The Cell in Development and Inheritance

Again, Boveri has observed that the segmenting ovum of Ascaris sometimes contains a supernumerary centrosome that does not enter Fig. 165. — Kggs oi Ascaris with supernumerary centrosome. [buVERl.] A. First cleavage-spindle above, isolated centrosome below. B. Result of the ensuing division. into connection with the chromosomes, but lies alone in the cytoplasm (Fig. 165). Such a centrosome forms an independent centre of divi- sion, the cell dividing into three parts, two of which are normal blastomeres, while the third contains only the centrosome and attrac- tion-sphere. The fate of such eggs was not determined, but they form a complete demonstration that it is in this case the centrosome and not the nucleus that determines the centres of division in the cell-body. Scarcely less conclusive is the case of dispermic eggs in sea-urchins. In such eggs both sperm-nuclei conjugate with the egg- nucleus, and both sperm-centrosomes divide (Fig. 166). The cleavage-nucleus, therefore, arises by the union of three nuclei and four centrosomes. Such eggs divide at the first cleavage into four equal blastomeres, each of which receives one of the centrosomes.

The latter must, therefore, be the centres of division ; ^ though it must not be forgotten that, in some cases at any rate, normal division requires the presence of nuclear matter (p. io8). The centrosome must, however, be something more than a mere division-centre ; for, on the one hand, in leucocytes and pigment-cells the astral system formed about it is devoted, as there is good reason to believe, not to cell-division, but to movements of the cell-body as a whole; and, on the other hand, as we have seen (pp. 165, 172), it is concerned in the formation of the flagella of the spermatozoa and spermatozoids, and probably also in that of cilia in epithelial cells. Strasburger ('97) was thus led to the conclusion that the centrosome is essentially a mass of kinoplasm, i.e. the active motor plasm, ^ and a nearly similar view has been adopted by several recent zoologists.

A. One sperm-nucleus has united with the egg-nucleus, shown at a. b. ; the other Hes above. Both sperm-asters have divided to form amphiasters (a. b. and c. d.). B. The cleavage-nucleus, formed by union of the three germ-nuclei, is surrounded by the four asters. C. Result of the first cleavage, the four blastomeres lettered to correspond with the four asters. Hennecfuv concludes that the centrosomes are " motor centres of the kinoplasm " both for external and for internal manifestations.'^ Lenhossek regards them as " motors " for the control of ciliary action as well as for that of the spermatozoon,"* and perhaps also for that of muscle-fibrillDe.^ Zimmerman concludes that *' the microcentrum is the motor centre of the cell, that is, the * kinocentrum ' opposed to the nucleus as the * chemocentrum.'" ^ Regarding their control of ciliary action, he makes the same suggestion as that of Henneguy and Lenhossek cited above. He adds the further very interesting sug- gestions that the centrosomes may be concerned with the pseudopodial movements in the epithelial cells of the intestine, and that they may

1 This phenomenon was first observed by Ilertwig, and afterward by Driesch. I have repeatedly observed the internal changes in the living eggs of Toxopneustes. also be concerned in the protoplasmic contraction of gland-cells by which the excretion is expelled. [This is based on the fact that the centrosomes are found in the free (pseudopodia-forming) ends of the epitheUal cells, and on the position of the centrosomes in goblet- cells (Fig. 23) and in those of the lachrymal gland.] Peter ('99) has attempted to test these conclusions experimentally by cuttino- or tear- ing off cilia from the cell-body (gut-epitheUum of Aiiodonta) and also by isolating the tails of spermatozoa. In groups consisting of only a few cilia, separated from the nucleus, the movements actively con- tinue, while those that are separated from the basal bodies cease to beat. Spermatozoon tails separated from the head also continue to

Fig. 167. — Centrosomes and cilia in spermatocytes of a butterfly. [Henneguy.] move, but only if they remain connected with the middle-piece. Peter, therefore, supports the above conclusions of Henneguy and Lenhossek. On the other hand, Meves ('99) finds that movements of the undulating membrane in the tails of salamander-spermatozoa continue if the middle-piece be entirely removed; while a number of earher observers^ have observed in flagellates that a flagellum separated from the body may actively continue its movements for a considerable time.

