RESUMEN

Using a transgenic zebrafish line harboring a heat-inducible dominant-interference pou5f3 gene (en-pou5f3), we reported that this PouV gene is involved in isthmus development at the midbrain-hindbrain boundary (MHB), which patterns the midbrain and cerebellum. Importantly, the functions of pou5f3 reportedly differ before and after the end of gastrulation. In the present study, we examined in detail the effects of en-pou5f3 induction on isthmus development during embryogenesis. When en-pou5f3 was induced around the end of gastrulation (bud stage), the isthmus was abrogated or deformed by the end of somitogenesis (24 hours post-fertilization). At this stage, the expression of MHB markers -- such as pax2a, fgf8a, wnt1, and gbx2 -- was absent in embryos lacking the isthmus structure, whereas it was present, although severely distorted, in embryos with a deformed isthmus. We further found that, after en-pou5f3 induction at late gastrulation, pax2a, fgf8a, and wnt1 were immediately and irreversibly downregulated, whereas the expression of en2a and gbx2 was reduced only weakly and slowly. Induction of en-pou5f3 at early somite stages also immediately downregulated MHB genes, particularly pax2a, but their expression was restored later. Overall, the data suggested that pou5f3 directly upregulates at least pax2a and possibly fgf8a and wnt1, which function in parallel in establishing the MHB, and that the role of pou5f3 dynamically changes around the end of gastrulation. We next examined the transcriptional regulation of pax2a using both in vitro and in vivo reporter analyses; the results showed that two upstream 1.0-kb regions with sequences conserved among vertebrates specifically drove transcription at the MHB. These reporter analyses confirmed that development of the isthmic organizer is regulated by PouV through direct regulation of pax2/pax2a in vertebrate embryos.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Factor de Transcripción PAX2 , Proteínas de Pez Cebra , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/embriología , Pez Cebra/metabolismo , Factor de Transcripción PAX2/metabolismo , Factor de Transcripción PAX2/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Gastrulación/genética , Animales Modificados Genéticamente , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Embrión no Mamífero/metabolismo , Factores del Dominio POU/genética , Factores del Dominio POU/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Desarrollo Embrionario/genética , Mesencéfalo/metabolismo , Mesencéfalo/embriología , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Somitos/metabolismo , Somitos/embriología , Factores de Crecimiento de Fibroblastos

RESUMEN

BACKGROUND: Elongation of the spinal cord is dependent on neural development from neuromesodermal progenitors in the tail bud. We previously showed the involvement of the Oct4-type gene, pou5f3, in this process in zebrafish mainly by dominant-interference gene induction, but, to compensate for the limitation of this transgene approach, mutant analysis was indispensable. pou5f3 involvement in the signaling pathways was another unsolved question. RESULTS: We examined the phenotypes of pou5f3 mutants and the effects of Pou5f3 activation by the tamoxifen-ERT2 system in the posterior neural tube, together confirming the involvement of pou5f3. The reporter assays using P19 cells implicated tail bud-related transcription factors in pou5f3 expression. Regulation of tail bud development by retinoic acid (RA) signaling was confirmed by treatment of embryos with RA and the synthesis inhibitor, and in vitro reporter assays further showed that RA signaling regulated pou5f3 expression. Importantly, the expression of the RA degradation enzyme gene, cyp26a1, was down-regulated in embryos with disrupted pou5f3 activity. CONCLUSIONS: The involvement of pou5f3 in spinal cord extension was supported by using mutants and the gain-of-function approach. Our findings further suggest that pou5f3 regulates the RA level, contributing to neurogenesis in the posterior neural tube.

