The Cell in Development and Inheritance

' This result is opposed to Boveri's earlier work on Ascaris (p. 355), and is modified by Ziegler ('98), who observed in a single case that an irregular cleavage occurred in the enucleated half after two or three divisions of the centrosome. On the other hand, it is sup- ported by Morgan's convincing experiments on the eggs oi Arbacia (p. 308). 2 Cf. the interesting photographic figures of Ziegler ('95). A still closer simulacrum of the amphiaster is produced by fine crystals of sulphate of quinine (a semiconductor) sus-

and Gallardo ('96, '97). It is impossible to regard this analogy as exact ; first, because it is inconsistent with the occurrence of tripolar astral figures ; second, as Meves has recently urged ^ the course of the astral fibres does not really coincide with the lines of force, the most important deviation being the crossing of the rays opposite the equa- torial region of the spindle, which is impossible in the magnetic or electric field. We must, however, remember that the amphiastcr is formed in a viscid medium, that it may perform various movements, and that its fibres probably possess the power of active growth. The

Fig- 53- — Division of dispermic eggs in sea-urchin eggs, schematic. [Boveri.] A. C. E. Eggs before division, showing various connections of the asters. B. D. F. Result- ing division in the three respective cases, showing cleavage only between centres connected by a physical or chemical effect of the centres, through which the amphias- tcr primarily arises, may thus be variously disturbed (^r modified in later stages, and the crossing of the rays is therefore not necessarily fatal to the assumption of dynamic centres. Biitschli ('92, '98) has, moreover, recently shown that a close sijuii/acntin of the amphiastcr, showing a distinct crossing of the rays, may be produced in an arti- ficial alveolar structure (coagulated gelatine) by tractive forces ccn-

pended in spirits of turpentine (a poor conductor) between two electric poles. This experi- ment, devised by Faraday, has recently been applied by Gallardo ('96, '97) to an analysis of the mitotic figure. ^ '96, p. 371. tring in two adjacent points. This result is obtained by warming and then cooling a film of thick gelatine-solution, filled with air-bubbles, and then coagulating the mass in chromic acid. Such a film shows a fine alveolar structure, which assumes a radial arrangement about the air-bubbles, owing to the traction exerted on the surrounding structure by shrinkage of the bubbles on cooling. The amphiastral simjilacra are produced about two adjacent bubbles, — a " spindle " being formed between them, and the " astral rays " sometimes showing a crossing like that seen in the actual amphiaster (Biitschli is himself unable to explain fully how the crossing arises). The protoplasmic asters are maintained by Biitschli to be, in like manner, no more than a radial configuration of the alveolar cell-substance caused by centripetal diffusion-currents toward the astral centres.^ The most interesting part of this view is the assumption that these currents are caused by specific chemical cJianges taking place in the centrosome which causes an absorption of liquid from the surrounding region. ** The astral bodies are structures which, under certain circumstances, function in a measure as centres from which emanate chemical actions upon pro- toplasm and nucleus ; and the astral phenomena which appear about the centrosomes are only a result incidental to this action of the central bodies upon the plasma." ^ Through centripetal currents thus caused arise the asters, and they may even account, in a measure, for the move- ments of the chromosomes.^ This latter part of BiitschU's conception is, I believe, quite inadequate ; but the hypothesis of definite chemical activity in the centrosome is a highly important one, which is sustained by the staining-reactions of the centrosome and by its definite morpho- logical changes during the cycle of cell-division.

More or less similar chemical hypotheses have been suggested by several other writers.^ Of these perhaps the most interesting is Strasburger's suggestion,^ that the movements of the chromosomes may be of a chemotactic character, which I suspect may prove to have been one of the most fruitful contributions to the subject. Beside this may be placed Carnoy's still earlier hypothesis ('85), that the asters are formed under the influence of specific ferments emanating from the poles of the nucleus. Mathews ('99, 2) has recently pointed out that there is a considerable analogy between the formation of the astral rays and that of fibrin-fibrils under the infiuence of fibrin-fer- ment, adding the suggestion that the centrosome may actually contain

1 Carnoy ('S5) and Plainer ('86) had previously held a similar view, suggesting that not only the spindle-formation, but also the movements of the chromosomes, might be explained as the result of protoplasmic currents. fibrin-ferment. Attention may be called here to the fact, now definitely determined by experiment,^ that cell-division may be incited by chemi- cal stimulus. In most of the cases thus far experimentally examined the divisions so caused are pathological in character, but in others they are quite normal, as shown in Loeb's remarkable results on the production of parthenogenesis in sea-urchin eggs by chemical stimulus as described at pages 215 and 308. While these experiments by no means show that division is itself merely a chemical process, thev strongly suggest that it cannot be adequately analyzed without reckon- ing with the chemical changes involved m it.

