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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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Expression of G1- and S-phase cell-cycle genes fails to correlate with the status of Shh signaling in fin buds. Wild-type embryos were treated with 100 μM cyclopamine (cyA) (G-L, T-Y) or with the carrier 0,5% ethanol (EtOH) (A-F, M-S) for 6 hours from 34 to 40 hpf (A-L) or for 13 hours from 34 to 47 hpf (M-Y) and analysed for the expression of ptc1, pea3, cyclinD1, pcna, mcm5 and ra1. Expression of ptc1 was nearly completely lost after both 6 hours (A, G) and 13 hours (M, T) inhibition pulses. A 6-hour Hedgehog signaling inhibition led to a small change in pea3 expression in fin buds (B, H). Comparably small changes in expression after 6-hour Hedgehog signaling inhibition were observed for cyclinD1, pcna, mcm5 and ra1 (C-F, I-L). After 13-hour Hedgehog signaling inhibition, fin bud pea3 expression was strongly decreased (N, U). Likewise, expression of cyclinD1, pcna and mcm5 in fin buds was strongly downregulated (O-R, V-X). Expression of ra1gene was only mildly affected by 13-hour cyclopamine treatment (S, Y).
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Figure 2: Expression of G1- and S-phase cell-cycle genes fails to correlate with the status of Shh signaling in fin buds. Wild-type embryos were treated with 100 μM cyclopamine (cyA) (G-L, T-Y) or with the carrier 0,5% ethanol (EtOH) (A-F, M-S) for 6 hours from 34 to 40 hpf (A-L) or for 13 hours from 34 to 47 hpf (M-Y) and analysed for the expression of ptc1, pea3, cyclinD1, pcna, mcm5 and ra1. Expression of ptc1 was nearly completely lost after both 6 hours (A, G) and 13 hours (M, T) inhibition pulses. A 6-hour Hedgehog signaling inhibition led to a small change in pea3 expression in fin buds (B, H). Comparably small changes in expression after 6-hour Hedgehog signaling inhibition were observed for cyclinD1, pcna, mcm5 and ra1 (C-F, I-L). After 13-hour Hedgehog signaling inhibition, fin bud pea3 expression was strongly decreased (N, U). Likewise, expression of cyclinD1, pcna and mcm5 in fin buds was strongly downregulated (O-R, V-X). Expression of ra1gene was only mildly affected by 13-hour cyclopamine treatment (S, Y).

Mentions: Since the loss of G1- and S-phase cell-cycle genes in shh mutant fin buds occurs relatively late, and only after Fgf signaling is lost, we decided to use selective inhibition of Hh signaling using the plant alcaloid cyclopamine [40] to determine the time period of inhibition necessary to affect cell-cycle progression. Cyclopamine inhibits the action of Smoothened protein, which transduces the Hh signal after it becomes released from Patched1-mediated inhibition [41]. The use of cyclopamine allows inhibition of Hedgehog signaling for varying periods of time, and thereby temporal control over the signaling inhibition. Our aim was to find a duration of cyclopamine treatment sufficient to inhibit Hedgehog signaling, but leaving Fgf signaling largely unaffected, thereby uncoupling the two pathways from each other. We find that a 6-hour treatment from 34 to 40 hpf with 100 μM cyclopamine is sufficient to inhibit patched1 expression almost completely (Fig. 2A, G), but has little effect on expression of the Fgf-target pea3 (Fig. 2B, H). Likewise, this treatment has little or no effect on cyclinD1, pcna, mcm5 and ra1 expression (Fig. 2C–F, I–L). In contrast, however, a 13-hour cyclopopamine treatment from 34 to 47 hpf leads to loss of both ptc1 and pea3 expression (Fig. 2M, N, T, U), and also leads to strong reduction of cyclinD1, pcna, and mcm5 expression, but without affecting ra1 (Fig. 2O–S, V–Y). These results show that loss of Shh signaling leads to loss of cell-cycle gene expression only after a 13-hour delay, indicating that this is likely to be an indirect effect. Since after this delay period cell-cycle gene expression loss correlates closely with reduction of Fgf signaling in response to Shh inhibition, Fgfs are very good candidates for mediating the effect of Shh on cell-cycle progression in the fin bud.


