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Keywords:

  • gastrulation;
  • left-right axis;
  • mouse;
  • Fgf signaling

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Fgf signaling plays pivotal roles in mouse gastrulation and left-right axis formation. However, although genetic analyses have revealed important aspects of Fgf signaling in these processes, the temporal resolution of genetic studies is low. Here, we combined whole-embryo culture with application of chemical compounds to inhibit Fgf signaling at specific time points. We found that sodium chlorate and PD173074 are potent inhibitors of Fgf signaling in early mouse embryos. Fgf signaling is required for the epithelial-to-mesenchymal transition of the primitive streak before the onset of gastrulation. Once gastrulation begins, Fgf signaling specifies mesodermal fates via the Ras/MAPK downstream cascade. Finally, Fgf signaling on the posterior side of the embryo during gastrulation induces Nodal expression in the node via Tbx6-Dll1, the initial event required for Nodal expression in the left lateral plate mesoderm. Developmental Dynamics 239:1768–1778, 2010. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Fibroblast growth factor (Fgf) signaling plays essential roles in both gastrulation and left–right (L-R) axis formation in vertebrates. In mouse, genetic analyses have revealed that Fgf8 and its receptor, Fgfr1, are involved in both events (Deng et al.,1994; Yamaguchi et al.,1994; Meyers and Martin,1999; Sun et al.,1999). Mouse gastrulation begins at embryonic day (E) 6.5 and produces the three germ layers of embryo proper: the definitive endoderm, the ectoderm, and the mesoderm. Gastrulation is initiated by formation of the primitive streak on the posterior side of the epiblast, where a dynamic cellular reorganization known as an epithelial-to-mesenchymal transition (EMT) occurs. Epiblast cells ingress through the primitive streak by EMT and either give rise to the mesodermal layer between the epiblast and visceral endoderm or intercalate into the visceral endoderm layer to become definitive endoderm. While Fgf8 is expressed in the primitive streak and visceral endoderm during gastrulation, Fgfr1 is expressed in the embryonic ectoderm (Yamaguchi et al.,1992; Crossley and Martin,1995; Lin et al.,2002). Fgf8 null mice (Fgf8−/−) show severe gastrulation defects, with epiblast cells accumulating in an inwardly protruding streak region (Sun et al.,1999). Similar defects are also observed in Fgfr1−/− embryos (Deng et al.,1994; Yamaguchi et al.,1994). In chimeric embryos consisting of wild-type and Fgfr1−/− cells, the Fgfr1−/− cells are unable to ingress through the primitive streak and accumulate in the streak region due to a failure to down-regulate E-cadherin (Ciruna et al.,1997; Ciruna and Rossant,2001). Fgf8−/− embryos also exhibit anterior patterning defects that expand the expression domain of marker genes for the anterior visceral endoderm (AVE) and anterior neuroectoderm (ANE). It remains unclear, however, which phenotypes in these mutants are a direct result of Fgf signaling defects and which are secondary defects.

At E7.5, L–R axis formation is triggered by nodal flow, a leftward fluid flow on the ventral surface of the node (Nonaka et al.,1998,2002). Nodal, a member of the TGF-β superfamily of molecules, is then produced in the node crown cells and is transferred to the lateral plate mesoderm (LPM) to induce its own expression on the left side (Collignon et al.,1996; Lowe et al.,1996; Brennan et al.,2002; Saijoh et al.,2003; Oki et al.,2007,2009). The L–R axis is established in the LPM through the reaction-diffusion of Nodal and Lefty, followed by asymmetric organogenesis (Nakamura et al.,2006 and reviewed in Hamada et al.,2002; Shiratori and Hamada,2006). Recently, small vesicles containing retinoic acid and Shh protein known as nodal vesicle parcels (NVPs) were found to be released on the ventral surface of the node and to be transported by nodal flow towards the left side of the node (Tanaka et al.,2005). The release of NVPs from the apical surface of the node cells is dependent on Fgf signaling (Tanaka et al.,2005). Embryos treated with SU5402, a chemical inhibitor of Fgfr1, eliminated both NVP release and the subsequent calcium ion influx around the node, which is stronger on the left side than on the right side in wild-type embryos (McGrath et al.,2003). However, it is unclear whether NVPs are indispensable for L–R axis formation or lead to Nodal expression on the left side of the LPM. Notably, Nodal expression in the node is lost or weak in half of the hypomorphic Fgf8 mutant embryos (Fgf8neo/−, Meyers and Martin,1999). Subsequent Nodal expression in the LPM is also lost, resulting in right isomerism of the thoracic organs. However, it remains unclear why reduced Fgf8 expression impairs Nodal expression.