Resume. A review of the foregoing facts and theories shows how far we still are from any real understanding of the process involved either in the origin or in the mode of action of the mitotic figure. The evidence seems well-nigh demonstrative, in case of the mantle-fibres and the astral rays, that Van Beneden's hypothesis contains an element of truth, but we must now recognize that it was formulated in too simple a form for the solution of so complex a problem. No satisfac- tory hypothesis can, I believe, be reached that does not reckon with the chemical changes occurring at the spindle-poles and in the nucleus ; and these changes are probably concerned not only with the origin of the amphiaster, but also with the movements of the chromosomes. In cases where the centrosome persists from cell to cell we may perhaps regard it as the vehicle of specific substances (ferments ?) which become active at the onset of mitosis, and run through a definite cycle of changes, to initiate a like cycle in the following generation ; and it is quite conceivable that such substances may persist at the nuclear poles, or may be re-formed there as an after-effect, even though the formed centrosome disappears.^ In this consideration we may find a clue to the strange fact — should it indeed prove to be a fact — that the cen- trosome may divide, yet afterward disappear without discoverable connection with the centrosomes of the succeeding mitosis, as several recent observers have maintained. ^ When all is said, we must admit that the mechanism of mitosis in every phase still awaits adequate physiological analysis. The suggestive experiments of Biitschli and Heidenhain lead us to hope that a partial solution of the problem may be reached along the lines of physical and chemical experiment.

At present we can only admit that none of the conclusions thus far reached, whether by observation or by experiment, are more than the first 7iazz'e attempts to analyze a group of most complex phenomena of which we have little real understanding. In de\'eloping his theory of fibrillar contractility, Van Benedcn expressed the view — only, however, as a possibility — that the splitting of the chromosomes might be passively caused by the con- tractions of the two sets of opposing s{)indle-fibres to which each is attached.^ Later observations have demonstrated that this sugges- tion cannot be sustained ; for in many cases the chromatin-thread splits before division of the centrosome and the formation of the achromatic figure — sometimes during the spireme-stage, or even in the reticulum, while the nuclear membrane is still intact. Boveri showed this to be the case in Ascaris, and a similar fact has been observed by many observers since, both in plants and in animals.

A. From the endosperm of the lily, showing true nucleoli. [FLEMMING.] B. Spermatocyte of salamander. Segmented double spireme-thread composed of chromo- meres and completely split. Two centrosomes and central spindle at s. [Hkrmann.] C. Spireme-thread completely split, with six nucleoli. Endosperm of Fritillaria. [FLEM- The splitting of the chromosomes is therefore, in Bovcri's words, " an independent vital manifestation, an act of reproduction on the part of tJic chromosomes'''^

All of the recent researches in this field point to the conclusion that this act of division must be -referred to the fission of the chromatin-granules or chromomeres of which the chromatin-thread is built. These granules were first clearly described by Balbiani ('76) in the chromatin-network of epithelial cells in the insect- ovary, and he found that the spireme-thread arose by the linear arrangement of these granules in a single row like a chain of bacte- ria."^ Six years later Pfitzner ('82) added the interesting discovery

that during the mitosis of various tissue-cells of the salamander, the granules of the spireme-thread divide by fission and thus determine the longitudinal splitting of the entire cJironiosoine. This discovery was confirmed by Flemming in the following year ('82, p. 219), and a simi- lar result has been reached by many other observers (Fig. 54). The division of the chromatin-granules may take place at a very early period. Flemming observed as long ago as 1881 that the chromatin-

