RESUMEN
Gene/transcript model sets predicted from decoded genome sequences are an important resource for a wide range of biological studies. Accuracy of gene models is therefore critical for deducing accurate conclusions. Computationally predicted models are sometimes inconsistent with experimental data from cDNA clones and RNA-sequencing. In an ascidian, Ciona robusta (Ciona intestinalis type A), a manually curated gene/transcript model set, which was constructed using an assembly in which 68% of decoded sequences were associated with chromosomes, had been used during the last decade. Recently a new genome assembly was published, in which over 95% of decoded sequences are associated with chromosomes. In the present study, we provide a high-quality version of the gene/transcript model set for the latest assembly. Because the Ciona genome has been used in a variety of studies such as developmental biological studies, evolutionary studies, and physiological studies, the current gene/transcript model set provides a fundamental biological resource.
Asunto(s)
Ciona intestinalis , Animales , Secuencia de Bases , Evolución Biológica , Cromosomas , Ciona intestinalis/genética , GenomaRESUMEN
The ascidian larval tail contains muscle cells for swimming. Most of these muscle cells differentiate autonomously. The genetic program behind this autonomy has been studied extensively and the genetic cascade from maternal factors to initiation of expression of a muscle structural gene, Myl.c, has been uncovered; Myl.c expression is directed initially by transcription factor Tbx6-r.b at the 64-cell stage and then by the combined actions of Tbx6-r.b and Mrf from the gastrula to early tailbud stages. In the present study, we showed that transcription of Myl.c continued in late tailbud embryos and larvae, although a fusion protein of Tbx6-r.b and GFP was hardly detectable in late tailbud embryos. A knockdown experiment, reporter assay, and in vitro binding assay indicated that an essential cis-regulatory element of Myl.c that bound Tbx6-r.b in early embryos bound Tbx15/18/22 in late embryos to maintain expression of Myl.c. We also found that Tbx15/18/22 was controlled by Mrf, which constitutes a regulatory loop with Tbx6-r.b. Therefore, our data indicated that Tbx15/18/22 was activated initially under control of this regulatory loop as in the case of Myl.c, and then Tbx15/18/22 maintained the expression of Myl.c after Tbx6-r.b had disappeared. RNA-sequencing of Tbx15/18/22 morphant embryos revealed that many muscle structural genes were regulated similarly by Tbx15/18/22. Thus, the present study revealed the mechanisms of maintenance of transcription of muscle structural genes in late embryos in which Tbx15/18/22 takes the place of Tbx6-r.b.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Expresión Génica , Músculos/embriología , Músculos/metabolismo , Proteínas de Dominio T Box/metabolismo , Urocordados/embriología , Urocordados/genética , Animales , Sitios de Unión , Diferenciación Celular/genética , Femenino , Gástrula/metabolismo , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes , Células Musculares/citología , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Oviparidad/genética , Proteínas de Dominio T Box/genética , Transcripción Genética/genéticaRESUMEN
Zic-r.a, a maternal transcription factor, specifies posterior fate in ascidian embryos. However, its direct target, Tbx6-r.b, does not contain typical Zic-r.a-binding sites in its regulatory region. Using an in vitro selection assay, we found that Zic-r.a binds to sites dissimilar to the canonical motif, by which it activates Tbx6-r.b in a sub-lineage of muscle cells. These sites with non-canonical motifs have weak affinity for Zic-r.a; therefore, it activates Tbx6-r.b only in cells expressing Zic-r.a abundantly. Meanwhile, we found that Zic-r.a expressed zygotically in late embryos activates neural genes through canonical sites. Because different zinc-finger domains of Zic-r.a are important for driving reporters with canonical and non-canonical sites, it is likely that the non-canonical motif is not a divergent version of the canonical motif. In other words, our data indicate that the non-canonical motif represents a motif distinct from the canonical motif. Thus, Zic-r.a recognizes two distinct motifs to activate two sets of genes at two timepoints in development. This article has an associated 'The people behind the papers' interview.
