RESUMEN

The advent of facile genome engineering technologies has made the generation of knock-in gene-expression or fusion-protein reporters more tractable. Fluorescent protein labeling of specific genes combined with surface marker profiling can more specifically identify a cell population. However, the question of which fluorescent proteins to utilize to generate reporter constructs is made difficult by the number of candidate proteins and the lack of updated experimental data on newer fluorescent proteins. Compounding this problem, most fluorescent proteins are designed and tested for use in microscopy. To address this, we cloned and characterized the detection sensitivity, spectral overlap, and spillover spreading of 13 monomeric fluorescent proteins to determine utility in multicolor panels. We identified a group of five fluorescent proteins with high signal to noise ratio, minimal spectral overlap, and low spillover spreading making them compatible for multicolor experiments. Specifically, generating reporters with combinations of three of these proteins would allow efficient measurements even at low-level expression. Because the proteins are monomeric, they could function either as gene-expression or as fusion-protein reporters. Additionally, this approach can be generalized as new fluorescent proteins are developed to determine their usefulness in multicolor panels. © 2018 International Society for Advancement of Cytometry.

Asunto(s)

Citometría de Flujo/métodos , Colorantes Fluorescentes , Genes Reporteros , Animales , Línea Celular , Humanos , Ratones , Microscopía Confocal

RESUMEN

The homeobox gene hhex is one of the earliest markers of the anterior endoderm, which gives rise to foregut organs such as the liver, ventral pancreas, thyroid, and lungs. The regulatory networks controlling hhex transcription are poorly understood. In an extensive cis-regulatory analysis of the Xenopus hhex promoter, we determined how the Nodal, Wnt, and BMP pathways and their downstream transcription factors regulate hhex expression in the gastrula organizer. We show that Nodal signaling, present throughout the endoderm, directly activates hhex transcription via FoxH1/Smad2 binding sites in the proximal -0.44 Kb promoter. This positive action of Nodal is suppressed in the ventral-posterior endoderm by Vent 1 and Vent2, homeodomain repressors that are induced by BMP signaling. Maternal Wnt/ß-catenin on the dorsal side of the embryo cooperates with Nodal and indirectly activates hhex expression via the homeodomain activators Siamois and Twin. Siamois/Twin stimulate hhex transcription through two mechanisms: (1) they induce the expression of Otx2 and Lim1 and together Siamois, Twin, Otx2, and Lim1 appear to promote hhex transcription through homeobox sites in a Wnt-responsive element located between -0.65 to -0.55 Kb of the hhex promoter. (2) Siamois/Twin also induce the expression of the BMP-antagonists Chordin and Noggin, which are required to exclude Vents from the organizer allowing hhex transcription. This study reveals a complex network regulating anterior endoderm transcription in the early embryo.

Asunto(s)

Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes/fisiología , Proteínas de Homeodominio/genética , Transcripción Genética , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Animales , Factores de Transcripción Forkhead/fisiología , Proteínas de Homeodominio/fisiología , Proteína Nodal/fisiología , Factores de Transcripción Otx/fisiología , Elementos de Respuesta , Transducción de Señal , Proteína Smad2/fisiología , Proteínas Wnt/fisiología , Proteínas de Xenopus/fisiología , beta Catenina/fisiología

RESUMEN

Current models of canonical Wnt signaling assume that a pathway is active if beta-catenin becomes nuclearly localized and Wnt target genes are transcribed. We show that, in Xenopus, maternal LRP6 is essential in such a pathway, playing a pivotal role in causing expression of the organizer genes siamois and Xnr3, and in establishing the dorsal axis. We provide evidence that LRP6 acts by degrading axin protein during the early cleavage stage of development. In the full-grown oocyte, before maturation, we find that axin levels are also regulated by Wnt11 and LRP6. In the oocyte, Wnt11 and/or LRP6 regulates axin to maintain beta-catenin at a low level, while in the embryo, asymmetrical Wnt11/LRP6 signaling stabilizes beta-catenin and enriches it on the dorsal side. This suggests that canonical Wnt signaling may not exist in simple off or on states, but may also include a third, steady-state, modality.