Fig- 55- — Formation of chromosomes and early splitting of the chromatin-granules in sperma- togonia oi As car is megalocephala, var. bivalens. [BraUER.] A. Ver}^ early prophase; granules of the nuclear recticulum already divided. B. Spireme; the continuous chromatin-thread split throughout. C. Later spireme. A Shortening of the thread. E. Spireme-thread divided into two parts. F. Spireme-thread segmented into four si)lit chromosomes. thread might spHt in the spireme-stage (epithelial cells of the sala- mander), and this has since been shown to occur in many other cases ; for instance, by Guignard in the mother-cells of the pollen in the lily ('91). Brauer's recent work on the spermatogenesis of Ascaris shows that the fission of the chromatin-granules here takes place even before the spireme-stage, when the chromatin is still in the form of a reticulum, and long before the division of the centrosome (Fig. 55). He therefore concludes : " With Boveri I regard the splitting as an

independent reproductive act of the chromatin. The reconstruction of the nucleus, and in particular the breaking up of the chromosomes after division into small granules and their uniform distribution through the nuclear cavity, is, in the first place, for the purpose of allowing a uniform growth to take place ; and in the second place, after the granules have grown to their normal size, to admit of tJicir precisely equal gnautitative and qualitative division. I hold that all the succeeding phenomena, such as the grouping of the granules in threads, their union to form larger granules, the division of the thread into segments and finally into chromosomes, are of secondary importance ; all these are only for the purpose of bringing about in the simplest and most certain manner the transmission of the daugh- ter-granules (Spalthalften) to the daughter-cells."^ " In my opinion the chromosomes are not independent individuals, but only groups of numberless minute chromatin-granules, which alone have the value of individuals." ^

These obser\'ations certainly lend strong support to the view that the chromatin is to be regarded as a morphological aggregate — as a congeries or colony of self-propagating elementary organisms capable of assimilation, growth, and division. They prove, more- over, that mitosis involves two distinct though closely related factors, one of which is the fission of the chromatic nuclear substance, while the other is the distribution of that substance to the daughter-cells. In the first of these it is the chromatin that takes the active part ; in the second it would seem that the main role is played by the amphiastcr.

We turn now to the rarer and simpler mode of division known as amitosis ; but as Flemming has well said, it is a somewhat trying task to give an account of a subject of which the final outcome is so unsatisfactory as this ; for in spite of extensive investigation, we still have no very definite conclusion in regard either to the mechan- ism of amitosis or its biological meaning. Amitosis, or direct division, differs in two essential respects from mitosis. First, the nucleus remains in the resting state (reticulum), and there is no formation of a spireme or of chromosomes. Second, division occurs without the formation of an amphiaster; hence the centrosome is not con- cerned with the nuclear division, which takes place by a simple constriction. The nuclear substance, accordingly, undergoes a divi-

sion of its total mass, but not of its individual elements or chromatin- granules (Fig. 56). Before the discovery of mitosis, nuclear division was generally assumed to take place in accordance with Remak's scheme (p. 63). The rapid extension of our knowledge of mitotic division between the years 1875 and 1885 showed, however, that such a mode of division was, to say the least, of rare occurrence, and led to doubts as to whether it ever actually took place as a normal process. As soon, however, as attention was especially directed to the subject, many cases of amitotic division were accurately determined, though

Fig. 56,— Group of cells with amitotically dividing nuclei ; ovarian follicular epithelium of the cockroach. [WHEELER.] very few of them conformed precisely to Remak's scheme. One such case is that described by Carnoy in the follicle-cells of the Qgg in the mole-cricket, where division begins in the fission of the nucleolus, followed by that of the nucleus. Similar cases have been since described, by Hoyer ('90) in the intestinal epithelium of the nematode Rhabdonema, by Korschelt in the intestine of the annelid Ophryotrocha, and in a few other cases. In many cases, how- ever, no preliminary fission of the nucleolus occurs; and Remak's scheme must, therefore, be regarded as one of the rarest forms of cell-division (!).

