PATTERN FORMATION IN THE BLUE-GREEN ALGA ANABAENA · out that these stages do not correspond to the 7 stages of heterocyst differentiation denned by Fogg (1951) from cytological observations

J. Cell Sd. 13, 637-649 (1973) 637Printed in Great Britain

PATTERN FORMATION IN THE BLUE-GREEN

ALGA ANABAENA

II. CONTROLLED PROHETEROCYST REGRESSION

MICHAEL WILCOX, G. J. MITCHISON AND R. J. SMITHMRC Laboratory of Molecular Biology, Hills Road, Cambridge, CBi 2QH,England

SUMMARY

We present further evidence for an interactive mechanism in the formation of the spacedpattern of heterocysts in Anabaena. The evidence comes from experiments which are an exten-sion 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 ofan inhibitory substance: when the proheterocyst is deprived of these supporting vegetativecells it will be forced to regress. We showed earlier that such regressions do occur in earlyproheterocysts when a filament is broken on one side only. We now find that advanced pro-heterocysts can be made to regress when double breakages are performed to leave smallfragments containing the proheterocysts.

The probability of a proheterocyst regressing is correlated with its stage of development andwith the size of the fragment: the smaller the fragment, the more advanced is the stage at whichregression will occur. To formulate this we have defined developmental stages in terms ofultrastructure and compiled the results of a diversity of breakage operations with the cells atthese specified stages.

Certain compounds affect the spacing of the heterocyst pattern, causing it to become wideror narrower. These compounds have the predicted effect upon regression frequencies, up-holding our assumption that regressions express an underlying competitive mechanism.

INTRODUCTION

In a previous paper (Wilcox, Mitchison & Smith, 1973) we showed that the forma-tion of a spaced heterocyst pattern in Anabaena can be described by a combination ofan inhibitory zone mechanism and an interactive mechanism which prevents pro-heterocysts (presumptive heterocysts) from developing too close together. To accountfor the latter mechanism we assumed that a proheterocyst requires the 'support' ofadjacent vegetative cells in order to develop, and that 2 close proheterocysts depriveeach other of essential support. To test this hypothesis we showed that a prohetero-cyst can be made to reverse its development and regress to the state of a vegetativecell when its support is physically removed by breaking the filament nearby. Theseexperiments used proheterocysts at a very early stage of development. In the presentpaper we describe an extension of these experiments in which the support is furtherreduced by making breaks on both sides of a proheterocyst - in the extreme caseproducing an isolated cell. We then find that proheterocysts at a much higher level ofdevelopment can be made to regress. Moreover, it is possible to correlate the stage of

41 CEL 13

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 givensize. To make this precise we have defined stages of proheterocyst development interms of ultrastructure, and correlated these with the light-microscopic appearance ofthe same cells. This we describe in the first section. In the second, we summarize therelationship 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), wasgrown in liquid culture in a defined salts medium, pH 80 (Allen & Arnon, 1955), or on plates ofthe 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 breakingof 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 hin 4 % glutaraldehyde/o' 1 M potassium cacodylate, pH 7, and stained with 2 % aqueous potas-sium permanganate for 15 h. Each operation was carried out at 3 CC. After rinsing with water,small agar blocks containing filaments were cut, dehydrated using a graded ethanol seriesfollowed by propylene oxide, and embedded in Epon 812 after equilibrating first in 1:1 and 3 :1Epon/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 wereobserved in a Siemens Elmiskop electron microscope.

For correlation of light and electron micrographs, 6-8 filaments were aligned on agar platesinto a parallel bundle, using micromanipulators. Light micrographs were then taken before thefilaments were fixed. After sectioning, electron micrographs were taken which could be com-pared 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 distinc-tive ultrastructural features. Under the electron microscope the first sign of differentia-tion (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 vegetativecell (Fig. 1). Note the microplasmadesmata (m), which can be seen in the cross-wallbetween the cells, and which appear to be identical to those between more advancedproheterocysts or heterocysts and their adjacent vegetative cells (Figs. 2, 6). In a stageII proheterocyst the fibrous layer is complete and the junction between the cell andits neighbours is 'squaring off' (Fig. 2), this being the first stage in the formation ofthe specialized polar structure of the mature heterocyst. The junction is drawn outinto a neck during stages III-IV; the beginning of this process is seen as an elongationor 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, andelectron-transparent spaces appear between the twin membranes of the photosyntheticlamellae. These spaces increase to a maximum in the stage IV proheterocyst andaccompany contortion and apparent fragmentation of the lamellae (Fig. 4). By stage V

