The Cell in Development and Inheritance

nucleus with no trace of a centrosome. In this same form, as described above, the ordinary vegetative mitoses are quite of the metazoan type, with a persistent extranuclcar centrosome. The history of the chromatin in the mitosis of unicellular forms shows some interesting modifications. In a considerable number of forms a more or less clearly marked spireme-stage precedes the forma- tion of chromosomes (diatoms, Infusoria, dinoflagellates, Iuio/yp/i(i)\ in others, long chromosomes are formed without a distinct spireme- stage (N'octihtca). It has been clearly demonstrated that in some cases these chromosomes split lengthwise, as in Metazoa {A^octilnca,

Fig. 43. — Mitosis in Parafnccba. [SCJIAUDINN.] At the left, amoeboid phase, showing nucleus and " Nebenkorper." At the right, four stages of division in the swarm-spores. diatoms, Actinophrys, probably in EuglypJia) ; but in some cases they are stated to divide transversely in the middle (Infusoria according to Hertwig, Ccratiuin according to Lauterborn). These chromosomes appear always to arise, as in Metazoa, through the linear arrangement of chromatin-granules {^Noctihica, Actinospluvriuni, Euglciia\ wliich themselves in many cases arise by the preliminary fragmentation of one or more large chromatin-masses {^c.g. in Noctiluca or ActiuosplicB- rijun). In other forms no such linear aggregates are formed, and direct fission of the chromatin-granules appears to take place without the formation of bodies morphologically comparable with the chromo- somes of such forms as Noctiluca. This is apparently the case in Tetrajnitus, and Achromatium, other forms having a distributed

nucleus,! ^nd in such forms as CJiilomouas and Tmchclomonas, where the <^ranules are permanently aggret^ated about a central body. Too little is known of the facts to justify a very positive statement; but on the whole they point toward the conclusion that in the simplest Fig. 44. — Mitosis in the rhizoped Actinospluvrium. [Braukr.] A. Nucleus and surrounding structures in the early prophase; above and below the reticular nucleus lie the semilunar " pole-plates." and outside these the cytoplasmic masses in which the asters afterward develop. B. Later stage of the nucleus. D. Mitotic figure in the metaphase, showing equatorial plate, intra-nuclear spindle, and pole-plates {p.p.). C. Equatorial plate, viewed ^////r*", consisting of double chromatin-granules. E. Early anaphase. E.G. Later ana- phases. H. F^inal anaphase. /. Telophase; daughter-nucleus forming, chromatin in loop-shaped threads; outside the nuclear membrane the centrosome, already divided, and the aster. J. 1-ater stage; the daughter-nucleus established; divergence of the centrosomes. Beyond this point the centrosomes have not been followed.

types of mitosis no true chromosome-formation occurs, thus sustaininp^ Brauer's conclusion that the es.sential fact in the history of the chro- matin in mitosis is the fission of the individual granules.^ 1 The fission of the individual granules is carefully described and figured by Schewiakoff in Achromatimn. ■ '-^ For speculations on the historical origin of the centrosome, etc., see p. 315. Under certain circumstances the delicate mechanism of cell-division may become deranged, and so give rise to various forms of patho- logical mitoses. Such a miscarriage may be artificially produced, as Hertwig, Galeotti, and others have shown, by treating the dividing cells with poisons and other chemical substances (quinine, chloral, nicotine, potassic iodide, etc.). Pathological mitoses may, however,

Fig. 45. — Mitosis in Actinosphcerium. [R. Hertwig.] A. Encysted form, with resting nucleus; chromatin aggregated into large nucleolus-like body. B. prophase of division of the encysted form, showing chromosome-like bodies formed of granules, and spindle without centrosomes. C. Earlier prophase of the first maturation division, showing extrusion of chromatic substance to form the centrosome. D. Later stage, showing centrosome and aster. occur without discoverable external cause ; and it is a very interesting fact, as Klebs, Hansemann, and Galeotti have especially pointed out, that they are of frequent occurrence in abnormal growths such as cancers and tumours.

