Regulatory elements controlling Ci-msxb tissue-specific expression during Ciona intestinalis embryonic development

Laboratory of Biochemistry and Molecular Biology, Stazione Zoologica "A. Dohrn", Naples 80121, Italy.
Developmental Biology (Impact Factor: 3.55). 04/2004; 267(2):517-28. DOI: 10.1016/j.ydbio.2003.11.005
Source: PubMed


The msh/Msx family is a subclass of homeobox-containing genes suggested to perform a conserved function in the patterning of the early embryo. We had already isolated a member of this gene family (Ci-msxb) in Ciona intestinalis, which has a very complex expression pattern during embryogenesis. To identify the regulatory elements controlling its tissue-specific expression, we have characterized the gene structure and the regulatory upstream region. By electroporation experiments, we demonstrated that a 3.8-kb region located upstream of the gene contains all the regulatory elements able to reproduce its spatial expression pattern. Analyzing progressively truncated fragments of this region, three discrete and separate regions driving LacZ reporter gene expression in the ventral epidermis, primordial pharynx and neural territories have been identified. We further investigated the element(s) necessary for Ci-msxb activation in the nervous system during embryonic development by in vivo and in vitro experiments. Both electroporation and gel-shift assays of overlapping wild type and mutated oligonucleotides demonstrated that a unique sequence of 30 bp is involved in Ci-msxb neural activation from neurula to larva stage. This sequence contains consensus binding sites for various ubiquitous transcription factors such as TCF11 whose possible implication in formation of the regulatory complexes is discussed.

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    • "The importance of the FGF, Notch, and Wnt/β-catenin signaling systems in A7.6/TLC induction has been previously demonstrated (Kawaminani and Nishida, 1997; Shimauchi et al., 2001; Imai et al., 2002a, 2003, 2006). Ascidian homologues of NC-RGN tier 2 genes (msx1/2, pax7, dlx3, and zic) are expressed along the margin of the neural plate (Ma et al., 1996; Wada et al., 1997; Caracciolo et al., 2000; Imai 2002b; Satou et al., 2002b; Gostling and Shimeld, 2003; Russo et al., 2004). We investigated the Ciona orthologues of msx (Ci-msxb), pax3 and 7 (Ci-pax3/7), dlx3 (Ci- dll3), and zic (Ci-zicL and Ci-macho) in the A7.6/TLC lineage. "
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    ABSTRACT: Neural crest-like cells (NCLC) that express the HNK-1 antigen and form body pigment cells were previously identified in diverse ascidian species. Here we investigate the embryonic origin, migratory activity, and neural crest related gene expression patterns of NCLC in the ascidian Ciona intestinalis. HNK-1 expression first appeared at about the time of larval hatching in dorsal cells of the posterior trunk. In swimming tadpoles, HNK-1 positive cells began to migrate, and after metamorphosis they were localized in the oral and atrial siphons, branchial gill slits, endostyle, and gut. Cleavage arrest experiments showed that NCLC are derived from the A7.6 cells, the precursors of trunk lateral cells (TLC), one of the three types of migratory mesenchymal cells in ascidian embryos. In cleavage arrested embryos, HNK-1 positive TLC were present on the lateral margins of the neural plate and later became localized adjacent to the posterior sensory vesicle, a staging zone for their migration after larval hatching. The Ciona orthologues of seven of sixteen genes that function in the vertebrate neural crest gene regulatory network are expressed in the A7.6/TLC lineage. The vertebrate counterparts of these genes function downstream of neural plate border specification in the regulatory network leading to neural crest development. The results suggest that NCLC and neural crest cells may be homologous cell types originating in the common ancestor of tunicates and vertebrates and support the possibility that a putative regulatory network governing NCLC development was co-opted to produce neural crest cells during vertebrate evolution.
    Developmental Biology 10/2008; 324(1):152-60. DOI:10.1016/j.ydbio.2008.08.022 · 3.55 Impact Factor
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    • "Transient transgenesis can be used for efficient expression of exogenous genes in ascidian embryos and larvae (Corbo et al., 1997; Hikosaka et al., 1992). cis-Regulatory DNAs of a number of tissuespecific genes have been studied by introducing reporter fusion constructs into ascidian embryos (Bertrand et al., 2003; Corbo et al., 1997; Di Gregorio et al., 2001; Erives and Levine, 2000; Fanelli et al., 2003; Hikosaka et al., 1994; Katsuyama et al., 2002; Kusakabe et al., 1995; Mitani et al., 2001; Oda-Ishii and Saiga, 2003; Russo et al., 2004; Satou and Satoh, 1996; Yoshida et al., 2004). These studies have suggested that relatively short 5V flanking sequences are generally sufficient to recapitulate the endogenous gene expression patterns of most tissuespecific genes in ascidians. "
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    ABSTRACT: Temporally and spatially co-expressed genes are expected to be regulated by common transcription factors and therefore to share cis-regulatory elements. In the ascidian Ciona intestinalis, the whole-genome sequences and genome-scale gene expression profiles allow the use of computational techniques to investigate cis-elements that control transcription. We collected 5' flanking sequences of 50 tissue-specific genes from genome databases of C. intestinalis and a closely related species Ciona savignyi. We searched for DNA motifs over-represented in upstream regions of a group of co-expressed genes. Several motifs were distributed predominantly in upstream regions of photoreceptor, pan-neuronal, or muscle-specific gene groups. One muscle-specific motif, M2, was distributed preferentially in regions from -200 to -100 bp relative to the translational start sites. Promoters of muscle-specific genes of C. intestinalis were isolated, connected with a green fluorescent protein gene (GFP), and introduced into C. intestinalis embryos. In muscle cells, these promoters specifically drove GFP expression, which mutations of the M2 sites greatly reduced. When M2 sites were located upstream of a basal promoter, the reporter GFP was specifically expressed in muscle cells. These results suggest the validity of our computational prediction of cis-regulatory elements. Thus, bioinformatics can help identify cis-regulatory elements involved in chordate development.
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