The search for more effective drugs for the management of common hair growth disorders remains a top priority, both for clinical dermatology and industry. In this pilot study, we report a pragmatic organotypic assay for basic and applied hair research. The patented technique produces microdroplets, which generate human folliculoid microspheres (HFMs), consisting of human dermal papilla fibroblasts and outer root sheath keratinocytes within an extracellular matrix that simulates elements of the hair follicle mesenchyme. Studying a number of different markers (for example, proliferation, apoptosis, cytokeratin-6, versican), we show that these HFMs, cultured under well-defined conditions, retain several essential epithelial-mesenchymal interactions characteristic for human scalp hair follicle. Selected, recognized hair growth-modulatory agents modulate these parameters in a manner that suggests that HFMs allow the standardized preclinical assessment of test agents on relevant human hair growth markers under substantially simplified in vitro conditions that approximate the in vivo situation. Furthermore, we show by immunohistochemistry, reverse transcriptase-PCR, and DNA microarray techniques that HFMs also offer a useful discovery tool for the identification of target genes and their products for candidate hair drugs. HFM thus represent an instructive modern experimental and screening tool for basic and applied hair research in the human system.

• MeSH
• apoptóza MeSH
• cyklosporin farmakologie   MeSH
• cytokiny genetika   MeSH
• epitelové buňky cytologie   MeSH
• financování organizované MeSH
• hepatocytární růstový faktor farmakologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezibuněčná komunikace MeSH
• mezoderm cytologie   MeSH
• mikrosféry MeSH
• proliferace buněk MeSH
• stanovení celkové genové exprese MeSH
• vlasový folikul cytologie   růst a vývoj   MeSH
• Check Tag
• lidé MeSH

Novel mRNA isoforms encoding the enamel matrix proteins amelin-1, amelin-2 and ameloblastin have been recently described. We have applied detailed immunohistochemical as well as non-radioactive in situ hybridization analyses to follow amelin-1 expression in developing rat incisors and molars. We constructed an expression vector, overproduced recombinant amelin in Escherichia coli and prepared an antibody. In addition to the previously reported amelin mRNA expression patterns in ameloblasts, the amelin message was also detected in pulpal mesenchymal cells including preodontoblasts and young odontoblasts. The signal in these cells persisted until deposition of mantle dentin became evident. The immunolocalization of amelin-1 in preodontoblasts and ameloblasts essentially followed the pattern of mRNA expression. The most intense staining was found in the enamel matrix adjacent to secretory ameloblasts. Focal accumulations of immunoreactive material were found at the dentinoenamel junction during the maturation stage. Also, using 5'-RACE (Rapid Amplification of cDNA Ends) we could confirm only amelin-1 and ameloblastin messages in the total RNA pool from rat molars and conclude that amelin-2 is a truncated form of ameloblastin. The sequential expression of amelin in mesenchymal and epithelial cells suggests it plays a role in cell differentiation during early tooth development.

• MeSH
• ameloblasty metabolismus   MeSH
• DNA primery genetika   MeSH
• epitelové buňky metabolismus   MeSH
• hybridizace in situ MeSH
• imunohistochemie MeSH
• komplementární DNA genetika   MeSH
• krysa rodu rattus MeSH
• messenger RNA genetika   MeSH
• mezoderm metabolismus   MeSH
• odontoblasty metabolismus   MeSH
• odontogeneze * genetika   MeSH
• potkani Sprague-Dawley MeSH
• proteiny zubní skloviny * genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny genetika   MeSH
• sekvence nukleotidů MeSH
• vývojová regulace genové exprese MeSH
• zubní sklovina cytologie   metabolismus   růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Clinical evidence suggests that healing is faster and almost scarless at an early neonatal age in comparison with that in adults. In this study, the phenotypes of neonatal and adult dermal fibroblasts and keratinocytes (nestin, smooth muscle actin, keratin types 8, 14 and 19, and fibronectin) were compared. Furthermore, functional assays (proliferation, migration, scratch wound closure) including mutual epithelial‑mesenchymal interactions were also performed to complete the series of experiments. Positivity for nestin and α smooth muscle actin was higher in neonatal fibroblasts (NFs) when compared with their adult counterparts (adult fibroblasts; AFs). Although the proliferation of NFs and AFs was similar, they significantly differed in their migration potential. The keratinocyte experiments revealed small, poorly differentiated cells (positive for keratins 8, 14 and 19) in primary cultures isolated from neonatal tissues. Moreover, the neonatal keratinocytes exhibited significantly faster rates of healing the experimentally induced in vitro defects in comparison with adult cells. Notably, the epithelial/mesenchymal interaction studies showed that NFs in co-culture with adult keratinocytes significantly stimulated the adult epithelial cells to acquire the phenotype of small, non-confluent cells expressing markers of poor differentiation. These results indicate the important differences between neonatal and adult cells that may be associated with improved wound healing during the early neonatal period.

