Zebrafish Ext2 is necessary for Fgf and Wnt signaling, but not for Hh signaling
© Fischer et al; licensee BioMed Central Ltd. 2011
Received: 14 February 2011
Accepted: 5 September 2011
Published: 5 September 2011
Heparan sulfate (HS) biosynthesis is tightly regulated during vertebrate embryo development. However, potential roles for HS biosynthesis in regulating the function of paracrine signaling molecules that bind to HS are incompletely understood.
In this report we have studied Fgf, Wnt and Hedgehog (Hh) signaling in ext2 mutants, where heparan sulfate content is low. We found that Fgf targeted gene expression is reduced in ext2 mutants and that the remaining expression is readily inhibited by SU5402, an FGF receptor inhibitor. In the ext2 mutants, Fgf signaling is shown to be affected during nervous system development and reduction of Fgf ligands in the mutants affects tail development. Also, Wnt signaling is affected in the ext2 mutants, as shown by a stronger phenotype in ext2 mutants injected with morpholinos that partially block translation of Wnt11 or Wnt5b, compared to injected wild type embryos. In contrast, Hh dependent signaling is apparently unaffected in the ext2 mutants; Hh targeted gene expression is not reduced, the Hh inhibitor cyclopamine is not more affective in the mutants and Hh dependent cell differentiation in the retina and in the myotome are normal in ext2 mutants. In addition, no genetic interaction between ext2 and shha during development could be detected.
We conclude that ext2 is involved in Fgf and Wnt signaling but not in Hh signaling, revealing an unexpected specificity for ext2 in signaling pathways during embryonic development. Thus, our results support the hypothesis that regulation of heparan sulfate biosynthesis has distinct instructive functions for different signaling factors.
KeywordsZebrafish Ext2 HSPG Heparan Fgf Wnt Hh
Heparan Sulfate Proteoglycans (HSPG) consist of proteoglycan core proteins to which long linear heparan sulfate (HS) chains carrying sulfate groups in different positions are attached [1–3]. They are ubiquitous components of cell surfaces and of the extracellular matrix. Extensive biochemical and genetic studies have shown that HSPGs influence the extracellular transport and activity of paracrine signaling molecules such as Fgf, Wnt, and Hh. Several excellent reviews on the subject of the complex role of HSPGs in developmental processes have been written [4–7].
Fgf proteins are thoroughly studied HSPG binding proteins with important roles in cell migration, proliferation and differentiation during development . In Drosophila, the functions of Fgf receptors are abnormal in embryos with defective HSPG biosynthesis [9, 10]. Mice Ugdh mutants (lazy mesoderm) have reduced synthesis of HS, chondroitin sulfate and hyaluronan, strongly suggesting that glycosaminoglycans are required for Fgf signaling . Moreover, a specific inhibition of HSPG biosynthesis during brain development results in defective Fgf8 function . It is suggested that the HSPG Glypcan4 enhances Fgf signaling during Xenopus neurulation  and HSPG biosynthesis is shown to influence Fgf function during limb development in zebrafish  and lens development and lacrimal gland induction in mice [15, 16].
Wnt and Hh proteins are secreted signaling morphogens with functions in numerous developmental processes. A large number of experiments in Drosophila have revealed a crucial role for HSPGs in regulating these functions (reviewed in ). In vertebrates, several studies demonstrate an essential role for the HSPG Glypican in Wnt11 signaling during gastrulation [17–19]. Decreased HSPG sulfation reduces Shh signaling in mice  and decreased HS polymerization is suggested to interfere with the function of Indian hedgehog (Ihh) (discussed below).
Several different mechanisms may be used by HSPGs to effect their regulation of Fgf, Wnt and Hh function. Cell surface HSPGs can serve as co-receptors for Fgf ligands [21, 22] and biochemical studies have suggested that HSPGs participate in Fgf signaling by directly interacting with Fgf ligands and their receptors to form ligand-receptor complexes [23, 24]. HSPGs are probably enhancing Fgf signaling by facilitating interactions of ligands and receptors or by stabilizing the signaling complex . Experiments in Drosophila suggest that HSPGs also act as co-receptors for the binding of Hh and Wg/Wnt to the receptors Patched and Frizzled respectively [26–29], in a similar way to the function of HSPGs in Fgf signaling. However, many observations support models where HSPGs are instead required for proper tissue distribution of Wg/Wnt and Hh proteins [30–32]. HSPGs might protect Hh and Wg/Wnt protein from degradation and increase the local concentration by reducing the dimensionality of ligand diffusion from three to two dimensions, thereby increasing the concentration of Wnt and Hh ligands close to cell surfaces (reviewed in ). Both Hh and Wnt proteins are lipid modified and they are unlikely to freely diffuse between cells. Instead, their movement probably involves additional molecules such as lipoprotein particles . Interestingly, a recent study suggests that HSPGs can influence lipid-linked morphogen signaling by a direct interaction with lipoprotein particles .