The abnormal forms of mitoses are arranged by Hansemann in two general groups, as follows: (i) asymmetrical mitoses, in which the chromosomes are unequally distributed to the daughter-cells, and (2) multipolar mitoses, in which the number of centrosomes is more than two, and more than one spindle is formed. Under the first group are inckided not only the cases of unequal distribution of the daughter- chromosomes, but also those in which chromosomes fail to be drawn into the equatorial })late and hence are lost in the cytoplasm.

Klebs first pointed out the occurrence of asymmetrical mitoses in carcinoma-cells, where they have been carefully studied by Hanse- mann and Galeotti. The inequality is here often extremely marked, so that one of the daughter-cells may receive more than twice as much chromatin as the other (Fig. 46). Hansemann, whose conclu- Fig. 46. — Pathological mitoses in human cancer-cells. [Galeotti.] A. Asymmetrical mitosis with unequal centrosomes. B. Later stage, showing unequal distri- bution of the chromosomes. C. Quadripolar mitosis. D. Tripolar mitosis. E. Later stage. F. Trinucleate cell resulting.

sions are accepted by Galeotti, believes that this asymmetry of mito- sis gives an explanation of the familiar fact that in cancer-cells many of the nuclei are especially rich in chromatin (hyperchromatic cells), while others are abnormally poor (hypochromatic cells). Lustig and Gale(^tti ('93) showed that the unequal distribution of chromatin is correlated with and probably caused by a corresponding inequality in the centrosomes which causes an asymmetrical development of the amphiaster. A very interesting discovery made by Galeotti ('93) is that asymmetrical mitoses, exactly like those seen in carcinoma, may be artificially produced in the epithelial cells of salamanders (Fig. 47) by treatment with dilute solutions of various drugs (antipyrin, cocaine, quinine).

Normal multipolar mitoses, though rare, sometimes occur, as in the division of the pollen-mother-cells and the endosperm-cells of flower- ing plants (Strasburger) ; but such mitotic figures arise through the union of two or more bipolar amphiasters in a syncytium and are due to a rapid succession of the nuclear divisions unaccompanied by fission of the cell-substance. These are not to be confounded with pathological mitoses arising by premature or abnormal division of the centrosome. If one centrosome divide, while the other does not, triasters are produced, from which may arise three cells or a tri- nucleated cell. If both centrosomes divide, tetrasters or polyasters are formed. Here again the same result has been artificially attained by chemical stimulus {cf. Schottlander, '^S). Multipolar mitoses are

Fig. 47. — Pathological mitoses in epidermal cells of salamander caused by poisons. [Galeotti.] A. Asymmetrical mitosis after treatment with 0.05 % antipyrin solution. B. Tripolar mitosis after treatment with 0.5% potassic iodide solution. also common in regenerating tissues after irritative stimulus (Strobe); but it is uncertain whether such mitoses lead to the formation of normal tissue.^ The frequency of abnormal mitoses in pathological growths is a most suggestive fact, but it is still wholly undetermined whether the abnormal mode of cell-division is the cause of the disea.se or the reverse. The latter seems the more probable alternative, since nor- mal mitosis is certainly the rule in abnormal growths ; and Galeotti's experiments suggest that the pathological mitoses in such growths may be caused by the presence of deleterious chemical products in the diseased tissue, and perhaps point the way to their medical treatment.

1 The remarkable polyasters formed in polyspermia fertilization of the egg are de- scribed at page 198. We now pass to a consideration of the forces at work iii mitotic division, which leads us into one of the most debatable helds of All observers agree that the amphiaster is in some manner an expression of the forces by which cell- division is caused, and many accept, in one form or another, the first view clearly stated by Fol,^ that the asters represent in some manner centres of attractive forces focussed in the centrosome or dv- namic centre of the cell. Regarding the nature of these forces, there is, how- ever, so wide a divergence of opinion as to compel the admission that we have thus far accomplished little more than to clear the ground for a ])recise in- vestigation of the subject ;

