Following the p63/Keratin5 basal cells in the sensory and non-sensory epithelia of the vomeronasal organ.

The vomeronasal organ (VNO) is a part of the accessory olfactory system, which detects pheromones and chemical factors that trigger a spectrum of sexual and social behaviors. The vomeronasal epithelium (VNE) shares several features with the epithelium of the main olfactory epithelium (MOE). However, it is a distinct neuroepithelium populated by chemosensory neurons that differ from the olfactory sensory neurons in cellular structure, receptor expression, and connectivity. The vomeronasal organ of rodents comprises a sensory epithelium (SE) and a thin non-sensory epithelium (NSE) that morphologically resembles the respiratory epithelium. Sox2-positive cells have been previously identified as the stem cell population that gives rise to neuronal progenitors in MOE and VNE. In addition, the MOE also comprises p63 positive horizontal basal cells, a second pool of quiescent stem cells that become active in response to injury. Immunolabeling against the transcription factor p63, Keratin-5 (Krt5), Krt14, NrCAM, and Krt5Cre tracing experiments highlighted the existence of horizontal basal cells distributed along the basal lamina of SE of the VNO. Single cell sequencing and genetic lineage tracing suggest that the vomeronasal horizontal basal cells arise from basal progenitors at the boundary between the SE and NSE proximal to the marginal zones. Moreover, our experiments revealed that the NSE of rodents is, like the respiratory epithelium, a stratified epithelium where the p63/Krt5+ basal progenitor cells self-replicate and give rise to the apical columnar cells facing the lumen of the VNO.

Local tissue interactions govern pLL patterning in medaka.

Vertebrate organs are arranged in a stereotypic, species-specific position along the animal body plan. Substantial morphological variation exists between related species, especially so in the vastly diversified teleost clade. It is still unclear how tissues, organs and systems can accommodate such diverse scaffolds. Here, we use the distinctive arrangement of neuromasts in the posterior lateral line (pLL) system of medaka fish to address the tissue-interactions defining a pattern. We show that patterning in this peripheral nervous system is established by autonomous organ precursors independent of neuronal wiring. In addition, we target the keratin 15 gene to generate stuck-in-the-midline (siml) mutants, which display epithelial lesions and a disrupted pLL patterning. By using siml/wt chimeras, we determine that the aberrant siml pLL pattern depends on the mutant epithelium, since a wild type epithelium can rescue the siml phenotype. Inducing epithelial lesions by 2-photon laser ablation during pLL morphogenesis phenocopies siml genetic mutants and reveals that epithelial integrity defines the final position of the embryonic pLL neuromasts. Our results using the medaka pLL disentangle intrinsic from extrinsic properties during the establishment of a sensory system. We speculate that intrinsic programs guarantee proper organ morphogenesis, while instructive interactions from surrounding tissues facilitates the accommodation of sensory organs to the diverse body plans found among teleosts.

Fine-tuned repression of Drp1-driven mitochondrial fission primes a 'stem/progenitor-like state' to support neoplastic transformation.

Gene knockout of the master regulator of mitochondrial fission, Drp1, prevents neoplastic transformation. Also, mitochondrial fission and its opposing process of mitochondrial fusion are emerging as crucial regulators of stemness. Intriguingly, stem/progenitor cells maintaining repressed mitochondrial fission are primed for self-renewal and proliferation. Using our newly derived carcinogen transformed human cell model, we demonstrate that fine-tuned Drp1 repression primes a slow cycling 'stem/progenitor-like state', which is characterized by small networks of fused mitochondria and a gene-expression profile with elevated functional stem/progenitor markers (Krt15, Sox2 etc) and their regulators (Cyclin E). Fine tuning Drp1 protein by reducing its activating phosphorylation sustains the neoplastic stem/progenitor cell markers. Whereas, fine-tuned reduction of Drp1 protein maintains the characteristic mitochondrial shape and gene-expression of the primed 'stem/progenitor-like state' to accelerate neoplastic transformation, and more complete reduction of Drp1 protein prevents it. Therefore, our data highlights a 'goldilocks' level of Drp1 repression supporting stem/progenitor state dependent neoplastic transformation.