The exostosin gene family contains glycosyltransferases required for HS biosynthesis, and include, in mouse, Ext1, Ext2, Extl1, Extl2 and Extl3. During HS biosynthesis in the Golgi compartment, a serine residue in the proteoglycan core protein is modified by stepwise addition of monosaccharides to form a linkage tetrasaccharide which constitutes the substrate for Ext enzymes to initiate and polymerize the HS polysaccharide chain (reviewed in ). Ext1 and Ext2 together form a copolymerase which is responsible for the polymerization process where repeating units of N-acetylglucosamine and glucuronic acid are incorporated in the growing linear polysaccharide chain. During polymerization, the HS chains are modified by the addition of sulfate groups in tissue-specific patterns [35, 36]. The relative expression of HSPG biosynthesis genes may sometimes determine the outcome of the process, but the regulation of HSPG biosynthesis is far from understood [37–39]. Drosophila exostosins genes include ttv, sotv and botv (encoding homologues of mouse Ext1, Ext2 and Extl3, respectively), which have been demonstrated to be important for the signaling activity of Hh and Wg/Wnt and for shaping extracellular morphogen gradients (reviewed by ). Ext1-null and Ext2-null mice both fail to gastrulate, pointing to the early essential roles for Ext genes in developing embryos [40, 41]. Reduced expression of Ext1 results in delayed hypertrophic differentiation and endochondral ossification of the chondrocytes of limb growth plates, probably caused by increased Ihh diffusion [42, 43]. Mutations in Ext genes is the cause of the human disease Multiple Hereditary Exostoses (MHE) [44, 45].
The zebrafish ext2 and extl3 genes are ubiquitously expressed during zebrafish development and they are disrupted in dackel (dak) and boxer (box) mutants, respectively . In ext2 and extl3 mutants, maternally deposited mRNA provides sufficient levels of HSPGs to allow normal gastrulation of the embryo while HS polymerization is subsequently reduced in all tissues . ext2 and extl3 mutants were originally isolated based on their defective limb development  and we have previously shown that Fgf10 signaling during limb development requires ext2 and extl3 . ext2 and extl3 mutants also show defects in cartilage and pharyngeal arch morphogenesis [48, 49], development of the ear , and axon sorting in the optic tract [46, 51].
The role of ext2 in regulating paracrine signaling is of specific interest for understanding the molecular mechanisms of the human disease MHE, but is also of general interest for elucidating the role of the HS portion of the HSPGs, as opposed to the functions of the proteoglycan core protein. In this study we systematically investigate the general role of ext2 in FGF, Wnt and Hh signaling during zebrafish tissue patterning and organogenesis. We find that Fgf signaling is generally reduced in ext2 mutants, based on the reduced expression of target genes and the observation that remaining expression of Fgf target genes is sensitized for treatment with a pharmacological Fgf inhibitor, suggesting that all Fgf signaling is dependent on ext2 function. We also found that a smaller decrease of Wnt11 and Wnt5b translation is required in ext2 mutants, compared with siblings, to interfere with Wnt dependent processes, indicating that ext2 is also involved in Wnt signaling. In contrast, we find that Hh signaling does not act on the same pathways as ext2. We base this conclusion on the observation that Hh signaling in ext2 mutants (1) induces normal expression of target genes, (2) functions in Hh signaling dependent cell differentiation and (3) is not sensitized by partial inhibition of Hh signaling compared to control embryos. We propose that the ext2 gene, as well as any gene influencing levels of HS in zebrafish tissues, is a candidate for regulation of aspects of Fgf and Wnt function, while Hh signaling is likely to be largely independent of ext2 function.
