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Formation of the embryonic organizer is restricted by the competitive influences of Fgf signaling and the SoxB1 transcription factors.

Kuo CL, Lam CM, Hewitt JE, Scotting PJ - PLoS ONE (2013)

Bottom Line: We show that ectopic expression of organizer genes induced solely by the inhibition of SoxB1 function is dependent upon the activation of fgf expression.These data allow us to describe a comprehensive signaling network in which the SoxB1 factors restrict organizer formation by inhibiting Fgf, Nodal and Wnt signaling, as well as independently repressing the targets of that signaling.The organizer therefore forms only where Nodal-induced Fgf signaling overlaps with Wnt signaling and the SoxB1 proteins are absent.

View Article: PubMed Central - PubMed

Affiliation: Centre for Genetics and Genomics, School of Biology, University of Nottingham, QMC, Nottingham, United Kingdom.

ABSTRACT
The organizer is one of the earliest structures to be established during vertebrate development and is crucial to subsequent patterning of the embryo. We have previously shown that the SoxB1 transcription factor, Sox3, plays a central role as a transcriptional repressor of zebrafish organizer gene expression. Recent data suggest that Fgf signaling has a positive influence on organizer formation, but its role remains to be fully elucidated. In order to better understand how Fgf signaling fits into the complex regulatory network that determines when and where the organizer forms, the relationship between the positive effects of Fgf signaling and the repressive effects of the SoxB1 factors must be resolved. This study demonstrates that both fgf3 and fgf8 are required for expression of the organizer genes, gsc and chd, and that SoxB1 factors (Sox3, and the zebrafish specific factors, Sox19a and Sox19b) can repress the expression of both fgf3 and fgf8. However, we also find that these SoxB1 factors inhibit the expression of gsc and chd independently of their repression of fgf expression. We show that ectopic expression of organizer genes induced solely by the inhibition of SoxB1 function is dependent upon the activation of fgf expression. These data allow us to describe a comprehensive signaling network in which the SoxB1 factors restrict organizer formation by inhibiting Fgf, Nodal and Wnt signaling, as well as independently repressing the targets of that signaling. The organizer therefore forms only where Nodal-induced Fgf signaling overlaps with Wnt signaling and the SoxB1 proteins are absent.

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Sox3 can directly bind to a region upstream of fgf3.(A) Synteny in the region of the genome flanking the fgf3 gene. Coloured boxes indicate different genes and direction of transcription. Not to scale. Absence of line indicates incomplete genomic scaffold information. (B) Diagram showing the upstream region of fgf3. Green bars indicate regions of homology among different species. The position of potential Sox binding sites (A/T)(A/T)CAA(A/T)G within these homologous regions are shown as black bars and similar potential Sox binding sites lacking the final 3′ “G” are shown as gray bars. The red bars show the PCR products, including the Sox binding sites that would be produced by different primer pairs. (C) 25 pg sox3-HA and sox3N40I-HA RNA were injected at the 1–2 cell stage embryos and harvested at 4.5 hpf. ChIP analysis using an anti-HA antibody to precipitate Sox3 and bound DNA. PCR results after ChIP procedure showed that the DNA fragments pulled down by Sox3-HA can be amplified only by primer pair 2. tubb5 was included as a negative control. (D) Quantitative PCR results of precipitated chromatin using primer pairs 2 and 3 showed that the target sequence for primer pair 2 was significantly enriched following IP of WT Sox3 whereas the target for primer pair 3 was not. Values represented as fold change compared to the uninjected value.
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pone-0057698-g002: Sox3 can directly bind to a region upstream of fgf3.(A) Synteny in the region of the genome flanking the fgf3 gene. Coloured boxes indicate different genes and direction of transcription. Not to scale. Absence of line indicates incomplete genomic scaffold information. (B) Diagram showing the upstream region of fgf3. Green bars indicate regions of homology among different species. The position of potential Sox binding sites (A/T)(A/T)CAA(A/T)G within these homologous regions are shown as black bars and similar potential Sox binding sites lacking the final 3′ “G” are shown as gray bars. The red bars show the PCR products, including the Sox binding sites that would be produced by different primer pairs. (C) 25 pg sox3-HA and sox3N40I-HA RNA were injected at the 1–2 cell stage embryos and harvested at 4.5 hpf. ChIP analysis using an anti-HA antibody to precipitate Sox3 and bound DNA. PCR results after ChIP procedure showed that the DNA fragments pulled down by Sox3-HA can be amplified only by primer pair 2. tubb5 was included as a negative control. (D) Quantitative PCR results of precipitated chromatin using primer pairs 2 and 3 showed that the target sequence for primer pair 2 was significantly enriched following IP of WT Sox3 whereas the target for primer pair 3 was not. Values represented as fold change compared to the uninjected value.

