abnormal (either 0 or ≥ 2 SG per HF) in 63±13% of PP HFs (n=14 HF) vs 0% in PN HFs (n=12 HF). Relative to epidermal volume, these results predict a reduction in SG and HF cell populations of 97% and 24%, consonant with observed down-regulation of SG vs HF module genes (avg 34.5% vs 11.6%, respectively). Taken together, our results establish connections between psoriasis microanatomy and global gene expression, and suggest that loss of SG function, rather than follicular atrophy, may contribute to the reversible hair loss seen in psoriatic lesions.

        250 Master regulators involved in human dermal papilla induction of hair follicles

        E Wang,1 C Higgins,1 J Chen,1,2 C Jahoda4 and A Christiano1,31 Dermatology, Columbia University, New York City, NY, 2 Systems Biology, Columbia University, New York City, NY, 3 Genetics and Development, Columbia University, New York City, NY and 4 Biology and Biological Sciences, Durham University, Durham, United Kingdom
        While rodent hair follicle dermal papilla (DP) cells reproducibly induce hair growth in recipient skin, human DP cells are far less inductive in vitro. The gene expression of human DP cells changes dramatically when isolated and cultured. This occurs almost immmediately, with p0 cells showing loss of hair-inducing properties. Recently, our group has shown that partial recovery of gene expression and inductive potential of DP cells can be achieved when placed in spheroid culture, suggesting that the spatial relationship and intercellular communication of DP cells are important in maintaining their inductive potential. DP spheroids at third passage were able to induce a non-pigmented hair on a neonatal foreskin xenograft on a SCID mouse at six weeks, and were shown by microarray analysis to have approximately 22% recovery of the gene expression of intact DP cells. We used GEDI (Gene Expression Dynamics Inspector) analysis of DP cells at various stages of culture, including spheroids, to identify potential master regulators of the intact DP signature, and postulate that these master regulators will also improve the inductive potential of DP cells. We transfected selected master regulators, including Fli-1, LEF-1, NR3C2 and ZBTB16 into cultured DP cells and are assessing their effect on clustering of cultured DP cells and hair-inductive potential of DP cells. While master regulators are commonly described in the context of cancer pathways, here we use systems biology approaches to model a developmental process with the goal of improving tissue engineering of complex tissues using regulatory pathways. Adapting the methods designed to dismantle targeted tissues, such as cancer, via context-specific molecular regulators to the reconstruction of hair and skin in vitro provides a novel systems biological perspective in the pursuit of engineering fully functioning complex tissues.

        251 Atoh1 overexpression is sufficient to direct the keratinocyte lineage to the Merkel cell fate

        S Ostrowski,1,2 A Bolock,2 C Smolak,2 M Wright2 and S Maricich21 Dermatology, Case Western Reserve University, Cleveland, OH and 2 Pediatrics, University of Pittsburgh, Pittsburgh, PA
        During embryonic development, Merkel cells descend from the keratinocyte lineage, a process that requires the transcription factor Atoh1. To test whether Atoh1 expression is sufficient to direct Merkel cell specification, we utilized a doxycycline inducible system to ectopically express Atoh1 in the keratinocyte lineage of transgenic mice. In postnatal day 21-24 mouse skin, induction of the Atoh1 transgene for one day led to detectable levels of nuclear Atoh1 protein expression throughout the epidermis, leading to the appearance of scattered ectopic “Merkel-like” cells that expressed the Merkel cell specific cytokeratins 8, 18 and 20. When transgenic mouse skin was examined two weeks and three months after Atoh1 induction, many of these ectopic Merkel-like cells persisted and exhibited key features of mature Merkel cells, such as expression of Rab3c and vesicular glutamate transporter 2 (VGLUT2) and uptake of FM1-43 dye. Together, these findings demonstrate that overexpression of Atoh1 is sufficient to direct adult keratinocytes and/or their precursors to adopt a “Merkel-like” cell fate, which may have direct implications regarding the pathogenesis of human Merkel cell carcinoma.

        252 Complex changes in the apoptotic and cell differentiation programs during initiation of the hair follicle response to chemotherapy

        TY Sharova,1 K Poterlowicz,3 NV Botchkareva,3 NA Kondratiev,1 A Aziz,1 JH Spiegel,2 VA Botchkarev3,1 and A Sharov11 Dermatology, Boston University School of Medicine, Boston, MA, 2 Otolaryngology, Head and Neck Surgery, Boston University School of Medicine, Boston, MA and 3 Centre for Skin Sciences, School of Life Sciences, University of Bradford, Bradford, United Kingdom
        Chemotherapy has severe side-effects for normal rapidly proliferating organs, such as hair follicle, and causes massive apoptosis in hair matrix keratinocytes followed by hair loss. To define the molecular signature of hair follicle response to chemotherapy, human scalp hair follicles cultured ex vivo were treated with doxorubicin and global microarray analysis was performed 3 hours after treatment. Microarray data revealed changes in expression of 504 genes in doxorubicin-treated hair follicles versus the controls. Among these genes, upregulations of several tumor necrosis factor family of apoptotic receptors (FAS, TRAIL receptors 1/2), as well as of a large number of the keratin-associated protein genes were seen after doxorubicin treatment. Hair follicle apoptosis induced by doxorubicin was significantly inhibited by either TRAIL or TRAIL-receptor 1/2 neutralizing antibodies, or by caspase 8 inhibitor, thus suggesting a novel role for TRAIL receptor signaling in mediating doxorubicin-induced hair loss. These data demonstrate that the early phase of the hair follicle response to doxorubicin includes upregulation of apoptosis-associated markers, as well as substantial re-organization of the terminal differentiation programs in hair follicle keratinocytes. These data provide an important platform for further studies towards the design of novel approaches for management of chemotherapy-induced hair loss.

        253 Low-level laser treatment in a rat model of chemotherapy-induced alopecia

        S Damanpour,1 K Nouri,1,2 J Jimenez1 and TC Wikramanayake1,21 Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL and 2 Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL
        Chemotherapy-Induced Alopecia (CIA) is one of the most distressing side effects of cancer chemotherapy, affecting approximately 65% of patients receiving treatment. Whereas CIA is almost always reversible, hair regrowth can take 3-6 months. Hair loss can negatively impact patient perceptions of appearance, body image, sexuality, and self-esteem, as well as depriving patients the privacy of having cancer. Some patients would even consider declining chemotherapy for fear of CIA. In recent years, low-level laser therapy (LLLT) has been shown to promote hair growth in other hair loss situations such as male and female pattern hair loss. In this study, we investigated the effects of LLLT on CIA in a rat model. Young Long-Evans rats were given chemotherapeutic agents to induce total alopecia, treated with LLLT or sham, and monitored for hair loss and regrowth. They were also injected with Shay chloroleukemic cells to determine the effects of LLLT on chemotherapy efficacy. All the rats that received chemotherapeutic agents, whether treated with LLLT or sham, developed total alopecia. These observations indicated that with our treatment regimen, LLLT did not prevent CIA. However, LLLT-treated rats showed significantly accelerated hair regrowth than sham-treated rats. Histological analysis confirmed that hair follicles in the LLLT-treated skin entered the growth phase (anagen) much earlier than control. Additionally, there was no significant difference in the survival rates between LLLT- and sham-treated rats that received cancer cells and chemotherapy, indicating that LLLT treatment did not compromise chemotherapy efficacy. Our results suggest that LLLT should be explored for the treatment of CIA in clinical trials because LLLT devices for home use provide a safe and user-friendly approach that could be translated to increased patient compliance and improved efficacy for chemotherapy.