Asunto(s)
Linaje de la Célula/genética , Linaje de la Célula/fisiología , Expresión Génica , Dedos de Zinc/genética , Animales , Sitios de Unión , Ciona intestinalis/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/metabolismo , Urocordados/embriología , Urocordados/genéticaRESUMEN
In ascidian embryos, the earliest transcription from the zygotic genome begins between the 8-cell and 16-cell stages. Gata.a, a maternally expressed Gata transcription factor, activates target genes specifically in the animal hemisphere, whereas the complex of ß-catenin and Tcf7 antagonizes the activity of Gata.a and activates target genes specifically in the vegetal hemisphere. Here, we show that genes zygotically expressed at the 16-cell stage have significantly more Gata motifs in their upstream regions. These genes included not only genes with animal hemisphere-specific expression but also genes with vegetal hemisphere-specific expression. On the basis of this finding, we performed knockdown experiments for Gata.a and reporter assays, and found that Gata.a is required for the expression of not only genes with animal hemisphere-specific expression, but also genes with vegetal hemisphere-specific expression. Our data indicated that weak Gata.a activity that cannot induce animal hemisphere-specific expression can allow ß-catenin/Tcf7 targets to be expressed in the vegetal cells. Because genes zygotically expressed at the 32-cell stage also had significantly more Gata motifs in their upstream regions, Gata.a function may not be limited to the genes expressed specifically in the animal or vegetal hemispheres at the 16-cell stage, and Gata.a may play an important role in the earliest transcription of the zygotic genome.
Asunto(s)
Ciona intestinalis/embriología , Factores de Transcripción GATA/metabolismo , Animales , Tipificación del Cuerpo/genética , Ciona intestinalis/metabolismo , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/metabolismo , Factores de Transcripción GATA/genética , Regulación del Desarrollo de la Expresión Génica/genética , Factor 1 de Transcripción de Linfocitos T/genética , Factor 1 de Transcripción de Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Urocordados/embriología , Cigoto/metabolismoRESUMEN
Striated muscle cells in the tail of ascidian tadpole larvae differentiate cell-autonomously. Although several key regulatory factors have been identified, the genetic regulatory pathway is not fully understood; comprehensive understanding of the regulatory pathway is essential for accurate modeling in order to deduce principles for gene regulatory network dynamics, and for comparative analysis on how ascidians have evolved the cell-autonomous gene regulatory mechanism. Here, we reveal regulatory interactions among three key regulatory factors, Zic-r.b, Tbx6-r.b and Mrf, and elucidate the mechanism by which these factors activate muscle structural genes. We reveal a cross-regulatory circuit among these regulatory factors, which maintains the expression of Tbx6-r.b and Mrf during gastrulation. Although these two factors combinatorially activate muscle structural genes in late-stage embryos, muscle structural genes are activated mainly by Tbx6-r.b before gastrulation. Time points when expression of muscle structural genes become first detectable are strongly correlated with the degree of Tbx6-r.b occupancy. Thus, the genetic pathway, starting with Tbx6-r.b and Zic-r.b, which are activated by maternal factors, and ending with expression of muscle structural genes, has been revealed.