Many efforts have focused on understanding Fgf signaling at the molecular level (reviewed in Eswarakumar et al.,2005; Thisse and Thisse,2005). To date, 22 Fgf ligands and four receptors have been identified, and these form ligand-receptor complexes with some specificity. Fgfr1 and Fgf8 are thought to be specific partners in gastrulating embryos, due to the similarity of their mutant phenotypes. Fgf ligands require heparin or heparan sulfate (HS) for their signaling. Heparin and HS are sulfated glycosaminoglycans (GAGs) that serve as Fgf coreceptors that facilitate interaction with Fgfrs. Upon complex formation, dimerized Fgfrs auto-phoshorylate their tyrosine residues and trigger intracellular signaling, such as the Ras/MAPK, PLCγ, and PI3K pathways. However, which cascades are involved in mouse gastrulation and L–R axis formation is largely unknown. To address this question, proteomic or transcriptomic analysis together with temporal inhibition of specific pathways at specific time points by Cre-mediated conditional knock-outs or specific inhibitor treatment are needed. The use of a specific Fgf inhibitor has the advantage of being easy, high throughput, and more immediate in effect. However, it is crucial that the inhibitor be proven to be specific for Fgf signaling and that its molecular action be well-characterized.

In this report, we show that sodium chlorate and PD173074, but not SU5402, are potent inhibitors of Fgf signaling in mouse embryos from gastrulation to early somite stages. Culturing embryos with these inhibitors not only mimics the phenotype of Fgf8 and Fgfr1 mutants, but also provides new insights into the action of Fgf signaling in gastrulation and L–R axis formation.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Timed Treatment of Sodium Chlorate Can Be Used to Dissect the Role of Fgf Signal During Gastrulation

Sulfated glycosaminoglycans (GAGs) are distributed in the basement membrane of the epiblast and the extracellular matrix of mesoderm cells in gastrulating mouse embryos (Garcia-Garcia and Anderson,2003). Because heparan sulfate (HS), a sulfated GAG, is necessary for Fgf signaling (Bernfield et al.,1999), we used sodium chlorate as an inhibitor of sulfated GAG biosynthesis to examine the role of Fgf signaling during gastrulation (Greve et al.,1988; Ullrich and Huber,2001). Thus, we collected mouse embryos at embryonic day (E) 6.2 or E6.5, and cultured for them for 16–20 hr with 15 mM chlorate, a concentration that we previously showed to inhibit sulfated GAG biosynthesis at early somite stages (Oki et al.,2007). Chlorate-treated embryos were fixed and subjected to morphological and histological analysis. When pre-gastrulation E6.2 embryos were cultured with chlorate, the primitive streak exhibited an irregular shape, with the epiblast layer protruding inwardly into the proamniotic cavity and a mass of cells accumulating in the protrusion, which was not seen in control cultures (Fig. 1A, E, C, G). The cells in the protrusion expressed Lim1, a marker gene for nascent mesoderm (Shawlot and Behringer,1995), but failed to downregulate E-cadherin, suggesting that EMT was impaired (see Fig. S1, which is available online). On the other hand, embryos that were cultured with chlorate from E6.5 (at the onset of gastrulation) onward showed normal ingression of mesodermal cells through the streak and lateral migration (Fig. 1D, H; compare with Fig. 1B, F). In both cases, the synthesis of HS was significantly inhibited in chlorate-treated embryos, whereas HS was normal in control cultures (Fig. 1I–L).

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Figure 1. Loss of heparan sulfate causes impaired epithelial-to-mesenchymal transition similar to that in Fgf8 and Fgfr1 mutant embryos. A–D: Lateral views of control (A, B) and chlorate-treated (C, D) embryos, with the anterior to the left. The embryonic stages at which the culture was started are indicated on the left. When the embryos are cultured with chlorate from E6.2 for 16 hr, severe defects are observed in the embryonic portion (C). E–L: Transverse sections of control (E, F, I, J) and chlorate-treated (G, H, K, L) embryos at the level indicated in A–D. Anterior is to the left. F-actin (phalloidin, green) and nuclei (DAPI, magenta) are shown in E–H, and heparan sulfate (anti-HS IgM, red) and nuclei (DAPI, blue) are shown in I–L. Arrowheads in G indicate the protruded ectoderm occupying the amniotic cavity. The amount of heparan sulfate is dramatically reduced in chlorate-treated embryos (K, L). a, anterior; p, posterior.