Expression of the Fgf signaling target gene etv5b is reduced in ext2mutants
ext2is a general enhancer of Fgf target gene expression
The transcription factor pea3 is, like etv5b, dependent on Fgf signaling for its expression [52, 53]. Surprisingly, with the exception of a reduction in midbrain-hindbrain boundary expression, pea3 expression is, in contrast to etv5b expression, largely similar in control embryos and ext2 mutants (compare panels E-F and G-H in Figure 1). Does this mean that some Fgf signaling, which predominantly induces pea3 expression rather than etv5b expression, is in fact independent of ext2 function? This was tested by exposing the embryos to the Fgf signaling inhibitor SU5402 which blocks both etv5b and pea3 expression [52, 53]. We reasoned that if pea3 expression in ext2 mutants was the result of HS independent fgf signaling, it should be equally sensitive to treatment with SU5402 as control embryos. However, while exposure of 8 mM SU5402 3 h prior to fixation only marginally reduces pea3 expression in 38 hpf control embryos (Figure 1I-J), pea3 expression in ext2 mutants is nearly eliminated by the same treatment. (Figure 1K-L). A similar difference in sensitivity was observed between 48 hpf and 60 hpf ext2 mutants and control embryos (data not shown). Moreover, the weak etv5b expression in ext2 mutants (Figure 1C-D) was blocked by 8 mM SU5402 exposure (Figure 1O-P), while etv5b expression was only mildly affected in SU5402 treated control embryos (Figure 1M-N) compared to non-treated control embryos (Figure 1A-B). We conclude that no expression of the Fgf signaling target genes etv5b and pea3 is independent of ext2 in the developing zebrafish embryo.
Impaired brain patterning in ext2embryos
Based on the reduced expression of Fgf target genes in ext2 mutants (Figure 1A-H), we hypothesized that not only is Fgf10 signaling defective in the developing limb  but that ext2 is enhancing Fgf signaling in general. To test this hypothesis we investigated the function of Fgf signaling in tissues other than the developing limb.
The midbrain-hindbrain boundary (MHB) is important for the patterning of the vertebrate brain and Fgf protein expressed in the MHB, in particular Fgf8a but not Fgf10, fulfill crucial organizing functions . Lee and co-workers have reported normal fgf8a expression in MHB of ext2 mutants at 24 hpf  and since fgf8a is known to regulate its own expression in MHB , they concluded that Fgf signaling during early brain patterning was normal. However, the reduced expression of etv5b and pea3 in MHB at 38 hpf in ext2 mutants compared to control embryos (Figure 1A-H) indicates that Fgf signaling in MHB at later stages of development is reduced. We then compared the expression of two marker genes for MHB signaling, eng1a and fgf8a, in ext2 mutants and control embryos. At 38 hpf a majority of ext2 mutants (60%, n = 14) display reduced expression of eng1a in MHB (Figure 2A-D) while in contrast a majority (76%, n = 17) of 38 hpf ext2 mutants express normal levels of fgf8a in MHB (additional files 1G-H). However, at 48 hpf all ext2 mutants express reduced levels of eng1a (2G-H) and fgf8a (Figure 2E-F). Thus, a portion of ext2 mutants express reduced levels of eng1a and fgf8a at 38 hpf and the reduction is fully penetrant at 48 hpf. Notably, the discovery that the reduction in etv5b and pea3 expression in the MHB of ext2 mutants (Figure 1A-H) precedes the fully penetrant reduction of eng1a and fgf8a expression (Figure 2E-H) suggests that a reduction of Fgf signaling is a possible cause of the reduction in MHB signaling. We conclude that although early brain patterning is normal, ext2 mutants show a gradually increased prevalence of abnormal brain patterning at later stages, consistent with a decline in Fgf signaling in the MHB after 24 hpf.
ext2interacts genetically with Fgf signaling during tail development
ext2has a role in Wnt signaling during zebrafish development
ptc1 expression and Hh dependent cell differentiation in the zebrafish retina and myotome are normal in ext2mutants
In contrast to Fgf and Wnt signaling, Hh signaling is not sensitized in ext2mutants
We next investigated whether Hh signaling in ext2 mutants is more susceptible to a reduction in Hh expression than in control embryos, in a similar way to the results presented above for Fgf and Wnt signaling. Nasevisius and co-workers have reported that inhibiting Hh function in zebrafish embryos by injecting a shha (sonic you) translation blocking morpholino (MO1-shha) results in embryos with U-shaped somites  which phenocopies the somite phenotype of the shha mutants . We injected 14 ng MO1-shha morpholino in offspring (n = 153) derived from crossings of heterozygous ext2 +/- individuals, and we scored the presence of U-shaped somites in ext2 mutants at 3 dpf (additional file 2A-D). However, MO1-shha injected ext2 mutants displayed a similar proportion of individuals with U-shaped somites as siblings (47% and 43%, respectively). Repeating the experiment with 7 ng of MO1-shha morpholino (n = 201) resulted in fewer individuals with U-shaped somites but the proportion of ext2 mutants and siblings with U-shaped somites was still similar (14% and 11%, respectively). Even a lower concentration of MO1-shha morpholino (3, 5 ng) did not result in any individuals with U-shaped somites. Taken together, these data indicate that the MO1-shha is equally effective in inducing U-shaped somites in ext2 mutants as in siblings.
