RESUMO
While interactions between neural crest and placode cells are critical for the proper formation of the trigeminal ganglion, the mechanisms underlying this process remain largely uncharacterized. Here, by using chick embryos, we show that the microRNA (miR)-203, whose epigenetic repression is required for neural crest migration, is reactivated in coalescing and condensing trigeminal ganglion cells. Overexpression of miR-203 induces ectopic coalescence of neural crest cells and increases ganglion size. By employing cell-specific electroporations for either miR-203 sponging or genomic editing using CRISPR/Cas9, we elucidated that neural crest cells serve as the source, while placode cells serve as the site of action for miR-203 in trigeminal ganglion condensation. Demonstrating intercellular communication, overexpression of miR-203 in the neural crest in vitro or in vivo represses an miR-responsive sensor in placode cells. Moreover, neural crest-secreted extracellular vesicles (EVs), visualized using pHluorin-CD63 vector, become incorporated into the cytoplasm of placode cells. Finally, RT-PCR analysis shows that small EVs isolated from condensing trigeminal ganglia are selectively loaded with miR-203. Together, our findings reveal a critical role in vivo for neural crest-placode communication mediated by sEVs and their selective microRNA cargo for proper trigeminal ganglion formation.
Assuntos
Comunicação Celular , Vesículas Extracelulares , MicroRNAs , Crista Neural , Gânglio Trigeminal , Crista Neural/metabolismo , Crista Neural/embriologia , Crista Neural/citologia , Animais , MicroRNAs/metabolismo , MicroRNAs/genética , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/embriologia , Gânglio Trigeminal/citologia , Vesículas Extracelulares/metabolismo , Embrião de Galinha , Comunicação Celular/genética , Movimento Celular/genética , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
The neural plate border (NPB) of vertebrate embryos is segregated from the neural plate (NP) and epidermal regions, and comprises an intermingled group of progenitors with multiple fate potential. Recent studies have shown that, during the gastrula stage, TFAP2A acts as a pioneer factor in remodeling the epigenetic landscape required to activate components of the NPB induction program. Here, we show that chick Tfap2a has two highly conserved binding sites for miR-137, and both display a reciprocal expression pattern at the NPB and NP, respectively. In addition, ectopic miR-137 expression reduced TFAP2A, whereas its functional inhibition expanded their territorial distribution overlapping with PAX7. Furthermore, we demonstrate that loss of the de novo DNA methyltransferase DNMT3A expanded miR-137 expression to the NPB. Bisulfite sequencing revealed a markedly elevated presence of non-canonical CpH methylation within the miR-137 promoter region when comparing NPB and NP samples. Our findings show that miR-137 contributes to the robustness of NPB territorial restriction in vertebrate development.
Assuntos
Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs , Placa Neural , Fator de Transcrição AP-2 , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Embrião de Galinha , Metilação de DNA/genética , Placa Neural/metabolismo , Placa Neural/embriologia , Fator de Transcrição AP-2/metabolismo , Fator de Transcrição AP-2/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A/metabolismo , Regiões Promotoras Genéticas/genética , Sítios de LigaçãoRESUMO
Environmental changes alter the sex fate in about 15% of vertebrate orders, mainly in ectotherms such as fish and reptiles. However, the effects of temperature changes on the endocrine and molecular processes controlling gonadal sex determination are not fully understood. Here, we provide evidence that thyroid hormones (THs) act as co-players in heat-induced masculinization through interactions with the stress axis to promote testicular development. We first demonstrated that the thyroid axis (through thyroid-related genes and T3 levels) is highly active in males during the gonadal development in medaka (Oryzias latipes). Similarly, T3 treatments promoted female-to-male sex reversal in XX embryos. Subsequently, embryonic exposure to temperature-induced stress up-regulated the genes related to the thyroid and stress axes with a final increase in T3 levels. In this context, we show that blocking the stress axis response by the loss of function of the corticotropin-releasing hormone receptors suppresses thyroid-stimulating hormone expression, therefore, heat-induced activation of the thyroid axis. Thus, our data showed that early activation of the stress axis and, in consequence, the TH axis, too, leaves us with that both being important endocrine players in inducing female-to-male reversal, which can help predict possible upcoming physiological impacts of global warming on fish populations.