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, andsome polarization of the lamellae, which are by now heavily contorted. From thesepolar regions in the maturing heterocyst there is a great proliferation of what appearto be new lamellae (Figs. 7, 8), which differ from those found in vegetative cells in thatthe 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 showingvariable staining with permanganate is formed in the neck of the junction. Traces ofthis 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 com-posed of tightly packed membranes. In other micrographs (e.g. Fig. 9) where the plughas contracted and pulled away from the cell plasmalemma, there is evidence that theplug is itself bounded by membranes (arrow) which are apparently continuous withthe lamellae. It should be emphasized that it is not clear from this study if any of thesemature heterocyst features are concerned more with the ageing of the heterocyst thanwith the maturing process. For a fuller discussion of the various ultrastructuralfeatures, most of which are common to all the Anabaena species which have beeninvestigated, 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 (seeMaterials and Methods), we have been able to determine the appearance under thelight microscope of proheterocysts at each of the 7 stages (a sequence of light micro-graphs is shown in Fig. 12A-G). Initially, an increase in greyness (under phase-contrast optics) and a loss of granularity in the cell can be seen (stage I). The cell thenelongates beyond the stage at which a new septum would normally appear (stage II)and subsequently enlarges greatly and 'rounds up', its diameter eventually reaching1-5 times that of a vegetative cell (stages III-V). Finally, the cell becomes very re-fractile, and distinctive polar structures are formed (stages VI-VII). We would pointout that these stages do not correspond to the 7 stages of heterocyst differentiationdenned by Fogg (1951) from cytological observations. In the main, these latter arestages 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 forma-tion in Anabaena in which proheterocysts arise from cells lying outside inhibitoryzones surrounding each heterocyst. This model also requires a ' competitive' mecha-nism for preventing 2 cells from developing simultaneously when they are too closetogether. This competitive mechanism can only be inferred indirectly from eventsseen in normal growth, so we devised an experiment to demonstrate it more clearly.We proposed that, in order to develop, proheterocysts require 'supporting' adjacentvegetative cells (which destroy a hypothetical inhibitory substance produced by theproheterocyst), so that proheterocysts close to each other will compete for support. Thiscan also be described in terms of a 'mirror image' argument (Wilcox et al. 1973) with-out 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


were performed on A. cylindrica filaments, and proheterocysts regressed only whenthey were chosen at the earliest possible stage. By making 2 breaks, so as to reduce thesupport on both sides of the proheterocyst, we have been able to obtain regressions ata much more advanced state of development. A. catemila was used for these experi-ments; similar results were obtained, however, in a Limited number of experimentsusing A. cylindrica.

Table 1. Proheterocyst regressions at increasing stages of development

Regression frequency, %, at stage

Fragment II III IV V VI VII

3PI3P2 P I2PI P II Pp

3 15'2

17-33 2 14 9 0

9 3 3ioo-o

0

0

0

12-s31-577-89 2 9

———0

0

2 8 9

6SS

—————0

29-2

Single filaments were transferred to agar plates and fragments obtained by puncturing chosencells 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 nomencla-ture for fragments and for proheterocyst stages is given in the text. A minimum of 25 operationswas 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 (fragmentswith three vegetative cells on one side of the proheterocyst and one on the other) stageII proheterocysts regressed only in some 3 % of cases, but, in 2P1 fragments thisfrequency increased to 17 % (the rate of spontaneous regression of stage II prohetero-cysts in control filaments was 0-5 %). Stage III proheterocysts, which did not regressin either of these cases, could be induced to regress in 1P1 fragments (regressionfrequency 31 %; a sequence of photographs illustrating such an operation is shown inFig. 13). In a similar way, stage IV proheterocysts regressed 29% of the time in iPfragments, 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 toobtain any regressions, however, with stage VI or VII maturing heterocysts (see theDiscussion).

From the results obtained with 3P and 2P fragments it appears that a proheterocystat the end of a fragment has a somewhat lower probability of regressing than oneplaced internally. For example, regression frequencies for stage II proheterocysts in3P fragments and for stage II and III proheterocysts in 2P fragments were lower thanthose obtained when the cells were placed internally in the approximately equivalentfragments, 2P1 and 1P1, respectively (Table 1).

In all cases, regression and subsequent division led to the formation of normal


vegetative cells from which a new proheterocyst might eventually be chosen. Whenearly proheterocysts regressed, the envelope usually disintegrated (Fig. 13), or wasdiscarded essentially in one piece (Figs. 10, 15). With more advanced cells it tendedto 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.

