PATTERN FORMATION IN THE BLUE-GREEN ALGA ANABAENA

Transcription

1 J. Cell Sd. 13, (1973) 637 Printed in Great Britain PATTERN FORMATION IN THE BLUE-GREEN ALGA ANABAENA II. CONTROLLED PROHETEROCYST REGRESSION MICHAEL WILCOX, G. J. MITCHISON AND R. J. SMITH MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CBi 2QH, England SUMMARY We present further evidence for an interactive mechanism in the formation of the spaced pattern of heterocysts in Anabaena. The evidence comes from experiments which are an extension of those described earlier, in which filaments of the alga are broken near to a proheterocyst. We argue that a proheterocyst depends upon neighbouring vegetative cells for the removal of an inhibitory substance: when the proheterocyst is deprived of these supporting vegetative cells it will be forced to regress. We showed earlier that such regressions do occur in early proheterocysts when a filament is broken on one side only. We now find that advanced proheterocysts can be made to regress when double breakages are performed to leave small fragments containing the proheterocysts. The probability of a proheterocyst regressing is correlated with its stage of development and with the size of the fragment: the smaller the fragment, the more advanced is the stage at which regression will occur. To formulate this we have defined developmental stages in terms of ultrastructure and compiled the results of a diversity of breakage operations with the cells at these specified stages. Certain compounds affect the spacing of the heterocyst pattern, causing it to become wider or narrower. These compounds have the predicted effect upon regression frequencies, upholding our assumption that regressions express an underlying competitive mechanism. INTRODUCTION In a previous paper (Wilcox, Mitchison & Smith, 1973) we showed that the formation of a spaced heterocyst pattern in Anabaena can be described by a combination of an inhibitory zone mechanism and an interactive mechanism which prevents proheterocysts (presumptive heterocysts) from developing too close together. To account for the latter mechanism we assumed that a proheterocyst requires the 'support' of adjacent vegetative cells in order to develop, and that 2 close proheterocysts deprive each other of essential support. To test this hypothesis we showed that a proheterocyst can be made to reverse its development and regress to the state of a vegetative cell when its support is physically removed by breaking the filament nearby. These experiments used proheterocysts at a very early stage of development. In the present paper we describe an extension of these experiments in which the support is further reduced by making breaks on both sides of a proheterocyst - in the extreme case producing an isolated cell. We then find that proheterocysts at a much higher level of development can be made to regress. Moreover, it is possible to correlate the stage of 41 CEL 13

2 638 M. Wilcox, G. J. Mitchison and R. J. Smith development of a cell with the probability of its regressing in a fragment of a given size. To make this precise we have defined stages of proheterocyst development in terms of ultrastructure, and correlated these with the light-microscopic appearance of the same cells. This we describe in the first section. In the second, we summarize the relationship between the stage of development, fragment size and regression frequency. MATERIALS AND METHODS The organism used for these studies, A. catemda (Cambridge Culture Collection 1403/1), was grown in liquid culture in a defined salts medium, ph 80 (Allen & Arnon, 1955), or on plates of the same medium solidified with 1 % agar. Detailed culture conditions are given in an earlier paper (Wilcox et al. 1973). Details of light microscopy, and of the handling and controlled breaking of single algal filaments are given in the legend to Table 1 (p. 640). Electron microscopy Algal filaments growing on plates were covered with a layer of agar, then fixed in situ for 6 h in 4 % glutaraldehyde/o' 1 M potassium cacodylate, ph 7, and stained with 2 % aqueous potassium permanganate for 15 h. Each operation was carried out at 3 C C. After rinsing with water, small agar blocks containing filaments were cut, dehydrated using a graded ethanol series followed by propylene oxide, and embedded in Epon 812 after equilibrating first in 1:1 and 3 :1 Epon/propylene oxide mixtures. Following polymerization (40 C for 16 h, 60 C for 72 h) sections were cut on a Porter-Blum HT-i ultramicrotome, placed on Formvar-coated grids, and post-stained briefly in 5 % aqueous uranyl acetate and 4 % lead citrate. Sections were observed in a Siemens Elmiskop electron microscope. For correlation of light and electron micrographs, 6-8 filaments were aligned on agar plates into a parallel bundle, using micromanipulators. Light micrographs were then taken before the filaments were fixed. After sectioning, electron micrographs were taken which could be compared directly with the light micrographs of the same cells. RESULTS Definition of stages in proheterocyst development We have denned 7 stages in proheterocyst development in terms of certain distinctive ultrastructural features. Under the electron microscope the first sign of differentiation (stage I) is the laying down of the outer fibrous layer of the heterocyst envelope (the terminology is that of Lang & Fay, 1971) around an otherwise normal vegetative cell (Fig. 1). Note the microplasmadesmata (m), which can be seen in the cross-wall between the cells, and which appear to be identical to those between more advanced proheterocysts or heterocysts and their adjacent vegetative cells (Figs. 2, 6). In a stage II proheterocyst the fibrous layer is complete and the junction between the cell and its neighbours is 'squaring off' (Fig. 2), this being the first stage in the formation of the specialized polar structure of the mature heterocyst. The junction is drawn out into a neck during stages III-IV; the beginning of this process is seen as an elongation or drawing out of the cell wall in the polar region by stage III (Fig. 3). By this time, too, the envelope is thickening as the middle homogeneous layer begins to form, and electron-transparent spaces appear between the twin membranes of the photosynthetic lamellae. These spaces increase to a maximum in the stage IV proheterocyst and accompany contortion and apparent fragmentation of the lamellae (Fig. 4). By stage V