Further research is therefore required to test these suggestions. The intimate connection of the centrosomes with the formation, on the one hand, of the astral rays, on the other of contractile organs, such as cilia, flagella, and pseudopodia,^ the centrosomes in ciliated cells and spermatozoa, and in the swarm-spores of Noctiluca, is, however, a most striking fact, and is one of the strongest indirect arguments in favour of the general theory of fibrillar contractility in mitosis.

The facts reviewed in the foregoing pages converge to the conclu- sion that the differentiation of the cell-substance into nucleus and cytoplasm is the expression of a fundamental physiological division of labour in the cell. Experiments upon unicellular forms demonstrate that, in the entire absence of a nucleus, protoplasm is able for a considerable time to liberate energy and to manifest coordinated activities dependent on destructive metabolism. There is here sub- stantial ground for the view that the cytoplasm is the principal seat of these activities in the normal cell. On the other hand, there is strong cumulative evidence that the nucleus is intimately concerned in the constructive or synthetic processes, whether chemical or morphological.

That the nucleus has such a significance in synthetic metaboHsm is proved by the fact that digestion and absorption of food and growth soon cease with its removal from the cytoplasm, while destruc- tive metabolism may long continue as manifested by the phenomena of irritability and contractiHty. It is indicated by the position and movements of the nucleus in relation to the food-supply and to the formation of specific cytoplasmic products. It harmonizes with the fact, now universally admitted, that active exchanges of material go on between nucleus and cytoplasm. The periodic changes of staining-capacity undergone by the chromatin during the cycle of cell- life, taken in connection with the researches of physiological chemists on the chemical composition and staining-reactions of the nuclein series, indicate that the phosphorus-rich substance known as nucleinic acid plays a leading part in the constructive process. During the vegetative phases of the cell this substance is combined with a large amount of the albumin radicles histon, protamin, and related sub- stances, and probably in part with albumin itself, to form nuclein. During the mitotic or reproductive processes this combination appears to be dissolved, the albuminous elements being in large part split off, leaving the substance of the chromosomes with a high percentage of nucleinic acid, as is shown by direct analysis of the sperm-nucleus and is indicated by the staining-reactions of the chromosomes. There is, therefore, considerable ground for the hypothesis that in a chemi- cal sense this substance is the most essential nuclear element handed

on from cell to cell, whether by cell-division or by fertilization ; and that it may be a primary factor in the constructive processes of the nucleus and through these be indirectly concerned with those of the cytoplasm. The role of the nucleus in constructive metabolism is intimately related with its role in morphological synthesis, and thus in inheri- tance ; for the recurrence of similar morphological characters must in the last analysis be due to the recurrence of corresponding forms of metabohc action of which they are the outward expression. That the nucleus is in fact a primary factor in morphological as well as chemi- cal synthesis is demonstrated by experiments on unicellular plants and animals, which prove that the power of regenerating lost parts disap- pears with its removal, though the enucleated fragment may continue to live and move for a considerable period. That the nuclear sub- stance, and especially the chromatin, is a leading factor in inheritance is powerfully supported by the facts of maturation, fertilization, and cell-division. In maturation the germ-nuclei are by an elaborate process prepared for the subsequent union of equivalent chromatic elements from the two sexes. By fertilization these elements are brought together, and by mitotic division distributed with exact equal- ity to the embryonic cells. The result, which is especially striking in the case of hybrid-fertilization, proves that the spermatozoon is as potent in inheritance as the ovum, though the latter contributes an amount of cytoplasm which is but an infinitesimal fraction of that supplied by the ovum.