        254 Chromatin organizer and AT-rich binding protein Satb1 controls re-organization of lineage-specific differentiation programs during keratinocyte reprogramming towards the induced pluripotent state

        TY Sharova,1 M Ahmed,4 H Han,3 K Poterlowicz,4 G Mostoslavsky,2 T Kohwi-Shigematsu,3 VA Botchkarev4,1 and A Sharov11 Dermatology, Boston University, Boston, MA, 2 Center for Regenerative Medicine, Boston University, Boston, MA, 3 Lawrence Berkeley National Laboratory, University of California Berkeley, San Francisco, MA and 4 Centre for Skin Sciences, School of Life Sciences, University of Bradford, Bradford, United Kingdom
        Reprogramming of adult somatic cells towards the induced pluripotent state (iPS) is associated with marked changes in the genome organization and gene expression programs. Here, we analyzed global changes in gene expression during iPS reprogramming of primary epidermal keratinocytes (KCs) isolated from transgenic mice expressing the Dox-inducible cassette of four mouse pluripotency genes (Oct4, Sox2, Klf4 and c-myc) under Col1a1 promoter. Microarray analyses of the iPS colonies shows that KC-specific genes that constitute keratin type I/II loci, as well as Epidermal Differentiation Complex locus, are markedly downregulated during KC reprogramming towards iPS, while expression of the pluripotency gene Nanog is upregulated. KC transition towards iPS is accompanied by marked changes in expression of several genes involved in the control of chromatin organization and remodelling including the genome organiser Satb1, which was markedly downregulated during reprogramming. Interestingly, ChIP-qPCR revealed Oct4 binding to regulatory regions of the Satb1 gene indicating that pluripotency factors might directly repress Satb1 during reprogramming. Furthermore, treatment of KCs with lentiviruses expressing Satb1 shRNA significantly accelerated appearance of Nanog+ cells and iPS colonies compared to the controls. Acceleration of iPS colony formation under shRNA-mediated Satb1 knockdown was accompanied by more rapid decrease in the expression of KC-specific genes, compared to controls. Thus, these data demonstrate that Oct4-mediated downregulation of Satb1 serves as an essential step in switching-off KC-specific gene expression programs during their reprogramming towards the induced pluripotent state.

        255 A lncRNA-MAF:MAFB transcription factor network regulates epidermal differentiation

        V Lopez-Pajares,1 K Qu,1 J Zhang,1 D Webster,1 B Barajas,1 B Zarnegar,1 L Boxer,1 M Kretz1 and P Khavari1,21 Stanford University, Palo Alto, CA and 2 VA Palo Alto, Palo Alto, CA
        Progenitor differentiation requires reprogramming of genomic expression, however, in many tissues, such as epidermis, the spectrum of reprogrammed genes and the transcription factors (TFs) that control them are not fully defined. We thus performed kinetic transcriptome analysis during regeneration of differentiated epidermal tissue to define the transcriptional landscape involved in the regeneration of differentiated epidermal tissue from epidermal progenitors. We identified gene sets enriched in progenitors (594 genes), in early (159 genes), and in late differentiation (387 genes). Module mapping of 1046 TFs identified 100 TFs with predicted influence, 36 of which encompass presently known TF regulators of epidermal gene expression. Among newly identified TFs were MAF and MAFB. Both MAF and MAFB were induced >35-fold during epidermal differentiation, where they controlled the expression of 393 genes altered in this setting. In tissue, combined MAF:MAFB loss led to a failure of terminal differentiation whereas enforced MAF:MAFB expression in the epidermal basal layer produced premature differentiation and tissue collapse. Integrative analysis identified ANCR and TINCR long non-coding RNAs (lncRNAs) as essential upstream MAF:MAFB regulators. The ANCR lncRNA targeted EZH2 to MAF and MAFB genes to repress their expression and TINCR bound MAF mRNA and enhanced its stability. ChIP-seq analysis found significant co-localization of MAF and MAFB peaks (p<1x10-200), identified MAF and MAFB DNA binding motifs, and demonstrated enriched binding at genes implicated in human skin diseases characterized by abnormal epidermal differentiation. ChIP-seq also demonstrated MAF:MAFB binding to known epidermal differentiation TFs whose expression they controlled, including GRHL3, ZNF750, KLF4, and PRDM1. Each of these TFs rescued expression of specific MAF:MAFB target gene subsets in the setting of MAF:MAFB loss, indicating they act downstream of MAF:MAFB. These data thus identify a new lncRNA-TF network as essential for epidermal differentiation.

        256 Various skin abnormalities and greatly reduced adipose depots in Mpzl3 knockout mice

        AG Leiva, AL Chen, P Devarajan, Z Chen, S Damanpour, JA Hall, AC Bianco, J Li, E Badiavas, J Zaias, M Miteva, P Romanelli, K Nouri and TC WikramanayakeUniversity of Miami Miller School of Medicine, Miami, FL
        The rough coat (rc) spontaneous mutation causes sebaceous gland hypertrophy, hair loss and extracutaneous abnormalities including growth retardation. The rc mice have a missense mutation in the predicted immunoglobulin protein Mpzl3. In this study, we generated Mpzl3 knockout mice in order to determine the Mpzl3 functions in the skin. Homozygous Mpzl3 null mice appeared normal at birth, but showed unkempt and greasy hair coat and hair loss soon after. Severe abnormalities were detected in their hair shaft. Histological analysis revealed severe sebaceous gland hypertrophy and increased dermal thickness, but did not detect significant changes in the hair cycle. Mpzl3 null mice frequently developed inflammatory skin lesions. These abnormalities resemble closely those observed in the rc/rc mice, as well as mice heterozygous for both the rc and Mpzl3 knockout alleles, indicating that rc and Mpzl3 are allelic. Using a reporter gene, we investigated Mpzl3 promoter activity during the hair cycle. We also determined whether the skin abnormalities in the knockout mice were the results of defects in the immune system. Interestingly, loss of MPZL3 function caused a striking reduction in cutaneous and overall adipose tissue. Our results suggest an important role for Mpzl3 in both skin function and adiposity.

