The Cell in Development and Inheritance

In many worms and mollusks the obliquity of cleavage appears still earlier, at the second cleavage, the four cells being so arranged that two of them meet along a ''cross-furrow" at the lower pole of the ^gg, while the other two meet at the upper pole along a similar, though often shorter, cross-furrow at right angles to the lower {e.g. in Nereis, Fig. 171). It is a curious fact that the direction of the dis- placement is quite constant, the first or upper quartet in the eight- cell stage being rotated to the right, or with the hands of a watch, the second quartet to the left, the third to the right, and so on. Crampton ('94) has discovered the remarkable fact that in P/iysa, a gasteropod having a reversed or sinistral shell, the whole order of displacement is likewise reversed, and the same has recently been shown by Holmes ('99) to be true of Aiicylus.

The spiral or alternating type of cleavage beautifully illustrates Sachs's second rule as affected by modifying conditions ; for, as may be seen by an inspection of Figs. 170, 171, each division-plane is approximately at right angles to the preceding and succeeding (whence the " alternation . of the spirals" described by students of cell-lineage), while they are so directed that each cell as it is formed is placed at once in the position of least resistance in the mass, i.e. in the position of minimal surface-contact. It is impossible to resist the conclusion that one of the factors by which the position of the cells (and hence the direction of cell-division) is determined is a purely mechanical one, identical with that which determines the arrangement of soap-bubbles and the like.

Very little acquaintance with the facts of development is however required to show that this purely mechanical factor, though doubtless real, must be subordinate to some other. This is strikingly shown, for example, in the development of annelids and mollusks, where the spiral cleavage, strictly maintained during the earlier stages, finally gives way more or less completely to a bilateral type of division in which the rule of minimal surface-contact is often violated. We see here a tendency operating directly against, and finally overcoming,

Fig. 171. — Cleavage oi Nereis. An example of a spiral cleavage, unequal from tlie beginning and of a marked determinate character. ^.Two-cell stage (the circles are oil-drops). B. Four-cell stage ; the second cleavage-plane passes through the future median plane. C. The same from the right side. D. Eight-cell stage. E. Sixteen cells; from the cells marked / arises the prototroch or larval ciliated belt, from X the ventral nerve-cord and other structures, from D the mcsoblast-bands, the germ-cells, and a part of the alimentary canal. F. Twenty-nine-cell stage, from the right side ; /. girdle of prototrochal cells which give rise to the ciliated belt.

the mechanical factor which predominates in the earlier stages ; and in some cases, e.g. in the ^g% of Clavclina (Fig. 177) and other tuni- cates, this tendency predominates from the beginning. In both these cases this *' tendency " is obviously related to the growth-jirocess to which the future bilateral embryo will owe its form ; ^ and every attempt to explain the position of the cells and the direction of cleav- age must reckon with the morphogenic process taken as a whole. The blastomere is not merely a cell dividing under the stress of rude

mechanical conditions ; it is beyond this " a builder which lays one stone here, another there, each of which is placed with reference to future development."^ The third class of modifications, due to unequal division of the cells, not only leads to the most extreme types of cleavage but also to its Fig. 172. — The eight-cell stage of four different animals showing gradations in the inequality of the third cleavage. A. The leech Clepsine (Whitman). B. The oh^Xo'^o^ Rhynchelmis (Vejdovsky). C. The lamellibranch Unto (Lillie). D. Amphioxus.

most difficult problems. Unequal divisions appear sooner or later in all forms of cleavage, the perfect equality so long maintained in Synapta being a rare phenomenon. The period at which the in- equality first appears varies greatly in different forms. In Polygordius (Fig. 170) the first marked inequality appears at the fifth cleavage; in sea-urchins it appears at the fourth (Fig. 3); in AmpJiioxus at the third (Fig. 172); in the tunicate Clavclina at the second ( Fig. 177); in Nereis at the first division (Figs. 60, 171). The extent of the in- equality varies in like manner. Taking the third cleavage as a type, we may trace every transition from an equal division (echinoderms, Polygordiiis), through forms in which it is but slightly marked {Avi- phioxns, frog), those in which it is conspicuous {Nereis, Lymniea, poly- clades, Petroinyzon, etc.), to forms such as Clepsine, where the cells of the upper quartet are so minute as to appear Hke mere buds from the four large lower cells (Fig. 172). At the extreme of the series we reach the partial or meroblastic cleavage, such as occurs in the ceph- alopods, in many fishes, and in birds and reptiles. Here the lower hemisphere of the ^gg does not divide at all, or only at a late period, segmentation being confined to a disc-hke region or blastoderm at one pole of the ^gg (Fig. 173).

