The Cell in Development and Inheritance

The visible organs of the cell fall under two categories, according as they are merely temporary structures, formed anew in each successive cell-generation out of the common structural basis, or permanent struc- tures whose identity is never lost, since they are directly handed on by division from cell to cell.^ To the former category belong, in general, such structures as cilia, pseudopodia, and the like ; to the latter, the nucleus, perhaps also the centrosomes, and the plastids of plant-cells. A peculiar interest attaches to the permanent cell-organs. Closely interrelated as these organs are, they nevertheless have a remarkable degree of morphological independence. They assimilate food, grow, divide, and perform their own characteristic actions like coexistent but independent organisms, of a lower grade than the cell, living together in colonial or symbiotic association. So striking is this morphological and physiological autonomy in the case of the green plastids or chro- matophores that neither botanists nor zoologists are as yet able to dis- tinguish with absolute certainty between those that form an integral part of the cell, as in the higher green plants, and those that are actually independent organisms living symbiotically within it, as is probably the case with the yellow cells of Radiolaria. Even so acute an investigator as Watase ('93, i) has seriously propounded the view that the nucleus itself — or rather the chromosome — should be regarded as a distinct organism living in symbiotic association with the cytoplasm, but having had, in an historical, sense, a different origin. This rather fantastic view has not found much favour, and even were it true w^ould teach us nothing of the origin of the power of division, which must for the present be taken as an elementary process forming one of the primary data of biology.

Yet we may still inquire whether the power of division shown by such protoplasmic masses as plastids, chromosomes, centrosomes, nucleoli, and nuclei may not have its root in a like power residing in ultimate protoplasmic units of which they are made up. Could we accept such a view, we might much more easily meet some puzzling cytological difficulties. For under this assumption the difference between transient and permanent cell- organs would become only one of degree, depending on the degree of cohesion between their structural components ; and we could thus con- ceive, for example, how such a body as a centrosome might form, per- sist by division for a number of generations, and finally disintegrate. In connection with this it may be pointed out that even such a typical permanent organ as the nucleus does not persist as siicJi during the ordinary form of division ; for it loses its boundary and many of its other structural characters, becoming resolved into a group of sepa- rate chromosomes. What persists is here not the structural unit, but the characteristic substance which forms its essential constituent, and

a large part even of this substance may be lost in the process. The term ''persistent organ" is therefore used in rather a figurative sense, and if too literally understood may easily mislead us. With the foregoing considerations in mind let us turn to the actual structural relation of the cell-orgfans. In Chapter I. some of the reasons have been given for the conclu- sion that none of the obvious structural features of protoplasm (fibrillce, alveoli, granules, and the like) can be regarded as necessary or uni- versal ; and we may now inquire whether there is any evidence that such structures may have such a common structural basis as De \'ries's theory assumes. I shall here take as a point of departure my observa- tions on the structure of protoplasm in echinoderm-eggs,' already briefly reviewed at page 28. The beautiful alveolar structure of these eggs is entirely of secondary origin, and all the visible structural elements arise during the growth of the eggs by the deposit and subsequent enlargement of minute spherical bodies, all apparently liquid drops, in a homogeneous or finely granular basis which is itself a liquid. Some of these spheres enlarge to form the alveolar spheres, whiie the homogeneous basis or continuous substance remains as the interalve- olar material. Others remain much smaller to constitute the " micro- somes " scattered through the interalveolar walls ; and these bodies, like the alveolar spheres, are perfectly visible in life, as well as in section ; they are therefore not coagulation-products or artifacts. From these three elements arise all the other structures observed in these eggs, deutoplasm-spheres {Ophiiira) and pigment-bodies {Arbncia) being formed by further enlargement and chemical alteration of the alveolar spheres, while astral rays and spindle-fibres are differentiated out of the inter-alveolar material and microsomes.^ These various elements show a continuous gradation in size from the largest deuto- plasm-spheres down to the smallest visible granules, the latter being the source of all the larger elements, and in their turn emerging into view from the " homogeneous " basis.

