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Distinct roles of Shh and Fgf signaling in regulating cell proliferation during zebrafish pectoral fin development.

Prykhozhij SV, Neumann CJ - BMC Dev. Biol. (2008)

Bottom Line: Correlating with this reduction, Fgf signaling is normal at early stages, but is later lost in shh mutants.Furthermore, pharmacological inhibition of Hh signaling for short periods has little effect on either Fgf signaling, or on expression of G1- and S-phase cell-cycle genes, whereas long periods of inhibition lead to the downregulation of both.The results presented here show that the role of Shh in this process is indirect, and is mediated by its effect on Fgf signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany. prykhozh@embl.de

ABSTRACT

Background: Cell proliferation in multicellular organisms must be coordinated with pattern formation. The major signaling pathways directing pattern formation in the vertebrate limb are well characterized, and we have therefore chosen this organ to examine the interaction between proliferation and patterning. Two important signals for limb development are members of the Hedgehog (Hh) and Fibroblast Growth Factor (Fgf) families of secreted signaling proteins. Sonic hedgehog (Shh) directs pattern formation along the anterior/posterior axis of the limb, whereas several Fgfs in combination direct pattern formation along the proximal/distal axis of the limb.

Results: We used the genetic and pharmacological amenability of the zebrafish model system to dissect the relative importance of Shh and Fgf signaling in regulating proliferation during development of the pectoral fin buds. In zebrafish mutants disrupting the shh gene, proliferation in the pectoral fin buds is initially normal, but later is strongly reduced. Correlating with this reduction, Fgf signaling is normal at early stages, but is later lost in shh mutants. Furthermore, pharmacological inhibition of Hh signaling for short periods has little effect on either Fgf signaling, or on expression of G1- and S-phase cell-cycle genes, whereas long periods of inhibition lead to the downregulation of both. In contrast, even short periods of pharmacological inhibition of Fgf signaling lead to strong disruption of proliferation in the fin buds, without affecting Shh signaling. To directly test the ability of Fgf signaling to regulate proliferation in the absence of Shh signaling, we implanted beads soaked with Fgf protein into shh mutant fin buds. We find that Fgf-soaked beads rescue proliferation in the pectoral find buds of shh mutants, indicating that Fgf signaling is sufficient to direct proliferation in zebrafish fin buds in the absence of Shh.

Conclusion: Previous studies have shown that both Shh and Fgf signaling are crucial for outgrowth of the vertebrate limb. The results presented here show that the role of Shh in this process is indirect, and is mediated by its effect on Fgf signaling. By contrast, the activity of the Fgf pathway affects proliferation directly and independently of its effect on Shh. These results show that Fgf signaling is of primary importance in directing outgrowth of the limb bud, and clarify the role of the Shh-Fgf feedback loop in regulating proliferation.

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Fgf signaling inhibition does not cause global downregulation of G1- and S-phase cell-cycle gene expression. Wild-type embryos were treated with 10 μM SU5402 (A-E) or the carrier 0,125% DMSO (F-J) for 3 hours from 20 to 23 hpf. SU5402 treatment caused a strong downregulation of pea3 gene expression (A, F), but expression of cyclinD1, pcna, mcm5 and ra1 genes was not changed in SU5402-treated embryos compared to control ones (B-E, G-J).
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Figure 4: Fgf signaling inhibition does not cause global downregulation of G1- and S-phase cell-cycle gene expression. Wild-type embryos were treated with 10 μM SU5402 (A-E) or the carrier 0,125% DMSO (F-J) for 3 hours from 20 to 23 hpf. SU5402 treatment caused a strong downregulation of pea3 gene expression (A, F), but expression of cyclinD1, pcna, mcm5 and ra1 genes was not changed in SU5402-treated embryos compared to control ones (B-E, G-J).

Mentions: Since we observed that blockage of Fgf signaling with SU5402 leads to rapid loss of G1- and S-phase gene expression both in the pectoral fin buds and in the branchial arches, we also checked whether Fgf signaling is required for proliferation in other tissues. We therefore performed an inhibitor treatment at 20 hpf, a stage at which many embryonic cells are still proliferative. After 3 hours of treatment with 10 μM of SU5402, expression of pea3 is almost completely lost in these embryos, but cyclinD1, pcna, mcm5 and ra1 expression is unaltered (Fig. 4A–E, F–J). Furthermore, while SU5402 treatment at 39hpf leads to loss of cell-cycle genes from both the pectoral fin buds and the branchial arches, it has no effect on the same genes expressed in the retina and the optic tectum (data not shown). These results indicate that Fgf signaling is not generally required for proliferation in the whole embryo, but that it instead directs expression of cell-cycle genes specifically in the pectoral fin buds and in the branchial arches.


