1 Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-8602 Japan
2 Early Embryogenesis, Department of Developmental Genetics, National Institute of Genetics, Yata 1111, Mishima, 411-8540 Japan
3 Kondoh Differentiation Signalling Project ERATO, JST, Kinkichihou Hatsumei Centre, 14 Kawaramachi, Yoshida, Sakyo-ku, Kyoto, 606-8305 Japan
* Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
Author for correspondence (e-mail: htakeda{at}biol.s.u-tokyo.ac.jp)
Accepted August 8, 2001
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SUMMARY |
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Key words: fgf3, Ras, ERK, morpholino, SU5402, regionalisation, Zebrafish
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INTRODUCTION |
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We address this question in zebrafish embryos by direct observation of the response to Fgfs. Fgf receptors are members of the receptor tyrosine kinase (RTK) superfamily (Klint and Claesson-Welsh, 1999; Ornitz, 2000). At present, four members are known and each of them has several splicing isoforms (Klint and Claesson-Welsh, 1999). In general, RTKs trigger, by way of Ras, a sequential activation of the mitogen-activated protein kinase (MAPK) (or extracellular regulated kinase (ERK)) signalling cascade (Cobb and Goldsmith, 1995). ERK is activated by dual phosphorylation of threonine and tyrosine residues by MAPK/ERK kinase (MEK) (Crews et al., 1992). A monoclonal antibody raised against a dually phosphorylated form of ERK (dpERK) has been successfully applied to Drosophila and Xenopus, and reveals a dynamic dpERK staining pattern in early embryos (Gabay et al., 1997a; Gabay et al., 1997b; Christen and Slack, 1999). In Drosophila, several different RTKs participate in ERK activation, while, in Xenopus, the Fgf family seems to be responsible for the full pattern of dpERK in early development as activation can be blocked by the expression of a dominant-negative form of Fgf receptor (Christen and Slack, 1999).
We find that ERK is activated in the zebrafish ANB. Like Xenopus, the activation in the ANB is attributable to an Fgf signal. Interestingly, ERK activation in the ANB is maintained in ace mutants, suggesting other Fgfs function in this region. We also examined the function of Fgf signalling in zebrafish forebrain patterning by use of several inhibitors of the Fgf/"Ras/MAPK signalling cascade, such as dominant-negative form of Ras (RasN17) and the Fgf receptor, and a chemical inhibitor of Fgfr, SU5402. In treated embryos, the induction of telencephalic territory proceeds normally but the development of the subpallial telencephalon is suppressed, indicating that Fgf signalling is required for the regionalisation within the telencephalon. Finally, we show using antisense that both Fgf3 and Fgf8 are required for normal development of the subpallial region.
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MATERIALS AND METHODS |
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mRNA and morpholino injection
Capped sense RNAs were synthesised using the mMESSAGE mMACHINETM large scale in vitro transcription kit (Ambion) from the plasmid containing full-length human RasN17 (Deng and Karin, 1994), Xenopus bFR4 (Hongo et al., 1999), and zebrafish fgf3, fgf3-myc, GFP and lacZ. GFP and lacZ RNA were used as a control. The mRNAs were diluted to 0.8-1.0 µg/µl with distilled water and 200-500 ng/µl (RasN17), 400 ng/µl (b
FR4), 5 ng/µl (fgf3-myc) or/and 5 or 100 ng/µl (fgf3) mRNAs were injected into one- to two-cell stage embryos. Morpholino oligonucleotides were solubilised in water at the concentration of 50 µg/µl. The resulting stock solution was diluted to working concentrations in water before injection into one- to two-cell stage embryos. Injected embryos were cultured in 1/3 Ringers solution until use. The sequences of morpholino oligonucleotides used in the present study were as follows (see Fig. 9N): fgf8-MO, 5'-TCAACCGTGAAGGTATGAGTCTC-3'; fgf3-MO, 5'-TATAACCATTGTGGCATGGCGGGAT-3'; 4-mis-MO, 5'-TATTACCTTTGTGGCATGCCGGCAT-3'; and fgf3-MO, 5'-CAACAAGAGCAGAATTATAACCATT-3'.
