Faculty Bibliography

Thanks to their transparent and rapidly developing mosaic embryos, ascidians (or sea squirts) have been a model system for embryological studies for over a century. Recently, ascidians have entered the postgenomic era, with the sequencing of the Ciona intestinalis genome and the accumulation of molecular resources that rival those available for fruit flies and mice. One strength of ascidians as a model system is their close similarity to vertebrates. Literature reporting molecular homologies between vertebrate and ascidian tissues has flourished over the past 15 years, since the first ascidian genes were cloned. However, it should not be forgotten that ascidians diverged from the lineage leading to vertebrates over 500 million years ago. Here, we review the main similarities and differences so far identified, at the molecular level, between ascidian and vertebrate tissues and discuss the evolution of the compact ascidian genome.

A screen for the systematic identification of cis-regulatory elements within large (>100 kb) genomic domains containing Hox genes was performed by using the basal chordate Ciona intestinalis. Randomly generated DNA fragments from bacterial artificial chromosomes containing two clusters of Hox genes were inserted into a vector upstream of a minimal promoter and lacZ reporter gene. A total of 222 resultant fusion genes were separately electroporated into fertilized eggs, and their regulatory activities were monitored in larvae. In sum, 21 separable cis-regulatory elements were found. These include eight Hox linked domains that drive expression in nested anterior-posterior domains of ectodermally derived tissues. In addition to vertebrate-like CNS regulation, the discovery of cis-regulatory domains that drive epidermal transcription suggests that C. intestinalis has arthropod-like Hox patterning in the epidermis.

Homeobox-containing genes play crucial roles in various developmental processes, including body-plan specification, pattern formation and cell-type specification. The present study searched the draft genome sequence and cDNA/EST database of the basal chordate Ciona intestinalis to identify 83 homeobox-containing genes in this animal. This number of homeobox genes in the Ciona genome is smaller than that in the Caenorhabditis elegans, Drosophila melanogaster, human and mouse genomes. Of the 83 genes, 76 have possible human orthologues and 7 may be unique to Ciona. The ascidian homeobox genes were classified into 11 classes, including Hox class, NK class, Paired class, POU class, LIM class, TALE class, SIX class, Prox class, Cut class, ZFH class and HNF1 class, according to the classification scheme devised for known homeobox genes. As to the Hox cluster, the Ciona genome contains single copies of each of the paralogous groups, suggesting that there is a single Hox cluster, if any, but genes orthologous to Hox7, 8, 9 and 11 were not found in the genome. In addition, loss of genes had occurred independently in the Ciona lineage and was noticed in Gbx of the EHGbox subclass, Sax, NK3, Vax and vent of the NK class, Cart, Og9, Anf and Mix of the Paired class, POU-I, III, V and VI of the POU class, Lhx6/7 of the LIM class, TGIF of the TALE class, Cux and SATB of the Cut class, and ZFH1 of the ZFH class, which might have reduced the number of Ciona homeobox genes. Interestingly, one of the newly identified Ciona intestinalis genes and its vertebrate counterparts constitute a novel subclass of HNF1 class homeobox genes. Furthermore, evidence for the gene structures and expression of 54 of the 83 homeobox genes was provided by analysis of ESTs, suggesting that cDNAs for these 54 genes are available. The present data thus reveal the repertoire of homeodomain-containing transcription factors in the Ciona genome, which will be useful for future research on the development and evolution of chordates.

Hox genes are organized in genomic clusters. In all organisms where their role has been studied, Hox genes determine developmental fate along the antero-posterior axis. Hence, these genes represent an ideal system for the understanding of relationships between the number and expression of genes and body organization. We report in this paper that the ascidian Ciona intestinalis genome appears to contain a single Hox gene complex which shows absence of some of the members found in all chordates investigated up to now. Furthermore, the complex appears to be either unusually long or split in different subunits. We speculate that such an arrangement of Hox genes does not correspond to the chordate primordial cluster but occurred independently in the ascidian lineage.

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains approximately 16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.

Ascidians are marine protochordates at the evolutionary boundary between invertebrates and vertebrates. Ascidian larvae provide a simple system for unraveling gene regulation networks underlying the formation of the basic chordate body plan. After being used for over a century as a model for embryological studies, ascidians have become, in the past decade, an increasingly popular organism for studying gene regulation. Part of the renewed appeal of this system is the use of electroporation to introduce transgenic DNAs into developing embryos. This method is considerably more efficient than conventional microinjection assays and permits the simultaneous transformation of hundreds of embryos. Electroporation has allowed the identification and characterization of cis-regulatory DNAs that mediate gene expression in a variety of tissues, including the notochord, tail muscles, CNS, and endoderm. Electroporation has also provided a simple method for misexpressing patterning genes and producing dominant mutant phenotypes. Recent studies have used electroporation to create "knock-out" phenotypes by overexpressing dominant negative forms of particular proteins. Here we review the past and present uses of electroporation in ascidian development, and speculate on potential future uses.

We present evidence that notochord and muscle differentiation are crucial for morphogenesis of the ascidian tail. We developed a novel approach for embryological manipulation of the developing larval tissues using a simple method to introduce DNA into Ciona intestinalis and the several available tissue-specific promoters. With such promoters, we misexpressed the Xenopus homeobox gene bix in notochord or muscle of Ciona embryos as a means of interfering with development of these tissues. Ciona embryos expressing bix in the notochord from the 64-cell stage develop into larvae with very short tails, in which the notochord precursors fail to intercalate and differentiate. Larvae with mosaic expression of bix have intermediate phenotypes, in which a partial notochord is formed by the precursor cells that did not receive the transgene while the precursors that express the transgene cluster together and fail to undergo any of the cell-shape changes associated with notochord differentiation. Muscle cells adjacent to differentiated notochord cells are properly patterned, while those next to the notochord precursor cells transformed by bix exhibit various patterning defects. In these embryos, the neural tube extends in the tail to form a nerve cord, while the endodermal strand fails to enter the tail region. Similarly, expression of bix in muscle progenitors impairs differentiation of muscle cells, and as a result, notochord cells fail to undergo normal extension movements. Hence, these larvae have a shorter tail, due to a block in the elongation of the notochord. Taken together, these observations suggest that tail formation in ascidian larvae requires not only signaling from notochord to muscle cells, but also a "retrograde" signal from muscle cells to notochord.

The Ciona forkhead/HNF-3beta gene (Ci-fkh) is expressed in the primary axial tissues of the developing tadpole, including the notochord, endoderm, and rudimentary floor plate of the CNS. In an effort to determine the basis for this complex pattern of expression we have conducted a detailed analysis of the Ci-fkh 5'-regulatory region. Different 5' sequences were attached to a lacZ reporter gene and analyzed in electroporated Ciona embryos. A short regulatory sequence (AS) located approximately 1.7 kb upstream of the transcribed region is shown to be essential for expression in all three axial tissues. The proximal 20 bp of the AS contains overlapping Snail repressor elements and a T-box motif. Deleting these sequences causes the loss of reporter gene expression in the endoderm, as well as expanded expression in the neural tube. These results suggest that a T-box gene such as Ci-VegTR activates Ci-fkh expression in the endoderm, while the Ci-Sna repressor excludes expression from the lateral ependymal cells and restricts the Ci-fkh pattern to the rudimentary floor plate in ventral regions of the neural tube. We also present evidence for Ci-fkh positive autofeedback, whereby the Ci-Fkh protein binds to critical activator sites within the Ci-fkh 5'-regulatory region and helps maintain high levels of expression. We discuss these results with respect to forkhead/HNF-3beta regulation in vertebrates.