Fig. 48. — Slightly schematic figures of dividing eggs ^ . ' . o{ Ascaris, illustrating Van Bencdcn's theory of mitosis, and the mccnanism Ot mi- A. Early anaphase; each chromosome has divided qj-j^. ^f ^\^^^ moSt fascinating into two. B. Later anaphase during divergence of the ^ . daughter-chromosomes, a.c. Antipodal cone of astral problems 01 CytOlOgy. rays; f.s. cortical zone of the attraction-sphere ; /. inter- (^(ji\ 'J'Jw TllCOiy of Fl- p.c. principal cone, forming one-half of the contractile vicVV that haS taken the

spindle (the action of these fibres is reenforced by that of cf-rnno'est hold On rCCCnt tlie antipodal cone) ; s.e.c. subequatorial circle, to which t> ^ _ of fibrillar contractility. First suggested by Klein in 1878, this hypothesis was independ- ently put forward by Van Beneden in 1883, and fully outlined by him four years later in the following words: *' In our opinion all the internal movements that accompany cell-division have their immediate cause in the contractility of the protoplasmic fibrilla:^ and their arrangement in a kind of radial muscular system, composed of antagonizing groups" {i.e. the asters with their rays). " In this sys- tem the central corpuscle (centrosome) plays the part of an organ of insertion. It is the first of all the various organs of the cells to divide, and its division leads to the grouping of the contractile elements in two systems, each having its own centre. The presence of these two systems brings about cell-division, and actively determines the paths of the secondary chromatic asters" {i.e. the daughter-groups of chro- mosomes) '' in opposite directions. An important part of the phe- nomena of (karyo-) kinesis has its efficient cause, not in the nucleus, but in the protoplasmic body of the cell." ^ This beautiful hypothesis was based on very convincing evidence derived from the study of the Ascaris eofo:, and it was here that Van Beneden first demonstrated the fact, already suspected by Flemming, that the daughter-chromosomes move apart to the poles of the spindle and give rise to the two respective daughter-nuclei.^

Van Beneden's general hypothesis was accepted in the following year by Boveri ('88, 2), who contributed many important additional facts in its support, though neither his observations nor those of later investigators have sustained Van Beneden's account of the grouping of the astral rays. Boveri showed in the clearest manner that, during the fertilization of Ascaris, the astral rays become attached to the chromosomes of the germ-nuclei ; that each comes into connection with rays from both the asters ; that the chromosomes, at first irregu- larly scattered in the ^gg, are drawn into a position of equilibrium in the equator of the spindle by the shortening of these rays (Figs. 90. 147); and that tJie rays thicke^i as tJiey sJiorten. He showed that as the chromosome splits, each half is connected only with rays (spindle- fibres) from the aster on its own side ; and he followed, step by step. the shortening and thickening of these rays as the daughter-chromo- somes diverge. In all these operations the behaviour of the rays is

2 '83, p. 544. Van Beneden describes the astral rays, both in Ascaris and in tunicates, as differentiated into several groups. One set, forming the "jirincipal cone." are attached to the chromosomes and form one-half of the spindle, and, l)y the contractions of these lilires, the chromosomes are passively dragged apart. An opposite group, forming the " antipodal cone," extend from the centrosome to the cell-periphery, the base of the cone forming the "polar circle." These rays, opposing the action of the principal cones, not only hold the centrosomes in place, but, by their contractions, drag them apart, and thus cause an actual divergence of the centres. The remaining astral rays are attached to the cell-periphery and

are limited by a subequatorial circle (Fig. 48). Later observations indicate, however, that this arrangement of the astral rays is not of general occurrence, and that the rays often do not reach the periphery, but lose themselves in the general reticulum. precisely like that of muscle-fibres ; and it is difficult to study Bovcri's beautiful figures and clear descriptions without sharing his conviction that "of the contractility of the tibrilke there can be no doubt." ^

