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Human intronic enhancers control distinct sub-domains of Gli3 expression during mouse CNS and limb development.

Abbasi AA, Paparidis Z, Malik S, Bangs F, Schmidt A, Koch S, Lopez-Rios J, Grzeschik KH - BMC Dev. Biol. (2010)

Bottom Line: Limb bud specificity is also found in chicken but had not been detected in zebrafish embryos.Even though fish, birds, and mammals share an ancient repertoire of gene regulatory elements within Gli3, the functions of individual enhancers from this catalog have diverged significantly.These results not only demonstrate the high level of complexity in the genetic mechanisms controlling Gli3 expression, but also reveal the evolutionary significance of cis-acting regulatory networks of early developmental regulators in vertebrates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Human Genetics, Philipps-Universit├Ąt Marburg, 35037 Marburg, Germany. abbasiam@qau.edu.pk

ABSTRACT

Background: The zinc-finger transcription factor GLI3 is an important mediator of Sonic hedgehog signaling and crucial for patterning of many aspects of the vertebrate body plan. In vertebrates, the mechanism of SHH signal transduction and its action on target genes by means of activating or repressing forms of GLI3 have been studied most extensively during limb development and the specification of the central nervous system. From these studies it has emerged, that Gli3 expression must be subject to a tight spatiotemporal regulation. However, the genetic mechanisms and the cis-acting elements controlling the expression of Gli3 remained largely unknown.

Results: Here, we demonstrate in chicken and mouse transgenic embryos that human GLI3-intronic conserved non-coding sequence elements (CNEs) autonomously control individual aspects of Gli3 expression. Their combined action shows many aspects of a Gli3-specific pattern of transcriptional activity. In the mouse limb bud, different CNEs enhance Gli3-specific expression in evolutionary ancient stylopod and zeugopod versus modern skeletal structures of the autopod. Limb bud specificity is also found in chicken but had not been detected in zebrafish embryos. Three of these elements govern central nervous system specific gene expression during mouse embryogenesis, each targeting a subset of endogenous Gli3 transcription sites. Even though fish, birds, and mammals share an ancient repertoire of gene regulatory elements within Gli3, the functions of individual enhancers from this catalog have diverged significantly. During evolution, ancient broad-range regulatory elements within Gli3 attained higher specificity, critical for patterning of more specialized structures, by abolishing the potential for redundant expression control.

Conclusion: These results not only demonstrate the high level of complexity in the genetic mechanisms controlling Gli3 expression, but also reveal the evolutionary significance of cis-acting regulatory networks of early developmental regulators in vertebrates.

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GLI3 specific, spatiotemporal pattern of reporter gene expression in transgenic mouse embryos is evoked by intronic conserved non-coding sequence elements (CNEs) acting in a complementary fashion. (A) Human chromosome 7 coordinates along with the graphical representation of exons (numbered) and introns of GLI3. (B) Graphical plot depicting evolutionary conservation of human GLI3 across multiple mammalian vertebrates (blue) and Fugu (green) generated using sequence alignment tools in the UCSC comparative genomics alignment pipeline. http://genome.ucsc.edu. (C) 1-12: Human/Fugu CNEs characterized as enhancers through functional assays by employing human cell lines and zebrafish embryos [26,28]. A, R: activating or repressory potential of these enhancers in cell culture. (D) Subset of 6 intronic CNEs whose spatiotemporal regulatory potential is defined in transgenic mouse assay. Selected embryos are shown at representative time points (E11.5 or E12.5) along with their primary target sites.
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Figure 1: GLI3 specific, spatiotemporal pattern of reporter gene expression in transgenic mouse embryos is evoked by intronic conserved non-coding sequence elements (CNEs) acting in a complementary fashion. (A) Human chromosome 7 coordinates along with the graphical representation of exons (numbered) and introns of GLI3. (B) Graphical plot depicting evolutionary conservation of human GLI3 across multiple mammalian vertebrates (blue) and Fugu (green) generated using sequence alignment tools in the UCSC comparative genomics alignment pipeline. http://genome.ucsc.edu. (C) 1-12: Human/Fugu CNEs characterized as enhancers through functional assays by employing human cell lines and zebrafish embryos [26,28]. A, R: activating or repressory potential of these enhancers in cell culture. (D) Subset of 6 intronic CNEs whose spatiotemporal regulatory potential is defined in transgenic mouse assay. Selected embryos are shown at representative time points (E11.5 or E12.5) along with their primary target sites.

Mentions: Multi-species alignment of human GLI3 genomic sequence with orthologous intervals from other vertebrate species localized 12 intronic conserved non-coding elements, showing at least 50% identity over a 60 bp window down to Fugu. These elements are distributed across almost the entire GLI3 interval (Figure 1A and 1B), with 2 elements in each of introns 2, 3, 4, and 10 and one in each of introns 1, 6, and 13 [25]. The GLI3 specific gene regulatory functions of 11 of these putative human enhancers had previously been determined using human cell lines (Figure 1C). The elements which could activate reporter gene expression in cell cultures functioned likewise in zebrafish embryos [25]. Additionally, the spatiotemporal aspects of one ultraconserved element, CNE2, were analyzed in mouse embryos [26]. However, the spatiotemporal functionality of other GLI3 associated enhancers in a mammalian model remained to be defined.