Asunto(s)

Factores de Transcripción , Pez Cebra , Animales , Regulación del Desarrollo de la Expresión Génica , Ácido Retinoico 4-Hidroxilasa/genética , Ácido Retinoico 4-Hidroxilasa/metabolismo , Médula Espinal/metabolismo , Factores de Transcripción/metabolismo , Tretinoina/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo

RESUMEN

Mammalian Nanog is critical in pluripotency acquisition and maintenance. Nonetheless, a recent report from zebrafish (Danio rerio) suggests that Nanog is not required for embryonic cells which is not like the mammalian homologs, but is necessary for the proper formation of the extra-embryonic yolk syncytial layer (YSL). However, whether its biological function in other fishes is conservative remains to be investigated. Our previous work shows that Nanog from Nile tilapia (Oreochromis niloticus) (termed as Ong thereafter) displays differential spatiotemporal expression patterns from the other teleost fishes including zebrafish. In this study, Ong co-expression with Pou5f3 (another core pluripotent transcription factor), transcriptional regulation and its biological functions during embryonic development and in the survival and proliferation of embryonic cells were investigated. At the blastula stage, both Ong and Pou5f3 were highly expressed in embryonic cells and co-located in the nucleus. After that, the expression of both Ong and Pou5f3 began to decrease at the gastrula stage (24 haf) and then exhibited a differential expression profile at the segmentation stage (28-36 haf). Ong disappeared in embryonic cells and was limited to YSL, whilst Pou5f3 was highly expressed in embryonic cells even some with obvious cytoplasmic distribution. Luciferase assay indicated that Ong was negatively regulated by Pou5f3 and positively regulated by androgen and itself. Ong depletion in fertilized one-cell embryos through CRISPR/Cas9 led to blastula blockage or death, and the survival and proliferation of blastula-derived embryonic cells in vitro failed. Collectively, Ong has similar expression and biological function to Pou5f3 at the blastula stage, which is similar to mammalian homolog but different from zebrafish homolog. These data suggest that the expression patterns and functions of Nanog are not conservative in fishes and vary from species to species. This study enriches our understanding about Nanog and its evolution.

Asunto(s)

Cíclidos , Pez Cebra , Animales , Cíclidos/genética , Cíclidos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Blástula , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Desarrollo Embrionario/genética , Mamíferos/metabolismo , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo

RESUMEN

POUV is a relatively newly emerged class of POU transcription factors present in jawed vertebrates (Gnathostomata). The function of POUV-class proteins is inextricably linked to zygotic genome activation (ZGA). A large body of evidence now extends the role of these proteins to subsequent developmental stages. While some functions resemble those of other POU-class proteins and are related to neuroectoderm development, others have emerged de novo. The most notable of the latter functions is pluripotency control by Oct4 in mammals. In this review, we focus on these de novo functions in the best-studied species harbouring POUV proteins-zebrafish, Xenopus (anamniotes) and mammals (amniotes). Despite the broad diversity of their biological functions in vertebrates, POUV proteins exert a common feature related to their role in safeguarding the undifferentiated state of cells. Here we summarize numerous pieces of evidence for these specific functions of the POUV-class proteins and recap available loss-of-function data.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Pez Cebra , Animales , Mamíferos/genética , Xenopus laevis/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Cigoto/metabolismo

RESUMEN

In vertebrate embryogenesis, elongation of the posterior body is driven by de novo production of the axial and paraxial mesoderm as well as the neural tube at the posterior end. This process is presumed to depend on the stem cell-like population in the tail bud region, but the details of the gene regulatory network involved are unknown. Previous studies suggested the involvement of pou5f3, an Oct4-type POU gene in zebrafish, in axial elongation. In the present study, we first found that pou5f3 is expressed mainly in the dorsal region of the tail bud immediately after gastrulation, and that this expression is restricted to the posterior-most region of the elongating neural tube during somitogenesis. This pou5f3 expression was complementary to the broad expression of sox3 in the neural tube, and formed a sharp boundary with specific expression of tbxta (orthologue of mammalian T/Brachyury) in the tail bud, implicating pou5f3 in the specification of tail bud-derived cells toward neural differentiation in the spinal cord. When pou5f3 was functionally impaired after gastrulation by induction of a dominant-interfering pou5f3 mutant gene (en-pou5f3), trunk and tail elongation were markedly disturbed at distinct positions along the axis depending on the stage. This finding showed involvement of pou5f3 in de novo generation of the body from the tail bud. Conditional functional abrogation also showed that pou5f3 downregulates mesoderm-forming genes but promotes neural development by activating neurogenesis genes around the tail bud. These results suggest that pou5f3 is involved in formation of the posterior spinal cord.