It remains to be seen whether the chromatin can actually be re- garded as the idioplasm or physical basis of inheritance, as maintained by Hertwig and Strasburger. Verworn has justly urged that the nucleus cannot be regarded as the sole vehicle of inheritance, since the cooperation of both nucleus and cytoplasm is essential to com- plete cell-life; and, as will be shown in Chapter IX., the cytoplasmic organization plays an important role in shaping the course of devel- opment. Considered in all their bearings, however, the facts seem to accord best with the hypothesis that the cytoplasmic organization is itself determined, in the last analysis, by the nucleus ; ^ and the principle for which Hertwig and Strasburger contended is thus sus- tained.

Bernard, Claude. — Lepons sur les Phenomenes de la \'ie : ist ed. 1878: 2d ed. 1885. Paris. Chittenden, R. H. — Some Recent Chemico-phvsiological Discoveries regarding tlie Haberlandt, G. — tjber die Beziehungen zwischen Funktion und Lage des Zellkerns. Fi seller, 1887. Id. — Physiologische Pflanzenatomie. Leipzig, 1896. Halliburton, W. D. — A Text-book of Cliemical Physiology and Pathology. London^ Id. — The Chemical Physiology of the Cell (^Gouldstonian Lectures): B?'it. Med.

Joitrn. 1893. Hammarsten, 0. — Lehrbuch der physiologische Chemie. 3d ed. Wiesbaden, 1895. Hertwig, 0. and R. — Uber den Befruchtungs- und Teilungsvorgang des tierischen Eies unter dem Einfluss ausserer Agentien. Jena, 1887. Kolliker, A. — Das Karyoplasma und die \'ererbung, eine Kritik der Weismann'schen Theorie von der Kontinuitat des Keimplasmas : Zeitschr. iviss. ZooL, XLIV. Korschelt, E. — Beitrage sur Morphologic und Physiologic des Zellkernes : Zool.

JaJirb. Atiat. it. Oniog., IV. 1889. Kossel, A. — Uber die chemische Zusammensetzung der Zelle : Arch. Anat. u. P/iys. Id. — Uber die basischen Stoffe des Zellkernes: ZeiL P/iys. C/ieni., XXII., 1896. Lilienfeld, L. — Uber die Wahlverwandtschaft der Zellelemente zu Farbstoffen : Arch. Anat. n. Phys. 1893. Malfatti, H. — Beitrage zur Kenntniss der Nucleine : Zeitschr. Phys. Cheju., XVI. Mathews, A. P. — The Metabolism of the Pancreas Cell : Journ. Morph., XV. SuppL

Miescher, F. — Physiologisch-chemische Untersuchungen liber die Lachsmilch : Arch. Exp. Path. u'. Pharm., XXXVII., 1896. Prenant, A. — See Literature VI. Riickert, J. — Zur Entwicklungsgeschichte des Ovarialeies bei Selachiern : An. Anz., Sachs, J. — Vorlesungen liber Pflanzen-physiologie. Leipzig, 1882. Id. — Stofif und Form der Pflanzen-organe : Gesanimelte Abhandlnngen, II. 1893. Strasburger. — See footnote, p. 269. Verworn, M. — Die Physiologische Bedeutung des Zellkerns : Arch, fur die Ges.

Zacharias, E. — Uber des Verhalten des Zellkerns in wachsenden Zellen : Flora, 81. " Wir konnen demnach endlich den Satz aufstellen, dass samnitliche im cntwickelten Zustande vorhandenen Zellen oder Aequivalente von Zellen durch eine f(jrtschreitende Gliederung der Eizelle in morphologisch ahnliche Elemente entstehen, und dass die in einer embryonischen Organ-Anlage enthaltenden Zellen, so gering auch ihre Zahl scin mag, dennoch die ausschliessliche ungegliederte Anlage fiir sammtliche Formbestandtheile der spateren Organe enthalten," Remak.^

Since the early work of Kolliker and Remak it has been recog- nized that the cleavage or segmentation of the ovum, with which the development of all higher animals begins, is nothing other than a rapid series of mitotic cell-divisions by which the Qgg splits up into the elements of the tissues. This process is merely a contin- uation of that by which the germ-cell arose in the parental body. A long pause, however, intervenes during the latter period of its ovarian life, during which no divisions take place. Throughout this period the egg leads, on the whole, a somewhat passive existence, devoting itself especially to the storage of potential energy to be used during the intense activity that is to come. Its power of division remains dormant until the period of full maturity approaches. The entrance of the spermatozoon arouses in the egg a new phase of activity. Its power of division, which may have lain dormant for months or years, is suddenly raised to the highest pitch of intensity, and in a very short time it gives rise by division to a myriad of de- scendants which are ultimately differentiated into the elements of the tissues.