The behaviour of the centrosome in amitosis forms an interesting question on account of its bearing on the mechanics of cell-division. Fk^iiming obser\-ed ('91 ) that the nucleus of leucocytes might in some cases divide directly without the formation of an amphiaster, the attraction-sphere remaining undivided mean- while. Heidenhain showed in the following year, however, that in some cases leucocytes containing two nuclei (doubtless formed by amitotic division) might also contain two asters connected by a si)indle. Both Heidenhain and Flemming drew from this the conclusion that direct division of the uulU'hs is in this case inde- pendent of the centrosome, but that the latter might be concerned in the division of the cell-body, though no such process was observed. A little later, however, Meves jniblished remarkable observations that seem to indicate a functional activity of the attraction-sphere during amitotic nuclear division in the "spermatogonia"' of the salamander.! Krause and Flemming observed that in the autumn many of these cells show peculiarly lobed and irregular nuclei (the "polymorphic nuclei " of Bellonci). These were, and still are by some writers, regarded as degenerating nuclei. Meves, however, asserts — and the accuracy of his observations is in the main vouched for by Flemming — that in the ensuing spring these nuclei become uniformly rounded, and may then divide amitotically. In the autumn the attraction- sphere is represented by a diffused and irregular granular mass, which more or less completelv surrounds the nucleus. In the spring, as the nuclei become rounded, the granular substance draws together to form a definite rounded sphere, in which a distinct centrosome may sometimes be made out. Division takes place in the following extraordinary manner: The nucleus assumes a dumb-bell shape, while the attraction-si)here becomes drawn out into a band which surrounds the central part of the nucleus, and finally forms a closed ring, encircling the nucleus.

After this the nucleus divides into two, while the ring-shaped attraction-sphere (-archo- plasm") is again condensed into a sphere. The appearances suggest that the ring- shaped sphere actually compresses the nucleus and cuts it through. In a later paper (94) Meves shows that the diffused "archoplasm" of the autumn-stage arises by the breaking down of a definite spherical attraction-sphere, which is re-formed again in the spring in the manner described, and in this condition the cells mav divide cither uiitotically or amitotically. He adds the interesting observa- tion, since confirmed by Rawitz ('94), that in the spermatocytes of the salamander the attraction-spheres of adjoining cells are often connected by intercellular bridges, but the meaning of this has not yet been determined.

It is certain that the remarkable transformation of the sphere into a ring during amitosis is not of universal, or even of general, occurrence, as shown by the later studies of \'om Rath (95, 3). In leucocytes, for example, the sphere persists in its typical form, and contains a centrosome, during every stage of the division: but it is an interesting fact that during all these stages the sphere lies on the concave side of the nucleus in the bay which finally cuts through the entire nucleus. Again, in the liver-cells of the isopod Porcellio, the nucleus divides, not by constriction, as in the leucocyte, but by the appearance of a nuclear plate, in the formation of which the attraction sphere is apparently not concerned.'-^ The relations of the centro- some and archoplasm in amitosis are, therefore, still in doubt; but, on the whole, the evidence goes to show that they take no essential part in the process.

A survey of the known cases of amitosis brings out the following siirnificant' facts. It is of extreme rarity, if indeed it ever occurs in embryonic cells or such as are in the course of rapid and contniued 2 Such a mode of amitotic division was first described by Saliatier in the Crustacea ('89), and a similar mode has been observed by Carnoy and Van der Stricht. multiplication. It is frequent in pathological growths and in cells such as those of the vertebrate decidua, of the embryonic envelopes of insects, or the yolk-nuclei (periblast, etc.), zvJiicJi arc on the luay toivard degeneration. In many cases, moreover, direct nuclear divi- sion is not followed by fission of the cell-body, so that multinuclear cells and polymorphic nuclei are thus often formed. These and many similar facts led Flemming in 1891 to express the opinion that so far as the higher plants and animals are concerned amitosis is "a process which does not lead to a new production and multiplication of cells, but wherever it occurs represents either a degeneration or an aberration, or perhaps in many cases (as in the formation of multi- nucleated cells by fragmentation) is tributary to metabolism through the increase of nuclear surface." ^ In this direction Flemming sought an explanation of the fact that leucocytes may divide either mitotically or amitotically (/. Peremeschko, Lowit, Arnold, Flemming). In the normal lymph-glands, where new leucocytes are continually regenerated, mitosis is the prevalent mode. Elsewhere (wandering- cells) both processes occur. '* Like the cells of other tissues the leucocytes find their normal physiological origin (Neubildung) in mitosis ; only those so produced have the power to live on and repro- duce their kind through the same process."^ Those that divide ami- totically are on the road to ruin. Amitosis in the higher forms is thus conceived as a purely secondary process, not a survival of a primitive process of direct division from the Protozoa, as Strasburger ('82) and Waldeyer i^^'^) had conceived it.