Asunto(s)

Proteínas de Pez Cebra , Pez Cebra , Animales , Desarrollo Embrionario , Mesodermo , Médula Espinal , Pez Cebra/genética , Proteínas de Pez Cebra/genética

RESUMEN

Enucleated oocytes can reprogram differentiated nuclei to totipotency after somatic cell nuclear transfer (SCNT), which is valuable in understanding nuclear reprogramming and generating genetically modified animals. To date, reprogramming efficiency is low and the development of SCNT embryos is not going as well as anticipated. To further disclose the reprogramming mechanisms during SCNT zebrafish embryo development, we examined the expression patterns of transcription regulation factors and regulated them by mRNA and morpholino microinjection. In this study, we show that stem cell-related transcription factors are downregulated in zebrafish SCNT embryos at the blastula stage. Exogenous expression of pou5f3 at the single-cell stage improves SCNT embryo development from the blastula to the gastrula stage. We also found that exogenous expression of klf4 or sox2 decreases SCNT embryo development from the blastula to the gastrula stage, while expression of nanog is necessary for the development of SCNT embryos. Our results conclude that zebrafish pou5f3 facilitates the development of SCNT embryos from the blastula to gastrula stage.

Asunto(s)

Blastocisto/citología , Reprogramación Celular , Embrión no Mamífero/citología , Desarrollo Embrionario , Técnicas de Transferencia Nuclear , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Blastocisto/metabolismo , Embrión no Mamífero/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Pez Cebra , Proteínas de Pez Cebra/genética

RESUMEN

Three POU family class V gene homologues are expressed in the development of Xenopus. In contrast to the expression of Pou5f3.1 and Pou5f3.2 in organogenesis, Pou5f3.3 is expressed during oogenesis in ovary. We investigated the expression and function of Pou5f3.3 in organogenesis of Xenopus laevis. RT-PCR and immunohistochemical analysis indicated that Pou5f3.3 was expressed in a small number of adult liver cells and blood cells. Immunocytochemical investigation proved that Bmi1, a marker for hematopoietic progenitor cells, was co-expressed in Pou5f3.3-expressing small spherical cells in the peripheral blood. In anemic induction by intraperitoneal injection of phenyl hydrazine, the number of Pou5f3.3-expressing cells significantly increased within 3 days after phenyl hydrazine injection. In CRISPR/Cas mutagenesis of Pou5f3.3, Bmi1-positive hematopoietic progenitor cell count decreased in the hematopoietic dorsal-lateral plate (DLP) region, resulting in a considerable reduction in peripheral blood cells. CRISPR/Cas-induced hematopoietic deficiency was completely rescued by Pou5f3.3 supplementation, but not by Pou5f3.1 or Pou5f3.2. Transplantation experiments using the H2B-GFP transgenic line demonstrated that DLP-derived Pou5f3.3-positive and Bmi1-positive cells were translocated into the liver and bone through the bloodstream. These results suggest that Pou5f3.3 plays an essential role in the establishment and maintenance of hematopoietic progenitor cells during Xenopus development.