Mentions: In order to investigate whether fgfs could be direct targets of the SoxB1 transcription factors, we analysed binding of Sox3 to the fgf3 promoter using ChIP-PCR. Comparison of genome organization between species in the region of fgf3 using the ENSEMBL database showed that a significant degree of synteny had been retained across a wide diversity of species from coelacanth to human, including zebrafish (Fig. 2A). This allowed us to identify regions around the fgf3 transcription unit suitable for comparison to find conserved potential regulatory sequences. Previous studies have found that highly conserved non-coding sequences 5′ to a gene often harbour functional transcription factor binding sites [12]. We therefore used PipMaker [13] to indentify such regions of conservation. This identified two regions (regions ‘C’ and ‘D’ in Fig. 2B) that were highly conserved between fish, birds, lizard, frogs, platypus and opposum and a third region only conserved among fish (region ‘B’ in Fig. 2B) within 4 kb of the fgf3 transcription start site (TSS). A fourth region (region ‘A’ in Fig. 2B) positioned approximately 23 Kb upstream of the TSS, was also highly conserved including in mouse and human (see Fig. S3 in the supplementary material). All of these regions contained potential Sox-binding sites (Fig. 2B and see Fig. S4 in the supplementary material for full sequence comparisons). We therefore designed primers to detect all of these regions for use in ChIP-PCR experiments (as labelled in Fig. 2B).


Formation of the embryonic organizer is restricted by the competitive influences of Fgf signaling and the SoxB1 transcription factors.

Kuo CL, Lam CM, Hewitt JE, Scotting PJ - PLoS ONE (2013)

Sox3 can directly bind to a region upstream of fgf3.(A) Synteny in the region of the genome flanking the fgf3 gene. Coloured boxes indicate different genes and direction of transcription. Not to scale. Absence of line indicates incomplete genomic scaffold information. (B) Diagram showing the upstream region of fgf3. Green bars indicate regions of homology among different species. The position of potential Sox binding sites (A/T)(A/T)CAA(A/T)G within these homologous regions are shown as black bars and similar potential Sox binding sites lacking the final 3′ “G” are shown as gray bars. The red bars show the PCR products, including the Sox binding sites that would be produced by different primer pairs. (C) 25 pg sox3-HA and sox3N40I-HA RNA were injected at the 1–2 cell stage embryos and harvested at 4.5 hpf. ChIP analysis using an anti-HA antibody to precipitate Sox3 and bound DNA. PCR results after ChIP procedure showed that the DNA fragments pulled down by Sox3-HA can be amplified only by primer pair 2. tubb5 was included as a negative control. (D) Quantitative PCR results of precipitated chromatin using primer pairs 2 and 3 showed that the target sequence for primer pair 2 was significantly enriched following IP of WT Sox3 whereas the target for primer pair 3 was not. Values represented as fold change compared to the uninjected value.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585176&req=5

