The Cell in Development and Inheritance

When all these facts are placed in connection, we find it difficult to escape the conclusion that no definite line can be drawn between the cytoplasmic granules at one extreme and the chromatin-granules at the other. And inasmuch as the latter are certainly capable of growth and division, we cannot deny the possibility that the former may themselves have, or arise from elements having like powers. But while we may take this as a fair working hypothesis, we should clearly recognize that the base of well-determined fact on which it rests is approached by a circuitous route ; that in case of most of the cytoplasmic granules there is not the slightest evidence that they multiply by division ; and that even though some of them may have such powers, we cannot regard them as the ultimate structural units, for the latter must be bodies far more minute.

From our present point of view the centrosome possesses a peculiar interest as a cell-organ which may be scarcely larger than a cytomi- crosome, yet possesses specific physiological properties, assimilates, grows, divides, and may persist from cell to cell without loss of identity. Nearly all observers of the centrosome have found it lying in the cytoplasm, outside the nucleus; but apart from the Protozoa (p. 94) there is at least one well-established case in which it lies within the nucleus, namely, that of Ascaris, where Brauer made the interesting discovery that in one variety {iinivalens) the centrosome lies inside the nnclens^ in the other variety {bivalens) outside — a fact which proves that its position is non-essential ((/. Figs. 120 and 148).

An intra-nuclear origin of the centrosome has also been asserted by Julin ('93) in the primary spermatocytes of Styleopsis, by Riickert ('94) in the eggs of Cyclops, Mathews ('95) in those of Asterias, Car- noy and Le Brun ('97, 2) in Ascaris, Van der Stricht ('98) in the eggs of Thysanozodn, by R. Hertwig ('98) in ActinospJicerinm, Calkins ('98, I ) in Noctiluca, and Schaudinn ('96, 3) in spore-producing buds of Acanthocystis, though in the last-named form the centrosome of the vegetative forms is extra-nuclear (p. 92).

As already stated, ^ it is still undetermined whether a true centro- some may ever arise de novo, but the evidence in favour of such a possibility has of late rapidly increased. Carnoy ('86) long since showed that the ^g^ of Ascaris, during the formation of the polar bodies, sometimes showed numerous accessory asters scattered through the cytoplasm. Reinke ('94) described somewhat similar asters in peritoneal cells of the salamander, distinguishing among them three orders of magnitude, the largest containing distinct centrosomes or "primary centres," while the smaller contained "secondary" and "tertiary" centres, the last named being single

Fig. 148. — Mitosis witli intra-nuclear centrosome, in the spermatocytes of Ast\iris tttfx<^h- cephala, var. univalens. [Brauer.] A. Nucleus containing a quadruple group or tetrad of chromosomes (/). nucleolus («). and centrosome {c). B. C. Division of the centrosome, D. E.F. G. Formation of the mitotic tiijure. centrosomes escaping from the nucleus in G. microsomes at the nodes of the cytoreticulum. By successive aggre- gations of the tertiary and secondary centres arise true centrosomes as new formations. Watase ('94-95) ^^^o finds in the q^^ of Mturo- bdella, besides the normal aster containing an undoubted centrosome. numerous smaller asters graduating downwards to such "tertiary asters" as Reinke describes with a microsome at the centre of each, and on this basis concludes that the true centrosome differs from a microsome only in degree and may arise dc novo. iMottier ('97. 2) finds in pollen-mother-cells numerous minute " cyto-asters " having no direct relation to the spindle-formation (Fig. 133). Again Juel

('97) finds that an isolated chromosome, accidentally separated from the equatorial plate (pollen-mother-cells of Hcmei'ocallis), may give rise to a small vesicular nucleus which may subsequently divide by mitosis, though it is quite out of relation to the spindle-poles of the preceding mitosis (Fig. 149). Strong evidence of the same character as the last is given by the facts in the heliozoon AauitJiocystis, as shown by Schaudinn ('96, 3), the ordinary vegetative cells containing a persistent extra-nuclear centrosome, while in the bud-formation of the swarm-spores a centrosome is formed dc novo, ivitJiojit relation to that of the mother-cell, inside the nucleus of the bud (Fig. 41).