Clearly, then, none of these bodies can be regarded as the ultimate structural units ; for the latter, if they exist, must lie in a region at present inaccessible to the micro- scope. This fact, however, no more disproves their existence than it does that of molecules and atoms. It only shows the futiHty of such attempts as those of Altmann and his predecessors to identify " gran- ules " or ''microsomes " as final morphological units, and compels us to turn to indirect instead of direct evidence. It may, however, again be pointed out that it would be quite irrational to conclude that the small-

est visible granules first come into existence when they first come within view of the microscope. The ** homogeneous " substance must itself contain or consist of granules still smaller. The real question is not whether such ultra-microscopical bodies exist, but whether they are permanent organize dho^i^?. possessing besides the power of growth also the power of division. This question can be only indirectly ap- proached ; and we shall find it convenient to do so by beginning at the opposite end of the series, through a reconsideration of the phenomena of nuclear division.

(a) HypotJiesis of the Individuality of the CJiromosomes. — It may now be taken as a well-estabhshed fact that the nucleus is never formed de novo, but always arises by the division of a preexisting nucleus. In the typical mode of division by mitosis the chromatic substance is resolved into a group of chromosomes, always the same in form and number in a given species of cell, and having the power of assimilation, growth, and division, as if they were morphological individuals of a lower order than the nucleus. That they are such individuals or units has been maintained as a definite hypothesis, es- pecially by Rabl and Boveri. As a result of careful study of mitosis in epithelial cells of the salamander, Rabl ('85) concluded that the cJironiosomes do not lose their individuality at the close of division, but persist in the chromatic reticnhim of the resting nnclens. The reticu- lum arises through a transformation of the chromosomes, which give off anastomosing branches, and thus give rise to the appearance of a network. Their loss of identity is, however, only apparent. They come into view again at the ensuing division, at the beginning of which "the chromatic substance flows back, through predetermined paths, into the primary chromosome-bodies " (Kernfaden), which re- appear in the ensuing spireme-stage in nearly or quite the same posi- tion they occupied before. Even in the resting nucleus, Rabl believed that he could discover traces of the chromosomes in the configuration of the network, and he described the nucleus as showing a distinct polarity having a ''pole" corresponding with the point toward which the apices of the chromosomes converge {i.e. toward the centrosome), and an '' antipole" (Gegenpol) at the opposite point {i.e. toward the equator of the spindle) (Fig. 22).

Rabl's hypothesis was precisely formulated and ardently advocated by Boveri in 1887 and 1888, and again in 1891, on the ground of his own studies and those of Van Beneden on the early stages of Ascaris. The hypothesis was supported by extremely strong evidence, derived especially from a study of ab- normal variations in the early development of Ascaris, the force of which has, I think, been underestimated by the critics of the hypothesis. Some of this evidence may here be briefly reviewed. In some cases, through a miscarriage of the mitotic mechanism, one or both of the chromosomes destined for the second polar body are accidentally left

Fig. 143. — Evidence of the individuality of the chromosomes. Abnormalities in the fertiliza- tion of Ascaris. [BOVERI.J A. The two chromosomes of the egg-nucleus, accidentally separated, have given rise each to a reticular nucleus (?, ?) ; the sperm-nucleus below (-T). B. Later stage of the same, a single chromosome in each egg-nucleus, two in the sperm-nucleus. C. An egg in which the second polar body has been retained; p.b'^ the two chromosomes arising from it; 9 the egg-chromo- somes; d" the sperm-chromosomes. D. Resulting equatorial plate with six chromosomes.

in the ^gg. These chromosomes give rise in the Q.gz to a reticular nu- cleus, indistinguishable from the egg-nucleus. At a later period this nucleus gives rise to the same number of chromosomes as those that entered into its formation, i.e. either one or two. These are drawn into the equatorial plate along with those derived from the germ- nuclei, and mitosis proceeds as usual, the number of chromosomes being, however, abnormally increased from four to five or six (Fig. 143,

C, D). Again, the two chromosomes left in the ^gg after removal of the second polar body may accidentally become separated. In this case each chromosome gives rise to a reticular nucleus of half the usual size, and from each of these a single chromosome is afterward formed (Fig. 143, A, B). Finally, it sometimes happens that the two germ-nuclei completely fuse, while in the reticular state, as is normally the case in sea-urchins and some other animals (p. 188). From the cleavage-nucleus thus formed arise four chromosomes.

