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Conservation of shh cis-regulatory architecture of the coelacanth is consistent with its ancestral phylogenetic position.

Lang M, Hadzhiev Y, Siegel N, Amemiya CT, Parada C, Strähle U, Becker MB, Müller F, Meyer A - Evodevo (2010)

Bottom Line: The functionality of the putative Latimeria enhancers was confirmed by reporter gene expression analysis in transient transgenic zebrafish and chick embryos.In contrast to lineage-specific losses and differentiations in more derived lineages, Latimeria shh enhancers reveal low levels of sequence diversification.High overall sequence conservation of shh conserved noncoding elements (CNE) is consistent with the general trend of high levels of conservation of noncoding DNA in the slowly evolving Latimeria genome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Konstanz, 78457 Konstanz, Germany. f.mueller@bham.ac.uk.

ABSTRACT

Background: The modern coelacanth (Latimeria) is the extant taxon of a basal sarcopterygian lineage and sister group to tetrapods. Apart from certain apomorphic traits, its morphology is characterized by a high degree of retention of ancestral vertebrate structures and little morphological change. An insight into the molecular evolution that may explain the unchanged character of Latimeria morphology requires the analysis of the expression patterns of developmental regulator genes and their cis-regulatory modules (CRMs).

Results: We describe the comparative and functional analysis of the sonic hedgehog (shh) genomic region of Latimeria menadoensis. Several putative enhancers in the Latimeria shh locus have been identified by comparisons to sarcopterygian and actinopterygian extant species. Specific sequence conservation with all known actinopterygian enhancer elements has been detected. However, these elements are selectively missing in more recently diverged actinopterygian and sarcopterygian species. The functionality of the putative Latimeria enhancers was confirmed by reporter gene expression analysis in transient transgenic zebrafish and chick embryos.

Conclusions: Latimeria shh CRMs represent the ancestral set of enhancers that have emerged before the split of lobe-finned and ray-finned fishes. In contrast to lineage-specific losses and differentiations in more derived lineages, Latimeria shh enhancers reveal low levels of sequence diversification. High overall sequence conservation of shh conserved noncoding elements (CNE) is consistent with the general trend of high levels of conservation of noncoding DNA in the slowly evolving Latimeria genome.

No MeSH data available.


Functional assay of Latimeria ar-A, ar-B, and ar-D enhancers in zebrafish. Two fluorescent images are shown for each embryo. Low magnification displays the whole embryo and high magnification focuses on the trunk above the yolk extension. Schematic representations of the injected zebrafish (z) and Latimeria (l) promoter (pr) and enhancer reporter constructs are shown on the left side of each panel (a-d) VISTA plot comparisons of the zebrafish and Latimeria enhancer regions are shown below reporter constructs and indicate the degree of conservation. Conservation identity greater than 70% is highlighted in color. (a) Embryo injected with control construct containing the 0.8 kb (form the transcriptional start site) zebrafish shh promoter, linked to GFP. (b-d) Embryos injected with reporter constructs containing the minimal zebrafish shh promoter and one of the Latimeria shh enhancers ar-D (b) ar-A (c) and ar-B (d). GFP expression in the floorplate is indicated by arrows and the arrowheads point at expression in the notochord.
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Figure 3: Functional assay of Latimeria ar-A, ar-B, and ar-D enhancers in zebrafish. Two fluorescent images are shown for each embryo. Low magnification displays the whole embryo and high magnification focuses on the trunk above the yolk extension. Schematic representations of the injected zebrafish (z) and Latimeria (l) promoter (pr) and enhancer reporter constructs are shown on the left side of each panel (a-d) VISTA plot comparisons of the zebrafish and Latimeria enhancer regions are shown below reporter constructs and indicate the degree of conservation. Conservation identity greater than 70% is highlighted in color. (a) Embryo injected with control construct containing the 0.8 kb (form the transcriptional start site) zebrafish shh promoter, linked to GFP. (b-d) Embryos injected with reporter constructs containing the minimal zebrafish shh promoter and one of the Latimeria shh enhancers ar-D (b) ar-A (c) and ar-B (d). GFP expression in the floorplate is indicated by arrows and the arrowheads point at expression in the notochord.