Human gastrointestinal epithelia of the esophagus, stomach, and duodenum resolved at single-cell resolution.

The upper gastrointestinal tract, consisting of the esophagus, stomach, and duodenum, controls food transport, digestion, nutrient uptake, and hormone production. By single-cell analysis of healthy epithelia of these human organs, we molecularly define their distinct cell types. We identify a quiescent COL17A1high KRT15high stem/progenitor cell population in the most basal cell layer of the esophagus and detect substantial gene expression differences between identical cell types of the human and mouse stomach. Selective expression of BEST4, CFTR, guanylin, and uroguanylin identifies a rare duodenal cell type, referred to as BCHE cell, which likely mediates high-volume fluid secretion because of continual activation of the CFTR channel by guanylin/uroguanylin-mediated autocrine signaling. Serotonin-producing enterochromaffin cells in the antral stomach significantly differ in gene expression from duodenal enterochromaffin cells. We, furthermore, discover that the histamine-producing enterochromaffin-like cells in the oxyntic stomach express the luteinizing hormone, yet another member of the enteroendocrine hormone family.

Severe epidermolysis bullosa simplex phenotype caused by codominant mutations p.Ile377Thr in keratin 14 and p.Gly138Glu in keratin 5.

Epidermolysis bullosa simplex (EBS) is a rare skin disease usually inherited in an autosomal dominant pattern. EBS is resulting from mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes encoding the keratins 5 and 14 proteins expressed in the keratinocytes of the basal layer of the epidermis. To date, seven pathogenic mutations have been reported to be responsible for EBS in the Canadian population from the province of Quebec: p.Pro25Leu, p.Leu150Pro, p.Met327Thr and p.Arg559X in KRT5; p.Arg125Ser, p.Ile377Thr and p.Ile412Phe in KRT14. Here, we present a novel French-Canadian patient diagnosed with EBS confined to the soles but presenting a severe complication form including blisters, hyperkeratosis, skin erosions and toenail abnormalities. Mutation screening was performed by direct sequencing of the entire coding regions of KRT5 and KRT14 genes and revealed the previously reported missense heterozygous mutation c. 1130T > C in KRT14 (p.Ile377Thr). Furthermore, this patient is carrying a second mutation in KRT5, c.413G > A (p.Gly138Glu), which has been linked to an increased risk of basal cell carcinoma in the literature. We suspect an impact of the p.Gly138Glu variant on the EBS phenotype severity of the studied patient. The pathogenicity and consequences of both genetic variations were simulated by in silico tools.

KRT 15 as a prognostic biomarker is highly expressed in esophageal carcinoma.

Aim: To investigate the expression and prognostic value of KRT 15 in esophageal carcinoma. Materials & methods: The expression levels of KRT 15 were measured in 128 cases of esophageal carcinoma and matched adjacent normal tissues by immunohistochemistry and Western blot assays. Results & conclusion: Western blot analysis shown the expression levels of KRT 15 in esophageal carcinoma were significantly higher compared with those in matched adjacent normal tissues (p < 0.001). immunohistochemistry result shown the high-expression rate of KRT 15 in esophageal carcinoma were 56.3%, which was significantly higher than those in normal tissues (35.9%; p = 0.002). KRT 15 high-expression correlated with T stage, lymph node metastasis, tumor node metastasis stage and prognosis (p < 0.05). These data indicate KRT 15 as a prognostic biomarker is highly expressed in esophageal carcinoma.

KRT15 overexpression predicts poor prognosis in colorectal cancer.

