Wellcome/CRC Institute, Tennis Court Road, Cambridge, CB2 1QR, and Department of Zoology, University of Cambridge, UK
*Author for correspondence (e-mail: j.b.gurdon{at}welc.cam.ac.uk)
Accepted 21 February 2002
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SUMMARY |
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Key words: Morphogen, TGFß, Smad2, Xenopus
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INTRODUCTION |
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We have analysed this problem with respect to the TGFß factor activin, which is currently believed to simulate the action of Xnrs in Nieuwkoop signalling during the first few hours of Amphibian development (Clements et al., 1999; Whitman, 2001
). We concentrate here on signal transduction from the cell surface to the nucleus, thereby extending our previous work on the analysis of activin action at the cell surface.
A large amount of work has analysed the TGFß signalling pathway, of which Smads 2, 3 and 4 are key members. Part of this work has been carried out on Xenopus cells (Chen et al., 1996; Chen et al., 1997
; Germain et al., 2000
; Graff et al., 1996
; Hill, 2001
; Howell and Hill, 1997
; Howell et al., 1999
; Howell et al., 2001
; Whitman, 2001
; Yeo et al., 1999
), but most has been conducted on stable lines of transformed mammalian cells, which respond to TGFß stimulation by growth arrest (for reviews, see Massague, 2000
; Massague et al., 2000
). The case we analyse is different for two reasons: (1) A small, threefold, change in activin concentration causes animal cap cells to choose between two different zygotic gene responses, both of which are distinct from their behaviour in the absence of activin; (2) Cells must not only choose their correct response, but they must also achieve this when the signal factor concentration changes with time as the source starts to emit its signal. These aspects of signal transduction do not take place in cultured cell lines and have not so far been investigated in embryonic cells.
Our results lead us to the following concept of how cells respond to changing signal factor concentration during development. Soon after exposure to activin, a small part of a large cytoplasmic pool of Smad2 is phosphorylated and flows rapidly to the nucleus, where it is degraded. When the extracellular concentration of signal factor rises, the volume of flow of activated Smad2 increases in proportion, and so determines the steady state concentration of nuclear Smad2. This flow is maintained by an activated receptor complex. It is by the volume of this flow and the consequent Smad2 concentration in the nucleus that cells interpret their position in a morphogen gradient.
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MATERIALS AND METHODS |
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Cell dissociation and activin treatment
Animal caps (five/assay) from blastulae at about stage 8-8.5 were dissociated by incubation in Ca2+- and Mg2+-free 1x MBS supplemented with EDTA (0.5 mM) and with 0.1% BSA, for 10-15 minutes. Cells were dispersed by gentle pipetting, incubated for 10 minutes in the desired concentration of activin (R&D Systems), washed three times in the above dissociation medium, and then washed once in 1x MBS with 0.5% BSA. Cells were then spun briefly and incubated in 1x MBS with 0.5% BSA for reaggregation. Protein synthesis was inhibited by cycloheximide treatment (10 µg/ml) for 1 hour before activin treatment. To inhibit serine-threonine kinase, after activin treatment, cells were reaggregated in 1x MBS with 0.5% BSA supplemented with the Ser-Thr kinase inhibitor H7 (Calbiochem) at 500 µM.
RNase protection
RNA was prepared and RNase protection assays were performed as previously described (Ryan et al., 1996). Quantitation was achieved by use of a Fujifilm Phosphoimager and MacBAS2.5 software.
RNA expression constructs
Capped mRNAs were synthesised in vitro using Ambion Megascript (Ambion). Myc-Smad2 was prepared from pCS2+Myc6-Smad2 and GST-Smad2 from pT7-HA-GST-Smad2 as described before (Shimizu and Gurdon, 1999). pT7-GFP-Smad2 was constructed by replacing the HA2 tag sequence in pT7-HA2-Smad2 by a DNA fragment encoding the GFP, linearised by XbaI, and transcribed to GFP-Smad2 mRNA by T7. pT7-GR-GFP-Smad2 was constructed by inserting a DNA fragment encoding the hormone binding domain of the glucocorticoid receptor (GR) with the GFP in pT7-GFP-Smad2, linearised by XbaI and transcribed to GR-GFP-Smad2 mRNA by T7. pT7-HA2-Smad2* was constructed by mutating the Ser465 and Ser467 into Glu.