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Beads transplantation and SU5402 treatment
Mouse recombinant Fgf8b protein (R&D Systems) or BSA was used at the concentration of 0.25 µg/µl. Preparation of the beads soaked in Fgf8b or BSA were performed as described in Makita et al. (Makita et al., 1998). Dechorionated embryos at tailbud stage were placed in 3% methylcellulose in 1/3 Ringers solution and transplanted into the forebrain using a tungsten needle. After transplantation, embryos were cultured at 28.5°C in 1/3 Ringers solution until use.
For injection, 10 mg/ml SU5402 (Calbiochem) in DMSO was used. Dechorionated embryos were placed in 1.5% methylcellulose in 1/3 Ringers solution and injected into thick head region at tailbud stage. DMSO was injected as a control. Alternatively, dechorionated embryos at proper stages were soaked for 10 minutes in SU5402/1/3 Ringer solution at a concentration of 0.1 mg/ml, and washed several times with 1/3 Ringers solution. Treated embryos were cultured at 28.5°C in 1/3 Ringers solution until use. We found that the activity of SU5402 varied a great deal, depending on the lot number. Thus, the incubation was sometimes carried out at a concentration of 0.2 mg/ml.
Detection of apoptosis
Apoptosis in zebrafish whole-mounts was detected according to a protocol given by the manufacturer with some modifications (Dead EndTM Colorimetric Apoptosis Detection System, Promega). After fixation overnight in 4% paraformaldehyde (PFA) in phosphate-buffered saline (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. Then the embryos were 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. After incubation for 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 2x saline sodium citrate (SSC) and PBST. Biotin was detected by horseradish-peroxidase-labelled streptavidin with diaminobenzidine (DAB).
Whole-mount in situ hybridisation and histological analysis
Digoxigenin-labelled probes were synthesised by in vitro transcription using T3, T7 and SP6 polymerases. Whole-mount in situ hybridisation was performed by the standard protocol with some modifications (Schulte-Merker et al., 1992).
For histological analysis, the specimens were embedded in Technovit 8100 (Heraeus Kulzer, Wehrheim) and cut at 7 µm.
Immunohistochemistry and western blot
For whole-mount immunostaining, embryos were fixed with 3.7% formaldehyde/0.2% glutaraldehyde/PBS for 1 hour at room temperature. After washing with PBS, they were dehydrated with methanol and transferred to PBS. Then, they were washed with MABT (MAB/0.1% TritonX-100; MAB, 100mM maleic acid and 150 mM NaCl) 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 and ERK2 (MAPK-YT) antibody (Sigma) for overnight at 4°C. They were then washed with MABDT three times for 5 minutes, four times for 30 minutes, and incubated in blocking solution again for 30 minute, followed by incubation with the second antibody (1:500 anti-mouse IgG biotin conjugated antibody, Vector Laboratory) for 2 hours at room temperature. After the washing with MABDT as described above, the signals were detected with ABC staining kit according to the manufacturers instructions (Vector Laboratory). To examine ERK activation in ace homozygous mutants at the five-somite stage, embryos obtained from two heterozygous ace carriers were cut into two pieces at the level of the hindbrain, and they were fixed immediately. The anterior and posterior halves were then processed for dpERK or myoD staining, respectively.
Western blot analysis was performed following the standard method for ECL western blotting detection reagent (Amersham Pharmacia Biotech). Proteins from three embryos 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 in immunostaining. The homogenates were prepared from whole embryos at 75% epiboly, bud and the six- to seven-somite stage. ace homozygotes could be distinguished by morphology by the six-somite stage.
For detection of Fgf3-Myc, 15 blastoderms taken from sphere-stage embryos injected with RNAs were loaded in each lane. The same staining protocol was applied except for that 9E10 (1:100 dilution, kindly provided by Dr Tatsumi Hirata, National Institute of Genetics) and 2% skim milk were used as the first antibody and as a blocking solution, respectively.
