RESUMO
wnt8a Is essential for normal patterning during vertebrate embryonic development, and either gain or loss-of-function gene dysregulation results in severe axis malformations. The zebrafish wnt8a locus is structured such that transcripts may possess two regulatory 3' untranslated regions (UTRs), raising the possibility of post-transcriptional regulation as an important mode of wnt8a signaling control. To determine whether both UTRs contribute to post-transcriptional wnt8a gene regulation, each UTR (UTR1 and UTR2) was tested in transient and transgenic reporter assays. Both UTRs suppress EGFP reporter expression in cis, with UTR2 exhibiting a more pronounced effect. UTR2 contains a 6 base sequence necessary for UTR2 regulatory function that is complementary to the seed of the microRNA, miR-430. A target protector morpholino that overlaps the seed complement stabilizes both reporter mRNAs and wnt8a mRNAs, and produces phenotypic abnormalities consistent with wnt8a gain-of-function. In rescue assays, specific functions can be attributed to each of the two wnt8a proteins encoded by the locus. An interplay of wnt8a.1 and wnt8a.2 regulates neural and mesodermal patterning and morphogenesis as well as patterning between brain subdivisions. Thus, post-transcriptional control of wnt8a is essential to fine tune the balance of the signaling outputs of the complex wnt8a locus.
Assuntos
Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica no Desenvolvimento , Processamento Pós-Transcricional do RNA , Proteínas Wnt/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Animais , Sequência de Bases , Padronização Corporal , Proteínas de Fluorescência Verde/metabolismo , MicroRNAs/metabolismo , Dados de Sequência Molecular , Neurônios/metabolismo , Fenótipo , RNA Mensageiro/metabolismo , Transdução de Sinais , Transgenes , Peixe-Zebra/genéticaRESUMO
The sheep genome contains multiple copies of endogenous betaretroviruses highly related to the exogenous and oncogenic jaagsiekte sheep retrovirus (JSRV). The endogenous JSRVs (enJSRVs) are abundantly expressed in the uterine luminal and glandular epithelia as well as in the conceptus trophectoderm and are essential for conceptus elongation and trophectoderm growth and development. Of note, enJSRVs are present in sheep and goats but not cattle. At least 5 of the 27 enJSRV loci cloned to date possess an intact genomic organization and are able to produce viral particles in vitro. In this study, we found that enJSRVs form viral particles that are released into the uterine lumen of sheep. In order to test the infectious potential of enJSRV particles in the uterus, we transferred bovine blastocysts into synchronized ovine recipients and allowed them to develop for 13 days. Analysis of microdissected trophectoderm of the bovine conceptuses revealed the presence of enJSRV RNA and, in some cases, DNA. Interestingly, we found that RNAs belonging to only the most recently integrated enJSRV loci were packaged into viral particles and transmitted to the trophectoderm. Collectively, these results support the hypothesis that intact enJSRV loci expressed in the uterine endometrial epithelia are shed into the uterine lumen and could potentially transduce the conceptus trophectoderm. The essential role played by enJSRVs in sheep reproductive biology could also be played by endometrium-derived viral particles that influence development and differentiation of the trophectoderm.
Assuntos
Blastocisto/virologia , Retrovirus Jaagsiekte de Ovinos/patogenicidade , Infecções por Retroviridae/veterinária , Trofoblastos/virologia , Útero/virologia , Vírion/isolamento & purificação , Animais , Bovinos , Doenças dos Bovinos/virologia , DNA Viral/isolamento & purificação , Transferência Embrionária , Feminino , Retrovirus Jaagsiekte de Ovinos/crescimento & desenvolvimento , Retrovirus Jaagsiekte de Ovinos/isolamento & purificação , Gravidez , Ovinos , Doenças dos Ovinos/virologia , Transdução Genética , Eliminação de Partículas ViraisRESUMO
Interferon τ (IFNT), the pregnancy recognition signal in ruminants, abrogates the luteolytic mechanism for maintenance of the corpus luteum for production of progesterone (P(4)). This study examined the expression of DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 (DDX58) and phospholipid scramblase 1 (PLSCR1) mRNAs in the ovine uterus as these genes were increased most in 2fTGH (STAT1 positive) cells by IFNT. The results of this study indicated that IFNT regulates expression of DDX58 and PLSCR1 mRNAs in the ovine uterus, which confirmed the results of the in vitro transcriptional profiling experiment with the 2fTGH (parental STAT1 positive) and U3A (STAT1 null) cell lines. Steady-state levels of DDX58 and PLSCR1 mRNAs increased in cells of the ovine uterus between days 12 and 20 of pregnancy, but not between days 10 and 16 of the estrous cycle. The expression of DDX58 and PLSCR1 mRNAs was greatest in endometrial stromal cells, but there was transient expression in uterine luminal and superficial glandular epithelial cells. P(4) alone did not induce expression of DDX58 and PLSCR1 mRNAs; however, intrauterine injections of IFNT did induce expression of DDX58 and PLSCR1 mRNAs in the endometria of nonpregnant ewes independent of effects of P(4). These results indicate that IFNT induces expression of DDX58 and PLSCR1 in ovine endometrial cells via the classical STAT1-mediated cell signaling pathway. Based on their known biological effects, DDX58 and PLSCR1 are IFN-stimulated genes, which may increase the antiviral status of cells of the pregnant uterus to protect against viral infection and/or enhance secretion of type I IFNs that inhibit viral replication.
Assuntos
RNA Helicases DEAD-box/metabolismo , Implantação do Embrião , Embrião de Mamíferos/metabolismo , Endométrio/enzimologia , Interferon Tipo I/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas da Gravidez/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Implantação do Embrião/genética , Feminino , Regulação da Expressão Gênica , Humanos , Ovariectomia , Proteínas de Transferência de Fosfolipídeos/genética , Gravidez , Progesterona/metabolismo , RNA Mensageiro/metabolismo , Receptores Imunológicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/metabolismo , Ovinos , Fatores de TempoRESUMO
Interferon tau (IFNT), the maternal recognition of pregnancy signal in sheep and other ruminants, is secreted by the conceptus and regulates the expression of a number of genes in a cell-specific manner within the uterus. The response of different endometrial cell types to IFNT appears to be specified by IFN regulatory factors (IRFs). IRF2, a potent repressor of gene transcription, is expressed only by luminal (LE) and superficial glandular epithelia (sGE), whereas IRF1 and IRF9, activators of gene transcription, are expressed only in GE and stromal cells of the uterus during early pregnancy. In the present study, IRF6 was found to be expressed in LE/sGE and middle GE of the ovine uterine endometrium as well as conceptus trophectoderm. IRF family members can regulate transcription via IFN-stimulated response elements (ISREs). Transient transfection analyses found that IRF6 enhanced basal activity of ISRE-containing promoters, but did not enhance IFNT stimulation of ISRE-containing promoters in variety of different cell types. Further, IRF6 did not cooperate with IRF1 or reduce IRF2 repression of ISRE-containing promoter activity. These results establish that IRF6 is a transcriptional activator that is preferentially expressed in the endometrial epithelia and conceptus trophectoderm. IRF6 is hypothesized to play critical roles in endometrial gene expression as well as in conceptus trophectoderm growth and differentiation.