3 Pattern formation in Anabaena 639 (Fig. 5) the first traces of the innermost laminated layer of the envelope are visible, and at about the same time, great enlargement and rounding up of the cell is seen, and some polarization of the lamellae, which are by now heavily contorted. From these polar regions in the maturing heterocyst there is a great proliferation of what appear to be new lamellae (Figs. 7, 8), which differ from those found in vegetative cells in that the distance between the adjacent membranes of each lamella is much reduced (Lang & Fay, 1971; compare Fig. 7). At the same time as this, a 'plug' of material showing variable staining with permanganate is formed in the neck of the junction. Traces of this material, in this case electron-transparent, can be seen in the stage VI cell (Fig. 6, arrow). Often the plug stains strongly (Fig. 8) and, on occasion, appears to be composed of tightly packed membranes. In other micrographs (e.g. Fig. 9) where the plug has contracted and pulled away from the cell plasmalemma, there is evidence that the plug is itself bounded by membranes (arrow) which are apparently continuous with the lamellae. It should be emphasized that it is not clear from this study if any of these mature heterocyst features are concerned more with the ageing of the heterocyst than with the maturing process. For a fuller discussion of the various ultrastructural features, most of which are common to all the Anabaena species which have been investigated, the reader is referred to other reports (Lang & Fay, 1971; Lang, 1965). By the direct correlation of light and electron micrographs of the same cell (see Materials and Methods), we have been able to determine the appearance under the light microscope of proheterocysts at each of the 7 stages (a sequence of light micrographs is shown in Fig. 12A-G). Initially, an increase in greyness (under phasecontrast optics) and a loss of granularity in the cell can be seen (stage I). The cell then elongates beyond the stage at which a new septum would normally appear (stage II) and subsequently enlarges greatly and 'rounds up', its diameter eventually reaching 1-5 times that of a vegetative cell (stages III-V). Finally, the cell becomes very refractile, and distinctive polar structures are formed (stages VI-VII). We would point out that these stages do not correspond to the 7 stages of heterocyst differentiation denned by Fogg (1951) from cytological observations. In the main, these latter are stages in heterocyst maturation and ageing (i.e. our stages VI-VII). Controlled proheterocyst regression In an earlier paper (Wilcox et al. 1973) we put forward a model for pattern formation in Anabaena in which proheterocysts arise from cells lying outside inhibitory zones surrounding each heterocyst. This model also requires a ' competitive' mechanism for preventing 2 cells from developing simultaneously when they are too close together. This competitive mechanism can only be inferred indirectly from events seen in normal growth, so we devised an experiment to demonstrate it more clearly. We proposed that, in order to develop, proheterocysts require 'supporting' adjacent vegetative cells (which destroy a hypothetical inhibitory substance produced by the proheterocyst), so that proheterocysts close to each other will compete for support. This can also be described in terms of a 'mirror image' argument (Wilcox et al. 1973) without reference to a specific model. When a filament is broken close to a proheterocyst, its support is cut down, and the cell may regress. In the earlier paper, single breaks 41-2

