1 Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
2 Laboratoire de Génétique et Physiologie du Développement, Institut de Biologie du Développement de Marseille, CNRS-INSERM-Université de la Méditerranée-AP de Marseille, Campus de Luminy, Case 907, F-13288 Marseille Cedex 9, France
*Author for correspondence (e-mail: darras{at}ibdm.univ-mrs.fr)
Accepted April 25, 2001
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SUMMARY |
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Key words: Notochord induction, BMP, Chordin, bFGF, Ascidian embryo, Asymmetric division, Halocynthia roretzi
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INTRODUCTION |
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In the present study, we provide evidence for the role of the bone morphogenetic protein (BMP) pathway in notochord induction. First, overexpression of the BMP antagonist chordin prevented notochord development. Second, BMP-4 was able to induce primary notochord formation from isolated notochord precursors. We could also address the temporal requirement for notochord induction by bFGF and BMP-4. Although isolated notochord precursors responded to bFGF during the 32-cell stage, they responded to BMP-4 during the 44-cell stage, i.e. when HrBMPb transcripts were first detected in the anterior endoderm. Interestingly, notochord induction by BMP-4 required active FGF signaling. In a similar manner, bFGF and BMP-4 acted sequentially to induce secondary notochord. The inducing blastomeres were identified and enabled us to draw a common scenario for induction of both primary and secondary lineages. From the 24-cell stage to the 44-cell stage, active FGF signaling would be a prerequisite for enabling the BMP pathway to complete notochord induction between the 44-cell stage and the 64-cell stage.
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MATERIALS AND METHODS |
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Injection of synthetic mRNA
mRNA was synthesized according to the manufacturer protocol (mMessage mMachine, Ambion) with T3 polymerase for pBSRN3Hrchordin, pBSRN3HrBMPb and pBSRN3Xlnoggin3'. Ascidian eggs were injected with mRNAs during the second phase of ooplasmic segregation before the first cleavage (Miya et al., 1997).
In situ hybridization and immunohistochemistry
Whole-mount in situ hybridization was performed as described previously (Miya et al., 1997) except that the DIG-probes were not hydrolyzed by alkaline treatment. The Not-1 monoclonal antibody specifically recognizes notochord cells at the mid-tailbud stage (Nishikata and Satoh, 1990). The Mu-2 monoclonal antibody recognizes the myosin heavy chain in tail muscle cells of larvae (Nishikata et al., 1987). Samples were fixed in methanol for 10 minutes at 20°C and stained by indirect immunofluorescence with an Alexa 488-conjugated secondary antibody (Molecular Probes).
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RESULTS |
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Primary notochord induction by BMP-4 requires FGF signaling
bFGF is also a potent primary notochord inducer (Nakatani et al., 1996). The sensitive period to bFGF ends when isolated notochord precursors divide at the 44-cell stage (Nakatani et al., 1996). Therefore, notochord precursors have different sensitive periods to bFGF and BMP-4 treatment. We examined the relationship of these two signaling pathways with drugs that specifically block the FGF pathway: inhibitor of FGF receptor 1 (SU5402) and inhibitor of MEK-1 (U0126). These inhibitors blocked notochord formation when applied to whole embryos from the 24-cell stage to the 64-cell stage as revealed by the loss of As-T expression (SU5402: 3%, n=62; U0126: 0%, n=54) and Not-1 staining (SU5402: 0%, n=22; U0126: 9%, n=22; in G. J. Kim and H. N., unpublished). Then we isolated A6.2/A6.4 blastomeres and treated them with BMP-4 in the presence of SU5402 or U0126. In both cases, notochord induction by BMP-4 was suppressed (Figs 4C, 5; Table 1A). Similarly in A7.3/A7.7 notochord precursors isolated at the 44-cell stage, inhibition of the FGF pathway prevented induction of notochord by BMP-4 (Fig. 5; Table 1B). Thus, induction of notochord by BMP-4 required a functional FGF signaling pathway.
An asymmetric division separates the notochord and nerve cord lineages
We examined the competence of notochord and nerve cord precursors to bFGF treatment at the 44-cell stage. Unexpectedly, bFGF treatment induced the notochord precursors isolated at the 44-cell stage to form notochord (Table 1B; Fig. 5). However, the mother cells of these blastomeres that are isolated at the 24-cell stage and cultured in isolation do not respond to bFGF after the 44-cell stage (Nakatani et al., 1996). Nerve cord precursors isolated at the 44-cell stage never formed notochord after treatment with bFGF, BMP-4 or a combination of both. When a notochord/nerve cord precursor was isolated at the 24-cell stage and treated immediately with either BMP-4 or bFGF, all cells within a partial embryo differentiated into notochord (present study and Nakatani et al., 1996). However, when the daughters of this blastomere were isolated after their division at the 44-cell stage, only the notochord precursor was competent to form notochord. These results suggest that the competence to form notochord is restricted to only one of the two daughter cells (the notochord precursors) through an asymmetric division.
