The Cell in Development and Inheritance

Although these accounts have been supported by some other workers,^ and have recently been in part reiterated by Guignard himself ('98, i), they have not been sustained by some of the best and most careful later observers, who describe a mode of spindle-formation differing radically from that seen in thal- lophytes and in animals generally.-^ According to these observations, begun by Farmer and Belajcff, and strongly sustained by the care- ful studies of Osterhout, Mottier, Nemec, and others, the achromatic figure is almost wholly of cytoplasmic origin, arising from a fibrillar material (" kinoplasm " or " filar plasm," of Strasburger), which at the beginning of mitosis forms a net-like mass surrounding the nucleus, from which fibrilloe radiate out into the cytoplasm. As the nuclear membrane fades, these fibrillae, continually increasing, invade the nuclear area, gather themselves into bundles, converging to a number

1 See especially Swingle ('97) on Sphacelariacea, Strasburger ('97) on Fucus, Mottier ('98) on Dictyota ; cf. also Harper ('97) on Erysiphe and Peziza. of centres (without centrosomes), and thus give rise to an irregular multipolar figure (Figs. 36, 133). This figure finally resolves itself into a definite bipolar spindle which is devoid of centrosomes, and in the earlier stages also of asters, though in the later phases some- what irregular asters are formed. On the basis of these observations Mottier^ proposes to distinguish provisionally two well-defined types of mitosis in plants which he designates as the ''thallophyte " and the "cormophyte" types. The latter seems wholly irreconcilable with the process observed in animal-cells ; for the whole course of spindle- formation seems diametrically opposed in the two cases, and should the cormophyte-type be established it would, to say the least, greatly restrict the application of the centrosome-theory of Van Beneden and Boveri. Only future re- search can definitely de- termine the question. There can be no doubt that the descriptions of Guignard and his follow- ers do not rest upon pure imagination ; for it is easy to observe at the spindle- poles in some prepara- tions {e.g. sections of root- tips of AlliuDi, Liliuin, etc.) deeply staining- bodies such as these authors describe. These ''centrosomes" seem, however, to be of quite inconstant occurrence ; and the careful studies of Osterhout, Mottier, and Nemec seem to give good ground for the conclusion that they have no such significance as the centrosomes of lower plants or of animals. It should nevertheless be borne in mind that true centrosomes (*' blepharoplasts ") have been demonstrated in the spermatogenic divisions of some of the vascular cryptogams, and that analogous bodies occur in the corresponding divisions of the cycads (p. 175).

We should therefore still hold open the possibility that centrosomes may occur in the vegetative mitoses of the higher plants, their apparent absence being possibly due to lack of staining- capacity or similar conditions rendering their demonstration difificult.- Fig. 33. — Mitosis in ascus-nuclei of a fungus, Erysiphe. [Harper.] . A. Resting nucleus with disc-shaped centrosome [c^. B. Early prophase with aster. C. Later prophase; amphi- aster; intranuclear spindle forming. D. Spindle estab- lished. E. Daughter-nucleus after division ; spore-mem- brane developing from astral rays.

2 Mention may here be made of the barrel-shaped truncated spindles described m some of the plants. In Basidiobolus, Fairchild ('97) finds spindles of this type, having no asters A no less remarkable mode of spindle-formation, which is in a cer- tain way intermediate between the cormophyte-type and the usual animal type is described by Mead ('97, '98, i) in the first maturation- division of CJuctoptcnis. Here the completed am]:)hiaster is of quite typical form, and the centrosomes persist for the followin^^ mitosis; yet Mead is convinced that the amphiaster is synthetically formed by the union of two separate asters and centrosomes (Fig. 150) which

Fig- 34- — Division of pollen-mother-cells in the lily as described by Gl'IGNARD. A. An.ii)hase of the first division, showing the twelve daughter-chromosomes on eacli side, the interzonal fibres stretching between them, and the centrosomes, already double, at the spindle- poles. D. Later stage, showing the cell-plate at the equator of the spindle and the daughter- spiremes (dispireme-stage of Flemming). C. Division completed; double centrosomes in the resting cell. D. Ensuing division in progress; the upper cell at the close of the proj^hases, the chromosomes and centrosomes still undivided; lower cell in the late anaphase, cell-plate not yet formed.

have no genetic connection, arising independently dc novo in the cytoplasm.^ Improbable as such a conclusion may seem on a priori grounds, it is supported by very strong evidence,^ and, taken together and nearly parallel fibres, each of which terminates in a deeply staining granule. Nearly similar spindles have been described by Strasburger ('So) in Spirogyra, and in the embryo- sac o{ Monoiropa. It is not impossible that such spindles may represent a type intermediate between the "cormoptyte " and " thallophyte " types of Mottier.