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The irregular streak formation in cultured embryos lacking HS resembles the phenotype of Fgf8−/− and Fgfr1−/− embryos (Deng et al.,1994; Yamaguchi et al.,1994; Sun et al.,1999; Ciruna and Rossant,2001), suggesting that chlorate successfully inhibited Fgf signaling. We next investigated to what extent the chlorate culture system was able to mimic the phenotype of Fgf8 and Fgfr1 mutants. Because Fgf8−/− and Fgfr1−/− embryos show perturbed expression of posterior and axial marker genes (Deng et al.,1994; Yamaguchi et al.,1994; Sun et al.,1999; Ciruna and Rossant,2001), we cultured E6.5 embryos for 16–24 hr with 15 mM chlorate and analyzed gene expression. As expected, chlorate-treated embryos showed the same altered marker gene expression as Fgf8−/− and Fgfr1−/− embryos. T, a marker of the primitive streak and axial mesoderm at E7.5 (Wilkinson et al.,1990; Herrmann,1991), was expressed in the primitive streak, but its expression was not extended anteriorly (Fig. 2A; n=12/12). Tbx6, which is expressed in the primitive streak and nascent mesoderm (Chapman et al.,1996), was lost (Fig. 2B; n=7/7). The expression of Snail, a zinc finger transcription factor that is essential for EMT during gastrulation (Nieto et al.,1992; Smith et al.,1992; Carver et al.,2001), was also absent from the primitive streak (Fig. 2C; n=9/9). FoxA2, which is expressed in the anterior streak at E6.75 and in the node and notochord from E7.5 (Ang et al.,1993; Sasaki and Hogan,1993), was expressed in the anterior streak, although they were at the equivalent stage of E7.5 (Fig. 2D; n=4/5). Nodal expression, which is observed in the posterior embryonic ectoderm and visceral endoderm at E6.5 and in the node at E7.5 (Conlon et al.,1994), persisted in the ectoderm without being confined to the node (Fig. 2E; n=6/6). Shh, which marks the anterior midline structures, such as the notochord, at E7.5 (Echelard et al.,1993; Roelink et al.,1994), was broadly expressed at the distal tip of the embryo without extending anteriorly (Fig. 2F; n=2/6). We also examined the expression of Sef, a probable target gene of Fgf signaling (Lin et al.,2002). In chlorate-treated embryos, the expression of Sef in the streak region was significantly diminished (Fig. 2G; n=7/7). These alterations in gene expression indicate that mesodermal patterning is severely affected in chlorate-treated embryos. Importantly, the shape of the primitive streak and the migration of mesodermal cells were relatively normal in the embryos cultured with chlorate from E6.5 (Fig. 1H). However, the embryos exhibited perturbed marker expression similar to that seen in Fgf8 and Fgfr1 mutants, suggesting that the perturbation of gene expression seen in both mutants is independent of the EMT defects and is directly regulated by Fgf signaling.

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Figure 2. Chlorate treatment at gastrulation causes mesodermal patterning defects similar to those in Fgf8 and Fgfr1 mutant embryos. A–G: Embryos were cultured from E6.5 for 16 hr (A–E, G) or 24 hr (F) in the absence (top) or presence (bottom) of 15 mM chlorate. Expression of various marker genes was examined by whole-mount in situ hybridization. The genes examined are indicated in each panel. Anterior is to the left (A–E, G) or at the top (F). l, left; r, right.

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Interestingly, Fgf8−/− embryos show patterning defects in anterior tissues at E7.5, even though Fgfr1 is not expressed in the visceral endoderm (Yamaguchi et al.,1992; Sun et al.,1999). For example, the expression of anterior visceral endoderm (AVE) markers Hex and Hesx1 (Hermesz et al.,1996; Thomas et al.,1998) is expanded, and anterior neuroectoderm (ANE) markers Six3 and Otx2 (Simeone et al.,1993; Oliver et al.,1995) are broadly expressed. In embryos cultured with chlorate from E6.5, the expression domain of AVE and ANE marker genes was expanded, reaching the distal tip of the embryo (Fig. 3A–D; n=4/4 for each gene), suggesting that the perturbation of these anterior markers is also independent of EMT defects and results indirectly from loss of Fgf signaling during gastrulation (see Discussion section). In summary, the gene expression patterns of chlorate-treated embryos resemble that of Fgf mutants, suggesting that chlorate treatment causes loss of HS, resulting in inhibition of Fgf signaling in gastrulating embryos.