The divisions of the egg during cleavage are exactly comparable with those of tissue-cells, and all of the essential phenomena of mitosis are of the same general character in both. But for two reasons the cleavage of the egg possesses a higher interest than any other case of cell-division. First, the egg-cell gives rise by divi- sion not only to cells like itself, as is the case with most tissue-cells, but also to many other kinds of cells. The operation of cleavage is therefore immediately connected with the process of differentiation, which is the most fundamental phenomenon in development. Second, definite relations may often be traced between the planes of division and the structural axes of the adult body, and these relations are

sometimes so clearly marked and appear so early that with the very first cleavage the position in which the embryo will finally appear in the ^g% may be exactly predicted. Such *' promorphological " rela- tions of the segmenting ^^^ possess a very high interest in their bearing on the theory of germinal localization and on account of the light which they throw on the conditions of the formative process. The present chapter is in the main a prelude to that which follows, its purpose being to sketch some of the external features of early development regarded as particular expressions of the gen- eral rules of cell-division. For this purpose we may consider the cleavage of the ovum under two heads, namely : — -

1. TJie Geometrical Relations of Cleavage-forms, with reference to the general rules of cell-division. 2. TJie Promoi'p ho logical Relations of the blastomeres and cleav- age-planes to the parts of the adult body to which they give rise. The geometrical relations of the cleavage-planes and the relative size and position of the cells vary endlessly in detail, being modified by innumerable mechanical and other conditions, such as the amount and distribution of the inert yolk or deutoplasm, the shape of the ovum as a whole, and the like. Yet all the forms of cleavage can be referred to a single type which has been moulded this way or that by special conditions, and which is itself an expression of two general rules of cell-division, first formulated by Sachs in the case of plant- cells. These are : —

2. Each new plane of division tends to intersect the pi'cce ding plane at a right angle. In the simplest and least modified forms the direction of the cleavage-planes, and hence the general configuration of the cell- system, depends on the general form of the dividing mass ; for, as Sachs has shown, the cleavage-planes tend to be either vertical to the surface {anticlines) or parallel to it {periclines). Ideal schemes of division may thus be constructed for various geometrical figures. In a flat circular disc, for example, the anticlinal planes pass through the radii; the periclines are circles concentric with the periphery. If the disc be elongated to form an ellipse, the periclines also become ellipses, while the anticlines are converted into hyperbolas confocal with the periclines. If it have the form of a parabola, the periclines and anticlines form two systems of confocal parabolas intersecting at right angles. All these schemes are mutatis mutandis, directly con- vertible into the corresponding solid forms in three dimensions.

Sachs has shown in the most beautiful manner that all the above ideal types are closely approximated in nature, and Rauber has applied the same principle to the cleavage of animal-cells. The discoid or spheroid form is more or less nearly realized in the thalloid growths of Fig. i68. — Geometrical relations of cleavage-planes in growing plant-tissues. [From f^AClls. after various authors.] A. Flat ellipsoidal germ-disc of Melobesia (Rosanoff) ; nearly typical relation of eiiiptic periclines and hyperbolic anticlines. B. C. Apical view of terminal knob on epidermal liair of Pinguicola. B. shows the ellipsoid type, C. the circular (spherical type), somewhat modified (only anticlines present). D. Growing point of Salvhiia (Pringsheim), typical ellipsoid type; the single pericline is, however, incomplete. E. Growing point of Azolla (Strasburger) ; circular or spheroidal type transitional to ellipsoidal. F. Root-cap of E(]uisrtinn (Xageli and Leitgeb) ; modified circular type. G. Cross-section of leaf-vein, Trichowanes (Prantl) ; ellipsoidal type with incomplete periclines. H. Embryo oi Alisma ; typical elliyisoid type, pericline incomplete only at lower side. /. Growing point of bud of the pine {AHes) ; typical paraboloid type, both anti- clines and periclines having the form of parabolas (Sachs).