The combination of bFGF and BMP-4 promotes B-line notochord induction
chordin overexpression reduced the formation of both primary (A-line) and secondary notochord (B-line). Therefore, we investigated the role of the BMP pathway in secondary notochord formation, as its mechanism of induction is poorly understood. We first tested if BMP could be a B-line notochord inducer. Contrary to A4.1 blastomeres cultured alone, when B4.1 blastomeres were isolated at the eight-cell stage notochord formation did not occur (Table 2; Nakatani and Nishida, 1994). Treatment of B4.1 with BMP-4 induced notochord formation. A similar result was obtained with B5.1 isolates, taken from 16-cell stage embryos. In contrast, BMP-4 was unable to induce notochord in B6.2 blastomeres isolated at the 24-cell stage (Fig. 6B,F; Table 2).
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B7.3 blastomeres isolated at the 44-cell stage did not form notochord (Table 3), while they autonomously formed notochord when isolated at the 64-cell stage (Table 3; Nakatani and Nishida, 1994). Thus, signals might be emitted between the 44-cell and the 64-cell stages to complete notochord induction. BMP-4 alone can replace these signals because it induced notochord in B7.3 blastomeres isolated at the 44-cell stage (Table 3), probably because B7.3 already received an FGF-like endodermal signal during the 32-cell stage.
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B-line notochord is induced by two signals spatially separated
We tried to see if we could attribute each signal to a defined blastomere neighboring the secondary notochord precursor. At the 24-cell stage, the B6.2 blastomere is surrounded by precursors of anterior endoderm/TLC (A6.3), posterior endoderm (B6.1), muscle/mesenchyme (B6.4) and ectoderm (b5.3) (Fig. 7A). None of the possible co-isolations of two blastomeres (B6.2+B6.1, B6.2+A6.3) resulted in notochord formation (Fig. 7A; Nakatani and Nishida, 1994). However, co-isolation of the three blastomeres (B6.2+B6.1+A6.3) promoted notochord differentiation. This induction was unlikely to be due to a mass effect, as isolations of three blastomeres or more, excluding the A6.3 blastomeres, did not promote notochord formation (B6.2+B6.1+b5.3, B6.2+B6.1+B6.3+B6.4, B6.2+B6.1+B6.3+B6.4+b5.3+b5.4) (Fig. 7A). The signal from the A6.3 blastomere could be substituted by BMP-4 and the signal from B6.1 by bFGF, although with a weaker efficiency (Fig. 7A). We then performed the same type of co-isolations at the 44-cell stage (Fig. 7B). We found that, at this stage, the posterior endoderm precursors were no longer required. The co-isolation of B7.3 notochord precursor with either of the A6.3 descendants (B7.3+A7.5, B7.3+A7.6) or with both daughter cells (B7.3+A7.5+A7.6) was sufficient to induce notochord formation, although the A7.6 blastomere appeared to be more efficient (Fig. 7B). The results of these co-isolation experiments are in agreement with those of bFGF and BMP-4 treatment experiments, and allow us to propose the following model. B-line notochord is induced by two signals. The first signal, which emanates from the posterior endoderm precursor (B6.1), acts from the 24-cell stage to the 44-cell stage and can be substituted by bFGF. The second signal, which emanates from the anterior endoderm and TLC precursors (A7.5 and A7.6), acts from the 44-cell stage and can be substituted by BMP-4.
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DISCUSSION |
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First step: acquisition of competence
The first step occurs at the 32-cell stage. We showed that, in B-line notochord, BMP-4 could not induce notochord if the presumptive notochord blastomere has not received a bFGF-like signal first (Figs 6, 7). However, at the 44-cell stage, BMP-4 alone or anterior endoderm is sufficient to induce B7.3 to form notochord (Table 3; Fig. 7). In the primary notochord, we did not address this question directly. However, the ability of A-line notochord precursors to induce each other (Nakatani and Nishida, 1994) suggests that they possess an autonomous FGF secretion. Thus, we propose that a bFGF-like signal is required during the 32-cell stage to make notochord precursors competent to respond to BMP-4. Moreover, in the primary lineage at the 44-cell stage, the competence to form notochord is restricted only to notochord precursors through an asymmetric division that separates notochord and nerve cord lineages. FGF-like molecules secreted from endoderm blastomeres that are located next to the notochord precursors are likely to control this asymmetric division. Owing to this restriction of competence at the 44-cell stage, the secreted molecules that complete notochord induction should be considered as permissive signals.