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Figure 3. Chlorate treatment at gastrulation causes disturbed AVE and ANE marker gene expression similar to that in Fgf8 and Fgfr1 mutant embryos. A–D: Embryos were cultured from E6.5 for 16 hr in the absence (top) or presence (bottom) of 15 mM chlorate. Expression of AVE and ANE marker genes was examined by whole-mount in situ hybridization. The genes examined are indicated in each panel. Anterior is to the left.

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PD173074, But Not SU5402, Is a Potent Inhibitor of Fgf Signaling in Gastrulating Mouse Embryos

Although our data indicate that chlorate causes gastrulation defects, probably by inhibiting HS-dependent Fgf signaling, HS is also known to mediate other signaling molecules, such as BMPs and Wnts (Hacker et al.,2005). HS secreted into the extracellular space interacts with these proteins and serves as a scaffold for establishing molecular gradients, as shown in Drosophila (Belenkaya et al.,2004; Kirkpatrick et al.,2004; Kreuger et al.,2004). Although the gastrulation defects in the chlorate-treated embryos differ from those of BMP and Wnt signaling mutants, we nonetheless examined whether Fgfr-specific inhibitors have the same effect on gastrulation as chlorate.

We first tested a synthetic Fgfr1 inhibitor, SU5402 (Mohammadi et al.,1997). This compound is widely used to inhibit Fgf signaling in gastrulating Xenopus and zebrafish embryos and causes severe defects in mesoderm patterning (e.g., Griffin and Kimelman,2003; Fletcher and Harland,2008). We cultured E6.5 embryos with 40 μM SU5402 for 16 hr and found that Tbx6 expression was normal (data not shown). We also tested a higher dose of SU5402 (100 μM), but the expression of T, Nodal, and Snail was still unaffected (data not shown). It is unlikely that the SU5402 we used had decreased inhibitory activity, since we were able to reproduce the published data with this compound (Corson et al.,2003; Maretto et al.,2008). Erk phosphorylation was diminished in the extra-embryonic ectoderm when E6.2 embryos were cultured in 40 μM SU5402 for 1.5 hr, and Tbx6 expression was downregulated when E7.2 embryos were cultured in the same concentration of SU5402 for 20 hr (data not shown). These results suggest that the action of SU5402 is context-dependent, and this compound is not able to perturb gene expression in the same ways as chlorate treatment.

We next tested another compound, PD173074, which inhibits auto-phosphorylation of Fgfr1 with higher specificity and affinity than does SU5402 (Mohammadi et al.,1998). Although this compound is widely used in cell biological experiments and is able to substitute for SU5402 at lower doses (e.g., Ying et al.,2008), to our knowledge there has been no report of PD173074 treatment in early vertebrate development. We cultured E6.5 embryos for 16 hr in various concentration of PD173074 (0.2–5 μM) and examined the expression of Tbx6. Based on the staining level, Tbx6 transcripts were reduced by about half with 0.5 μM PD173074, and were completely abolished at more than 1 μM PD173074 (Fig. 4B; see also Fig. 6A, D, and data not shown). We, therefore, used 1 μM of the compound to test whether it could recapitulate the perturbed gene expression observed in Fgf8 and Fgfr1 mutants. Notably, PD173074 treatment of E6.5 embryos resulted in expression patterns that were indistinguishable from those of chlorate-treated and Fgf8 and Fgfr1 mutant embryos (Fig. 4A–J). In addition, embryos cultured under these conditions did not exhibit EMT defects, consistent with chlorate-treated E6.5 embryos (data not shown). These results indicate that unlike SU5402, PD173074 is able to inhibit Fgfr1 in gastrulating embryos.

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Figure 4. PD173074 treatment at gastrulation phenocopies Fgf8 and Fgfr1 mutant embryos. A–J: Embryos were cultured from E6.5 for 16 hr (A–E, G–J) or 24 hr (F) with DMSO (top) or 1 μM PD173074 (bottom). Expression of various marker genes was examined by whole-mount in situ hybridization. The genes examined are indicated in each panel. Anterior is to the left (A–E, G–J) or at the top (F). The number of embryos with perturbed marker gene expression is as follows: 3/3 (A), 10/10 (B), 3/3 (C), 3/5 (D), 3/3 (E), 4/4 (F), 7/7 (G), 6/6 (H), 3/3 (I), 3/3 (J).