Asunto(s)

Oocitos/metabolismo , Receptores de LDL/metabolismo , Proteínas Represoras/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriología , Xenopus/metabolismo , beta Catenina/metabolismo , Animales , Proteína Axina , Secuencia de Bases , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Femenino , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Modelos Biológicos , Oocitos/crecimiento & desarrollo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de LDL/genética , Transducción de Señal , Xenopus/genética

RESUMEN

The segregation of the vertebrate embryo into three primary germ layers is one of the earliest developmental decisions. In Xenopus, where the process is best understood, the endoderm is specified by a vegetally localized transcription factor, VegT, which releases nodal signals that specify the adjacent marginal zone of the blastula to become mesoderm. However, little is known about how the ectoderm becomes specified. In this paper, we show that the forkhead protein FoxI1e (also known as Xema) is required at the blastula stage for normal formation of both the central nervous system and epidermis, the two early derivatives of the ectoderm. In addition, FoxI1e is required to maintain the regional identity of the animal cells of the blastula, the cells that are precursors of ectodermal structures. In its absence, they lose contact with the animal cap, mix with cells of other germ layers and differentiate according to their new positions. Because FoxI1e is initially expressed in the animal region of the embryo and is rapidly downregulated in the neural plate, its role in neural and epidermal gene expression must precede the division of the ectoderm into neural and epidermal. The work also shows that FoxI1e plays a role in the embryo in the poorly understood process of differential adhesion, which limits cell mixing as primary germ layers become specified.

Asunto(s)

Blástula/citología , Ectodermo/citología , Factores de Transcripción/fisiología , Proteínas de Xenopus/fisiología , Animales , Adhesión Celular , Diferenciación Celular , Sistema Nervioso Central/citología , Sistema Nervioso Central/embriología , Inducción Embrionaria , Células Epidérmicas , Epidermis/embriología , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/genética , Xenopus , Proteínas de Xenopus/genética

RESUMEN

Wnt signaling pathways have essential roles in developing embryos and adult tissue, and alterations in their function are implicated in many disease processes including cancers. The major nuclear transducers of Wnt signals are the Tcf/LEF family of transcription factors, which have binding sites for both the transcriptional co-repressor groucho, and the co-activator beta-catenin. The early Xenopus embryo expresses three maternally inherited Tcf/LEF mRNAs, and their relative roles in regulating the expression of Wnt target genes are not understood. We have addressed this by using antisense oligonucleotides to deplete maternal XTcf1 and XTcf4 mRNAs in oocytes. We find that XTcf1 represses expression of Wnt target genes ventrally and laterally, and activates their expression dorsally. Double depletions of XTcf1 and XTcf3 suggest that they act cooperatively to repress Wnt target genes ventrally. In contrast, XTcf4 has no repressive role but is required to activate expression of Xnr3 and chordin in organizer cells at the gastrula stage. This work provides evidence for distinct roles for XTcfs in regulating Wnt target gene expression.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Factor 1 de Transcripción de Linfocitos T/metabolismo , Factores de Transcripción TCF/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Embrión no Mamífero/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor 1 de Transcripción de Linfocitos T/genética , Factores de Transcripción TCF/genética , Proteína 1 Similar al Factor de Transcripción 7 , Proteína 2 Similar al Factor de Transcripción 7 , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Wnt/genética , Xenopus/embriología , Xenopus/metabolismo , Proteínas de Xenopus/genética

RESUMEN

Xenopus Vg 1, a transforming growth factor beta (Tgfbeta) family member, was one of the first maternally localized mRNAs identified in vertebrates. Its restriction to the vegetal pole of the egg made it the ideal candidate to be the mesoderm-inducing signal released by vegetal cells, but its function in vivo has never been resolved. We show that Vg 1 is essential for Xenopus embryonic development, and is required for mesoderm induction and for the expression of several key Bmp antagonists. Although the original Vg 1 transcript does not rescue Vg 1-depleted embryos, we report that a second allele is effective. This work resolves the mystery of Vg 1 function, and shows it to be an essential maternal regulator of embryonic patterning.