4 640 M. Wilcox, G. J. Mitchison and R. J. Smith were performed on A. cylindricafilaments,and proheterocysts regressed only when they were chosen at the earliest possible stage. By making 2 breaks, so as to reduce the support on both sides of the proheterocyst, we have been able to obtain regressions at a much more advanced state of development. A. catemila was used for these experiments; similar results were obtained, however, in a Limited number of experiments using A. cylindrica. Table 1. Proheterocyst regressions at increasing stages of development Regression frequency, %, at stage Fragment II III IV V VI VII 3PI 3P 2PI 2P IPI IP p 31 5' ioo-o s SS Single filaments were transferred to agar plates and fragments obtained by puncturing chosen cells using Zeiss (Jena) micro-manipulators. After a coverslip had been placed in position, fragments were observed using phase-contrast optics and 400 x magnification. The nomenclature for fragments and for proheterocyst stages is given in the text. A minimum of 25 operations was performed in cases where no regressions occurred, and a minimum of 50 in other cases. We have found a correlation between the stage of development of a proheterocyst, the number of vegetative cells in the isolated fragment, and the frequency of regression. The results are summarized in Table 1. For example, in 3P1 fragments (fragments with three vegetative cells on one side of the proheterocyst and one on the other) stage II proheterocysts regressed only in some 3 % of cases, but, in 2P1 fragments this frequency increased to 17 % (the rate of spontaneous regression of stage II proheterocysts in control filaments was 0-5 %). Stage III proheterocysts, which did not regress in either of these cases, could be induced to regress in 1P1 fragments (regression frequency 31 %; a sequence of photographs illustrating such an operation is shown in Fig. 13). In a similar way, stage IV proheterocysts regressed 29% of the time in ip fragments, and, in the case of a completely isolated proheterocyst, or P fragment, stage V cells regressed, also with a frequency of 29 % (see Fig. 14). We were unable to obtain any regressions, however, with stage VI or VII maturing heterocysts (see the Discussion). From the results obtained with 3P and 2P fragments it appears that a proheterocyst at the end of a fragment has a somewhat lower probability of regressing than one placed internally. For example, regression frequencies for stage II proheterocysts in 3P fragments and for stage II and III proheterocysts in 2P fragments were lower than those obtained when the cells were placed internally in the approximately equivalent fragments, 2P1 and 1P1, respectively (Table 1). In all cases, regression and subsequent division led to the formation of normal

5 Pattern formation in Anabaena 641 vegetative cells from which a new proheterocyst might eventually be chosen. When early proheterocysts regressed, the envelope usually disintegrated (Fig. 13), or was discarded essentially in one piece (Figs. 10, 15). With more advanced cells it tended to remain intact, the new filament emerging from one or both poles (Figs. 11, 15). Proheterocysts which failed to regress developed normally in all cases. Table 2. Effect of ammonia and j-azatryptophan on regression frequency Fragment Control Regression frequency, % + Ammonia + 7-azatryptophan 3P ip Control fragments were obtained as described in the legend to Table 1. For + ammonia or + azatryptophan operations, filaments were incubated for h in salts medium supplemented with 0-2 % (3-5 x io~ 3 M) NH 4 C1, or for 4 h in medium containing 2 x IO~ 6 M DL-7- azatryptophan, and then transferred to plates containing 0-2% NH 4 C1 or 5 x IO~ 6 M DL-7- azatryptophan, respectively. All proheterocysts were picked when at stage II. A minimum of 60 operations was performed in each case. To rule out the possibility that regressions were caused by some non-specific effect, unrelated to the pattern-forming process, we tested 2 compounds known to influence the heterocyst pattern to see whether they affected the frequency of proheterocyst regression. It has been known for some time that, in the presence of ammonia, heterocyst development is affected, so that a pattern consisting largely of proheterocysts, rather than mature heterocysts, is formed (Talpasayi & Bahal, 1967; Wilcox, 1970). Eventually the pattern widens considerably and becomes very indistinct (Fogg, 1949). We have recently found that the tryptophan analogue 7-azatryptophan has the opposite effect on heterocyst spacing in Anabaena, leading to the formation of a much closer and less regular pattern with many multiple and terminal heterocysts (G. J. Mitchison & M. Wilcox, in preparation; see Fig. 16). Both these compounds markedly affect regression frequencies, as shown in Table 2. For example, when 3P fragments containing stage II proheterocysts were isolated in the presence of ammonia, the proheterocysts regressed in 76 % of cases (the frequency in controls was 5 %). Similar increases were found in the regression frequencies of proheterocysts in 3P1 and 2P1 fragments. Even in the presence of ammonia, however, we were unable to induce the regression of any stage VI or VII cells. When ip fragments were isolated in the presence of low concentrations (2-5 x io~ s M) of DL-7-azatryptophan the regression frequency of stage II proheterocysts was dramatically reduced from 93% (in controls) to 11 %. DISCUSSION The experiments described in this paper provide convincing evidence in support of an interactive mechanism which serves to prevent proheterocysts from arising too

