1 Division of Early Embryogenesis, National Institute of Genetics, Mishima, 411-8540 Japan
2 Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan
3 Vertebrate Development Laboratory, Imperial Cancer Research Fund, 44 Lincolns Inn Fields, London WC2A 3PX, UK
* Present address: Department of Biological Sciences, Graduate School of Science, The University of Tokyo. Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
Author for correspondence (e-mail: htakeda{at}biol.s.u-tokyo.ac.jp)
Accepted August 29, 2001
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SUMMARY |
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Key words: Fgf, Somite, Segmentation, Tailbud, fused somites, after eight, her1, Zebrafish
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INTRODUCTION |
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The understanding of the molecular mechanisms creating periodicity of somite formation has been greatly advanced in the last few years. The remarkably cyclic expression pattern of chick hairy1, a hairy-related bHLH transcription factor, in the PSM provided the first evidence for an intrinsic molecular clock linked to somitogenesis (Palmeirim et al., 1997). The presence of the clock in vertebrate PSM was further supported by the periodic expression of several genes including other hairy-related genes in mouse (Jouve et al., 2000) and zebrafish (Sawada et al., 2000; Holley et al., 2000), and genes implicated in the Notch signalling pathway, lunatic fringe in mouse (Aulehla and Johnson, 1999; Forsberg et al., 1998) and chick (McGrew et al., 1998) or deltaC and deltaD in zebrafish (Jiang et al., 1998). The expression stripes of these genes appear in the tailbud each somite cycle, sweep caudorostrally across the PSM, and finally stabilize in the anterior PSM before segment border formation. Recent genetic studies in mouse and zebrafish demonstrated that Notch/Delta signalling is required for synclonization of the segmentation clock in the PSM (Jiang et al., 1998; Jiang et al., 2000; Pourquié, 1999).
Existence of the clock in the PSM has been predicted by theoretical models such as clock and wavefront (Cooke and Zeeman, 1976; Dale and Pourquié, 2000). In this model, the clock creates a temporal periodicity, such as a cyclic wave of gene expression, which would later be interpreted by the wavefront to generate spatial periodicity of the somites. The wavefront that exists in the anterior PSM gradually moves back as somitogenesis proceeds. Thus, the stepwise interaction between the clock and the wavefront (or periodic entry of the wave into the wavefront) leads to regularly spaced furrow formation. The model also predicts the presence of positional information restricting the wavefront to the anterior PSM. In facts, PSM cells, born in an immature state in the tailbud, become matured as they pass the intermediate to the anterior PSM, and finally acquire the wavefront activity that arrests the cyclic gene expression and initiates somite furrow formation (Holley et al., 2000). The zebrafish fused somites (fss) mutation bocks this maturation process, leading to no segmentation in the paraxial mesoderm (Holley et al., 2000; van Eeden et al., 1998). Although accumulated data of vertebrate somitogenesis support the clock-and-wavefront model, the presence and molecular nature of the positional information within the PSM are totally unknown.
Fgf receptor (Fgfr)-mediated signalling is implicated in somitogenesis: Fgfr1 is expressed in the PSM and the anterior part of segmented somites in mice and zebrafish (Sawada et al., 2000; Yamaguchi et al., 1992) and Fgfr1 knockout mice produce embryos with disturbed segment borders (Yamaguchi et al., 1994). Mice and zebrafish fgf8 mutants, however, do not proceed through gastrulation and do not show severe somitogenesis phenotypes, respectively (Sun et al., 1999; Reifers et al., 1998). Thus, in part due to early vital function and/or genetic redundancy of Fgf family and receptor genes, the precise role of Fgf signalling in segmentation has remained unclear. We report that Fgf/mitogen-activated protein kinase (MAPK) signalling activated in the posterior PSM is a crucial positional cue in restricting the maturation wavefront in the anterior PSM and maintains posterior PSM cells in an immature state in an after eight/deltaD- and fss-independent manner.