Very interesting is the case of the teloblasts or pole-cells character- istic of the development of many annelids and mollusks and found in some arthropods. These remarkable cells are large blastomeres, set aside early in the development, which bud forth smaller cells in reg- ular succession at a fixed point, thus giving rise to long cords of cells (Fig. 175). The teloblasts are especially characteristic of apical growth, such as occurs in the elongation of the body in annelids, and they are closely analogous to the apical cells situated at the growing point in many plants, such as the ferns and stoneworts.

Still more suggestive is the formation of rudinientaiy cells, arising as minute buds from the larger blastomeres, and, in some cases, appar- ently taking no part in the formation of the embryo (Fig. 174).^ We are as far removed from an explanation of unequal division as from that of the rhythm and direction of division. Inequality of divi- sion, like difference of rhythm, is often correlated with inequalities in the distribution of metaplasmic substances — a fact generalized by Balfour in the statement ('80) that the size of the cells formed in cleavage varies inversely to the relative amount of protoplasm in the region of the ^gg from which they arise. Thus, in all telolecithal ova, where the deutoplasm is mainly stored in the lower or vegetative hemisphere, as in many worms, mollusks, and vertebrates, the cells of the upper or protoplasmic hemisphere are smaller than those of the lower, and may be distinguished as micrGineres from the larger viacro- meres of the lower hemisphere. The size-ratio between micromeres and macromeres is on the whole directly proportional to the ratio between protoplasm and deutoplasm. Partial or discoidal cleavage occurs when the mass of deutoplasm is so great as entirely to prevent cleavage in the lower hemisphere. This has been beautifully con-

firmed by O. Hertwig ('98), who, by placing frogs' eggs in a centrifu- gal machine, has caused them to undergo a meroblastic cleavage through the artificial accumulation of yolk at the lower ^^'ole, due to the centrifugal force. While doubtless containing an element of truth, this explanation is, however, no more adequate than Balfour's rule regarding the relation between deutoplasm and rhythm (p. 366); for innumerable cases are known in which no correlation can be made out between the distribu- tion of inert substance and the inequality of division. This is the case, for example, with the teloblasts mentioned above, which contain no deutoplasm, yet regularly divide unequally. It seems to be inap-

Fig. 173. — Partial or meroblastic cleavage in the squid LoUgo. [Watase.] plicable to the inequalities of the first two divisions in annelids and gasteropods. It is conspicuously inadequate in the history of indi- vidual blastomeres, where the history of division has been accurately determined. In Nereis, for example, a large cell known as the first somatoblast, formed at the fourth cleavage (X, Fig. 171, E\ under- goes an invariable order of division, three unequal divisions being fol- lowed by an equal one, then by three other unequal divisions, and again by an equal. This cell contains little or no deutoplasm and undergoes no perceptible changes of substance.

The collapse of the rule is most complete in case of the rudi- mentary cells referred to above. In some of the annelids, e.g. in Aricia, where they were first observed,^ these cells are derived from the very large primary mesoblast-cell, which first divides into equal halves. Each of these then buds forth a cell so small as to be no larger than a polar body, and then immediately proceeds to give rise to the mesoblast-bands by continued divisions, always in the same plane at right angles to that in which the rudimentary cells are formed (Fig. 174). The cause of the definite succession of equal and unequal divisions is here wholly unexplained. No less difficult is the extreme inequality of division involved in the formation of the polar bodies. We cannot explain this through the fact that deutoplasm is collected in the lower hemisphere ; for, on the one hand, the succeed- ing divisions (first cleavages) are often equal, while, on the other hand, the inequality is no less pronounced in eggs having equally

Fig, 174. — Rudimentary blastomeres in the embryo of an aiuK-liu. Aruta. A. From lower pole ; rudimentary cells at^. <?; the heavy outline is the lip of tli D. The same in sagittal optical section, showing rudimentary cell (f). piimnv im and mesoblast-band (w), distributed deutoplasm, or in those, like echinoderm-eggs, which are "alecithal." Such cases prove that Balfour's law is only a partial exi>lanati<m. being probably the expression of a more deeply lying cause, and :here is reason to believe that this cause lies outside the immediate mechanism of mitosis, Conklin (94) has called attention to the facti that the immediate cause of the inequality j^robably does not lie either in the nucleus or in the amphiaster ; tor not only the chromatin-halves, but also tlic asters, are exactly ecpial in the early prophases, and the inequality of the a.sters only appears as the division proceeds. Probably, therefore, the cause lies in some rela- tion between the mitotic figure and the cell-body in which it lies.