Assuntos
Temperatura Alta , Glândula Tireoide , Feminino , Masculino , Animais , Temperatura , Gônadas , Folhas de PlantaRESUMO
While interactions between neural crest and placode cells are critical for the proper formation of the trigeminal ganglion, the mechanisms underlying this process remain largely uncharacterized. Here, we show that the microRNA-(miR)203, whose epigenetic repression is required for neural crest migration, is reactivated in coalescing and condensing trigeminal ganglion cells. Overexpression of miR-203 induces ectopic coalescence of neural crest cells and increases ganglion size. Reciprocally, loss of miR-203 function in placode, but not neural crest, cells perturbs trigeminal ganglion condensation. Demonstrating intercellular communication, overexpression of miR-203 in the neural crest in vitro or in vivo represses a miR-responsive sensor in placode cells. Moreover, neural crest-secreted extracellular vesicles (EVs), visualized using pHluorin-CD63 vector, become incorporated into the cytoplasm of placode cells. Finally, RT-PCR analysis shows that small EVs isolated from condensing trigeminal ganglia are selectively loaded with miR-203. Together, our findings reveal a critical role in vivo for neural crest-placode communication mediated by sEVs and their selective microRNA cargo for proper trigeminal ganglion formation.
RESUMO
Folate is an essential vitamin for vertebrate embryo development. Methotrexate (MTX) is a folate antagonist that is widely prescribed for autoimmune diseases, blood and solid organ malignancies, and dermatologic diseases. Although it is highly contraindicated for pregnant women, because it is associated with an increased risk of multiple birth defects, the effect of paternal MTX exposure on their offspring has been largely unexplored. Here, we found MTX treatment of adult medaka male fish (Oryzias latipes) causes cranial cartilage defects in their offspring. Small non-coding RNA (sncRNAs) sequencing in the sperm of MTX treated males identify differential expression of a subset of tRNAs, with higher abundance for specific 5' tRNA halves. Sperm RNA methylation analysis on MTX treated males shows that m5C is the most abundant and differential modification found in RNAs ranging in size from 50 to 90 nucleotides, predominantly tRNAs, and that it correlates with greater testicular Dnmt2 methyltransferase expression. Injection of sperm small RNA fractions from MTX-treated males into normal fertilized eggs generated cranial cartilage defects in the offspring. Overall, our data suggest that paternal MTX exposure alters sperm sncRNAs expression and modifications that may contribute to developmental defects in their offspring.
Assuntos
Metotrexato , Pequeno RNA não Traduzido , Animais , Masculino , Gravidez , Humanos , Feminino , Metotrexato/efeitos adversos , Metotrexato/metabolismo , Sêmen , Espermatozoides/metabolismo , Ácido Fólico/metabolismo , Pequeno RNA não Traduzido/genética , RNA de Transferência/genética , RNA de Transferência/metabolismoRESUMO
The neural plate border (NPB) of vertebrate embryos segregates from the neural and epidermal regions, and it is comprised of an intermingled group of multipotent progenitor cells. Folate is the precursor of S-adenosylmethionine, the main methyl donor for DNA methylation, and it is critical for embryonic development, including the specification of progenitors which reside in the NPB. Despite the fact that several intersecting signals involved in the specification and territorial restriction of NPB cells are known, the role of epigenetics, particularly DNA methylation, has been a matter of debate. Here, we examined the temporal and spatial distribution of the methyl source and analyzed the abundance of 5mC/5 hmC and their epigenetic writers throughout the segregation of the neural and NPB territories. Reduced representation bisulfite sequencing (RRBS) on Reduced Folate Carrier 1 (RFC1)-deficient embryos leads to the identification of differentially methylated regions (DMRs). In the RFC1-deficient embryos, we identified several DMRs in the Notch1 locus, and the spatiotemporal expression of Notch1 and its downstream target gene Bmp4 were expanded in the NPB. Cell fate analysis on folate deficient embryos revealed a significant increase in the number of cells coexpressing both neural (SOX2) and NPB (PAX7) markers, which may represent an enhancing effect in the cellular potential of those progenitors. Taken together, our findings propose a model where the RFC1 deficiency drives methylation changes in specific genomic regions that are correlated with a dysregulation of pathways involved in early development such as Notch1 and BMP4 signaling. These changes affect the potency of the progenitors residing in the juncture of the neural plate and NPB territories, thus driving them to a primed state.
