Brad Davidson

Ciona intestinalis: an emerging model for whole-genome analyses

Abstract The tadpole larvae of the ascidian Ciona intestinalis possess the most simplified chordate body plan. Analysis of the Ciona draft genome indicates that the ∼153–159Mb genome contains ∼16 000 protein-coding genes. Among these is a fundamental set of conserved chordate proteins involved in cell signaling and development. A thorough examination of Ciona gene expression (the transcriptome) is ongoing, including large-scale expressed sequence tag analyses, cDNA sequencing and in situ hybridization screens. Together with recent advances in the methodology used to investigate gene regulation and function, these make Ciona an attractive experimental system for genome level analysis of chordate developmental genetics.

Evolutionary origins of the vertebrate heart: Specification of the cardiac lineage in Ciona intestinalis

Here we exploit the extensive cell lineage information and streamlined genome of the ascidian, Ciona intestinalis, to investigate heart development in a basal chordate. Several cardiac genes were analyzed, including the sole Ciona ortholog of the Drosophila tinman gene, and tissue-specific enhancers were isolated for some of the genes. Conserved sequence motifs within these enhancers facilitated the isolation of a heart enhancer for the Ciona Hand-like gene. Altogether, these studies provide a regulatory framework for the differentiation of the cardiac mesoderm, beginning at the 110-cell stage, and extending through the fusion of cardiac progenitors during tail elongation. The cardiac lineage shares a common origin with the germ line, and zygotic transcription is first detected in the heart progenitors only after its separation from the germ line at the 64-cell stage. We propose that germ-line determinants influence the specification of the cardiac mesoderm, both by inhibiting inductive signals required for the development of noncardiac mesoderm lineages, and by providing a localized source of Wnt-5 and other signals required for heart development. We discuss the possibility that the germ line also influences the specification of the vertebrate heart.

The transcription/migration interface in heart precursors of Ciona intestinalis.

Gene regulatory networks direct the progressive determination of cell fate during embryogenesis, but how they control cell behavior during morphogenesis remains largely elusive. Cell sorting, microarrays, and targeted molecular manipulations were used to analyze cardiac cell migration in the ascidian Ciona intestinalis. The heart network regulates genes involved in most cellular activities required for migration, including adhesion, cell polarity, and membrane protrusions. We demonstrated that fibroblast growth factor signaling and the forkhead transcription factor FoxF directly upregulate the small guanosine triphosphatase RhoDF, which synergizes with Cdc42 to contribute to the protrusive activity of migrating cells. Moreover, RhoDF induces membrane protrusions independently of other cellular activities required for migration. We propose that transcription regulation of specific effector genes determines the coordinated deployment of discrete cellular modules underlying migration.

Uncoupling heart cell specification and migration in the simple chordate Ciona intestinalis

The bHLH transcription factor Mesp has an essential but ambiguous role in early chordate heart development. Here, we employ the genetic and morphological simplicity of the basal chordate Ciona intestinalis to elucidate Mesp regulation and function. Characterization of a minimal cardiac enhancer for the Ciona Mesp gene demonstrated direct activation by the T-box transcription factor Tbx6c. The Mesp enhancer was fused to GFP, permitting high-resolution visualization of heart cells as they migrate and divide. The enhancer was also used to drive targeted expression of an activator form of Mesp, which induces heart formation without migration. We discuss the implications of Tbx6-Mesp interactions for the evolution of cardiac mesoderm in invertebrates and vertebrates.

FoxF is essential for FGF-induced migration of heart progenitor cells in the ascidian Ciona intestinalis

Heart development requires precise coordination of morphogenetic movements with progressive cell fate specification and differentiation. In ascidian embryos, FGF/MAPK-mediated activation of the transcription factor Ets1/2 is required for heart tissue specification and cell migration. We found that FoxF is one of the first genes to be activated in heart precursors in response to FGF signaling. We identified the FoxF minimal heart enhancer and used a cis-trans complementation test to show that Ets1/2 can interact with the FoxF enhancer in vivo. Next, we found that FoxF function is required downstream and in parallel to the FGF/MAPK/Ets cascade for cell migration. In addition, we demonstrated that targeted expression of a dominant-negative form of FoxF inhibits cell migration but not heart differentiation, resulting in a striking phenotype: a beating heart at an ectopic location within the body cavity of juveniles. Taken together, our results indicate that FoxF is a direct target of FGF signaling and is predominantly involved in the regulation of heart cell migration.

Cytoskeletal polarity mediates localized induction of the heart progenitor lineage.

Cells must make appropriate fate decisions within a complex and dynamic environment. In vitro studies indicate that the cytoskeleton acts as an integrative platform for this environmental input. External signals regulate cytoskeletal dynamics and the cytoskeleton reciprocally modulates signal transduction. However, in vivo studies linking cytoskeleton/signalling interactions to embryonic cell fate specification remain limited. Here we show that the cytoskeleton modulates heart progenitor cell fate. Our studies focus on differential induction of heart fate in the basal chordate Ciona intestinalis. We have found that differential induction does not simply reflect differential exposure to the inductive signal. Instead, pre-cardiac cells employ polarized, invasive protrusions to localize their response to an ungraded signal. Through targeted manipulation of the cytoskeletal regulator CDC42, we are able to depolarize protrusive activity and generate uniform heart progenitor fate specification. Furthermore, we are able to restore differential induction by repolarizing protrusive activity. These findings illustrate how bi-directional interactions between intercellular signalling and the cytoskeleton can influence embryonic development. In particular, these studies highlight the potential for dynamic cytoskeletal changes to refine cell fate specification in response to crude signal gradients.

