In this issue of Developmental Cell, Lopez-Pajares et al. (2015) identify regulatory networks that are controlled by the transcriptional regulators MAF:MAFB. These networks play a crucial and previously unrecognized role in epidermal differentiation, functioning both to repress progenitor genes and to activate early and late differentiation genes.

Mammary gland branching morphogenesis and ductal homeostasis relies on mammary stem cell function for the maintenance of basal and luminal cell compartments. The mechanisms of transcriptional regulation of the basal cell compartment are currently unknown. We explored these mechanisms in the basal cell compartment and identified the Co-factor of LIM domains (CLIM/LDB/NLI) as a transcriptional regulator that maintains these cells. Clims act within the basal cell compartment to promote branching morphogenesis by maintaining the number and proliferative potential of basal mammary epithelial stem cells. Clim2, in a complex with LMO4, supports mammary stem cells by directly targeting the Fgfr2 promoter in basal cells to increase its expression. Strikingly, Clims also coordinate basal-specific transcriptional programs to preserve luminal cell identity. These basal-derived cues inhibit epidermis-like differentiation of the luminal cell compartment and enhance the expression of luminal cell-specific oncogenes ErbB2 and ErbB3. Consistently, basal-expressed Clims promote the initiation and progression of breast cancer in the MMTV-PyMT tumor model, and the Clim-regulated branching morphogenesis gene network is a prognostic indicator of poor breast cancer outcome in humans.

Mutations in interferon regulatory factor 6 (IRF6) account for ∼70% of cases of Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate. In 8 of 45 VWS-affected families lacking a mutation in IRF6, we found coding mutations in grainyhead-like 3 (GRHL3). According to a zebrafish-based assay, the disease-associated GRHL3 mutations abrogated periderm development and were consistent with a dominant-negative effect, in contrast to haploinsufficiency seen in most VWS cases caused by IRF6 mutations. In mouse, all embryos lacking Grhl3 exhibited abnormal oral periderm and 17% developed a cleft palate. Analysis of the oral phenotype of double heterozygote (Irf6(+/-);Grhl3(+/-)) murine embryos failed to detect epistasis between the two genes, suggesting that they function in separate but convergent pathways during palatogenesis. Taken together, our data demonstrated that mutations in two genes, IRF6 and GRHL3, can lead to nearly identical phenotypes of orofacial cleft. They supported the hypotheses that both genes are essential for the presence of a functional oral periderm and that failure of this process contributes to VWS.

Agr2 is a putative protein disulfide isomerase (PDI) initially identified as an estrogen-responsive gene in breast cancer cell lines. While Agr2 expression in breast cancer is positively correlated with estrogen receptor (ER) expression, it is upregulated in both hormone dependent and independent carcinomas. Several in vitro and xenograft studies have implicated Agr2 in different oncogenic features of breast cancer; however, the physiological role of Agr2 in normal mammary gland development remains to be defined. Agr2 expression is developmentally regulated in the mammary gland, with maximum expression during late pregnancy and lactation. Using a mammary gland specific knockout mouse model, we show that Agr2 facilitates normal lobuloalveolar development by regulating mammary epithelial cell proliferation; we found no effects on apoptosis in Agr2(-/-) mammary epithelial cells. Consequently, mammary glands of Agr2(-/-) females exhibit reduced expression of milk proteins, and by two weeks post-partum their pups are smaller in size. Utilizing a conditional mouse model, we show that Agr2 constitutive expression drives precocious lobuloalveolar development and increased milk protein expression in the virgin mammary gland. In vitro studies using knock down and overexpression strategies in estrogen receptor positive and negative mammary epithelial cell lines demonstrate a role for Agr2 in estradiol-induced cell proliferation. In conclusion, the estrogen-responsive Agr2, a candidate breast cancer oncogene, regulates epithelial cell proliferation and lobuloalveolar development in the mammary gland. The pro-proliferative effects of Agr2 may explain its actions in early tumorigenesis.