• MeSH
• aktiny metabolismus   MeSH
• buněčná diferenciace MeSH
• crista neuralis cytologie   MeSH
• dárci tkání * MeSH
• dospělí MeSH
• epitelové buňky cytologie   metabolismus   MeSH
• fenotyp MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fibronektiny biosyntéza   MeSH
• imunohistochemie MeSH
• keratinocyty cytologie   metabolismus   MeSH
• kmenové buňky metabolismus   MeSH
• kokultivační techniky MeSH
• lidé MeSH
• mezoderm cytologie   MeSH
• myofibroblasty cytologie   MeSH
• nestin metabolismus   MeSH
• neuroplasticita MeSH
• novorozenec MeSH
• pohyb buněk MeSH
• proliferace buněk MeSH
• stanovení celkové genové exprese MeSH
• stárnutí fyziologie   MeSH
• vývojová regulace genové exprese MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH

The proper positioning of organs during development is essential, yet little is known about the regulation of this process in mammals. Using murine tooth development as a model, we have found that cell migration plays a central role in positioning of the organ primordium. By combining lineage tracing, genetic cell ablation, and confocal live imaging, we identified a migratory population of Fgf8-expressing epithelial cells in the embryonic mandible. These Fgf8-expressing progenitors furnish the epithelial cells required for tooth development, and the progenitor population migrates toward a Shh-expressing region in the mandible, where the tooth placode will initiate. Inhibition of Fgf and Shh signaling disrupted the oriented migration of cells, leading to a failure of tooth development. These results demonstrate the importance of intraepithelial cell migration in proper positioning of an initiating organ.

• MeSH
• epitelové buňky cytologie   metabolismus   MeSH
• fibroblastové růstové faktory metabolismus   MeSH
• mezoderm cytologie   metabolismus   MeSH
• moláry cytologie   embryologie   metabolismus   MeSH
• morfogeneze fyziologie   MeSH
• myši MeSH
• odontogeneze fyziologie   MeSH
• pohyb buněk fyziologie   MeSH
• vývojová regulace genové exprese fyziologie   MeSH
• zuby cytologie   embryologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Despite recent progress in recognizing the importance of mesenchymal cells for the homeostasis of the intestinal system, the current picture of how these cells communicate with the associated epithelial layer remains unclear. To describe the relevant cell populations in an unbiased manner, we carried out a single-cell transcriptome analysis of the adult murine colon, producing a high-quality atlas of matched colonic epithelium and mesenchyme. We identify two crypt-associated colonic fibroblast populations that are demarcated by different strengths of platelet-derived growth factor receptor A (Pdgfra) expression. Crypt-bottom fibroblasts (CBFs), close to the intestinal stem cells, express low levels of Pdgfra and secrete canonical Wnt ligands, Wnt potentiators, and bone morphogenetic protein (Bmp) inhibitors. Crypt-top fibroblasts (CTFs) exhibit high Pdgfra levels and secrete noncanonical Wnts and Bmp ligands. While the Pdgfralow cells maintain intestinal stem cell proliferation, the Pdgfrahigh cells induce differentiation of the epithelial cells. Our findings enhance our understanding of the crosstalk between various colonic epithelial cells and their associated mesenchymal signaling hubs along the crypt axis-placing differential Pdgfra expression levels in the spotlight of intestinal fibroblast identity.