Keratin-15 (KRT15) is a type I keratin lacking a defined type II partner and plays a key role in maintaining cytoplasmic stability. Recently, studies have reported that KRT15 was correlated with tumor formation and progression. However, the clinical significance of KRT15 in colorectal cancer is unclear. In this study, we aimed to investigate the expression of KRT15 and its clinical significance in colorectal cancer. KRT15 expression was examined in 98 cases of colorectal cancer and matched adjacent normal tissues by quantificational real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC), respectively. Then, the clinical significance of KRT15 expression was evaluated in colorectal cancer. QRT-PCR results revealed that the mRNA levels of KRT15 in colorectal cancer tissues were significantly higher compared with those in normal tissues (p<0.0001). The rates of KRT15 high-expression in colorectal cancer and normal tissues were 57.1% and 8.9%, respectively, and the difference was statistically significant (p<0.0001). KRT15 high-expression correlated with differentiation, T stage, lymph node metastasis and clinical stage in colorectal cancer (p<0.05). Meanwhile, KRT15 overexpression predicted poor prognosis and could be used as an independent prognostic factor. These data indicate KRT15 is highly expressed in colorectal cancer and may serve as a prognostic biomarker.

Potential regulatory SNPs in the ATXN7L3B and KRT15 genes are associated with gender-specific colorectal cancer risk.

Aim: According to the current data, a major factor for phenotypic variation of complex traits and disease susceptibility is the cis-acting effects of noncoding variants on gene expression. Our purpose was to evaluate the association between colorectal cancer (CRC) and six single nucleotide polymorphisms identified using our original bioinformatics approach as regulatory and putatively related to CRC. Materials: One hundred and sixty CRC patients and 185 healthy controls have been genotyped for rs590352, rs2072580, rs78317230, rs3829202, rs11542583 and rs4796672. Results: Genotypes and alleles distributions of rs590352 of ATXN7L3B gene were significantly different between the male CRC subjects and controls. Significant correlation of genotype with CRC is observable for women only for the rs4796672 of KRT15 gene. Analysis of haplotypes reveals that rs2072580 of the ISCU and SART3 genes can be also associated with CRC. Conclusion: We have identified three SNPs associated with CRC risk and demonstrated a gender specificity of rs590352 and rs4796672.

Krt5+ urothelial cells are developmental and tissue repair progenitors in the kidney.

Congenital urinary tract obstruction (UTO) is the leading cause of chronic kidney disease in children; however, current management strategies do not safeguard against progression to end-stage renal disease, highlighting the need for interventions to limit or reverse obstructive nephropathy. Experimental UTO triggers renal urothelial remodeling that culminates in the redistribution of basal keratin 5-positive (Krt5+) renal urothelial cells (RUCs) and the generation of uroplakin-positive (Upk)+ RUCs that synthesize a protective apical urothelial plaque. The cellular source of Upk+ RUCs is currently unknown, limiting the development of strategies to promote renal urothelial remodeling as a therapeutic approach. In the present study, we traced the origins of adult Upk+ RUCs during normal development and in response to UTO. Fate mapping analysis demonstrated that adult Upk+ RUCs derive from embryonic and neonatal Krt5+ RUCs, whereas Krt5+ RUCs lose this progenitor capacity and become lineage restricted by postnatal day 14. However, in response to UTO, postnatal day 14-labeled adult Krt5+ RUCs break their lineage restriction and robustly differentiate into Upk+ RUCs. Thus, Krt5+ RUCs drive renal urothelial formation during normal ontogeny and after UTO by differentiating into Upk+ RUCs in a temporally restricted manner.

Krt5+/Krt15+ foregut basal progenitors give rise to cyclooxygenase-2-dependent tumours in response to gastric acid stress.

