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Genome wide analysis reveals Zic3 interaction with distal regulatory elements of stage specific developmental genes in zebrafish.

Winata CL, Kondrychyn I, Kumar V, Srinivasan KG, Orlov Y, Ravishankar A, Prabhakar S, Stanton LW, Korzh V, Mathavan S - PLoS Genet. (2013)

Bottom Line: Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray.Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers.This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.

View Article: PubMed Central - PubMed

Affiliation: Human Genetics, Genome Institute of Singapore, Singapore, Singapore.

ABSTRACT
Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.

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Zebrafish phenotype as a result of Zic3 knock-down.A, MF-20 antibody staining of heart, showing normal, bilateral, and reversal of looping at 48 hpf. Curvature of body axis and edema of the heart in Zic3 morphants at 96 hpf. B, percentage of embryos with heart laterality and axis curvature defects observed at 48 hpf in embryos injected with Zic3 MO alone, Zic3 MO and Zic3 mRNA, and Zic3 mRNA alone. C, expression levels of selected marker genes measured by qRT-PCR. Assay was performed in three independent groups of embryos injected with 3.4 ng of Zic3 morpholino. Marker genes oep through invs was assayed at 8 hpf, neurog1 at 10 hpf, and pax3 through her9 at 24 hpf.
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pgen-1003852-g004: Zebrafish phenotype as a result of Zic3 knock-down.A, MF-20 antibody staining of heart, showing normal, bilateral, and reversal of looping at 48 hpf. Curvature of body axis and edema of the heart in Zic3 morphants at 96 hpf. B, percentage of embryos with heart laterality and axis curvature defects observed at 48 hpf in embryos injected with Zic3 MO alone, Zic3 MO and Zic3 mRNA, and Zic3 mRNA alone. C, expression levels of selected marker genes measured by qRT-PCR. Assay was performed in three independent groups of embryos injected with 3.4 ng of Zic3 morpholino. Marker genes oep through invs was assayed at 8 hpf, neurog1 at 10 hpf, and pax3 through her9 at 24 hpf.

Mentions: To identify Zic3 target genes during gastrulation and early neural development, we profiled the transcriptome of 8 hpf embryos after Zic3 morpholino (MO)-mediated knockdown. Embryos injected with the same MO dosage as in Cast et al.[10] exhibited similar gastrulation and convergent extension (C-E) defects (data not shown). However, to minimize the detection of non-direct targets in microarray, we injected the embryos with a lower dose of MO (1.7 ng in our experiments versus 7.5 ng in [10]) which did not cause visible morphological defects during gastrulation (refer to Methods section), but affected heart laterality and caused curvature of the A-P axis at later stages (Fig. 4A). These phenotypes were rescued by co-injection with Zic3 mRNA which, when injected alone, had little effect (Fig. 4B). This confirmed the specificity of the phenotypes caused by Zic3 MO injection.


Genome wide analysis reveals Zic3 interaction with distal regulatory elements of stage specific developmental genes in zebrafish.

Winata CL, Kondrychyn I, Kumar V, Srinivasan KG, Orlov Y, Ravishankar A, Prabhakar S, Stanton LW, Korzh V, Mathavan S - PLoS Genet. (2013)

Zebrafish phenotype as a result of Zic3 knock-down.A, MF-20 antibody staining of heart, showing normal, bilateral, and reversal of looping at 48 hpf. Curvature of body axis and edema of the heart in Zic3 morphants at 96 hpf. B, percentage of embryos with heart laterality and axis curvature defects observed at 48 hpf in embryos injected with Zic3 MO alone, Zic3 MO and Zic3 mRNA, and Zic3 mRNA alone. C, expression levels of selected marker genes measured by qRT-PCR. Assay was performed in three independent groups of embryos injected with 3.4 ng of Zic3 morpholino. Marker genes oep through invs was assayed at 8 hpf, neurog1 at 10 hpf, and pax3 through her9 at 24 hpf.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3814314&req=5

pgen-1003852-g004: Zebrafish phenotype as a result of Zic3 knock-down.A, MF-20 antibody staining of heart, showing normal, bilateral, and reversal of looping at 48 hpf. Curvature of body axis and edema of the heart in Zic3 morphants at 96 hpf. B, percentage of embryos with heart laterality and axis curvature defects observed at 48 hpf in embryos injected with Zic3 MO alone, Zic3 MO and Zic3 mRNA, and Zic3 mRNA alone. C, expression levels of selected marker genes measured by qRT-PCR. Assay was performed in three independent groups of embryos injected with 3.4 ng of Zic3 morpholino. Marker genes oep through invs was assayed at 8 hpf, neurog1 at 10 hpf, and pax3 through her9 at 24 hpf.
Mentions: To identify Zic3 target genes during gastrulation and early neural development, we profiled the transcriptome of 8 hpf embryos after Zic3 morpholino (MO)-mediated knockdown. Embryos injected with the same MO dosage as in Cast et al.[10] exhibited similar gastrulation and convergent extension (C-E) defects (data not shown). However, to minimize the detection of non-direct targets in microarray, we injected the embryos with a lower dose of MO (1.7 ng in our experiments versus 7.5 ng in [10]) which did not cause visible morphological defects during gastrulation (refer to Methods section), but affected heart laterality and caused curvature of the A-P axis at later stages (Fig. 4A). These phenotypes were rescued by co-injection with Zic3 mRNA which, when injected alone, had little effect (Fig. 4B). This confirmed the specificity of the phenotypes caused by Zic3 MO injection.

Bottom Line: Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray.Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers.This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.

View Article: PubMed Central - PubMed

Affiliation: Human Genetics, Genome Institute of Singapore, Singapore, Singapore.

ABSTRACT
Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.

Show MeSH
Related in: MedlinePlus