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Published online before print
January 12, 2004, 10.1101/gr.1819204 Genome Res. 14:228-238, 2004 ©2004 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/04 $5.00
Letter Expression Profiling and Comparative Genomics Identify a Conserved Regulatory Region Controlling Midline Expression in the Zebrafish Embryo1 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 10142, F-67404 Illkirch Cedex, C.U. de Strasbourg, France 2 Max Planck Institut für Molekulare Genetik, D-14195 Berlin (Dahlem), Germany
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.
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
Despite enormous progress in our understanding of transcriptional regulation in vertebrates (Davidson 2001
Comparison of genomes of related species was shown to provide a possible shortcut in the identification of regulatory regions as they form islands of partially conserved sequence in noncoding regions (Hardison 2000
Several studies reported conservation of regulatory sequences in comparisons of mammalian and Takifugu loci (Aparicio et al. 1995
Because the number of zebrafish genes whose regulatory structure has been analyzed in depth is rather small, it remains to be seen whether this high conservation of regulatory sequences of teleost genomes is indeed a general phenomenon and can be exploited as a rapid approach to elucidate the regulatory structure of genomes. In fact, there is evidence that regulatory sequences in zebrafish genes were not maintained structurally during evolution. The structure of an enhancer that was mapped to the second intron of the zebrafish netrin1 gene and that mediates floor plate expression in zebrafish has not been conserved (Rastegar et al. 2002
To assess the wider applicability of comparative approaches to the zebrafish genome sequence, we carried out a systematic search for homologies in nonconserved regions of the zebrafish and Takifugu loci and tested the regulatory activity of these regions by transient transgenesis in the zebrafish. We chose these two teleost genomes to carry out the comparison as we expected a higher yield of conserved sequences within the teleost lineage. We first identified genes expressed in the embryonic midline comprising the axial mesoderm, notochord, and prechordal plate. In addition to providing mechanical support, these midline structures have important regulatory activity that controls cell differentiation in the adjacent neural tube, endoderm, and somites (Pourquie et al. 1993
Preparation of a Complex Probe From Midline Cells To isolate a representative set of genes expressed in the midline of zebrafish embryos, we used a subtraction strategy that was previously used to identify genes acting downstream from Nodal signals in the gastrula (Dickmeis et al. 2001a  ). This approach entails several steps of enrichment and selection to minimize the scale of the final screening by in situ hybridization (Fig. 1). We used transgenic embryos that express the green fluorescent protein (gfp) gene under control of the regulatory elements of the shh gene (Albert et al. 2003). At the 6-10-somite stage, these transgenic embryos express the gfp reporter gene in the notochord, the prechordal plate, and in the ventral midline of the neural plate comprising the prospective floor plate and hypothalamus (Fig. 1). Several hundred shh:gfp embryos were dissociated into single cells at the 6-10-somite stage, and GFP-positive cells were enriched with a fluorescence-activated cell sorter (FACS). As a second further enrichment, we carried out a cDNA subtraction using cDNA prepared from the shh:gfp cells as tester cDNA. The driver cDNA was prepared from the GFP-positive cells sorted from a second transgenic line that expressed GFP under control of the ngn1 gene (-8.4ngn1:gfp;Blader et al. 2003). GFP marks primary neurons and is also weakly detectable in the epidermis of these embryos at the 6-10-somite stage (Fig. 1). The forward subtraction (S) of the two cDNA pools normalized the abundance of transcripts and enriched midline-specific genes (Fig. 1). The reverse subtraction (N) was carried out as a control.