6 642 M. Wilcox, G. J. Mitchison and R. J. Smith close together. We have interpreted the single break experiments, in which early proheterocysts were induced to regress (Wilcox et al. 1973), as demonstrating that a proheterocyst can have an inhibitory effect upon itself. In this way we suppose that the regression experiments show the same interactive process which occurs in normal growth. It is possible, however, that non-specific effects (e.g. damage during the operation) are causing the regressions. This is made unlikely by the finding that compounds such as ammonia and 7-azatryptophan, which affect the pattern during normal growth, have a marked and parallel effect on the probability of regression. There is a further alternative explanation for regressions of early proheterocysts, which assumes that there is a ' preliminary phase' of development where proheterocysts are neither producing inhibitor, nor committed to development. We have given statistical arguments against this view (Wilcox et al. 1973), but a much stronger argument is now available, since very advanced proheterocysts are found to regress in the double-break experiments. Proheterocysts at these advanced stages have a well denned inhibitory zone and must therefore be supposed to be sources of the hypothetical inhibitor. We must conclude that proheterocysts can be both a source of inhibitor and, at the same time, susceptible to its effect. This implies that, whatever form the model takes, it will have to incorporate competition. We are still faced with the problem of accounting for the anomalous results obtained with fragments containing terminal proheterocysts. According to our model, when reasonable assumptions are made for the kinetics of morphogen metabolism, a 1P1 fragment should be no more efficient than a 2P fragment (or a 2P1 than a 3P) in causing regressions; i.e. one vegetative cell on each side of a proheterocyst should provide at least as efficient a support system as two cells on one side of the proheterocyst. This prediction is not borne out. A proheterocyst left in a terminal position has a lower probability of regressing than one placed internally. This is exactly analogous to the finding with terminal proheterocysts in the single-break experiments, and we would offer the same explanation (Wilcox et al. 1973), of inhibitor leakage through an exposed junction. The events which take place when proheterocysts regress may provide an interpretation for what has been termed 'heterocyst germination'. This phenomenon, if confirmed, would have a bearing on the question of heterocyst function. Wolk (1965) reports that, under certain conditions, germlings are seen emerging from heavy and well formed envelopes, supposedly those of mature heterocysts. We have grown cultures under these conditions, and although we do see germlingg emerging from envelope material, we have also found that the conditions make it impossible foi us to determine their origin, and to assert that they arise from heterocysts. However, these germlings closely resemble the filaments emerging from regressing proheterocysts, where there is often a well defined envelope discarded even from an early proheterocyst (see, for example, Fig. 15). Since there is a large amount of filament breakage under the germination conditions, we would suggest that' heterocyst germination' is in reality the result of breakage near to proheterocysts. With this assumption, germlings would probably never come from maturing (stage VI) or mature (stage VII)

7 Pattern formation in Anabaena 643 heterocysts, since such heterocysts cannot be made to regress in our experiments, even when the cells have been completely isolated in the presence of ammonia. Having said that mature heterocysts cannot be induced to regress, it should be emphasized that stage V proheterocysts, which are at an advanced level of development and morphologically very different from their precursor vegetative cells, will regress if completely isolated. This adds to a considerable body of evidence (Hay, 1968) that cells may not be committed to a particular course of differentiation even when they have developed much of the appropriate morphology. What our fragment experiments provide is a method for putting upon a cell a precise amount of pressure to dedifferentiate. This method is, moreover, one which is directly related to the pattern-forming process in the organism. We are grateful to Keith Roberts for his expert advice, and to Sydney Brenner, Francis Crick and Peter Lawrence for helpful discussions. REFERENCES ALLEN, M. B. & ARNON, D. I. (1955). Studies on nitrogen-fixing blue-green algae. PL Physiol., Lancaster 30, FOGG, G. E. (1949). Growth and heterocyst production in Anabaena cylindrica Lemm. II. In relation to carbon and nitrogen metabolism. Ann. Bot., N.S. 13, FOGG, G. E. (1951). The cytology of heterocysts. Ann. Bot., N.S. 15, HAY, E. D. (1968). Dedifferentiation and metaplasia in vertebrate and invertebrate regeneration. In The stability of the Differentiated State (ed. H. Ursprung), pp Berlin, Heidelberg, New York: Springer-Verlag. LANG, N. J. (1965). Electron microscopic study of heterocyst development in A. azollae. J. Phycol. 1, LANG, N. J. & FAY, P. (1971). The heterocysts of blue-green algae. Proc. R. Soc. B 178, TALPASAYI, E. R. S. & BAHAL, M. R. (1967). Cellular differentiation in A. cylindrica. Z. Pfl. Physiol. 56, WILCOX, M. (1970). One-dimensional pattern found in blue-green algae. Nature, Lond. 228, WILCOX, M., MITCHISON, G. J. & SMITH, R. J. (1973). Pattern formation in the blue-green alga Anabaena. I. Basic mechanisms. J. Cell Set. 12, WOLK, C. P. (1965). Heterocyst germination under defined conditions. Nature, Lond. 205, {Received 13 February 1973)