        257 Transfer of alopecia areata to C3H/HeJ mice using cultured lymph node derived cells

        EH Wang and KJ McElwee Dermatology and Skin Science, University of British Columbia, Vancouver, BC, Canada
        The C3H/HeJ mouse strain is one of the most well defined animal models for alopecia areata (AA), a cell-mediated, inflammatory, autoimmune hair loss disease. Both CD4 and CD8 T cells are important for the onset and progression of AA both in humans and rodent models. C3H/HeJ mice are an inbred strain, but only a small subset of mice spontaneously develops AA-like symptoms. Currently, grafting full thickness skin from an AA affected C3H/HeJ mouse to healthy recipients is the most practical method to generate larger quantities of AA mice for research. However, skin grafting is an invasive procedure which can only be conducted by someone with anesthesia and surgical experience. The procedure is time consuming and expensive, skin grafts are prone to rejection; and only one donor source of skin is used per surgical session. As a result, skin grafting success can be unpredictable within and between different litters. In our investigation, we isolated skin-draining lymph nodes from AA affected or normal haired C3H/HeJ mice and obtained the lymph node cells (LNCs) in a single-cell suspension. The LNCs were cultured with the presence of magnetic beads coated with anti-CD3 and anti-CD28 crosslinking antibodies for unspecific cell stimulation and expansion. At the end of a five-day expansion period, ∼10 million of either AA or control LNCs were injected intradermally into the back skin of healthy mice. Within 7-19 weeks of a 20 week observation period, 9/10 mice receiving AA LNCs developed AA while 9/10 mice receiving control LNCs stayed fully haired. H&E staining showed the typical clustering of lymphocytes around the dystrophic hair follicles in the skin of AA mice similar to mice that have developed AA spontaneously or via skin grafts. This result reconfirms the cell-mediated mechanism of AA pathogenesis and presents a novel, simplified way to generate AA mice.

        258 Genome-wide analysis of P300 binding reveals enrichment of layer-specific enhancers within exons of epidermal gene clusters

        S Kumar, S Krishnaswami, A Grainger, C Adase, C Ricker and BD YuDivision of Dermatology, Institute for Genomic Medicine, UCSD School of Medicine, La Jolla, CA
        The conserved organization of gene clusters is thought to reflect the presence of critical cis-regulatory elements important for coordinated gene expression. To identify novel cis-regulatory elements active during skin development, we performed next-generation sequencing of sites associated with P300, a histone acetyltransferase known to associate with enhancer elements. 92,095 loci associated with P300 were found in the mouse epidermis of which 284 localized to keratin or epidermal differentiation complex (EDC) gene clusters. Surprisingly, analysis of gene clusters (type I/II keratin, EDC, HOX) revealed 3-4 fold enrichment of P300-associated loci within genes and exons (15-26% vs. 6% within whole genome). Reporter assays in differentiating keratinocytes and in engrafted keratinocytes reveal a number of exonic P300-linked sites act during keratinocyte differentiation and as spatially restricted enhancers. These findings indicate that cis-regulatory activity of genes within clustered loci may themselves act to coordinate gene expression in the epidermis and may contribute to selective maintenance of gene clusters during vertebrate evolution.

        259 Roles of mesenchymal pdgf signaling during hair follicle formation

        A Rezza, R Sennett, C Clavel and M Rendl Developmental and Regenerative Biology, Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY
        Although embryonic hair follicle formation is dependent on successive signaling crosstalk between epithelial stem cells and underlying specialized mesenchymal cells in dermal condensates, the precise signals involved in this process remain unclear. Platelet Derived Growth Factor (PDGF) signaling has been shown to be instrumental in embryonic development and adult tissue function of several tissues, including gonads, lung, kidney, intestine, brain and skin. Global deletion of the PDGF receptors, PDGFRα and PDGFRβ, in knockout mice results in early embryonic lethality with specific defects suggesting unique physiological functions. However, both receptors mostly share overlapping expression patterns suggesting functional compensation in several tissues. In the skin, mice lacking the PDGF receptor PDGFRα exhibit widespread dermal hypoplasia, while PDGF-A null mice show increasing loss of dermal mesenchyme and reduced hair development after birth, and embryonic skin treated with blocking antibodies against PDGFRα failed to form hair follicles. In order to study the role of PDGF signaling specifically in dermal condensates during hair follicle formation, we ablated both PDGFRα and PDGFRβ in these mesenchymal cells using Tbx18Cre mice. We show that Tbx18Cre efficiently ablates the expression of both PDGFRα and PDGFRβ in dermal condensates. Hair follicle formation seems to start normally, as Sox2GFP-positive dermal condensates are present in knockout embryos, suggesting that PDGF signaling in dermal condensates is not required for the initiation of hair follicle formation. Further characterization of older embryos will show the potential importance of PDGF signaling in late hair follicle formation.

        260 Reprogramming regular skin fibroblasts into hair inducing dermal papilla cells

        C Clavel,1,2 A Rezza,1,2 R Sennett1,2 and M Rendl1,2,31 Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 2 Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY and 3 Dermatology, Icahn School of Medicine at Mount Sinai, New York, NY
        Dermal papilla (DP) cells can induce new hair follicles together with skin stem cells, but they lose their hair inducing activity during cell culture expansion. For this reason cell-based regenerative therapies are still unavailable to restore hair follicles in hair loss patients and burn victims. To generate sufficient cell quantities for hair regenerative therapies we sought to reprogram regular fibroblasts into DP cells. Overexpression of previously identified DP signature transcription factors (TFs) in freshly isolated fibroblasts significantly upregulated several DP signature genes. In addition, upregulation of DP signature genes is enhanced by inhibitors of histone modifiers and 3D aggregation clustering. Furthermore, TF overexpressing fibroblast lines isolated from double-transgenic Sox2GFP/Lef1-RFP reporter mice activated reporter activity and induced the DP molecular identity. Our preliminary data suggest that the right combination of DP TFs can reprogram DP niche fate in regular fibroblasts that can potentially be utilized in future hair restoration efforts.