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

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

Expression of G1- and S-phase cell-cycle genes fails to correlate with the status of Shh signaling in fin buds. Wild-type embryos were treated with 100 μM cyclopamine (cyA) (G-L, T-Y) or with the carrier 0,5% ethanol (EtOH) (A-F, M-S) for 6 hours from 34 to 40 hpf (A-L) or for 13 hours from 34 to 47 hpf (M-Y) and analysed for the expression of ptc1, pea3, cyclinD1, pcna, mcm5 and ra1. Expression of ptc1 was nearly completely lost after both 6 hours (A, G) and 13 hours (M, T) inhibition pulses. A 6-hour Hedgehog signaling inhibition led to a small change in pea3 expression in fin buds (B, H). Comparably small changes in expression after 6-hour Hedgehog signaling inhibition were observed for cyclinD1, pcna, mcm5 and ra1 (C-F, I-L). After 13-hour Hedgehog signaling inhibition, fin bud pea3 expression was strongly decreased (N, U). Likewise, expression of cyclinD1, pcna and mcm5 in fin buds was strongly downregulated (O-R, V-X). Expression of ra1gene was only mildly affected by 13-hour cyclopamine treatment (S, Y).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Expression of G1- and S-phase cell-cycle genes fails to correlate with the status of Shh signaling in fin buds. Wild-type embryos were treated with 100 μM cyclopamine (cyA) (G-L, T-Y) or with the carrier 0,5% ethanol (EtOH) (A-F, M-S) for 6 hours from 34 to 40 hpf (A-L) or for 13 hours from 34 to 47 hpf (M-Y) and analysed for the expression of ptc1, pea3, cyclinD1, pcna, mcm5 and ra1. Expression of ptc1 was nearly completely lost after both 6 hours (A, G) and 13 hours (M, T) inhibition pulses. A 6-hour Hedgehog signaling inhibition led to a small change in pea3 expression in fin buds (B, H). Comparably small changes in expression after 6-hour Hedgehog signaling inhibition were observed for cyclinD1, pcna, mcm5 and ra1 (C-F, I-L). After 13-hour Hedgehog signaling inhibition, fin bud pea3 expression was strongly decreased (N, U). Likewise, expression of cyclinD1, pcna and mcm5 in fin buds was strongly downregulated (O-R, V-X). Expression of ra1gene was only mildly affected by 13-hour cyclopamine treatment (S, Y).
Mentions: Since the loss of G1- and S-phase cell-cycle genes in shh mutant fin buds occurs relatively late, and only after Fgf signaling is lost, we decided to use selective inhibition of Hh signaling using the plant alcaloid cyclopamine [40] to determine the time period of inhibition necessary to affect cell-cycle progression. Cyclopamine inhibits the action of Smoothened protein, which transduces the Hh signal after it becomes released from Patched1-mediated inhibition [41]. The use of cyclopamine allows inhibition of Hedgehog signaling for varying periods of time, and thereby temporal control over the signaling inhibition. Our aim was to find a duration of cyclopamine treatment sufficient to inhibit Hedgehog signaling, but leaving Fgf signaling largely unaffected, thereby uncoupling the two pathways from each other. We find that a 6-hour treatment from 34 to 40 hpf with 100 μM cyclopamine is sufficient to inhibit patched1 expression almost completely (Fig. 2A, G), but has little effect on expression of the Fgf-target pea3 (Fig. 2B, H). Likewise, this treatment has little or no effect on cyclinD1, pcna, mcm5 and ra1 expression (Fig. 2C–F, I–L). In contrast, however, a 13-hour cyclopopamine treatment from 34 to 47 hpf leads to loss of both ptc1 and pea3 expression (Fig. 2M, N, T, U), and also leads to strong reduction of cyclinD1, pcna, and mcm5 expression, but without affecting ra1 (Fig. 2O–S, V–Y). These results show that loss of Shh signaling leads to loss of cell-cycle gene expression only after a 13-hour delay, indicating that this is likely to be an indirect effect. Since after this delay period cell-cycle gene expression loss correlates closely with reduction of Fgf signaling in response to Shh inhibition, Fgfs are very good candidates for mediating the effect of Shh on cell-cycle progression in the fin bud.

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