Finding meaning in chaos: a selection signature for functional interactions and its use in molecular biology.

A primary goal of biomedical research is to elucidate molecular mechanisms, particularly those responsible for human traits, either normal or pathological. Yet achieving this goal is difficult if not impossible when the traits of interest lack tractable models and so cannot be dissected through time-honoured approaches like forward genetics or reconstitution. Arguably, no biological problem has hindered scientific progress more than this: the inability to dissect a trait's mechanism without a tractable likeness of the trait. At root, forward genetics and reconstitution are powerful approaches because they assay for specific molecular functions. Here, we discuss an alternative way to uncover important mechanistic interactions, namely, to assay for positive natural selection. If an interaction has been selected for, then it must perform an important function, a function that significantly promotes reproductive success. Accordingly, selection is a consequence and indicator of function, and uncovering multimolecular selection will reveal important functional interactions. We propose a selection signature for interactions and review recent selection-based approaches through which to dissect traits that are not inherently tractable. The review includes proof-of-principle studies in which important interactions were uncovered by screening for selection. In sum, screens for selection appear feasible when screens for specific functions are not. Selection screens thus constitute a novel tool through which to reveal the mechanisms that shape the fates of organisms.

Modeling by disruption and a selected-for partner for the nude locus.

A long-standing problem in biology is how to dissect traits for which no tractable model exists. Here, we screen for genes like the nude locus (Foxn1)-genes central to mammalian hair and thymus development-using animals that never evolved hair, thymi, or Foxn1. Fruit flies are morphologically disrupted by the FOXN1 transcription factor and rescued by weak reductions in fly gene function, revealing molecules that potently synergize with FOXN1 to effect dramatic, chaotic change. Strong synergy/effectivity in flies is expected to reflect strong selection/functionality (purpose) in mammals; the more disruptive a molecular interaction is in alien contexts (flies), the more beneficial it will be in its natural, formative contexts (mammals). The approach identifies Aff4 as the first nude-like locus, as murine AFF4 and FOXN1 cooperatively induce similar cutaneous/thymic phenotypes, similar gene expression programs, and the same step of transcription, pre-initiation complex formation. These AFF4 functions are unexpected, as AFF4 also serves as a scaffold in common transcriptional-elongation complexes. Most likely, the approach works because an interaction's power to disrupt is the inevitable consequence of its selected-for power to benefit.

Direct control of regulatory T cells by keratinocytes.

Environmental challenges to epithelial cells trigger gene expression changes that elicit context-appropriate immune responses. We found that the chromatin remodeler Mi-2ß controls epidermal homeostasis by regulating the genes involved in keratinocyte and immune-cell activation to maintain an inactive state. Mi-2ß depletion resulted in rapid deployment of both a pro-inflammatory and an immunosuppressive response in the skin. A key target of Mi-2ß in keratinocytes is the pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP). Loss of TSLP receptor (TSLPR) signaling specifically in regulatory T (Treg) cells prevented their activation and permitted rapid progression from a skin pro-inflammatory response to a lethal systemic condition. Thus, in addition to their well-characterized role in pro-inflammatory responses, keratinocytes also directly support immune-suppressive responses that are critical for re-establishing organismal homeostasis.

A composite enhancer regulates p63 gene expression in epidermal morphogenesis and in keratinocyte differentiation by multiple mechanisms.

p63 is a crucial regulator of epidermal development, but its transcriptional control has remained elusive. Here, we report the identification of a long-range enhancer (p63LRE) that is composed of two evolutionary conserved modules (C38 and C40), acting in concert to control tissue- and layer-specific expression of the p63 gene. Both modules are in an open and active chromatin state in human and mouse keratinocytes and in embryonic epidermis, and are strongly bound by p63. p63LRE activity is dependent on p63 expression in embryonic skin, and also in the commitment of human induced pluripotent stem cells toward an epithelial cell fate. A search for other transcription factors involved in p63LRE regulation revealed that the CAAT enhancer binding proteins Cebpa and Cebpb and the POU domain-containing protein Pou3f1 repress p63 expression during keratinocyte differentiation by binding the p63LRE enhancer. Collectively, our data indicate that p63LRE is composed of additive and partly redundant enhancer modules that act to direct robust p63 expression selectively in the basal layer of the epidermis.

Skin as a living coloring book: how epithelial cells create patterns of pigmentation.