The same general result is given by the observations of Zur Strassen ('98) on the history of giant embryos in Ascaris. These embryos arise by the fusion, either before or after the fertilization, of previ- ously separate eggs, and have been shown to be capable of develop- ment up to a late stage. Not only in the first but also in some, at least, of the later mitoses, such eggs show an increased number of chromosomes proportional to the number of nuclei that have united. Thus in monospermic double eggs (variety bi- valeiis) the number is six instead of four ; in dispermic double eggs the number is increased to eight (Fig. 144).

These remarkable observations show that whatever be the imniber of eJironio- sonies entering into the forniation of a reticular nnclens, the same nnniber after- ward issnes from it — a result which de- monstrates that the number of chromo- somes is not due merely to the chemical composition of the chromatin-substance, but to a morphological organization of Fig. 144. — Giant-embryo of y4j^ar/>, the nuclcus. A bcautiful Confirmation of this conclusion was afterward made by Boveri ('93, '95, i) and IMorgan (95, 4), in the case of echinoderms, by rear- ing larvae from enucleated egg-fragments, fertilized by a single sper- matozoon (p. 194). All the nuclei of such larvae contain but half the typical number of chromosomes, — i.e. in Echinns nine instead of eighteen, — since all are descended from one germ-nucleus instead of two !

Equally striking is the remarkable fact, described at page 275, that all of the cells in the sexual generation (oophore) of the higher cryptogams show half the number of chromosomes characteristic of the sporophyte, the explanation being that while reduction occurs at the time of spore-formation, the spores develop without fertilization, the reduced chromosome-number persisting until fertilization occurs var. bivaleiis, arising from a double- fertilized double egg, showing eight chromosomes {7.ur Strasseti).

long afterward. Attention may be again called to the surprising case of Arteniia, described at page 281, which gives a strong argument in favour of the hypothesis. In addition to the foregoing evidence, Van Beneden and Boveri were able to demonstrate in Ascaris that in the formation of the spireme the chromosomes reappear in the same position as those which entered into the formation of the reticulum, precisely as Rabl Fig. 145. — Evidence of the individuality of the chromosomes in the egg of Ascaris. [Boveri.]

E. Anaphase of the first cleavage. F. Two-cell stage with lobed nuclei, the lobes formed by the ends of the chromosomes. G. Early prophase of the ensuing division ; chromosomes re-form- ing, centrosomes dividing. H. Later prophase, the chromosomes lying with their ends in the same position as before ; centrosomes divided. maintained. As the long chromosomes diverge, their free ends arc always turned toward the middle plane (Fig. 31), and upon the re- construction of the daughter-nuclei these ends give rise to corresjiond- ing lobes of the nucleus, as in Fig. 145, which persist throughout the resting state. At the succeeding division the chromosomes reappear exactly in the same position, their ends lyiuj^ in the nuclear lobes as before {¥ig. 145, G, H). On the strength of these facts Boveri con- cluded that the chromosomes must be regarded as " individuals " or '' elementary organisms," that have an independent existence in the

cell. During the reconstruction of the nucleus they send forth pseu- dopodia which anastomose to form a network in which their identity is lost to view. As the cell prepares for division, however, the chro- mosomes contract, withdraw their processes, and return to their "resting state," in which fission takes place. Applying this con- clusion to the fertilization of the Qgg, Boveri expressed his belief that Fig. 146.— Independence of paternal and maternal chromatin in the segmenting eggs of Cyclops. [A-C, from RucKERT; D, from Hacker.]

