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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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G1- and S-phase cell-cycle gene expression in fin buds of sonic-you mutant correlates with the Fgf signaling status. Wild-type embryos and sonic-you mutant embryos (in which the zebrafish shh gene is disrupted) at 32 hpf (A-L) and 38 hpf (M-Y) were analysed for expression of the Shh target patched1(ptc1) (A, G, M; T), the Fgf target pea3 (B, H, N, U), the cell-cycle genes cyclinD1, pcna, and mcm5 (C-E, I-K, O-R, V-X), and replication protein A1(ra1) (F, L, S, Y). The Shh target ptc1 was expressed in the posterior part of wild-type fin buds at 32 and 38 hpf stages (A, M), but its expresssion was absent in sonic-you mutant fin buds (G, T). The Fgf signaling target pea3 was expressed at comparable levels in wild-type and sonic-you fin buds at 32 hpf stage (B, H). At 38 hpf pea3 was still strongly expressed in the wild-type fin buds (N), but almost completely downregulated in the sonic-you mutant fin buds (U). cyclinD1, pcna and mcm5 were expressed strongly in both wild-type and sonic-you fin buds at 32 hpf stage (C-E, I-K). At 38 hpf these genes were still strongly expressed in the wild-type fin buds (O-R), but downregulated in the sonic-you mutant fin buds (V-X). Expression of ra1 was similar in both wild-type and sonic-you fin buds at 32 and 38 hpf stages.
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Figure 1: G1- and S-phase cell-cycle gene expression in fin buds of sonic-you mutant correlates with the Fgf signaling status. Wild-type embryos and sonic-you mutant embryos (in which the zebrafish shh gene is disrupted) at 32 hpf (A-L) and 38 hpf (M-Y) were analysed for expression of the Shh target patched1(ptc1) (A, G, M; T), the Fgf target pea3 (B, H, N, U), the cell-cycle genes cyclinD1, pcna, and mcm5 (C-E, I-K, O-R, V-X), and replication protein A1(ra1) (F, L, S, Y). The Shh target ptc1 was expressed in the posterior part of wild-type fin buds at 32 and 38 hpf stages (A, M), but its expresssion was absent in sonic-you mutant fin buds (G, T). The Fgf signaling target pea3 was expressed at comparable levels in wild-type and sonic-you fin buds at 32 hpf stage (B, H). At 38 hpf pea3 was still strongly expressed in the wild-type fin buds (N), but almost completely downregulated in the sonic-you mutant fin buds (U). cyclinD1, pcna and mcm5 were expressed strongly in both wild-type and sonic-you fin buds at 32 hpf stage (C-E, I-K). At 38 hpf these genes were still strongly expressed in the wild-type fin buds (O-R), but downregulated in the sonic-you mutant fin buds (V-X). Expression of ra1 was similar in both wild-type and sonic-you fin buds at 32 and 38 hpf stages.

Mentions: In order to investigate the role of Shh in regulating cell-cycle progression in the limb bud, we analyzed the expression of cell-cycle genes in the pectoral fin buds of zebrafish shh mutants. We focused on cyclinD1, pcna and mcm5, which are generally used as markers of proliferating cells in zebrafish [35,36]. Expression of cyclinD1 is necessary for G1-progression and S-phase entry, while pcna and mcm5 are necessary for DNA replication during S-phase [37]. As a control we analyzed expression of replication protein A1 gene (ra1), which is expressed constitutively in all cells of the fin bud. We find that at 32 hpf, cyclinD1, pcna, mcm5 and ra1 are expressed at indistinguishable levels in wild-type and in shh mutant fin buds (Fig. 1C–F, I–L). Since expression of the Shh-target patched1 [38] is absent from shh mutant fin buds at all stages (Fig. 1A, M), these results indicate that expression of G1- and S-phase cell-cycle genes is independent of Shh at 32hpf. Examination of these cell-cycle genes at 38hpf, however, reveals that cyclinD1, pcna, and mcm5 expression are lost in shh mutant fin buds, while ra1 remains unaltered (Fig. 1O–R, V–X), suggesting that cell-cycle progression becomes dependent on Shh signaling at later stages. Since the expression of Fgf ligands in the AER depends on Shh activity [1,8], we also tested whether the activity of the Fgf signaling pathway in shh mutant fin buds correlates with the observed reduction in cell-cycle gene expression. Using the Fgf-target pea3 as a marker for Fgf signaling [39], we find that pea3 expression in shh mutant pectoral fin buds is identical to wild-type fin buds at 32hpf, but is strongly reduced at 38hpf (Fig. 1B, H, N, U). This result is consistent with the observation that Shh is necessary for maintenance of Fgf expression in the AER, and suggests a correlation between the activity of Fgf signaling and the expression of cell-cycle genes in shh mutant fin buds. Taken together, these results show that expression of G1- and S-phase cell-cycle genes is initially normal in shh mutant pectoral fin buds, but is later lost, and that this shift correlates with a similar loss of Fgf signaling activity at later stages.