Very convincing evidence in the same direction is afforded by pigment-cells and leucocytes or wandering cells, in both of which there is a very large permanent aster (attraction-sphere) even in the resting cell. The structure of the aster in the leucocyte, where it was first discovered by Flemming in 1891, has been studied very carefully by Heidenhain in the salamander. The astral rays here extend throughout nearly the whole cell (Fig. 49), and are believed

Fig. 49. — Leucocytes or wandering cells of the salamander. [Heidenhain.] A. Cell with a single nucleus containing a very coarse network of chromatin and two nucleoli (plasmosomes) ; s. permanent aster, its centre occupied by a double centrosome surrounded by an attraction-sphere. i9. Similar cell, with double nucleus; the smaller dark masses in the latter are oxychromatin-granules (linin), the larger masses are basichromatin (chromatin proper). by Heidenhain to represent the contractile elements by means of which the cell changes its form and creeps about. A similar con- clusion was reached by Solger ('91) and Zimmermann ('93, 2) in the case of pigment-cells (chromatophores) in fishes. These cells have, in an extraordinary degree, the power of changing their form and of actively creeping about. Solger and Zimmermann have shown that the pigment-cell contains an enormous aster, whose rays extend in every direction through the pigment-mass, and it is almost impos- sible to doubt that the aster is a contractile apparatus, like a radial muscular system, by means of which the active changes of form are produced (Fig. 50). This interpretation of the aster receives addi- tional support through Schaudinn's ('96, 3) highly interesting dis-

covery that the ''central granule " of the Heliozoa is to be identified with the centrosome and plays the same role in mitosis (Fig. 41). In these animals the axial filaments of the radiating pseudopodia con- verge to the central granule during the vegetative state of the cell, thus forming a permanent aster which Schaudinn's observations prove to be directly comparable to that of a leucocyte or of a mitotic figure. There is in this case no doubt of the contractility of the rays, and a

Fig. 50. — Pigment-cells and asters from the epidermis of fishes. [ZiMMERMAN'N.] A. Entire pigment-cell, from Bleimius. The central clear space is the central mass of the aster from which radiate the pigment-granules; two nuclei below. D. Nucleus (//) and aster after ex- traction of the pigment, showing reticulated central mass. C. Two nuclei and aster with rod- shaped central mass, from Sargus. strong, if indirect, argument is thus given in favour of contractility in other forms of asters.^ The contraction-hypothesis is beautifully illustrated by means of a simple and easily constructed model, devised by Heidenhain ('94, '96), which closely simulates some of the phenom- ena of mitosis. In its simplest form the model consists of a circle, marked on a flat surface, to the periphery of which are attached at equal

1 For an interesting discussion and develoj ment of the contraction-hypothesis see Watase, '94. Fig. 51. — Heidenhain's model of mitosis (mainly from Heidenhain). A. Dotted lines show position of the rays upon sever- ing connection between the small rings. B. Position upon insertion of " nuclcu=." C. D. Models with fiexiblc hinged hoops, showing division. intervals a series of rub- ber bands (astral rays). At the other ends these bands are attached to a pair of small rings (cen- trosomes) fastened to- gether. In the position of equilibrium, when the rays are stretched at equal tension, the rays form a symmetrical aster with the centrosomes at the centre of the circle (Fig. 51, A). If the con- nection between the cen- trosomes be severed, they are immediately dragged apart to a new position of equilibrium with the rays grouped in two asters, as in the actual cell (dotted lines in Fig. 51, A). If a round pasteboard box of suitable size (nucleus) be inserted between two of the ravs, it assumes an eccentric position, the cell-axis being formed by a line passing through its centre and that of the \)?i\x of small rings {cf. the epithelial cell, p. 57), and upon division of the aster it takes up a position between the two asters. In a second form of the models the circle is formed of two half rings of flexible steel, joined by hinges ; the diver- gence of the small rings is here accompanied by an elongation and partial constriction of the model