        261 Wnt-dependent initiation and maintenance of postnatal hair growth by hair-inducing dermal papilla cells

        L Zhou, Y Yang, M Taketo, SE Millar and Y Zhang University of Cincinnati, Cincinnati, OH
        The development and function of the skin epidermis and hair follicles rely on cell-cell communication among their distinct stem/progenitor cell populations. The two critical cell types necessary to produce and maintain hair follicles are fibroblasts residing in the dermal papilla (DP) and dermal sheath, which are derived from the mesenchyme, and hair follicle stem cells (HFSCs), which are derived from the surface ectoderm. Although regeneration of the hair structure and pigmentation is accomplished by HFSCs, the DP is well recognized as the commanding force to drive the hair cycle. However, little is known of the molecular mechanism by which dermal papilla cells (DPCs) exert influence over heterogeneous HFSCs and their progeny to coordinate hair growth and pigmentation To definitely address this question, we generated a Prom1+/C-L; Rosa-rtTA; tetO-Ctnnb1ΔN mouse, in which a stabilized form of β-catenin can be expressed exclusively in hair-inducing DPCs upon induction. By inducing expression of mutant β-catenin either during embryonic anagen or before the induction of a synchronized hair growth cycle by depilation, the growth of pigmented hair follicles was significantly accelerated in mutant mice than those in control littermates. To determine the underlying cause, we discovered that there was an increased proliferation and differentiation of matrix keratinocytes in mutant hair follicles. In vitro 3D culture of sorted CD133-positive DPCs revealed that, after activating β-catenin signaling, sphere formed by DPCs from mutant mice were much bigger than those from control ones. We then performed skin patch assays by injecting cultured DPCs subcutaneously into the back skin of immunodeficient nude mice. Experimental results showed that DPCs with up-regulated β-catenin activity lead to much quicker hair follicle reconstruction than control DPCs did. These data provide the evidence that the initiation and maintenance of postnatal hair growth and pigmentation can be coordinately controlled by prom1-positive hair-inducing DPCs through the modulation of Wnt/β-catenin activity.

        262 Histone deacetylase 3 is essential for stepwise differentiation of the epidermis

        F Liu,1 M Leboeuf,1 M Lazar2 and S MIllar11 Dermatology, University of Pennsylvania, Philadelphia, PA and 2 2Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
        Continuous renewal of the epidermis relies on basal progenitor cells that both self-renew and give rise to progeny that undergo step-wise differentiation to produce a cornified layer. Histone deacetylases (HDACs) are major chromatin remodeling enzymes that cause chromatin compaction and transcriptional repression. Type I HDACs, HDAC1, 2 and 3 associate with chromatin by interacting with transcription factors in large repression complexes. HDAC1 and HDAC2 are generally found in the same complexes, but HDAC3 is uniquely recruited to the NCoR/SMART complex, suggesting distinct functions. We showed previously that embryos lacking both Hdac1 and Hdac2 in surface ectoderm fail to form a stratified epidermis and exhibit progressive defects in progenitor cell proliferation. To determine the function of HDAC3 in skin development, we generated mice constitutively lacking epidermal Hdac3. Hdac3 mutation did not significantly affect progenitor cell proliferation or survival. Instead Hdac3-null epidermis displayed disruption of step-wise differentiation of the epidermis. Hdac3-null spinous layer keratinocytes prematurely expressed terminal differentiation proteins, resulting in formation of a disorganized cornified layer, and severe barrier defects that caused perinatal lethality. Nile red and Oil Red O staining revealed excessive accumulation of lipids in mutant epidermis. Microarray profiling experiments identified upregulation of a subset of terminal epidermal differentiation genes. In addition, genes in the PPAR signaling pathway that regulate fatty acid biosynthesis and metabolism were significantly upregulated. We found that HDAC3 forms complexes with PPARγ in embryonic epidermis. Furthermore, treatment in utero with the PPARγ inhibitor GW9662 partially rescued excessive lipid accumulation in Hdac3-null epidermis. Thus, HDAC3 performs a central role in establishing the epidermal barrier by coordinating stratification and lipid metabolism.

        263 Lipoproteins and bovine milk peptides stimulate lipogenesis and inflammatory response in human sebocytes

        RK Sivamani,1 W Burney,1 G Killer,1 S Sharma,1 D Dallas,2 B German2 and R Isseroff11 Dermatology, UC Davis, Sacramento, CA and 2 Food Science & Technology, UC Davis, Davis, CA
        The correlation between dietary factors and acne has been controversial with epidemiological studies associating skim milk with worsening acne. Although insulin and IGF-1 stimulate sebocyte lipogenesis (Smith et al., JID, 2006) few other studies have evaluated the role of diet-associated peptides on human sebocytes. Here, we investigated the influence of postdigestive bovine milk peptides (bMPs) and lipoproteins on human SEB-1 sebocyte lipogenesis and inflammatory responses. Casein based post-digestive bMPs (0.01 mg/mL) and lipoproteins subfractions (100 ug/mL LDL, 100 ug/mL VLDL, and 100 ug/mL HDL) were incubated with human sebocytes. Modulation in neutral lipid content was assessed with Nile Red and with evaluation of the expression of the lipid gene transcription factor Sterol Regulatory Element Binding Protein-1 (SREBP-1) through western blotting. Pro-inflammatory cytokine IL-6 release from sebocytes was measured with ELISA. While bMPs, VLDL, and LDL increased intracellular lipid content by 20.2% (p<0.001), 35% (p=0.01), and 38% (p=0.005) respectively, HDL did not have any effect. Western blots revealed that bMPs, VLDL, and LDL increased expression of SREBP-1. bMP elevated the production of IL-6 by 10.2-fold (p<0.001) while VLDL elevated IL-6 production by 2.1-fold (p=0.003) in comparison to untreated control sebocytes. Although TNF elevated IL-6 production by 4-fold (p<0.001), the combination of TNF and bMPs synergistically elevated IL-6 production by 22.8-fold (p=0.001). Although LDL by itself did not alter IL-6 production, the combination of TNF and LDL synergistically elevated IL-6 production by 19.6-fold (p=0.002). Taken together we conclude that bMPs and lipoproteins activate lipogenesis and the inflammatory response in sebocytes, especially in an inflammatory TNF-rich microenvironment. Our findings may provide new therapeutic targets and stimulate further dietary research in acne.

        264 Expansion of specialized epidermis induced by hormones and mechanical strain

        T Easwaran,1 C Offutt,1 H Wu,1 DF Spandau,2 S Koyama3,1 and JG Foley1,21 Medical Sciences, Indiana University School of Medicine, Bloomington, IN, 2 Dermatology, Indiana University School of Medicine, Indianapolis, IN and 3 Psychology, Indiana University School of Medicine, Bloomington, IN
        In mammals, sites of specialized skin such as the palms, soles, and lips grow proportionally with the animal. However, specialized skin, such as the nipple, expands dramatically with changes of circulating hormones. The specific mechanisms that mediate the growth of the nipple have not been identified. This manuscript characterizes the formation of functional areola in mice during the first pregnancy and lactation period that entailed the expansion of a thickened, specialized epidermis into the region surrounding the nipple, changes in connective tissue, and hair shaft loss. Although epidermal and sebaceous gland proliferation increased in the areola during pregnancy and lactation, high levels of proliferation did not occur in underlying connective tissue cells, but instead, Collagen III and fibronectin gene expression was activated suggesting the addition of matrix within the region. To determine the role of pregnancy and lactation hormonal milieu, as well as localized mechanical strain on areola formation, we created models that separated these stimuli to evaluate changes in gross structure and histology. While modest increases of epidermal proliferation and remodeling of connective tissue occurred as a result of individual stimuli, areola formation required exposure to pregnancy and lactation hormones, as well as mechanical strain.