8 644 M. Wilcox, G. J. Mitchison and R. J. Smith Fig. 1. The stage I proheterocyst differs from its adjacent vegetative cell only in the presence of traces of the fibrous layer (/) of the envelope; the other features normally found in vegetative cells (e.g. photosynthetic lamellae (Ja), cyanophycin granules (c), polyhedral bodies (b)) are present in both cells. Also indicated are the thin microplasmadesmata (m) between the cells, x Fig. 2. In a stage II cell the fibrous layer (/) is complete and the proheterocystvegetative cell junction is 'squaring off'. Note again the microplasmadesmata (m). x Fig. 3. By stage III, the homogeneous layer (h) of the envelope is forming inside the fibrous layer (/), and the cell is drawn out (arrowed) as the specialized junction begins to form, x Fig. 4. Stage IV proheterocyst showing fibrous (/) and extensive homogeneous (h) layers, and contorted lamellae often containing electron-transparent intralamellar spaces (e). x Fig. 5. By stage V, traces of the innermost laminated envelope layer (I) are visible. Note apparent concentration of fragmented contorted membranes in polar regions. /, fibrous layer; h, homogeneous layer, x Fig. 6. In a stage VI maturing heterocyst the 3 envelope layers (/, h, I) are clearly seen. Note again the apparent polarization of the lamellae, traces of electron-transparent 'plug' material (arrowed) in the pore channel of the junction, and microplasmadesmata (m). x

9 Pattern formation in Anabaena 645 * 6

10 646 M. Wilcox, G. J. Mitchison and R. J. Smith Fig. 7. In a mature (stage VII) heterocyst there is a proliferation of lamellae. The plug in this case is electron-transparent, x Fig. 8. A mature heterocyst showing extensive proliferation of lamellae and heavily stained plug region. Compare with Fig. 7. x Fig. 9. The pore region of a mature heterocyst showing membranes (arrowed) enclosing the electron-transparent plug and apparently continuous with the lamellae, x Fig. 10. A Pi fragment containing a regresssing stage II proheterocyst, fixed 15 h after its isolation. The discarded proheterocyst envelope is arrowed, x Fig. 11. A regressing stage IV proheterocyst, fixed 20 h after its isolation. The elongating cell is emerging from both ends of the cell, leaving the envelope (arrowed) intact, x

11 Pattern formation in Anabaena 647

12 648 M. Wilcox, G. J. Mitchison and R. J. Smith Fig. 12. Light-microscopic appearance of the 7 stages of heterocyst development in A. catenula. A-G are a sequence of photographs of the same cell at stages I-VII, taken at o, 3, 6, 9, 12, 14 and 18 h, respectively, x 800. Fig. 13. Sequence of light micrographs showing regression of a stage III proheterocyst in a 1P1 fragment; the proheterocyst is arrowed. Alongside is a 2P1 fragment containing a stage II proheterocyst which failed to regress. A, B, C were taken at o, 10 and 17 h, respectively, after isolation of the fragments, x 800. Fig. 14. Regression of a completely isolated stage V proheterocyst. Light micrographs A, B, c, D were taken at o, 22, 44 and 68 h, respectively, after isolation. A proheterocyst (arrowed) is developing in the new filament in D. x 800. Fig. 15. Light-microscopic appearance of a regressed stage II proheterocyst (isolated in a Pi fragment 24 h earlier) showing the discarded proheterocyst envelope (arrowed), x1000. Fig. 16. A, an A. catenula filament from a culture grown for 40 h in the presence of 2 x io~ 6 M DL-7-azatryptophan. B, a filament from a control culture. Proheterocysts and heterocysts are arrowed, x 600.

13 Pattern formation in Anabaena 649

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