The antagonistic actions of Polycomb and Trithorax are responsible for proper cell fate determination in mammalian tissues. In the epidermis, a self-renewing epithelium, previous work has shown that release from Polycomb repression only partially explains differentiation gene activation. We now show that Trithorax is also a key regulator of epidermal differentiation, not only through activation of genes repressed by Polycomb in progenitor cells, but also through activation of genes independent of regulation by Polycomb. The differentiation associated transcription factor GRHL3/GET1 recruits the ubiquitously expressed Trithorax complex to a subset of differentiation genes.

The epidermis is a highly regenerative barrier protecting organisms from environmental insults, including UV radiation, the main cause of skin cancer and skin aging. Here, we show that time-restricted feeding (RF) shifts the phase and alters the amplitude of the skin circadian clock and affects the expression of approximately 10% of the skin transcriptome. Furthermore, a large number of skin-expressed genes are acutely regulated by food intake. Although the circadian clock is required for daily rhythms in DNA synthesis in epidermal progenitor cells, RF-induced shifts in clock phase do not alter the phase of DNA synthesis. However, RF alters both diurnal sensitivity to UVB-induced DNA damage and expression of the key DNA repair gene, Xpa. Together, our findings indicate regulation of skin function by time of feeding and emphasize a link between circadian rhythm, food intake, and skin health.

Deregulation of canonical Wnt/CTNNB1 (beta-catenin) pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells.

The genetic basis of human neural tube defects (NTDs), such as anencephaly and spina bifida (SB), is complex and heterogeneous. Grainyhead-like genes represent candidates for involvement in NTDs based on the presence of SB and exencephaly in mice carrying loss-of-function alleles of Grhl2 or Grhl3. We found that reinstatement of Grhl3 expression, by bacterial artificial chromosome (BAC)-mediated transgenesis, prevents SB in Grhl3-null embryos, as in the Grhl3 hypomorphic curly tail strain. Notably, however, further increase in expression of Grhl3 causes highly penetrant SB. Grhl3 overexpression recapitulates the spinal NTD phenotype of loss-of-function embryos, although the underlying mechanism differs. However, it does not phenocopy other defects of Grhl3-null embryos such as abnormal axial curvature, cranial NTDs (exencephaly) or skin barrier defects, the latter being rescued by the Grhl3-transgene. Grhl2 and Grhl3 can form homodimers and heterodimers, suggesting a possible model in which defects arising from overexpression of Grhl3 result from sequestration of Grhl2 in heterodimers, mimicking Grhl2 loss of function. This hypothesis predicts that increased abundance of Grhl2 would have an ameliorating effect in Grhl3 overexpressing embryo. Instead, we observed a striking additive genetic interaction between Grhl2 and Grhl3 gain-of-function alleles. Severe SB arose in embryos in which both genes were expressed at moderately elevated levels that individually do not cause NTDs. Furthermore, moderate Grhl3 overexpression also interacted with the Vangl2Lp allele to cause SB, demonstrating genetic interaction with the planar cell polarity signalling pathway that is implicated in mouse and human NTDs.

The circadian clock coordinates a variety of immune responses with signals from the external environment to promote survival. We investigated the potential reciprocal relationship between the circadian clock and skin inflammation. We treated mice topically with the Toll-like receptor 7 (TLR7) agonist imiquimod (IMQ) to activate IFN-sensitive gene (ISG) pathways and induce psoriasiform inflammation. IMQ transiently altered core clock gene expression, an effect mirrored in human patient psoriatic lesions. In mouse skin 1 d after IMQ treatment, ISGs, including the key ISG transcription factor IFN regulatory factor 7 (Irf7), were more highly induced after treatment during the day than the night. Nuclear localization of phosphorylated-IRF7 was most prominently time-of-day dependent in epidermal leukocytes, suggesting that these cell types play an important role in the diurnal ISG response to IMQ. Mice lacking Bmal1 systemically had exacerbated and arrhythmic ISG/Irf7 expression after IMQ. Furthermore, daytime-restricted feeding, which affects the phase of the skin circadian clock, reverses the diurnal rhythm of IMQ-induced ISG expression in the skin. These results suggest a role for the circadian clock, driven by BMAL1, as a negative regulator of the ISG response, and highlight the finding that feeding time can modulate the skin immune response. Since the IFN response is essential for the antiviral and antitumor effects of TLR activation, these findings are consistent with the time-of-day-dependent variability in the ability to fight microbial pathogens and tumor initiation and offer support for the use of chronotherapy for their treatment.