pone-0057698-g002: Sox3 can directly bind to a region upstream of fgf3.(A) Synteny in the region of the genome flanking the fgf3 gene. Coloured boxes indicate different genes and direction of transcription. Not to scale. Absence of line indicates incomplete genomic scaffold information. (B) Diagram showing the upstream region of fgf3. Green bars indicate regions of homology among different species. The position of potential Sox binding sites (A/T)(A/T)CAA(A/T)G within these homologous regions are shown as black bars and similar potential Sox binding sites lacking the final 3′ “G” are shown as gray bars. The red bars show the PCR products, including the Sox binding sites that would be produced by different primer pairs. (C) 25 pg sox3-HA and sox3N40I-HA RNA were injected at the 1–2 cell stage embryos and harvested at 4.5 hpf. ChIP analysis using an anti-HA antibody to precipitate Sox3 and bound DNA. PCR results after ChIP procedure showed that the DNA fragments pulled down by Sox3-HA can be amplified only by primer pair 2. tubb5 was included as a negative control. (D) Quantitative PCR results of precipitated chromatin using primer pairs 2 and 3 showed that the target sequence for primer pair 2 was significantly enriched following IP of WT Sox3 whereas the target for primer pair 3 was not. Values represented as fold change compared to the uninjected value.
Mentions: In order to investigate whether fgfs could be direct targets of the SoxB1 transcription factors, we analysed binding of Sox3 to the fgf3 promoter using ChIP-PCR. Comparison of genome organization between species in the region of fgf3 using the ENSEMBL database showed that a significant degree of synteny had been retained across a wide diversity of species from coelacanth to human, including zebrafish (Fig. 2A). This allowed us to identify regions around the fgf3 transcription unit suitable for comparison to find conserved potential regulatory sequences. Previous studies have found that highly conserved non-coding sequences 5′ to a gene often harbour functional transcription factor binding sites [12]. We therefore used PipMaker [13] to indentify such regions of conservation. This identified two regions (regions ‘C’ and ‘D’ in Fig. 2B) that were highly conserved between fish, birds, lizard, frogs, platypus and opposum and a third region only conserved among fish (region ‘B’ in Fig. 2B) within 4 kb of the fgf3 transcription start site (TSS). A fourth region (region ‘A’ in Fig. 2B) positioned approximately 23 Kb upstream of the TSS, was also highly conserved including in mouse and human (see Fig. S3 in the supplementary material). All of these regions contained potential Sox-binding sites (Fig. 2B and see Fig. S4 in the supplementary material for full sequence comparisons). We therefore designed primers to detect all of these regions for use in ChIP-PCR experiments (as labelled in Fig. 2B).

Bottom Line: We show that ectopic expression of organizer genes induced solely by the inhibition of SoxB1 function is dependent upon the activation of fgf expression.These data allow us to describe a comprehensive signaling network in which the SoxB1 factors restrict organizer formation by inhibiting Fgf, Nodal and Wnt signaling, as well as independently repressing the targets of that signaling.The organizer therefore forms only where Nodal-induced Fgf signaling overlaps with Wnt signaling and the SoxB1 proteins are absent.

View Article: PubMed Central - PubMed

Affiliation: Centre for Genetics and Genomics, School of Biology, University of Nottingham, QMC, Nottingham, United Kingdom.

ABSTRACT
The organizer is one of the earliest structures to be established during vertebrate development and is crucial to subsequent patterning of the embryo. We have previously shown that the SoxB1 transcription factor, Sox3, plays a central role as a transcriptional repressor of zebrafish organizer gene expression. Recent data suggest that Fgf signaling has a positive influence on organizer formation, but its role remains to be fully elucidated. In order to better understand how Fgf signaling fits into the complex regulatory network that determines when and where the organizer forms, the relationship between the positive effects of Fgf signaling and the repressive effects of the SoxB1 factors must be resolved. This study demonstrates that both fgf3 and fgf8 are required for expression of the organizer genes, gsc and chd, and that SoxB1 factors (Sox3, and the zebrafish specific factors, Sox19a and Sox19b) can repress the expression of both fgf3 and fgf8. However, we also find that these SoxB1 factors inhibit the expression of gsc and chd independently of their repression of fgf expression. We show that ectopic expression of organizer genes induced solely by the inhibition of SoxB1 function is dependent upon the activation of fgf expression. These data allow us to describe a comprehensive signaling network in which the SoxB1 factors restrict organizer formation by inhibiting Fgf, Nodal and Wnt signaling, as well as independently repressing the targets of that signaling. The organizer therefore forms only where Nodal-induced Fgf signaling overlaps with Wnt signaling and the SoxB1 proteins are absent.

Show MeSH
Related in: MedlinePlus