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Fgf Signaling Is Required for the Expression of Nodal and Its Upstream Genes

It has been reported that Nodal expression in the node and LPM disappears in hypomorphic Fgf8 mutants (Fgf8neo/−) (Meyers and Martin,1999). Because Nodal expression in the node is required for its subsequent expression in the LPM, it is likely that loss of Fgf8 principally affects Nodal expression in the node. However, it was unclear why reduced Fgf8 expression resulted in impaired expression of Nodal in the node. To address this question, we first examined whether Nodal expression in the node was diminished when cultured with Fgf signaling inhibitors. We collected embryos at the late-bud stage, just prior to the generation of nodal flow and Nodal expression in the node, and cultured them with PD173074 or chlorate. In control cultures, Nodal expression became apparent in the node when embryos were cultured until the two- to three-somite stage (for 12–14 hr; Fig. 5A; n=4/4). When the embryos were cultured further, Nodal expression also appeared in the left LPM, and eventually expanded throughout the LPM by the five- to six-somite stage (for 16–20 hr; data not shown and Fig. 5B; n=8/10). In contrast, embryos cultured with 1 μM PD173074 failed to express Nodal in the node at the two- to three-somite stage or in the LPM at the five- to six-somite stage (Fig. 5E, F; n=4/4 and 10/10, respectively). Similar results were obtained by culturing until the two- to three-somite stage with 50 mM chlorate, although culturing at this concentration until the five- to six-somite stage caused severe growth retardation (Fig. 5I; n=6/6 and data not shown). Altogether, these results are consistent with the impaired Nodal expression observed in Fgf8 hypomorphic mutants.

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Figure 5. Inhibition of Fgf signaling affects the Tbx6-Dll1 cascade and results in diminished Nodal expression in the node. A–K: Expression of Nodal (A, B, E, F, I), Dll1 (C, G, J), and Tbx6 (D, H, K) in embryos cultured with DMSO (A–D), 1 μM PD173074 (E–H), or 50 mM chlorate (I–K). Embryos collected at the late-bud stage were cultured until the two- to three-somite stage, except in B and F, which show embryos cultured until the five- to six-somite stage. Anterior is at the top.

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We next investigated the expression of genes upstream of Nodal. Previous studies suggested that Nodal expression in the node requires the Notch ligand Dll1, which activates the node-specific enhancer of Nodal via the Notch signal-dependent transcription factor Rbp-Jκ (Krebs et al.,2003; Raya et al.,2003). Furthermore, genetic and biochemical analyses have revealed that Dll1 is a direct target of Tbx6 (White and Chapman,2005; Hadjantonakis et al.,2008). We, therefore, investigated the expression of Dll1 and Tbx6 in PD173074- or chlorate-treated embryos. In control cultured embryos at the late-bud stage to the 2- to 3-somite stage, Dll1 and Tbx6 are expressed in the pre-somitic mesoderm encircling the node crown cells (Fig. 5C, D; n=4 for each gene). However, in embryos cultured with PD173074 or chlorate, the expression of both genes was greatly reduced in this region (Fig. 5G, H, J, K; n=5 for each case). Because the reduction in Dll1 expression seems to be more severe than observed in Tbx6−/− embryos (Hadjantonakis et al.,2008), Dll1 may be regulated by other transcription factors in addition to Tbx6. Nonetheless, these results suggest that Fgf signaling induces Nodal expression in the node by the Tbx6-Dll1 cascade. In addition, this cascade probably still functions even after Nodal expression in the node has been established, because the expression of Nodal in the node, as well as that of Tbx6 and Dll1, is significantly reduced when embryos are cultured with PD173074 to the five- to six-somite stage from head-fold stage, when Nodal expression is already apparent in the node (data not shown). Thus, Fgf signaling is necessary for both initiating and maintaining Nodal expression in the node through the Tbx6-Dll1 cascade.