Second step: completion of induction
At the 44-cell stage, treatment with BMP-4 alone was sufficient to induce notochord in both A-line and B-line precursors that are not yet specified. This indicates the involvement of BMP in the second step. However, blocking the FGF pathway blocks this effect at least in the primary lineage. Several explanations are possible (as follows).
The requirement of the FGF signaling at the 44-cell stage, as shown by the inhibition of BMP-4 action by U0126, suggests that MEK activity is still needed at the 44-cell stage. One possibility is that it takes some time to complete intracellular signal after FGF signal has been received at the cell surface during the 32-cell stage. Another possibility might be that secreted FGF molecules act at the cell surface during the 44-cell stage. This might be explained by the presence of a certain amount of FGF molecules on the notochord precursor surface, as FGF protein is known to bind to the cell surface heparan sulfate proteoglycans (Ornitz, 2000). The bound FGF might not be sufficient to complete notochord induction, but might be required at the 44-cell stage when BMP acts. An additive effect of FGF and BMP would then be sufficient to promote notochord induction (Fig. 8). Alternatively, BMP-4 might promote the immediate autocrine secretion of FGF from the notochord blastomere at the 44-cell stage, and this secreted FGF might achieve notochord specification.
The canonical BMP pathway involves activation of Smad proteins through the serine-threonine kinase activity of BMP receptors (for a review, see Kretschmar and Massagué, 1998). However, there is evidence for a crosstalk between the Ras/MAPK pathway and the transforming growth factor ß (TGFß) pathway. Smad activity has been shown to be regulated via phosphorylation by protein kinases downstream of MEK-1 (de Caestecker et al., 1998; Massague and Chen, 2000 and references therein). It has also been shown that TGFß action could be transduced through the Ras/MAPK pathway (reviewed by Mulder, 2000). Therefore, it is possible that, in our case, the BMP and FGF pathways also converge to the regulation of the signaling mediators of the BMP pathway, namely Smad 1,5 or 8, although there is no direct evidence for this in ascidian embryos.
Comparison with vertebrate mesoderm formation
In vertebrate embryos, FGF and TGFß superfamily growth factors have been shown to be required for mesoderm formation. Both types of molecules can induce mesoderm in vitro (Slack, 1994), but the inducing signals emitted from the endoderm are likely to be TGFß of the activin/Nodal subfamilies (Kimelman and Griffin, 2000). However, the actual nature of inducers, especially notochord inducers, is not known. In ascidians, while bFGF is a potent notochord inducer, activin is not (Nakatani et al., 1996). Here, we have shown that both BMP and FGF are required for notochord formation, and we would like to compare this observation with mesoderm formation in vertebrates.
In vertebrates, an early role for FGF concerns the competence to respond to TGFß-like signals (Cornell et al., 1995). As we described, a similar situation may be found in ascidians, although a main divergence is observed: BMP rather than activin/Nodal would act as a notochord inducer. Moreover, in the case of A-line notochord induction, while a 10 minute pulse of bFGF is sufficient during the 32-cell stage to trigger notochord differentiation (Nakatani et al., 1996), FGF signal during the same period does not appear to be sufficient in vivo, as blastomeres isolated at the 44-cell stage (about 60 minutes after the beginning of the 24-cell stage) did not form notochord. We propose that the early action of FGF, during the 32-cell stage, is to make cells competent to respond to BMP, similar to the early role of FGF in Xenopus.
In Xenopus, eFGF acts within a positive regulatory loop with Brachyury, which is necessary for notochord formation during gastrulation (Schulte-Merker and Smith, 1995; Casey et al., 1998). In ascidian, notochord precursors are determined between the 44-cell and the 64-cell stage, as they autonomously differentiate into notochord when they are isolated about 20-30 minutes after the beginning of the 44-cell stage. This timing coincides precisely with the initiation of As-T expression revealed by in situ hybridization (not shown). Moreover, blocking FGF signaling from this stage no longer inhibits notochord formation (G. J. Kim and H. N., unpublished). These observations suggest that a positive feedback loop between FGF and As-T is unlikely to take place in ascidians (see also Kim et al., 2000).