        265 Wnt10a regulates proliferation of multiple epithelial stem and progenitor populations

        M Xu and S Millar Department of Dermatology, University of Pennsylvania, Philadelphia, PA
        Wnt/β-catenin signaling is required for embryonic development of ectodermal appendages, and regulates proliferation of adult stem and progenitor cells in hair follicles, interfollicular epidermis and non-hairy epithelia. However, definitive genetic loss of function evidence for the involvement of specific Wnt ligands is lacking. To begin to address this question, we generated mice with constitutive or inducible deletion of Wnt10a, which is specifically expressed in epidermis and its appendages. Constitutive loss of Wnt10a did not affect embryonic hair follicle or taste placode development, possibly due to functional redundancy with the related ligand Wnt10b. However, Wnt10a mutants displayed malformed and absent teeth, indicating a non-redundant role for Wnt10a in embryonic tooth development. Inducible postnatal deletion of epithelial Wnt10a resulted in delayed onset of the first postnatal hair growth cycle and shortened anagen, revealing roles for Wnt10a in stimulating stem cell proliferation at anagen onset and maintaining anagen. Postnatal Wnt10a deletion also caused decreased thickness and proliferation of ventral paw epidermis between the footpads, decreased proliferation of tongue filiform papillae, and defective renewal of taste buds. Mutant mice displayed severely impaired sweating ability associated with degeneration of sweat gland ducts and defects in myoepithelial cells. Similar defects in sweat duct maintenance were observed in mice with inducible postnatal deletion of epithelial β-catenin, revealing a previously unknown requirement for Wnt10a/β-catenin signaling in sweat duct maintenance. Consistent with these results, WNT10A mutations are associated with non-syndromic tooth agenesis and ectodermal dysplasia syndromes in humans. These data identify WNT10A as a key ligand required for proliferation of adult epithelial progenitor cells and maintenance of appendage structures. Furthermore, Wnt10a mutant mice provide a faithful model for the human condition and can be used to test potential therapeutic approaches.

        266 Intraflagellar transport 27 (Ift27) is essential for hedgehog signaling but dispensable for ciliogenesis during hair follicle formation

        N Yang,1 L Li,2 JP Sundberg,3 GJ Pazour4 and J Chen11 Pathology, Stony Brook University, Stony Brook, NY, 2 Dermatology, Peking Union Medical College Hospital, Beijing, China, 3 The Jackson Laboratory, Bar Harbor, ME and 4 University of Massachusetts Medical School, Worcester, MA
        The development of hair follicles requires hedgehog (Hh) signaling. The activation of the Hh signaling pathway requires the presence of the primary cilium, an antenna-like protrusion of the cell. Disrupting genes essential for ciliogenesis, such as Ift88, Fuz and Intu, caused hair follicle development defects due to attenuated Hh signaling. However, the loss of cilia in these cilium mutants makes it impossible to determine whether the hair follicle phenotypes are caused by the loss of cilia, defects in the transduction of Hh signals, or a combination of both. In this study, we characterized the function of Ift27, a subunit of the intraflagellar transport (IFT) complex B. We discovered that the development of hair follicles of Ift27 null mice was arrested, which is reminiscent of that of previously reported cilium and Hh mutants. Furthermore, we confirmed that the Hh signaling pathway was attenuated in Ift27 mutants. Surprisingly, the assembly of primary cilia was unaffected. In addition, we found that the expression and distribution of ciliogenic proteins were unaffected in Ift27 null cells; whereas Ift25, the binding partner of Ift27, was dislocated in Ift27 null cells. This study demonstrated that Ift27 is essential for Hh signaling but dispensable for ciliogenesis. The function of Ift27 described herein not only demonstrated that the molecular machineries underlying the transduction of Hh signaling could be separated from ciliogenesis during hair follicle formation, but also provided important insight on a novel venue of targeting Hh signaling, such as for the development of targeted therapies for basal cell carcinoma (BCC).

        267 Acne vulgaris disease progression is associated with perturbation of cellular differentiation at the hair follicle canal / infundibulum

        U Amadi, YR Helfrich and S Wong Dermatology, University of Michigan, Ann Arbor, MI
        Faulty cellular function and aberrant signaling have been implicated in the formation of acne-like cystic lesions. However, despite being the most common skin disorder, the precise mechanisms involved in the development of acne remain unknown. This study was undertaken to examine whether acne vulgaris progression is associated with abnormal cellular differentiation at the hair follicle canal, or infundibulum, where acne arises. We recently identified a novel keratin (Keratin 79, or K79), which is preferentially expressed at the infundibulum in normal hair follicles. Other markers including Keratin 10 (K10) and cornifin-α were also shown to be expressed in this domain. We obtained full thickness acne skin lesion biopsies from the faces of healthy subjects of mixed racial, ethnic, and gender background with acne vulgaris. We evaluated open comedones, closed comedones, and pustules for the aforementioned infundibular markers in addition to the basal epidermal marker Keratin 5 (K5). We found that among the cellular markers examined, K79 expression was significantly decreased in open comedone acne lesions. In contrast, K79 was frequently maintained or increased in closed comedone and pustule acne lesions. Expression of K10, cornifin-α and K5 were not significantly changed. Taken together, these findings suggest that aberrant cellular differentiation at the infundibulum is a manifestation of acne vulgaris and that expression of K79 may be altered during disease progression.