GFP-Smad2 observation by confocal microscopy
Animal cap cells from embryos injected with 0.25 ng of GFP-Smad2 mRNA were cultured on fibronectin slides, as described previously, and observed by confocal microscopy (Gurdon et al., 1999). To release the GR-GFP-Smad2 protein, dexamethasone (Sigma) was added to the medium at 10 µM.
Protein analysis
Dispersed cells from GST-Smad2 injected embryos were homogenised in buffer A (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% NP40; 0.1 mM PMSF; 0.2 mM NaF; 2 mM Na3VO4) supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Homogenates were spun three times and incubated with 40 µl glutathione sepharose slurry (pre-equilibrated in buffer A; Pharmacia Biotech) for 1 hour at 4°C. Bound proteins were resolved on SDS-PAGE and transferred to nitrocellulose. Phosphorylation of GST-Smad2 was detected by western blotting using the enhanced chemiluminescence immunoblotting-detection system (Pharmacia Biotech) with an anti-phosphoserine antibody (Zymed). Anti-GST blotting was performed according to the same protocol. Smad2 western blotting was performed as described (Faure et al., 2000).
Smad2 metabolic phosphorylation
Dissociated cells from animal caps of Myc-Smad2 injected embryos were treated with the indicated amount of activin, washed and cultured in 1x MBS with 0.5% BSA supplemented with 32P-orthophosphate (0.5 mCi/ml) until control embryos reached stage 10.5. Cells were lysed in TNE buffer containing protease inhibitors (Roche) and phosphatase inhibitor (Sigma), and the lysates pre-cleared with protein-A-Sepharose beads and subjected to anti-Myc immunoprecipitation. The immunoprecipitates were subjected to SDS-PAGE and visualised by autoradiography. Anti-Myc blotting was performed according to the protocol described above.
Labelling experiment
Dissociated cells from stage 8-8.5 embryos animal caps were cultured in Ca2+- and Mg2+-free 1x MBS with EDTA (0.5 mM) and with 0.1% BSA supplemented with [35S]methionine/cysteine (0.5 mCi/ml) for 20 minutes or with [32P]orthophosphate (0.5 mCi/ml) for 1 hour. The cells were washed extensively in the dissociating medium, treated with activin as described above and cultured for the indicated lengths time in 1x MBS with 0.5% BSA supplemented with cold methionine/cysteine (2 mM) or cold PO4 (10 mM). The cells were lysed in TNE buffer containing protease inhibitors (Roche), and the lysates pre-cleared with protein-A-Sepharose beads and subjected to anti-Smad2 (Transduction Laboratories) immunoprecipitation. The immunoprecipitates were subjected to SDS-PAGE, visualised and quantitated by use of a Fujifilm Phosphoimager.
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RESULTS |
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Modified Smad2s are valid markers of concentration-dependent activin transduction
Smad2 is a key component of the activin transduction pathway, and we need to know whether we can legitimately use modified Smad2s to follow small concentration-dependent variations in the use of this pathway. We have used Myc- or GST-Smad2 for immunoprecipitation and GFP-Smad for viewing living cells (Fig. 2A). To test whether these tagged forms correctly represent the activity of endogenous Smad2, we have compared their effectiveness in inducing Xbra and Eomes in animal cap cells. We find myc-Smad2, GST-Smad2 and GFP-Smad2 are almost equally efficient (Fig. 2B), and at most half as efficient than the wild-type Smad2 for the induction of Xbra (Fig. 2C), per amount of mRNA injected. Most importantly, these modified Smad2s induce Xbra much more strongly than Eomes at low doses, as does overexpression of unmodified Smad2 (Shimizu and Gurdon, 1999).
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Signal transduction immediately follows activin treatment
We need to know how cells time their response to changing signal factor concentration. Do cells always transduce a signal as soon as receptors are activated, or do they delay transduction until they are ready to make a gene response? One mechanism would be for cells to bind and transduce signal factors only at a particular time or stage in development, defined by developmental age. Cells would be able to receive or transduce a signal only at this time. As signal factor release and subsequent build-up is probably related to developmental age, cells would be provided with the right concentration of signal at their competent time. This is not an unreasonable idea, because, as noted above, cells express genes such as Xbra in response to activin signalling mainly according to a developmental clock, and not according to when they were exposed to the signal. The alternative idea is that cells initiate intracellular transduction whenever the signal is presented, but reveal a choice of gene response only at the appropriate stage of development, defined by developmental age.
