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1.  Guidelines for the Nomenclature of Genetic Elements in Tunicate Genomes 
Tunicates are invertebrate members of the chordate phylum, and are considered to be the sister group of vertebrates. Tunicates are composed of ascidians, thaliaceans, and appendicularians. With the advent of inexpensive high-throughput sequencing, the number of sequenced tunicate genomes is expected to rise sharply within the coming years. To facilitate comparative genomics within the tunicates, and between tunicates and vertebrates, standardized rules for the nomenclature of tunicate genetic elements need to be established. Here we propose a set of nomenclature rules, consensual within the community, for predicted genes, pseudogenes, transcripts, operons, transcriptional cis-regulatory regions, transposable elements, and transgenic constructs. In addition, the document proposes guidelines for naming transgenic and mutant lines.
PMCID: PMC4308547  PMID: 25220678
tunicates; genome annotation; gene; transposable element; cis-regulatory sequences
2.  NK4 Antagonizes Tbx1/10 to Promote Cardiac versus Pharyngeal Muscle Fate in the Ascidian Second Heart Field 
PLoS Biology  2013;11(12):e1001725.
Cross inhibition between NK4 and TBX1 transcription factors specifies heart versus pharyngeal muscle fates by promoting the activation of tissue-specific regulators in distinct precursors within the cardiopharyngeal lineage of the ascidian, Ciona intestinalis.
The heart and head muscles share common developmental origins and genetic underpinnings in vertebrates, including humans. Parts of the heart and cranio-facial musculature derive from common mesodermal progenitors that express NKX2-5, ISL1, and TBX1. This ontogenetic kinship is dramatically reflected in the DiGeorge/Cardio-Velo-Facial syndrome (DGS/CVFS), where mutations of TBX1 cause malformations in the pharyngeal apparatus and cardiac outflow tract. Cardiac progenitors of the first heart field (FHF) do not require TBX1 and segregate precociously from common progenitors of the second heart field (SHF) and pharyngeal muscles. However, the cellular and molecular mechanisms that govern heart versus pharyngeal muscle specification within this lineage remain elusive. Here, we harness the simplicity of the ascidian larva to show that, following asymmetric cell division of common progenitors, NK4/NKX2-5 promotes GATAa/GATA4/5/6 expression and cardiac specification in the second heart precursors by antagonizing Tbx1/10-mediated inhibition of GATAa and activation of Collier/Olf/EBF (COE), the determinant of atrial siphon muscle (ASM) specification. Our results uncover essential regulatory connections between the conserved cardio-pharyngeal factor Tbx1/10 and muscle determinant COE, as well as a mutual antagonism between NK4 and Tbx1/10 activities upstream of GATAa and COE. The latter cross-antagonism underlies a fundamental heart versus pharyngeal muscle fate choice that occurs in a conserved lineage of cardio-pharyngeal progenitors. We propose that this basic ontogenetic motif underlies cardiac and pharyngeal muscle development and evolution in chordates.
Author Summary
Mutations in the regulatory genes encoding the transcription factors NKX2-5 and TBX1, which govern heart and head muscle development, cause prevalent congenital defects. Recent studies using vertebrate models have shown that the heart and pharyngeal head muscle cells derive from common progenitors in the early embryo. To better understand the genetic mechanisms by which these progenitors select one of the two developmental trajectories, we studied the activity of these transcription factors in a simple invertebrate chordate model, the sea squirt Ciona intestinalis. We show that the sea squirt homolog of NKX2-5 promotes early heart specification by inhibiting the formation of pharyngeal muscles. Conversely, the TBX1 homolog determines pharyngeal muscle fate by inhibiting GATAa and thereby the heart program it instructs, as well as promoting the pharyngeal muscle program through activation of COE (Collier/Olf-1/EBF), a recently identified regulator of skeletal muscle differentiation. Finally, we show that the NKX2-5 homolog protein directly binds to the COE gene to repress its activity. Notably, these antagonistic interactions occur in heart and pharyngeal precursors immediately following the division of their pluripotent mother cells, thus contributing to their respective fate choice. These mechanistic insights into the process of early heart versus head muscle specification in this simple chordate provide the grounds for establishing the etiology of human congenital cardio-craniofacial defects.
PMCID: PMC3849182  PMID: 24311985
3.  Cis-Regulatory Timers for Developmental Gene Expression 
PLoS Biology  2013;11(10):e1001698.
How does a fertilized egg decode its own genome to eventually develop into a mature animal? Each developing cell must activate a battery of genes in a timely manner and according to the function it will ultimately perform, but how? During development of the notochord—a structure akin to the vertebrate spine—in a simple marine invertebrate, an essential protein called Brachyury binds to specific sites in its target genes. A study just published in PLOS Biology reports that if the target gene contains multiple Brachyury-binding sites it will be activated early in development but if it contains only one site it will be activated later. Genes that contain no binding site can still be activated by Brachyury, but only indirectly by an earlier Brachyury-dependent gene product, so later than the directly activated genes. Thus, this study shows how several genes can interpret the presence of a single factor differently to become active at distinct times in development.