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Materials and Methods |
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Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed as described in Nikaido et al. (Nikaido et al., 1997).
Immunohistochemistry and western blot
For whole-mount immunostaining, embryos were fixed with 3.7% formaldehyde/0.2% glutaraldehyde/phosphate-buffered saline (PBS) for 1 hour at the room temperature. After washing with PBS, they were dehydrated with methanol and transferred to PBS. They were washed with MABT (Maleic buffer (0.15 M maleic acid, 0.1 M NaCl pH. 7.5 (MAB)/0.1% Triton X-100)) three times for 10 minutes, and MABDT (MAB/0.1% Triton X-100/1% DMSO) twice for 30 minutes. After blocking with 2% FCS/MABDT, the embryos were incubated in the blocking solution containing 1:10000 anti-di-phosphorylated ERK1 abd ERK2 (MAPK-YT) antibody (Sigma) at 4°C overnight. They were then washed with MABDT three times for 5 minutes, four times for 30 minutes, and incubated in blocking solution again for 30 minutes, followed by incubation with the second antibody (1:500 anti-mouse IgG biotin conjugated antibody) for 2 hours at the room temperature. After the washing with MABDT as described above, the signals were detected with ABC staining kit according to the manufacturers instruction (Vector Laboratory). For labelling mitotic cells, 1:200 dilution of anti-phosphorylated histone H3 (Upstate Biotechnology) was used as the first antibody (Saka and Smith, 2001).
Western blot analysis was performed following of the standard method for ECL western Blotting Detection Reagent (Amersham Pharmacia Biotech). Protein (10 µg/lane) were separated by 12.5% polyacrylamide gels and transferred to HybondTM ECLTM membranes (Amersham Pharmacia Biotech) by electroblotting. Monoclonal antibody against dpERK was used at the same concentration as used for immunostaining. Yolk cell was removed from embryos before homogenization. The results were analysed by use of Limi-Imager and Lumi Analyst (Roche Molecular Biochemicals).
Beads transplantation and SU5402 treatment
Heparin-insolubilized acrylic beads (Sigma) were washed three times in PBS and soaked in 0.5 µg/µl mouse recombinant FGF8b protein (R&D Systems) or 0.5 µg/µl BSA (Sigma) for two hours in room temperature. Transplantation was performed with tungsten needle into the posterior PSM of decorionated embryos at the two-somite stage.
SU5402 (Calbiochem) treatment was performed with manually dechorionated embryos at the two-somite stage. SU5402 of 10 mg/ml in DMSO was used as a stock solution and diluted before use. Embryos were soaked for 8 minutes in the medium containing SU5402 at a concentration of 0.2 mg/ml (2% DMSO), followed by intense wash.
Detection of apoptosis
Apoptosis in zebrafish whole-mount was detected according to a protocol given by the manufacturer with some modifications (Dead EndTM Colorimetric Apoptosis Detection System; Promega) (Gacrieli et al., 1992). After fixation overnight in 4% paraformaldehyde (PFA) in PBS, embryos were transferred in methanol and then rehydrated in PBST (PBS/0.1% Tween 20). Subsequently, embryos were digested in 5 µg/ml proteinase K in PBS for 5 minutes and postfixed for 20 minutes in 4% PFA in PBS. The embryos were then immersed in acetone for 7 minutes at 20°C and incubated in the equilibration buffer (provided in the kit) for 10 minutes at the room temperature (RT). After incubation 3 hours at 37°C in working strength terminal deoxynucleotidyl transferase (TdT) enzyme, the DNA end-labelling reaction using biotinylated dUTP was stopped by washing in 2xsaline sodium citrate (SSC) and PBST. Biotin was detected by horseradish-peroxidase-labelled streptavidin with diaminobenzidine (DAB).