Asunto(s)

Tipificación del Cuerpo/fisiología , Inducción Embrionaria/genética , Glicoproteínas/metabolismo , Mesodermo/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Xenopus/embriología , Xenopus/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Inducción Embrionaria/fisiología , Hibridación in Situ , Datos de Secuencia Molecular , Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Xenopus

RESUMEN

The Xenopus T box family member VegT is expressed maternally in the vegetal hemisphere of the embryo. Mis-expression of VegT in prospective ectodermal tissue causes ectopic activation of mesodermal and endodermal markers, and ablation of VegT transcripts prevents proper formation of the mesendoderm, with the entire embryo developing as epidermis. These observations define VegT as a key initiator of mesendodermal development in the Xenopus embryo, and in an effort to understand how it exerts its effects we have used microarray analysis to compare gene expression in control animal caps with that in ectodermal tissue expressing an activated form of VegT. This procedure allowed the identification of 99 potential VegT targets, and we went on to study the expression patterns of these genes and then to ask, for those that are expressed in mesoderm or endoderm, which are direct targets of VegT. The putative regulatory regions of the resulting 14 genes were examined for T domain binding sites, and we also asked whether their expression is down-regulated in embryos in which VegT RNA is ablated. Finally, the functions of these genes were assayed by both over-expression and by use of antisense morpholino oligonucleotides. Our results provide new insights into the function of VegT during early Xenopus development.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Análisis por Micromatrices/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Dominio T Box/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/genética , Animales , Sitios de Unión , ADN Complementario/metabolismo , Regulación hacia Abajo , Endodermo/metabolismo , Técnicas Genéticas , Genoma , Hibridación in Situ , Mesodermo/metabolismo , Modelos Genéticos , Hibridación de Ácido Nucleico , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Proteínas de Xenopus/genética

RESUMEN

Wnt signaling pathways play essential roles in patterning and proliferation of embryonic and adult tissues. In many organisms, this signaling pathway directs axis formation. Although the importance of intracellular components of the pathway, including beta-catenin and Tcf3, has been established, the mechanism of their activation is uncertain. In Xenopus, the initiating signal that localizes beta-catenin to dorsal nuclei has been suggested to be intracellular and Wnt independent. Here, we provide three lines of evidence that the pathway specifying the dorsal axis is activated extracellularly in Xenopus embryos. First, we identify Wnt11 as the initiating signal. Second, we show that activation requires the glycosyl transferase X.EXT1. Third, we find that the EGF-CFC protein, FRL1, is also essential and interacts with Wnt11 to activate canonical Wnt signaling.

Asunto(s)

Tipificación del Cuerpo/fisiología , Proteínas del Citoesqueleto/metabolismo , Glicoproteínas/metabolismo , Transducción de Señal/fisiología , Transactivadores/metabolismo , Xenopus/metabolismo , Animales , Tipificación del Cuerpo/genética , Proteínas del Citoesqueleto/genética , Cartilla de ADN/genética , Embrión no Mamífero/metabolismo , Femenino , Proteínas Ligadas a GPI , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Glicoproteínas/genética , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas de la Membrana/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Unión Proteica , ARN Mensajero/genética , Transducción de Señal/genética , Transactivadores/genética , Factores de Transcripción/metabolismo , Proteínas Wnt , Xenopus/embriología , Proteínas de Xenopus/metabolismo , beta Catenina

RESUMEN

FoxH1 (Fast1) was first characterized as the transcriptional partner for Smad proteins. Together with Smad2/4, it forms the activin response factor (ARF) that binds to the Mix.2 promoter in Xenopus embryos. Foxh1 is expressed maternally in Xenopus. Depletion of maternal Foxh1 mRNA results in abnormalities of head and dorsal axis formation. We show that FoxH1 is required, together with XTcf3/beta catenin, to activate the zygotic expression of the nodal gene, Xnr3 in a Smad2-independent manner. In contrast, maternal FoxH1 acts as an inhibitor of Xnr5 and 6 transcription, preventing their upregulation on the ventral side of the embryo, by the maternal T-box transcription factor VegT. We conclude that maternal FoxH1 has essential, context-dependent roles in regulating the pattern of zygotic gene expression in the early embryo.

Asunto(s)

Tipificación del Cuerpo/fisiología , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Femenino , Factores de Transcripción Forkhead , Genes Reporteros , Cabeza/embriología , Proteína Nodal , Ligandos de Señalización Nodal , ARN Mensajero/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/metabolismo , Xenopus , Proteínas de Xenopus/metabolismo