Fragment

Effect of ammonia and

Control

•j-azatryptophan on regression frequency

Regression frequency, %

+ Ammonia + 7-azatryptophan

3P 52 75-5 —iP 933 — 107

Control fragments were obtained as described in the legend to Table 1. For + ammonia or+ azatryptophan operations, filaments were incubated for 16-18 h in salts medium supple-mented with 0-2 % (3-5 x io~3

M) NH4C1, or for 4 h in medium containing 2 x IO~6M DL-7-azatryptophan, and then transferred to plates containing 0-2% NH4C1 or 5 x IO~6M DL-7-azatryptophan, respectively. All proheterocysts were picked when at stage II. A minimum of60 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 influencethe heterocyst pattern to see whether they affected the frequency of proheterocystregression. It has been known for some time that, in the presence of ammonia, hetero-cyst 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 oppositeeffect on heterocyst spacing in Anabaena, leading to the formation of a much closerand less regular pattern with many multiple and terminal heterocysts (G. J. Mitchison& M. Wilcox, in preparation; see Fig. 16). Both these compounds markedly affectregression frequencies, as shown in Table 2. For example, when 3P fragments con-taining stage II proheterocysts were isolated in the presence of ammonia, the pro-heterocysts regressed in 76 % of cases (the frequency in controls was 5 %). Similarincreases were found in the regression frequencies of proheterocysts in 3P1 and 2P1fragments. Even in the presence of ammonia, however, we were unable to induce theregression of any stage VI or VII cells. When iP fragments were isolated in the pre-sence of low concentrations (2-5 x io~s M) of DL-7-azatryptophan the regressionfrequency 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 ofan interactive mechanism which serves to prevent proheterocysts from arising too


close together. We have interpreted the single break experiments, in which earlyproheterocysts were induced to regress (Wilcox et al. 1973), as demonstrating that aproheterocyst can have an inhibitory effect upon itself. In this way we suppose thatthe regression experiments show the same interactive process which occurs in normalgrowth. It is possible, however, that non-specific effects (e.g. damage during theoperation) are causing the regressions. This is made unlikely by the findingthat compounds such as ammonia and 7-azatryptophan, which affect the patternduring normal growth, have a marked and parallel effect on the probability ofregression.

There is a further alternative explanation for regressions of early proheterocysts,which assumes that there is a ' preliminary phase' of development where prohetero-cysts are neither producing inhibitor, nor committed to development. We have givenstatistical arguments against this view (Wilcox et al. 1973), but a much strongerargument is now available, since very advanced proheterocysts are found to regress inthe double-break experiments. Proheterocysts at these advanced stages have a welldenned inhibitory zone and must therefore be supposed to be sources of the hypo-thetical inhibitor. We must conclude that proheterocysts can be both a source ofinhibitor and, at the same time, susceptible to its effect. This implies that, whateverform the model takes, it will have to incorporate competition.

We are still faced with the problem of accounting for the anomalous results obtainedwith fragments containing terminal proheterocysts. According to our model, whenreasonable assumptions are made for the kinetics of morphogen metabolism, a 1P1fragment should be no more efficient than a 2P fragment (or a 2P1 than a 3P) in causingregressions; i.e. one vegetative cell on each side of a proheterocyst should provide atleast as efficient a support system as two cells on one side of the proheterocyst. Thisprediction is not borne out. A proheterocyst left in a terminal position has a lowerprobability of regressing than one placed internally. This is exactly analogous to thefinding with terminal proheterocysts in the single-break experiments, and we wouldoffer the same explanation (Wilcox et al. 1973), of inhibitor leakage through anexposed junction.

The events which take place when proheterocysts regress may provide an interpre-tation for what has been termed 'heterocyst germination'. This phenomenon, if con-firmed, would have a bearing on the question of heterocyst function. Wolk (1965)reports that, under certain conditions, germlings are seen emerging from heavy andwell formed envelopes, supposedly those of mature heterocysts. We have growncultures under these conditions, and although we do see germlingG emerging fromenvelope material, we have also found that the conditions make it impossible foi us todetermine their origin, and to assert that they arise from heterocysts. However, thesegermlings closely resemble the filaments emerging from regressing proheterocysts,where there is often a well defined envelope discarded even from an early prohetero-cyst (see, for example, Fig. 15). Since there is a large amount of filament breakageunder the germination conditions, we would suggest that ' heterocyst germination' isin reality the result of breakage near to proheterocysts. With this assumption, germ-lings would probably never come from maturing (stage VI) or mature (stage VII)


heterocysts, since such heterocysts cannot be made to regress in our experiments, evenwhen the cells have been completely isolated in the presence of ammonia.

Having said that mature heterocysts cannot be induced to regress, it should beemphasized that stage V proheterocysts, which are at an advanced level of develop-ment and morphologically very different from their precursor vegetative cells, willregress 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 evenwhen they have developed much of the appropriate morphology. What our fragmentexperiments provide is a method for putting upon a cell a precise amount of pressureto dedifferentiate. This method is, moreover, one which is directly related to thepattern-forming process in the organism.