In another experiment we tested Hh signaling in ext2 mutants for its sensitivity to cyclopamine, a drug which blocks Hh signaling by direct binding to the Hh signal transduction component Smoothened . If Hh signaling was less robust as a result of lowered HS content in ext2 mutants, it should be possible to block the expression of the Hh signaling downstream target ptc1 with a lower concentration of cyclopamine in ext2 mutants than in the siblings. However, when we treated the offspring (n = 100) from crossings between adult heterozygous ext2 mutant carriers with 50 μM cyclopamine 32-38 hpf, ptc1 expression was similarly reduced at 38 hpf in both ext2 mutants (identified by their smaller limb buds) and siblings (additional file 2E-H). Repeating this experiment with 25 μM cyclopamine did not result in an apparent reduction in ptc1 expression in either ext2 mutants or siblings and taken together these data indicate that cyclopamine is equally effective in reducing ptc1 expression in ext2 mutants and siblings. We finally tested whether ext2 and shha interact genetically by crossing the ext2 tw25e allele into the shha t4 background. To reduce the shha gene dose in ext2 mutants we crossed heterozygous ext2 +/- ;shha +/- double carriers with ext2 +/- carriers resulting in 50% of ext2 mutants lacking one functional shha gene. We reasoned that if the reduced gene dose of ssha in ext2 mutant did affect hedgehog signaling, then 50% of the isolated ext2 mutants in this cross would display changes in ptc1 expression. However, ptc1 expression was indistinguishable in all ext2 mutants at 38 hpf (n = 34, additional file 3A) and 48 hpf (n = 23, additional file 3B) indicating that one or two copies of a functional shha gene did not affect hedgehog signaling in ext2 mutants.
We conclude that reducing Hh signaling in ext2 mutants by several different methods does not affect expression of Hh signaling target genes or the severity of morphological phenotypes associated with defective Hh signaling. This is in strong contrast to Fgf and Wnt signaling. Taken together these data suggest that ext2 differently modulates the function of HS binding paracrine signaling factors.
In this study we present evidence for differences in the role of ext2 in Fgf, Wnt and Hh signaling during zebrafish embryo development. While ext2 functions as an enhancer of Fgf and Wnt signaling, Hh signaling is apparently not affected by the decreased HS levels caused by the ext2 deficiency.
Fgf signaling in ext2mutants
We previously reported that ext2 is required for Fgf10 signaling but not for Fgf24 or Fgf4 signaling during limb bud development, which suggested that different Fgfs have specific requirements for HSPGs in vivo . In this study we addressed the follow up question of whether ext2 has a general role in Fgf signaling. We show that the two Fgf signaling target genes pea3 and etv5b are reduced in tissues other than the developing limb and that all remaining expression in the mutants is sensitised for inhibition of Fgf signaling. For example, in the MHB where Fgf10 is not expressed, pea3 and etv5b expression are reduced in ext2 mutants at 38 hpf (Figure 1C, D, G, H). Fgf8 is a key mediator of MHB signaling and mice with the conditionally disrupted Ext1 gene, encoding one component of the HS copolymerase complex EXT1/EXT2 , exhibit reduced HS polymerization in the brain and display similar abnormal expression of Fgf and Engrailed proteins  as ext2 mutants (Figure 2F, H). MHB expression of etv5b and pea3 is reduced in ext2 mutants at 38 hpf (Figure 1D, H) but marker genes of MHB signaling such as fgf8a and eng1a are not generally reduced until 48 hpf (Figure 2E-H), suggesting that a reduction in Fgf signaling precedes and possibly causes the reduction in MHB signaling at 48 hpf. Notably, the patterning of the MHB contains several feedback loops (reviewed in ) and the reduced Fgf signaling in MHB of ext2 mutants (Figure 1A-H) might well cause a complex mispatterning of this tissue. Notably, although eng1a expression is subsequently reduced in the MHB, it is never absent (Figure 5 and data not shown) indicating that the MHB retains some signaling activity during embryo development. Moreover, in zebrafish, both Fgf8 and Fgf24 are required to promote posterior mesodermal development. Removal of both gene functions significantly impairs development of posterior tissues . The tail development in ext2 mutants is disturbed when either Fgf24 or Fgf8 is removed (Figure 3E, F) which suggests a role for ext2 in maintaining Fgf signaling pathways. Taken together, our data suggest that ext2 acts as a general enhancer of Fgf signaling after 24 hpf.
