Cis-regulatory interfaces reveal the molecular mechanisms underlying the notochord gene regulatory network of Ciona.

Tissue-specific gene expression is fundamental in development and evolution, and is mediated by transcription factors and by the cis-regulatory regions (enhancers) that they control. Transcription factors and their respective tissue-specific enhancers are essential components of gene regulatory networks responsible for the development of tissues and organs. Although numerous transcription factors have been characterized from different organisms, the knowledge of the enhancers responsible for their tissue-specific expression remains fragmentary. Here we use Ciona to study the enhancers associated with ten transcription factors expressed in the notochord, an evolutionary hallmark of the chordate phylum. Our results illustrate how two evolutionarily conserved transcription factors, Brachyury and Foxa2, coordinate the deployment of other notochord transcription factors. The results of these detailed cis-regulatory analyses delineate a high-resolution view of the essential notochord gene regulatory network of Ciona, and provide a reference for studies of transcription factors, enhancers, and their roles in development, disease, and evolution.

Xbp1 and Brachyury establish an evolutionarily conserved subcircuit of the notochord gene regulatory network.

Gene regulatory networks coordinate the formation of organs and structures that compose the evolving body plans of different organisms. We are using a simple chordate model, the Ciona embryo, to investigate the essential gene regulatory network that orchestrates morphogenesis of the notochord, a structure necessary for the proper development of all chordate embryos. Although numerous transcription factors expressed in the notochord have been identified in different chordates, several of them remain to be positioned within a regulatory framework. Here, we focus on Xbp1, a transcription factor expressed during notochord formation in Ciona and other chordates. Through the identification of Xbp1-downstream notochord genes in Ciona, we found evidence of the early co-option of genes involved in the unfolded protein response to the notochord developmental program. We report the regulatory interplay between Xbp1 and Brachyury, and by extending these results to Xenopus, we show that Brachyury and Xbp1 form a cross-regulatory subcircuit of the notochord gene regulatory network that has been consolidated during chordate evolution.

A Guide for Undergraduates to the Society for Neuroscience Annual Meeting.

The annual meeting of the Society for Neuroscience (SfN) attracts over 30,000 attendees, including many of the world's most accomplished researchers. Although it can be intimidating to attend a conference of this scale, there are many rewards for undergraduates. Based on surveys of young neuroscientists, we provide planning strategies to ensure attendees maximize their exposure and retention of the breadth and depth offered by this large conference format without becoming overwhelmed.

Brachyury, Foxa2 and the cis-Regulatory Origins of the Notochord.

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.

Functional Brachyury binding sites establish a temporal read-out of gene expression in the Ciona notochord.

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.

From notochord formation to hereditary chordoma: the many roles of Brachyury.

Chordoma is a rare, but often malignant, bone cancer that preferentially affects the axial skeleton and the skull base. These tumors are both sporadic and hereditary and appear to occur more frequently after the fourth decade of life; however, modern technologies have increased the detection of pediatric chordomas. Chordomas originate from remnants of the notochord, the main embryonic axial structure that precedes the backbone, and share with notochord cells both histological features and the expression of characteristic genes. One such gene is Brachyury, which encodes for a sequence-specific transcription factor. Known for decades as a main regulator of notochord formation, Brachyury has recently gained interest as a biomarker and causative agent of chordoma, and therefore as a promising therapeutic target. Here, we review the main characteristics of chordoma, the molecular markers, and the clinical approaches currently available for the early detection and possible treatment of this cancer. In particular, we report on the current knowledge of the role of Brachyury and of its possible mechanisms of action in both notochord formation and chordoma etiogenesis.

Tbx2/3 is an essential mediator within the Brachyury gene network during Ciona notochord development.

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.

The identification of transcription factors expressed in the notochord of Ciona intestinalis adds new potential players to the brachyury gene regulatory network.

The notochord is the distinctive characteristic of chordates; however, the knowledge of the complement of transcription factors governing the development of this structure is still incomplete. Here we present the expression patterns of seven transcription factor genes detected in the notochord of the ascidian Ciona intestinalis at various stages of embryonic development. Four of these transcription factors, Fos-a, NFAT5, AFF and Klf15, have not been directly associated with the notochord in previous studies, while the others, including Spalt-like-a, Lmx-like, and STAT5/6-b, display evolutionarily conserved expression in this structure as well as in other domains. We examined the hierarchical relationships between these genes and the transcription factor Brachyury, which is necessary for notochord development in all chordates. We found that Ciona Brachyury regulates the expression of most, although not all, of these genes. These results shed light on the genetic regulatory program underlying notochord formation in Ciona and possibly other chordates.