Distinct roles of Shh and Fgf signaling in regulating cell proliferation during zebrafish pectoral fin development
© Prykhozhij and Neumann; licensee BioMed Central Ltd. 2008
Received: 30 April 2008
Accepted: 23 September 2008
Published: 23 September 2008
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.
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.
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.
During the development of multicellular organisms, pattern formation must be precisely coordinated with proliferation and differentiation. Given that only a relatively small number of signaling pathways are used to direct both pattern formation and cell proliferation during development, it is clear that cell fate specification and cell division are highly context-dependent read-outs of signaling in a given tissue or organ. Activation of a particular signaling pathway, such as the Hedgehog pathway, can stimulate proliferation in one cell type, while activation of the same pathway in another cell type has no effect on proliferation. Moreover, the observation that identical signaling pathways can regulate both pattern formation and cell proliferation provides a mechanism for coordination of these distinct behaviours.
The vertebrate limb is an excellent model system in which to study the interplay between pattern formation and cell proliferation. Limb development is highly amenable to experimental and genetic manipulation in several model organisms, and the main signaling pathways that direct limb development are well characterized (reviewed in [1–3]). Three signaling centers are required for pattern formation and growth in the developing limb bud, two of which we chose to study in this work. One of these is the zone of polarizing activity (ZPA), a small group of cells in the posterior mesenchyme, which controls polarity along the anterior/posterior axis . The secreted signaling protein Sonic hedgehog (Shh) is expressed in the ZPA, and has been shown to mediate the effect of the ZPA during limb development [5–8].
The apical ectodermal ridge (AER) is another major signaling center of the limb bud which runs along its distal margin, and which is the site of expression of several Fgf genes (reviewed in ). The AER is required for outgrowth and patterning of the limb along its proximal/distal axis, and can be functionally replaced by FGF-soaked beads in chicken embryos, indicating that Fgf signaling can mediate AER function [10, 11]. Furthermore, conditional inactivation of both Fgf4 and Fgf8 in the mouse AER leads to failure of proximal/distal outgrowth , thus identifying these members of the Fgf family as the main mediators of AER signaling. Factors from the AER and ZPA form a mutual feedback loop, thereby allowing growth and patterning of the different axes to be coordinated. Thus fgf-4, which is expressed in the posterior AER, can be induced in the anterior AER of the chicken limb bud by ectopic Shh protein [13, 14]. Furthermore, removal of Shh activity from the zebrafish fin buds leads to loss of fgf4 and fgf8 expression in the AER , and, conversely, removal of Fgf4 and Fgf8 activity from the mouse AER leads to loss of shh expression in the ZPA , indicating that each signaling pathway is required for the maintenance of the other pathway.
Members of both the Hh and Fgf family of signaling proteins have been shown to function as mitogens in several contexts. Indeed, Fgf1 and Fgf2 were initially identified as mitogenic factors in fibroblast tissue culture, and subsequently, other members of the FGF protein family were found to have a similar activity . Furthermore, Fgf signaling has also been shown to have mitogenic activity in vivo during embryonic development. Thus FGF-4 is necessary for proliferation of the inner cell mass during early post-implantation development in the mouse , and FGF-8 and FGF-17 are required for proliferation in the mouse dorsal midbrain . Additionally, Fgf signaling promotes proliferation of osteoblasts , of lens cells , and during hematopoiesis .
Like the Fgf family, members of the Hh family function as mitogens in a number of contexts. The Hh signaling pathway has been linked to several cancers, including basal cell carcinoma, pancreatic tumors, and digestive tract tumors, and may be upregulated in as many as 25% of tumors [21–25]. In addition to this oncogenic effect, Hedgehog signaling also directs proliferation during normal development, including in the mouse cerebellum , in the Drosophila eye , in mammalian keratinocyctes , and in the mammalian kidney . In several cases Hh signaling has been shown to stimulate cell-cycle progression by causing transcriptional upregulation of D-type and E-type cyclins in target cells [27, 30–32]. This transcriptional up-regulation of cell-cycle genes in some instances has been shown to occur as a direct response to promoter binding of members of the GLI family, the zinc-finger transcription factors which transduce Hh signaling to the nucleus [27, 32, 33].