Fgf Signaling Regulates Tbx6 Expression Through the Ras/MAPK Cascade

Fgfr signals mainly through three pathways: the Ras/MAPK, PLCγ, and PI3K cascades (reviewed in Eswarakumar et al.,2005; Thisse and Thisse,2005). While Fgf signaling is necessary for gastrulation and mesoderm patterning, it is largely unknown which cascade Fgf signaling uses in these developmental processes. To investigate this question, we tested available chemical compounds that specifically inhibit these cascades. E6.5 embryos were cultured for 16 hr with inhibitors for Mek (PD98059 at 100 μM or U0126 at 100 μM), PLCγ (U73122 at 8 μM), or PI3K (LY294002 at 100 μM) and were examined for Tbx6 expression. However, no alterations in Tbx6 expression were observed with either compound (Fig. 6A–C; n=5 for each compound; and data not shown). We next tested the combination of these compounds with a low dose of Fgfr inhibitor that partially impairs Fgf signaling. Although no effects were observed by either inhibitor in conjunction with 40 μM SU5402 (data not shown), culturing with PD173074 alone at 0.5 μM decreased Tbx6 expression to about half that of controls (Fig. 6A, D; n=5). Under these sensitized conditions (0.5 μM PD173074), treatment with the Mek inhibitors (100 μM PD98059 or 100 μM U0126) completely eliminated Tbx6 expression (Fig. 6E, F; n=8 for each compound). In contrast, the PLCγ or PI3K inhibitor (20 μM U73122 or 100 μM LY294002) had no effect on Tbx6 expression (data not shown). This is the first evidence suggesting that Fgf signaling exerts its effects through the Ras/MAPK cascade during gastrulation.

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Figure 6. Synergistic effect of Fgfr and Mek inhibitors on Tbx6 expression. A–F: E6.5 embryos were cultured for 16 hr with DMSO (A, D), 100 μM PD98059 (B,E) or 100 μM U0126 (C,F) in the absence (A–C) or presence (D–F) of 100 μM PD173074. Anterior is to the left. PD98059 (B) or U0126 (C) alone does not affect Tbx6 expression, but in combination with a low dose of PD173074, they eliminate Tbx6 expression (E, F).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Fgf Signaling in L–R Axis Formation

Although hypomorphic Fgf8 mutants lack Nodal expression in the node and the left LPM (Meyers and Martin,1999), the mechanism by which Fgf8 and Nodal expression are linked was unclear. Here, we provided evidence that Fgf signaling is necessary for the expression of Tbx6 and Dll1, genes upstream of Nodal in the node. In addition, although Tbx6 expression is lost in Fgfr1 mutant embryos, it was not clear whether this is caused by EMT defects (Ciruna and Rossant,2001). We demonstrated that Fgf signaling continuously induces Tbx6 expression throughout gastrulation and at the early-somite stage, irrespective of EMT defects. Taken together with previous findings (Krebs et al.,2003; Raya et al.,2003; White and Chapman,2005; Hadjantonakis et al.,2008), our data suggest a model that explains how Fgf signaling induces Nodal expression in the node: Fgf signaling initially induces Tbx6 expression in the streak region, which in turn enhances Dll1 expression. The resulting Notch signal induces Nodal expression in the node by activating an enhancer located in the 5′ region of Nodal.

How, then, is Fgf signaling involved in Nodal expression in the left LPM? The following three models have been proposed to date: First, Fgf8 protein produced in the primitive streak diffuses asymmetrically to the LPM and induces Nodal expression in the left LPM. This model is supported by the induction of Nodal expression in the right LPM by implantation of Fgf8-soaked beads (Meyers and Martin,1999). However, it is unclear whether Fgf8 is the endogenous inducer of Nodal expression in the LPM. In fact, Fgf8 is not only expressed in the primitive streak, but also shows bilaterally symmetrical expression in the anterior LPM (Crossley and Martin,1995; data not shown), making it unlikely that Fgf8 directly induces Nodal expression in the LPM. Instead, it is more likely that Fgf signaling in the LPM is involved in the expression of Nodal signaling components, such as Gdf1, Cryptic, and Smads, so that the cells around Fgf8-soaked beads are more sensitive to Nodal signals from the node and ectopically express Nodal as a result. To test this model, Nodal expression in the LPM of conditional mutants specifically lacking Fgfr1 in the LPM should be examined.

In the second model, Fgf signaling allows the release of NVPs containing Shh and retinoic acid, which in turn enhances calcium ion influx on the left side of the node and eventually induces Nodal expression in the left LPM. However, a recent report has demonstrated that Nodal is expressed normally in mutant embryos that cannot respond to Hedgehog signaling specifically in the node region, indicating that Shh carried by NVPs is dispensable for Nodal expression in the LPM (Tsiairis and McMahon,2009). Furthermore, we found that Nodal was expressed normally in the left LPM when late bud stage embryos were cultured with 20 μM SU5402 (data not shown), a concentration at which NVP release is considerably reduced (Tanaka et al.,2005). This suggests that the NVP-calcium cascade may not play a prominent role in L–R axis formation.

In the third model, Fgf signaling is required for Nodal expression only in the node. Nodal produced in the node likely diffuses to the LPM to induce Nodal expression there via a Nodal positive-regulatory loop (Brennan et al.,2002; Saijoh et al.,2003; Oki et al.,2007,2009). This is the simplest model and does not require Fgf signaling in the LPM. This model is supported by our observation that PD173074-treated embryos that lost Nodal expression in the LPM always failed to correctly express Nodal in the node.