Laser-assisted uncaging
Labelling PSM cells using caged fluorescein-dextran was performed mainly following that described by Gristsman et al. (Gristsman et al., 2000). 2% solution of dextran, DMNB-caged fluorescein and biotin (D-7146, Molecular probe) was injected into one- to two-cell-stage embryos. The embryos were left to develop under dark condition until use. Uncaging was performed with a few second pulse of 365 nm pulsed nitrogen laser (Laser Science) focused through a 40x dry objective of Zeiss Axioskop2 microscope. To ensure labeling of PSM cells at different levels of depth, the focus of the objective was changed at several times. Fluorescent and Nomarski images were sequentially acquired using chilled CCD (Hamamatsu Photonics), adjusted individually and overlaid.
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RESULTS |
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Transient manipulation of Fgf signalling alters somite size
To explore the role of Fgf/MAPK signal in the PSM, we manipulated the level of Fgf signalling and observed the consequences upon somite formation. Treatment at the two-somite stage with SU5402 for 8 minutes resulted in the formation of abnormally large somites after a period of four to five rounds of normal somite formation (Fig. 2A,B) (about 10 to 12 cells wide instead of six to eight cells wide in normal somites; the axial length of the seventh somite was 72 µm±3.8, n=10 after SU5402 treatment but 49 µm±5.7 for control treatment). Increase in somite size (Fig. 2E,F), however, is limited to one or two consecutive somites, and, thereafter, normal somite formation resumes, although the somites just posterior to the large ones are sometimes smaller in size (Fig. 2C,D). Histological sections confirm that the large somites do not show any cellular abnormalities such as apoptosis or increase in cell volume, but simply contain a larger number of somitic cells (Fig. 2E,F), except for a restricted cell death occasionally observed in the tailbud at later stages. Furthermore, the segmental expression of mesp and papc, markers for the anterior part of prospective and/or segmented somites, confirms that anteroposterior specification within large somites normally takes place (Fig. 3G-I).
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Alterations in somite size are caused by an altered pace of wavefront progression
According to the clock-and-wavefront (Cooke and Zeeman, 1976; Dale and Pourquié, 2000) model, the somite size is a function of the frequency of the segmentation clock and of the velocity of the maturation wavefront. Increase in somite size could therefore be achieved either by slowing down the oscillation or by accelerating the wavefront progression. To test this, we examined the cyclic expression of her1, a gene encoding a hairy-related bHLH transcription factor, in treated embryos. As shown in Fig. 3A, her1 expression usually appears as three stripes in the PSM (referred to as posterior, intermediate and anterior stripes) (Fig. 4A). A new wave of her1 expression appears in the tailbud every 30 minutes (the duration of one-somite formation in zebrafish), becomes narrower as it moves rostrally and finally stabilizes at the future segmentation point in the anterior PSM before decaying (Sawada et al., 2000; Holley et al., 2000). The expression pattern of her1 was examined at the six-somite when the prospective large somites were forming in the PSM after treatment at the two-somite stage. Comparison with control embryos reveals that the her1 stripe in the anterior PSM is always undetectable with the posterior two stripes intact (Fig. 3A-C), indicating that, after SU5402 treatment, the her1 cyclic expression prematurely terminates in the intermediate PSM, instead of the anterior PSM (the length from tailbud to the anterior border of the expression stripe (L)=566±23.3 µm for control (n=10) and 446±28.8 µm for SU5402 treated embryos (n=10)). The operation of the oscillator seems to be unaffected because a variety of posterior expression patterns are seen in treated embryos fixed at the same stage, implying a cyclic expression (data not shown). More importantly, somites are regularly specified (compare Fig. 2B,D with 2A,C), despite size difference, and an interval between intermediate and posterior her1 stripes is unchanged in treated embryos at any stage examined (Fig. 3B,C), indicating a normal pace of oscillation.