The strongest case in favour of the independent origin of centro- somes is, however, given by the observations of Mead on CJiatopteriis ('98) and the remarkable experiments of R. Hertwig ('95, '96) and Fig. 149. — Abnormal mitosis in pollen-mother-cells of Hemerocallis, showing formation of small nucleus from one or two stray chromosomes and its subsequent division. [JUEL.] Morgan ('96, i ; '99, i)on the eggs of echinoderms and other animals. When eggs of CJicEtopterns are taken from the body-cavity and placed in sea-water, a multitude of small asters appear in the cytoplasm, two of which are believed to persist as those of the polar spindle, while the others degenerate (Fig. 150). Mead is therefore convinced that the polar centrosomes arise in this case separately and de novo} R. Hertwig showed that when unfertilized eggs of sea-urchins { Strongylocentrotus , EcJiiniis) are kept for some time in sea-water or treated with dilute solutions of strychnine the nuclei undergo some of

1 A number of other authors {^e.g. Griffin, Thalassema, Coe, Cerebratuhis) have likewise found the first polar asters widely separated at their first appearance. On the other hand, Mathews ('95), whose preparations I have seen, finds the polar centrosomes in Asto-ias close together, and Francotte ('97, '98) has demonstrated that in Cydopoi-iis and Prosthece- ra:iis\\v^v arise by the division of a single primary centrosome. The same is stated by Gar- diner ('98) to be the case in Polychccrus. It should be noted, further, that Mead could find no undoubted centrosomes save in the " primary " or definitive polar asters.

the changes of mitosis, the chromatin-nctwork giving rise to a group of chromosomes and a spindle, or more frequently a fan-shaped half-spindle, arising from the achromatic substance. In some cases not only a complete spindle appeared but also asters at the poles, though no centrosomes were observed (Fig. 151). Morgan's experi- ments along the same lines were mainly performed upon the sea- urchin Arbacia, but included also the eggs of Astcrias, Sipunculus, and Cerebratnlus (Figs. 150, 151). In these eggs numerous asters may arise in the cytoplasm, if they are allowed to lie some time in sea-

A. Unfertilized egg of ChcBtopterus with " secondary asters " developed a few minutes after the egg is placed in sea-water. D. Slightly later stage with two definitive polar asters and centrosomes. C. Large "sun" (transformed polar aster) containing numerous small "secondary asters " .in <1 centrosomes, from unfertilized egg of Cerebratnlus after 22 hours in 1.5 '•; sodium chlorult- water or treated by weak solutions of sodium or magnesium chloride. These asters often contain deeply staining, central granules indistin- guishable from the centrosomes of the normal asters ; and, what is ot high interest, such of them as lie near the nucleus take part in the irregular nuclear division that ensues, forming centres toward which the chromosomes pass. These divisions continue for some time, the chromosomes being irregularly distributed through the it^^^, and giving rise to nuclei of various sizes apparently dependent upon the number of chromosomes each receives. After a variable number of such

divisions the asters disappear, yet the irregular nuclear divisions con- tinue, nuclear spindles with distinct centrosomes being formed at each division, but apparently without relation to the older asters, and they Fig. 151. — Formation of centrosomes and asters in unfertilized echinoderm-eggs. [A, B, Morgan ; C-E, R. Hertwig.] A. Arbacia, after 4V2 hours in 1.5 % solution of sodium chloride, then 5 hours in sea-water; scattered chromosomes and asters. B. Asters formed after 6^2 hours in XaCl. C-E. Echinus after treatment with 0.5 % strychnine-solution, showing various forms of astral formations (fan- shaped aster, half spindle, and complete mitotic figure).

are believed by Morgan to arise de novo from the Qgg substance.^ In the meantime irregular cleavage of the Qgg occurs, though no embryo is produced.'^ Loeb, however, in the remarkable experiments - Morgan makes the important observation, which harmonizes with that of Boveri, reported at page 108, that the divisions occur with respect to the nujuber and position of the nuclei, not of the asters, concluding that the former must therefore play an essential rdle as centres of division, and that the activity of the asters is in itself not sufficient to account for division of the cytoplasm.