        268 Since human facial sebaceous glands contain prostamide F2α receptors these may play a role in sebaceous gland physiology in health and disease

        M Shalbaf,1 JW Wang,2 D Woodward2 and V Randall11 Centre for Skin Sciences, University of Bradford, Bradford, United Kingdom and 2 Biological and Chemical Sciences, Allergan Inc., Irvine, CA
        Existing therapeutic approaches for acne have significant limitations. Unfortunately, the physiology of the sebaceous gland and the pathogenesis of acne are not fully understood. Acne is generally associated with androgen-stimulated increased sebum production, blocked ducts and inflammation of sebaceous glands involving prostaglandin mediators. Prostamides are a new group of paracrine molecules, closely related to prostaglandins. We recently identified receptors for prostamide F2α in human scalp follicles which can stimulate hair growth (Khidhir et al 2013). Therefore, we aimed to determine whether this new class of signalling molecules could be involved in sebaceous glands by investigating the presence of prostamide F2α receptors in human facial sebaceous glands using molecular biological methods and immunohistochemistry. To identify gene expression, sebaceous glands were microdissected from female facial skin (n=5; aged 47-72) after digestion with dispase for 18 hours at 4oC. Individual glands were collected into RNAlater, total RNA extracted and cDNA synthesised. RT-PCR using primers for all variants of prostaglandin F2α receptor (FP) revealed 3 bands. Further analysis using specific primers identified the gene expression of FP and two splice variants, altFP4 & altFP1; their identity was confirmed by sequencing. Immunohistochemistry of frozen sections of female human facial skin (n=3) also located FP protein expression in the sebaceous glands in the basal sebocytes around the edges of the acini and in the larger mid-differentiated sebocytes. Therefore, human facial sebaceous glands contain prostamide F2α receptors, which are believed to be heterodimers of FP and its splice variants, in the cells which are involved in the alteration of sebum production. Clarification of their role in sebaceous glands may lead to new approaches for acne treatments.



        270 Cxcr4 is transiently expressed in both mesenchymal and epithelial compartments of the nascent hair follicle but is not required for follicle formation

        R Sennett, A Rezza, C Clavel and M Rendl Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY
        Hair follicle (HF) formation and early growth relies on the coordinated exchange of signals between mesenchymal and epithelial compartments of embryonic skin. In efforts to identify the key molecular players driving HF formation, we investigated gene expression in specialized dermal condensate (DC) cells, thought to be crucial for HF formation. Our analysis identified enriched expression of chemokine receptor Cxcr4 suggesting a specialized role in promoting DC function during embryonic HF formation. Condensate-specific expression in the dermis was confirmed by real-time PCR analysis and immunofluorescence staining, which additionally revealed transient Cxcr4 expression in incipient epithelial hair placodes. Placodal Cxcr4 appeared prior to detection in the DC, representing a unique “switch” of expression between epithelial and mesenchymal compartments. To functionally explore the role of this receptor in both compartments we conditionally ablated Cxcr4 with DC-targeting Tbx18Cre knock-in and epithelial-targeting K14-Cre transgenic mice. Conditional knockouts for both crosses were viable throughout embryogenesis and into adulthood. Preliminary results suggest that neither dermal nor epithelial Cxcr4 expression is required for early HF formation, as comparable numbers of placodes and condensates appeared at all embryonic HF waves. While Cxcr4 expression in embryonic skin is dispensable for HF formation, this study reveals a novel marker that highlights either compartment of nascent HFs in a stage-dependent manner.

        271 Expression of Tlr4 is enhanced by co-expression of CD34 and CD49f in murine keratinocyte stem cells

        Y Li,1 A Singh,1 J Akech,2 M Wang,2 N Readio,1 K Johnson,1 B Li1 and RJ Morris11 Stem Cells and Cancer, Hormel Institute, Austin, MN and 2 Advanced Cell Diagnostics, Inc., Hayward, CA
        Epidermal keratinocytes (KCs) normally express several Toll-like receptors (Tlr) including Tlr4, the latter widely recognized as an essential element in the triggering of innate immunity, binding pathogen-associated molecules such as lipopolysaccharide (LPS), and initiating a cascade of pro-inflammatory events. However, the regulation of Tlr4 expression in KCs, particularly CD34+/CD49f+ keratinocyte hair follicle stem cells (KSCs), remains unclear. To address this problem, four subpopulations were sorted from freshly harvested Tlr4 wild-type and mutant KCs by fluorescence activated cell sorting followed by quantitative real-time polymerase chain reaction. Tlr4 mRNA was visualized by RNAscope hybridization to sections of skin. We found that the expression of CD34 and its transcription factors such as Sox9, Lhx2, Nfatc1 was similar between CD34+/CD49f+ (KSCs) and CD34+/CD49f- from Tlr4 wild-type and mutant mice (p=0.9097 for CD34, 0.0611 for Sox9, 0.8734 for Lhx2, 0.1124 for Nfatc1). The expression of those genes in bulge stem cells was significantly higher than that of CD34-/CD49f+ basal KCs (p=0.00038 for CD34, 0.000203 for Sox9, 1.784E-05 for Lhx2, 4.806E-05 for Nfatc1). However, Tlr4 expression in KSCs was at least 30-fold higher than that of CD34-/CD49f+ (p=0.000189) and at least 15-fold higher than that of CD34+CD49f- (p=0.000483). In situ hybridization in skin sections confirmed the enrichment of Tlr4 mRNA signal in cells of the hair follicle bulge. After LPS stimulation, the expression of Cxcl1, Tnf, and IL1-alpha in KSCs was respectively 5-, 3-, and 25-fold lower than that of the CD34-/CD49f+ population. These results suggest that expression of Tlr4 mRNA is enhanced by co-expression of CD34 and CD49f in murine KSCs. We conclude that their lack of LPS responsiveness may be a consequence of KSC immune privilege.

        272 Mouse models of cicatricial alopecia

        C Potter,1 CH Pratt,1 H Hama,2 K Wolk,1,3 KA Silva,1 VE Kennedy,1 HB Everts,3 LE King4 and JP Sundberg1,41 The Jackson Laboratory, Bar Harbor, ME, 2 Medical University of South Carolina, Charleston, SC, 3 The Ohio State University, Columbus, OH and 4 The Skin Disease Research Center, Vanderbilt University, Nashville, TN
        Cicatricial alopecia's (CA) are a group of skin diseases in which there is a progressive and permanent destruction of the hair follicle followed by replacement with fibrous tissue. Unfortunately, in human patients by the time they seek clinical evaluation their skin disease is already inflamed and/or scarred, essentially well into the pathogenesis of the disease with little hope for a return to normal hair growth. Animal models, and in particular laboratory mice, have been a valued resource in advancing understanding of CA. Here we compare and contrast current laboratory mouse models with mutations in the following genes, resulting in scarring alopecia: Scd1, Pparg, Gsdma3, Foxn1, Hoxc13, and Tal1. Each of these mutant lines is a valuable tool to advance understanding of the complex fundamental pathophysiology of analogous human primary CA diseases. These mutant mice, many of which are accessible through The Jackson Laboratory Mutant Mouse Repository, are also critical in the battle to identify new targeted therapeutic approaches to treating patients with CA.