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We further examined the effect of exogenous Fgfs on her1 expression. Embryos were implanted with Fgf-soaked beads into the tailbud at the two-somite stage, and were fixed at various times after implantation. The bead does not significantly affect the pattern of her1 expression when located in the posterior to intermediate PSM (Fig. 3J,K). However, as the bead passes the intermediate to anterior PSM, the anterior boundary of the intermediate her1 stripe is anteriorly displaced when compared with that on the control side (Fig. 3L). In another words, the anterior part of the intermediate stripe does not reduce the slowing down rate and moves more anteriorly, resulting in an anteriorly broaden stripe. Transplantation of Fgf beads into fss embryos confirms that the affected her1 stripe is fss independent (Fig. 3M). Eventually, on the transplanted side, the her1 expression reaches more anteriorly than it does on the control side (Fig. 3L), which may cause an anterior shift in furrow formation, explaining the formation of smaller somites anterior to the transplanted beads. Taken together, the prolonged activation of Fgf/MAPK signalling delays maturation of PSM cells, and anteriorizes the domain transition of her1 expression (Fig. 4B), resulting in an anterior shift in furrow formation.
Fgf/MAPK signalling functions independently of the Notch or Fss pathway
To determine whether the Fgf-mediated positioning of PSM maturation is influenced by Notch/Delta pathway, we performed SU5402 treatment in after eight (aei) mutants, in which deltaD gene is defective (Holley et al., 2000). In aei embryos, the synchronized wave of her1 is lost (Jiang et al., 2000), while the mature anterior PSM expresses her1, deltaC and mesp (Fig. 3N) (Holley et al., 2000; Jiang et al., 2000; Sawada et al., 2000). As in wild-type (Fig. 3A-C) and fss (Fig. 3D,E) embryos, SU5402 treatment shifts the expression domains of her1 (Fig. 3N-P) to the intermediate region in aei mutants (L=520±22.6 µm for control aei (n=10) and 441±27.3 µm for SU5402-treated aei (n=10)). Furthermore, the activation pattern of ERK and the expression pattern of fgf8 in the PSM do not significantly change in fss and aei mutants (Fig. 1D-F). Their patterns in the segmented somites, however, varies in mutant embryos; nearly undetectable in fss (Fig. 1E,H,K) but tending to increase in aei mutants (arrowheads in Fig. 1F,I,L). Thus, the action of Fgf signalling in the PSM is likely to be independent of Notch/Delta and Fss pathways.
Manipulation of Fgf/MAPK signalling does not affect proliferation or behaviour of PSM cells
Finally, we tested whether the manipulation of Fgf/MAPK signalling affects cell death, cell proliferation and cell migration in the PSM. The numbers of apoptotic and mitotic cells were counted in histological sections of treated embryos by a modified TUNEL method (Gavrieli et al., 1992) and immunohistochemical staining with anti-phosphorylated histone H3 that recognizes cells in M phase (Saka and Smith, 2001). As summarized in Table 1, no significant difference was observed in cell death and proliferation in the PSM after manipulation of an Fgf signal (Fig. 5A-D). Cell behaviour in the PSM was examined by a photoactivation technique of caged substances (Gritsman et al., 2000). One- to two-cell embryos were injected with caged fluorescein-dextran dye and left to develop to the two-somite stage, when Fgf8-soaked beads were transplanted into the tailbud region. We then labelled, by laser-assisted uncaging, a group of PSM cells located anterior to the beads, as well as cells at the same axial level on the control side (Fig. 5E). The positions of those labelled cells were examined after 5 hours incubation (12-somite stage). As shown in Fig. 5F, although the somite boundaries are anteriorly displaced on the transplanted side, the axial position of labelled cells does not significantly change between the control and transplanted sides, indicating that no specific cell migration is induced by exogenous Fgfs.