Human intronic enhancers control distinct sub-domains of Gli3 expression during mouse CNS and limb development.

Abbasi AA, Paparidis Z, Malik S, Bangs F, Schmidt A, Koch S, Lopez-Rios J, Grzeschik KH - BMC Dev. Biol. (2010)

GLI3 specific, spatiotemporal pattern of reporter gene expression in transgenic mouse embryos is evoked by intronic conserved non-coding sequence elements (CNEs) acting in a complementary fashion. (A) Human chromosome 7 coordinates along with the graphical representation of exons (numbered) and introns of GLI3. (B) Graphical plot depicting evolutionary conservation of human GLI3 across multiple mammalian vertebrates (blue) and Fugu (green) generated using sequence alignment tools in the UCSC comparative genomics alignment pipeline. http://genome.ucsc.edu. (C) 1-12: Human/Fugu CNEs characterized as enhancers through functional assays by employing human cell lines and zebrafish embryos [26,28]. A, R: activating or repressory potential of these enhancers in cell culture. (D) Subset of 6 intronic CNEs whose spatiotemporal regulatory potential is defined in transgenic mouse assay. Selected embryos are shown at representative time points (E11.5 or E12.5) along with their primary target sites.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875213&req=5

Figure 1: GLI3 specific, spatiotemporal pattern of reporter gene expression in transgenic mouse embryos is evoked by intronic conserved non-coding sequence elements (CNEs) acting in a complementary fashion. (A) Human chromosome 7 coordinates along with the graphical representation of exons (numbered) and introns of GLI3. (B) Graphical plot depicting evolutionary conservation of human GLI3 across multiple mammalian vertebrates (blue) and Fugu (green) generated using sequence alignment tools in the UCSC comparative genomics alignment pipeline. http://genome.ucsc.edu. (C) 1-12: Human/Fugu CNEs characterized as enhancers through functional assays by employing human cell lines and zebrafish embryos [26,28]. A, R: activating or repressory potential of these enhancers in cell culture. (D) Subset of 6 intronic CNEs whose spatiotemporal regulatory potential is defined in transgenic mouse assay. Selected embryos are shown at representative time points (E11.5 or E12.5) along with their primary target sites.
Mentions: Multi-species alignment of human GLI3 genomic sequence with orthologous intervals from other vertebrate species localized 12 intronic conserved non-coding elements, showing at least 50% identity over a 60 bp window down to Fugu. These elements are distributed across almost the entire GLI3 interval (Figure 1A and 1B), with 2 elements in each of introns 2, 3, 4, and 10 and one in each of introns 1, 6, and 13 [25]. The GLI3 specific gene regulatory functions of 11 of these putative human enhancers had previously been determined using human cell lines (Figure 1C). The elements which could activate reporter gene expression in cell cultures functioned likewise in zebrafish embryos [25]. Additionally, the spatiotemporal aspects of one ultraconserved element, CNE2, were analyzed in mouse embryos [26]. However, the spatiotemporal functionality of other GLI3 associated enhancers in a mammalian model remained to be defined.

Bottom Line: Limb bud specificity is also found in chicken but had not been detected in zebrafish embryos.Even though fish, birds, and mammals share an ancient repertoire of gene regulatory elements within Gli3, the functions of individual enhancers from this catalog have diverged significantly.These results not only demonstrate the high level of complexity in the genetic mechanisms controlling Gli3 expression, but also reveal the evolutionary significance of cis-acting regulatory networks of early developmental regulators in vertebrates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Human Genetics, Philipps-Universit├Ąt Marburg, 35037 Marburg, Germany. abbasiam@qau.edu.pk

ABSTRACT

Background: The zinc-finger transcription factor GLI3 is an important mediator of Sonic hedgehog signaling and crucial for patterning of many aspects of the vertebrate body plan. In vertebrates, the mechanism of SHH signal transduction and its action on target genes by means of activating or repressing forms of GLI3 have been studied most extensively during limb development and the specification of the central nervous system. From these studies it has emerged, that Gli3 expression must be subject to a tight spatiotemporal regulation. However, the genetic mechanisms and the cis-acting elements controlling the expression of Gli3 remained largely unknown.

Results: Here, we demonstrate in chicken and mouse transgenic embryos that human GLI3-intronic conserved non-coding sequence elements (CNEs) autonomously control individual aspects of Gli3 expression. Their combined action shows many aspects of a Gli3-specific pattern of transcriptional activity. In the mouse limb bud, different CNEs enhance Gli3-specific expression in evolutionary ancient stylopod and zeugopod versus modern skeletal structures of the autopod. Limb bud specificity is also found in chicken but had not been detected in zebrafish embryos. Three of these elements govern central nervous system specific gene expression during mouse embryogenesis, each targeting a subset of endogenous Gli3 transcription sites. Even though fish, birds, and mammals share an ancient repertoire of gene regulatory elements within Gli3, the functions of individual enhancers from this catalog have diverged significantly. During evolution, ancient broad-range regulatory elements within Gli3 attained higher specificity, critical for patterning of more specialized structures, by abolishing the potential for redundant expression control.

Conclusion: These results not only demonstrate the high level of complexity in the genetic mechanisms controlling Gli3 expression, but also reveal the evolutionary significance of cis-acting regulatory networks of early developmental regulators in vertebrates.

Show MeSH