        273 Squamous epithelial organization and quiescent stem cells: The concept of the “reserve cell layer”

        T Andl,1 G LeBras,2 H Loomans,2 KA Clay1 and CD Andl21 Medicine, Vanderbilt University, Nashville, TN and 2 Surgery, Vanderbilt University, Nashville, TN
        Studies of mouse epidermis have been pivotal to our understanding of epidermal biology. However, in striking contrast to other epithelia such as the colon, little is known about the stem cells of the epidermis and whether a defined stem cell population actually exists. The same is true for all the other squamous epithelia. Here, we compared the organization of different squamous epithelia from rodents and other mammals to identify a common core of basal cell layer markers. Since the basal layer is the putative home of squamous epithelial stem cells such knowledge will help to shed light on the regulation of stemness. In contrast to rodents, all other mammals tested (human, macaque, opossum, dog and pig) exhibited a quiescent basal cell layer, the reserve cell layer, in their oral mucosae. On the other hand, mouse mucosa showed characteristics similar to the epidermis: a basal cell layer that contains the majority of the proliferating cells. This separation of proliferation and basal cell features cannot be detected in epidermi of any species. The markers, we have identified for the reserve cell layer (RCMs) support the notion that this cell layer is maintained in a quiescent state by TGFbeta signaling. Of the 144 reserve cell markers that we have identified, 68 are TGFbeta target genes, while only 16 of 126 parabasal markers are associated with TGFbeta signaling. Using systems biology and bioinformatics tools, we identified MAPK9 (JNK2) as a key hub protein of the reserve cell marker set linking TGFbeta, Hippo and MAPK signaling. Surprisingly, the majority of the reserve cell markers are also expressed in the basal layer of the epidermis, however mixed with cells expressing proliferation markers. Treatment of keratinocytes with chemotherapeutic drugs induced a subset of RCMs while inhibiting proliferation marker expression. This may have implications for the treatment of hyper-proliferative diseases and cancer, which focus on inhibition of growth.

        274 Regulation of ear morphogenesis by macroenvironmental signals from hair follicles

        R Ramos, T Hsi, J Oh, CF Guerrero-juarez and M Plikus Developmental and Cell Biology, University of California, Irvine, Irvine, CA
        Hair follicle (HF) regeneration involves not only intrinsic signals from different HF compartments, but also inputs coming from the extra-follicular macroenvironment. Such signals can come from neighboring HFs, adipocytes, pre-adipocytes, and possibly other tissue-resident skin cells, allowing for collective regenerative behavior. We posit that skin also provides an ideal model to study the macroenvironmental regulation of morphogenesis. In the mouse external ear, a 2-3 cell-thick sheet of chondrocytes located in the center, comes in direct contact with HFs of the ear skin on both sides. We show that in mouse ear, development of HFs and cartilage closely overlaps in time and in space, providing a novel model to study signaling crosstalk between their respective morphogenetic programs. In the HF, WNT signaling acts to promote early morphogenesis and cyclic regeneration. In the cartilage, WNTs have been implicated in chondroblast proliferation and chondrocyte differentiation. Here, we evaluated the possibility of WNT signaling crosstalk in the shared ear skin / cartilage macroenvironment by modulating WNT signaling outputs using transgenic approaches. We first analyzed the effects of epithelial WNT overexpression on cartilage morphogenesis. Because ear shape and size are directly proportional to cartilage cell number, ear morphology can be used as a sensitive rheostat of chondrocyte population size. Mice overexpressing epithelial Wnts have significantly larger ears compared to WT control; consistent with the known function of WNT in delaying the onset of terminal chondrocyte differentiation. However, in a mouse model expressing soluble WNT antagonist in skin epithelia, ears become misshapen and notably elongated, but, surprisingly, not smaller. These results suggest that ear HFs and cartilage morphogenesis occur in shared signaling macroenvironment and reveal HF-derived WNT signaling inputs as functionally important modulators of auricular chondrogenesis.

        275 Skin microbiome – metabolome interplay in the pathogenesis of frontal fibrosing alopecia

        S Shah,1,2 R Subbaiah,1,2 MC Consolo,1,2 P Mirmirani,1,2 VH Price1,2 and P Karnik1,21 Dermatology, Case Western Reserve University, Cleveland, OH and 2 Dermatology, Univ Hosp Case Med Center, Cleveland, OH
        The incidence of Frontal Fibrosing Alopecia (FFA), a lymphocyte mediated cicatricial alopecia, is thought to be rapidly increasing both among pre- and post-menopausal women. However, the etiology and pathogenic mechanisms of FFA remain poorly defined. Here, we report a global mass spectrometry (MS) based metabolomics study that demonstrates the effects of the skin ‘‘microbiome’’ on the host metabolic pathways in FFA. Scalp skin extracts from paired unaffected and affected FFA (N=12) were compared with normal healthy controls (N=6) by using LC/MS and GC/MS-based platforms. Welch's two-sample t-test was used to identify metabolites that differed significantly between unaffected or affected FFA from normal controls. Significant metabolic differences were noted in unaffected and affected FFA scalp skin and normal controls. Increased oxidative stress, elevated glycolytic flux and lactate production, and decreased TCA cycle activity suggestive of mitochondrial dysfunction were observed in FFA. Intriguingly, a number of bacterial metabolites derived from the breakdown of host aromatic amino acids were significantly increased in FFA. Tryptophan, phenylalanine and tyrosine derived bioactive indole and phenyl metabolites including indolelactate, indoleacetate and phenol sulfate were significantly increased in unaffected and affected FFA compared to normal controls. The uremic toxin 3-carboxy-4-methyl-5-pentyl-2-furanpropionic acid (CMPF), a known inhibitor of mitochondrial respiration was also significantly increased in FFA. The presence of sulfated (phenol sulfate) and glucuronide (CMPF) metabolites suggests a xenobiotic response of the host to the bacterial metabolites in FFA. Our metabolomics data provides novel insights into the interplay between bacterial and host metabolism in the skin and in the pathogenesis of FFA.

        276 Hair cycles of transplanted human hairs in immunocompromised mice

        J Oh,1,2 M Kim,2 M Plikus1 and J Kim21 Developmental and Cell Biology, University of California, Irvine, Irvine, CA and 2 Department of Immunology and Hair Research Center, Kyungpook National University School of Medicine, Daegu, Republic of Korea
        Growth of human scalp hair follicles is characterized by short telogen and prolonged anagen, often lasting many years. This makes it difficult to reliably capture the event of telogen-to-anagen transition. Because anagen initiation is a clinically relevant event in the human hair growth cycle, there is an unmet need for a better experimental model. In this study we aimed to characterize temporal dynamics of human scalp hair follicle regeneration in human-on mouse xenotransplantation model. We transplanted human scalp follicles into the skin of immuno-compromised nude (Foxn1 null) and SCID mice and carefully analyzed temporal dynamics of post-transplantation telogen-to-anagen transitions on serial-section histology and immunohistochemistry. Hair follicles were analyzed for up to one year after xenotransplantation, initially with 7-day intervals. Based on our histological studies and mathematical analysis we were able to construct a detailed probabilistic timeline of post-transplantation hair cycle phases. We distinguished the following phases: transplantation-induced catagen stages I, II, III, transitional stage (aka new anagen initiation), anagen II, III, IV, V, VI. Probabilistically, the peak in transitional stage hair follicles occured on post-transplantations day 33 in SCID mice and day 37 in nude mice. Our results provide a detailed experimental blueprint for future studies tasked with studying signaling regulation of human anagen initiation in human-on-mouse xenotransplantation model. Importantly, they also reveal that the choice of host (nude vs. SCID mouse) as an important experimental parameter to consider.