2 I have had the privilege of examining some of Mead's beautiful preparations. with the facts described in plants, it indicates that the forces involved in sjDindle-formation are far more complex than Van Ik'neden's and Boveri's hypothesis would lead one to suppose.^ The centrosome and centrosphere appear to present great varia- tions that have not yet been thoroughly cleared up and will be more critically discussed beyond. ^ They are known to underg(j extensive changes in the cycle of cell-division and to vary greatly in different forms (Fig. 152). In some cases the aster contains at its centre nothing more than a minute deeply staining granule, which doubtless

^^g- 36- — Division of spore-mother-cells in Eqiiisetum, sliowing spindle-formation. [OsTERHOfT.] A. Early prophase, " kinoplasmic " fibrillae in the cytoplasm. B. Multipolar fibrillar figure invad- ing the nuclear area, after disappearance of the nuclear membrane. C. Multipolar spindle. D. Quadripolar spindle which finally condenses into a bipolar one. represents the centrosome alone. In other cases the granule is sur- rounded by a larger body, which in turn lies within the centrosphere or attraction-sphere. In still other cases the centre of the aster is occupied by a large reticular mass, within which no smaller body can be distinguished {e.g. in pigment-cells); this mass is sometimes called the centrosome, sometimes the centrosphere. Sometimes, again, the spindle-fibres are not focus.sed at a single point, and the spindle

^ See p. 276 for the peculiar spindles, devoid of asters, observed during the maturation of the egg in certain forms. Cf. also Morgan's experiments on the artificial production of asters and centrosomes, p. 307. - See p. 304. appears truncated at the ends, its fibres terminating in a transverse row of granules (maturation-spindles of Ascaris, and some plant-cells). It is not entirely certain, however, that such spindles observed in ]:)reparations represent the normal structure during life.

b. Tlic CJinwiatic Fii^mr. — The variations ot the chromatic figure must for the most part be considered in the more special parts of this work. There seems to be no doubt that a single continuous spireme-thread may be formed {rf. p. 113). ^^'^it it is equally certain that the thread may appear from the beginning in a number of distinct segments, i.e. as a segmented spireme, and there are some cases in which no distinct spireme can be seen, the reticulum resolving itself directly into the chromosomes. The chromosomes, when fully formed, vary greatly in appear- ance. In many of the tissues of adult plants and animals they are rod-shaped and are often bent in the middle like a V (Figs. 28, 131 ). Thev often have this form, too, in embryonic cells, as in the segmentation-stages of the Qgg in Ascaris (Fig. 31) and other forms. The rods may, however, be short and straight (seg- menting eggs of echinoderms, etc), and may be reduced to spheres, as in the maturation-stages of the germ-cells. In the equatorial plate the V-shaped chromosomes are placed with the apex of the V turned toward the spindle (Fig. 28), while the straight rods are placed with one end toward the spindle. In either case the daughter- chromosomes first begin to move apart at the point nearest the spindle, the separation proceeding thence toward the free portion. The V-shaped chromosomes, opening apart from the apex, thus give rise in the early anaphase to <>-shaped figures; while rod-shaped chromosomes often produce A- ^'I'ld i-shaped figures (the stem of the 1 being double). The latter, opening farther apart, form straight rods twice the length of the original chromosome (since each consists of two daughter-chromosomes joined at one end). This rod finally breaks across the middle, thus giving the deceptive appearance of a transverse instead of a longitudinal division (Fig. 52).

The <>- shaped figures referred to above are nearly related to those that occur in the so-called Jictcrotypical mitosis. Under this ^name Flem- ming ('87) first described a peculiar modification of the division of the chromosomes that has since been shown to be of very great impor- tance in the early history of the germ-cells, though it is not confined to them. In this form the chromosomes split at an early period, but the halves remain united by their ends. Each double chromosome then opens out to form a closed ring (Fig. 37), which by its mode of origin is shown to represent two daughter-chromosomes, each forming half of the ring, united by their ends. The ring finally breaks in two to form two U-shaped chromosomes which diverge to opposite poles

of the spindle as usual. As will be shown in Chapter V.,the divisions by which the germ-cells are matured are in many cases of this type ; but the primary rings here in many cases represent not two but four chromosomes, into which they afterward break up. Fig- 37- ~ Heterotypical mitosis in spermatocytes of the salamander. [Flemminc] A. Prophase, chromosomes in the form of scattered rings, each of which represents lui. daughter-chromosomes joined end to end. B. The rings ranged about the equator of the spindle and dividing ; the swellings indicate the ends of the chromosomes. C. The same viewed from the spindle-pole. D.. Diagram (Hermann) showing the central spindle, asters, and centrosomes,and the contractile mantle-fibres attached to the rings (one of the latter dividing).