The possibility that cells are able to bind activin to their receptors only at a particular developmental age can be eliminated. 35S-activin binds to the activin type II receptor at any time over the whole 5 hour period from stage 8 to stage 10.5, and this binding is competable and specific (Dyson and Gurdon, 1998). Moreover, phosphorylation of Smad2 can be detected before MBT, in embryos injected with activin mRNA at the two-cell stage (Faure et al., 2000
). The first intracellular step of activin transduction, namely the phosphorylation of Smad2, is recognisable by Western analysis with an antibody that recognises phosphoserine. Dissociated animal cap cells were prepared from embryos injected with GST-Smad2 mRNA, treated with activin (3 ng/ml for 15 minutes), washed and frozen for analysis 20 to 45 minutes after activin treatment. Western blots (Fig. 3A) show that the total amount of GST-Smad2 recognised by an anti-GST antibody is the same in all the samples. An anti-phosphoserine antibody applied to the same blot shows that Smad2 phosphorylation is already detectable 20 minutes after the first activin addition, and is seen strongly by 45 minutes (Fig. 3A). This rapid phosphorylation response is observed whether cells are treated with activin at a mid (stage 8) or late (stage 9.5) blastula stage (not shown), and therefore takes place at the same rate whether or not there is a subsequent transcriptional delay.
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We need also to know whether the nuclear Smad2 response to activin signalling is concentration related. From Fig. 3C, we see that, while the total amount of Myc-Smad2 remains the same, the amount of phosphorylated Smad2 increases proportionately, with threefold increments in activin concentration, in agreement with Shimizu and Gurdon (Shimizu and Gurdon, 1999). Real-time observation of nuclear GFP-Smad2 shows the same concentration related response, whenever cells are treated with activin between stage 9 and stage 10.5 (Fig. 3D).
In conclusion, we can eliminate the idea that an activin signal is transduced only at a particular developmental age. Rather we see that Smad2 phosphorylation and nuclear concentration follow directly and rapidly after activin exposure at any time during mid- and late-blastula stages, whether or not there is a transcriptional delay. We also see that the magnitude of this response is related to activin concentration.
Nuclear Smad2 concentration is inherited through mitosis
During the course of normal development between stages 8 and 10.5, each cell goes through several rounds of division. Fig. 4A shows the rate of increase in cell number during the early stages of activin response. Is the memory of activin treatment, and specifically the nuclear content of Smad2, inherited through mitosis? Low-density cultures on fibronectin slides show a uniformity of gene expression among cells after brief activin treatment (Gurdon et al., 1999). This suggests that all cells have remembered the activin concentration that they experienced, including those which have gone through a round of division. To trace nuclear Smad2 directly through mitosis, we have used cells from GFP-Smad2-injected embryos, treated with two different concentrations of activin (3 and 9 ng/ml). We have followed the intracellular movement of GFP-Smad2 protein by confocal microscopy in real time, and have looked at individual dividing cells containing GFP-Smad2 in the nucleus (Fig. 4B). Cells that received a 10 minute exposure to activin were cultured after washing to remove free activin and observed under the microscope as they underwent mitosis. For the two concentrations of activin that we used, both daughter cells deriving from a cell with nuclear GFP-Smad2 contained a similar amount of GFP-Smad2 localised in the nucleus (Fig. 4B). The quantitation of nuclear GFP-Smad2 shows no decrease in concentration after cell division (Fig. 4B). After division, daughter cells are half the volume of their parent cell, as there is no growth in these early embryo cells. Therefore the concentration of nuclear GFP-Smad2 should be the same in mother and daughter cells if receptors are working with the same efficiency. We conclude that the memory of activin concentration is inherited through, or reconstituted after, mitosis.
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In mammalian tissue culture experiments, phosphorylated Smad2 peaks 1 hour after a short exposure to TGFß, and then declines (Lo and Massague, 1999). With Xenopus animal cap cells exposed to activin for 10 minutes, and then washed, the result is different. Phosphorylation of the Smad2 C-terminal serines, as measured by western analysis, reaches a maximum level 45 minutes after activin treatment, and then shows no decline for at least the next 4 hours (Fig. 4C). Likewise, the nuclear concentration of Smad2 that follows a 10 minute activin exposure lasts at least 4 hours in a non-dividing cell (Fig. 4D, quantitation).