various lower plants, in the embryos of flowering plants, and else- where (Fig. 1 68). The paraboloid form is according to Sachs charac- teristic of the growing points of many higher plants ; and here, too, the actual form is remarkably similar to the ideal scheme (Fig. i68, /). For our purpose the most important form is the sphere, which is the typical shape of the egg-cell ; and all forms of cleavage may be related to the typical division of a sphere in accordance with Sachs's rules. The ideal form of cleavage would here be a succession of rectangular cleavages in the three dimensions of space, the anticlines passing through the centre so as to split the ^gg in the initial stages successively into halves, quadrants, and octants, the periclines being parallel to the surface so as to separate the inner ends of these cells from the outer. No case is known in which this order is accurately followed throughout, and the periclinal cleavages are of compara- tively rare occurrence, being found as a regular feature of the early cleavage only in those cases where the primary germ-layers are sepa- rated by delamination. The simplest and clearest form of egg- cleavage occurs in eggs like those of echinoderms, which are of spherical form, and in which the deutoplasm is small in amount and equally distributed through its substance. Such a cleavage is beauti- fully displayed in the ^gg of the holothurian Synapta, as shown in the diagrams, Fig. 169, constructed from Selenka's drawings. The first cleavage is vertical, or meridional, passing through the egg-axis and dividing the ^gg into equal halves. The second, which is also meridional, cuts the first plane at right angles and divides the ^gg into quadrants. The third is horizontal, or equatorial , dividing the ^gg into equal octants. The order of division is thus far exactly that demanded by Sachs's rule and agrees precisely with the cleavage of various kinds of spherical plant-cells.

The later cleavages depart from the ideal type in the absence of periclinal divisions, the embryo becoming hollow, and its walls consisting of a single layer of cells in which anticHnal cleavages occur in regular rectangular succession. The fourth cleavage is again meridional, giving two tiers of eight cells each ; the fifth is horizontal, dividing each tier into an upper and a lower layer. The regular alternation is continued up to the ninth division (giving 512 cells), when the divisions pause while the gastrulation begins. In later stages the regularity is lost.

Hertwig s Development of Sachs's Rules. — Beside Sachs's rules may be placed two others formulated by Oscar Hertwig in 1884, which bear directly on the facts just outHned and which lie behind Sachs's principle of the rectangular intersection of successive division- planes. These are : — 1 . The nucleus tends to take np a position at the centre of its sphere of influence, i.e. of the protoplasmic mass in zvhich it lies. 2. The axis of the 7nitotic figtires typically lies in the longest axis of the protoplasmic mass, and division therefore tends to cut this axis at a right a^tgle.

The second rule explains the normal succession of the division- planes according to Sachs's second rule. The first division of a homo- geneous spherical ^^^^, for example, is followed by a second division at right angles to it, since each hemisphere is twice as Ion- in the plane of division as in any plane vertical to it. The mitotic fi-ure of the second division lies therefore parallel to the first plane which forms the base of the hemisphere, and the ensuing division is vertical to It. The same applies to the third division, since each quadrant is as long as the entire ^gg while at most only half its diameter. Divi- sion is therefore transverse to the long axis and vertical to the first two planes.

