Gata is ubiquitously required for the earliest zygotic gene transcription in the ascidian embryo

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 β-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 β-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

Temporal Regulation of the Muscle Gene Cascade by Macho1 and Tbx6 Transcription Factors in Ciona Intestinalis

For over a century, muscle formation in the ascidian embryo has been representative of \u27mosaic\u27 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

Eph regulates dorsoventral asymmetry of the notochord plate and convergent extension-mediated notochord formation.

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

The regulatory pathway from genes directly activated by maternal factors to muscle structural genes in ascidian embryos

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

Two distinct motifs for Zic-r.a drive specific gene expression in two cell lineages

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

Dynamics of two key maternal factors that initiate zygotic regulatory programs in ascidian embryos

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/β-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 β-catenin, and expression profiles of their target genes precisely. β-catenin began to translocate into the nuclei at the 16-cell stage, and thus expression of β-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 β-catenin targets may be repressed by the same mechanism in early embryos, because β-catenin targets were not expressed in 4-cell embryos treated with a GSK inhibitor, in which β-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

Antagonism between β-catenin and Gata.a sequentially segregates the germ layers of ascidian embryos

Many animal embryos use nuclear β-catenin (nβ-catenin) during the segregation of endomesoderm (or endoderm) from ectoderm. This mechanism is thus likely to be evolutionarily ancient. In the ascidian embryo, nβ-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, nβ-catenin activates endomesoderm genes in the vegetal hemisphere. At the same time, nβ-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 nβ-catenin and Gata.a also operates during the binary fate switch at the 32-cell stage. Namely, in marginal cells where nβ-catenin is absent, Gata.a directly activates its target, Zic-r.b (ZicL), to specify the marginal cell lineages. Thus, the antagonistic action between nβ-catenin and Gata.a is involved in two consecutive stages of germ layer segregation in ascidian embryos

Tbx15/18/22 shares a binding site with Tbx6-r.b to maintain expression of a muscle structural gene in ascidian late embryos

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