RESUMO
Trichomonas vaginalis is an extracellular parasite that colonizes the human urogenital tract, causing a highly prevalent sexually transmitted infection. The parasite must change its transcriptional profile in order to establish and maintain infection. However, few core regulatory elements and transcription factors have been identified to date and little is known about other mechanisms that may control these rapid changes in gene expression during parasite infection. In the last years, epigenetic mechanisms involved in the regulation of gene expression have been gaining major attention in this parasite. In this review, we summarize and discuss the major advances of the last few years with regard to epigenetics (DNA methylation, post-translational histone modifications, and histone variants) in the parasite T. vaginalis. These studies can shed light into our current understanding of this parasite's biology with far-reaching implications for the prognosis and treatment of trichomoniasis.
Assuntos
Epigênese Genética , Tricomoníase/parasitologia , Trichomonas vaginalis/genética , Animais , Metilação de DNA , Humanos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trichomonas vaginalis/metabolismoRESUMO
Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract causing infections that range from asymptomatic to highly inflammatory. Recent works have highlighted the importance of histone modifications in the regulation of transcription and parasite pathogenesis. However, the nature of DNA methylation in the parasite remains unexplored. Using a combination of immunological techniques and ultrahigh-performance liquid chromatography (UHPLC), we analyzed the abundance of DNA methylation in strains with differential pathogenicity demonstrating that N6-methyladenine (6mA), and not 5-methylcytosine (5mC), is the main DNA methylation mark in T. vaginalis Genome-wide distribution of 6mA reveals that this mark is enriched at intergenic regions, with a preference for certain superfamilies of DNA transposable elements. We show that 6mA in T. vaginalis is associated with silencing when present on genes. Interestingly, bioinformatics analysis revealed the presence of transcriptionally active or repressive intervals flanked by 6mA-enriched regions, and results from chromatin conformation capture (3C) experiments suggest these 6mA flanked regions are in close spatial proximity. These associations were disrupted when parasites were treated with the demethylation activator ascorbic acid. This finding revealed a role for 6mA in modulating three-dimensional (3D) chromatin structure and gene expression in this divergent member of the Excavata.
Assuntos
Adenina/metabolismo , Cromatina/química , Metilação de DNA/genética , Trichomonas vaginalis/genética , Ácido Ascórbico/farmacologia , Técnicas de Cultura de Células , Cromatina/genética , Cromatina/metabolismo , Biologia Computacional , Metilação de DNA/efeitos dos fármacos , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Conformação Molecular , Análise de Sequência de DNARESUMO
miR-203 is a tumor-suppressor microRNA with known functions in cancer metastasis. Here, we explore its normal developmental role in the context of neural crest development. During the epithelial-to-mesenchymal transition of neural crest cells to emigrate from the neural tube, miR-203 displays a reciprocal expression pattern with key regulators of neural crest delamination, Phf12 and Snail2, and interacts with their 3'UTRs. We show that ectopic maintenance of miR-203 inhibits neural crest migration in chick, whereas its functional inhibition using a 'sponge' vector or morpholinos promotes premature neural crest delamination. Bisulfite sequencing further shows that epigenetic repression of miR-203 is mediated by the de novo DNA methyltransferase DNMT3B, the recruitment of which to regulatory regions on the miR-203 locus is directed by SNAIL2 in a negative-feedback loop. These findings reveal an important role for miR-203 in an epigenetic-microRNA regulatory network that influences the timing of neural crest delamination.