The last paragraph leads to the consideration of certain varia- tions in the number of the chromosomes. Boveri discovered that the species Ascaris mcgaloccpJiala comprises two varieties which differ in no visible respect save in the number of chromosomes, the germ-nuclei of one form (" variety bivalens " of Hertwig) having two chromosomes, while in the other form (" variety iinivalens ") there is but one. Braiier discovered a similar fact in the phyllopod Artcmia, the number of somatic chromosomes being i68 in some individuals, in others only

It will appear hereafter that in some cases the primordial germ- cells show only half the usual number of chromosomes, and in Cyclops the same is true, according to Hacker, of all the cells of the early cleavage-stages. In all cases where the number of chromosomes is apparently reduced ("pseudo-reduction" of Riickert) it is highly probable that each chromatin-rod represents not one but two or more chromosomes united together, and Hacker has accordingly proposed the terms bivalent and plnrivaloit for such chromatin-rods.^ • The truth of this view, which originated with Vom Rath, is, I think, conclusively shown by the case of Artcniia described at page 281, and by many facts in the maturation of the germ-cells hereafter considered. In Ascaris we may regard the chromosomes of Hertwig's "variety univalens " as really bivalent or double, i.e. equivalent to two such chromosomes as appear in " variety bivalens." These latter, however, are probably in their turn plurivalent, i.e. represent a number of units of a lower order united together; for, as described at page 148, each of these normally breaks up in the somatic cells into a large number of shorter chromosomes closely similar to those of the related species Ascaris liunbricoides, where the normal number is 24.

Hacker has called attention to the striking fact that plurivalent mitosis is very often of the heterotypical form, as is very common in the maturation-mitoses of many animals (Chapter V.), and often occurs in the early cleavages of Ascaris ; but it is doubtful whether this is a universal rule. The process of mitosis in the one-celled plants and animals has a peculiar interest, for it is here that we must look for indications of its historical origin. But although traces of mitotic division were seen in the Infusoria by Balbiani ('58-61), Stein ('59), and others long before it was known in the higher forms, it has only recently received adequate attention and is still imperfectly understood.

Mitotic division has now been observed in many of the main divi- sions of Protozoa and unicellular plants ; but in the present state of 1 The words bivalent and uui-'alcni have been used in precisely the opposite sense by Hertwig in the case of Ascaris. the former term being applied to that variety having two chromosomes in the germ-cells, the latter to the variety with one. These terms certainly have priority, but were applied only to a specific case. Hacker's use of the words, which is strictly in accordance with their etymology, is too valuable for general descriptive purposes to be rejected.

the subject it must be left an open question whether it occurs in all. In some of the gregarines and Heliozoa, the process is of nearly or quite the same type as in the Metazoa. From such mitoses, how- ever, various gradations may be traced toward a much simpler pro- cess, such as occurs in Aniceba and the lower flagellates ; and it is not improbable that we have here representatives of more primitive con- ditions. Among the more interesting of these modifications may be mentioned : —

I. Even in forms that nearly approach the mitosis of higher types A-C. jSIacronucleus of Spirochona, showing pole-plates. D-H. Successive stagres in the division of the micronucleus of Paravicecium. D. The earliest stage, showing reticulum. G. Fol- lowing stage (" sickle-form ") with nucleolus. E. Chromosomes and pole-plates. /•: Late ana- phase. H. Final phase. the nuclear membrane may persist more or less completely through every stage {Noctiliica, EuglypJia, ActinospJicerium).

2. Asters maybe present (Heliozoa, gregarines) or wanting (In- fusoria, Radiolaria). 3. In one series of forms the centrosome or sphere is represented by a persistent intranuclear body (nucleolo-centrosome) of consider- able size, which divides to form a kind of central spindle { Huo^/ina Aniocbuy Infusoria.''). 4. In a second series the centrosome or sphere is a persistent cxtranuclear body, as in most Metazoa {Heliozoa, Noctilucay Para- moeba ).