As a first assessment of the distribution of cDNAs in the two resulting probe pools, S and N, we performed Southern blot analysis with a series of genes whose expression patterns are known (Fig. 2A). The pool S that was expected to contain genes expressed in the midline hybridized strongly to cDNAs such as one-eyed pinhead (oep), no tail (ntl), floating head (flh), collagen 2  1, and forkhead4 (fkd4) that are expressed in the midline of the zebrafish embryo (Schulte-Merker et al. 1992;Talbot et al. 1995; Yan et al. 1995;Odenthal and Nusslein-Volhard 1998;Gritsman et al. 1999). Much weaker or no signals were detected with probes that are derived from genes expressed predominantly in the neuroectoderm such as pax2a (Krauss et al. 1991), elav (Kim et al. 1996), msxB (Ekker et al. 1997), deltaA (dla), and deltaD (dld) (Haddon et al. 1998). The reverse pattern of hybridization signals was obtained when the probe pool N was hybridized: The neuroectoderm-specific cDNAs hybridized strongly, whereas the midline genes gave rise to only weak signals (Fig. 2A, bottom row, N). These results indicate that the subtraction resulted in the anticipated enrichment of genes. As a second evaluation, cDNA fragments from the S pool were subcloned and 37 fragments were sequenced (GenBank Accession numbers CD777505
[GenBank]
-CD777543). These 37 clones corresponded to 33 genes, indicating a low level of redundancy in the complex probe. Next, in situ hybridization was carried out to assess the tissue-specific expression of individual S clones. Of the clones, 40% were expressed in midline tissues including the prechordal plate-derived hatching glands (Fig. 2B-G;data not shown). Taken together, these tests indicate that the subtraction successfully enriched clones that are expressed in the midline.
Macroarray Analysis As a next step, the S and N probes were hybridized to a macroarray containing 25,102 cDNAs from 24-h-old whole embryos and adult liver cDNA (MPMGp609; Clark et al. 2001 ). This library contains a minimally redundant clone set derived from initially 75,000 clones by normalization with oligonucleotide fingerprinting (Clark et al. 1999). Hybridizations with the two complex probes were carried out in triplicate to membranes with two distinct arrangements of spotted clones. The resulting signals were averaged, and the differential intensity of each signal was calculated. A total of 662 cDNAs with a 1.8-fold or higher representation in the hybridizations with the S probe in comparison to the N probe were chosen for further analysis. The cDNAs were sequenced (GenBank accession numbers CD777544
[GenBank]
-CD778191), and the subsequent BLAST analysis revealed that 487 clones represented 152 genes with homologs in the protein database and that 93 clones were without such homologs. On average, every gene was represented 3.2 times in the set of 487 clones that yielded homology scores in the databases. Large differences in abundance of clones representing the same gene existed, with cathepsin l being the biggest cluster (55 clones) and 78 genes being represented only by one clone. Grouping the genes with homologies according to functional aspects (Fig. 3) revealed that the largest classes are the cytoskeletal/cell adhesion/extracellular matrix (27.6%) and the protein metabolism group (23.7%), followed by proteins in cell signaling (15.1%) and proteins of unknown function (15.1%). Smaller groups contain proteins involved in transcription (4.6%) and RNA metabolism (2.6%), and 4.6% of genes fall into various other functional categories.
Groups of Coexpressed Genes Identified by In Situ Hybridization Several of the identified genes were shown previously to be expressed in the axial mesoderm. These include shh (Krauss et al. 1993 ), no tail (Schulte-Merker et al. 1992), semaphorin Z2 (Halloran et al. 1998), calreticulin (Rubinstein et al. 2000), treb5 (Liang et al. 2001), col2a1 (Yan et al. 1995), calymmin (Cerda et al. 2002), cathepsin l (Mangos et al. 2000), hsp47 (Lele and Krone 1997), and brain subtype creatine kinase (Dickmeis et al. 2001b). To assess the pattern of expression of the remaining clones, an in situ hybridization screen with the entire initial set of cDNA clones was performed. The in situ analysis provided an independent line of proof for the clustering of the clones, assuming that the clones assigned to the same cluster yield the same pattern of expression. Expression was analyzed at 8- and 18-somite stages, and in situ hybridization was carried out using an in situ hybridization robot.