BrdU labelling and detection
To analyse cell mitosis at early segmentation stages, about 0.5 nl of 25 mM of BrdU (SIGMA) was injected into the yolk at tailbud stage. After 3 hours incubation, injected embryos were fixed with 4% PFA/PBS for 2 hours at room temperature. After washing with PBS, the embryos were dehydrated with 25%, 75% and 100% methanol, and rehydrates in PBST. They were then treated with proteinase K (5 µg/ml in PBS) for 5 minutes at 37°C and with glycine-HCl (2mg/ml, pH 2.2) for 5 minutes at room temperature, followed by washing with PBST and post-fixation with 4%PFA/PBS for 20 minutes at room temperature. After washing with PBST, the embryos were treated with 2N HCl for 20 minutes at room temperature to relax chromatin and facilitate immunodetection of incorporated BrdU. Acid-treated embryos were then washed in PBSDT (1%DMSO, 0.1%Tween20 in PBS), incubated with PBSDNT (PBSDT+2% foetal calf serum) for 30 minutes at room temperature, followed by overnight incubation with anti-BrdU antibody (1:200 dilution in PBSDNT, Sigma) at 4°C. After intense washing with PBSDT and blocking with PBSDNT, the embryos were incubated overnight with the second antibody (1:500 dilution in PBADNT, anti-mouse IgG antibody biotin-conjugated, Vector Laboratory) at 4°C. After intense washing in PBSDT, the signals were detected with ABC staining kit as described above.
For cell count, the stained embryos were embedded in Technovit 8100 and serially sectioned at 5 µm. Three sagittal sections (more than 10 µm apart to each other) at the midline of the telencephalon from each treated embryo were selected, and the total number of positive and negative cells in each section was counted (six to seven embryos for each treatment). The ventral corresponds approximately to the region that is positive for ERK staining.
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RESULTS |
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ERK is activated in the ANB of ace mutants
We then examined ERK activation in ace mutants. The ace mutation is a G to A transition in the splice site after the second exon of the fgf8 gene, which leads to incorrect splicing generating a truncated non-functional protein (Reifers et al., 1998). Because, at the five-somite stage, homozygous ace mutants can not be distinguished morphologically from wild-type embryos, we examined the expression pattern of myoD in the trunk region. It has been reported that the adaxial cells of homozygous ace mutants show reduced and fragmented expression of myoD (Reifers et al., 1998). Although the intensity of ERK staining varies to some degree and tends to be weaker as compared with wild-type siblings, a substantial amount of positive reactions are reproducibly observed in the ANB (7/10, Fig. 3A,B) of ace mutants. Western blot analysis revealed that the level of ERK activation was about half of the wild type in ace mutants. These results indicated that Fgf signalling remains activated in the ANB in the absence of Ace/Fgf8.
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The loss of the subpallial telencephalon was detected as early as segmentation stages. In normal embryos, nk2.1b expression in the telencephalon starts to be detected around the 14-somite stage (Rohr et al., 2001). However, nk2.1b expression was not detected throughout development in RasN17-injected embryos (20/20; Fig. 5Q,R). Consistently, dlx2 expression in the telencephalon, which starts to be activated at around the 15-somite stage (Akimenko et al., 1994), was not observed in RasN17-injected embryos at any stages (30/36, data not shown). In normal embryos, emx1 is first expressed in the telencephalic region, and, by the 10-somite stage, the expression is downregulated in the anteriormost of the forebrain, which may be fated to the subpallial telencephalon (Morita et al., 1995; Shanmugalingam et al., 2000). This downregulation, however, was never observed in RasN17-injected embryos (29/29; Fig. 5T). Thus, the regionalisation within the telencephalon was stopped at its early phase (at least as early as 10-somite stage of development) when Ras/MAPK cascade was blocked. During these early stages, inhibition of Ras pathway may affect cell growth in the telencephalic region. To test this, we injected BrdU into RasN17-injected embryos at tailbud stage and examined the proportion of labelled cells after 3 hours incubation (8-10 somite stage). However, we did not find any significant difference in cell proliferation in the telencephalon, either in ventral or dorsal region (Fig. 5Y,Z; Table 1).
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Inhibition of Ras-dependent pathway does not affect the formation of telencephalic territory
In contrast to the regionalisation within the telencephalon, early overall patterning of the forebrain appeared normal in RasN17-injected embryos. As described above, emx1, an early telencephalic marker, was normally activated at the tailbud stage in the prospective telencephalic region of the RasN17-injected embryos (21/22; Fig. 6A,B). At this stage, we observed normal expression of gsc (Stachel et al, 1993) (21/21; Fig. 6C,D), dkk-1 (Shinya et al., 2000) (28/28) and hlx1 (Fjose et al., 1994) (20/20) in injected embryos, indicating that RasN17 did not affect the development of the underlying prechordal plate that is known to be important in establishment of the telencephalic region (Shinya et al., 2000). The expression of another early telencephalic marker, bf-1, which is expressed in the telencephalon and a part of eyes (Toresson et al., 1998), was not altered by RasN17 injection when examined at the five-somite stage (20/20; Fig. 6E,F) and 24 hpf (41/41, data not shown). Furthermore, downregulation of otx2 at the mid-somite stage in the region fated to the telencephalon and ventral diencephalon (Mori et al., 1994) normally occurred in injected embryos (33/34; Fig. 6G,H). Thus, the establishment of telencephalic and diencephalic territories normally takes place even when the Ras/MAPK cascade is suppressed.