The interfollicular epidermis (IFE) forms a water-tight barrier that is often disrupted in inflammatory skin diseases. During homeostasis, the IFE is replenished by stem cells in the basal layer that differentiate as they migrate toward the skin surface. Conventionally, IFE differentiation is thought to be stepwise as reflected in sharp boundaries between its basal, spinous, granular and cornified layers. The transcription factor GRHL3 regulates IFE differentiation by transcriptionally activating terminal differentiation genes. Here we use single cell RNA-seq to show that murine IFE differentiation is best described as a single step gradualistic process with a large number of transition cells between the basal and spinous layer. RNA-velocity analysis identifies a commitment point that separates the plastic basal and transition cell state from unidirectionally differentiating cells. We also show that in addition to promoting IFE terminal differentiation, GRHL3 is essential for suppressing epidermal stem cell expansion and the emergence of an abnormal stem cell state by suppressing Wnt signaling in stem cells.

The epidermis is the outermost layer of skin. Here, we used targeted lipid profiling to characterize the biogeographic alterations of human epidermal lipids across 12 anatomically distinct body sites, and we used single-cell RNA-Seq to compare keratinocyte gene expression at acral and nonacral sites. We demonstrate that acral skin has low expression of EOS acyl-ceramides and the genes involved in their synthesis, as well as low expression of genes involved in filaggrin and keratin citrullination (PADI1 and PADI3) and corneodesmosome degradation, changes that are consistent with increased corneocyte retention. Several overarching principles governing epidermal lipid expression were also noted. For example, there was a strong negative correlation between the expression of 18-carbon and 22-carbon sphingoid base ceramides. Disease-specific alterations in epidermal lipid gene expression and their corresponding alterations to the epidermal lipidome were characterized. Lipid biomarkers with diagnostic utility for inflammatory and precancerous conditions were identified, and a 2-analyte diagnostic model of psoriasis was constructed using a step-forward algorithm. Finally, gene coexpression analysis revealed a strong connection between lipid and immune gene expression. This work highlights (a) mechanisms by which the epidermis is uniquely adapted for the specific environmental insults encountered at different body surfaces and (b) how inflammation-associated alterations in gene expression affect the epidermal lipidome.

Palmoplantar skin is structurally and functionally unique, but the transcriptional programs driving this specialization are unclear. Here, we use bulk and single-cell RNA sequencing of human palm, sole, and hip skin to describe the distinguishing characteristics of palmoplantar and non-palmoplantar skin while also uncovering differences between palmar and plantar sites. Our approach reveals an altered immune environment in palmoplantar skin, with downregulation of diverse immunological processes and decreased immune cell populations. Further, we identify specific fibroblast populations that appear to orchestrate key differences in cell-cell communication in palm, sole, and hip. Dedicated keratinocyte analysis highlights major differences in basal cell fraction among the three sites and demonstrates the existence of two spinous keratinocyte populations constituting parallel, site-selective epidermal differentiation trajectories. In summary, this deep characterization of highly adapted palmoplantar skin contributes key insights into the fundamental biology of human skin and provides a valuable data resource for further investigation.

Hair follicles undergo recurrent cycling of controlled growth (anagen), regression (catagen), and relative quiescence (telogen) with a defined periodicity. Taking a genomics approach to study gene expression during synchronized mouse hair follicle cycling, we discovered that, in addition to circadian fluctuation, CLOCK-regulated genes are also modulated in phase with the hair growth cycle. During telogen and early anagen, circadian clock genes are prominently expressed in the secondary hair germ, which contains precursor cells for the growing follicle. Analysis of Clock and Bmal1 mutant mice reveals a delay in anagen progression, and the secondary hair germ cells show decreased levels of phosphorylated Rb and lack mitotic cells, suggesting that circadian clock genes regulate anagen progression via their effect on the cell cycle. Consistent with a block at the G1 phase of the cell cycle, we show a significant upregulation of p21 in Bmal1 mutant skin. While circadian clock mechanisms have been implicated in a variety of diurnal biological processes, our findings indicate that circadian clock genes may be utilized to modulate the progression of non-diurnal cyclic processes.