Since there is clear evidence that both Shh and Fgf signaling are important for outgrowth of the vertebrate limb bud, and since both signaling pathways are known to have a mitogenic effect during development, this raises the question of the relative contribution of Shh and Fgf signaling to regulation of proliferation in the limb bud. This issue is complicated by the feedback loop operating between the two signals, as inhibition of either signaling pathway leads to loss of the other signaling pathway. Laufer and colleagues have previously addressed this issue by removing the AER from chicken wing buds and adding back either FGF4-soaked beads, or shh-expressing virus [13, 14]. Their results show that Shh alone is insufficient to induce mesodermal proliferation, whereas FGF4 alone is sufficient to do so, leading to them to conclude that the effect of Shh on mesodermal proliferation is indirect, and due to the induction of Fgfs in the AER. However, a recently published study  shows that Shh is sufficient to induce cyclin D1 expression in the mesoderm of chicken wing buds after AER removal. This observation raises a third possibility: that Shh and Fgf signaling both contribute to the regulation of limb bud proliferation.
To distinguish between these possibilities, we have made use of the genetic, embryological, and pharmacological tools of the zebrafish model system to uncouple the activity of Shh and Fgf signaling in the pectoral fin buds, and to investigate their individual effects on proliferation. In order to categorically uncouple the effect of Fgf signaling from Shh, we implanted FGF4-soaked beads into the limb buds of shh mutants. Our data confirm that Fgf signaling is of direct and crucial importance for growth and cell-cycle progression in the limb bud, whereas the effect of Shh on proliferation is indirect, and is mediated via its effect on Fgf expression in the AER.
Expression of G1- and S-phase cell-cycle genes in shhmutant fin buds is initially normal, but is lost at later stages of development
Loss of expression of G1- and S-phase genes after inhibition of Shh signaling correlates closely with reduction of Fgf signaling
Inhibition of Fgf signaling with SU5402 leads to rapid loss of G1- and S-phase cell-cycle genes in the fin bud
Fgf signaling is not generally required for cell-cycle progression in the zebrafish embryo
Implantation of Fgf4-soaked beads is sufficient to restore expression of G1- and S-phase cell-cycle genes and S-phase progression in shhmutant fin buds
The cell-cell signaling events that direct vertebrate limb development have been the subject of intense research for more than a hundred years. This provides an excellent foundation for investigating the mechanisms whereby pattern formation is integrated with proliferation. In this study we have focused on two of the main signals important for patterning and growth of the vertebrate limb: the Shh and Fgf signaling pathways. While both signals are crucial for outgrowth of the limb bud, it has been very challenging to uncouple these signals from each other, since expression of Shh depends on Fgf signaling, and vice versa. For example, while AER ablation experiments have been interpreted as causing failure of limb outgrowth because they remove the source of Fgf signaling from the limb bud [10, 11, 43], AER ablation simultaneously leads to loss of Shh expression from the ZPA [13, 14], and so cannot be used to separate the effect of Fgf on proliferation from the effect of Shh. To overcome this challenge, we have used a combination of loss-of-function and gain-of-function experiments in the the zebrafish model system to uncouple Shh from Fgf signaling in the pectoral fin bud, and have assessed the effect of each signal on fin bud proliferation independently of the other signal.
Our results show that the effect of Shh on proliferation during limb development is indirect, and is mediated by its effect on Fgf expression in the AER. Inhibition of Shh signaling leads to loss of cell-cycle progression only after a relatively long delay period of around 13 hours, and this correlates with a concomitant loss of Fgf signaling. Inhibition of Fgf signaling, on the other hand, leads to loss of cell-cycle progression very rapidly, after only 3 hours of inhibitor treatment, and this occurs even though activity of the Shh pathway is still present. This rapid effect of Fgf on cell-cycle progression suggests a direct transcriptional response of cell-cycle genes to the Fgf pathway in the limb bud, which is consistent with the direct mitogenic effect of Fgf signaling shown on several cell types in tissue culture.
Since Hh signaling has also been shown to have a direct mitogenic effect on some cell types, it is perhaps surprising that Shh directs proliferation indirectly in the vertebrate limb bud. However, there is at least one previous example of such an indirect effect of Hh signaling on proliferation. During Drosophila wing development, Hh is necessary for growth of the wing imaginal disc, but this effect is mediated via the Hh-dependent expression of Decapentaplegic (Dpp), a member of the Tgf-β family of secreted signaling proteins [44–46]. Furthermore, the proliferative response of different cell types to Hh is clearly context-dependent, and Hh signaling can even function as a negative regulator of the cell-cycle in some cell types . Interestingly, this negative effect of Hh signaling on proliferation also appears to be indirect in some cases. In the rodent colonic epithelium, for example, Hh signaling stimulates cell-cycle exit by antagonizing the Wnt pathway . In the Drosophila retina, on the other hand, Hh signaling has both positive and negative effects on cell-cycle progression . Cell-cycle arrest of cells in front of the retinal differentiation wave depends on Hh signaling in combination with Dpp, while cell-cycle re-entry behind the wave front also depends on Hh signaling, but in this case mediated by the Notch signaling pathway [49, 50].