referred to at page 215, finds that after treatment with magnesium chloride unfertilized sea-urchin eggs {Arbacia) may give rise to perfect Phiteus larvae — a result which if well founded seems to place the new formation of true centrosomes beyond question. Taken together, these researches give strong ground for the con- clusion that true {i.e. physiological) centrosomes may arise de novo from either the cytoplasmic or the nuclear substance and may play the usual role (whatever that may be) in mitosis. If this conclusion be sustained by future research, we shall no longer be able to accept Van Beneden's and Boveri's conception of the centrosome as a per- sistent organ in the same sense as the nucleus ; but on the other hand we shall have gained important ground for further inquiry into the nature and source of that power of division which is so characteristic of living things and upon which the law of genetic continuity rests.

Morphology of the Centrosome. — In its simplest form (Fig. 152, A) the centrosome appears under the highest powers as nothing more than a single granule of extraordinary minuteness which stains intensely with iron-haematoxylin, and can scarcely be distinguished from the cyto-microsomes except for the fact that it lies at the focus of the astral rays. In this form it always appears at the centre of the very young sperm-asters during fertilization (Figs. 97, 99), in the earlv phases of ordinary mitosis (Figs. 27, 32), and in some cases also in the resting cell, for example, in leucocytes and connective tissue corpuscles (Figs. 8, 49), where, however, it is often triple or quadruple. In the course of division the centrosome often increases in size and assumes a more complex form, becoming also surrounded by various structures involved in the aster-formation. The relation of these structures to the centrosome itself has not yet been fully cleared up and there is still much divergence of opinion regarding the cycle of changes through which the centrosome passes. It is, therefore, not yet possi- ble to give a very consistent account of the centrosome, still less to frame a satisfactory morphological definition of it.

It is convenient to take up as a starting-point Boveri's ('88) account of the centrosomes in the Qgg of Asearis, supplemented by Brauer's ('93) description of those in the spermatocytes of the same animal. During the early prophases of the first cleavage Boveri found the centrosome as a minute granule which steadily enlarges as the spin- dle forms, until shortly before the metaphase it becomes a rather large, well-defined sphere in the centre of which a minute central gratni I c or ceiitriole appears (Fig. 152, B, C). From this time onward the cen- trosome decreases in size until in the daughter-cells it is again reduced to a small granule which divides into two and goes through a similar cycle during the second cleavage and so on. The centrosome is at all stages surrounded by a clear zone ("Heller Hot") in which

the astral rays are thinner and stain less deeply than farther out. Brauer's account is substantially the same, though no definite "Heller Hof " was found, and the astral rays were traced directly in to the boundary of the centrosome. He added, however, two impor- tant observations, viz. (i) that the central granule is visible at every period ; and (2) division of tJie centrosome is preceded by division of tJie centi'al granule {Y\g. 148) — an observation recently extended by Boveri to the division of the egg-centrosome.^ Van Beneden and Neyt {^Zj\ on the other hand, gave a quite different account of the

t ig. 152. — Diagrams illustrating various accounts of centrosome and aster, A. Centrosome, a simple granule at the centre of the aster; ex. sperm-aster in various animals. B. "Centrosome," a sphere enclosing a central granule or centriole; ex. Brauer's account of spermatocytes oi Ascaris. C Like the last, but " centrosome " surrounded by a "Heller Hof"; ex. Boveri's account of the centrosome of the Ascaris egg. D. Central granule surrounded by a radial sphere ("centrosome") bounded by a microsome-circle, and lying in a "Heller Hof"; ex. polar spindles of Thysatiozodn, Van der Stricht. E. Central granule (" centrosome ") sur- rounded by medullary and cortical radial zones, each bounded by a microsome-circle ; ex. polar spindle of Unio, Lillie, F. Van Beneden's representation of aster of the Ascaris egg; like the last, but the " corpuscule central " consisting of a group of granules. G. " Centrosome," a group of granules surrounded by a "Heller Hof"; ex. the echinoderm-egg. //. "Centrosome" (central granule) surrounded by a vague larger body lying in a reticulated centrosphere ; ex. Thalasseyna. [Griffin.]