Mechanism of reduction of Fgf signaling in ext2mutant
So, by which mechanism is Fgf signaling reduced in ext2 mutants? HS binds to both Fgf ligands and receptors and facilitates receptor dimerization . In the most straightforward model, the reduced level of HS in the ext2 mutants would result in the formation of fewer signaling Fgf receptor dimers. However, the observation that etv5b expression is more greatly reduced than pea3 expression (Figure 1A-H), suggests more complex mechanisms. During zebrafish development, etv5b is expressed further away from cells expressing Fgf ligands than pea3, suggesting that cells typically require less Fgf ligands to elicit etv5b than pea3 expression . Therefore, if the only mechanism for reduction of Fgf signaling in ext2 mutants was a decreased participation of HS in ligand/receptor complexes, then pea3 expression would be expected to be more reduced than etv5b expression. In fact, the opposite was observed (Figure 1A-H). A role for HSPGs in Fgf transport, as has previously been described for Wnt and Hh transport , could possibly explain this result. If HS participates in Fgf transport, then cells positioned at greater distances from Fgf expressing cells, such as many etv5b expressing cells, would be exposed to a lower concentration of Fgf ligands, resulting in the observed effect in etv5b expression (Figure 1A-D). In contrast, pea3 is normally only expressed in cells close to Fgf expressing cells and would be less affected by reduced Fgf transport (Figure 1E-H). It should also be remembered that HSPGs, as well as Fgf receptors and ligands, interact with a large number of extracellular molecules  and the mechanisms by which ext2 enhances Fgf signaling might turn out to be complex.
Wnt signaling in ext2mutants
In Drosophila, sotv (ext2) and other ext genes are necessary for normal Wt function during wing development , but no study has yet reported a role for vertebrate ext genes in Wnt signaling. However, indirect evidence suggests that HS biosynthesis is necessary for vertebrate Wnt function since the HSPG glypican is required for vertebrate Wnt signaling [17–19]. Topoczewski and co-workers have shown that the zebrafish glypican gpc4 (knypek) potentiates non-canonical Wnt signaling and removal of gpc4 in the wnt11 mutant silberblick (slb) is correlated with defective anterior extension of midline cells, leading to failure of eye field separation and cyclopia. However, the glypican core protein can promote paracrine signaling independent of HS modification . Our results demonstrate that inhibition of Wnt11 translation in ext2 mutants phenocopies (Figure 4E-H) the gpc4 -/- ;wnt11 -/- double mutant eye phenotype  suggesting that it is indeed the HS modification of gpc4 that exhibit the Wnt signaling promoting function. We also studied the function of wnt5b in ext2 mutants. Wnt5b is known to physically interact with the the zebrafish glypican gpc3  and the wnt5b mutant pipetail (ppt) displays defects in axis elongation and cartilage differentiation which can be phenocopied by injection of a morpholino that blocks the Wnt5b translation . We found that a partial reduction of Wnt5b translation, insufficient to interfere with Wnt5b function in control embryos (Figure 4 I-J), still elicits the shorter body axis and hammerhead-like phenotype of ppt mutants  when injected in ext2 mutants (Figure 4K-L). Our data thus shows that ext2 potentiates vertebrate Wnt signaling and support the hypothesis that defects in Wnt5b function could be the explanation for the similar chondrocyte stacking phenotype in wnt5b and ext2 mutants .
Hh signaling in ext2mutants
Several studies have shown the requirement for ext2 (sotv) and other ext genes for proper distribution and signaling activity of Hh signaling molecules in Drosophila . However, in vertebrates the picture is less clear. In mice, the diffusion of Ihh during bone development is affected by a reduction of EXT1 expression [42, 43] but not of EXT2 . Previous studies of zebrafish ext2 mutants have not found evidence for defective Hh signaling during early brain patterning , limb development  or chondrocyte stacking . In this study we find further evidence for the lack of effects on Hh signaling in ext2 mutants; Hh signaling induces normal expression of the target gene ptc1 in ext2 mutants and Hh dependent cell differentiation in the retina and myotome is identical in control and ext2 mutants. In addition, control and ext2 mutant embryos respond to the same extent to reduction of Hh signaling, when Hh target gene expression and phenotypes associated with defective Hh signaling are studied. Since ext2 mutants have a fraction of the normal HS content in their tissues , we cannot exclude the possiblility that an even stronger reduction of HSPG biosynthesis would affect Hh signaling in zebrafish or even that more subtle aspects of Hh signaling are in fact altered in ext2 mutants. However, we conclude that, in comparison to the significant role for ext2 in Fgf and Wnt signaling, Hh signaling is virtually independent of ext2 function.