By observing animal cap cells dissociated from GFP-Smad2-injected embryos and treated with activin, we have distinguished between the burst and the continuous turnover explanations for the maintenance of phosphorylated nuclear Smad2 (Fig. 4E). These cells contain different amounts of GFP, as some cells received, by chance, more mRNA, and hence more GFP-Smad2 protein (Fig. 4E, red arrows) than others (Fig. 4E, yellow arrows). Because there is such a low amount of GFP-Smad2 in some cells (Fig. 4E, yellow arrows), a small decrease of GFP-Smad2 renders them invisible quicker than cells with a bigger pool of GFP-Smad2 (Fig. 4E, red arrows). After activin treatment, nuclear GFP-Smad2 can be easily seen to disappear in cells with a lower amount of GFP (Fig. 4E, yellow arrows). This favours the idea of continuous nuclear Smad2 turnover and replacement.
To determine whether the long lasting high level of nuclear Smad2 is maintained by turnover, we labelled activin-treated cells with 35S-methionine for 20 minutes. Two and a half hours after the end of the labelling period, immunoprecipitation showed that half of the labelled Smad2 pool had been degraded (Fig. 5A, black line), whereas in non-activin treated cells the amount of labelled Smad2 remained constant (Fig. 5A, grey line). To determine the turnover of activated (as opposed to total) Smad2, we labelled cells with 32P-orthophosphate which marks only activated Smad. After immunoprecipitation, the Smad2 signal was quantitated (Fig. 5B). Phosphorylation of Smad2 can not be detected more than two hours after the removal of activin. The Smad2 turnover must have a half-life of 1 hour or less, because the 32P-orthophosphate pool is not effectively chased in our experiments. These results are inconsistent with the burst hypothesis and support the idea of a continuous turnover and replacement.
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To demonstrate more directly the continuing activation and nuclear entry of Smad2 in response to activin, we fused GFP-Smad2 to the hormone-binding domain of the glucocorticoid receptor (GR-GFP-Smad2) (Fig. 5D) to provide a conditional release of Smad2. The glucocorticoid domain causes sequestration of the fusion protein in the heat-shock apparatus; this sequestration is relieved and the protein released by addition of dexamethasone (DEX) (Kolm and Sive, 1995; Mattioni et al., 1994
). Nuclear accumulation of GR-GFP-Smad2 can be observed in cells treated with dexamethasone more than 3 hours after the end of activin treatment (Fig. 5E). We conclude that the concentration of Smad2 is maintained in the nucleus of activin treated cells by continuous entry and degradation.
The Smad2 flow changes up but not down
In normal development, Nieuwkoop centre signalling is thought to start at the 250-cell stage (Jones and Woodland, 1987) and to become increasingly strong during stages 8 and 9 (Wylie et al., 1996
). Animal cap cells near the Nieuwkoop signalling source must therefore experience a continuous increase in the concentration of the factor(s) during this time. As already shown by bead-exchange experiments (Gurdon et al., 1995
) and confirmed in Fig. 6A, cells activated genes according to the highest concentration of activin they received. Cells exposed to 1 ng/ml activin for 15 minutes expressed Xbra but not Eomes at stage 10.5 (Fig. 6A, track 1). Cells given a 15 minute treatment of activin at 4 ng/ml expressed Eomes more strongly than Xbra (Fig. 6A, track 2). Exactly the same ratio of Eomes:Xbra expression was induced by 1 ng/ml followed by 4 ng/ml, as well as by 4 ng/ml followed by 1 ng/ml (Fig. 6A, tracks 3 and 4, respectively). Because an extracellular concentration of activin is transduced to the nucleus in 20 minutes, and as cells remember an activin concentration for several hours, we need to understand how cells react at the level of transduction to external activin concentrations that go up with time.