Distinct roles of Shh and Fgf signaling in regulating cell proliferation during zebrafish pectoral fin development.

Prykhozhij SV, Neumann CJ - BMC Dev. Biol. (2008)

Fgf signaling inhibition does not cause global downregulation of G1- and S-phase cell-cycle gene expression. Wild-type embryos were treated with 10 μM SU5402 (A-E) or the carrier 0,125% DMSO (F-J) for 3 hours from 20 to 23 hpf. SU5402 treatment caused a strong downregulation of pea3 gene expression (A, F), but expression of cyclinD1, pcna, mcm5 and ra1 genes was not changed in SU5402-treated embryos compared to control ones (B-E, G-J).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562996&req=5

Figure 4: Fgf signaling inhibition does not cause global downregulation of G1- and S-phase cell-cycle gene expression. Wild-type embryos were treated with 10 μM SU5402 (A-E) or the carrier 0,125% DMSO (F-J) for 3 hours from 20 to 23 hpf. SU5402 treatment caused a strong downregulation of pea3 gene expression (A, F), but expression of cyclinD1, pcna, mcm5 and ra1 genes was not changed in SU5402-treated embryos compared to control ones (B-E, G-J).
Mentions: Since we observed that blockage of Fgf signaling with SU5402 leads to rapid loss of G1- and S-phase gene expression both in the pectoral fin buds and in the branchial arches, we also checked whether Fgf signaling is required for proliferation in other tissues. We therefore performed an inhibitor treatment at 20 hpf, a stage at which many embryonic cells are still proliferative. After 3 hours of treatment with 10 μM of SU5402, expression of pea3 is almost completely lost in these embryos, but cyclinD1, pcna, mcm5 and ra1 expression is unaltered (Fig. 4A–E, F–J). Furthermore, while SU5402 treatment at 39hpf leads to loss of cell-cycle genes from both the pectoral fin buds and the branchial arches, it has no effect on the same genes expressed in the retina and the optic tectum (data not shown). These results indicate that Fgf signaling is not generally required for proliferation in the whole embryo, but that it instead directs expression of cell-cycle genes specifically in the pectoral fin buds and in the branchial arches.

Bottom Line: Correlating with this reduction, Fgf signaling is normal at early stages, but is later lost in shh mutants.Furthermore, pharmacological inhibition of Hh signaling for short periods has little effect on either Fgf signaling, or on expression of G1- and S-phase cell-cycle genes, whereas long periods of inhibition lead to the downregulation of both.The results presented here show that the role of Shh in this process is indirect, and is mediated by its effect on Fgf signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany. prykhozh@embl.de

ABSTRACT

Background: Cell proliferation in multicellular organisms must be coordinated with pattern formation. The major signaling pathways directing pattern formation in the vertebrate limb are well characterized, and we have therefore chosen this organ to examine the interaction between proliferation and patterning. Two important signals for limb development are members of the Hedgehog (Hh) and Fibroblast Growth Factor (Fgf) families of secreted signaling proteins. Sonic hedgehog (Shh) directs pattern formation along the anterior/posterior axis of the limb, whereas several Fgfs in combination direct pattern formation along the proximal/distal axis of the limb.

Results: We used the genetic and pharmacological amenability of the zebrafish model system to dissect the relative importance of Shh and Fgf signaling in regulating proliferation during development of the pectoral fin buds. In zebrafish mutants disrupting the shh gene, proliferation in the pectoral fin buds is initially normal, but later is strongly reduced. Correlating with this reduction, Fgf signaling is normal at early stages, but is later lost in shh mutants. Furthermore, pharmacological inhibition of Hh signaling for short periods has little effect on either Fgf signaling, or on expression of G1- and S-phase cell-cycle genes, whereas long periods of inhibition lead to the downregulation of both. In contrast, even short periods of pharmacological inhibition of Fgf signaling lead to strong disruption of proliferation in the fin buds, without affecting Shh signaling. To directly test the ability of Fgf signaling to regulate proliferation in the absence of Shh signaling, we implanted beads soaked with Fgf protein into shh mutant fin buds. We find that Fgf-soaked beads rescue proliferation in the pectoral find buds of shh mutants, indicating that Fgf signaling is sufficient to direct proliferation in zebrafish fin buds in the absence of Shh.

Conclusion: Previous studies have shown that both Shh and Fgf signaling are crucial for outgrowth of the vertebrate limb. The results presented here show that the role of Shh in this process is indirect, and is mediated by its effect on Fgf signaling. By contrast, the activity of the Fgf pathway affects proliferation directly and independently of its effect on Shh. These results show that Fgf signaling is of primary importance in directing outgrowth of the limb bud, and clarify the role of the Shh-Fgf feedback loop in regulating proliferation.

Show MeSH
Related in: MedlinePlus