Assuntos
Crista Neural/citologia , Crista Neural/metabolismo , Fatores de Transcrição/metabolismo , Animais , Embrião de Galinha , Epigenômica , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização In Situ , Fatores de Transcrição/genéticaRESUMO
Folate deficiency has been known to contribute to neural tube and neural crest defects, but why these tissues are particularly affected, and which are the molecular mechanisms involved in those abnormalities are important human health questions that remain unanswered. Here we study the function of two of the main folate transporters, FolR1 and Rfc1, which are robustly expressed in these tissues. Folate is the precursor of S-adenosylmethionine, which is the main donor for DNA, protein and RNA methylation. Our results show that knockdown of FolR1 and/or Rfc1 reduced the abundance of histone H3 lysine and DNA methylation, two epigenetic modifications that play an important role during neural and neural crest development. Additionally, by knocking down folate transporter or pharmacologically inhibiting folate transport and metabolism, we observed ectopic Sox2 expression at the expense of neural crest markers in the dorsal neural tube. This is correlated with neural crest associated defects, with particular impact on orofacial formation. By using bisulfite sequencing, we show that this phenotype is consequence of reduced DNA methylation on the Sox2 locus at the dorsal neural tube, which can be rescued by the addition of folinic acid. Taken together, our in vivo results reveal the importance of folate as a source of the methyl groups necessary for the establishment of the correct epigenetic marks during neural and neural crest fate-restriction.
Assuntos
Deficiência de Ácido Fólico/fisiopatologia , Crista Neural/metabolismo , Fatores de Transcrição SOXB1/fisiologia , Animais , Embrião de Galinha , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/genética , Repressão Epigenética/genética , Repressão Epigenética/fisiologia , Epigenômica , Receptor 1 de Folato , Ácido Fólico/metabolismo , Deficiência de Ácido Fólico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Histonas/metabolismo , Humanos , Tubo Neural/metabolismo , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/fisiopatologiaRESUMO
The central dogma of molecular biology statically says that the information flows from DNA to messenger RNA to protein. But the recent advances in mass spectrometry and high throughput technology have helped the scientists to view RNA as little more than a courier of genetic information encoded in the DNA. The dynamics of RNA modifications in coding and non-coding RNAs are just emerging as a carrier of non-genetic information, uncovering a new layer of complexity in the regulation of gene expression and protein translation. In this review, we summarize about the current knowledge of N6-methyladenosine (m6A), N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine (Ψ) modifications in RNA, and described how these RNA modifications are implicated in early animal development and in several human diseases.
Assuntos
RNA/genética , 5-Metilcitosina/metabolismo , Adenosina/análogos & derivados , Adenosina/genética , Animais , Expressão Gênica/genética , Humanos , Pseudouridina/genéticaRESUMO
Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Different T. vaginalis strains vary greatly in their adherence and cytolytic capacities. These phenotypic differences might be attributed to differentially expressed genes as a consequence of extra-genetic variation, such as epigenetic modifications. In this study, we explored the role of histone acetylation in regulating gene transcription and pathogenesis in T. vaginalis. Here, we show that histone 3 lysine acetylation (H3KAc) is enriched in nucleosomes positioned around the transcription start site of active genes (BAP1 and BAP2) in a highly adherent parasite strain; compared with the low acetylation abundance in contrast to that observed in a less-adherent strain that expresses these genes at low levels. Additionally, exposition of less-adherent strain with a specific histone deacetylases inhibitor, trichostatin A, upregulated the transcription of BAP1 and BAP2 genes in concomitance with an increase in H3KAc abundance and chromatin accessibility around their transcription start sites. Moreover, we demonstrated that the binding of initiator binding protein, the transcription factor responsible for the initiation of transcription of ~75% of known T. vaginalis genes, depends on the histone acetylation state around the metazoan-like initiator to which initiator binding protein binds. Finally, we found that trichostatin A treatment increased parasite aggregation and adherence to host cells. Our data demonstrated for the first time that H3KAc is a permissive histone modification that functions to mediate both transcription and pathogenesis of the parasite T. vaginalis.