ext2 modulates the function of specific HS binding signaling factors differently
HSPG biosynthesis is spatially and temporally varied during embryonic development [35, 75, 76] and numerous studies have suggested the possibility of instructive functions for HS biosynthesis in paracrine signaling (reviewed by ). Our results suggest that genes which, like ext2, affect the HS content in tissues, are far more likely to regulate Fgf and Wnt signaling than Hh signaling. The explanation for the abnormal Hh function in mice with reduced Ext1 expression [42, 43] - but not in mice or zebrafish with reduced Ext2 expression ([41, 46] and this study) - might be that HS polymerization is more effectively reduced by a reduction in Ext1 than Ext2. Alternatively, since relative expression levels of mouse EXT1 and EXT2 have distinct effects on subsequent HSPG sulfation  and Hh signaling is disturbed in mice with abnormal HS structures , it is possible that Hh signaling is sensitive to putative abnormal HS structure caused by EXT1 but not EXT2 deficiency. Expression of ext2 is ubiquitous during zebrafish development  but it is interesting to note that ext1 genes exhibits specific spatial and temporal expression , indicating that HS levels and structure might vary according to specific effects on Fgf, Wnt and Hh signaling.
Most paracrine signaling factors bind to HSPGs and genes affecting polymerization of HS are generally assumed to have important but permissive roles for paracrine signaling. In this study we have found an unexpected specificity in how zebrafish ext2 affects the function of paracrine signaling factors, suggesting possible instructive functions for genes regulating HS polymerization during animal development.
WIK and Tübingen were used as wild-type strains. Mutant strains used were: the fgf8a mutant acerebellar (ace), the shha mutant sonic you (syo t4 ) and the ext2 mutant dackel (dak tw25e ). Embryos were cultured in E3 medium, with or without the addition of 0.003% 1-Phenyl-2-thiourea (PTU, Sigma) to inhibit pigmentation. Embryos were staged according to hours post fertilisation (hpf) . In experiments including offspring from heterozygous adults, mutants and siblings were distinguished by previously described phenotypic characterisation: ext2 mutants were identified by the reduced size and signaling activity of the pectoral fin bud after 32 hpf [14, 64] or by the abnormal jaw morphology after 3 dpf . The fgf8a mutants were identified based on the absence of MHB 24 hpf .
Microinjection of morpholino oligonucleotides
The following morpholino oligonucleotides (MO) were purchased from GeneTools: MO2-fgf24 5'-AGGAGACTCCCGTACCGTACTTGCC-3' , MO1-wnt11 5'-GAAAGTTCCTGTATTCTGTCATGTC-3' , MO1-wnt5b 5'-GTCCTTGGTTCATTCTCACATCCAT-3' , MO1-shha 5'-CAGCACTCTCGTCAAAAGCCGCATT-3' . All oligonucleotides were solubilized in sterile water and injected into one-cell stage zebrafish embryos at concentrations ranging from 3,5-14 ng/embryo.
In situ hybridisation was performed as previously described . The following mRNA in situ probes were used: eng1a , etv5b , fgf4 , fgf8a (Reifers et al., 1998), fgf10a , fgf24 (, pea3 , ptc1 . Antibody labeling was carried out on 12 μm thick cryosections and analyzed with a Leica confocal microscope. The following antibodies were used: mouse anti-Isl1 (Developmental Studies Hybridoma Bank; 1:50) and mouse anti-Zpr1 (University of Oregon; 1:200).
Cyclopamine and SU5402 treatement
Cyclopamine (Toronto Research Chemicals, cat# C988400) was dissolved in ethanol at 20 mM. Treatment was performed with 50 μM or 25 μM solution of cyclopamine or a corresponding control ethanol solution in E3 embryo medium. 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.
We thank Carl Neumann for support and advice, Henry Roehl and William Norton for helpful discussions, Gillian Brunt for technical assistance, and Lena Kjellén for critical comments on the manuscript. J.L. is funded by the Human Frontier Science Program and Swedish Foundation for Strategic Research through the EMBL Alumni Association.
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