• MeSH
• analýza jednotlivých buněk metody   MeSH
• buněčná diferenciace fyziologie   MeSH
• epitelové buňky metabolismus   MeSH
• fibroblasty klasifikace   metabolismus   MeSH
• homeostáza MeSH
• kmenové buňky cytologie   MeSH
• kolon metabolismus   fyziologie   MeSH
• kostní morfogenetické proteiny metabolismus   MeSH
• mezoderm cytologie   fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• proliferace buněk fyziologie   MeSH
• signální transdukce MeSH
• stanovení celkové genové exprese metody   MeSH
• střeva fyziologie   MeSH
• střevní sliznice metabolismus   MeSH
• transkriptom genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Tooth formation requires complex signaling interactions both within the oral epithelium and between the epithelium and the underlying mesenchyme. Previous studies of the Wnt/β-catenin pathway have shown that tooth formation is partly inhibited in loss-of-function mutants, and gain-of-function mutants have perturbed tooth morphology. However, the stage at which Wnt signaling is first important in tooth formation remains unclear. Here, using an Fgf8-promoter-driven, and therefore early, deletion of β-catenin in mouse molar epithelium, we found that loss of Wnt/β-catenin signaling completely deletes the molar tooth, demonstrating that this pathway is central to the earliest stages of tooth formation. Early expression of a dominant-active β-catenin protein also perturbs tooth formation, producing a large domed evagination at early stages and supernumerary teeth later on. The early evaginations are associated with premature mesenchymal condensation marker, and are reduced by inhibition of condensation-associated collagen synthesis. We propose that invagination versus evagination morphogenesis is regulated by the relative timing of epithelial versus mesenchymal cell convergence regulated by canonical Wnt signaling. Together, these studies reveal new aspects of Wnt/β-catenin signaling in tooth formation and in epithelial morphogenesis more broadly.

• MeSH
• beta-katenin metabolismus   MeSH
• epitel metabolismus   MeSH
• epitelové buňky cytologie   metabolismus   MeSH
• mezoderm metabolismus   MeSH
• moláry cytologie   růst a vývoj   metabolismus   MeSH
• morfogeneze fyziologie   MeSH
• myši MeSH
• odontogeneze genetika   fyziologie   MeSH
• proliferace buněk MeSH
• signální dráha Wnt fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH

During molar development, apoptosis occurs in a well-characterised pattern suggesting several roles for cell death in odontogenesis. However, molecular mechanisms of dental apoptosis are only poorly understood. In this study, Apaf-1 and caspase-9 knockouts were used to uncover the engagement of these members of the apoptotic machinery during early tooth development, concentrating primarily on their function in the apoptotic elimination of primary enamel knot cells. Molar tooth germ morphology, proliferation and apoptosis were investigated on frontal histological sections of murine heads at embryonic days (ED) 15.5, the stage when the primary enamel knot is eliminated apoptotically. In molar tooth germs of both knockouts, no apoptosis was observed according to morphological (haematoxylin-eosin) as well as biochemical criteria (TUNEL). Morphology of the mutant tooth germs, however, was not changed. Additionally, knockout mice showed no changes in proliferation compared to wild type mice. According to our findings on knockout embryos, Apaf-1 and caspase-9 are involved in apoptosis during tooth development; however, they seem dispensable and not necessary for proper tooth shaping. Compensatory or other mechanisms of cell death may act to eliminate the primary enamel knot cells in the absence of Apaf-1 and caspase-9.

• MeSH
• apoptóza fyziologie   MeSH
• buněčný cyklus fyziologie   MeSH
• epitelové buňky cytologie   MeSH
• faktor 1 aktivující apoptotickou proteasu nedostatek   MeSH
• financování organizované MeSH
• kaspasa 9 nedostatek   MeSH
• mezoderm fyziologie   MeSH
• moláry embryologie   fyziologie   MeSH
• myši knockoutované MeSH
• myši MeSH
• proliferační antigen buněčného jádra analýza   MeSH
• zubní sklovina embryologie   fyziologie   MeSH
• zubní zárodek anatomie a histologie   embryologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH