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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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CNE1 and CNE9 govern lacZ expression along distinct domains of brain and spinal cord. (A-C) Whole mount views of transgenic mouse embryos expressing the reporter under control of CNE1 at E11.5 (A, B), and E12.5 (C). (F-H) Whole mount views of embryos carrying CNE9 as enhancer of lacZ expression at E11.5 (F-G), and E12.5 (H). (D, I) Transverse sections through the midbrain at the level shown with dotted lines in panels (C) and (H). (D) In the roofplate and dorsolateral part of alar-column of midbrain the CNE1-induced expression is apparent in marginal, mantle, and ependymal layers of neuroepithelium, whereas in medial section of alar- plate/entire basal-plate of midbrain, expression is restricted to the marginal layer. (I) CNE9-driven lacZ expression is present in ventral midline of caudal midbrain, whereas dorsally reporter signal is confined to the dorso-lateral marginal tissue. (E, J) Transverse sections through the spinal cord at the levels shown with dotted lines in the panels (C) and (F). (E) CNE1-generated transgene expression in the spinal cord is confined to the roofplate (RP), progenitors of Dp5, Dp6, Vp0, Vp1 interneurons, and progenitors of V3 interneurons (open arrowheads). (J) CNE9-induced lacZ expression in the spinal cord was present up-to embryonic day E11.5 and was confined to progenitors of motor neurons (pMN). amb, anterior midbrain; drg, dorsal root ganglia; dsnt, dorsal neural tube; hb, hindbrain; di, diencephalon; mb, midbrain; mge, medial ganglionic eminence; mhb, midbrain-hindbrain boundary; sc, spinal cord; tel, telencephalon. MV, mesencephalic vesicle; AP, alar-plate; BP, basal-plate; FP, floorplate.
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Figure 5: CNE1 and CNE9 govern lacZ expression along distinct domains of brain and spinal cord. (A-C) Whole mount views of transgenic mouse embryos expressing the reporter under control of CNE1 at E11.5 (A, B), and E12.5 (C). (F-H) Whole mount views of embryos carrying CNE9 as enhancer of lacZ expression at E11.5 (F-G), and E12.5 (H). (D, I) Transverse sections through the midbrain at the level shown with dotted lines in panels (C) and (H). (D) In the roofplate and dorsolateral part of alar-column of midbrain the CNE1-induced expression is apparent in marginal, mantle, and ependymal layers of neuroepithelium, whereas in medial section of alar- plate/entire basal-plate of midbrain, expression is restricted to the marginal layer. (I) CNE9-driven lacZ expression is present in ventral midline of caudal midbrain, whereas dorsally reporter signal is confined to the dorso-lateral marginal tissue. (E, J) Transverse sections through the spinal cord at the levels shown with dotted lines in the panels (C) and (F). (E) CNE1-generated transgene expression in the spinal cord is confined to the roofplate (RP), progenitors of Dp5, Dp6, Vp0, Vp1 interneurons, and progenitors of V3 interneurons (open arrowheads). (J) CNE9-induced lacZ expression in the spinal cord was present up-to embryonic day E11.5 and was confined to progenitors of motor neurons (pMN). amb, anterior midbrain; drg, dorsal root ganglia; dsnt, dorsal neural tube; hb, hindbrain; di, diencephalon; mb, midbrain; mge, medial ganglionic eminence; mhb, midbrain-hindbrain boundary; sc, spinal cord; tel, telencephalon. MV, mesencephalic vesicle; AP, alar-plate; BP, basal-plate; FP, floorplate.

Mentions: The spatiotemporal activities of CNE1, CNE2, and CNE9 complemented each other in the control of reporter expression reflecting part of the GLI3-specific pattern in the brain, spinal cord and craniofacial structures (Figure 1D, Figure 5). The mouse embryo expression patterns governed at E11.5 by CNE1 and CNE2, respectively, are independently reported in the Vista enhancer browser for sequence elements 1213 and 111 which include the sequences employed here http://enhancer.lbl.gov, adding credibility to the notion that the CNEs studied are bona fide GLI3 enhancers.


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)

CNE1 and CNE9 govern lacZ expression along distinct domains of brain and spinal cord. (A-C) Whole mount views of transgenic mouse embryos expressing the reporter under control of CNE1 at E11.5 (A, B), and E12.5 (C). (F-H) Whole mount views of embryos carrying CNE9 as enhancer of lacZ expression at E11.5 (F-G), and E12.5 (H). (D, I) Transverse sections through the midbrain at the level shown with dotted lines in panels (C) and (H). (D) In the roofplate and dorsolateral part of alar-column of midbrain the CNE1-induced expression is apparent in marginal, mantle, and ependymal layers of neuroepithelium, whereas in medial section of alar- plate/entire basal-plate of midbrain, expression is restricted to the marginal layer. (I) CNE9-driven lacZ expression is present in ventral midline of caudal midbrain, whereas dorsally reporter signal is confined to the dorso-lateral marginal tissue. (E, J) Transverse sections through the spinal cord at the levels shown with dotted lines in the panels (C) and (F). (E) CNE1-generated transgene expression in the spinal cord is confined to the roofplate (RP), progenitors of Dp5, Dp6, Vp0, Vp1 interneurons, and progenitors of V3 interneurons (open arrowheads). (J) CNE9-induced lacZ expression in the spinal cord was present up-to embryonic day E11.5 and was confined to progenitors of motor neurons (pMN). amb, anterior midbrain; drg, dorsal root ganglia; dsnt, dorsal neural tube; hb, hindbrain; di, diencephalon; mb, midbrain; mge, medial ganglionic eminence; mhb, midbrain-hindbrain boundary; sc, spinal cord; tel, telencephalon. MV, mesencephalic vesicle; AP, alar-plate; BP, basal-plate; FP, floorplate.
© Copyright Policy - open-access
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Figure 5: CNE1 and CNE9 govern lacZ expression along distinct domains of brain and spinal cord. (A-C) Whole mount views of transgenic mouse embryos expressing the reporter under control of CNE1 at E11.5 (A, B), and E12.5 (C). (F-H) Whole mount views of embryos carrying CNE9 as enhancer of lacZ expression at E11.5 (F-G), and E12.5 (H). (D, I) Transverse sections through the midbrain at the level shown with dotted lines in panels (C) and (H). (D) In the roofplate and dorsolateral part of alar-column of midbrain the CNE1-induced expression is apparent in marginal, mantle, and ependymal layers of neuroepithelium, whereas in medial section of alar- plate/entire basal-plate of midbrain, expression is restricted to the marginal layer. (I) CNE9-driven lacZ expression is present in ventral midline of caudal midbrain, whereas dorsally reporter signal is confined to the dorso-lateral marginal tissue. (E, J) Transverse sections through the spinal cord at the levels shown with dotted lines in the panels (C) and (F). (E) CNE1-generated transgene expression in the spinal cord is confined to the roofplate (RP), progenitors of Dp5, Dp6, Vp0, Vp1 interneurons, and progenitors of V3 interneurons (open arrowheads). (J) CNE9-induced lacZ expression in the spinal cord was present up-to embryonic day E11.5 and was confined to progenitors of motor neurons (pMN). amb, anterior midbrain; drg, dorsal root ganglia; dsnt, dorsal neural tube; hb, hindbrain; di, diencephalon; mb, midbrain; mge, medial ganglionic eminence; mhb, midbrain-hindbrain boundary; sc, spinal cord; tel, telencephalon. MV, mesencephalic vesicle; AP, alar-plate; BP, basal-plate; FP, floorplate.
Mentions: The spatiotemporal activities of CNE1, CNE2, and CNE9 complemented each other in the control of reporter expression reflecting part of the GLI3-specific pattern in the brain, spinal cord and craniofacial structures (Figure 1D, Figure 5). The mouse embryo expression patterns governed at E11.5 by CNE1 and CNE2, respectively, are independently reported in the Vista enhancer browser for sequence elements 1213 and 111 which include the sequences employed here http://enhancer.lbl.gov, adding credibility to the notion that the CNEs studied are bona fide GLI3 enhancers.

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
Related in: MedlinePlus