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bFR4, XFD and SU5402 produced the same phenotypes as those with RasN17
When specific inhibitors to Fgfr-mediated signal were employed, essentially the same results were obtained (Table 2). In bFR4-injected embryos, expression of dlx2 (Fig. 7A,B), nk2.1b (Fig. 7C,D) and islet-1 (20/30, Fig. 7E,F) in the subpallial telencephalon at 26 hpf was suppressed while pallial markers, emx1 (Fig. 7G,H), eom (23/29) and tbr1 (Fig. 7I,J) expression covered the entire telencephalon. These effects were already observed at mid-somite stage. In the b
FR4-injected embryos, nk2.1b expression was suppressed (15/18, Fig. 7L,M) at the 14-somite stage and emx1 was expressed throughout the telencephalon at the 15-somite stage (43/44, Fig. 7N,O). By contrast, the territory of the telencephalon was established normally, judging from the expression pattern of emx1 at tailbud stage, bf-1 at the five-somite stages and otx2 at the 15-somite stage (data not shown). The same phenotypes were obtained when XFD RNAs were injected (Table 2; Fig. 7K) (7/13 for tbr1).
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Among the RNAs injected, XFD seems to be less effective (Table 2). Especially, in the case of dlx2, only five out of 18 embryos injected with XFD RNAs showed reduced expression (Table 2). This could reflect the fact that the inhibitory effect of XFD on ERK activation does not last long (Fig. 2I). In spite of these, all inhibitors showed the similar tendency and, thus, we conclude that Fgf signalling through Ras/MAPK cascade is required for the development of the subpallial telencephalon.
The subpallial telencephalon requires Fgf signalling at early segmentation stages
Anti-dpERK staining revealed that ERK activation reached a level peak at the five-somite stage and, thereafter, gradually decreased. To address when Fgf signalling might be required for development of the subpallial telencephalon, we treated embryos with SU5402 at tailbud, five-somite, 10-somite and 15-somite stages, and its effect on dlx2 expression was examined. In this series of experiments, we soaked embryos in 0.1 mg/ml or 0.2 mg/ml SU5402 in 1/3 Ringers solution for 10 minutes. This worked well, and the resulting phenotype in the telencephalon was identical to that obtained with injection of SU5402.
As summarised in Table 3, early treatment (bud to 5-somite) with SU5402 led to loss or severe reduction of dlx2 in the subpallial telencephalon (Fig. 8E). By contrast, treatment from 10- and 15-somite stage gave rise to normal dlx2 expression in this region (Fig. 8F). Thus, Fgf signalling up to the five-somite stage is crucial for the establishment of the subpallial telencephalon but, thereafter, dlx2 expression can be maintained without Fgf signalling.
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In the MHB, the effects of fgf8-MO and fgf3-MO were different from each other. Injection of fgf8-MO reduced the expression of en2 (11/11; Fig. 9B') (Ekker et al., 1992) phenocopying the ace mutant, while that of fgf3-MO did not affect the expression (23/23; Fig. 9C').
To test the specificity of the phenotype, we made another fgf3-MO designed against more 3' sequence (fgf3-MO; Fig. 9N). fgf3-MO exhibits similar effects on nk2.1b expression in wild-type (32/41) and fgf8-MO-injected (22/26) embryos. We then constructed a cDNA encoding Fgf3 tagged with Myc epitope at the C terminus, because an antibody against zebrafish Fgf3 is not available. Western blot analyses were performed with the embryos injected with either fgf3-myc RNAs alone or fgf3-myc and fgf3-MO. Co-injection of fgf3-MO completely blocked the translation of injected fgf3-myc RNAs, while the degree of inhibition much decreased in the case of 4mis-MO in which a four-base mismatch was introduced into fgf3-MO (Fig. 9M). We also confirmed that 4mis-MO, when injected, did not affect nk2.1b expression (Fig. 9A). Furthermore, the effect of fgf3-MO was diminished when fgf3 RNAs (5 ng/µl, about 2.5 pg per embryo) were co-injected (9/18, Fig. 9E). We were unable to obtained 100% rescue because the increased amount of fgf3 RNAs suppressed the head development in injected embryos even in the presence of fgf3-MO, probably owing to strong posteriorising and dorsalizing effects of Fgf3 at the gastrulation stage (S. K., M. S., M. Nikaido, N. Ueno, S. Schulte-Merker, A. K. and H. T., unpublished). Finally, we injected fgf3-MO (10 µg/µl) in embryos obtained from ace heterozygous crosses. Seventeen out of 58 injected embryos showed no or faint nk2.1b expression (Fig. 9H), while such enhanced phenotype was not observed by injection of fgf8-MO (54/54; Fig. 9G).