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

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

G1- and S-phase cell-cycle gene expression in fin buds of sonic-you mutant correlates with the Fgf signaling status. Wild-type embryos and sonic-you mutant embryos (in which the zebrafish shh gene is disrupted) at 32 hpf (A-L) and 38 hpf (M-Y) were analysed for expression of the Shh target patched1(ptc1) (A, G, M; T), the Fgf target pea3 (B, H, N, U), the cell-cycle genes cyclinD1, pcna, and mcm5 (C-E, I-K, O-R, V-X), and replication protein A1(ra1) (F, L, S, Y). The Shh target ptc1 was expressed in the posterior part of wild-type fin buds at 32 and 38 hpf stages (A, M), but its expresssion was absent in sonic-you mutant fin buds (G, T). The Fgf signaling target pea3 was expressed at comparable levels in wild-type and sonic-you fin buds at 32 hpf stage (B, H). At 38 hpf pea3 was still strongly expressed in the wild-type fin buds (N), but almost completely downregulated in the sonic-you mutant fin buds (U). cyclinD1, pcna and mcm5 were expressed strongly in both wild-type and sonic-you fin buds at 32 hpf stage (C-E, I-K). At 38 hpf these genes were still strongly expressed in the wild-type fin buds (O-R), but downregulated in the sonic-you mutant fin buds (V-X). Expression of ra1 was similar in both wild-type and sonic-you fin buds at 32 and 38 hpf stages.
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Related In: Results  -  Collection

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Figure 1: G1- and S-phase cell-cycle gene expression in fin buds of sonic-you mutant correlates with the Fgf signaling status. Wild-type embryos and sonic-you mutant embryos (in which the zebrafish shh gene is disrupted) at 32 hpf (A-L) and 38 hpf (M-Y) were analysed for expression of the Shh target patched1(ptc1) (A, G, M; T), the Fgf target pea3 (B, H, N, U), the cell-cycle genes cyclinD1, pcna, and mcm5 (C-E, I-K, O-R, V-X), and replication protein A1(ra1) (F, L, S, Y). The Shh target ptc1 was expressed in the posterior part of wild-type fin buds at 32 and 38 hpf stages (A, M), but its expresssion was absent in sonic-you mutant fin buds (G, T). The Fgf signaling target pea3 was expressed at comparable levels in wild-type and sonic-you fin buds at 32 hpf stage (B, H). At 38 hpf pea3 was still strongly expressed in the wild-type fin buds (N), but almost completely downregulated in the sonic-you mutant fin buds (U). cyclinD1, pcna and mcm5 were expressed strongly in both wild-type and sonic-you fin buds at 32 hpf stage (C-E, I-K). At 38 hpf these genes were still strongly expressed in the wild-type fin buds (O-R), but downregulated in the sonic-you mutant fin buds (V-X). Expression of ra1 was similar in both wild-type and sonic-you fin buds at 32 and 38 hpf stages.
Mentions: In order to investigate the role of Shh in regulating cell-cycle progression in the limb bud, we analyzed the expression of cell-cycle genes in the pectoral fin buds of zebrafish shh mutants. We focused on cyclinD1, pcna and mcm5, which are generally used as markers of proliferating cells in zebrafish [35,36]. Expression of cyclinD1 is necessary for G1-progression and S-phase entry, while pcna and mcm5 are necessary for DNA replication during S-phase [37]. As a control we analyzed expression of replication protein A1 gene (ra1), which is expressed constitutively in all cells of the fin bud. We find that at 32 hpf, cyclinD1, pcna, mcm5 and ra1 are expressed at indistinguishable levels in wild-type and in shh mutant fin buds (Fig. 1C–F, I–L). Since expression of the Shh-target patched1 [38] is absent from shh mutant fin buds at all stages (Fig. 1A, M), these results indicate that expression of G1- and S-phase cell-cycle genes is independent of Shh at 32hpf. Examination of these cell-cycle genes at 38hpf, however, reveals that cyclinD1, pcna, and mcm5 expression are lost in shh mutant fin buds, while ra1 remains unaltered (Fig. 1O–R, V–X), suggesting that cell-cycle progression becomes dependent on Shh signaling at later stages. Since the expression of Fgf ligands in the AER depends on Shh activity [1,8], we also tested whether the activity of the Fgf signaling pathway in shh mutant fin buds correlates with the observed reduction in cell-cycle gene expression. Using the Fgf-target pea3 as a marker for Fgf signaling [39], we find that pea3 expression in shh mutant pectoral fin buds is identical to wild-type fin buds at 32hpf, but is strongly reduced at 38hpf (Fig. 1B, H, N, U). This result is consistent with the observation that Shh is necessary for maintenance of Fgf expression in the AER, and suggests a correlation between the activity of Fgf signaling and the expression of cell-cycle genes in shh mutant fin buds. Taken together, these results show that expression of G1- and S-phase cell-cycle genes is initially normal in shh mutant pectoral fin buds, but is later lost, and that this shift correlates with a similar loss of Fgf signaling activity at later stages.

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