This hypothesis has been carried still further by Ziegler and \o\Vi Rath ('91). In a paper on the origin of the blood in fishes, Ziegler i^'^y) showed that the periblast-nuclei in the ^gg of fishes divide ami- totically, and he was thus led like Flemming to the view that amitosis is connected with a high specialization of the cell and may be a fore- runner of degeneration. In a second paper ('91), published shortly after Flemming's, he points out the fact that amitotically dividing nuclei are usually of large size and that the cells are in many cases distinguished by a specially intense secretory or assimilative activity. Thus, Riige ('90) showed that the absorption of degenerate eggs in the Amphibia is effected by means of leucocytes which creej) into the egg-substance. The nuclei of these cells become enlarged, divide ami- totically, and then frequently degenerate. Other observers ( Korschelt, Carnoy) have noted the large size and amitotic division of the nuclei in the ovarian follicle-cells and nutritive cells surrounding the ovum in insects and Crustacea. Chun found in the entodermic cells of the radial canals of siphonophores huge cells filled with nests of nuclei amitotically produced, and suggested ('90) that the multiplication of

nuclei was for the purpose of increasing the nuclear surface as an aid to metabolic interchanges between nucleus and cytoplasm. Amitotic division leading to the formation of multinuclear cells is especially com- mon in gland-cells. Thus, Klein has described such divisions in the mucous skin-glands of Amphibia, and more recently Vom Rath has carefully described it in the huge gland-cells (probably salivary) of the isopod Aiiilocm ('95). Many other cases are known. Dogiel ('90) has ob.served exceedingly significant facts in this field that place the relations between mitosis and amitosis in a clear light. It is a well- known fact that in stratified epithelium new cells are continually formed in the deeper layers to replace those cast off from the super- ficial layers. Dogiel finds in the lining of the bladder of the mouse that the nuclei of the superficial cells, which secrete the mucus cover- ing the surface, regularly divide amitotically, giving rise to huge mul- tinuclear cells, which finally degenerate and are cast off. The new cells that take their place are formed in the deeper layers by mitosis alone. Especially significant, again, is the case of the ciliate Infu- soria, which possess two kinds of nuclei in the same cell, a macro- nucleus and a micronucleus. The former is known to be intimately concerned with the processes of metabolism {cf. p. 342). During con- jugation the macronucleus degenerates and disappears and a new one is formed from the micronucleus or one of its descendants. The macro- nucleus is therefore essentially metabolic, the micronucleus genera- tive in function.

In view of this contrast it is a significant fact that while both nuclei divide during the ordinary process of fission the mitotic phenomena are as a rule less clearly marked in the macronu- cleus than in the micronucleus, and in some cases the former appears to divide directly while the latter always goes through a process of mitosis. These conclusions received a very important support in the work of Vom Rath on amitosis in the testis ('93). On the basis of a compara- tive study of amitosis in the testis-cells of vertebrates, mollusks, and arthropods he concludes that amitosis never occurs in the sperm-pro- ducing cells (spermatogonia, etc.), but only in the supporting cells ( Randzellen, Stutzzellen). The former multiply through mitosis alone. The two kinds of cells have, it is true, a common origin in cells which divide mitotically. When, however, they have once become differen- tiated, they remain absolutely distinct ; amitosis never takes place in the series vvhich finally results in the formation of spermatozoa, and the amitotically dividing "supporting-cells" sooner or later perish. Vom Rath thus reached the remarkable conclusion that " when once a cell has undergone amitotic division it has received its death- warrant ; it may indeed continue for a time to divide by amitosis, but inevitably perishes in the end." ^

There is, however, strong evidence that this conckision is too extreme. Meves ('94) has given good reason for the conckision that in the salamander the nuclei of the sperm-producing cells (spermato- gonia) may divide by amitosis yet afterward undergo normal mitotic division, and Preusse ('95) has reached a similar result in the case of insect-ovaries. Perhaps the most convincing evidence in this direc- tion is afforded by Pfeffer's ('99) recent experiments on Spirogyra. If this plant be placed in water containing 0.5 to 1.0% of ether, active growth and division continue, but only by amitosis. If, however, the same individuals be replaced in water, viitotic division is resmiied and entirely normal growth continues. This seems to show conclusively that amitosis, in lower forms of Hfe at least, does not necessarily mean the approach of degeneration, but is a result of special conditions. Nevertheless, there can be no doubt that Flemming's hypothesis in a general way represents the truth, and that in the vast majority of cases amitosis is a secondary process which does not fall in the generative series of cell-divisions.