Isolation of genes involved in ascidian metamorphosis: epidermal growth factor signaling and metamorphic competence.

Abstract. Although embryonic development in ascidians has been studied for over a century, the signals involved in coordinating post-larval development and metamorphosis are just beginning to be investigated. In this paper, we demonstrate that transcription is necessary for both the acquisition of metamorphic competence and the completion of the initial events of metamorphosis in Boltenia villosa. Transcripts expressed during metamorphic competence were isolated by a suppressive PCR subtraction of Boltenia villosa larval cDNAs. One of these transcripts is homologous to cornichon. Cornichon has a crucial but undefined role in epidermal growth factor (EGF) signaling during Drosophila embryogenesis. In situ hybridization demonstrates that Boltenia cornichon (Cnib) is expressed in the anterior papillary region of larvae as they gain competence. Our hypothesis is that Cnib acts to potentiate EGF signaling, thereby allowing Boltenia larvae to respond to cues for metamorphosis. Further research into the role of Cnib in urochordate metamorphosis may provide insight into the function of cornichon in other organisms. A better molecular understanding of urochordate metamorphosis will also provide a foundation for exploring the role of metamorphosis in chordate evolution.

Spatio-temporal intersection of Lhx3 and Tbx6 defines the cardiac field through synergistic activation of Mesp

Mesp encodes a bHLH transcription factor required for specification of the cardiac mesoderm in Ciona embryos. The activities of Macho-1 and beta-catenin, two essential maternal determinants, are required for Mesp expression in the B7.5 blastomeres, which constitute the heart field. The T-box transcription factor Tbx6 functions downstream of Macho-1 as a direct activator of Mesp expression. However, Tbx6 cannot account for the restricted expression of Mesp in the B7.5 lineage since it is expressed throughout the presumptive tail muscles. Here we present evidence that the LIM-homeobox gene Lhx3, a direct target of beta-catenin, is essential for localized Mesp expression. Lhx3 is expressed throughout the presumptive endoderm and B7.5 blastomeres. Thus, the B7.5 blastomeres are the only cells to express sustained levels of the Tbx6 and Lhx3 activators. Like mammalian Lhx3 genes, Ci-Lhx3 encodes two isoforms with distinct N-terminal peptides. The Lhx3a isoform appears to be expressed both maternally and zygotically, while the Lhx3b isoform is exclusively zygotic. Misexpression of Lhx3b is sufficient to induce ectopic Mesp activation in cells expressing Tbx6b. Injection of antisense morpholino oligonucleotides showed that the Lhx3b isoform is required for endogenous Mesp expression. Mutations in the Lhx3 half-site of Tbx6/Lhx3 composite elements strongly reduced the activity of a minimal Mesp enhancer. We discuss the delineation of the heart field by the synergistic action of muscle and gut determinants.

A single GATA factor plays discrete, lineage specific roles in ascidian heart development

GATA family transcription factors are core components of the vertebrate heart gene network. GATA factors also contribute to heart formation indirectly through regulation of endoderm morphogenesis. However, the precise impact of GATA factors on vertebrate cardiogenesis is masked by functional redundancy within multiple lineages. Early heart specification in the invertebrate chordate Ciona intestinalis is similar to that of vertebrates but only one GATA factor, Ci-GATAa, is expressed in the heart progenitor cells and adjacent endoderm. Here we delineate precise, tissue specific contributions of GATAa to heart formation. Targeted repression of GATAa activity in the heart progenitors perturbs their transcriptional identity. Targeted repression of endodermal GATAa function disrupts endoderm morphogenesis. Subsequently, the bilateral heart progenitors fail to fuse at the ventral midline. The resulting phenotype is strikingly similar to cardia bifida, as observed in vertebrate embryos when endoderm morphogenesis is disturbed. These findings indicate that GATAa recapitulates cell-autonomous and non-cell-autonomous roles performed by multiple, redundant GATA factors in vertebrate cardiogenesis.

Initial deployment of the cardiogenic gene regulatory network in the basal chordate, Ciona intestinalis.

The complex, partially redundant gene regulatory architecture underlying vertebrate heart formation has been difficult to characterize. Here, we dissect the primary cardiac gene regulatory network in the invertebrate chordate, Ciona intestinalis. The Ciona heart progenitor lineage is first specified by Fibroblast Growth Factor/Map Kinase (FGF/MapK) activation of the transcription factor Ets1/2 (Ets). Through microarray analysis of sorted heart progenitor cells, we identified the complete set of primary genes upregulated by FGF/Ets shortly after heart progenitor emergence. Combinatorial sequence analysis of these co-regulated genes generated a hypothetical regulatory code consisting of Ets binding sites associated with a specific co-motif, ATTA. Through extensive reporter analysis, we confirmed the functional importance of the ATTA co-motif in primary heart progenitor gene regulation. We then used the Ets/ATTA combination motif to successfully predict a number of additional heart progenitor gene regulatory elements, including an intronic element driving expression of the core conserved cardiac transcription factor, GATAa. This work significantly advances our understanding of the Ciona heart gene network. Furthermore, this work has begun to elucidate the precise regulatory architecture underlying the conserved, primary role of FGF/Ets in chordate heart lineage specification.