Asunto(s)
Ciona intestinalis/embriología , Embrión no Mamífero/embriología , Gastrulación/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Redes Reguladoras de Genes/fisiología , Músculo Estriado/embriología , Animales , Ciona intestinalis/genética , Embrión no Mamífero/citología , Músculo Estriado/citologíaRESUMEN
In a multitude of organisms, transcription factors of the basic helix-loop-helix (bHLH) family control the expression of genes required for organ development and tissue differentiation. The functions of different bHLH transcription factors in the specification of nervous system and paraxial mesoderm have been widely investigated in various model systems. Conversely, the knowledge of the role of these regulators in the development of the axial mesoderm, the embryonic territory that gives rise to the notochord, and the identities of their target genes, remain still fragmentary. Here we investigated the transcriptional regulation and target genes of Bhlh-tun1, a bHLH transcription factor expressed in the developing Ciona notochord as well as in additional embryonic territories that contribute to the formation of both larval and adult structures. We describe its possible role in notochord formation, its relationship with the key notochord transcription factor Brachyury, and suggest molecular mechanisms through which Bhlh-tun1 controls the spatial and temporal expression of its effectors.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ciona/embriología , Ciona/genética , Redes Reguladoras de Genes , Notocorda/metabolismo , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Tipificación del Cuerpo/genética , Embrión no Mamífero/metabolismo , Elementos de Facilitación Genéticos/genética , Proteínas Fetales/genética , Proteínas Fetales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Notocorda/embriología , Reproducibilidad de los Resultados , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
In animal embryos, transcription is repressed for a definite period of time after fertilization. In the embryo of the ascidian, Ciona intestinalis (type A; or Ciona robusta), transcription of regulatory genes is repressed before the 8- or 16-cell stages. This initial transcriptional quiescence is important to enable the establishment of initial differential gene expression patterns along the animal-vegetal axis by maternal factors, because the third cell division separates the animal and vegetal hemispheres into distinct blastomeres. Indeed, maternal transcription factors directly activate zygotic gene expression by the 16-cell stage; Tcf7/ß-catenin activates genes in the vegetal hemisphere, and Gata.a activates genes in the animal hemisphere. In the present study, we revealed the dynamics of Gata.a and ß-catenin, and expression profiles of their target genes precisely. ß-catenin began to translocate into the nuclei at the 16-cell stage, and thus expression of ß-catenin targets began at the 16-cell stage. Although Gata.a is abundantly present before the 8-cell stage, transcription of Gata.a targets was repressed at and before the 4-cell stage, and their expression began at the 8-cell stage. Transcription of the ß-catenin targets may be repressed by the same mechanism in early embryos, because ß-catenin targets were not expressed in 4-cell embryos treated with a GSK inhibitor, in which ß-catenin translocated to the nuclei. Thus, these two maternal factors have different dynamics, which establish the pre-pattern for zygotic genetic programs in 16-cell embryos.
Asunto(s)
División Celular/genética , Cigoto/metabolismo , beta Catenina/metabolismo , Animales , Western Blotting , Ciona intestinalis/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismoRESUMEN
In embryos of ascidians, which are invertebrate chordates, the expression of a small number of genes from the zygotic genome begins between the 8- and 16-cell stages. They are considered the first zygotic genes to be expressed under the direct control of maternal factors. The initial transcriptional quiescence before the 8-cell stage is essential for establishing differential gene expression patterns between the animal and vegetal hemispheres, because these hemispheres are first segregated into distinct blastomeres at the 8-cell stage. While the mechanisms of this transcriptional silencing have not been understood, the mechanism by which maternal factors cooperatively establish the first differential gene expression patterns has been well understood. Here, we review the initial processes that occur until the 16-cell stage in ascidian embryos.
Asunto(s)
Blastómeros/citología , Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica/genética , Urocordados/citología , Cigoto/citología , Animales , Tipificación del Cuerpo/fisiología , Embrión no Mamífero/citología , HumanosRESUMEN
In many animal embryos, a specific gene expression pattern is established along the animal-vegetal axis soon after zygotic transcription begins. In the embryo of the ascidian Ciona intestinalis, soon after the division that separates animal and vegetal hemispheres into distinct blastomeres, maternal Gata.a and ß-catenin activate specific genes in the animal and vegetal blastomeres, respectively. On the basis of these initial distinct gene expression patterns, gene regulatory networks promote animal cells to become ectodermal tissues and vegetal cells to become endomesodermal tissues and a part of the nerve cord. In the vegetal hemisphere, ß-catenin directly activates Foxd, an essential transcription factor gene for specifying endomesodermal fates. In the present study, we found that Foxd also represses the expression of genes that are activated specifically in the animal hemisphere, including Dmrt1, Prdm1-r.a (Bz1), Prdm1-r.b (Bz2), and Otx. A reporter assay showed that Dmrt1 expression was directly repressed by Foxd, and a chromatin immunoprecipitation assay showed that Foxd was bound to the upstream regions of Dmrt1, Prdm1-r.a, Prdm1-r.b, and Otx. Thus, Foxd has a dual function of activating specific gene expression in the vegetal hemisphere and of repressing the expression of genes that are normally expressed in the animal hemisphere. This dual function stabilizes the initial patterning along the animal-vegetal axis by ß-catenin and Gata.a.