The pigmentation of mammalian skin and hair develops through the interaction of two basic cell types - pigment donors and recipients. The pigment donors are melanocytes, which produce and distribute melanin through specialized structures. The pigment recipients are epithelial cells, which acquire melanin and put it to use, collectively yielding the pigmentation visible to the eye. This review will focus on the pigment recipients, the historically less understood cell type. These end-users of pigment are now known to exert a specialized control over the patterning of pigmentation, as they identify themselves as melanocyte targets, recruit pigment donors, and stimulate the transfer of melanin. As such, this review will discuss the evidence that the skin is like a coloring book: the pigment recipients create a 'picture,' a blueprint for pigmentation, which is colorless initially but outlines where pigment should be placed. Melanocytes then melanize the recipients and 'color in' the picture.

A pair of transmembrane receptors essential for the retention and pigmentation of hair.

Hair follicles are simple, accessible models for many developmental processes. Here, using mutant mice, we show that Bmpr2, a known receptor for bone morphogenetic proteins (Bmps), and Acvr2a, a known receptor for Bmps and activins, are individually redundant but together essential for multiple follicular traits. When Bmpr2/Acvr2a function is reduced in cutaneous epithelium, hair follicles undergo rapid cycles of hair generation and loss. Alopecia results from a failure to terminate hair development properly, as hair clubs never form, and follicular retraction is slowed. Hair regeneration is rapid due to premature activation of new hair-production programs. Hair shafts differentiate aberrantly due to impaired arrest of medullary-cell proliferation. When Bmpr2/Acvr2a function is reduced in melanocytes, gray hair develops, as melanosomes differentiate but fail to grow, resulting in organelle miniaturization. We conclude that Bmpr2 and Acvr2a normally play cell-type-specific, necessary roles in organelle biogenesis and the shutdown of developmental programs and cell division.

A positive FGFR3/FOXN1 feedback loop underlies benign skin keratosis versus squamous cell carcinoma formation in humans.

Seborrheic keratoses (SKs) are common, benign epithelial tumors of the skin that do not, or very rarely, progress into malignancy, for reasons that are not understood. We investigated this by gene expression profiling of human SKs and cutaneous squamous cell carcinomas (SCCs) and found that several genes previously connected with keratinocyte tumor development were similarly modulated in SKs and SCCs, whereas the expression of others differed by only a few fold. In contrast, the tyrosine kinase receptor FGF receptor-3 (FGFR3) and the transcription factor forkhead box N1 (FOXN1) were highly expressed in SKs, and close to undetectable in SCCs. We also showed that increased FGFR3 activity was sufficient to induce FOXN1 expression, counteract the inhibitory effect of EGFR signaling on FOXN1 expression and differentiation, and induce differentiation in a FOXN1-dependent manner. Knockdown of FOXN1 expression in primary human keratinocytes cooperated with oncogenic RAS in the induction of SCC-like tumors, whereas increased FOXN1 expression triggered the SCC cells to shift to a benign SK-like tumor phenotype, which included increased FGFR3 expression. Thus,we have uncovered a positive regulatory loop between FGFR3 and FOXN1 that underlies a benign versus malignant skin tumor phenotype.

ABCA12 maintains the epidermal lipid permeability barrier by facilitating formation of ceramide linoleic esters.

Harlequin ichthyosis is a congenital scaling syndrome of the skin in which affected infants have epidermal hyperkeratosis and a defective permeability barrier. Mutations in the gene encoding a member of the ABCA transporter family, ABCA12, have been linked to harlequin ichthyosis, but the molecular function of the protein is unknown. To investigate the activity of ABCA12, we generated Abca12 null mice and analyzed the impact on skin function and lipid content. Abca12-/- mice are born with a thickened epidermis and die shortly after birth, as water rapidly evaporates from their skin. In vivo skin proliferation measurements suggest a lack of desquamation of the skin cells, rather than enhanced proliferation of basal layer keratinocytes, accounts for the 5-fold thickening of the Abca12-/- stratum corneum. Electron microscopy revealed a loss of the lamellar permeability barrier in Abca12-/- skin. This was associated with a profound reduction in skin linoleic esters of long-chain omega-hydroxyceramides and a corresponding increase in their glucosyl ceramide precursors. Because omega-hydroxyceramides are required for the barrier function of the skin, these results establish that ABCA12 activity is required for the generation of long-chain ceramide esters that are essential for the development of normal skin structure and function.

The kinase p38 alpha serves cell type-specific inflammatory functions in skin injury and coordinates pro- and anti-inflammatory gene expression.