in the equatorial plane ; and if, finally, the hinge-connection be re- moved, each half of the ring closes to form a complete ring.^ Heidenhain has fully worked out a theory of mitosis based upon the analogy of these pretty models. The astral rays of the cell (*' organic radii") are assumed to be in like manner of equal length and in a state of equal tonic contraction or tension, the centrosome forming the common insertion-point of the rays, and equilibrium of the system being maintained by turgor of the cell. Upon disappear- ance of the nuclear membrane and division of this insertion-point, the tension of the rays causes divergence of the centrosomes and forma- tion of the spindle between them, and by further contraction of the rays both the divergence of the daughter-chromosomes and the division of the cell-body are caused. A new condition of equilibrium is thus established in each daughter-cell until again disturbed by division of the centrosome.2 In some cases (leucocytes) the organic radii are visible at all periods. More commonly they are lost to view by breaking up into the cell-reticulum, without, however, losing their essential relations.

No one who witnesses the operation of Heidenhain's models can fail to be impressed with its striking simulation of actual cell-division. Closer study of the facts shows, however, that the contraction-hypothe- sis must be considerably restricted, as has been done by the successive modifications of Hermann ('91), Drijner('95), and others. Hermann, to whom the identiiication of the central spindle is due, pointed out that there is no evidence of contractility in the central spindle-fibres, which elongate instead of shorten during mitosis ; and he concluded that these fibres are non-contractile supporting elements, which form a basis on which the movements of the chromosomes take place. The ina7itle-fibres are the only contractile elements in the spindle, and it is by them that the chromosomes are brought into position about the central spindle and the daughter-chromosomes are dragged apart.^ Driiner ('95) still further restricts the hypothesis, maintaining that the progressive divergence of the spindle-poles is caused not by contrac- tion of the astral rays (''polar fibres"), as assumed by Heidenhain (following Van Beneden and Boveri), but by an active growth or elongation of the central spindle, which goes on throughout the whole period from the earliest prophases until the close of the ana- phases. This view is supported by the fact that the central spindle-

1 In a modification of the apparatus devised by Rhumbler ('97), the same effect is pro- duced without the hinges. 3 Belajeff ('94) and Strasburger ('95) have accepted a similar view as applied to mitosis in plant-cells. fibres are always contorted during the metaphases, as if pushing against a resistance ; and it harmonizes with the facts observed in the mitoses of infusorian nuclei, where no asters are present. This view has been accepted, with slight modifications, by Flemming, Boveri, Meves, Kostanecki, and also by Heidenhain. A nearly decisive argument in its favour is given by such cases as the polar bodies, or the mitosis of salamander spermatocytes as described by Meves ('96, '97, 3), where the spindle-poles are pushed out to the periphery of the cell, the polar astral rays meanwhile nearly or quite disappearing (Fig. 130). This not only strongly indicates the push of the central spindle, but also shows that the assumption of a pull by the polar rays is superfluous. But beyond this both Driincr and Meves have brought arguments against contractility in the other astral rays, endeavouring to show^ that these, like the spindle-fibres, are actively elongating elements, and that (Meves, '97, 3) the actual grouping of the rays during the anaphases is such as to suggest that even the division of the cell-body may be thus caused. A pushing function of the astral rays is also indicated by infolding of the nuclear membrane caused by the development of the aster as described by Platner, Watase, Braus, Griffin, and others.^ The contraction-hypothe- sis is thus restricted by Driiner and Meves to the mantle-fibres alone, though many others, among them Flemming and Kostanecki, still accept the contractility of the astral rays.

{b) OtJicr Facts and TJicorics. — Even in the restricted form indi- cated above the contraction-hypothesis encounters serious difficulties, one of which is the fact urged by me in an earlier paper ('95), and subsequently by Richard Hertwig ('98), that in the eggs of echino- derms and many other dividing cells the daughter-chromosome plates, extending through the whole substance of the spindle, wander to the extreme ends of the spindle — a process which demands a contraction of the fibres almost to the vanishing point, while in point of fact not even a shortening and thickening of the fibres can be seen (Fig. 52). Moreover, in these cases, no distinction can be seen between central spindle-fibres and mantle- fibres, and we can only save the contraction-hypothesis by the improbable assumption that fibres indistinguishably mingled, and having the same mode of origin, structure, and staining-reaction, have exactly opposite functions. The inadequacy of the general theory is sufficiently apparent from the fact that in amitosis cells many