Our results also show a context-dependent effect of Fgf signaling on cell-cycle progression. Thus Fgf signaling is clearly essential for cell-cycle progression in the pectoral fin buds and in the branchial arches, since expression of G1- and S-phase cell-cycle genes in these tissues is lost after only 3 hours of inhibition of the Fgf pathway. Inhibition of Fgf signaling fails to affect cell-cycle progression in other organs, however, such as the retina and the optic tectum, or at earlier stages of development. The Fgf signaling pathway is therefore not a global mitogenic signal in the zebrafish embryo, but instead directs proliferation in a highly tissue-specific manner. Altogether, the evidence thus indicates that both the Hh and Fgf pathway affect cell-cycle progression in some cell types but not in others, and that this effect can be either direct or indirect. The control of cell-proliferation in multicellular organisms can therefore only be understood in a context-dependent manner, and our results help to shed light on this question in the context of the vertebrate limb bud. The molecular mechanisms by which different cell types respond distinctly to the same signal are still poorly understood, but will undoubtedly be unravelled by future research.
Our data show that Shh and Fgf signaling have distinct effects on proliferation during vertebrate limb development. While both genes are necessary for outgrowth of the limb bud, the role of Shh in this process is indirect, and is mediated via its effect on expression of Fgf genes in the AER. In contrast, the effect of Fgf signaling on cell-cycle progression in the limb bud appears to be direct, and Fgf signaling is both necessary and sufficient to direct proliferation, even in the absence of Shh. Together with the work of others, our results indicate that the role of both signaling pathways in regulating proliferation is highly context dependent, and our results shed light on their function in directing proliferation in the context of limb development.
Fish stocks, maintenance and care
Wild-type Tupfel Long Fin (TLF) and sonic-yout4 heterozygous fish were used. Fish were maintained according to standard protocols. Embryos were grown in E3 embryo medium at 28°C with or without the addition of 0.003% 1-phenyl-2-thiourea (PTU, Sigma) to inhibit pigmentation. Staging was performed according to hours post-fertilization (hpf) .
Chemical inhibitors and treatment procedures
FGF signaling inhibitor SU5402 (Calbiochem, cat # 572630) was dissolved in DMSO at 8 mM. Treatment was performed with a 10 μM solution of SU5402 or a corresponding control DMSO solution in E3 embryo medium. Cyclopamine (Toronto Research Chemicals, cat# C988400) was dissolved in ethanol at 20 mM. Treatment was performed with 100 μM solution of cyclopamine or control ethanol solution in E3 embryo medium.
In situ hybridisation and probe synthesis
RNA in situ hybridisations were performed according to Jowett . Probes were made using Roche DIG RNA Labeling Kit (Cat # 11175025910). cDNAs used to make antisense probes: ptc1, pea3 , cyclin D1 , pcna (cDNA was ampplified using pcna_for: CCTACTCCAAACTAAGAAAGCAGCA and pcna_rev: ATCGGGAATCCATTGAACTGG), mcm5 (cDNA was amplified using mcm5_for: TGGTGGAGGAGAAAGCGTCG and mcm5_rev: GGCCTCATGGATTGCGACTC and cloned into pGEM-T-easy (Promega)), ra1 (NM199811) cDNA was ampplified using ra1_for: CCATTGGAGGAAACAGGAGA and T7_ra1_rev: TAATACGACTCACTATAGGGcagcgtgtacctggtaagca and transcribed from the PCR product using T7 polymerase).
Antibody stainings were performed on 14 μm cryosections. Cryosections were rehydrated in PBS with 0.1% Tween-20 (PBST), treated for 20 min with 4N HCl, washed several times in PBST and blocked for 1 hour with blocking solution (4% normal goat serum in PBST). Mouse anti-BrdU antibody (1:100) (Roche, cat# 1 170 376) and secondary anti-mouse Alexa Fluor 488 antibody (1:400) (Invitrogen, cat# A11001) were diluted in blocking solution and incubated with the sections for 2 hours each. Images were taken using Leica SP2 confocal microscope and processed using Adobe Creative Suite CS2.
Bead implantation was performed as described before . Recombinant human Fgf4 protein (R&D Systems, cat# 235-F4-025) was dissolved at a concentration 250 ng/μl in PBS with 0.1% bovine serum albumin and mixed in proportion 1:1 with the mix of beads filtered through a 70 μm Cell strainer (BD Falcon, cat# 352350).
We thank Sabine Fischer for technical assistance, Gill Brunt for fish care and Darren Gilmour for critical reading of the manuscript. SVP is grateful to Marlene Rau for a practical explanation of the bead implantation procedure and to William Norton for advice on the bead implantation protocol.
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