The mitogen-activated protein kinase p38 mediates cellular responses to injurious stress and immune signaling. Among the many p38 isoforms, p38 alpha is the most widely expressed in adult tissues and can be targeted by various pharmacological inhibitors. Here we investigated how p38 alpha activation is linked to cell type-specific outputs in mouse models of cutaneous inflammation. We found that both myeloid and epithelial p38 elicit inflammatory responses, yet p38 alpha signaling in each cell type served distinct inflammatory functions and varied depending on the mode of skin irritation. In addition, myeloid p38 alpha limited acute inflammation via activation of anti-inflammatory gene expression dependent on mitogen- and stress-activated kinases. Our results suggest a dual function for p38 alpha in the regulation of inflammation and show mixed potential for its inhibition as a therapeutic strategy.

Dedicated epithelial recipient cells determine pigmentation patterns.

Mammals generate external coloration via dedicated pigment-producing cells but arrange pigment into patterns through mechanisms largely unknown. Here, using mice as models, we show that patterns ultimately emanate from dedicated pigment-receiving cells. These pigment recipients are epithelial cells that recruit melanocytes to their position in the skin and induce the transfer of melanin. We identify Foxn1 (a transcription factor) as an activator of this "pigment recipient phenotype" and Fgf2 (a growth factor and Foxn1 target) as a signal released by recipients. When Foxn1 - and thus dedicated recipients - are redistributed in the skin, new patterns of pigmentation develop, suggesting a mechanism for the evolution of coloration. We conclude that recipients provide a cutaneous template or blueprint that instructs melanocytes where to place pigment. As Foxn1 and Fgf2 also modulate epithelial growth and differentiation, the Foxn1 pathway should serve as a nexus coordinating cell division, differentiation, and pigmentation.

Foxn1 promotes keratinocyte differentiation by regulating the activity of protein kinase C.

The transcription factor Foxn1 (the product of the nude locus) promotes the terminal differentiation of epithelial cells in the epidermis and hair follicles. Activated early in terminal differentiation, Foxn1 can modulate the timing or order of trait acquisition, as it induces early features of epidermal differentiation while suppressing late features. Here, we identify protein kinase C (PKC) as a key target of Foxn1 in keratinocyte differentiation control. Foxn1 has broad negative effects on the PKC family, as the loss of Foxn1 function leads to higher levels of total, primed, and activated PKC. Phosphorylated PKC substrates (the mediators of PKC function) rise when Foxn1 is inactivated and fall when Foxn1 is overproduced, suggesting that Foxn1 antagonizes PKC's effects. When PKC inhibitors are applied to nude (Foxn1 null) keratinocytes, nude defects are normalized or suppressed, as the inhibitors prevent nude cells from underproducing early differentiation markers and overproducing late markers. Taken together, the results suggest that Foxn1 acts as a restraint or brake on PKC signaling and that without this brake PKC disrupts differentiation. The results further suggest that Foxn1 modulates stage-specific markers by modulating PKC activity, providing control over the timing of steps in the differentiation program.

Bone morphogenetic protein signaling regulates the size of hair follicles and modulates the expression of cell cycle-associated genes.

Bone morphogenetic protein (BMP) signaling is involved in the regulation of a large variety of developmental programs, including those controlling organ sizes. Here, we show that transgenic (TG) mice overexpressing the BMP antagonist noggin (promoter, K5) are characterized by a marked increase in size of anagen hair follicles (HFs) and by the replacement of zig-zag and auchen hairs by awl-like hairs, compared with the age-matched WT controls. Markedly enlarged anagen HFs of TG mice show increased proliferation in the matrix and an increased number of hair cortex and medulla cells compared with WT HFs. Microarray and real-time PCR analyses of the laser-captured hair matrix cells show a strong decrease in expression of Cdk inhibitor p27(Kip1) and increased expression of selected cyclins in TG vs. WT mice. Similar to TG mice, p27(Kip1) knockout mice also show an increased size of anagen HFs associated with increased cell proliferation in the hair bulb. Primary epidermal keratinocytes (KC) from TG mice exhibit significantly increased proliferation and decreased p27(Kip1) expression, compared with WT KC. Alternatively, activation of BMP signaling in HaCaT KC induces growth arrest, stimulates p27(Kip1) expression, and positively regulates p27(Kip1) promoter activity, thus further supporting a role of p27(Kip1) in mediating the effects of BMP signaling on HF size. These data suggest that BMP signaling plays an important role in regulating cell proliferation and controls the size of anagen HFs by modulating the expression of cell-cycle-associated genes in hair matrix KC.