Fgf Signaling in Gastrulating Embryos

Fgf8 and Fgfr1 mutants show not only EMT defects during primitive streak formation, but also mesodermal patterning and AVE and ANE defects. Although the direct target genes of Fgf signaling are largely unknown, it has been shown that Fgf signaling in gastrulating embryos has two major effects: one is to induce Snail expression, which advances EMT by downregulating E-cadherin, and the other is to induce T and Tbx6 expression, which specify mesodermal fates (Ciruna and Rossant,2001). However, it was unclear whether these two processes are related to each other or independent.

The approach we used in this study, whole-embryo culture combined with application of chemical compounds, offered the advantage of inhibiting Fgf signaling at specific time points. This permitted us to dissect out the defects found in Fgf8 and Fgfr1 mutant embryos. For example, our results suggest that most of the gene expression perturbations seen in Fgf8 and Fgfr1 mutants are not due to EMT defects, since E6.5 embryos cultured with chlorate or PD173074 had normal EMT but abnormal gene expression patterns. This interpretation is supported by the complementary results in p38IP mutants (Zohn et al.,2006), which show EMT defects reminiscent of those seen in Fgf8 and Fgfr1 mutants, but do not appear to have altered Fgf signaling, based on their normal mesodermal patterning (Tbx6 expression).

Interestingly, we found that inhibiting Fgf signaling at E6.2, but not at E6.5, caused EMT defects, suggesting that Fgf signaling is required for EMT before the onset of gastrulation. Notably, inhibiting Fgf signaling at E6.5 decreased Snail expression, suggesting that the EMT defects in Fgf8 and Fgfr1 mutants are not caused solely by impaired Snail expression. This is consistent with the phenotype of Snail mutants, in which EMT in the primitive streak occurs normally (Carver et al.,2001). It is likely that EMT and correct cell movement during gastrulation require Snail and other unknown molecules that are downstream of Fgf signaling.

As shown here and in previous reports, embryos that lack Fgf signaling show widely perturbed marker gene expression. Because Fgf8 and Fgfr1 are expressed on the posterior side of the embryo (Yamaguchi et al.,1992; Crossley and Martin,1995), altered expression of the posterior markers T, Tbx6, and Snail is most simply explained by the loss of the Fgf signal on the posterior side. However, why is the expression of AVE and ANE marker genes altered in embryos with defective Fgf signaling? The simplest explanation is that Fgf8 produced in the visceral endoderm restricts the expansion of both marker genes. Fgfr1 is expressed in the embryonic ectoderm during gastrulation, whereas Fgfr2 expression appears in the AVE at E7.5 (Egea et al.,2008), possibly mediating Fgf8 signaling in these tissues. Alternatively, aberrant expression of AVE and ANE marker genes may be explained by the continued expression of Nodal throughout the embryonic ectoderm in these embryos. It has been shown that excessive Nodal signal expands the distal visceral endoderm, the precursor of the AVE (Yamamoto et al.,2009). In embryos with defective Fgf signaling, Nodal expression persists through the embryonic ectoderm, likely leading to expansion of the AVE. The ANE may then be expanded due to signals from the enlarged AVE. Because Nodal expression in the embryonic ectoderm and the node is regulated by different enhancers (Adachi et al.,1999; Norris and Robertson,1999), persistent expression of Nodal in embryos lacking Fgf signaling cannot be explained by the Tbx6-Dll1 cascade around the node.

Finally, our data suggest that Fgf signaling in mesodermal patterning functions through the Ras/MAPK cascade, as assessed by Tbx6 expression. This is consistent with previous studies in Xenopus and zebrafish showing that Fgf signaling acts via the Ras/MAPK cascade during gastrulation and mesoderm patterning (Christen and Slack,1999; Curran and Grainger,2000; Shinya et al.,2001). However, although phosphorylation of Erk during gastrulation in frogs and fish is robust, it is transient and patchy in the primitive streak of mouse embryos, probably due to the action of feedback inhibitors such as Sprouty and Sef (Corson et al.,2003). Therefore, the transient phosphorylation of Erk may be sufficient to induce Tbx6 expression in mouse embryos.