Mentions: To check whether the Latimeria conserved noncoding sequences had enhancer activity, reporter gene expression analysis was conducted in transient transgenic zebrafish embryos. This analysis had already been carried out with Latimeria ar-C in a previous study [34], and the aim here was to analyze other conserved regulatory regions that potentially drive shh midline expression. Conserved noncoding elements of Latimeria shh intron 1 located between exon 1 and the intronic enhancer ar-A did not drive specific reporter gene expression (data not shown). Latimeria putative enhancer orthologs ar-D, ar-A and ar-B were cloned into the above-mentioned shh minimal promoter constructs, containing GFP (Green Fluorescent Protein) instead of a LacZ reporter. Transient mosaic expression of GFP was measured as read out of reporter construct activity 24 hours after injection of zebrafish zygotes (Figure 3). The reporter expression directed by the zebrafish shh upstream region resembled the tissue-specific expression of the isolated ar-D enhancer (Additional file 5). GFP expression was observed in the ventral brain and in the anterior parts of the floorplate (data not shown) [33,40]. Similarly, Latimeria ar-D also directed floorplate-specific GFP expression (Figure 3b). However, the reporter expression was extended to the posterior parts of the floorplate. Among GFP-expressing embryos, fluorescence in the posterior floorplate cells (posterior to the start of the yolk extension) was detected in only 4% of specimens with the zebrafish upstream region but in 75% of corresponding embryos with Latimeria ar-D (Table 2). These results indicate that the CNE in the Latimeria upstream region is a functional midline enhancer which has similar but not identical activity in zebrafish to the zebrafish ar-D enhancer. On the basis of our DNA sequence comparison, Latimeria ar-D was found to contain sarcopterygian specific sequences with a puative FoxA2 element (92% match). Potentially, these elements can account for the posteriorly extended expression direction of Latimeria ar-D in the ventral neural tube of zebrafish.


Conservation of shh cis-regulatory architecture of the coelacanth is consistent with its ancestral phylogenetic position.

Lang M, Hadzhiev Y, Siegel N, Amemiya CT, Parada C, Strähle U, Becker MB, Müller F, Meyer A - Evodevo (2010)

Functional assay of Latimeria ar-A, ar-B, and ar-D enhancers in zebrafish. Two fluorescent images are shown for each embryo. Low magnification displays the whole embryo and high magnification focuses on the trunk above the yolk extension. Schematic representations of the injected zebrafish (z) and Latimeria (l) promoter (pr) and enhancer reporter constructs are shown on the left side of each panel (a-d) VISTA plot comparisons of the zebrafish and Latimeria enhancer regions are shown below reporter constructs and indicate the degree of conservation. Conservation identity greater than 70% is highlighted in color. (a) Embryo injected with control construct containing the 0.8 kb (form the transcriptional start site) zebrafish shh promoter, linked to GFP. (b-d) Embryos injected with reporter constructs containing the minimal zebrafish shh promoter and one of the Latimeria shh enhancers ar-D (b) ar-A (c) and ar-B (d). GFP expression in the floorplate is indicated by arrows and the arrowheads point at expression in the notochord.
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Related In: Results  -  Collection