An autoregulatory loop directs the tissue-specific expression of p63 through a long-range evolutionarily conserved enhancer.

p63, a p53 family member, is essential for the development of various stratified epithelia and is one of the earliest markers of many ectodermal structures, including the epidermis, oral mucosa, apical ectodermal ridge, and mammary gland. Genetic regulatory mechanisms controlling p63 spatial expression during development have not yet been defined. Using a genomic approach, we identified an evolutionarily conserved cis-regulatory element, located 160 kb downstream of the first p63 exon, which functions as a keratinocyte-specific enhancer and is sufficient to recapitulate expression of the endogenous gene during mouse embryogenesis. Dissection of the p63 enhancer activity revealed a positive autoregulatory loop in which the p63 proteins directly bind to and are essential regulators of the enhancer. Accordingly, transactivating p63 isoforms induce endogenous p63 expression in cells that do not normally express this gene, whereas dominant negative isoforms suppress p63 expression in keratinocytes. In addition the transcription factor AP-2 also binds to the enhancer and cooperates with p63 to induce its activity. These results demonstrate that a long-range autoregulatory loop is involved in the regulation of p63 expression during embryonic development and in adult cells.

Phosphoinositide 3-kinase signaling to Akt promotes keratinocyte differentiation versus death.

Signaling pathways regulating the differentiation program of epidermal cells overlap widely with those activated during apoptosis. How differentiating cells remain protected from premature death, however, is still poorly defined. We show here that the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated at early stages of mouse keratinocyte differentiation both in culture and in the intact epidermis in vivo. Expression of active Akt in keratinocytes promotes growth arrest and differentiation, whereas pharmacological blockade of PI3K inhibits the expression of "late" differentiation markers and leads to death of cells that would otherwise differentiate. Mechanistically, the activation of the PI3K/Akt pathway in keratinocyte differentiation depends on the activity of the epidermal growth factor receptor and Src families of tyrosine kinases and the engagement of E-cadherin-mediated adhesion. During this process, PI3K associates increasingly with cadherin-catenin protein complexes bearing tyrosine phosphorylated YXXM motifs. Thus, the PI3K signaling pathway regulates the choice between epidermal cell differentiation and death at the cross-talk between tyrosine kinases and cadherin-associated catenins.

Bone morphogenetic protein (BMP) signaling controls hair pigmentation by means of cross-talk with the melanocortin receptor-1 pathway.

Hair pigmentation is controlled by tightly coordinated programs of melanin synthesis and involves signaling through the melanocortin type 1 receptor (MC-1R) that regulates the switch between pheomelanogenesis and eumelanogenesis. However, the involvement of other signaling systems, including the bone morphogenetic protein (BMP) pathway, in the control of hair pigmentation remains to be elucidated. To assess the effects of BMP signaling on hair pigmentation, transgenic mice overexpressing the BMP antagonist noggin (promoter: keratin 5) were generated. Whereas wild-type C3H/HeJ mice have a subapical yellow band on otherwise black dorsal hairs, K5-Noggin mice are characterized by the absence of a yellow band and near-black pigment in dorsal coat. Noggin overexpression is accompanied by strongly reduced levels of Agouti signal protein and enhanced expression of microphthalmia transcription factor in the midphase of the hair-growth cycle. Wild-type color in K5-Noggin mice is restored by administration of a synthetic MC-1R antagonist resulting in the reappearance of a subapical yellow band. BMP-4 stimulates the expression of Agouti transcripts and protein in primary epidermal keratinocytes, and BMP signaling positively regulates dermal papilla-specific enhancer of the Agouti gene in primary dermal fibroblasts. Taken together, these data suggests that BMP signaling controls the expression of Agouti protein in the hair follicle and provide evidence for interaction between BMP and MC-1R signaling pathways to modulate the balance between pheomelanogenesis and eumelanogenesis during hair growth.

Ectodysplasin regulates pattern formation in the mammalian hair coat.