We are grateful to Keith Roberts for his expert advice, and to Sydney Brenner, FrancisCrick 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, 366-372.

FOGG, G. E. (1949). Growth and heterocyst production in Anabaena cylindrica Lemm. II . Inrelation to carbon and nitrogen metabolism. Ann. Bot., N.S. 13, 241-259.

FOGG, G. E. (1951). The cytology of heterocysts. Ann. Bot., N.S. 15, 23-35.HAY, E. D. (1968). Dedifferentiation and metaplasia in vertebrate and invertebrate regeneration.

In The stability of the Differentiated State (ed. H. Ursprung), pp. 85-108. Berlin, Heidelberg,New York: Springer-Verlag.

LANG, N. J. (1965). Electron microscopic study of heterocyst development in A. azollae. J.Phycol. 1, 127-134.

LANG, N. J. & FAY, P. (1971). The heterocysts of blue-green algae. Proc. R. Soc. B 178,193-203.

TALPASAYI, E. R. S. & BAHAL, M. R. (1967). Cellular differentiation in A. cylindrica. Z. Pfl.Physiol. 56, 100-101.

WILCOX, M. (1970). One-dimensional pattern found in blue-green algae. Nature, Lond. 228,686-687.

WILCOX, M., MITCHISON, G. J. & SMITH, R. J. (1973). Pattern formation in the blue-green algaAnabaena. I. Basic mechanisms. J. Cell Set. 12, 707-723.

WOLK, C. P. (1965). Heterocyst germination under defined conditions. Nature, Lond. 205,201-202.

{Received 13 February 1973)


Fig. 1. The stage I proheterocyst differs from its adjacent vegetative cell only in thepresence of traces of the fibrous layer (/) of the envelope; the other features normallyfound in vegetative cells (e.g. photosynthetic lamellae (Ja), cyanophycin granules (c),polyhedral bodies (b)) are present in both cells. Also indicated are the thin micro-plasmadesmata (m) between the cells, x 8000.Fig. 2. In a stage II cell the fibrous layer (/) is complete and the proheterocyst-vegetative cell junction is 'squaring off'. Note again the microplasmadesmata (m).x 12000.

Fig. 3. By stage III, the homogeneous layer (h) of the envelope is forming inside thefibrous layer (/), and the cell is drawn out (arrowed) as the specialized junction beginsto form, x 12000.

Fig. 4. Stage IV proheterocyst showing fibrous (/) and extensive homogeneous (h)layers, and contorted lamellae often containing electron-transparent intralamellarspaces (e). x 11300.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 15000.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 microplasma-desmata (m). x 15000.


* 6


Fig. 7. In a mature (stage VII) heterocyst there is a proliferation of lamellae. Theplug in this case is electron-transparent, x 17000.Fig. 8. A mature heterocyst showing extensive proliferation of lamellae and heavilystained plug region. Compare with Fig. 7. x 8200.Fig. 9. The pore region of a mature heterocyst showing membranes (arrowed)enclosing the electron-transparent plug and apparently continuous with the lamellae,x 10500.

Fig. 10. A Pi fragment containing a regresssing stage II proheterocyst, fixed 15 hafter its isolation. The discarded proheterocyst envelope is arrowed, x 11000.Fig. 11. A regressing stage IV proheterocyst, fixed 20 h after its isolation. The elon-gating cell is emerging from both ends of the cell, leaving the envelope (arrowed)intact, x 11000.



Fig. 12. Light-microscopic appearance of the 7 stages of heterocyst development inA. catenula. A-G are a sequence of photographs of the same cell at stages I-VII, takenat o, 3, 6, 9, 12, 14 and 18 h, respectively, x 800.Fig. 13. Sequence of light micrographs showing regression of a stage III prohetero-cyst in a 1P1 fragment; the proheterocyst is arrowed. Alongside is a 2P1 fragmentcontaining 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 micrographsA, B, c, D were taken at o, 22, 44 and 68 h, respectively, after isolation. A prohetero-cyst (arrowed) is developing in the new filament in D. x 800.Fig. 15. Light-microscopic appearance of a regressed stage II proheterocyst (isolatedin 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 of2 x io~6 M DL-7-azatryptophan. B, a filament from a control culture. Proheterocystsand heterocysts are arrowed, x 600.


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PATTERN FORMATION IN THE BLUE-GREEN ALGA ANABAENA · out that these stages do not correspond to the 7 stages of heterocyst differentiation denned by Fogg (1951) from cytological observations