Cells exposed to a weak concentration of 1 ng/ml activin for only 15 minutes show a visible but weak GFP accumulation in the nucleus, and this does not increase with time (Fig. 6B,C). The same 1 ng/ml activin for 15 minutes followed 30 minutes later by a concentration of 4 ng/ml activin results in a clear increase of the GPF nuclear accumulation (Fig. 6B,C). Not surprisingly, cells that received an initial concentration of 4 ng/ml, followed 30 minutes later by a 1 ng/ml concentration, do not show a decrease in nuclear GFP; the level of nuclear GFP remains at a high concentration (Fig. 6B,C).
We have shown above that a particular dose of activin, which equates to a constant number of activated receptor complexes and to continuous flow of Smad2 to the nucleus, elicits the same choice of gene response over several hours, and that there is no integration of number of occupied receptors with time. We now conclude that cells quite rapidly change their perception of activin concentration in an upwards direction, but not downwards, over the course of a few hours, by adapting the flow of Smad2 to the highest concentration of activin. We therefore suppose that, in the absence of anti-factors, when the concentration of signal factor has reached its highest level in part of an embryo, cells in that region will continue to express the appropriate genes, even if the concentration of factor subsequently decreases.
The Smad2 pool
We concluded that (1) cells remember the activin concentration for several hours, (2) the basis of this memory is reflected in the nuclear concentration of Smad2, and (3) this nuclear concentration of Smad2 is maintained in steady state by a continuous turnover and replacement. This model raises the following question: is the cytoplasmic pool of Smad2 big enough to refresh the nuclear pool of Smad2 for several hours, even in the presence of cycloheximide?
To answer this question, we analysed the Smad2 protein level in animal cap cells treated with activin in the presence or absence of cycloheximide (Fig. 7A). Cells were cultured for 3 hours after activin treatment and the total amount of Smad2 determined by western blot with an anti-Smad2 antibody. The quantitation of two separate experiments (Fig. 7B) shows that 3 hours after activin exposure and cycloheximide treatment, the amount of Smad2 decreases only by 25% from the initial level. We conclude that there is enough Smad2 in the cytoplasm to supply the Smad2 flow and to maintain the nuclear concentration for several hours, even in absence of protein synthesis.
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To test whether persistent receptor signalling is responsible for continuing Smad2 transduction, we have used the specific serine-threonine kinase inhibitor H7 to cause discontinuation of receptor signalling at various times after activin treatment. Animal cap cells were exposed to activin for 10 minutes and washed, as usual, and then treated with serial dilutions of the inhibitor H7. High concentrations of H7 damage cells, as judged by the inhibition of the FGF-R transcripts (Fig. 8A, tracks 3 and 4). A low concentration of inhibitor has no effect (Fig. 8A tracks 7 and 8). Middle concentrations (tracks 5 and 6) reduce or inhibit the transcription of Xbra, Eomes and Apod, but do not reduce the level of FGF-R.
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To confirm that H7 represses specifically the phosphorylation of Smad2, we used a constitutively active type I receptor (Alk4*) (Armes and Smith, 1997) or a constitutively activated Smad2 (Smad2*), where the C-terminal serines have been mutated to glutamine, to mimic the phosphorylation. The constitutively activated Smad2* activates gene transcription at a lower concentration of injected mRNA than Smad2 (data not shown). H7 should repress gene activation by Alk4* but not activation by Smad2*. Alk4*- or Smad2*-injected animal cap cells were treated, or not, with two concentrations of H7 and gene activation was analysed by RNase protection assay (Fig. 8C). The two effective concentrations of H7 repress the activation of Apod, Eomes and Xbra by Alk4* (lanes 6 and 7), but not their activation by Smad2* (lanes 3 and 4). We conclude that H7 specifically inhibits receptor signalling, but not downstream transduction.
A prediction from this result is that the inhibitor H7 should have no effect on gene expression the nearer it is administered to the time when gene transcription starts. This is because activin induces a continuing flow of Smad2 from the cytoplasm to the nucleus. The inhibitor arrests this flow, and the nuclear content of Smad2 will decrease in accord with its turnover, remaining at up to half of its normal level for 1-1.5 hours. To test this prediction, animal cap cells were treated with activin for 10 minutes, washed and the inhibitor added at different times. In Fig. 8D,E, we see that gene expression is progressively restored to the nil-inhibition level, the shorter the time between inhibitor addition and the assay of gene expression. We conclude that the continuing flow of Smad2 to the nucleus is maintained by a receptor, or ligand-receptor complex, that remains active in the absence of any extracellular supply of activin. An activated receptor complex is the most likely basis of the memory described above.
