The neural crest (NC) is a transient dynamic structure of ectodermal origin, found in early vertebrate embryos. The multipotential NC cells migrate along well defined routes, differentiate to various cell types including melanocytes and participate in the formation of various permanent tissues. As there is only limited information about the molecular mechanisms controlling early events in melanocyte specification and development, we exploited the AMV v-Myb transcriptional regulator, which directs differentiation of in vitro chicken NC cells to the melanocyte lineage. This activity is strictly dependent on v-Myb specifically binding to the Myb recognition DNA element (MRE). The two tamoxifen-inducible v-Myb alleles were constructed one which recognizes the MRE and one which does not. These were activated in ex ovo NC cells, and the expression profiles of resulting cells were analyzed using Affymetrix microarrays and RT-PCR. These approaches revealed up-regulation of the BMP antagonist Gremlin 2 mRNA, and down-regulation of mRNAs encoding several epithelial genes including KRT19 as very early events following the activation of melanocyte differentiation by v-Myb. The enforced v-Myb expression in neural tubes of chicken embryos resulted in detectable presence of Gremlin 2 mRNA. However, expression of Gremlin 2 in NC cells did not promote formation of melanocytes suggesting that Gremlin 2 is not the master regulator of melanocytic differentiation.

• Klíčová slova
• KRT19, Melanocyte development, PRDC, Tamoxifen-inducible v-Myb, v-Myb-dependent genes,
• MeSH
• aktivace transkripce * MeSH
• alely MeSH
• buněčná diferenciace * MeSH
• crista neuralis cytologie   MeSH
• keratin-19 genetika   metabolismus   MeSH
• kostní morfogenetický protein 5 genetika   metabolismus   MeSH
• kultivované buňky MeSH
• kuřecí embryo MeSH
• melanocyty fyziologie   MeSH
• mezibuněčné signální peptidy a proteiny genetika   metabolismus   MeSH
• onkogenní proteiny v-myb fyziologie   MeSH
• ptačí proteiny genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• keratin-19 MeSH
• kostní morfogenetický protein 5 MeSH
• mezibuněčné signální peptidy a proteiny MeSH
• onkogenní proteiny v-myb MeSH
• ptačí proteiny MeSH

Fibrotic diseases are a group of pathologies with high incidence and mortality. Despite extensive research efforts, effective therapies are still not available. Understanding the molecular mechanisms driving the onset, progression and possible resolution of fibrosis is a prerequisite to the development of successful therapies. The central role of the TGF-β pathway and myofibroblasts in the pathogenesis of fibrosis is now generally accepted. The possible mechanisms of myofibroblast elimination or dedifferentiation, on the other hand, are still almost uncharted territory. Here we show that sustained expression of some components of MAPK signaling pathway (PDGFB, Ha-Ras(G12V) or the transcription factor EGR4) in primary chicken embryo dermal myofibroblasts results in a loss of autocrine TGF-β signaling and suppression of the myofibroblastic phenotype, characterized by the loss of alpha smooth muscle actin fibers and a substantial reduction in the production of extracellular matrix. Detailed analysis of the possible molecular mechanisms employed by EGR4 revealed FOXG1, BAMBI, NAB1, NAB2 and DUSP5 genes forming an EGR4 regulated network counteracting autocrine TGF-β signaling. We have also found that a combination of chemical inhibition of TGF-β signaling and perturbation of MAPK signaling with phorbol ester mimics the anti-fibrotic effects of PDGFB, Ha-Ras(G12V) and EGR4.

• Klíčová slova
• EGR4, FOXG1, Ha-Ras(G12V), Microarrays, Myofibroblast, PDGFB, TGF-β,
• MeSH
• aktiny genetika   metabolismus   MeSH
• dediferenciace buněk * MeSH
• forbolové estery farmakologie   MeSH
• kuřecí embryo MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• myofibroblasty cytologie   metabolismus   MeSH
• signální transdukce MeSH
• transformující růstový faktor beta metabolismus   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• forbolové estery MeSH
• mitogenem aktivované proteinkinasy MeSH
• transformující růstový faktor beta MeSH

OBJECTIVE: The primary objective of this study was to identify and clone the first nonmammalian thrombopoietin (TPO), chicken TPO, and its receptor c-Mpl for the purpose of characterizing their activities both in vitro and in vivo. MATERIALS AND METHODS: Chicken TPO was cloned using the methods of reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Northern blotting and RNAse protection assays were employed to analyze the levels of RNA expression in a panel of tissues and cell lines. To study cell surface expression of c-Mpl, polyclonal antibodies were prepared against bacterially derived c-Mpl. Both baculovirus-derived recombinant TPO and retrovirally expressed TPO and c-Mpl were used for the in vivo experiments. RESULTS: Both chicken TPO and its receptor c-Mpl were identified and cloned. Expression of chicken TPO was restricted to only the liver and spleen, while c-mpl was expressed in the bone marrow, lung, and spleen. In vitro experiments with sorted multipotent chicken bone marrow-derived progenitors demonstrated that TPO plays a role in the commitment of these cells to the thrombocytic lineage. Furthermore, TPO in cooperation with stem cell factor also supports proliferation of multipotent progenitors. In experimental animals, the intravenous application of recombinant chicken TPO or overexpression of TPO and c-mpl via retroviral infection lead to erythroblastosis and thromboblastosis. CONCLUSION: The characterized chicken thrombopoietin and its receptor c-Mpl will be valuable tools to further study thrombocytic differentiation and hematopoietic stem cell development. Moreover, the introduced experimental model of the chicken bipotent thrombo-/erythropoietic-progenitor can be used to identify key regulators of cell fate determination.

• MeSH
• buněčný rodokmen MeSH
• Escherichia coli genetika   MeSH
• fibroblasty cytologie   MeSH
• hematopoéza fyziologie   MeSH
• klonování DNA MeSH
• kur domácí MeSH
• kuřecí embryo MeSH
• lidé MeSH
• messenger RNA biosyntéza   MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• receptory thrombopoetinu biosyntéza   genetika   fyziologie   MeSH
• rekombinantní proteiny genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• thrombopoetin biosyntéza   genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• receptory thrombopoetinu MeSH
• rekombinantní proteiny MeSH
• thrombopoetin MeSH