Altogether 68.5% of the genes showed tissue-restricted expression. The most frequently expressing tissue is the notochord, with 47% of the genes at the 6-10- somite stage (Table 1;Fig. 4;Supplemental material) and 33% at the 16-20-somite stage (Table 1;Supplemental material;data not shown). Expression was also frequently detected in the polster, a derivative of the prechordal plate that gives rise to the hatching glands in older stages. At the 6-10-somite and 16-20-somite stages, 30% and 29% of genes, respectively, were expressed in this tissue. Altogether, the genes expressing in axial tissues (notochord, polster) is 55.9% at the 6-10-somite stage and 53.3% at the 16-20-somite stage. At lower frequency, expression was noted in floor plate (11%) and hypochord (7%). Expression in these tissues was only scored in 16-20-somite-stage embryos for technical reasons and because the hypochord has not completely formed at early stages (Eriksson and Lofberg 2000
Comparative Sequence Analysis of Homologous Loci in the Takifugu Genome To delineate the putative regulatory sequences that are responsible for expression in the midline of the zebrafish embryo, the genomic sequence of the genes that are expressed in the midline were compared with the orthologous loci of the Takifugu genome. The Takifugu gene with the highest sequence similarity at the protein level was assumed to represent the ortholog, and only those genes (45 genes total) for which an unequivocal score was obtained in the annotated gene set of the Takifugu genome were analyzed further. For each locus, the coding region with the intronic sequences, 10 kb upstream of the ATG and 10 kb downstream from the stop codon, were searched for homologies with the "Compare" and "DotPlot" programs (GCG Wisconsin package). The settings of the search algorithm were kept at the default values, which allowed scoring the regulatory sequences of the zebrafish ngn1 (Blader et al. 2003 ) and shh genes (Muller et al. 1999, 2000;Albert et al. 2003). Among 45 pairwise comparisons, 18 homologies were detected in the noncoding regions of 10 genes (Fig. 5;Table 2). The size of these regions varied from  75 to 680 bp, and the degree of similarity ranged from 63.9% to 88.9% (Table 2). The positions of the regions of homology were found upstream and downstream of the coding regions (Fig. 5).
Functional Analysis of Identified Homology Regions To test whether these regions of homology mediate gene expression in the zebrafish embryo, we inserted PCR fragments spanning the regions of homology downstream from a reporter gene comprising the Herpes simplex virus thymidine kinase TATA-box directing expression of a green fluorescent reporter (gfp) gene (Rastegar et al. 2002 ). This expression vector works well in transient expression experiments and has a very low basal activity without inserted additional regulatory sequences (Rastegar et al. 2002). We inserted conserved fragments also downstream from an netrin1:gfp reporter gene. The promoter of the netrin1 gene is weakly active in the neural tube of the zebrafish and was used previously to map the regulatory sequences of the netrin1 locus (Rastegar et al. 2002).
The activity of the homology regions was tested by a transient expression protocol (Westerfield et al. 1992
The up1 homology of the ctgf gene is located immediately upstream of and overlaps with the first exon of the gene. This indicates that this region comprises the promoter of the gene. In agreement, a well-conserved TATA-box is located 69 bp upstream of the reported 5'-end of the mRNA (Fig. 6A). We thus inserted this region upstream of the gfp reporter gene in place of the netrin1 or tk promoters. We detected strong expression in the notochord (41%, n = 100) and floor plate (31%, n = 100) of 24-h-old embryos injected with the ctgf:gfp construct (Fig. 6C). These results indicate that the up1 region includes the promoter of the ctgf gene. This region seems to function, however, also as an enhancer because it can also act in a downstream position.
The other homology regions tested did not yield significant expression in injected embryos (Table 2;data not shown). Either these regions do not have transcription regulatory activity in the 24-h-old embryo or they are not active in the specific context of our reporter constructs as they are, for example, repressor elements. It is also possible that they have functions other than transcription regulation (Glazko et al. 2003
The Regulatory Region of the ctgf Gene Shares Homology With Other Floor Plate/Notochord Enhancers
It can be assumed that genes with similar expression profiles use similar sets of regulatory factors. Several regulatory regions with activity in the floor plate or notochord were previously identified in the zebrafish shh and netrin1 genes (Muller et al. 1999
Here we have tested the applicability of the zebrafish genome in comparative searches for cis-regulatory elements. We identified 85 genes that are expressed in midline tissues comprising notochord, prechordal plate, and floor plate. In a second step, we used the previously observed conservation of regulatory regions between orthologous loci in zebrafish and Takifugu. This approach was appealing as it has the advantage of yielding an enrichment of regulatory sequences over predicted nonfunctional sequences and was expected to facilitate detection of shared transcription-factor-binding sites. In a search of 45 genes expressed in the midline, we found 10 genes with significant homologies. One of these conserved regions directed gene expression in the midline of the zebrafish embryo. Our results demonstrate that the unbiased approach of comparing between orthologous loci of the zebrafish and Takifugu genomes can yield regulatory elements.