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DISCUSSION |
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The timing of gene response, reviewed by Cooke and Smith (Cooke and Smith, 1990) is a peculiar characteristic of animal development. The mechanism of this developmental timing is not understood. However, our results described here have eliminated one possible explanation for it. This is that it depends on the delayed transduction of a signal until a certain developmental age has been reached. We find that an activin signal is rapidly transduced to the level of nuclear Smad2 whenever the signal is presented. Evidently the timing mechanism resides at a level beyond that of nuclear Smad2 accumulation.
Response to changing morphogen concentration
The reason we initiated the work described here was to determine how cells make a correct response to changing signal factor concentration. Although there are only a few cases where this has been directly measured, nearly all signalling molecules believed to guide early vertebrate development have concentration-related effects, whether directly like members of the TGFß class that contribute to Nieuwkoop signalling, or indirectly like the anti-factors noggin and chordin in Spemann signalling. In the case of activin and probably in other early developmental signalling molecules, cells are competent to respond to signals over a wide time span of several hours. Therefore cells must experience factor concentrations that change with time, but must nevertheless select the correct concentration to which to make their major response.
Our results suggest the following process by which cells respond to changing signal factor concentration during development. As soon as signalling starts, cells bind the extracellular factor to their receptors, and quickly transduce the signal through activated Smad2 to create a nuclear concentration of Smad2. A steady-state nuclear Smad2 concentration is maintained by a continuous flow from the cytoplasm to the nucleus, where Smad2 is degraded. In transformed cell lines, phosphorylated Smad2 is degraded by ubiquitination (Lo and Massague, 1999). But in these cells, the significance of ubiquitin-dependent degradation of Smad2 remains to be defined. It may ensure a swift elimination of the TGFß signalling that regulates very dynamic physiological processes, or it may remove the surplus activated Smad from the nucleus (Massague and Chen, 2000
). In the case we have analysed here, it ensures a constant nuclear concentration of Smad2, which reflects the extracellular concentration of activin. A gene response, and hence cell-fate decision, is made at a particular developmental stage unrelated to when signalling commenced. Even in the absence of a continuing signal source, cells remember the highest concentration of signal that they have received by maintaining the same nuclear concentration of activated Smad through the continuing activity of an activated receptor complex. As the extracellular concentration of signal factor goes up with the continuing emission of a signal from its source in the embryo, the nuclear Smad2 concentration increases in proportion by an increase in the volume of intracellular Smad2 flow. In this way, cells always select their choice of gene response according to the highest signal factor concentration that they experience in their competent life.
Relationship to transduction response in normal development
All our experiments described here have treated blastula animal cells with activin. It is currently believed that activin reflects the activity of related TGFß signal molecules such as Xnr1-Xnr6, derriere, etc (Hill, 2001; Whitman, 2001
). Most animal cap cells do not normally receive TGFß signals, but nevertheless respond to experimentally supplied activin by following the same gene responses and cell fate pathways as equatorial blastula cells, thought to be the normal recipients of TGFß (Nieuwkoop) signalling. As activin protein is much easier to acquire than Xnr protein, and as animal cap cells have received no TGFß signals at the time of isolation, we use this protein and these cells to understand the principles of cell response to changing signal factor concentration in development.
We suggest that our model is likely to apply to signalling in normal development, except for the absence of anti-factors, such as Cerberus, that are secreted from the dorsal lip of gastrulae and are absent from animal cap cells. Such molecules may be responsible for the loss of phosphorylated Smad2 in the dorsal region of mid-gastrulae (Lee et al., 2001), and would presumably prevent an indefinite increase in extracellular concentration of TGFß factors. The decrease of phosphorylated Smad2 in the dorsal region compared with the ventral region occurs around stage 11 (Lee et al., 2001
). The loss of competence to respond to TGFß factors, which happens at stage 11 (Gurdon et al., 1985
; Jones and Woodland, 1987
), can also explain this loss of phosphorylated Smad2 in the dorsal region. In all other respects, we believe our conclusions are consistent with what is known about TGFß signalling in normal embryos, where the timing of signal factor exposure is not as accurately known as it is in our protein addition experiments.
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ACKNOWLEDGMENTS |
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