Finally, we examined emx1 and dlx2 expression in fgf3-MO-injected ace homozygous embryos. The expression of these two markers are relatively normal in ace mutants at 26 hpf. The ace homozygous embryos were identified by a lack of en2 expression in the MHB in double-stained embryos, as fgf3-MO alone does not affect en2 expression (Fig. 9C). Like SU5402 treatment, injection of fgf3-MO resulted in ventral expansion of emx2 (9/9; Fig. 9J) and reduction of dlx2 (11/18 for severe and 7/18 for mild reduction, Fig. 9L) expression in ace mutants, while no such effect was observed in control injections (18/18 for emx1 and 11/13 for dlx2; Fig. 9I,K). Thus, the expression of exm1 and dlx2 is more dependent on Fgf3.
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DISCUSSION |
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Fgf8 is not a sole Fgf involved in ERK activation in the ANB
As in other vertebrates, zebrafish fgf8 is expressed during gastrulation in the mesoderm, presumptive MHB and telencephalon. (Fürthauer et al., 1997; Reifers et al., 1998). Expression in the CNS is prominent in the MHB and in the anteroventral telencephalon at segmentation stages (Reifers et al., 1998; Shanmugalingam et al., 2000). ERK activation appears to follow the expression pattern of fgf8 up to mid-somite stage, suggesting that fgf8 is responsible for ERK activation. However, the analysis with ace mutants, in which no functional Fgf8 is produced (Reifers et al., 1998), suggests that Fgf8 is not a sole Fgf responsible for ERK activation. Although the level of activation tends to be weaker, a substantial amount of ERK activation persists in the ANB of ace homozygous mutant. Expression analysis and overexpression experiments suggest that Fgf3 is one of the candidates involved in ERK activation in the ANB.
After mid-somite stage, the level of ERK activation gradually decreases in the MHB and then in the ANB, whereas fgf8 expression remains high in these regions. This suggests that Fgfs, albeit expressed, do not necessarily activate their signalling pathway. Alternatively, ERK activation may not represent all Fgf activity, or antibody labelling may not detect low levels of ERK activation. In fact, Fgfs are known to use various signalling cascades other than Ras/MAPK such as phosphatidylinositol 3' kinase (Klint and Claesson-Welsh, 1999).
Roles of Fgf signalling in normal development of the subpallial telencephalon
Analysis with telencephalic markers reveals loss of the subpallial fate after treatment with inhibitors to Fgf receptors. In addition to region specific markers, disruption of Fgf signalling reduces or abolishes the expression of differentiation markers specific to putative subpallial neurones. The effects seem direct because the underlying prechordal plate develops normally in treated embryos as judged by gene expression. The present results, therefore, indicate that Fgf signalling in the ANB is required for differentiation of subpallial neurones as well as establishment of the subpallial region.
There are at least three possible explanations for the phenotype in affected embryos: (1) the ventral telencephalon fails to acquire subpallial fate and develops into the pallium; (2) the subpallial region is established but disappears due to cell death; and (3) cells in the subpallium fail to proliferate and remains in a small number, while development of the pallial telencephalon proceeds normally. As cell death in the telencephalon is detected only after loss or reduction of the subpallial markers, the second possibility seems less likely. The detailed analysis of cell proliferation and movements during normal development of the subpallial telencephalon has not been done in fish. Thus, we cannot rule out either the first or third explanations. However, the following two observations favour the first possibility. First, inhibition of Ras pathway does not affect cell proliferation in the telencephalon at early segmentation stages (Table 1). Second, in affected embryos, subpallial markers such as dlx2 and nk2.1b are never detected at any stages examined, suggesting that the subpallial fate is not induced from the beginning.