Structures at the centre of the aster. The " corpuscule central " (usually assumed by later writers to be the centrosome), described as a "mass of granules," is surrounded by two well-defined astral zones, formed as modifications of the inner part of the aster, and constitut- ing the "attraction-sphere." These are an inner "medullary zone," and an outer " cortical zone," each bounded by a very distinct layer of microsomes (Fig. 152, F). The discrepancy between these results on the part of the two pioneer investigators of the centrosome has led to great confusion in the terminology of the subject, which has not yet been fully cleared away. Many of the observers who followed Boveri (Flem- ming, Hermann, Van der Stricht, Heidenhain, etc.) found the centro- some, in various cells, as a much smaller body than he had described, often as a single or double minute granule, staining intensely with iron-haematoxylin. Heidenhain ('93, '94) and Druner ('94, '95) found further that the asters in leucocytes and other forms often show several concentric circles of microsomes, and that the sphere bounded by the innermost circle often stains more deeply than the outer por- tions and may appear nearly or quite homogeneous (Fig. 156). To this sphere, with its contained central granule or granules Heidenhain applies the term microcentrum ('94, p. 463), while Kostanecki and Siedlecki suggest the term microsphere ('96, p. 217). Still later Kostanecki and Siedlecki ('97) found that even in Ascaris, as in other forms, sufficient extraction of the colour (iron-haematoxylin) reduces the centrosome to a minute granule to which the astral rays converge, and which is presumably identical with Boveri's ''central granule." Heidenhain ('93, '94) found that in leucocytes the central granule is often double, triple, or even quadruple, while in giant-cells of certain kinds there are numerous deeply staining granules (Fig. 14). He therefore proposed to restrict the term centrosome to the individ- ual granules, whatever be their number, applying the term microccn- triim to the entire group ('94, p. 463).

With these facts in mind we can gain a clear view of the manner in which both the confusion of terminology and the contradiction of results has arisen. Brauer ('93) found in Ascaris (see above) that division of the central grmuile precedes division of the ^^ centrosome ,'' and therefore suggested that only the former is equivalent to Wan Beneden's '' corpuscule central," while the body called "centrosome" by Boveri is really the medullary astral zone, the " Heller Hof " being the cortical zone. This is substantially the same conclusion reached by Heidenhain, Rawitz, Lenhossek, Kostanecki and Siedlecki, Krlan- ger, Van der Stricht, Lillie, and several others. The confusion of the subject is owing, on the one hand, to the fact that those who have accepted this conclusion continue to use the word centrosome in two quite different senses, on the other hand to the fact that the conclusion is itself repudiated by Boveri ('95), MacFarland (97), and Fiirst ('98).

As regards the terminology we find that most recent writers agree with Heidenhain, Kostanecki and Siedlecki, in restricting the word centrosome to the minute, deeply staining granules, whether one or more, at the centre of the aster. On the other hand, Brauer, Fran- cotte, Van der Stricht, Meves, and others apply the term to the central granule or granules plus the surrounding sphere (" centrosome " of Boveri), which they regard as equivalent to the medullary zone of Van Beneden, the " corpuscule central" of the last-named author being identified with the central granule or "centriole" of Boveri, though the latter structure is considerably smaller than the former as described by Van Beneden.

The matter of fact turns largely on the question whether the astral rays traverse the larger sphere to the central granule. That such is the case in Ascaris is positively asserted by Kostanecki and Siedlecki, ('97) and as positively denied by Fiirst ('98) with whose observations Fig. 153. — Structure of the centiosome in the polar asters of a gasteropod, Dinulula. [MaC- Farland.] A. Mitotic figure, formation of first polar body. B. Inner aster at final anaphase; central granule double within the " centrosome." C. Elongation of old " centrosome " to form second polar spindle.

those of MacFarland ('97) on gasteropod-eggs agree. On the other hand, in the turbellarians the observations of Francotte ('97, '98) and Van der Stricht ('98, i) seem to leave no doubt that the larger sphere ("centrosome"), here very sharply defined and staining deeply in iron-haematoxylin, is traversed by well-defined astral rays converging to the central corpuscle, and both these observers agree further that hotJi the corpuscle and tJie sphere divide to persist as the ^^ eentrosonies " of tJie daughter-cells — a result in conformity with Van Beneden's con- clusion in the case of Ascaris.