A certain number of epithelial cells in intestinal crypts are DNA damage resistant and contribute to regeneration. However, the cellular mechanism underlying intestinal regeneration remains unclear. Using lineage tracing, we show that cells marked by an Msi1 reporter (Msi1+) are right above Lgr5high cells in intestinal crypts and exhibit DNA damage resistance. Single-cell RNA sequencing reveals that the Msi1+ cells are heterogeneous with the majority being intestinal stem cells (ISCs). The DNA damage-resistant subpopulation of Msi1+ cells is characterized by low-to-negative Lgr5 expression and is more rapidly cycling than Lgr5high radiosensitive crypt base columnar stem cells (CBCs). This enables an efficient repopulation of the intestinal epithelium at early stage when Lgr5high cells are not emerging. Furthermore, relative to CBCs, Msi1+ cells preferentially produce Paneth cells during homeostasis and upon radiation repair. Together, we demonstrate that the DNA damage-resistant Msi1+ cells are cycling ISCs that maintain and regenerate the intestinal epithelium.

The control principles behind robust cyclic regeneration of hair follicles (HFs) remain unclear. Using multi-scale modeling, we show that coupling inhibitors and activators with physical growth of HFs is sufficient to drive periodicity and excitability of hair regeneration. Model simulations and experimental data reveal that mouse skin behaves as a heterogeneous regenerative field, composed of anatomical domains where HFs have distinct cycling dynamics. Interactions between fast-cycling chin and ventral HFs and slow-cycling dorsal HFs produce bilaterally symmetric patterns. Ear skin behaves as a hyper-refractory domain with HFs in extended rest phase. Such hyper-refractivity relates to high levels of BMP ligands and WNT antagonists, in part expressed by ear-specific cartilage and muscle. Hair growth stops at the boundaries with hyper-refractory ears and anatomically discontinuous eyelids, generating wave-breaking effects. We posit that similar mechanisms for coupled regeneration with dominant activator, hyper-refractory, and wave-breaker regions can operate in other actively renewing organs.

Mammary and extramammary Paget's Diseases (PD) are a malignant skin cancer characterized by the appearance of Paget cells. Although easily diagnosed, its pathogenesis remains unknown. Here, single-cell RNA-sequencing identified distinct cellular states, novel biomarkers, and signaling pathways - including mTOR, associated with extramammary PD. Interestingly, we identified MSI1 ectopic overexpression in basal epithelial cells of human PD skin, and show that Msi1 overexpression in the epidermal basal layer of mice phenocopies human PD at histopathological, single-cell and molecular levels. Using this mouse model, we identified novel biomarkers of Paget-like cells that translated to human Paget cells. Furthermore, single-cell trajectory, RNA velocity and lineage-tracing analyses revealed a putative keratinocyte-to-Paget-like cell conversion, supporting the in situ transformation theory of disease pathogenesis. Mechanistically, the Msi1-mTOR pathway drives keratinocyte-Paget-like cell conversion, and suppression of mTOR signaling with Rapamycin significantly rescued the Paget-like phenotype in Msi1-overexpressing transgenic mice. Topical Rapamycin treatment improved extramammary PD-associated symptoms in humans, suggesting mTOR inhibition as a novel therapeutic treatment in PD.

Failure of neural tube closure leads to neural tube defects (NTDs), common congenital abnormalities in humans. Among the genes whose loss of function causes NTDs in mice, Grainyhead-like3 (Grhl3) is essential for spinal neural tube closure, with null mutants exhibiting fully penetrant spina bifida. During spinal neurulation Grhl3 is initially expressed in the surface (non-neural) ectoderm, subsequently in the neuroepithelial component of the neural folds and at the node-streak border, and finally in the hindgut endoderm. Here, we show that endoderm-specific knockout of Grhl3 causes late-arising spinal NTDs, preceded by increased ventral curvature of the caudal region which was shown previously to suppress closure of the spinal neural folds. This finding supports the hypothesis that diminished Grhl3 expression in the hindgut is the cause of spinal NTDs in the curly tail, carrying a hypomorphic Grhl3 allele. Complete loss of Grhl3 function produces a more severe phenotype in which closure fails earlier in neurulation, before the stage of onset of expression in the hindgut of wild-type embryos. This implicates additional tissues and NTD mechanisms in Grhl3 null embryos. Conditional knockout of Grhl3 in the neural plate and node-streak border has minimal effect on closure, suggesting that abnormal function of surface ectoderm, where Grhl3 transcripts are first detected, is primarily responsible for early failure of spinal neurulation in Grhl3 null embryos.