1 QC p. 68. It should be pointed out that the originator of the pushing theory was Watase ('93), who ingeniously developed an hypothesis exactly the opposite of Van Bene- den's, assuming both astral rays and spindle-fibres to be actively elongating fibres, dove-tailing in the spindle-region, and pushing the chromosomes apart. This hypothesis is, I believe, in- consistent with the phenomena observed in multiple asters and elsewhere, yet it probably contains a nucleus of truth that forms the basis of Driiner's conception of the central spindle.

divide without any amphiaster whatever. In Infusoria mitosis seems to occur in the entire absence of asters, althou^^h the cells divide by constriction, and the analogy with Heidenhain's model entirely fails. A. Metaphase; daughter-chronaosomes drawing apart but still united at one end. B. Daugh- ter-chromosomes separating. C. Late anaphase ; daughter-chromosomes lying near the spindle- poles. D. Final anaphase; daughter chromosomes converted into vesicles. E. Immediately after division, the asters undivided ; the spindle has disappeared. F. Resting 2-cell stage, the asters divided into tvvo in anticipation of the next division.

In Figs. A and /? the centrosome consists of a mass of intensely staining granules, which in Cand D elongates at right angles to the spindle-axis. In /-'the centrosome appears as a single or double granule, which in later stages gives rise to a pluricorpuscular centrum like that in .-/. The connection between D and F is not definitely determined. In Eiiglypha, according to Schewiakoff (Fig. 39), division of the cell- body appears to take place quite independently of the mitotic figure. Again, a considerable number of cases are now known in which dur- ing the fertilization of the ^gg a large amphiaster is formed, with

astral rays sometimes extending throughout almost the entire Qgg^ only to disappear or become greatly reduced without the occurrence of division, the ensuing cleavage being effected by a new am})hiaster or by the recrudescence of the old.^ For these and other reasons we must admit the probability that contractility of the astral fibrillx', if it exists, is but the expression or consequence of a more deeply lying phenomena of more general significance. The subtlety of the prob- lem is strikingly shown by Boveri's remarkable observations on abnormal sea-urchin eggs ('96), which show (i) that the periodic division of the centrosome and formation of the amphiaster may take place independently of the nucleus; (2) that the spindle, as well as the asters, is concerned in division of the cell-body ; and (3) that an amphiaster without chromosomes is unable to effect normal division of the cell-body. The first and third of these facts are shown by eggs in which during the first cleavage all of the chromatin passes to one pole of the spindle, so that one of the resulting halves of the tgg receives no nucleus, but only a centrosome and aster. In this half perfect amphiasters are formed simultaneously with each cleavage in the other half, yit no division of the protoplasmic mass occurs? The second fact is shown in polyspermic eggs, in which multipolar astral svstems are formed by union of the several sperm-asters (Figs. 53, loi ). In such eggs cleavages only occur between asters that are joined by a spindle. Normal cleavage of the cell-body thus requires the complete apparatus of mitosis, and even though the fibres be contractile they cannot fully operate in the absence of chromatin.

We may now turn to theories based on the hypothesis, first sug- gested by Fol in 1873, that the astral foci {i.e. centrosomes) represent dynamic centres of attractive or other forces. It should be noted that this hy])othesis involves two distinct questions, one relating to the origin of the amphiaster, the other to its mode of action ; and we have seen that some of the foremost advocates of the contraction-hypothesis, including Van Beneden and Boveri, have held the centrosomes to be attractive centres. Apart from the movements of the chromosomes, the most obvious indication that the centrosomes are dynamic centres is the extraordinary resemblance of the amphiaster to the lines of force in a magnetic field as shown by the arrangement of iron-filings about the poles of a horseshoe magnet — a resemblance pointed out by Fol himself, and urged by many later writers,'^ especially Ziegler ('95)