A. First cleavage-figure in C. strenuus ; complete independence of paternal and maternal chromosomes. B. Resulting two-cell stage with double nuclei. C. Second cleavage; chromosomes still in double groups. D. Blastomeres with double nuclei from the eight-cell stage of C brevicornis. " we may identify every chromatic element arising from a resting nucleus with a definite element that entered into the formation of that nucleus, from which the remarkable conclusion follows that in all cells derived in the regular course of division from the fertilized egg, one-half of the chromosojnes are of strictly pater^ial origin, the

Boveri's hypothesis has been criticised by many writers, especially by Hertwig, Guignard, and Brauer, and I myself have urged some objections to it. Recently, however, it has received a support so strong as to amount almost to a demonstration, through the remark- able observations of Ruckert, Hacker, Herla, and Zoja on the independence of the paternal and maternal chromosomes. These observations, already referred to at page 208, may be more fully re- view^ed at this point. Hacker ('92, 2) first showed that in Cyclops stre7iiiiis, as in Ascaris and other forms, the germ-nuclei do not fuse but give rise to two separate groups of chromosomes that lie side by side near the equator of the cleavage-spindle. In the two-cell stage (of Cyclops tciiiiicornis) each nucleus consists of two distinct thou^'-h closely united halves, which Hacker believed to be the derivatives of the two respective germ-nuclei. The truth of this surmise was demon- strated three years later by Ruckert ('95, 3) in a species of Cyclops, likewise identified as C. strcimiis (Fig. 146). The number of chromo- somes in each germ-nucleus is here twelve. Ruckert was able to trace the paternal and maternal groups of daughter-chromosomes not only into the respective halves of the daughter-nuclei of the two-cell stage, but into later cleavage -stages. From the bilobed nuclei of the two-cell stage arise, in each cell, a double spireme and a double group of chromosomes, from which are formed bilobed or double nuclei in the four-cell stage. This process is repeated at the ne.xt cleavage, and the double character of the nuclei was in nian\- cases distinctly recognizable at a late stage when the germ-layers were being formed.

Finally Victor Herla's ('93) and Zoja's ('95, 2) remarkable obser- vations on Ascaris showed that in Ascaris not only the chromatin of the germ-nuclei, but also the paternal and maternal cliroviosonics, remain perfectly distinct as far as the twelve-cell stage — certainly a brilliant confirmation of Boveri's conclusion. Just how far the dis- tinction is maintained is still uncertain, but Hacker's and Riickert's observations give some ground to believe that it may ])ersist through- out the entire life of the embryo. Both these observers have shown that the chromosomes of the germinal vesicle appear in ti^'o (lisfinct groups, and Ruckert suggests that these may represent the paternal and maternal elements that have remained distinct throughout the entire cycle of development, even down to the formation of the (t^^'g !

Leaving aside all doubtful cases (such as the above suggestion of Riickert's), the well-determined facts form an irresistible proof of the general hypothesis ; and it is one with which every general analysis of the cell has to reckon. I beheve, however, that the hypothesis has received an unfortunate name ; for, except in a few special cases,^ almost no direct evidence exists to show that the chromosomes persist as *' individuals " in the chromatin-reticulum of the resting cell. The facts indicate, on the contrary, that in the vast majority of cases the identity of the chromosomes is wholly lost in the resting nucleus, and the attempts to identify them through the polarity or other morpho- logical features of the nuclear network have on the whole been futile. It is therefore an abuse of language to speak of a persistent ''individ-

Fig. 147. — Hybrid fertilization of the egg oi As car is megalocephala, var. bivalens, by the sper- matozoon of van z^«/^'a/^«i'. [Herla.] A. The germ-nuclei shortly before union. B. The cleavage-figure forming; the sperm-nucleus has given rise to one chromosome (J'), the egg-nucleus to two (9). C. Two-cell stage dividing, showing the three chromosomes in each cell. D. Twelve-cell stage, with the three distinct chro- mosomes still shown in the primordial germ-cell or stem-cell.