The M3 muscarinic acetylcholine receptor is predominantly expressed in the basal epidermal layer where it mediates the effects of the autocrine/paracrine cytotransmitter acetylcholine. Patients with the autoimmune blistering disease pemphigus develop autoantibodies to M3 muscarinic acetylcholine receptor and show alterations in keratinocyte adhesion, proliferation, and differentiation, suggesting that M3 muscarinic acetylcholine receptor controls these cellular functions. Chmr3-/- mice display altered epidermal morphology resembling that seen in patients with pemphigus vulgaris. In this study, we characterized the cellular and molecular mechanisms through which M3 muscarinic acetylcholine receptor controls epidermal structure and function. We used single-cell RNA sequencing to evaluate keratinocyte heterogeneity and identify differentially expressed genes in specific subpopulations of epidermal cells in Chmr3-/- neonatal mice. We found that Chmr3-/- mice feature abnormal epidermal morphology characterized by accumulation of nucleated basal cells, shrinkage of basal keratinocytes, and enlargement of intercellular spaces. These morphologic changes were associated with upregulation of cell proliferation genes and downregulation of genes contributing to epidermal differentiation, extracellular matrix formation, intercellular adhesion, and cell arrangement. These findings provide, to our knowledge, previously unreported insights into how acetylcholine controls epidermal differentiation and lay a groundwork for future translational studies evaluating the therapeutic potential of cholinergic drugs in dermatology.

The migrating keratinocyte wound front is required for skin wound closure. Despite significant advances in wound healing research, we do not fully understand the molecular mechanisms that orchestrate collective keratinocyte migration. Here, we show that, in the wound front, the epidermal transcription factor Grainyhead like-3 (GRHL3) mediates decreased expression of the adherens junction protein E-cadherin; this results in relaxed adhesions between suprabasal keratinocytes, thus promoting collective cell migration and wound closure. Wound fronts from mice lacking GRHL3 in epithelial cells (Grhl3-cKO) have lower expression of Fascin-1 (FSCN1), a known negative regulator of E-cadherin. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on wounded keratinocytes shows decreased wound-induced chromatin accessibility near the Fscn1 gene in Grhl3-cKO mice, a region enriched for GRHL3 motifs. These data reveal a wound-induced GRHL3/FSCN1/E-cadherin pathway that regulates keratinocyte-keratinocyte adhesion during wound-front migration; this pathway is activated in acute human wounds and is altered in diabetic wounds in mice, suggesting translational relevance.

Transcription factor binding, chromatin modifications and large scale chromatin re-organization underlie progressive, irreversible cell lineage commitments and differentiation. We know little, however, about chromatin changes as cells enter transient, reversible states such as migration. Here we demonstrate that when human progenitor keratinocytes either differentiate or migrate they form complements of typical enhancers and super-enhancers that are unique for each state. Unique super-enhancers for each cellular state link to gene expression that confers functions associated with the respective cell state. These super-enhancers are also enriched for skin disease sequence variants. GRHL3, a transcription factor that promotes both differentiation and migration, binds preferentially to super-enhancers in differentiating keratinocytes, while during migration, it binds preferentially to promoters along with REST, repressing the expression of migration inhibitors. Key epidermal differentiation transcription factor genes, including GRHL3, are located within super-enhancers, and many of these transcription factors in turn bind to and regulate super-enhancers. Furthermore, GRHL3 represses the formation of a number of progenitor and non-keratinocyte super-enhancers in differentiating keratinocytes. Hence, chromatin relocates GRHL3 binding and enhancers to regulate both the irreversible commitment of progenitor keratinocytes to differentiation and their reversible transition to migration.