The effective prevention of tumor initiation, especially for potentially inoperable tumors, will be beneficial to obtain an overall higher quality of our health and life. Hence, thorough understanding of the pathophysiological mechanisms of early tumor formation arising from identifiable cellular origins is required to develop efficient preventative and early treatment options for each tumor type. Here, using genetically engineered mouse models, we provide preclinical experimental evidence for a long-standing open question regarding the pathophysiological potential of a microenvironmental and physiological stressor in tumor development, gastric acid-mediated regional microscopic injury in foregut squamous epithelia. This study demonstrates the association of gastric acid stress with Cyclooxygenase-2-dependent tumor formation originating from tumor-competent Krt5+/Krt15+ foregut basal progenitor cells. Our findings suggest that clinical management of microenvironmental stressor-mediated microscopic injury may be important in delaying tumor initiation from foregut basal progenitor cells expressing pre-existing tumorigenic mutation(s) and genetic alteration(s).

Intrinsic activation of cell growth and differentiation in ex vivo cultured human hair follicles by a transient endogenous production of ROS.

The emerging variety of signalling roles for ROS in eukaryotic cells and tissues is currently a matter of intense research. Here we make use of ex vivo cultured single human hair follicles as an experimental model to demonstrate that a transient production of non-lethal endogenous ROS levels in these mini-organs promotes efficiently the entry into the growth phase (anagen). The stimulatory process implicates the specific activation of the hair follicle stem cell niche, encompassing the induction of stem cell differentiation markers (Ck15), overall cell proliferation and sustained growth of the tissue associated with expression of gen targets (Ccnd1) concomitant with the inhibition of Wnt signaling antagonists and repressors (Dkk1, Gsk3ß) of Wnt signaling. As a whole, this observation indicates that, once activated, ROS signalling is an intrinsic mechanism regulating the hair follicle stem cell niche independently of any external signal.

KRT15, INHBA, MATN3, and AGT are aberrantly methylated and differentially expressed in gastric cancer and associated with prognosis.

AIM: The present study aims to identify aberrantly methylated and differentially expressed genes (DEGs) in gastric cancer (GC) and explore their potential role in the carcinogenesis and development of GC. METHODS: The original RNA-Seq, clinical information and Illumina Human Methylation 27 Chip data associated with GC were downloaded from The Cancer Genome Atlas (TCGA) database using the gdc-client tool. The DEGs and aberrantly methylated genes (AMGs) were screened with edgeR and limma package in R, respectively. The cut-off criteria for DEG identification were P < 0.05 and fold change (FC) >2.0, and for AMG identification were P < 0.05 and |t|>2.0. Genes which were both DEGs and AMGs were considered to be regulated by aberrant DNA methylation in GC. The common genes were used for further functional enrichment analysis in the categories of cellular component, molecular function, biological process and biological pathway. RESULTS: In total 465 genes including 336 down-regulated genes with hyper-methylation (DGs-Hyper) and 129 up-regulated genes with hypo-methylation (UGs-Hypo) were identified. Cellular component analysis showed that these genes were mainly expressed in the cytoplasm and plasma membrane. Molecular function and biological process analysis indicated that the genes primarily participate in cell communication, signal transduction, cell growth/maintenance and function as transcription factors, receptor, cell adhesion molecules, and transmembrane receptor protein tyrosine kinases. Biological pathway analysis revealed that the genes are involved in some crucial pathways including epithelial-to-mesenchymal transition, IL3-mediated signaling, mTOR signaling, VEGF/VEGFR and c-Met signaling. KRT15, INHBA, MATN3, and AGT are significantly associated with the prognosis of GC patients. CONCLUSION: Our study identified several DEGs regulated by aberrant DNA methylation in GC. The mechanism of DNA methylation in the carcinogenesis and development of GC could be further explored in these genes, especially KRT15, INHBA, MATN3, and AGT.

Molecular signature of the putative stem/progenitor cells committed to the development of the bovine mammary gland at puberty.