In the present study, inhibition of the Fgf signalling affects only a part of the telencephalon, the subpallial region. Cells in the subpallium are shown to derive from the ANB. The row 1 cells in the ANB contributes to the anteriormost subpallial telencephalon (Houart et al., 1998). The precise lineage analysis by Whitlock and Westerfield (Whitlock and Westerfield, 2000) has revealed that cells in the ventral telencephalon come from the anterior margin of the neural plate at the five-somite stage, whereas more posterior margin is fated to the dorsal telencephalon. Their fate map of the ventral telencephalon well fits with ERK activation domain that is narrower than emx1-positive domain (a maker for the entire telencephalon at early stages). Thus, it is reasonable to say that the effect of Fgf inhibition was observed in the region where Ras/MAPK pathway is activated.
Brief treatment of developing embryos with SU5402 reveals that Fgf signalling is not required for subpallial development after 10-somite stage. The crucial period coincides with the stage when the strongest activation of ERK is observed. However, it has been reported that the same treatment at 18-somite stage still causes some defects in axon guidance between anterior commissure and post-optic commissure (Shanmugalingam et al., 2000). Thus, it is likely that a crucial period for requirement of Fgf signalling varies, depending on the region and developmental processes of the telencephalon.
Fgf signalling may not be required for induction of the telencephalic territory in zebrafish
Studies in mice have provided evidence that the anterior neural ridge (ANR), which is located in the anterior boundary between the neural and non-neural ectoderm, plays an important role in induction of the telencephalon. Shimamura and Rubenstein (Shimamura and Rubenstein, 1997) demonstrated by in vitro culture method that the ANR in mice is necessary and sufficient for the induction of telencephalic bf-1 expression. In zebrafish, the most anterior neural cells in the ANB, called row 1 cells, were found to possess the similar inducing ability to the mouse ANR (Houart et al., 1998). The ablation of row 1 cells resulted in the loss of telencephalic markers and apoptosis in the anterior head region (Houart et al., 1998). As in vitro experiments in mice have demonstrated that Fgf8-soaked beads mimic nearly all activity of the ANR in mammals (Shimamura and Rubenstein, 1997), Fgf signalling has been proposed to account for the inducing ability of the ANR.
However, we observe in the present study that the establishment of the telencephalic and diencephalic territories normally proceeded in the RasN17-injected embryos, as indicated by normal expression patterns of bf1, emx1 and otx2 at early segmentation stages. This suggests more restricted functions of Fgf signalling in telencephalic development. Indeed, no expression of any fgf or activation of ERK is detected in the ANB at the gastrula stage when the overall patterning of the CNS is specified. Consistent with this, SU5402 treatment at mid-gastrula (75%-epiboly) stage did not affect emx1 expression at tailbud stage (data not shown). Therefore, although involvement of low level of Fgf signal can not be ruled out, our findings suggest that, in zebrafish, Fgf signalling is mainly required for regionalisation but not for induction of the telencephalon.
Overlapping and distinct functions of Fgfs expressed in the CNS
Our antisense experiments suggest that Fgf3 co-operates with Fgf8 in the ANB. The antisense experiments seemed to work well in the present study. We are able to generate a phenocopy of ace mutant by injecting fgf8-MO, and fgf3-MO is able to inhibit translation of injected fgf3 RNAs in a sequence-specific manner. Furthermore, we recently demonstrated that casanova (226D7)-MO injection phenocopied casanova mutant, a complete loss of endodermal precursors (Sakaguchi et al., 2001).