Assuntos
Adesão Celular/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Histonas/metabolismo , Vaginite por Trichomonas/patologia , Trichomonas vaginalis/genética , Trichomonas vaginalis/patogenicidade , Acetilação/efeitos dos fármacos , Adesão Celular/genética , Adesão Celular/fisiologia , Agregação Celular/fisiologia , Linhagem Celular Tumoral , Colo do Útero/citologia , Colo do Útero/metabolismo , Colo do Útero/parasitologia , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação da Expressão Gênica , Células HeLa , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Metaloendopeptidases/genética , Ligação Proteica/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Transcrição Gênica/genética , Ativação Transcricional/genética , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/metabolismoRESUMO
One of the earliest manifestations of neural induction is onset of expression of the neural marker Sox2, mediated by the activation of the enhancers N1 and N2. By using loss and gain of function, we find that Sox2 expression requires the activity of JmjD2A and the Msk1 kinase, which can respectively demethylate the repressive H3K9me3 mark and phosphorylate the activating H3S10 (H3S10ph) mark. Bimolecular fluorescence complementation reveals that the adaptor protein 14-3-3, known to bind to H3S10ph, interacts with JMJD2A and may be involved in its recruitment to regulatory regions of the Sox2 gene. Chromatin immunoprecipitation reveals dynamic binding of JMJD2A to the Sox2 promoter and N-1 enhancer at the time of neural plate induction. Finally, we show a clear temporal antagonism on the occupancy of H3K9me3 and H3S10ph modifications at the promoter of the Sox2 locus before and after the neural plate induction. Taken together, our results propose a series of epigenetic events necessary for the early activation of the Sox2 gene in neural progenitor cells.
Assuntos
Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Proteínas 14-3-3/metabolismo , Animais , Embrião de Galinha , Desenvolvimento Embrionário/genética , Elementos Facilitadores Genéticos/genética , Epigenômica , Regulação da Expressão Gênica no Desenvolvimento/genética , Placa Neural/metabolismo , Células-Tronco Neurais/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Neural crest cells form within the neural tube and then undergo an epithelial to mesenchymal transition (EMT) to initiate migration to distant locations. The transcriptional repressor Snail2 has been implicated in neural crest EMT via an as of yet unknown mechanism. We report that the adaptor protein PHD12 is highly expressed before neural crest EMT. At cranial levels, loss of PHD12 phenocopies Snail2 knockdown, preventing transcriptional shutdown of the adhesion molecule Cad6b (Cadherin6b), thereby inhibiting neural crest emigration. Although not directly binding to each other, PHD12 and Snail2 both directly interact with Sin3A in vivo, which in turn complexes with histone deacetylase (HDAC). Chromatin immunoprecipitation revealed that PHD12 is recruited to the Cad6b promoter during neural crest EMT. Consistent with this, lysines on histone 3 at the Cad6b promoter are hyperacetylated before neural crest emigration, correlating with active transcription, but deacetylated during EMT, reflecting the repressive state. Knockdown of either PHD12 or Snail2 prevents Cad6b promoter deacetylation. Collectively, the results show that PHD12 interacts directly with Sin3A/HDAC, which in turn interacts with Snail2, forming a complex at the Cad6b promoter and thus revealing the nature of the in vivo Snail repressive complex that regulates neural crest EMT.