The Embryonic Midline Expresses Collagens and Collagen-Processing Enzymes When examined for specific biochemical pathways, several collagens (col1a1,col2a1, col5a1, col9a1, col9a2, col9a3, and col11a1) and collagen-processing enzymes were detected in the somitogenesis but not in the gastrula stage screen. The processing genes include procollagen-lysine; 2-oxoglutarate 5-dioxygenase 1; prolyl-4-hydroxylase;the peptidyl-prolyl isomerase; fk506 binding protein;and the heat-shock proteins hsp47 and glucose regulated protein 78 (grp78). The abundance of these enzymes in the midline is likely to reflect the mechanical support function of the notochord for the early embryo.
The Promoter Region of the ctgf Gene Controls Floor Plate and Notochord Expression
The conserved up1 region confers notochord and floor plate expression to the tk:gfp reporter gene. The region overlaps with the first exon of the ctgf gene and is thus likely to represent the promoter of this gene. In agreement, it harbors a conserved TATA-box 69 bp upstream of the 5'-end of the isolated ctgf cDNA and is able to direct expression of gfp from ctgf:gfp constructs. The related region of human ctgf directs expression in tissue culture cells (Eguchi et al. 2001
The endogenous ctgf gene is not expressed at detectable levels in the notochord. This expression in the notochord in the transient expression assays used here appears thus to be an ectopic activity of the up1 region. This has previously been noted also for the promoter region of the tiggywinkle hedgehog (twhh) gene (Du et al. 1997
Several features in the up1 fragment are shared with previously identified notochord/floor plate enhancers. Motif1 overlaps with an FoxA2/HNF3
Zebrafish/Takifugu Locus Comparisons Permit Detection of Regulatory Elements
The frequency with which we detected homologous sequences is lower than expected from the previous analysis of a small sample of zebrafish genes (Zerucha et al. 2000
Thus other strategies have to be used to elucidate the cis-regulatory architecture of the zebrafish in a comprehensive fashion. It is possible to search the genome for clusters of known binding sites of transcription factors as used in the study of transcriptional networks in Drosophila (Markstein et al. 2002
In summary, however, our work shows that a comparative search for regulatory sequences using the Takifugu and zebrafish genomic sequences permits identification of regulatory sequences. The central question is why the regulatory sequences of certain genes are more conserved than others. It is intriguing that the zebrafish genes whose regulatory sequences were shown to be conserved so far are developmental key regulators (Zerucha et al. 2000
Fish Stocks The wild-type line is an intercross between the AB line (University of Oregon, Eugene) and the wtOX line, which was purchased from the Goldfish Bowl (Oxford, UK) and has been bred for several years in our laboratory. Fish were bred and transgenic embryos were collected as described (Westerfield 1993 ). The transgenic lines -2.2shh:gfpABC (shh:gfp) and -8.4ngn1:gfp (ngn1:gfp) were reported previously (Albert et al. 2003;Blader et al. 2003).
Cell sorting, Complex Probes, and Array Hybridization
Southern blotting and sequencing were carried out following standard procedures (Sambrook and Russel 2001
In Situ Hybridization
Sequence Comparison
Homology searches were carried out with the Compare, DotPlot, and BestFit programs of the GCG Wisconsin package using the default parameters of the program. Comparison of the ctgf up1, the 250-bp ar-C, the 500-bp ar-B, ar-A of shh (Muller et al. 1999
Expression Analysis
We thank D. Biellmann and C. Vialle for fish care and artwork. We are indebted to Nick Foulkes for a critical reading of the manuscript. We acknowledge the help of the project students Matthias Corrotte and Simon Jochum. This work was supported by Boehringer Ingelheim and AFM fellowships to T.D. We are also grateful to the Institut National de la Santé et de la Recherche Medicale, the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg, AFM, ARC, ACI, AICR, and the Max Planck Society. The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.1819204. Article published online before print in January 2004.
3 Present address: Max-Planck-Institut für Entwicklungsbiologie, Friedrich-Miescher-Laboratorium, D-72076 Tübingen, Germany
4 Present address: Department of Biochemistry and Biophysics, University of California-San Francisco, San Francisco, CA 94143-0448, USA.
5 Corresponding author. [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 [GenBank] -CD777543, and CD777544 [GenBank] -CD778191.]
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ABSTRACT
RESULTS
A), dld (
binding site. (B) Transient transgenic embryo expressing GFP under control of the up1 region from the tk:gfp cassette. (C) Transient transgenic embryo expressing GFP from the ctgf:gfp transgene. (f) Floor plate; (n) notochord.