Although the injection of fgf8-MO or fgf3-MO alone results in slight reduction of nk2.1b expression, severe reduction of nk2.1b is observed after co-injection of fgf8-MO and fgf3-MO. The results demonstrate that both Fgf8 and Fgf3 are required for normal nk2.1b expression of the subpallial telencephalon. It has also been revealed that emx1 and dlx2 expressions are more dependent on Fgf3. These data may account for relatively mild defects in the subpallial telencephalon of ace mutants. In the MHB, however, zebrafish Fgf8 has a major role as indicated by the phenotype of ace mutant, even though fgf3 expression is detected in the region (Fig. 4A,C). In support of this, the expression of en2 persists in fgf3-MO-injected embryos while the expression is reduced in fgf8-MO-injected embryos. Thus, Fgf8 and Fgf3 possess overlapping and distinct functions in the ANB and MHB, respectively. Considering that there are certainly further Fgfs in zebrafish that have yet to be identified, further molecular and genetic analyses will be required to elucidate full functions of Fgf signalling in the developing CNS.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Akimenko, M. A., Ekker, M., Wegner, J., Lin, W. and Westerfield, M. (1994). Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head. J. Neurosci. 14, 3475-3486.[Abstract]
Allende, M. L. and Weinberg, E. S. (1994). The expression pattern of two zebrafish achaete-scute homolog (ash) genes is altered in the embryonic brain of the cyclops mutant. Dev. Biol. 166, 509-530.[Medline]
Amaya, E., Musci, T. J. and Kirschner, M. W. (1991). Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell 66, 257-270.[Medline]
Campuzano, S. and Modolell, J. (1992). Patterning of the Drosophila nervous system: the achaete-scute gene complex. Trends Genet. 8, 202-208.[Medline]
Christen, B. and Slack, J. M. (1999). Spatial response to fibroblast growth factor signalling in Xenopus embryos. Development 126, 119-125.
Cobb, M. H. and Goldsmith, E. J. (1995). How MAP kinases are regulated. J. Biol. Chem. 270, 14843-14846.
Crews, C. M., Alessandrini, A. and Erikson, R. L. (1992). The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science 258, 478-480.[Medline]
Deng, T. and Karin, M. (1994). c-Fos transcriptional activity stimulated by H-Ras-activated protein kinase distinct from JNK and ERK. Nature 371, 171-175.[Medline]
Ekker, M., Wegner, J., Akimenko, M. A. and Westerfield, M. (1992). Coordinate embryonic expression of three zebrafish engrailed genes. Development 116, 1001-1010.
Fjose, A., Izpisua, B. J., Fromental, R. C. and Duboule, D. (1994). Expression of the zebrafish gene hlx-1 in the prechordal plate and during CNS development. Development 120, 71-81.
Fürthauer, M., Thisse, C. and Thisse, B. (1997). A role for FGF-8 in the dorsoventral patterning of the zebrafish gastrula. Development 124, 4253-4264.
Gabay, L., Seger, R. and Shilo, B. Z. (1997a). In situ activation pattern of Drosophila EGF receptor pathway during development. Science 277, 1103-1106.
Gabay, L., Seger, R. and Shilo, B. Z. (1997b). MAP kinase in situ activation atlas during Drosophila embryogenesis. Development 124, 3535-3541.
Gotoh, Y. and Nishida, E. (1996). Signals for mesoderm induction. Roles of fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK) pathway. Biochim. Biophys. Acta 1288, F1-F7.[Medline]
Hongo, I., Kengaku, M. and Okamoto, H. (1999). FGF signaling and the anterior neural induction in Xenopus. Dev. Biol. 216, 561-581.[Medline]
Houart, C., Westerfield, M. and Wilson, S. W. (1998). A small population of anterior cells patterns the forebrain during zebrafish gastrulation. Nature 391, 788-792.[Medline]
Inoue, A., Takahashi, M., Hatta, K., Hotta, Y. and Okamoto, H. (1994). Developmental regulation of islet-1 mRNA expression during neuronal differentiation in embryonic zebrafish. Dev. Dyn. 199, 1-11.[Medline]
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253-310.[Medline]
Klint, P. and Claesson-Welsh, L. (1999). Signal transduction by fibroblast growth factor receptors. Front. Biosci. 4, D165-D177.[Medline]
Koshida, S., Shinya, M., Mizuno, T., Kuroiwa, A. and Takeda, H. (1998). Initial anteroposterior pattern of the zebrafish central nervous system is determined by differential competence of the epiblast. Development 125, 1957-1966.
Krauss, S., Concordet, J. P. and Ingham, P. W. (1993). A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75, 1431-1444.[Medline]
Makita, R., Mizuno, T., Koshida, S., Kuroiwa, A. and Takeda, H. (1998). Zebrafish wnt11: pattern and regulation of the expression by the yolk cell and No tail activity. Mech. Dev. 71, 165-176.[Medline]
Maruoka, Y., Ohbayashi, N., Hoshikawa, M., Itoh, N., Hogan, B. L. M. and Furuta, Y. (1998). Comparison of the expression of three highly related genes, Fgf8, Fgf17 and Fgf18, in the mouse embryo. Mech. Dev. 74, 175-177.[Medline]
Meyers, E. N., Lewandoski, M. and Martin, G. R. (1998). An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat. Genet. 18, 136-141.[Medline]
Mione, M., Shanmugalingam, S., Kimelman, D. and Griffin, K. (2001). Overlapping expression of zebrafish T-brain-1 and eomesodermin during forebrain development. Mech. Dev. 100, 93-97.[Medline]
Mohammadi, M., McMahon, G., Sun, L., Tang, C., Hirth, P., Yeh, B. K., Hubbard, S. R. and Schlessinger, J. (1997). Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science 276, 955-960.