Lillie's valuable observations on the polar asters of Unio ('98) afford, I believe, conclusive evidence as to the nature of the sphere. In the the polar mitoses of C/n/o. [Lillie.] granule (centrosome) surrounded bv medullarv ^. Late anaphase of second polar mitosis raS oftt'oid'rS:,::--"^ ---.• ^ormatt' persist. The inner sphere is now bounded by a definite membrane, and its radiate structure becomes obscure, the astral rays extending only to the boundary of the sphere, though a few rays persist within it (Fig. 154, B). It is clear from this that the inner sphere and central granule pass through phases that bridge the gap between Van Beneden's and Boveri's descriptions. LilHe's observations fully sustain the conclusion that the cmtral gmniUe {^' ceiitriole'' of Boveri) corresponds to the '' corpiiscide cejitraV' of Van Beneden, and the inner spJiere {medullary zone) to BoverVs '' cejitrosomey A comparison of the polar aster of rm'o with that of TJiysanozoon, as described by Van der Stricht ('98), leaves hardly room for doubt that the cortical zone represents Boveri's " Heller Hof " ; for in both forms the rays of the cortical zone are much thinner and Hghter than the more peripheral portions, thus giving a clear zone, which in Uiiio is bounded by only a fairly definite microsome-circle and in TJiysanozoon by none. Lastly, we must recognize the justice of the view urged by Kos- tanecki. Griffin, Mead, Lillie, Coe, and others, that the term centro- some should be applied to the central granule and not to the sphere surrounding it (medullary zone), despite the fact that historically the word was first applied by Boveri to the latter structure. For in both Diauhila (MacFarland) and Unio (Lillie) the second polar spindle arises from the substance of the inner sphere, while the central granule, becoming double, gives rise to the centrosomes at its poles.

By following Boveri's terminology, therefore, MacFarland is driven to the strange conclusion that the second polar spindle is nothing other than an enormously enlarged '* centrosome " — a result little short of a reductio ad absnrdnm when we consider that in Ascaris the polar spindle arises by a direct transformation of the germinal vesicle (p. 277). The obvious interpretation is that the central granule is the only structure that should be called a centrosome, the surround- ing sphere being a part of the aster, or rather of the attraction-sphere. Thus regarded, the origin of the spindle in Dianlula presents nothing anomalous and a similar interpretation may be placed on the polar spindles of Ascaris as described by Fiirst ('98).^

1 In echinoderms the concurrent' results of Reinke ('95), Boveri ('95), myself ('96-'97), show that the " centrosome " is a well-defined sphere containing a large group (ten to twenty) of irregularly scattered, deeply staining granules. I have shown in this case that in the early prophases there is but one such granule, which then becomes double and finally multiple, forming a pluricorpuscular centrum (Fig. 52) not unlike that described by Heidenhain in giant-cells. Kostanecki, who asserts that the centrosome of echinoderms is a single granule ('96, i, '96, 2, p. 248), has not sufficiently studied the later phases of mitosis. Cf. also Erlanger ('98). The centrosomes described in nerve cells by Lenhossek ('95) are apparently of somewhat similar type. Until the facts are more fully known the exact nature of these "centrosomes" remains an open question. Lillie's observations on Unio show that here, too (first polar spindle), the centrosome divides to form a considerable number of

The genesis of the concentric spheres surrounding the centrosome will be considered in the following section. We may here only emphasize the remarkable fact that the centres of the dividino- system are bodies which are in many cases so small as to lie almost at the limits of microscopical vision, and which in the absence of the surrounding structures could not be distinguished from other proto- plasmic granules. Full weight should be given to this fact in every estimate of the centrosome theory, and it is no less interestin"- in its bearing upon the corpuscular theory of protoplasm.

Watase ('93, '94) made the very interesting suggestion that t/ie cen- trosome is itself notJiing other tJian a microsome of the same morj^ho- logical nature as those of the astral rays and the general meshwork, differing from them only in size and in its peculiar powers.^ Despite the vagueness of the word "microsome," which has no well-defined meaning, Watase's suggestion is full of interest, indicating as it does that the centrosome is morphologically comparable to other elemen- tary bodies existing in the cytoplasmic sti-ucture, and which, minute though they are, may have specific chemical and physiological prop- erties.