Taken together the rules of Sachs and Hertwig, applied to the ^gg, give us a kind of ideal type or model, well illustrated by the Fig. 169. — Cleavage of the ovum in the \\o\o\h\xx\^Xi Synapta (slightly schematized) f After Selenka.] '■ A-E. Successive cleavages to the 32-cell stage. F. Blastula of 128 cells. cleavage of Synapta, described above, to which all the forms of cleav- age may conveniently be referred as a basis of comparison. Numer- ous exceptions to all four of these rules are, however, known, and they are of little value save as a starting-point for a closer study of the facts. Cleavage of such schematic regularity as that of Synapta is extremely rare, both the form and the order of division being end- lessly varied and in extreme cases showing scarcely a discoverable connection with the ''type." We may conveniently consider these modifications under the following three heads : —

2. Displacement of the cells {iticlnding variations in the direction of cleavage). Nothing is more common than a departure from the regular rhythm of division. The variations are sometimes quite irregular, sometimes follow a definite rule, as, for instance, in the annelid N'eiris (Fig. 171), where the typical succession in the number of cells is with great constancy 2, 4, 8, 16, 20, 23, 29, 32, 37, 38, 41, 42, after which the order is more or less variable. The factors that determine such variations in the rhythm of division are very little understood. Bal- four, one of the first to consider the subject, sought an explanation in the varying distribution of metaplasmic substances, maintaining ('75, '80) that the rapidity of division in any part of the ovum is in general inversely proportional to the amount of deutoplasm that it contains. The entire inadequacy of this view has been demonstrated by a long series of precise studies on cell-lineage, which show that while the large deutoplasm-bearing cells often do divide more slowly than the smaller protoplasmic ones the reverse is often the case, while remarkable differences in the rhythm of division are often observed in cells which do not perceptibly differ in metaplasmic content.^ All the evidence indicates that the rhythm of division is at bottom deter- mined by factors of a very complex character which cannot be disentangled from those which control growth in general. LilHe ('95, '99) points out the very interesting fact, determined through an analysis of the cell-lineage of mollusks and annelids, that the rate of cleavage shows a direct relation to the period at which the prod- ucts become functional. Thus in Unio the more rapid cleavage of a certain large cell (" d.

2"), formed at the fourth cleavage, is obviously correlated with the early formation of the shell-gland to which it gives rise, while the relatively slow rate of division in the first ectomere- quartet is correlated with reduction of the prae-trochal region. The prospective character shown here w^ill be found to apply also to other characters of cleavage, as described beyond. When we turn to the factors that determine the direction of cleav- age or the displacement of cells subsequent to division, we find, as in the case of the division-rhythm, obvious mechanical factors com- bined with others far more complex. The arrangement of tissue-cells usually tends toward that of least resistance or greatest economy of space ; and in this regard they have been shown to conform, broadly speaking, with the behaviour of elastic spheres, such as soap-bubbles when massed together and free to move. Such bodies, as Plateau

and Lamarle have shown, assume a pol^^hedral form and tend toward such an arrangement that the area of surfacccontact /v '-. // a nun^uucn^. Spheres in a mass thus tend o aTsume The £^ Z mterlockm. polyhedrons so arranged that three pCsLterc in a hne, whde four Imes and six planes meet at a point If a ran .cd in a smgle layer on an extended surface, they a'sume the "."'0' hexagonal prisms, three planes meeting along a line as before. I^oth these forms are commonly shown in the arrangement of the cells of plant and animal tissues; and Berthold {'^^6) and Errcra {'^6, '%j) carefully analyzing the phenomena, have endeavoured to show that not only the form and relative position of cells, but also the direction of cell-division, is, partially at least, thus determined.

It is through displacements of the cells of this type that many of the most frequent modifications of cleavage arise. Sometimes, as in Synapta, the alternation of the cells is effected through displacement of the blastomeres after their formation. More commonly it arises during the division of the cells, and may even be predetermined by the position of the mitotic figures before the slightest external sign of division. Thus arises that form of cleavage known as the spiral, oblique, or alternating type, where the blastomeres interlock during their formation and lie in the position of least resistance from the beginning. This form of cleavage, especially characteristic of many worms and mollusks, is typically shown by the ^^^ of Polygordius (Fig. 170). The four-celled stage is nearly like that of Syiiapta, though even here the cells slightly interlock. The third division is, however, oblique, the four upper cells being virtually rotated to the right (with the hands of a watch) so as to alternate with the four lower ones. The fourth cleavage is likewise oblique, but at right angles to the third, so that all of the cells interlock as shown in Fig. 170, D. This alternation regularly recurs for a considerable period.