Mori, H., Miyazaki, Y., Morita, T., Nitta, H. and Mishina, M. (1994). Different spatio-temporal expressions of three otx homeoprotein transcripts during zebrafish embryogenesis. Mol. Brain Res. 27, 221-231.[Medline]
Morita, T., Nitta, H., Kiyama, Y., Mori, H. and Mishina, M. (1995). Differential expression of two zebrafish emx homeoprotein mRNAs in the developing brain. Neurosci. Lett. 198, 131-134.[Medline]
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene knockdown in zebrafish. Nat. Genet. 26, 216-220.[Medline]
Ornitz, D. M. (2000). FGFs, heparan sulfate and FGFRs: complex interactions essential for development. BioEssays 22, 108-112.[Medline]
Reifers, F., Böhli, H., Walsh, E. C., Crossley, P. H., Stainier, D. Y. and Brand, M. (1998). Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 125, 2381-2395.
Rohr, K. B., Barth, K. A., Vargam, Z. M. and Wilson, S. W. (2001). The Nodal pathway acts upstream of Hedgehog signaling to specify ventral telencephalic identity. Neuron 29, 341-351.[Medline]
Rubenstein, J. L., Shimamura, K., Martinez, S. and Puelles, L. (1998). Regionalization of the prosencephalic neural plate. Annu. Rev. Neurosci. 21, 445-477.[Medline]
Sakaguchi, T., Kuroiwa, A. and Takeda, H. (2001). A novel sox gene, 226D7, acts downstream of Nodal signaling to specify endoderm precursors in zebrafish. Mech. Dev. 107, 25-38.[Medline]
Schulte-Merker, S., Ho, R. K., Herrmann, B. G. and Nüsslein, V. C. (1992). The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 116, 1021-1032.
Shanmugalingam, S., Houart, C., Picker, A., Reifers, F., Macdonald, R., Barth, A., Griffin, K., Brand, M. and Wilson, S. W. (2000). Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon. Development 127, 2549-2561.
Shimamura, K., Hartigan, D. J., Martinez, S., Puelles, L. and Rubenstein, J. L. (1995). Longitudinal organization of the anterior neural plate and neural tube. Development 121, 3923-3933.
Shimamura, K. and Rubenstein, J. L. (1997). Inductive interactions direct early regionalization of the mouse forebrain. Development 124, 2709-2718.
Shinya, M., Eschbach, C., Clark, M., Lehrach, H. and Furutani-Seiki, M. (2000). Zebrafish dkk1, induced by the pre-MBT wnt signaling, is secreted from the prechordal plate and patterns the anterior neural plate. Mech Dev. 98, 3-17.[Medline]
Stachel, S. E., Grunwald, D. J. and Myers, P. Z. (1993). Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish. Development 117, 1261-1274.
Toresson, H., Martinez-Barbera, J. P., Bardsley, A., Caubit, X. and Krauss, S. (1998). Conservation of BF-1 expression in amphioxus and zebrafish suggests evolutionary ancestry of anterior cell types that contribute to the vertebrate telencephalon. Dev. Genes Evol. 208, 431-439.[Medline]
Whitlock, K. E. and Westerfield, M. (2000). The olfactory placodes of the zebrafish form by convergence of cellular fields at the edge of the neural plate. Development 127, 3645-3653.
Wilson, S. W. and Rubenstein, J. L. R. (2000). Induction and dorsoventral patterning of the telencephalon. Neuron 28, 641-651.[Medline]
Woo, K. and Fraser, S. E. (1997). Specification of the zebrafish nervous system by nonaxial signals. Science 277, 254-257.
Xu, J., Lawshé, A., MacArthur, C. A. and Ornitz, D. M. (1999). Genomic structure, mapping, activity and expression of fibroblast growth factor 17. Mech. Dev. 83, 165-178.[Medline]