All cells arise by division from preexisting cells, cell-body from cell-body, nucleus from nucleus, plastids (when these bodies are pres- ent) from plastids, and in some cases centrosomes from centrosomes. The law of genetic continuity thus applies not merely to the cell con- sidered as a whole, but also to some of its structural constituents. In mitosis, the usual and typical mode of division, the nucleus under- goes a complicated transformation, and, together with some of the cytoplasmic material, gives rise to the mitotic figure. Of this, the most characteristic features are the chromatic figure, consisting of chromosomes derived from the chromatin, and the achromatic figure, derived from the cytoplasm, the nucleus, or from both, and consisting of a spindle, at each pole of which, as a rule, is a centrosome and aster. There is, however, strong evidence that both these latter struc- tures may in some cases be wanting, and the spindle is therefore prob- ably to be regarded as the most essential element.

The chromosomes, always of the same number in a given species (with only apparent exceptions), arise by the transformation of the chromatin-reticulum into a thread which breaks into segments and splits lengthwise throughout its whole extent. The two halves arc thereupon transported in opposite directions along the spindle to its respective poles and there enter into the formation of the two corresponding daughter-nuclei. The spireme-thread, and hence the chromosome, arises from a single series of chromatin-granules or chromomeres which, by their fission, cause the splitting of the thread.

Every individual chromatin-j^ranule therefore contributes its quota to each of the daughter-nuclei, but it is uncertain whether they are persistent bodies or only temporary structures like the chromosomes themselves. The spindle may arise from the achromatic substance of the nucleus, from the cytoplasmic substance, or from both. When cen- trosomes are present it is they, as a rule, that lead the way in divi- sion. About the daughter-centrosomes as foci are formed the asters and between them stretches the spindle, forming an auipJiiastcr which is the most highly developed form of the achromatic figure. When centrosomes are absent, as now appears to be the case in the higher plants, the spindle is formed from fibrous protoplasmic ele- ments that gradually group themselves into a spindle.

The mechanism of mitosis is imperfectly understood. Experi- mental studies give ground for the conclusion that the changes undergone by the chromatic and the achromatic figures respectively are parallel but in a measure independent processes, which are how- ever so correlated that both must cooperate for complete cell-division. Thus there is strong evidence that the fission of the chromatin-gran- ules, and the splitting of the thread, is not caused by division of the centrosome or the formation of the spindle, but only accompanies it as a parallel phenomenon. The divergence of the daughter-chromo- somes, on the other hand, is in some manner determined by the spindle-fibres. There are cogent reasons for the view that some of these fibres are contractile elements which, like muscle-fibres, drae: the daughter-chromosomes asunder ; while other s])indle-fibres act as supporting and guiding elements, and probably by their elongation push the spindle-poles apart. The adequacy of this explanation is, however, doubtful, and it is not improbable that the centrosome or spindlc-poles are centres of chemical or other physiological activities that play an essential part in the process and are correlated with those taking place in the chromatin. The functions of the astral rays are likewise still involved in doubt, the rays being regarded by some investigators as contractile elements like muscle-fibres, by others as rigid supporting fibres, or even as actively pushing elements like those of the central spindle. It is generally believed further that they play a definite part in division of the cell-body — a conclusion sup- ported by the fact that the size of the aster is directly related to that of the resulting cell. On the other hand division of the cell-body may apparently occur in the absence of asters (as in amitosis, or among the Infusoria).

These facts show that mitosis is due to the coordinate play of an extremely complex system of forces which are as yet scarcely com- prehended. Its general significance is, however, obvious. The effect of mitosis is to produce a meristic division, as opposed to a viere mass- division, of the chromatin of the mother-cell, and its equal distribution to the nuclei of the daughter-cells. To this result all the operations of mitosis are tributary; and it is a significant fact that this process is characteristic of all embryonic and actively growing cells, while mass-division, as shown in amitosis, is equally characteristic of highly specialized or degenerating cells in which development is approaching its end.