5. In a few forms havin<; a scattered nucleus the chromatin-gran- ules are only collected about the apparently j)ersistent sphere or centrosome at the time of its division, and afterward scatter throuirh the cell, leaving the sphere lying in the general cell-substance ( Tctrainitiis). 6. The arrangement of the chromatin-granules to form chromo- somes appears to be of a secondary importance as compared with Fig- 39- — Mitosis in the rhizopod, Euglyplia. [SCHEWIAKOFF.] In this form the body is surrounded by a firm shell which prevents direct constriction of the cell-body. The latter therefore divides by a process of budding from the opening of the shell (the initial phase shown at .-/) ; the nucleus meanwhile divides, and one of the daughter-nuclei aftenvard wanders out into the bud.

A. Early prophase; nucleus near lower end containing a nucleolus and numerous chromo- somes. D. lu]u.-itorial plate and spindle formed insirle the nucleus; pole-bodies or pole-plates (i.e. attraction-spheres or centrosomes) at the spindle-poles. C. Metaphase. D. Late ana- phase, spindle dividing; after division of the spindle the outer nucleus wanders out into the bud. higher forms, and the essential feature in nuclear division a])pears to be the fission of the individual crranules.

We may first consider especially the achromatic figure. The basis of our knowledge in this field was laid by Richard Hertwig through his studies on an infusorian, SpirocJioiia { ^yy), and a rhizo})od, Actiiio- spJicenum{'?i/\). In both these forms a typical spindle and equatorial plate are formed inside tJie nuclear membrane by a direct transfor- mation of the nuclear substance. In SpirocJiona (Fig. 38, A-C) a hemispherical ''end-plate" or *' pole-plate " is situated at either pole of the spindle, and Hertwig's observations indicated, though they did not prove, that these plates arose by the division of a large "nucleolus." Nearly similar pole-plates were somewhat described by Schewiakoff ('88) in Eiiglypha (Fig. 39), and it seems clear that they are the analogues of the centrosomes or attraction-spheres in higher forms. In Euglcna, as shown by Keuten, the pole-plates, or their analoo-ues, certainly arise by division of a distinct and persistent intra- nuclear body ("nucleolus" or " nucleolo-centrosome ") which elon-

Fig. 40. — Mitosis in the flagellate, Eugleua. [KEUTEN.] A Preparing for division ; the nucleus contains a " nucleolus " or nucleolo-centrosome sur- rounded Ty a g-up of chromosomes. B. Division of the ;• nucleolus to form an mtranuclear spindle. C. Later stage. D. The nuclear division completed. gates to form a kind of central spindle around which the chromatin elements are grouped (Fig. 40); and Schaudinn ("95) described a similar process in Am(^ba. Richard Hertwig's latest work_ on Infusoria ('95) indicates that a similar process occurs m the micro- nuclei of Paramcccinm, which at first contain a large " nucleolus and afterward a conspicuous pole-plate at either end ot the spindle (Fio- :;8 D-H\ TJie origin of the pole-plates was not, however, po^tively determined. A corresponding dividing body is foupd m Ccratiinn (Lauterborn, '95), and as in the Infusoria the entire nucleus transforms itself into a fibrillar spindle-like body.

Still simpler conditions are found in some of the flagellates.^ In Chilonionas the sphere may still be regarded as intranuclear, since it lies in the middle of an irregular mass of chromatin-granules, though the latter are apparently not enclosed by a membrane. Nuclear division is here accomplished by fission of the sphere and the aggre- gation of the chromatin-granules around the two products. In Tctramitus, finally (Fig. i6), the nucleus is represented by chromatin- granules that are scattered irregularly through the cell and only at the time of division collect about the dividing sphere.