uality " of chromosomes. But this verbal difficulty should not blind us to the extraordinary interest and significance of the facts. It is difficult to suppose that the tendency of the chromatin to resolve itself into a particular number of chromosomes is directly due to its chemical or molecular structure, or is analogous to crystallization ; for in the chromatin of the same species, or even in that of the same ^^%^ this tendency varies, not with chemical, but with purely morphological

conditions, i.e. with the number of chromosomes that enter the nucleus. Neither can we assume that it is due merely to the total mass of the chromatin in each case ; for this varies in different nuclei of the same species, or even in the nucleus of the same cell at dif- ferent periods (as in the egg-cell), yet the same number of chromo- somes is characteristic of all. Indeed, we seek in vain for an analogy to these phenomena and can only admit our entire inability to explain them. No phenomena in the history of the cell more clearly indicate the existence of a morphological organization which, though resting upon, is not to be confounded with, the chemical and molecular structure that underlies it ; and this remains true even though we are wholly ignorant what that organization is.

{b^ Coviposition of the CJiromosonies. — We owe to Roux ^ the first clear formulation of the view that the chromosomes, or the chr(jmatin- thread, consist of successive regions or elements that are qualitatively different (p. 244). This hypothesis, which has been accepted by Weismann, Strasburger, and a number of others, lends a peculiar interest to the morphological composition of the chromatic substance. The facts are now well estabhshed (i ) that in a large number of cases the chromatin-thread consists of a series of granules (chromomeres) embedded in and held together by the linin-substance, (2) that the splitting of the chromosomes is caused by the division of these more elementary bodies, (3) that the chromatin-grains may divide at a time when the spireme is only just beginning to emerge from the reticulum of the resting nucleus. These facts point unmistakably to the conclu- sion that these granules are perhaps^ to be regarded as independent morphological elements of a lower grade than the chromosomes. That they are not artifacts or coagulation-products is proved by their uniform size and regular arrangement in the thread, especially when the thread is split. A decisive test of their morphological nature is, however, even more difficult than in the case of the chromosomes ; for the chromatin-grains often become apparently fused together so that the chromatin-thread appears perfectly homogeneous, and whether they lose their individuality in this close union is undetermined. Observations on their number are still very scanty, but they point to some very interesting conclusions. In Boveri's figures of the egg- maturation of Ascaris each element of the tetrad consists of six chro- matin-discs arranged in a linear series (Van Beneden's figures of the same object show at most five) which finally fuse to form an appar- ently homogeneous body.

In the chromosomes of the germ-nuclei the number is at least double this (Van Beneden). Their number has been more carefully followed out in the spermatogenesis of the same animal (variety bivalens) by Brauer. At the time the chromatin-grains divide, in the reticulum of the spermatocyte-nucleus, they are very numerous. His figures of the spireme-thread show at first nearly forty granules in linear series (Fig. 120, />). Just before the breaking of the thread into two the number is reduced to ten or twelve (Fig. 120, C). Just after the division to form the two tetrads the number is four or five (Fig. 120, Z>), which finally fuse into a homogeneous body.^

It is certain, therefore, that the number of chromomeres is not con- stant in a given species, but it is a significant fact that in Ascaris the final number, before fusion, appears to be nearly the same (four to six) both in the oogenesis and the spermatogenesis. The facts re- garding bivalent and plurivalent chromosomes (p. '^j) at once sug- gest themselves, and one cannot avoid the thought that the smallest chromatin-grains may successively group themselves in larger and larsfer combinations of which the final term is the chromosome. Whether these combinations are to be regarded as ** individuals " is a question which can only lead to a barren play of words. The fact that cannot be escaped is that the history of the chromatin-substance reveals to us, not a homogeneous substance, but a definite morpho- logical organization in which, as through an inverted telescope, we behold a series of more and more elementary groups, the last visible term of which is the smallest chromatin-granule, or nuclear microsome, beyond which our present optical appliances do not allow us to see. Are these the ultimate dividing units, as Brauer suggests (p. 113)? Here again we may well recall Strasburger's warning, and hesitate to identify the end of the series with the Hmits reached by our best lenses. Somewhere, however, the series must end in final chromatic units which cannot be further subdivided without the decomposition of chromatin into simpler chemical substances ; and these units must be capable of assimilation, growth, and division without loss of their specific character. It is in these ultimate units that we must seek the ** qualities," if they exist, postulated in Roux's hypothesis; but the existence of such qualitative differences is a physiological assumption that in no manner prejudices our conclusion regarding the ultimate vioi'pJiological composition of the chromatin.