Asunto(s)

Desarrollo Embrionario , Células Madre Hematopoyéticas/metabolismo , Factores del Dominio POU/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Anemia/patología , Animales , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Movimiento Celular , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis , Mutagénesis/genética , Factores del Dominio POU/sangre , Factores del Dominio POU/genética , Proteínas de Xenopus/sangre , Proteínas de Xenopus/genética , Xenopus laevis/genética

RESUMEN

Zyxin is a cytoskeletal LIM-domain protein that regulates actin cytoskeleton assembly and gene expression. In the present work, we find that zyxin downregulation in Xenopus laevis embryos reduces the expression of numerous genes that regulate cell differentiation, but it enhances that of several genes responsible for embryonic stem cell status, specifically klf4, pou5f3.1, pou5f3.2, pou5f3.3, and vent2.1/2. For pou5f3 family genes (mammalian POU5F1/OCT4 homologs), we show that this effect is the result of mRNA stabilization due to complex formation with the Y-box protein Ybx1. When bound to Ybx1, zyxin interferes with the formation of these complexes, thereby stimulating pou5f3 mRNA degradation. In addition, in zebrafish embryos and human HEK293 cells, zyxin downregulation increases mRNA levels of the pluripotency genes KLF4, NANOG, and POU5F1/OCT4. Our findings indicate that zyxin may play a role as a switch among morphogenetic cell movement, differentiation, and embryonic stem cell status.

Asunto(s)

Células Madre Embrionarias/metabolismo , Zixina/metabolismo , Zixina/fisiología , Animales , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Células Madre Embrionarias/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Células HEK293 , Humanos , Factor 4 Similar a Kruppel , Morfogénesis , Placa Neural/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Xenopus laevis/metabolismo , Pez Cebra/metabolismo

RESUMEN

In early vertebrate embryos, the dorsal ectoderm is induced by the axial mesendoderm to form the neural plate, which is given competence to form neural cells by soxB1 genes. Subsequently, neurogenesis proceeds in proneural clusters that are generated by a gene network involving proneural genes and Notch signaling. However, what occurs between early neural induction and the later initiation of neurogenesis has not been fully revealed. In the present study, we demonstrated that during gastrulation, the expression of the Oct4-related PouV gene pou5f3 (also called pou2), which is widely observed at earlier stages, was rapidly localized to an array of isolated spotted domains, each of which coincided with individual proneural clusters. Two-color in situ hybridization confirmed that each pou5f3-expressing domain included a proneural cluster. Further analysis demonstrated that anterior pou5f3 domains straddled the boundaries between rhombomere 1 (r1) and r2, whereas posterior domains were included in r4. The effects of forced expression of an inducible negative dominant-interfering pou5f3 gene suggested that pou5f3 activated early proneural genes, such as neurog1 and ebf2, and also soxB1, but repressed the late proneural genes atoh1a and ascl1b. Furthermore, pou5f3 was considered to repress her4.1, a Notch-dependent Hairy/E(spl) gene involved in lateral inhibition in proneural clusters. These results suggest that pou5f3 promotes early neurogenesis in proneural clusters, but negatively regulates later neurogenesis. Suppression of pou5f3 also altered the expression of other her genes, including her3, her5, and her9, further supporting a role for pou5f3 in neurogenesis. In vitro reporter assays in P19 cells showed that pou5f3 was repressed by neurog1, but activated by Notch signaling. These findings together demonstrate the importance of the pou5f3-mediated gene regulatory network in neural development in vertebrate embryos.

Asunto(s)

Placa Neural/embriología , Neurogénesis , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Tipificación del Cuerpo , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Placa Neural/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factores de Transcripción SOXB1/genética , Proteínas de Pez Cebra/genética