Asunto(s)
Ciona intestinalis/genética , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica , Animales , Tipificación del Cuerpo , Ciona intestinalis/embriología , Ciona intestinalis/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Many animal embryos use nuclear ß-catenin (nß-catenin) during the segregation of endomesoderm (or endoderm) from ectoderm. This mechanism is thus likely to be evolutionarily ancient. In the ascidian embryo, nß-catenin reiteratively drives binary fate decisions between ectoderm and endomesoderm at the 16-cell stage, and then between endoderm and margin (mesoderm and caudal neural) at the 32-cell stage. At the 16-cell stage, nß-catenin activates endomesoderm genes in the vegetal hemisphere. At the same time, nß-catenin suppresses the DNA-binding activity of a maternal transcription factor, Gata.a, through a physical interaction, and Gata.a thereby activates its target genes only in the ectodermal lineage. In the present study, we found that this antagonism between nß-catenin and Gata.a also operates during the binary fate switch at the 32-cell stage. Namely, in marginal cells where nß-catenin is absent, Gata.a directly activates its target, Zic-r.b (ZicL), to specify the marginal cell lineages. Thus, the antagonistic action between nß-catenin and Gata.a is involved in two consecutive stages of germ layer segregation in ascidian embryos.
Asunto(s)
Tipificación del Cuerpo/genética , Ciona intestinalis/embriología , Factor de Transcripción GATA1/antagonistas & inhibidores , Estratos Germinativos/embriología , beta Catenina/antagonistas & inhibidores , Animales , Animales Modificados Genéticamente , Linaje de la Célula/genética , Ciona intestinalis/genética , Embrión no Mamífero , Factor de Transcripción GATA1/genética , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Urocordados/embriología , Urocordados/genética , beta Catenina/genéticaRESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1006045.].
RESUMEN
Maternal factors initiate the zygotic developmental program in animal embryos. In embryos of the chordate, Ciona intestinalis, three maternal factors-Gata.a, ß-catenin, and Zic-r.a-are required to establish three domains of gene expression at the 16-cell stage; the animal hemisphere, vegetal hemisphere, and posterior vegetal domains. Here, we show how the maternal factors establish these domains. First, only ß-catenin and its effector transcription factor, Tcf7, are required to establish the vegetal hemisphere domain. Second, genes specifically expressed in the posterior vegetal domain have additional repressive cis-elements that antagonize the activity of ß-catenin/Tcf7. This antagonizing activity is suppressed by Zic-r.a, which is specifically localized in the posterior vegetal domain and binds to DNA indirectly through the interaction with Tcf7. Third, Gata.a directs specific gene expression in the animal hemisphere domain, because ß-catenin/Tcf7 weakens the Gata.a-binding activity for target sites through a physical interaction in the vegetal cells. Thus, repressive regulation through protein-protein interactions among the maternal transcription factors is essential to establish the first distinct domains of gene expression in the chordate embryo.