        277 Sonic hedgehog signaling is essential for touch dome Merkel cell development

        Y Xiao, JS Williams and I BrownellDermatology Branch, NCI, NIH, Bethesda, MD
        The touch dome (TD) is a specialized epidermal structure that contains Merkel cells (MCs) and mediates touch sensation. In embryonic day 14.5 (E14.5) mice, a primary wave of hair follicle induction takes place. These follicles produce guard hairs that comprise ∼2% of the adult mouse coat. Developing MCs are first detected ∼E15.5 in association with nascent guard hair germs. No MCs form in association with awl and auchene follicles induced ∼E16.6, or zig-zag follicles induced ∼E18.5. MCs do not form in Eda mutant Tabby mice that lack the primary wave of follicle induction, and thus lack guard hairs. We used genetically modified mice to investigate the developmental signaling requirements for TD and MC specification. The Eda receptor, Edar, is upregulated in primary hair placodes, and downless mice with mutant Edar lack guard hairs. We found that downless mice also failed to form TD MCs, confirming the requirement for Eda signaling to guard hair placodes. Interestingly, fate-mapping experiments with Shh-Cre mice demonstrated that TD MCs arise from tissue outside the hair follicle lineage. As Shh is one of the early signaling molecules produced by the hair placode, and Shh is specifically lost in Eda mutant mice, we considered its role in TD specification. Shh mutant mice have defective follicle development where hair germs form, but do not elongate into follicles. We found that Shh null mice lack TD MCs, suggesting that Shh from the primary hair follicles is necessary for the specification of TD MCs. In contrast, TDs with MCs formed in nude mice that have dystrophic hair follicles, and persisted in adult hairless mice after cystic degeneration of their hair follicles, demonstrating that not all disruptions of guard hair follicles result in a loss of TD MCs. These findings suggest that TD specification requires reciprocal signaling within the developing ectoderm including Eda signaling to primary hair placodes, and subsequent Shh signaling from primary hair germs to TD anlages.

        278 Inducible gene expression in the dermal papilla in vivo to study anagen initiation

        B MorganCutaneous Biology Research Center, Harvard Medical School, Boston, MA
        Loss of function studies have revealed the requirement for the activation of several growth factor signaling pathways in the dermal papilla to regenerate an anagen follicle in the adult mouse. Anagen re-entry results from complex integration of inputs from the different compartments within a follicle as well as signals from adjacent follicles, other components of the skin and beyond. A system for the reliable induction of signaling molecules in the DP allows assessment of the sufficiency of signaling inputs from the DP to alter anagen re-entry through broad areas of the skin. This exploits the different signaling environments in different regions of the skin. It also avoids the difficulties of achieving controlled and reliable delivery of growth factors and eliminates other stimuli that affect anagen entry associated with other methods of exogenous factor delivery. Here we report doxycycline inducible expression of growth factors in the DP of adult mice. Using this system we probe the function of FGFs expressed by the DP in the process of anagen initiation. Previous work had reported that FGF7 or 10 is not sufficient to initiate anagen in telogen skin. While our work confirms this conclusion in some contexts, we find induced expression accelerates anagen onset in a context dependent fashion. Once anagen has begun, augmented FGF expression does not alter the structure of the hair or the duration of the hair cycle.

        279 Endoplasmic reticulum (ER) chaperone GRP78 is required for epidermal self-renewal and differentiation during skin morphogenesis

        Y Leung,1,2 AS Lee3,4 and K Kobielak1,21 The Eli and Edythe Broad Center for Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, CA, 2 Pathology, University of Southern California, Los Angeles, CA, 3 Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles, CA and 4 Norris Cancer Center, University of Southern California, Los Angeles, CA
        Glucose regulated protein 78kDa (GRP78), is a major endoplasmic reticulum (ER) chaperone and a master regulator of ER stress signaling. As a key effector of the protective arm of the unfolded protein response (UPR), GRP78 facilitates protein folding protecting cells from unfolded and misfolded proteins in ER and exhibits potent anti-apoptotic properties. GRP78 deletion results in early embryo lethality at the peri-implantation stage at E3.5 from proliferation defects and apoptosis of the inner cell mass (ICM). However, even though GRP78 is essential for initial development of ICM, still little is known about the role of GRP78 in different tissue development and differentiation at later stages. In this study, we aim to unveil the role of GRP78 during tissue morphogenesis using the skin as a model system to delete this gene later specifically in the basal layer of the developing epithelium. Here, we report creation of a novel mouse model where GRP78 was specifically ablated in the basal layer of the skin. GRP78 knockout (KO) mice fail to form the stratified layers of the skin epidermis and in consequence skin barrier function was compromised. Here we showed that GRP78 KO cells lack proper epithelial differentiation as it undergoes apoptosis demonstrating that GRP78 is crucial for proper epithelial differentiation and skin development. Additionally, we revealed that GRP78 regulates epidermal self-renewal. Together, these studies add new insights into the role of GRP78 later in tissue morphogenesis.

        280 Sexually-dimorphic responses to pharmacologic inhibition of HSP90 by subpopulations of stem cells in the hair

        S Krishnaswami, C Adase, J Gutierrez and BD Yu Division of Dermatology, Stem Cell Program, Institute for Genomic Medicine, UCSD, La Jolla, CA
        Activation of the hair cycle requires coordinate regulation of components of the cyclin and cyclin-dependent kinases (CDK). To investigate the contribution of CDK4 chaperones during hair cycle activation, we investigated the hair cycle response to pharmacologic inhibition of HSP90 using topically applied geldanamycin-derivative (17-AAG). In female mice, HSP90 inhibition suppresses hair cycle activation in 80% of treated animals. Surprisingly, male mice demonstrate 100% resistance to 17-AAG. To rule-out differences in metabolism, mice were also treated with HSP90 inhibitors, which are resistant to differential expression of P-glycoprotein and NQO1-diaphorase, but resulted in similar findings. During hair cycle activation, HSP90 was found to be induced in the lower bulge region and the hair germ. HSP90 expression was sensitive to cyclosporine treatment, and in cell-based assays, a direct role for NFAT-mediated repression of HSP90 from distant cis-regulatory elements was identified. These studies reveal unexpected sexual dimorphism in the pharmacologic response of adult stem cells and may have major implications in the treatment of hair disease and cancer.