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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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Inhibition of Fgf signaling causes expansion of sox3 and sox19a expression into the vegetal margin.In situ hybridization for sox3 (A–D), sox19a (E–H) or ntl (I–L). Expression of sox3 and sox19a (dark purple) is seen throughout the entire animal hemisphere at the 30% epiboly stage, when only a very thin band of ntl expression was seen around the vegental margin (I). Although expression of ntl was completely lost following treatment with SU5402 (but not in the DMSO control) (I,J), it was not possible to detect any change in expression of sox3 or sox19a (A,B,E,F). However, treatment with SU5402 also resulted in loss of expression of ntl by the 60% epiboly stage (K,L) (position of vegetal margin identified with red bars), which was concomitant with expansion of the expression of sox3 and sox19a towards the vegetal margin (D,H) (region of expansion of expression shown with red brackets in panels C,D,G,H). Close analysis of the dorsal organizer region revealed that, as in untreated embryos (M), sox3 expression is absent from the region of the organizer. Treatment with SU5402 (or DMSO as a control) did not alter the exclusion of sox3 expression from this region (N,O). The expression of boz (dark purple) also remained unaltered with a gap in between the domains of sox3 and boz expression maintained (P,Q; arrow). All treatments were started at 3 hpf (the time that zygotic expression begins). All panels are lateral views, dorsal to the right except lower panels of M–Q, which are animal pole views. The proportion of embryos exhibiting these phenotypes is shown at the bottom right of each panel.
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pone-0057698-g006: Inhibition of Fgf signaling causes expansion of sox3 and sox19a expression into the vegetal margin.In situ hybridization for sox3 (A–D), sox19a (E–H) or ntl (I–L). Expression of sox3 and sox19a (dark purple) is seen throughout the entire animal hemisphere at the 30% epiboly stage, when only a very thin band of ntl expression was seen around the vegental margin (I). Although expression of ntl was completely lost following treatment with SU5402 (but not in the DMSO control) (I,J), it was not possible to detect any change in expression of sox3 or sox19a (A,B,E,F). However, treatment with SU5402 also resulted in loss of expression of ntl by the 60% epiboly stage (K,L) (position of vegetal margin identified with red bars), which was concomitant with expansion of the expression of sox3 and sox19a towards the vegetal margin (D,H) (region of expansion of expression shown with red brackets in panels C,D,G,H). Close analysis of the dorsal organizer region revealed that, as in untreated embryos (M), sox3 expression is absent from the region of the organizer. Treatment with SU5402 (or DMSO as a control) did not alter the exclusion of sox3 expression from this region (N,O). The expression of boz (dark purple) also remained unaltered with a gap in between the domains of sox3 and boz expression maintained (P,Q; arrow). All treatments were started at 3 hpf (the time that zygotic expression begins). All panels are lateral views, dorsal to the right except lower panels of M–Q, which are animal pole views. The proportion of embryos exhibiting these phenotypes is shown at the bottom right of each panel.

Mentions: Closer analysis of SU5402-treated embryos at 60% epiboly revealed that, rather than losing expression, the domain of sox3 and sox19a expression was expanded towards the vegetal margin (See Fig. S10A,B in the supplementary material; Fig. 6A–H), which was coincident with loss of expression of the mesoderm marker, ntl (Fig. 6A–H) (as described previously by Rodaway et al. (1999) [21] using a dominant-negative FGF). We also noted that inhibition of Fgf signaling resulted in stronger expression of sox3 and sox19a (but not sox19b) throughout the embryo at 30% epiboly (Fig. 6A,C, Fig. S10 in the supplementary material). sox19b expression was already weak by this stage so it was not possible to be certain if a similar expansion occurred.


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)

Inhibition of Fgf signaling causes expansion of sox3 and sox19a expression into the vegetal margin.In situ hybridization for sox3 (A–D), sox19a (E–H) or ntl (I–L). Expression of sox3 and sox19a (dark purple) is seen throughout the entire animal hemisphere at the 30% epiboly stage, when only a very thin band of ntl expression was seen around the vegental margin (I). Although expression of ntl was completely lost following treatment with SU5402 (but not in the DMSO control) (I,J), it was not possible to detect any change in expression of sox3 or sox19a (A,B,E,F). However, treatment with SU5402 also resulted in loss of expression of ntl by the 60% epiboly stage (K,L) (position of vegetal margin identified with red bars), which was concomitant with expansion of the expression of sox3 and sox19a towards the vegetal margin (D,H) (region of expansion of expression shown with red brackets in panels C,D,G,H). Close analysis of the dorsal organizer region revealed that, as in untreated embryos (M), sox3 expression is absent from the region of the organizer. Treatment with SU5402 (or DMSO as a control) did not alter the exclusion of sox3 expression from this region (N,O). The expression of boz (dark purple) also remained unaltered with a gap in between the domains of sox3 and boz expression maintained (P,Q; arrow). All treatments were started at 3 hpf (the time that zygotic expression begins). All panels are lateral views, dorsal to the right except lower panels of M–Q, which are animal pole views. The proportion of embryos exhibiting these phenotypes is shown at the bottom right of each panel.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585176&req=5

pone-0057698-g006: Inhibition of Fgf signaling causes expansion of sox3 and sox19a expression into the vegetal margin.In situ hybridization for sox3 (A–D), sox19a (E–H) or ntl (I–L). Expression of sox3 and sox19a (dark purple) is seen throughout the entire animal hemisphere at the 30% epiboly stage, when only a very thin band of ntl expression was seen around the vegental margin (I). Although expression of ntl was completely lost following treatment with SU5402 (but not in the DMSO control) (I,J), it was not possible to detect any change in expression of sox3 or sox19a (A,B,E,F). However, treatment with SU5402 also resulted in loss of expression of ntl by the 60% epiboly stage (K,L) (position of vegetal margin identified with red bars), which was concomitant with expansion of the expression of sox3 and sox19a towards the vegetal margin (D,H) (region of expansion of expression shown with red brackets in panels C,D,G,H). Close analysis of the dorsal organizer region revealed that, as in untreated embryos (M), sox3 expression is absent from the region of the organizer. Treatment with SU5402 (or DMSO as a control) did not alter the exclusion of sox3 expression from this region (N,O). The expression of boz (dark purple) also remained unaltered with a gap in between the domains of sox3 and boz expression maintained (P,Q; arrow). All treatments were started at 3 hpf (the time that zygotic expression begins). All panels are lateral views, dorsal to the right except lower panels of M–Q, which are animal pole views. The proportion of embryos exhibiting these phenotypes is shown at the bottom right of each panel.
Mentions: Closer analysis of SU5402-treated embryos at 60% epiboly revealed that, rather than losing expression, the domain of sox3 and sox19a expression was expanded towards the vegetal margin (See Fig. S10A,B in the supplementary material; Fig. 6A–H), which was coincident with loss of expression of the mesoderm marker, ntl (Fig. 6A–H) (as described previously by Rodaway et al. (1999) [21] using a dominant-negative FGF). We also noted that inhibition of Fgf signaling resulted in stronger expression of sox3 and sox19a (but not sox19b) throughout the embryo at 30% epiboly (Fig. 6A,C, Fig. S10 in the supplementary material). sox19b expression was already weak by this stage so it was not possible to be certain if a similar expansion occurred.

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