RESUMEN

Spermatogenesis is a process driven by stem cell, where germ cell cycle is under the control of a specific genotype species. Considering that Jundiá (Rhamdia quelen) is a Neotropical catfish with great economical importance and useful experimental model, little information is available on basic aspects of its reproductive biology, especially on spermatogenesis. As a result, this study aimed to characterize the male germ cells, estimate the duration of spermatogenesis and evaluate the expression of selected stem cell genes in Jundiá testis. Similar to other fish species, our results showed a remarkable decrease of germ cell nuclear volume during Jundiá spermatogenesis, particularly from type A undifferentiated to late type B spermatogonia and from diplotene to late spermatids. Using a S-phase marker, bromodeoxyuridine (BrdU), the combined duration of meiotic and spermiogenic phases in this species was estimated in approximately 7 days. This is considered very short when compared to mammals, where spermatogenesis last from 30 to 74 days. Selected stem cell genes were partially sequenced and characterized in Jundiá testis. Expression analysis showed higher plzf and pou5f3 mRNA levels in the cell fractions enriched by type A undifferentiated spermatogonia. These results were further confirmed by in situ hybridization that showed strong signal of plzf and pou5f3 mRNA in type A undifferentiated spermatogonia. Altogether, these information will expand our knowledge of the reproductive biology of this species, contributing to improve its production and management, and also for biotechnological applications, such as germ cell transplantation.

Asunto(s)

Biomarcadores/metabolismo , Bagres/metabolismo , Espermatogénesis , Espermatogonias/citología , Células Madre/metabolismo , Clima Tropical , Animales , Bagres/genética , Regulación del Desarrollo de la Expresión Génica , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducción , Espermátides/metabolismo , Espermatogénesis/genética , Espermatogonias/metabolismo , Espermatozoides/citología , Espermatozoides/metabolismo , Distribución Tisular

RESUMEN

Homeobox transcription factors play an essential role in cells differentiation. The function is realized by the proteins (not by the mRNA) and it is necessary to pay more attention to the protein patterns. In this study we were the first to obtain antibodies against the ved protein, tested their specificity by Western-blot analysis and performed a whole mount immunostaining of zebrafish embryos. It was shown that the spatial-temporal ved protein pattern did not differ from that of other vent-family factors. And moreover, its synthesis like that of vox and vent did not depend on pou5f3.

RESUMEN

Zebrafish pou5f3 (previously named pou2), a close homologue of mouse Oct4, encodes a PouV-family transcription factor. pou5f3 has been implicated in diverse aspects of developmental regulation during embryogenesis. In the present study, we addressed the molecular function of Pou5f3 as a transcriptional regulator and the mechanism by which pou5f3 expression is transcriptionally regulated. We examined the influence of effector genes on the expression of the luciferase gene under the control of the upstream 2.1-kb regulatory DNA of pou5f3 (Luc-2.2) in HEK293T and P19 cells. We first confirmed that Pou5f3 functions as a transcriptional activator both in cultured cells and embryos, which confirmed autoregulation of pou5f3 in embryos. It was further shown that Luc-2.2 was activated synergistically by pou5f3 and sox3, which is similar to the co-operative activity of Oct4 and Sox2 in mice, although synergy between pou5f3 and sox2 was less obvious in this zebrafish system. The effects of pou5f3 deletion constructs on the regulation of Luc-2.2 expression revealed different roles for the three subregions of the N-terminal region in Pou5f3 in terms of its regulatory functions and co-operativity with Sox3. Electrophoretic mobility shift assays confirmed that Pou5f3 and Sox3 proteins specifically bind to adjacent sites in the 2.1-kb DNA and that there is an interaction between the two proteins. The synergy with sox3 was unique to pou5f3-the other POU factor genes examined did not show such synergy in Luc-2.2 regulation. Finally, functional interaction was observed between pou5f3 and sox3 in embryos in terms of the regulation of dorsoventral patterning and convergent extension movement. These findings together demonstrate co-operative functions of pou5f3 and sox3, which are frequently coexpressed in early embryos, in the regulation of early development.