Assuntos
Proteínas Aviárias/genética , Epigênese Genética/genética , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/fisiologia , Crista Neural/metabolismo , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Proteínas Aviárias/metabolismo , Caderinas/genética , Caderinas/metabolismo , Embrião de Galinha , Galinhas/genética , Galinhas/metabolismo , Galinhas/fisiologia , Cromatina/genética , Cromatina/metabolismo , Cromatina/fisiologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Lisina/genética , Lisina/metabolismo , Crista Neural/fisiologia , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3 , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Ativação Transcricional/genéticaRESUMO
The neural crest (NC) is a multipotent, migratory cell population that arises from the dorsal neural fold of vertebrate embryos. NC cells migrate extensively and differentiate into a variety of tissues, including melanocytes, bone, and cartilage of the craniofacial skeleton, peripheral and enteric neurons, glia, and smooth muscle and endocrine cells. For several years, the gene regulatory network that orchestrates NC cells development has been extensively studied. However, we have recently begun to understand that epigenetic and posttranscriptional regulation, such as miRNAs, plays important roles in NC development. In this review, we focused on some of the most recent findings on chromatin-dependent mechanisms and miRNAs regulation during vertebrate NC cells development.
Assuntos
Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Embrião de Mamíferos/embriologia , Epigênese Genética/fisiologia , MicroRNAs/metabolismo , Crista Neural/embriologia , Animais , Embrião de Mamíferos/citologia , Humanos , Crista Neural/citologiaRESUMO
The epithelial-to-mesenchymal transition (EMT) is an important event converting compact and ordered epithelial cells into migratory mesenchymal cells. Given the molecular and cellular similarities between pathological and developmental EMTs, studying this event during neural crest development offers and excellent in vivo model for understanding the mechanisms underlying this process. Here, we review new and old insight into neural crest EMT in search of commonalities with cancer progression that might aid in the design of specific therapeutic prevention.
Assuntos
Transição Epitelial-Mesenquimal , Crista Neural/embriologia , Crista Neural/metabolismo , Junções Aderentes/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Epigênese Genética , Matriz Extracelular/metabolismo , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The central nervous system of adult teleost fish is peculiar because of the following features: (1) the persistence of radial glial cells, (2) an important neurogenic activity and (3) a high aromatase expression by radial cells. In this study, the proliferative zones of the forebrain were described using bromodeoxyuridine (BrdU) treatment in the brain of the pejerrey, an Acanthopterygian teleost fish. These cells were shown to have morphological and immunoreactive characteristics of radial cells and to express aromatase. Three different progenitor populations were identified based on the mobility and proliferation capacity 6 weeks after BrdU treatment: transit amplifying progenitors, slowly proliferating stem cells, and cells remaining in the proliferative zones showing no signs of mitotic activity. The proliferative cells were always located in the ventricular zone and were never observed in the brain parenchyma; however, 3 weeks later they were found away from these proliferative zones and displayed acetylated tubulin immunoreactivity. Other BrdU-positive cells showed astrocyte morphology and were immunoreactive to the S100 glial marker. These results show that in this fish, radial cells are true progenitors generating neurons and possibly astrocytes.
Assuntos
Peixes/anatomia & histologia , Neurogênese , Prosencéfalo/anatomia & histologia , Prosencéfalo/fisiologia , Células-Tronco/fisiologia , Animais , Aromatase/metabolismo , Bromodesoxiuridina/metabolismo , Contagem de Células/métodos , Diferenciação Celular , Movimento Celular/fisiologia , Proliferação de Células , Neurônios/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas S100/metabolismo , Fatores de TempoRESUMO
Although estrogens exert many functions on vertebrate brains, there is little information on the relationship between brain aromatase and estrogen receptors. Here, we report the cloning and characterization of two estrogen receptors, alpha and beta, in pejerrey. Both receptors' mRNAs largely overlap and were predominantly expressed in the brain, pituitary, liver, and gonads. Also brain aromatase and estrogen receptors were up-regulated in the brain of estradiol-treated males. In situ hybridization was performed to study in more detail, the distribution of the two receptors in comparison with brain aromatase mRNA in the brain of adult pejerrey. The estrogen receptors' mRNAs exhibited distinct but partially overlapping patterns of expression in the preoptic area and the mediobasal hypothalamus, as well as in the pituitary gland. Moreover, the estrogen receptor alpha, but not beta, were found to be expressed in cells lining the preoptic recess, similarly as observed for brain aromatase. Finally, it was shown that the onset expression of brain aromatase and both estrogen receptors in the head of larvae preceded the morphological differentiation of the gonads. Because pejerrey sex differentiation is strongly influenced by temperature, brain aromatase expression was measured during the temperature-sensitive window and was found to be significantly higher at male-promoting temperature. Taken together these results suggest close neuroanatomical and functional relationships between brain aromatase and estrogen receptors, probably involved in the sexual differentiation of the brain and raising interesting questions on the origin (central or peripheral) of the brain aromatase substrate.