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DISCUSSION |
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Modulating Fgf signalling resulted in alterations in somite size. Detailed analyses of gene expressions in manipulated wild-type and mutant embryos revealed a novel function of Fgf/MAPK signalling in the PSM, the maintenance of cells in an immature state that allows the her1 wave to sweep through the PSM. Suppression of Fgf signalling posteriorizes the domain shift of her1 expression, as well as the expression of other segmentation genes such as mesp and papc. This leads to a posterior shift in segment border formation and larger somites. These results are complementary to those obtained with transplantation of Fgf beads, strengthening the idea that an Fgf signal determines the position of segment border formation by negatively regulating the maturation of the PSM. As Fgf signal is known to have profound effects on many developmental processes such as cell growth and maintenance of progenitor cells (Szebenyi and Fallon 1999; Mathis et al., 2001), it is possible that manipulation of an Fgf signal locally changes the cell number in the PSM by regulating cell proliferation and/or cell migration within the mesoderm (axial, paraxial and lateral plate mesoderm). This could cause alterations in somite size. However, no such effect was observed in manipulated PSM, indicating that an Fgf signal in the PSM simply regulates the maturation status of cells without affecting cell proliferation or migration.
Our data are largely consistent with the clock-and-wavefront model in which a cyclic wave operates in conjunction with a maturation wavefront that gradually moves posteriorly, resulting in arrest of the cyclic wave and initiation of segment furrow formation (Cooke and Zeeman, 1976; Dale and Pourquié, 2000). Fgf/MAPK signalling negatively regulates the wavefront activity and restricts it to the anterior PSM that is devoid of MAPK activation (Fig. 4C). In zebrafish, the essential components of a conserved somite-making mechanism, the segmentation clock and wavefront, were shown to be Notch- and Fss-dependent, respectively. Zebrafish aei/deltaD mutation desychronizes the oscillation wave (Jiang et al., 2000), while, in the absence of Fss, the anterior PSM fails to acquire the wavefront activity (Holley et al., 2000). How could Fgf/MAPK signal interact with these components? In fact, it has been reported that the Ras/MAPK pathway interacts with the Notch pathway in C. elegans vulval development (Sundaram and Han, 1996) and malignant transformation of cultured cells (Fitzgerald et al., 2000). However, we failed to show any interaction between Fgf/MAPK and Notch or Fss pathways: modulating Fgf signalling exerts identical effects on wild-type and aei/DeltaD or fss mutants in terms of gene expression. Furthermore, the patterns of ERK activation and fgf8 expression in the PSM is not affected by aei/DeltaD and fss mutations. Thus, we conclude that the activation and action of Fgf/MAPK signalling in the PSM are not mediated by Notch or Fss pathway.
The fact that four to five somites are normally formed after SU5402 treatment indicates that the positioning of furrow formation is already specified or Fgf insensitive at least at the position -IV to -V in the PSM (Fig. 4C). The result also indicates that ERK activation in segmented somites (arrowheads in Fig. 1D,G,J,M) is not involved in segment border formation. Interestingly, the Fgf-sensitive region corresponds approximately to the heat-shock sensitive zone in zebrafish; that is, the initial defects in the segmental pattern of somite boundaries are observed five somites caudal to the forming somite at the time of heat shock (Roy et al., 1999). Our data suggest that position -IV to -V represents a position at which the level of Fgf/MAPK activation drops below a threshold, rendering the cells competent to maturation signals (Fig. 1A,B). In support of this, transplanted Fgf8 beads exert their effects only when they are located in the Fgf-negative anterior PSM (Fig. 3K,L). Importantly, the relative position of MAPK activation domain to the newly formed somite is kept constant in the PSM as the embryos extend. These observations are consistent with the idea that the level of Fgf/MAPK activation serves as a positional cue within the PSM. Recently, Dubrulle et al. (Dubrulle et al., 2001) have demonstrated that Fgf signalling controls somite boundary position in chick embryos, indicating conserved mechanisms in somite boundary determination among vertebrates. The existence of positional information along the anteroposterior axis in the PSM has been proposed by the clock-and-wavefront model (Cooke and Zeeman, 1976; Dale and Pourquié, 2000), and the present study is the first to provide evidence for the molecular identity of, at least, one positional cue that governs the positions of segment border formation, and thereby the size of somites.
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ACKNOWLEDGMENTS |
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