Asunto(s)
Desarrollo Embrionario/genética , Factor 1 de Transcripción de Linfocitos T/genética , Cigoto/crecimiento & desarrollo , beta Catenina/genética , Animales , Blastómeros/metabolismo , Tipificación del Cuerpo/genética , Ciona intestinalis/genética , Ciona intestinalis/crecimiento & desarrollo , Factores de Transcripción GATA/biosíntesis , Factores de Transcripción GATA/genética , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Mapas de Interacción de Proteínas , Análisis de Secuencia de ADN , Factor 1 de Transcripción de Linfocitos T/biosíntesis , Cigoto/metabolismo , beta Catenina/biosíntesisRESUMEN
A main challenge of modern biology is to understand how specific constellations of genes are activated to differentiate cells and give rise to distinct tissues. This study focuses on elucidating how gene expression is initiated in the notochord, an axial structure that provides support and patterning signals to embryos of humans and all other chordates. Although numerous notochord genes have been identified, the regulatory DNAs that orchestrate development and propel evolution of this structure by eliciting notochord gene expression remain mostly uncharted, and the information on their configuration and recurrence is still quite fragmentary. Here we used the simple chordate Ciona for a systematic analysis of notochord cis-regulatory modules (CRMs), and investigated their composition, architectural constraints, predictive ability and evolutionary conservation. We found that most Ciona notochord CRMs relied upon variable combinations of binding sites for the transcription factors Brachyury and/or Foxa2, which can act either synergistically or independently from one another. Notably, one of these CRMs contains a Brachyury binding site juxtaposed to an (AC) microsatellite, an unusual arrangement also found in Brachyury-bound regulatory regions in mouse. In contrast, different subsets of CRMs relied upon binding sites for transcription factors of widely diverse families. Surprisingly, we found that neither intra-genomic nor interspecific conservation of binding sites were reliably predictive hallmarks of notochord CRMs. We propose that rather than obeying a rigid sequence-based cis-regulatory code, most notochord CRMs are rather unique. Yet, this study uncovered essential elements recurrently used by divergent chordates as basic building blocks for notochord CRMs.
Asunto(s)
Proteínas Fetales/genética , Factor Nuclear 3-beta del Hepatocito/genética , Notocorda/crecimiento & desarrollo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas de Dominio T Box/genética , Animales , Sitios de Unión , Tipificación del Cuerpo/genética , Ciona intestinalis/genética , Ciona intestinalis/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Genoma , RatonesRESUMEN
The appearance of the notochord represented a milestone in Deuterostome evolution. The notochord is necessary for the development of the chordate body plan and for the formation of the vertebral column and numerous organs. It is known that the transcription factor Brachyury is required for notochord formation in all chordates, and that it controls transcription of a large number of target genes. However, studies of the structure of the cis-regulatory modules (CRMs) through which this control is exerted are complicated in vertebrates by the genomic complexity and the pan-mesodermal expression territory of Brachyury. We used the ascidian Ciona, in which the single-copy Brachyury is notochord-specific and CRMs are easily identifiable, to carry out a systematic characterization of Brachyury-downstream notochord CRMs. We found that Ciona Brachyury (Ci-Bra) controls most of its targets directly, through non-palindromic binding sites that function either synergistically or individually to activate early- and middle-onset genes, respectively, while late-onset target CRMs are controlled indirectly, via transcriptional intermediaries. These results illustrate how a transcriptional regulator can efficiently shape a shallow gene regulatory network into a multi-tiered transcriptional output, and provide insights into the mechanisms that establish temporal read-outs of gene expression in a fast-developing chordate embryo.
