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: During notochord formation in chordate embryos, the transcription factor Brachyury employs different regulatory strategies to ensure the sequential activation of downstream genes and thereby the deployment of a specific developmental program at the right time and place. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The transcription factor Ciona Brachyury (Ci-Bra) plays an essential role in notochord development in the ascidian Ciona intestinalis. We characterized a putative Ci-Bra target gene, which we named Ci-multidom, and analyzed in detail its expression pattern in normal embryos and in embryos where Ci-Bra was misexpressed. Ci-multidom encodes a novel protein, which contains eight CCP domains and a partial VWFA domain. We show that an EGFP-multidom fusion protein localizes preferentially to the endoplasmic reticulum (ER), and is excluded from the nucleus. In situ hybridization experiments demonstrate that Ci-multidom is expressed in the notochord and in the anterior neural boundary (ANB). We found that the expression in the ANB is fully recapitulated by an enhancer element located upstream of Ci-multidom. By means of misexpression experiments, we provide evidence that Ci-Bra controls transcription of Ci-multidom in the notochord; however, while Ci-Bra is homogeneously expressed throughout this structure, Ci-multidom is transcribed at detectable levels only in a random subset of notochord cells. The number of notochord cells expressing Ci-multidom varies among different embryos and is independent of developmental stage, lineage, and position along the anterior-posterior axis. These results suggest that despite its morphological simplicity and invariant cell-lineage, the ancestral notochord is a mosaic of cells in which the gene cascade downstream of Brachyury is differentially modulated., (Copyright 2007 Wiley-Liss, Inc.)

The ascidian Ciona intestinalis is one of the model organisms of choice for comparative investigations of chordate development and for unraveling the molecular mechanisms underlying morphogenesis and cell fate specification. Taking advantage of the availability of various genetically encoded fluorescent proteins and of defined cis-regulatory elements, we combined transient transgenesis with laser scanning confocal imaging to acquire and quantitate 3D time-lapse data from living Ciona embryos. We used Ciona tissue-specific enhancers to drive expression of spectrally distinct fluorescent protein reporters to label and simultaneously visualize axially and paraxially positioned mesodermal derivatives, as well as neural precursors in individual embryos. We observed morphogenetic movements, without perturbing development, from the early gastrula throughout the larval stage, including gastrulation, neurulation, convergent extension of the presumptive notochord, and tail elongation. These multidimensional data allowed us to establish a reference system of metrics to quantify key developmental events including blastopore closure and muscle extension. The approach we describe can be used to document morphogenetic cell and tissue rearrangements in living embryos and paves the way for a live digitized anatomical atlas of Ciona.

Otx (otd in Drosophila) is a well-conserved homeobox gene throughout animal phylogeny and commonly expressed in the anterior part of the embryo. In embryos of the ascidian Halocynthia roretzi, Hroth, the otx homologue in this species, is expressed in the endoderm and the sensory vesicle, the anterior part of the larval ascidian central nervous system (CNS), which has been thought to be homologous to vertebrate forebrain and midbrain. The developmental expression pattern of Hroth is very similar to that of vertebrate counterparts, which leads to a possibility that a similar mechanism may exist in the patterning of the CNS between ascidians and vertebrates. To better understand the mechanism, we decided to undertake analysis of the transcriptional regulatory regions of Hroth. We isolated and determined the nucleotide sequence of the 11.4-kbp region upstream of the translation start site of Hroth. We found that Hroth transcripts are modified likely with spliced leader RNA; therefore, we could not determine the transcription start site. However, first, we identified three introns that are unknown with vertebrate otx genes. Second, we found two regions that are capable of functioning as a promoter through deletion analysis, one of which appeared to be an endogenous promoter of Hroth. We analyzed the 5' upstream region 5402-1473bp, the region between 1473 and 5402 base pairs upstream from the translation start site of Hroth, including the putative endogenous promoter. This region was capable of driving Hroth expression in the sensory vesicle lineage cells as well as some other lineages at the early tail bud stage. Deletion analysis of this region suggested that three regions, 1659-1650bp, 1628-1613bp, and 1542-1473bp are responsible for regulating Hroth expression in the sensory vesicle cells at the tail bud stage. Among these regions, no apparent sequence conservation was observed. The present study has revealed a complex organization of transcription regulatory regions for the ascidian otx., (Copyright 2003 Wiley-Liss, Inc.)