Milk production is highly dependent on the extensive development of the mammary epithelium, which occurs during puberty. It is therefore essential to distinguish the epithelial cells committed to development from the related epithelial hierarchy. Using cell phenotyping and sorting, we highlighted four cell sub-populations within the bovine mammary gland at puberty. The CD49fhighCD24neg cells expressing CD10, KRT14, vimentin and PROCR corresponded to cells committed to the basal lineage. The CD49flow sub-population contained two cell subsets (CD49flowCD24neg and CD49flowCD24pos). Both subsets expressed hormone receptors including ER, PR and PRLR, as well as ALDH1 activity but only the CD49flowCD24pos subset expressed ELF5. These data indicated that the CD49flow sub-population is mainly composed of cells displaying a luminal phenotype and that this population comprises two luminal cell subsets, namely the CD24neg and CD24pos cells, likely committed to ductal and alveolar lineage, respectively. The putative mammary stem cell (MaSC) fraction was recovered in the CD49fhighCD24pos sub-population which were shown to form mammospheres in vitro. These cells differentially expressed CD10, KRT14 and KRT7, suggesting the existence of several putative MaSC sub-fractions. In-depth characterization of these epithelial sub-populations provides new insights into the bovine mammary epithelial cell lineage and suggests a common developmental lineage in mammals.

Whole exome sequencing to identify genetic markers for trastuzumab-induced cardiotoxicity.

Although trastuzumab-induced cardiotoxicity is an important determinant to limit the use of this drug, the molecular mechanism of risk for this toxicity is not well understood. To identify genetic variants determining the risk of trastuzumab-induced cardiotoxicity, we carried out whole exome sequencing of germline DNA samples from 9 patients with trastuzumab-induced cardiotoxicity, and conducted a case-control association study of 2258 genetic variants between 9 cases (with trastuzumab-induced cardiotoxicity) and general Japanese population controls registered in the Human Genetic Variation Database (HGVD). The top variant which showed the lowest P-value in the screening study was rs139503277 in PHD Finger Protein 3 (Pmin = .00012, odds ratio [OR] = 51.23). To further validate the result of screening study, we carried out a replication study of 10 variants showing Pmin < .001 in the screening study using 234 independent patients treated with trastuzumab, including 10 cases and 224 controls (without trastuzumab-induced cardiotoxicity). In the replication study, we observed that three variants had an effect in the same direction as in the screening study (rs78272919 in exon 2 of Keratin 15, rs5762940 in exon 2 of zinc and ring finger 3, and rs139944387 in exon 44 of Eyes shut homologs [EYS]). A combined result of the screening and the replication studies suggested an association of a locus on chromosome 6q12 with trastuzumab-induced cardiotoxicity (rs139944387 in EYS, combined Pmin = .00056, OR = 13.73). This finding provides new insights into personalized trastuzumab therapy for patients with human epidermal growth factor receptor 2 (HER2)-positive cancer.

Mapping the distribution of stem/progenitor cells across the mouse middle ear during homeostasis and inflammation.

The middle ear epithelium is derived from neural crest and endoderm, which line distinct regions of the middle ear cavity. Here, we investigate the distribution of putative stem cell markers in the middle ear, combined with an analysis of the location of label-retaining cells (LRCs) to create a map of the middle ear mucosa. We show that proliferating cells and LRCs were associated with specific regions of the ear epithelium, concentrated in the hypotympanum at the base of the auditory bulla and around the ear drum. Sox2 was widely expressed in the endodermally derived ciliated pseudostratified epithelium of the hypotympanum. This part of the middle ear showed high levels of Wnt activity, as indicated by the expression of Axin2, a readout of Wnt signalling. Keratin 5 showed a more restricted expression within the basal cells of this region, with very little overlap between the Sox2- and keratin 5-positive epithelium, indicating that these genes mark distinct populations. Little expression of Sox2 or keratin 5 was observed in the neural crest-derived middle ear epithelium that lined the promontory, except in cases of otitis media when this epithelium underwent hyperplasia. This study lays the foundation for furthering our understanding of homeostasis and repair in the middle ear.

microRNA-184 Induces a Commitment Switch to Epidermal Differentiation.