RESUMEN

The transcription factor VegT, is required in early Xenopus embryos for the formation of both the mesoderm and endoderm germ layers. Inherited as a maternal mRNA localized only in vegetal cells, VegT activates the transcription of a large number of transcription factors, as well as signaling ligands that induce cells in the vegetal mass to form endoderm, and the marginal zone to form mesoderm. It is important now to understand the extent to which transcription factors downstream of VegT play individual, or overlapping, roles in the specification and patterning of the endoderm and mesoderm. In addition, it is important to understand the mechanism that specifies the boundary between endoderm and mesoderm. One of the downstream targets of VegT, the homeodomain protein Mixer, is expressed at high levels at the mesoderm/endoderm boundary at the late blastula stage. We therefore examined its functions by blocking its translation using morpholino oligos. In Mixer-depleted embryos, the expression of many signaling ligands and transcription factors was affected. In particular, we found that the expression of several genes, including several normally expressed in mesoderm, was upregulated. Functional assays of Mixer-depleted vegetal cells showed that they have increased mesoderm-inducing activity. This demonstrates that Mixer plays an essential role in controlling the amount of mesoderm induction by the vegetal cells.

Asunto(s)

Tipificación del Cuerpo/fisiología , Embrión no Mamífero/fisiología , Proteínas de Homeodominio/fisiología , Factores de Transcripción/fisiología , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Animales , Secuencia de Bases , Blástula , Anomalías Congénitas/genética , Embrión no Mamífero/citología , Endodermo/fisiología , Gástrula/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Mesodermo/fisiología , Datos de Secuencia Molecular , Oligorribonucleótidos/química , Técnicas de Cultivo de Órganos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Cigoto/fisiología

RESUMEN

Convergent extension behaviour is critical for the formation of the vertebrate body axis. In Xenopus, components of the Wnt signaling pathway have been shown to be required for convergent extension movements but the relationship between cell fate and morphogenesis is little understood. We show by loss of function analysis that Xnr3 activates Xbra expression through FGFR1. We show that eFGF activity is not essential in the pathway, and that dishevelled acts downstream of Xnr3 and not in a parallel pathway. We provide evidence for the involvement of the EGF-CFC protein FRL1, and suggest that the pro-domain of Xnr3 may be required for its activity. Since Xnr3 is a direct target of the maternal betacatenin/XTcf3 signaling pathway, it provides the link between the initial, maternally controlled, allocation of cell fate, and the morphogenetic movements of cells derived from the organizer.

Asunto(s)

Tipificación del Cuerpo/fisiología , Movimiento Celular/fisiología , Glicoproteínas , Péptidos y Proteínas de Señalización Intercelular , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Xenopus laevis/embriología , Animales , Biomarcadores , Regulación del Desarrollo de la Expresión Génica , Oligonucleótidos Antisentido/metabolismo , Oocitos/fisiología , Organizadores Embrionarios/fisiología , Fenotipo , Proteínas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/genética , Transducción de Señal/fisiología , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factor de Crecimiento Transformador beta/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/fisiología

RESUMEN

In the early Xenopus embryo, the dorsal axis is specified by a Wnt signal transduction pathway, involving the movement of beta-catenin into dorsal cell nuclei and its functional association with the LEF-type transcription factor XTcf3. The subsequent function of XTcf3 is uncertain. Overexpression data has suggested that it can be both an activator and repressor of downstream genes. XTcf3 mRNA is synthesized during oogenesis in Xenopus and is stored in the egg. To identify its role in dorsal axis specification, we depleted this maternal store in full-grown oocytes using antisense deoxyoligonucleotides, and fertilized them. The developmental effects of XTcf3 depletion, both on morphogenesis and the expression of marker genes, show that primarily, XTcf3 is an inhibitor, preventing both dorsal and ventral cells of the late blastula from expressing dorsal genes. We also show that simple relief from the repression is not the only factor required for dorsal gene expression. To demonstrate this, we fertilized eggs that had been depleted of both XTcf3 and the maternal transcription factor VegT. Dorsal genes normally repressed by XTcf3 are not activated in these embryos. These data show that normal dorsal gene expression in the embryo requires the transcriptional activator VegT, whilst XTcf3 prevents their inappropriate expression on the ventral side of the embryo.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Proteínas HMGB/fisiología , Organizadores Embrionarios , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Proteínas de Xenopus , Animales , Proteína Axina , Blastocisto/fisiología , Tipificación del Cuerpo/fisiología , Proteínas del Citoesqueleto/metabolismo , Femenino , Proteína Goosecoide , Proteínas HMGB/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ligandos de Señalización Nodal , Oocitos/metabolismo , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/fisiología , Factores de Transcripción TCF , Transactivadores/metabolismo , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/metabolismo , Xenopus , beta Catenina