Fig. 41. — Mitosis in the Heliozoa. [SCHAUDINN.] A. Sp/ifrrastruiN : vegetative cell showing nucleus, "central gmnule" (centrosonic), and axial rays. li-G. Acanthocystis. B-D. Prophases of mitosis. E. Budding to form swarm-spores. F. Swarm-spores, devoid of centrosomes. G. Swarm-spores preparing for division ; intranuclear origin of centrosome. In a second series of forms, represented by Noctiliica (Ishikawa, '94, 98), (Calkins, '98, 2), raranurba (Schaudinn, '96, i), Actinophrys and Acanthoi-ystis (Schaudinn, '96, 2), and the diatoms (Lauterborn, '96), the sphere lies outside the nucleus in the cytoplasm and the mitosis is closely similar to that observed in most Metazoa. This is most striking in the Heliozoa, where the centrosome persists through the vegetative condition of the cell as the *• central granule," to which the axial filaments of the pseudopodia converge. Schaudinn ('96, 2) shows that by the division of this body a typical extranuclear amphi- aster and central spindle are formed (Fig. 41), while the chromatin

passes through a spireme-stage, breaks into very short rod-shaped chromosomes which spHt lengthwise and arrange themselves in the equator of the spindle, while the nuclear membrane fades away. Noctiluca (Fig. 42), as shown by Ishikawa and Calkins, agrees with this in the main points; but the nuclear membrane does not at any period wholly disappear, and a distinct centrosome is found at the centre of the sphere. The latter body, which is very large, gives

Fig. 42. — Mitosis in Noctiluca. [Calkins.] A. Prophase; division of the sphere to form the central spindle; chromosomes convertjing to the nuclear pole. B. Late anapliase. in horizontal section, showing centrosomes; the crntral spindle has sunk into the nucleus; nuclear membrane still intact except at the polos. C. \-A\\y anaphase; mantle-fibres connected with the diverging chromosomes. D. Kinal anaphase (which is also the initial prophase of the succeeding division of spore-forming mitosis) ; doubling of cen- trosome and splitting of chromosomes.

rise by a division to a fibrillated central spindle, about which the nucleus wraps itself while mantle-fibres are developed from the sphere-substance and become attached to the chromosomes, the nu- clear membrane fading away along the surface of contact with the central spindle (Calkins). Broadly speaking, the facts are similar in the diatoms {Surirclla, t. Lauterborn), where the central spindle, arisinf( by a peculiar ]:)rocess from an extranuclear centrosome, (sphere ?) sinks into the nucleus in a manner strongly suggesting that observed in Xoctiluca.

In the interesting form raiamaini, as described by Schaudinn ('96, I ), the sphere (*' Nebenkorper "), which is nearly as large as the nucleus, divides to form a central spindle, about the equator of which the chromatin-elements become arranged in a ring (Fig. 43); but no centrosome has yet been demonstrated in the sj^here. J\\rai)uvba appears to differ from Rugloia mainly in the fact that at the close of dixision the sphere is in the former left outside the daughter-nucleus and in the latter enclosed within it.^ The connecting link is perfectly given by Tctramitus, where no morphological nucleus is formed, and the sphere lies in the general cell-substance (p. 92); and we could have no clearer demonstration that the extra- or intranuclear position of sphere or centrosome is of quite secondary importance. As regards the formation of the spheres (pole-plates) ActinospJiceriuin (Figs. 44, 45) seems to show a simpler condition than any of the above forms, since no permanent sphere exists, and Brauer ('94) and R. riertwig ('98) agree that the pole-plates are formed by a gradual accumulation of the achromatic substance of the nucleus at opposite poles.

A distinct centrosome (centriole t) in the interior of the sphere has thus far only been observed in a few forms {Noctiluca, ActitiospJice- riu7ii), and neither its origin nor its relation to the sphere has yet been sufficiently cleared up. Both Ishikawa('94) and Calkins ('98, 2) somewhat doubtfully concluded that in Xoctiluca the centrosomes arise within the nucleus, migrating thence out into the extranuclear sphere. With this agree R. Hertwig's latest studies on Actiiiosphce- riiiDi ('98), the spindle-poles being first formed from the pole-plates (themselves of nuclear origin), and the centrosomes then passing into them from the nucleus. HertwMg reaches the further remarkable conclusion that the centrosomes arise as portions of the cJiromatin- iictivork extruded at the nuclear poles (Fig. 45), first forming a spongy irregular mass, but afterward condensing into a deeply staining pair of granules which pass to the respective poles of the spindle. It is a remarkable fact that these centrosomes are only found in the two maturation-divisions, and are absent from the ordi- nary vegetative mitoses where the spindle-poles arc formed by two cytoplasmic masses derived, as Hertwig believes, from the intra- nuclear plates. Schaudinn ('96, 3) likewise describes and clearly figures an intranuclear origin of the centrosome in buds of AcantJio- cystis (Fig. 41), which are derived by direct division of the mother-