Assuntos
Aromatase/biossíntese , Encéfalo/fisiologia , Perciformes/fisiologia , Receptores de Estrogênio/biossíntese , Sequência de Aminoácidos , Animais , Aromatase/genética , Aromatase/metabolismo , Sequência de Bases , Encéfalo/anatomia & histologia , Encéfalo/enzimologia , Clonagem Molecular , Estradiol/farmacologia , Feminino , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Perciformes/anatomia & histologia , Perciformes/genética , Perciformes/metabolismo , Filogenia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
Most vertebrates express two gonadotropin releasing hormone (GnRH) variants in brain tissue but there is an increasing number of fish species for which a third GnRH form has been detected. We characterized the precursors (cDNAs) of all three forms expressed in the brain of the pejerrey (silverside) fish, Odontesthes bonariensis (Atheriniformes): type I (GnRH-I; 440 bp), type II (GnRH-II; 529 bp), and type III (GnRH-III; 515 bp). The expression of these GnRHs precursors was also observed in peripheral tissues related to reproduction (gonads), visual and chemical senses (eye and olfactory epithelium), and osmoregulation (gill), suggesting that in teleost fish and possibly other vertebrates GnRH mediates directly or indirectly many other functions besides reproduction. We also present a comprehensive phylogenetic analysis including representatives of all chordate GnRH precursors characterized to date that supports the idea of two main paralogous GnRH lineages with different function. A "forebrain lineage" separates evolutionarily from the "midbrain lineage" as a result of an ancient duplication (ca. 600 million years ago). A third, fish-only clade of GnRH genes seems to have originated before the divergence of fish and tetrapods but retained only in fish. Phylogenetic analyses of GnRH precursors (DNA and protein sequences) under different optimality criteria converge on this result. Although alternative scenarios could not be statistically rejected in this study due to the relatively short size of the analyzed molecules, this hypothesis also receives support from chromosomal studies of synteny around the GnRH genes in vertebrates.
Assuntos
DNA Complementar/genética , Evolução Molecular , Peixes/genética , Hormônio Liberador de Gonadotropina/genética , Precursores de Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/metabolismo , Funções Verossimilhança , Modelos Genéticos , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The brain-type aromatase (CYP19A2) cDNA from pejerrey Odontesthes bonariensis was characterized. Its sequence differs from the ovarian-derived aromatase (CYP19A1) previously reported for the same species. The cDNA is 2305bp in length and the deduced protein comprises 501 amino-acids. The percentage of identity was higher when compared to other brain-derived aromatase proteins (85-63%) and lower with ovarian-derived aromatases (64-57%). Pejerrey aromatases share 61% of identity. The tissue expression analysis showed that CYP19A2 was expressed in the kidney, brain, and pituitary gland of both sexes and also in the ovary, but not in the eye, spleen, liver, gill, and testis. Semi-quantitative RT-PCR analysis of different brain areas revealed that CYP19A2 was expressed significantly higher in anterior male brain areas than in the corresponding female areas, and also when compared to posterior brain areas from both sexes. An immunological analysis using a polyclonal anti-teleost aromatase showed immunoreactive aromatase cells bordering the telencephalic ventricle and a strong signal in the ependymal cells of the preoptic area and the hypothalamus. In the optic tectum immunoreactive aromatase cells were labeled in the ventral wall and in the ependymal layer of the third and fourth ventricle with lateral projections. In the pituitary gland immunoreactive aromatase cells could be found in the rostral and proximal pars distalis. In this gland, aromatase fibers were also detected in different areas; many of them concentrated around blood vessels.