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Figure 3: Functional assay of Latimeria ar-A, ar-B, and ar-D enhancers in zebrafish. Two fluorescent images are shown for each embryo. Low magnification displays the whole embryo and high magnification focuses on the trunk above the yolk extension. Schematic representations of the injected zebrafish (z) and Latimeria (l) promoter (pr) and enhancer reporter constructs are shown on the left side of each panel (a-d) VISTA plot comparisons of the zebrafish and Latimeria enhancer regions are shown below reporter constructs and indicate the degree of conservation. Conservation identity greater than 70% is highlighted in color. (a) Embryo injected with control construct containing the 0.8 kb (form the transcriptional start site) zebrafish shh promoter, linked to GFP. (b-d) Embryos injected with reporter constructs containing the minimal zebrafish shh promoter and one of the Latimeria shh enhancers ar-D (b) ar-A (c) and ar-B (d). GFP expression in the floorplate is indicated by arrows and the arrowheads point at expression in the notochord.
Mentions: To check whether the Latimeria conserved noncoding sequences had enhancer activity, reporter gene expression analysis was conducted in transient transgenic zebrafish embryos. This analysis had already been carried out with Latimeria ar-C in a previous study [34], and the aim here was to analyze other conserved regulatory regions that potentially drive shh midline expression. Conserved noncoding elements of Latimeria shh intron 1 located between exon 1 and the intronic enhancer ar-A did not drive specific reporter gene expression (data not shown). Latimeria putative enhancer orthologs ar-D, ar-A and ar-B were cloned into the above-mentioned shh minimal promoter constructs, containing GFP (Green Fluorescent Protein) instead of a LacZ reporter. Transient mosaic expression of GFP was measured as read out of reporter construct activity 24 hours after injection of zebrafish zygotes (Figure 3). The reporter expression directed by the zebrafish shh upstream region resembled the tissue-specific expression of the isolated ar-D enhancer (Additional file 5). GFP expression was observed in the ventral brain and in the anterior parts of the floorplate (data not shown) [33,40]. Similarly, Latimeria ar-D also directed floorplate-specific GFP expression (Figure 3b). However, the reporter expression was extended to the posterior parts of the floorplate. Among GFP-expressing embryos, fluorescence in the posterior floorplate cells (posterior to the start of the yolk extension) was detected in only 4% of specimens with the zebrafish upstream region but in 75% of corresponding embryos with Latimeria ar-D (Table 2). These results indicate that the CNE in the Latimeria upstream region is a functional midline enhancer which has similar but not identical activity in zebrafish to the zebrafish ar-D enhancer. On the basis of our DNA sequence comparison, Latimeria ar-D was found to contain sarcopterygian specific sequences with a puative FoxA2 element (92% match). Potentially, these elements can account for the posteriorly extended expression direction of Latimeria ar-D in the ventral neural tube of zebrafish.

Bottom Line: The functionality of the putative Latimeria enhancers was confirmed by reporter gene expression analysis in transient transgenic zebrafish and chick embryos.In contrast to lineage-specific losses and differentiations in more derived lineages, Latimeria shh enhancers reveal low levels of sequence diversification.High overall sequence conservation of shh conserved noncoding elements (CNE) is consistent with the general trend of high levels of conservation of noncoding DNA in the slowly evolving Latimeria genome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Konstanz, 78457 Konstanz, Germany. f.mueller@bham.ac.uk.

ABSTRACT

Background: The modern coelacanth (Latimeria) is the extant taxon of a basal sarcopterygian lineage and sister group to tetrapods. Apart from certain apomorphic traits, its morphology is characterized by a high degree of retention of ancestral vertebrate structures and little morphological change. An insight into the molecular evolution that may explain the unchanged character of Latimeria morphology requires the analysis of the expression patterns of developmental regulator genes and their cis-regulatory modules (CRMs).

Results: We describe the comparative and functional analysis of the sonic hedgehog (shh) genomic region of Latimeria menadoensis. Several putative enhancers in the Latimeria shh locus have been identified by comparisons to sarcopterygian and actinopterygian extant species. Specific sequence conservation with all known actinopterygian enhancer elements has been detected. However, these elements are selectively missing in more recently diverged actinopterygian and sarcopterygian species. The functionality of the putative Latimeria enhancers was confirmed by reporter gene expression analysis in transient transgenic zebrafish and chick embryos.

Conclusions: Latimeria shh CRMs represent the ancestral set of enhancers that have emerged before the split of lobe-finned and ray-finned fishes. In contrast to lineage-specific losses and differentiations in more derived lineages, Latimeria shh enhancers reveal low levels of sequence diversification. High overall sequence conservation of shh conserved noncoding elements (CNE) is consistent with the general trend of high levels of conservation of noncoding DNA in the slowly evolving Latimeria genome.

No MeSH data available.