PMCID: PMC3812112  PMID: 24204213
4.  Genetic and Genomic Toolbox of the Chordate Ciona intestinalis 
Genetics  2012;192(1):55-66.
The experimental malleability and unique phylogenetic position of the sea squirt Ciona intestinalis as part of the sister group to the vertebrates have helped establish these marine chordates as model organisms for the study of developmental genetics and evolution. Here we summarize the tools, techniques, and resources available to the Ciona geneticist, citing examples of studies that employed such strategies in the elucidation of gene function in Ciona. Genetic screens, germline transgenesis, electroporation of plasmid DNA, and microinjection of morpholinos are all routinely employed, and in the near future we expect these to be complemented by targeted mutagenesis, homologous recombination, and RNAi. The genomic resources available will continue to support the design and interpretation of genetic experiments and allow for increasingly sophisticated approaches on a high-throughput, whole-genome scale.
PMCID: PMC3430545  PMID: 22964837
ascidian; development; transgenesis; electroporation
5.  A cis-Regulatory Signature for Chordate Anterior Neuroectodermal Genes 
PLoS Genetics  2010;6(4):e1000912.
One of the striking findings of comparative developmental genetics was that expression patterns of core transcription factors are extraordinarily conserved in bilaterians. However, it remains unclear whether cis-regulatory elements of their target genes also exhibit common signatures associated with conserved embryonic fields. To address this question, we focused on genes that are active in the anterior neuroectoderm and non-neural ectoderm of the ascidian Ciona intestinalis. Following the dissection of a prototypic anterior placodal enhancer, we searched all genomic conserved non-coding elements for duplicated motifs around genes showing anterior neuroectodermal expression. Strikingly, we identified an over-represented pentamer motif corresponding to the binding site of the homeodomain protein OTX, which plays a pivotal role in the anterior development of all bilaterian species. Using an in vivo reporter gene assay, we observed that 10 of 23 candidate cis-regulatory elements containing duplicated OTX motifs are active in the anterior neuroectoderm, thus showing that this cis-regulatory signature is predictive of neuroectodermal enhancers. These results show that a common cis-regulatory signature corresponding to K50-Paired homeodomain transcription factors is found in non-coding sequences flanking anterior neuroectodermal genes in chordate embryos. Thus, field-specific selector genes impose architectural constraints in the form of combinations of short tags on their target enhancers. This could account for the strong evolutionary conservation of the regulatory elements controlling field-specific selector genes responsible for body plan formation.
Author Summary
Regional identity in embryos is defined by a few specific transcription factors that activate a large number of target genes through binding to common tags in regulatory sequences. In chordates it is unclear if such tags can be identified in the cis-regulatory regions of regionally expressed genes. To address this question we focused on the anterior nervous system where Otx codes for a transcription factor that triggers expression of many other head-specific genes. We analyzed an element that is active in the region bordering the anterior nervous system in the marine invertebrate Ciona intestinalis. We found that the crucial binding sites have to be duplicated and close enough. One of the pairs is bound by OTX. We showed that anterior nervous system genes are often flanked by duplicated OTX binding sites. We confirmed by transgenic assays that about half of these genomic sequences are active and drive expression anteriorly. This study unravels a simple regulatory logic in the anterior enhancers. It indicates that although there are major changes in the organization of the binding sites at short evolutionary range, conserved expression patterns are partly generated by a duplicated organization of conserved binding sites for region-specific transcription factors.
PMCID: PMC2855326  PMID: 20419150
6.  Fibroblast growth factor signalling controls nervous system patterning and pigment cell formation in Ciona intestinalis 
Nature Communications  2014;5:4830.
During the development of the central nervous system (CNS), combinations of transcription factors and signalling molecules orchestrate patterning, specification and differentiation of neural cell types. In vertebrates, three types of melanin-containing pigment cells, exert a variety of functional roles including visual perception. Here we analysed the mechanisms underlying pigment cell specification within the CNS of a simple chordate, the ascidian Ciona intestinalis. Ciona tadpole larvae exhibit a basic chordate body plan characterized by a small number of neural cells. We employed lineage-specific transcription profiling to characterize the expression of genes downstream of fibroblast growth factor signalling, which govern pigment cell formation. We demonstrate that FGF signalling sequentially imposes a pigment cell identity at the expense of anterior neural fates. We identify FGF-dependent and pigment cell-specific factors, including the small GTPase, Rab32/38 and demonstrated its requirement for the pigmentation of larval sensory organs.
The fibroblast growth factor (FGF) signalling pathway specifies the fate of pigmented cells in the ascidian Ciona intestinalis. Here, the authors obtain lineage-specific transcription profiles of pigment precursor cells and identify FGF downstream genes involved in central nervous system patterning, and the specification and differentiation of pigmented cells.
PMCID: PMC4164782  PMID: 25189217

Results 1-6 (6)