Asunto(s)

Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Factor 3 de Transcripción de Unión a Octámeros/genética , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Embrión no Mamífero/citología , Células HEK293 , Humanos , Técnicas In Vitro , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/metabolismo

RESUMEN

Xenopus primordial germ cells (PGCs) are determined by the presence of maternally derived germ plasm. Germ plasm components both protect PGCs from somatic differentiation and begin a unique gene expression program. Segregation of the germline from the endodermal lineage occurs during gastrulation, and PGCs subsequently initiate zygotic transcription. However, the gene network(s) that operate to both preserve and promote germline differentiation are poorly understood. Here, we utilized RNA-sequencing analysis to comprehensively interrogate PGC and neighboring endoderm cell mRNAs after lineage segregation. We identified 1865 transcripts enriched in PGCs compared with endoderm cells. We next compared the PGC-enriched transcripts with previously identified maternal, vegetally enriched transcripts and found that ∼38% of maternal transcripts were enriched in PGCs, including sox7 PGC-directed sox7 knockdown and overexpression studies revealed an early requirement for sox7 in germ plasm localization, zygotic transcription and PGC number. We identified pou5f3.3 as the most highly expressed and enriched POU5F1 homolog in PGCs. We compared the Xenopus PGC transcriptome with human PGC transcripts and showed that 80% of genes are conserved, underscoring the potential usefulness of Xenopus for understanding human germline specification.

Asunto(s)

Células Germinativas/metabolismo , Factores de Transcripción SOXF/metabolismo , Transcriptoma/fisiología , Proteínas de Xenopus/metabolismo , Cigoto/metabolismo , Animales , Células Germinativas/citología , Humanos , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción SOXF/genética , Proteínas de Xenopus/genética , Xenopus laevis , Cigoto/citología

RESUMEN

Mammalian POU5F1 (also known as OCT4) is an essential transcription factor that induces and controls stemness in the inner cell mass and embryonic stem (ES) cells. Its expression results from intricate regulatory networks involving its 5' upstream DNA elements and numerous transcription factors. Pou5f3, the ortholog of POU5F1, has been identified in non-mammalians including fish. However, little is known about the molecular mechanisms controlling its expression up to date. Here we report the promoter activity and regulation of Nile tilapia (Oreochromis niloticus) pou5f3 (Onpou5f3) in fish early-stage embryos and ES cells. A 3.1-kb Onpou5f3 promoter region was cloned, analyzed and constructed into pT2AL-GFP vector. Multiple potential regulatory elements including potential octamer sequence for Pou domain and retinoic acid-responsive elements were found in the 5' upstream region. In vivo and in vitro transfection assays reveal that the 3.1-kb DNA sequence was sufficient to drive strong GFP expression in blastula-stage embryos and ES cells, but low or undetectable expression in either late developmental stage embryos or differentiated cells, suggesting the feasibility as a tool to monitor the pluripotency state in fish stem cells. Deletion luciferase assays reveal that the region from -726 to -219 contains positive regulatory elements, whereas both the regions from -3056 to -1306 and -1306 to -729 contain negative regulatory elements. Notably, just like mammalian POU5F1, OnPou5f3 significantly enhanced its own expression in a dose-dependent manner, whereas RA treatment dramatically reduced its expression. Taken together, our study not only provides a tool for monitoring the pluripotency state of fish stem cells in vitro, but also experimentally demonstrates the molecular mechanisms underlying the Pou5f1 homolog expression might be conserved to some content between mammals and fish.

Asunto(s)

Cíclidos/embriología , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Células Cultivadas , Cíclidos/genética , Clonación Molecular , Secuencia Conservada , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factor 3 de Transcripción de Unión a Octámeros/química , Filogenia , Elementos Reguladores de la Transcripción , Tretinoina/metabolismo