Asunto(s)
Ciona intestinalis/genética , Proteínas Fetales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Notocorda/metabolismo , Proteínas de Dominio T Box/metabolismo , Animales , Sitios de Unión , Ciona intestinalis/crecimiento & desarrollo , Secuencia de Consenso/genética , Notocorda/crecimiento & desarrollo , Unión Proteica/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Reproducibilidad de los Resultados , Especificidad de la Especie , Factores de TiempoRESUMEN
T-box genes are potent regulators of mesoderm development in many metazoans. In chordate embryos, the T-box transcription factor Brachyury (Bra) is required for specification and differentiation of the notochord. In some chordates, including the ascidian Ciona, members of the Tbx2 subfamily of T-box genes are also expressed in this tissue; however, their regulatory relationships with Bra and their contributions to the development of the notochord remain uncharacterized. We determined that the notochord expression of Ciona Tbx2/3 (Ci-Tbx2/3) requires Ci-Bra, and identified a Ci-Tbx2/3 notochord CRM that necessitates multiple Ci-Bra binding sites for its activity. Expression of mutant forms of Ci-Tbx2/3 in the developing notochord revealed a role for this transcription factor primarily in convergent extension. Through microarray screens, we uncovered numerous Ci-Tbx2/3 targets, some of which overlap with known Ci-Bra-downstream notochord genes. Among the Ci-Tbx2/3 notochord targets are evolutionarily conserved genes, including caspases, lineage-specific genes, such as Noto4, and newly identified genes, such as MLKL. This work sheds light on a large section of the notochord regulatory circuitry controlled by T-box factors, and reveals new components of the complement of genes required for the proper formation of this structure.
Asunto(s)
Ciona intestinalis/embriología , Proteínas Fetales/metabolismo , Notocorda/embriología , Proteínas de Dominio T Box/metabolismo , Animales , Sitios de Unión , Linaje de la Célula , Ciona intestinalis/fisiología , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Hibridación in Situ , Mutación , Notocorda/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Dominio T Box/fisiología , TransgenesRESUMEN
BACKGROUND: The notochord is a signaling center required for the patterning of the vertebrate embryonic midline, however, the molecular and cellular mechanisms involved in the formation of this essential embryonic tissue remain unclear. The urochordate Ciona intestinalis develops a simple notochord from 40 specific postmitotic mesodermal cells. The precursors intercalate mediolaterally and establish a single array of disk-shaped notochord cells along the midline. However, the role that notochord precursor polarization, particularly along the dorsoventral axis, plays in this morphogenetic process remains poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the notochord preferentially accumulates an apical cell polarity marker, aPKC, ventrally and a basement membrane marker, laminin, dorsally. This asymmetric accumulation of apicobasal cell polarity markers along the embryonic dorsoventral axis was sustained in notochord precursors during convergence and extension. Further, of several members of the Eph gene family implicated in cellular and tissue morphogenesis, only Ci-Eph4 was predominantly expressed in the notochord throughout cell intercalation. Introduction of a dominant-negative Ci-Eph4 to notochord precursors diminished asymmetric accumulation of apicobasal cell polarity markers, leading to defective intercalation. In contrast, misexpression of a dominant-negative mutant of a planar cell polarity gene Dishevelled preserved asymmetric accumulation of aPKC and laminin in notochord precursors, although their intercalation was incomplete. CONCLUSIONS/SIGNIFICANCE: Our data support a model in which in ascidian embryos Eph-dependent dorsoventral polarity of notochord precursors plays a crucial role in mediolateral cell intercalation and is required for proper notochord morphogenesis.
Asunto(s)
Tipificación del Cuerpo/fisiología , Ciona intestinalis/embriología , Notocorda/embriología , Receptores de la Familia Eph/genética , AnimalesRESUMEN
For over a century, muscle formation in the ascidian embryo has been representative of 'mosaic' development. The molecular basis of muscle-fate predetermination has been partly elucidated with the discovery of Macho1, a maternal zinc-finger transcription factor necessary and sufficient for primary muscle development, and of its transcriptional intermediaries Tbx6b and Tbx6c. However, the molecular mechanisms by which the maternal information is decoded by cis-regulatory modules (CRMs) associated with muscle transcription factor and structural genes, and the ways by which a seamless transition from maternal to zygotic transcription is ensured, are still mostly unclear. By combining misexpression assays with CRM analyses, we have identified the mechanisms through which Ciona Macho1 (Ci-Macho1) initiates expression of Ci-Tbx6b and Ci-Tbx6c, and we have unveiled the cross-regulatory interactions between the latter transcription factors. Knowledge acquired from the analysis of the Ci-Tbx6b CRM facilitated both the identification of a related CRM in the Ci-Tbx6c locus and the characterization of two CRMs associated with the structural muscle gene fibrillar collagen 1 (CiFCol1). We use these representative examples to reconstruct how compact CRMs orchestrate the muscle developmental program from pre-localized ooplasmic determinants to differentiated larval muscle in ascidian embryos.
