Expression Profiling and Comparative Genomics Identify a Conserved Regulatory Region Controlling Midline Expression in the Zebrafish Embryo

Abstract

Differential gene transcription is a fundamental regulatory mechanism of biological systems during development, body homeostasis, and disease. Comparative genomics is believed to be a rapid means for the identification of regulatory sequences in genomes. We tested this approach to identify regulatory sequences that control expression in the midline of the zebrafish embryo. We first isolated a set of genes that are coexpressed in the midline of the zebrafish embryo during somitogenesis stages by gene array analysis and subsequent rescreens by in situ hybridization. We subjected 45 of these genes to Compare and DotPlot analysis to detect conserved sequences in noncoding regions of orthologous loci in the zebrafish and Takifugu genomes. The regions of homology that were scored in nonconserved regions were inserted into expression vectors and tested for their regulatory activity by transient transgenesis in the zebrafish embryo. We identified one conserved region from the connective tissue growth factor gene (ctgf), which was able to drive expression in the midline of the embryo. This region shares sequence similarity with other floor plate/notochord-specific regulatory regions. Our results demonstrate that an unbiased comparative approach is a relevant method for the identification of tissue-specific cis-regulatory sequences in the zebrafish embryo.[Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to GenBank under accession nos. AY428026-AY428035, CD777505-CD777543, and CD777544-CD778191.]Differential gene transcription is essential for many developmental processes and physiological responses. Moreover, evolution of the body plan is thought to have been driven to a large extent by changes in gene expression, and differences in gene expression are believed to determine disease risk (Davidson 2001; Ludwig 2002 and references therein). A detailed knowledge of the cisregulatory architecture of genomes is thus a central prerequisite for a comprehensive understanding of many fundamental biological processes and has also medical implications.Despite enormous progress in our understanding of transcriptional regulation in vertebrates (Davidson 2001), we are not able to unequivocally predict the pattern and responsiveness of expression by mere sequence analysis of a gene. This is largely because of the structure and the scattered location of cisregulatory regions in the huge vertebrate genomes (Davidson 2001). In vertebrates, promoter regions are rarely sufficient to drive faithful gene transcription. Frequently, other regulatory regions contribute to the correct spatial and temporal expression of genes. These regions can be scattered over large distances in noncoding sequences, and they can be located upstream, downstream, and in intronic regions (Davidson 2001). Regulatory regions are usually a composite of multiple transcription-factorbinding sites. The small size and degenerate DNA sequence of these binding sites renders a genome-wide computational analys...

Duplicated connective tissue growth factor genes in hypoxia-sensitive blunt snout bream Megalobrama amblycephala and their in vivo expression

“…During embryogenesis, the blunt snout bream Ctgfa mRNA was shown to be transcribed at the adaxial cells of the somites, the floor plate, and at the tailbud at 24 hpf, and expressed at the notochord and at ethmoid plate at 36 hpf. These results are in line with the expression pattern found in zebrafish, where Ctgfa transcripts were also found to transcribe in the adaxial cells and the floor plate at the 8-somite stage and in 18-h-old embryos, and in the tailbud, axial vasculature and ethmoid plate at 30 or 48 hpf (Dickmeis et al, 2004;Fernando et al, 2010). Moreover, zebrafish Ctgfa mRNA was also observed at the notochord in transgenic embryos injected with Ctgfa-promoter-driven GFP plasmids (Chiou et al, 2006).…”

Identification of direct T-box target genes in the developing zebrafish mesoderm

“…The use of comparative genomics to identify zebrafish regulatory sequence has been successfully implemented in the past (Dickmeis et al, 2004;Yang et al, 2007;Allende et al, 2006). One difficulty with this approach is that the positions of regulatory elements and of transcription factor binding sites within them often change over evolution, making them difficult to identify through alignments, particularly across large evolutionary distances (Sanges et al, 2006;Moses et al, 2006).…”

Ancient duplicated conserved noncoding elements in vertebrates: A genomic and functional analysis

“…CNEs, and other similar highly conserved noncoding sequences, are found to cluster in the vicinity of genes implicated in transcriptional regulation and early development (Bejerano et al 2004a;Sandelin et al 2004;Woolfe et al 2005) and the majority of those tested in vivo (5/7 in mice, Nobrega et al 2003; 23/25 in fish, Woolfe et al 2005) drive expression of reporter genes in a temporal and spatial specific manner during early development. Many other studies around specific developmental genes have also identified highly conserved noncoding sequences between humans and fish that have enhancer activity (Zerucha et al 2000;Barton et al 2001;Lien et al 2002;Blader et al 2003; Lettice et al 2003;Dickmeis et al 2004;Kimura-Yoshida et al 2004;de la Calle-Mustienes et al 2005;Goode et al 2005). The association of these highly conserved sequences to genes implicated in the regulation in early development is most likely a result of the fundamental nature of the developmental process in vertebrates.…”