RESUMEN

Octamer-binding transcription factor 4 (Oct4) is a member of POU (Pit-Oct-Unc) transcription factor family Class V that plays a crucial role in maintaining the pluripotency and self-renewal of stem cells. Though it has been deeply investigated in mammals, its lower vertebrate homologue, especially in the marine fish, is poorly studied. In this study, we isolated the full-length sequence of Paralichthys olivaceus pou5f3 (Popou5f3), and we found that it is homologous to mammalian Oct4. We identified two transcript variants with different lengths of 3'-untranslated regions (UTRs) generated by alternative polyadenylation (APA). Quantitative real-time RT-PCR (qRT-PCR), in situ hybridization (ISH) and immunohistochemistry (IHC) were implemented to characterize the spatial and temporal expression pattern of Popou5f3 during early development and in adult tissues. Our results show that Popou5f3 is maternally inherited, abundantly expressed at the blastula and early gastrula stages, then greatly diminishes at the end of gastrulation. It is hardly detectable from the heart-beating stage onward. We found that Popou5f3 expression is restricted to the adult gonads, and continuously expresses during oogenesis while its dynamics are downregulated during spermatogenesis. Additionally, numerous cis-regulatory elements (CRE) on both sides of the flanking regions show potential roles in regulating the expression of Popou5f3. Taken together, these findings could further our understanding of the functions and evolution of pou5f3 in lower vertebrates, and also provides fundamental information for stem cell tracing and genetic manipulation in Paralichthys olivaceus.

Asunto(s)

Secuencia Conservada/genética , Desarrollo Embrionario/genética , Evolución Molecular , Lenguado/embriología , Lenguado/genética , Genómica , Gónadas/embriología , Factor 3 de Transcripción de Unión a Octámeros/genética , Región de Flanqueo 5'/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas/genética , Biología Computacional , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Gónadas/metabolismo , Inmunohistoquímica , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Regiones Promotoras Genéticas , Análisis de Secuencia de Proteína , Homología de Secuencia de Ácido Nucleico , Sintenía/genética

RESUMEN

PouV class transcription factor Oct4/Pou5f1 is a central regulator of indefinite pluripotency in mammalian embryonic stem cells (ESCs) but also participates in cell lineage specification in mouse embryos and in differentiating cell cultures. The molecular basis for this versatility, which is shared between Oct4 and its non-mammalian homologs Pou5f1 and Pou5f3, is not yet completely understood. Here, I review the current understanding of the evolution of PouV class transcription factors and discuss equivalent and diverse roles of Oct4 homologs in pluripotency, differentiation, and cell behavior in different vertebrate embryos. This article is part of a Special Issue entitled: The Oct Transcription Factor Family, edited by Dr. Dean Tantin.

Asunto(s)

Evolución Biológica , Regulación del Desarrollo de la Expresión Génica , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/metabolismo , Proteínas de Pez Cebra/genética , Animales , Diferenciación Celular , Linaje de la Célula/genética , Embrión de Mamíferos , Embrión no Mamífero , Ratones , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Filogenia , Células Madre Pluripotentes/citología , Xenopus laevis/clasificación , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrollo , Pez Cebra/clasificación , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/metabolismo

RESUMEN

The transcription factors Pou5f1, Sox2, and Nanog are central regulators of pluripotency in mammalian ES and iPS cells. In vertebrate embryos, Pou5f1/3, SoxB1, and Nanog control zygotic genome activation and participate in lineage decisions. We review the current knowledge of the roles of these genes in developing vertebrate embryos from fish to mammals and suggest a model for pluripotency gene regulatory network functions in early development.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Células Madre Pluripotentes , Cigoto , Animales , Linaje de la Célula , Evolución Molecular , Femenino , Humanos , Mamíferos/embriología , Mamíferos/genética , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo

RESUMEN

The transcription factor of pluripotency Pou5f3 is considered to enhance the Vox expression. This conclusion was based on the study of mRNA expression, but the expression of the Vent-family proteins was not analyzed. We compare spatiotemporal distribution of the Vox and Vent mRNAs and the proteins in embryos of wild type zebrafish (WT) and MZspg (spiel ohne grenzen) mutants devoid of both maternal and embryonic Pou5f3 functions. We revealed the Vox mRNA and its protein in both the WT and mutant embryos during the cleavage period. They were probably prestored maternally. The quantity of the prestored protein, unlike the mRNA, in the mutants was visibly less than that in the WT embryos. The Pou5f3, therefore, had no influence on the Vox mRNA maternal synthesis, but it affected the maternal Vox protein synthesis. During the blastula and gastrula periods the MZspg mutants, but not the WT, failed to synthesize the new Vox mRNA, while the prestored maternal mRNA was gradually degrading. At these stages the WT and mutant embryos displayed minor visual quantitative difference in staining of Vox protein. The Vent mRNA was not maternally prestored and its zygote synthesis slightly depended on the Pou5f3. The Vent protein in mutants and WT was synthesized on the new zygote mRNAs. By the gastrula period, the Vent staining of the WT and mutant embryos were almost comparable. The data obtained suggest the existence of mechanisms sustaining a required Vox and Vent proteins level, but these mechanisms are not directly dependent on the Pou5f3.