What, now, is the relation of the chromatin-grains to the linin-net- work and the cytoplasm } Van Beneden long ago maintained ^ that 1 Eisen ('99) finds that the chromosomes of the spermatogonia of Batrachoseps always consist of six "chromomeres," each of which consists of three smaller granules or " chromi- oles." The latter persist as the chromatin-granules of the resting nucleus; and it is through their successive aggregation that the chromumeres and chromosomes are formed.

the achromatic network, the nuclear membrane, and the cell-mesh- work have essentially the same structure, all consisting of microsomes united by connective substance, and being only " parts of one and the same structure." But, more than this, he asserted that the chromatic and acJiroinatic 7nicrosomcs might be transformed into 07ie another, and were therefore of essentially the same morphological nature. " They pass successively, in the course of the nuclear evolution, through a chromatic or an achromatic stage, according as they imbibe or *^nve off the chromophilous substance." ^ Both these conclusions are borne out by recent researches. Heidenhain ('93, '94), confirmed by Reinke and Schloter, finds that the nuclear network contains granules of two kinds differing in their staining-capacity. The first are the basi-chro- matin granules, which stain with the true nuclear dyes (basic tar-col- ours, etc.), and are identical with the '' chromatin-granules " of other authors. The second are the oxychromatin-granules of the linin-net- work, which stain with the plasma-stains (acid colours, etc.), and are closely similar to those of the cytoreticulum. These two forms gradu- ate into one ajiother, and are conjectured to be diffeirnt phases of the same elements. This conception is furthermore supported by many observations on the behaviour of the nuclear network as a whole. The chromatic substance is known to undergo very great changes in staining-capacity at different periods in the life of the nucleus (p. ll^\ and is known to vary greatly in bulk. In certain cases a very large amount of the original chromatic network is cast out of the nucleus at the time of the division, and is converted into cytoplasm.

And, finally, in studying mitosis in sea-urchin eggs I found reason to con- clude ('95, 2) that a considerable part of the linin-network, from which the spindle-fibres are formed, is actually derived from the chromatin. From the time of the earlier writings of Frommann ('65, '67), Arnold {^^j\ Heitzmann ('73), and Klein {^J^\ down to the present, an increasing number of observers have held that the nuclear reticu- lum is to be conceived as a modification of the same structural basis as that which forms the cytoplasm. The latest researches indicate, indeed, that true chromatin (nuclein) is confined to the nucleus.- I^ut the whole weight of the evidence now goes to show that the linin- network is of the same nature as the cell-mesh work, and that the achromatic nuclear membrane is formed as a condensation of the same substance. Many investigators, among whom may be named From- mann, Leydig, Klein, Van Beneden, Carnoy, and Reinke, have de- scribed the fibres of both the intra- and extra-nuclear network as terminating in the nuclear membrane ; and the membrane itself is described by these and other observers as being itself reticular in structure, and by some (Van Beneden) as consisting of closely crowded

microsomes arranged in a network. The clearest evidence is, however, afforded by the origin of the spindle-fibres in mitotic division ; for it is now well established that these may be formed either inside or out- side the nucleus, and at the close of mitosis the central portion of the spindle appears always to give rise to a portion of the cytoplasm lying between the daughter-nuclei. In such a case as that of the sea-urchin (see above) we have, therefore, evidence of a direct trans- formation of chromatin into linin-substance, of the latter into spindle- fibres, and, finally, of these into cytoplasm.