In mammalian skin, hair follicles develop at regular intervals and with site-specific morphologies. This process generates distinct patterns of hair, but the mechanisms that establish these patterns remain largely unknown. Here we present evidence of follicular patterning by ectodysplasin-A1 (Eda-A1), a signaling protein necessary for the proper development of hair and other appendages. In transgenic mice, Eda-A1 was targeted to the epithelial compartment of the developing skin. At periodic locations, multiple hair follicles were induced side by side, without any interfollicular space. These follicles grew into the dermis as a fusion and subsequently branched to create discrete stalks and hair bulbs. Thus, at sites where interfollicular skin normally forms, hair follicles developed instead. This result shows that Eda-A1 can regulate basic developmental decisions, as cells were switched from interfollicular to follicular fates. Given these effects, it is likely that Eda-A1 is among the key regulators of pattern formation in the skin.

Noggin overexpression inhibits eyelid opening by altering epidermal apoptosis and differentiation.

Contact of developing sensory organs with the external environment is established via the formation of openings in the skin. During eye development, eyelids first grow, fuse and finally reopen, thus providing access for visual information to the retina. Here, we show that eyelid opening is strongly inhibited in transgenic mice overexpressing the bone morphogenetic protein (BMP) antagonist noggin from the keratin 5 (K5) promoter in the epidermis. In wild-type mice, enhanced expression of the kinase-inactive form of BMPR-IB mediated by an adenovirus vector also inhibits eyelid opening. Noggin overexpression leads to reduction of apoptosis and retardation of cell differentiation in the eyelid epithelium, which is associated with downregulation of expression of the apoptotic receptors (Fas, p55 kDa TNFR), Id3 protein and keratinocyte differentiation markers (loricrin, involucrin). BMP-4, but not EGF or TGF-alpha, accelerates opening of the eyelid explants isolated from K5-Noggin transgenic mice when cultured ex vivo. These data suggest that the BMP signaling pathway plays an important role in regulation of genetic programs of eyelid opening and skin remodeling during the final steps of eye morphogenesis.

Redefining the skin's pigmentary system with a novel tyrosinase assay.

In mammalian skin, melanin is produced by melanocytes and transferred to epithelial cells, with the epithelial cells thought to receive pigment only and not generate it. Melanin formation requires the enzyme tyrosinase, which catalyzes multiple reactions in the melanin biosynthetic pathway. Here, we reassess cutaneous melanogenesis using tyramide-based tyrosinase assay (TTA), a simple test for tyrosinase activity in situ. In the TTA procedure, tyrosinase reacts with biotinyl tyramide, causing the substrate to deposit near the enzyme. These biotinylated deposits are then visualized with streptavidin conjugated to a fluorescent dye. In the skin and eye, TTA was highly specific for tyrosinase and served as a sensitive indicator of pigment cell distribution and status. In clinical skin samples, the assay detected pigment cell defects, such as melanocytic nevi and vitiligo, providing confirmation of medical diagnoses. In murine skin, TTA identified a new tyrosinase-positive cell type--the medullary cells of the hair--providing the first example of cutaneous epithelial cells with a melanogenic activity. Presumably, the epithelial tyrosinase originates in melanocytes and is acquired by medullary cells during pigment transfer. As tyrosinase by itself can generate pigment from tyrosine, it is likely that medullary cells produce melanin de novo. Thus, we propose that melanocytes convert medullary cells into pigment cells by transfer of the melanogenic apparatus, an unusual mechanism of differentiation that expands the skin's pigmentary system.

Role of the nude gene in epithelial terminal differentiation.

Loss-of-function mutations in Whn (Hfh11, Foxn1), a winged-helix/forkhead transcription factor, cause the nude phenotype, which is characterized by the abnormal morphogenesis of the epidermis, hair follicles, and thymus. To delineate the biochemical pathway of Whn, we investigated its upstream regulation and downstream effects using primary keratinocytes from wild-type and transgenic mice. The transgenic animals express whn from the involucrin promoter, which is active in keratinocytes undergoing terminal differentiation. In wild-type cultures, as in the epidermis, Whn was induced during the early stages of terminal differentiation and declined during later stages. In transgenic keratinocytes, whn overexpression altered the terminal differentiation program, stimulating an early differentiation marker (keratin 1) and suppressing later markers (profilaggrin, loricrin, and involucrin). These results suggest a role for Whn in the stepwise or temporal regulation of differentiation, as Whn can ensure that the differentiation program is carried out in proper sequence. Before the start of differentiation, Whn levels were suppressed by the p42/p44 mitogen-activated protein kinase cascade, and this signaling pathway was rapidly inactivated as differentiation began. Thus, as keratinocytes commit to terminal differentiation, mitogen-activated protein kinase signaling decreases, which permits the induction of Whn; Whn then activates early features of the differentiation program.