It should be noted that mesodermal patterning is unaffected in Fgfr1ΔFrs homozygous mutants, which lack the binding site for Frs2/3, adaptor proteins indispensable for Fgfr1 signaling via the Ras/MAPK and PI3K cascades (Hoch and Soriano,2006). How can the discrepancy between this report and our data be explained? When only Mek inhibitor was added in our whole-embryo cultures, Tbx6 expression was not affected, whereas Mek inhibitor together with a low dose of Fgfr inhibitor eliminated Tbx6 expression. Thus, mesodermal patterning in mouse embryos may require all three intracellular Fgf signaling cascades, though the Ras/MAPK cascade appears to be of primary importance in this process. Further studies are required to uncover the role of each Fgf signaling cascade during gastrulation.

In summary, we used whole-embryo culture combined with application of chemical inhibitors to dissect the role of Fgf signaling during gastrulation and left–right axis formation. Although this approach cannot be applied to tissue-specific analysis, unlike Cre-mediated conditional knock-outs, it offers the great advantage of stage-specific and high-throughput analysis. Thus, this system is a powerful tool for analyzing the role of Fgf signaling with high temporal resolution.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Whole-Embryo Culture

The ICR mouse strain was used in this study. Noon on the day of the vaginal plug was designated as E0.5. Pregnant mice were sacrificed by cervical dislocation, and embryos were then collected in Hepes-buffered Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Embryos were cultured in DMEM with 75% rat serum. Embryos were cultured in a 50-ml centrifuge tube rotating in a humidified incubator containing 5% CO2 and 95% air. The following compounds were added to the culture medium at the concentrations indicated in the results section: the PAPS synthetase inhibitor chlorate (Nacalai), the Fgfr inhibitors SU5402 (Wako, Richmond, VA) and PD173074 (Sigma-Aldrich, St. Louis, MO), the Mek inhibitors PD98059 (Calbiochem, San Diego, CA) and U0126 (Santa Cruz, Santa Cruz, CA), the PLCγ inhibitor U73122 (Santa Cruz), the PI3K inhibitor, LY294002 (Wako), and DMSO as a vehicle control (maximally at 0.64%). The culture was terminated by treating with 4% paraformaldehyde at the time indicated in the Results section.

Whole-Mount In Situ Hybridization

Whole-mount in situ hybridization was performed according to a standard protocol. Briefly, fixed embryos were washed with phosphate-buffered saline (PBS) containing 0.1% Tween20 (PBS-Tween) and dehydrated with methanol. After treating with 6% H2O2/methanol, the rehydrated embryos were processed with 10 μg/ml proteinase K/PBS-Tween for 20 min. The embryos were then treated with 2 mg/ml glycine/PBS-Tween and re-fixed with 4% paraformaldehyde/0.2% glutaraldehyde. After prehybridization, embryos were incubated overnight in hybridization solution including digoxigenin-labeled riboprobe. The hybridized embryos were washed several times with increasing stringency, and finally incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche, Nutley, NJ). After extensive washing, the color was developed with NBT/BCIP (Roche). Probes used in this study were Tbx6, Snail, T, Shh, FoxA2, Hex, Hesx1, Six3, Otx2, Sef, Dll1, and Nodal.

Immunohistochemistry

Embryos fixed with 4% paraformaldehyde were washed with PBS containing 0.1% Triton X 100 (PBS-Triton), cryoprotected with 30% sucrose solution, and embedded in OCT compound (Sakura), followed by cryosectioning at a thickness of 6 μm. The sections were put on MAS-coated slide glasses (Matsunami) and air-dried. The sections were then washed with PBS-Triton, blocked with 5% skim milk, immunoreacted with mouse anti-HS antibody (10E4, Seikagaku) conjugated with Atto565 fluorescent dye (Lightning-Link Atto565 Conjugation Kit, Innova Biosciences), and incubated with Alexa Fluor 488 Phalloidin and DAPI (both from Molecular Probes, Eugene, OR). Photos of the sections were taken with a digital camera (Leica DFC 300FX, Wetzlar, Germany) attached to a fluorescence microscope (Leica).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

We thank Drs. H. Hamada, H. Sasaki, and T. Fujimori for critical reading of the manuscript. Probes for in situ hybridization were kind gifts from Drs. A. Nieto, H. Sasaki, S. Aizawa, P. Gruss, T. Gridley, H. Hamada, V. Papaioannou, R. Beddington, S. Ang, and S. Wilson. We also appreciate the technical support provided by the Research Support Center, Graduate School of Medical Sciences, Kyushu University. This work was supported by grants from the MEXT (C.M.), the JSPS (S.O.), and the Naito Foundation (C.M.).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY_22282_sm_suppfigS1.tif2222KSupporting Figure 1

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