Asunto(s)
Ciona intestinalis/metabolismo , Proteínas del Huevo/metabolismo , Factores de Transcripción/metabolismo , Animales , Ciona intestinalis/embriología , Ciona intestinalis/genética , Clonación Molecular , Proteínas del Huevo/genética , Embrión no Mamífero , Desarrollo Embrionario/genética , Colágenos Fibrilares/genética , Colágenos Fibrilares/metabolismo , Regulación del Desarrollo de la Expresión Génica , Desarrollo de Músculos/genética , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Transgenes/genéticaRESUMEN
The notochord is a defining feature of the chordate body plan. Experiments in ascidian, frog and mouse embryos have shown that co-expression of Brachyury and FoxA class transcription factors is required for notochord development. However, studies on the cis-regulatory sequences mediating the synergistic effects of these transcription factors are complicated by the limited knowledge of notochord genes and cis-regulatory modules (CRMs) that are directly targeted by both. We have identified an easily testable model for such investigations in a 155-bp notochord-specific CRM from the ascidian Ciona intestinalis. This CRM contains functional binding sites for both Ciona Brachyury (Ci-Bra) and FoxA (Ci-FoxA-a). By combining point mutation analysis and misexpression experiments, we demonstrate that binding of both transcription factors to this CRM is necessary and sufficient to activate transcription. To gain insights into the cis-regulatory criteria controlling its activity, we investigated the organization of the transcription factor binding sites within the 155-bp CRM. The 155-bp sequence contains two Ci-Bra binding sites with identical core sequences but opposite orientations, only one of which is required for enhancer activity. Changes in both orientation and spacing of these sites substantially affect the activity of the CRM, as clusters of identical sites found in the Ciona genome with different arrangements are unable to activate transcription in notochord cells. This work presents the first evidence of a synergistic interaction between Brachyury and FoxA in the activation of an individual notochord CRM, and highlights the importance of transcription factor binding site arrangement for its function.
Asunto(s)
Ciona intestinalis/embriología , Proteínas Fetales/metabolismo , Notocorda/metabolismo , Proteínas de Dominio T Box/metabolismo , Transactivadores/metabolismo , Animales , Elementos de Facilitación GenéticosRESUMEN
The optic vesicle is a multipotential primordium of the retina, which becomes subdivided into the neural retina and retinal pigmented epithelium domains. Although the roles of several paracrine factors in patterning the optic vesicle have been studied extensively, little is known about cell-autonomous mechanisms that regulate coordinated cell morphogenesis and cytodifferentiation of the retinal pigmented epithelium. Here we demonstrate that members of the SoxB1 gene family, Sox1, Sox2 and Sox3, are all downregulated in the presumptive retinal pigmented epithelium. Constitutive maintenance of SoxB1 expression in the presumptive retinal pigmented epithelium both in vivo and in vitro resulted in the absence of cuboidal morphology and pigmentation, and in concomitant induction of neural differentiation markers. We also demonstrate that exogenous Fgf4 inhibits downregulation all SoxB1 family members in the presumptive retinal pigment epithelium. These results suggest that retinal pigment epithelium morphogenesis and cytodifferentiation requires SoxB1 downregulation, which depends on the absence of exposure to an FGF-like signal.