In Xenopus embryos, chordin has been shown to induce a secondary axis while BMP-2 or BMP-4 overexpression suppresses axis and notochord formation (Sasai et al., 1994; reviewed by Dale and Jones, 1999). Loss-of-function experiments in mouse and zebrafish show that blocking BMP pathway is necessary for proper notochord formation, however, the notochord induction by itself is not affected in embryos mutant for BMP antagonists (Hammerschmidt et al., 1996; Schulte-Merker et al., 1997; Bachiller et al., 2000). We were surprised to observe that the effect of Chordin and BMP in ascidian appeared to be opposite to their effect in vertebrates. In mesoderm of vertebrate embryos, BMP-2 and BMP-4 are involved in dorsoventral patterning through a gradient of activity (Dosch et al., 1997) but not in mesoderm formation itself. In contrast, mesoderm patterning in ascidians, i.e. the definition of major mesodermal tissues that are notochord, mesenchyme and muscle, is mainly controlled by localized cytoplasmic factors (Kim et al., 2000). The secreted growth factors then act rather as permissive than instructive factors.
chordin as a positive target of the BMP pathway
chordin expression was initiated at the 44-cell stage in all vegetal blastomeres surrounding the anterior endoderm expressing BMPb. We showed that chordin might be a positive target of the BMP pathway in the A-line notochord precursors (Fig. 3). Therefore, it appears that the BMP pathway activates its own antagonist. It has been proposed that noggin and other inhibitors of the BMP pathway are involved in negative feedback regulatory loops (reviewed by Massagué and Chen, 2000). One could consider that chordin activation is important for restricting the range of BMPb action, for example, by preventing the nerve cord precursors from receiving the BMP signal and forming notochord. But this hypothesis is unlikely, as we have shown that nerve cord precursors are no longer competent to respond to BMP-4 in isolation at the 44-cell stage. Chordin binds directly to BMP protein (Piccolo et al., 1996) and consequently, it might prevent BMP from diffusing away, creating a high concentration of BMP close to the Chordin-secreting cells. Therefore, in ascidians, chordin might be involved in generating the highest BMPb concentration close to the notochord precursors. The significance of chordin expression during this period of development will require further experiments.
Unequal cleavage of B7.3 blastomere
As mentioned above, the primary and secondary notochord inductions share common mechanisms. But some differences were also noted. First, the precursor of primary notochord isolated at the 24-cell stage could be induced to form notochord cells by treatment with either FGF or BMP. In contrast, similar treatment of the precursor of secondary notochord isolated at the 24-cell stage did not result in notochord formation. Second, B7.3 blastomere at the 64-cell stage, which is when this cell acquires cell autonomy to form notochord, is still fated to form mesenchyme and secondary notochord. The next division separates these two fates so that B8.6 (smaller daughter cell; Fig, 1C,D) is restricted to form notochord.
B7.3 blastomeres isolated at the 44-cell stage divided equally and never formed notochord. When these blastomeres were treated with BMP-4 or isolated at the 64-cell stage, they divided unequally and formed four notochord cells in most cases. The unequal cleavage of B7.3 is likely to be an intrinsic property of this cell when it receives an extracellular signal; because it is difficult to imagine how BMP-4 protein, dissolved in seawater and thus present all around the cell surface, could behave as an asymmetric clue to control an unequal cleavage. BMP should be considered a permissive signal for unequal cleavage, rather than an instructive molecule that polarizes the cell. Therefore, BMP signaling might not be the sole mechanism controlling notochord specification and unequal cleavage of B7.3 blastomere in this case; something else is also involved.
In this study, we have described the involvement of the FGF and the BMP pathways in ascidian notochord induction. chordin overexpression reduced formation of both primary and secondary notochord, indicating that BMP signaling is required for induction of notochord. Two distinct steps in notochord induction can be distinguished. During the 32-cell stage, FGF would act as a competence factor to allow BMP to complete notochord induction from the 44-cell stage. This induction by BMP, however, requires active FGF signaling. By comparison with vertebrates embryogenesis, these results may appear surprising. While the BMP pathway is undoubtedly involved in ventralization of the early vertebrate embryo, we would like to speculate that it might also have a separate function in notochord formation. The present study may open new perspectives in understanding vertebrates mesoderm formation.
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ACKNOWLEDGMENTS |
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