An interesting hypothesis regarding the historical origin of centrosome is that of Rutschli ("91) and R. Hertwig ("92), who suggest that it may be a derivative of a body comparable with the micro-nucleus of Infusoria, which has lost its chromatin but retained the power of division ; and the last-named author has suggested further that the so-called " archoplasmic loops'' discovered by Platner in puhnonates mav be remnants of the chromatic elements. A similar view has been advocated bv Heidenhain (''93, '94) and Lauterborn ('96). Heidenhain regards central spindle and centrosomes as forming essentially a unit ("-microc^entrum ") homologous with the micro-nucleus of the Infusoria, the centrodesmus (p. 79) representing a part of the original achromatic elements. The metazoan nucleus is compared to the proto- zoan macro-nucleus. The improbability of a direct derivation of the Metazoa from Infusoria, urged by Boveri ('95) and Hertwig ("96), has led Lauterborn ("96) to the view that the metazoan centrosome and nucleus are respectively derivatives of two equivalent nuclei, such as Schaudinn ("95) describes in Ainivba hinucUdta, the ='Nebenkorper" of Paraifia'ba (cf. p. 94), being regarded as an intermediate step, and the micro-nucleus of Infusoria a side-branch. R. Hertwig ('96), on the other hand, regards the metazoon centrosome as a derivative of an intra-nuciear botlv such as the '' nucleolo-centrosome " of Euglena (p. 91), which has itself arisen through a condensation of the general achromatic substance. With this view Calkins (98), on the whole, agrees; but he regards it as probable that the •• nucleoIo-ccntrDsome "

granules of which one or two remain as the persistent centrosome. while others are eonverted into microsomes or other cytoplasmic structures. It is probable that somethinj; similar occurs in the echinoderms. 1 The microsome is conceived, if I understand Watase rightly, not as a permanenl mor- phological body, but as a temporary varicosity of the thread, which may lose its i.lentity in the thread and reappear when the thread contracts. The centrosome is in like manner not a permanent organ like the nucleus, but a temporary body formed at the focus of the astral rays. Once formed, however, it may long persist even after disappearance o\ the aster, and serve as a centre of formation for a new aster.

of Eitgleiia and Ainaba and the sphere of N'octiluca and Paramooba are to be com- pared with the attraction-sphere, while the centrosome may have had a different origin. It appears to me that none of these views rests upon a very substantial basis, and they must be taken rather as suggestions for further work than as w^ell-grounded conclusions. F. The Archoplasmic Structures I. Hypothesis of Fibrillar Persistence The asters and attraction-spheres have a special interest for the study of cell-organs ; for they are structures that may divide and persist from cell to cell or may lose their identity and re-form in suc- cessive cell-generations, and we may here trace with the greatest clearness the origin of a cell-organ by differentiation out of the struc- tural basis. Two sharply opposing views of these structures have been held, represented among the earlier observers on the one hand by Boveri, on the other by Blitschli, Klein, Van Beneden, and Carnoy. The latter observers held that the astral rays and spindle-fibres, and hence the attraction-sphere, arise through a morphological rearrange- ment of the preexisting protoplasmic meshwork, under the influence of the centrosome. This view, which may be traced back to the early work of Fol ('73) and Auerbach ('74), was first clearly formulated by Biitschli ('76), who regarded the aster as the optical expression of a peculiar physico-chemical alteration of the protoplasm primarily caused by diffusion-currents converging to the central area of the aster.^ An essentially similar view is maintained in Biitschli's recent great work on protoplasm,^ the astral " rays " being regarded as nothing more than the meshes of an alveolar structure arranged radially about the centrosomes (Fig. 10, B). The fibrous appearance of the astral rays is an optical illusion, for they are not fibres, but i^at lamellae forming the walls of elongated closed chambers. This view has recently been urged, especially by Erlanger ('97, 4, etc.), who sees in all forms of asters and spindles nothing more than a modified alveolar structure.