miR-184 is a highly evolutionary conserved microRNA (miRNA) from fly to human. The importance of miR-184 was underscored by the discovery that point mutations in miR-184 gene led to corneal/lens blinding disease. However, miR-184-related function in vivo remained unclear. Here, we report that the miR-184 knockout mouse model displayed increased p63 expression in line with epidermal hyperplasia, while forced expression of miR-184 by stem/progenitor cells enhanced the Notch pathway and induced epidermal hypoplasia. In line, miR-184 reduced clonogenicity and accelerated differentiation of human epidermal cells. We showed that by directly repressing cytokeratin 15 (K15) and FIH1, miR-184 induces Notch activation and epidermal differentiation. The disease-causing miR-184C57U mutant failed to repress K15 and FIH1 and to induce Notch activation, suggesting a loss-of-function mechanism. Altogether, we propose that, by targeting K15 and FIH1, miR-184 regulates the transition from proliferation to early differentiation, while mis-expression or mutation in miR-184 results in impaired homeostasis.

Identification of gene pairs through penalized regression subject to constraints.

BACKGROUND: This article concerns the identification of gene pairs or combinations of gene pairs associated with biological phenotype or clinical outcome, allowing for building predictive models that are not only robust to normalization but also easily validated and measured by qPCR techniques. However, given a small number of biological samples yet a large number of genes, this problem suffers from the difficulty of high computational complexity and imposes challenges to the accuracy of identification statistically. RESULTS: In this paper, we propose a parsimonious model representation and develop efficient algorithms for identification. Particularly, we derive an equivalent model subject to a sum-to-zero constraint in penalized linear regression, where the correspondence between nonzero coefficients in these models is established. Most importantly, it reduces the model complexity of the traditional approach from the quadratic order to the linear order in the number of candidate genes, while overcoming the difficulty of model nonidentifiablity. Computationally, we develop an algorithm using the alternating direction method of multipliers (ADMM) to deal with the constraint. Numerically, we demonstrate that the proposed method outperforms the traditional method in terms of the statistical accuracy. Moreover, we demonstrate that our ADMM algorithm is more computationally efficient than a coordinate descent algorithm with a local search. Finally, we illustrate the proposed method on a prostate cancer dataset to identify gene pairs that are associated with pre-operative prostate-specific antigen. CONCLUSION: Our findings demonstrate the feasibility and utility of using gene pairs as biomarkers.

Retinoic acid signaling regulates Krt5 and Krt14 independently of stem cell markers in submandibular salivary gland epithelium.

BACKGROUND: Retinoic acid (RA), the active metabolite of vitamin A, has been demonstrated to be important for growth and branching morphogenesis of mammalian embryonic salivary gland epithelium. However, it is not known whether RA functions directly within epithelial cells or in associated tissues that influence morphogenesis of salivary epithelium. Moreover, downstream targets of RA regulation have not been identified. RESULTS: Here, we show that canonical RA signaling occurs in multiple tissues of embryonic mouse salivary glands, including epithelium, associated parasympathetic ganglion neurons, and nonneuronal mesenchyme. By culturing epithelium explants in isolation from other tissues, we demonstrate that RA influences epithelium morphogenesis by direct action in that tissue. Moreover, we demonstrate that inhibition of RA signaling represses cell proliferation and expression of FGF10 signaling targets, and upregulates expression of basal epithelial keratins Krt5 and Krt14. Importantly, we show that the stem cell gene Kit is regulated inversely from Krt5/Krt14 by RA signaling. CONCLUSIONS: RA regulates Krt5 and Krt14 expression independently of stem cell character in developing salivary epithelium. RA, or chemical inhibitors of RA signaling, could potentially be used for modulating growth and differentiation of epithelial stem cells for the purpose of re-populating damaged glands or generating bioengineered organs. Developmental Dynamics 246:135-147, 2017. © 2016 Wiley Periodicals, Inc.