RESUMEN

POU class V (POU-V) transcription factors play the important role in maintenance of pluripotency and cell differentiation. Pou5f3.2 (Oct25), one of Xenopus POU-V transcription factors, shows the zygotic expression prior to gastrulation. In order to know the molecular mechanism of pou5f3.2 expression at gastrula stage, we examined a responsiveness of pou5f3.2 to Nodal signaling. Animal cap assay demonstrated that Xnr2 activates the gene expression of pou5f3.2. In comparative analysis of the 5'-flanking region of pou5f3.2 between Xenopus laevis and X. tropicalis, two conserved regions were detected within the flanking region. Reporter analyses showed that one of the conserved regions contained an enhancer region, which had several Smad2/3 and FoxH1 binding motifs. ChIP assay demonstrated that Smad2 binds to the enhancer region. These results suggest that Nodal signaling induces zygotic expression of pou5f3.2 at gastrula stage. To understand a role of pou5f3.2 in gastrula embryos, morpholino oligo DNA of pou5f3.2 was injected into the lateral side of one blastomere at the 2-cell stage. The morphant embryos showed diminution of Xbra1 expression and gastrulation defect in the injection side, suggesting the essential role of pou5f3.2 at the gastrula stage. Xbra1 expression and gastrulation were also inhibited by injecting with the synthesized RNAs of pou5f3.2. Furthermore, in the pou5f3.2-injected embryo, gene expression of p27Xic1 was drastically suppressed, and the number of dividing cells increased in the injection side. These results suggest that one role of pou5f3.2 is to keep the embryonic cells in undifferentiated and proliferative state during gastrulation.

Asunto(s)

Proliferación Celular/fisiología , Gastrulación , Factores del Dominio POU/fisiología , Xenopus laevis/embriología , Animales , Regulación del Desarrollo de la Expresión Génica , Factores del Dominio POU/genética

RESUMEN

BACKGROUND: Pou5f1/Oct4 is a transcription factor essential for maintenance of pluripotency in mammals and for control of blastula and gastrula stage gene regulatory networks in zebrafish. Information on Pou5f1 protein distribution was before this study not available for zebrafish. Therefore, we generated polyclonal antibodies that selectively recognize Pou5f1 and analyzed its protein distribution and modification during development. RESULTS: Pou5f1 protein is present in unfertilized oocytes, and persists in all embryonic and enveloping layer cell nuclei until the end of gastrulation, but is absent from yolk syncytial nuclei. Pou5f1 is subject to multiple developmentally regulated phosphorylations, with the higher phosphorylated forms prevailing in the oocyte and during late gastrulation. CONCLUSIONS: The developmental protein profile correlates with the stages during which deep cells are not committed to a specific germ layer. The posttranslational modification by phosphorylation opens the possibility that Pou5f1 may be subject to temporal or region specific modulation of its activity or stability by embryonic signaling mechanisms.

Asunto(s)

Gástrula/metabolismo , Gastrulación/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Procesamiento Proteico-Postraduccional/fisiología , Transducción de Señal/fisiología , Proteínas de Pez Cebra/biosíntesis , Pez Cebra/embriología , Animales , Gástrula/citología , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Oocitos/citología , Oocitos/metabolismo , Fosforilación/fisiología , Pez Cebra/genética , Proteínas de Pez Cebra/genética