I believe there is reason to accept the conclusion that this relation is one of position, however caused. A central position of the mitotic Fig 175 - Embryos of the earthworm Allolobophora fc.tida, showing teloblasts or apical cells. fio-ure results in an equal division ; an eccentric position caused by a radial movement of the mitotic figure, in the direction of its axis toward the periphery, leads to unequal division, and the greater the eccentricity, the greater the inequality, an extreme form being beauti- fully shown m the formation of the polar bodies. Here the orjcrinal amphiaster is perfectly symmetrical, with the asters of equal^'size (Fig. 97, A). As the spindle rotates into its radial position and approaches the periphery, the development of the outer aster be- comes, as it were, suppressed, while the central aster becomes enor- mously large. The str.e of the aster, in other ivords, depends upon the extent of the cytoplasmic area that falls zvithin the sphere of influence . of the centrosome ; and this area depends upon the position of the centrosome. If, therefore, the polar amphiaster could be artificially prevented from moving to its peripheral position, the <t^^g would probably divide equally. ^

This leads us to a further consideration of the attempts that have been made to explain the movements of the mitotic figure through mechanical or other causes.^ Highly interesting experiments have been made by Pflliger ('84), Roux ('85), Driesch ('92), and a number of later investigators which show that the direction of cleavage may be determined, or at least modified, by such a purely meclianical cause as pressure, through which the form of the dividing mass is changed.

Thus, Driesch has shown that when the eggs of sea-urchins are flattened by pressure, the amphiasters all assume the position of least resistance, i.e. parallel to the flattened sides, so that the cleavages are all vertical, and the ^gg segments as a flat plate of eight, sixteen, or thirty-two cells (Fig. 186). This is totally different from the nor- mal form of cleavage ; yet such eggs, when released from pressure, are capable of development and give rise to normal embryos. This interesting experiment makes it highly probable that the disc-like cleavage of meroblastic eggs, like that of the squid or bird, is in some degree a mechanical result of the accumulation of yolk by which the formative protoplasmic region of the ovum is reduced to a thin layer at the upper pole; and it indicates, further, that the unequal cleavage of less modified telolecithal eggs, like those of the frog or snail, are in like manner due to the displacement of the mitotic figures toward the upper pole.

The results of Pfliiger's and Driesch's pressure experiments obvi- ously harmonize with Hertw^ig's second rule, for the position of least resistance for the spindle is obviously in the long axis of the proto- plasmic mass which is here artificially modified ; and it harmonizes further with Driiner's hypothesis of the active elongation of the spindle in mitosis (p. 105). There are, however, a large number of facts which show that neither the form of the protoplasmic mass nor

^ Cf. P>ancotte on the polar l)odies of Turhellaria, p. 235. ^ For a good review and critique, see Jennings. '97. the distribution of metaplasmic materials is sufficient to explain the position of the spindle, whether with reference to the direction or the inequality of the cleavage. As regards the direction of the spindle, Berthold i^'^^) long since clearly pointed out that prismatic or cylindrical vegetable cells, for instance, those of the cambium, often divide lengthwise ; and numer- ous contradictions of Hertwig's '' law " have since been observed by students of cell-lineage with such accuracy that all attempts to explain them away have failed.^ In some of these cases the position of the spindle is not that of least but of greatest resistance,"^ the spindle ac-

A. Early prophase of second division, showing double centrosomes. B. Second cleavage in progress; upper blastomere dividing parallel to long axis of the cell. tually pushing away the adjoining cell to make way for itself. Simi- lar difficulties, some of which have been already considered (p. 372), stand in the way of the attempt to explain the eccentricity of the spindle in unequal division. All these considerations drive us to the view that the simpler mechanical factors, such as pressure, form, and the like, are subordinate to far more subtle and complex operations involved in the general development of the organism, a conclusion strikingly illustrated by the phenomena of teloblastic division (p. 371), where the constant succession of unequal divisions, always in the

2 See especially the case observed by Mead ('94, '97, 2), in the egg oi Amphiij-ite. same plane, is correlated with a deeply lying law of growth affecting the entire formation of the body. We cannot comprcJiend the forms of cleavage witJiont reference to the end-resnlt ; and thus these phe- nomena acquire a certain teleological character so happily expressed by Lillie (p. 370). This has been clearly recognized in various ways by a number of recent writers. Roux ('94), while seeking to explain many of the operations of mitosis on a mechanical basis, holds that the position of the spindle is partly determined by "immanent" nuclear tendencies. Braem ('94) recognizes that the position of the spindle is determined not merely as that of least resistance for the mitotic figure, but also for that of the resulting products. I pointed out ('92) that the bilateral form of cleavage in annelids must be regarded as a "forerunner" of the adult bilaterality. Jennings ('97) concludes that the form and direction of cleavage are related to the later morphogenetic processes ; and many similar expressions occur in the works of recent students of cell-lineage.^

The clearest and best expression of this view is, however, given bv Lillie ('95, '99), who not only correlates the direction and rate of cleavage, but also the size-relations of the cleavage-cells with the arrangement of the adult parts, pointing out that in general the size, as well as the position, of the blastomeres is directly correlated with that of the parts to which they give rise, and showing that on this basis " one can thus go over every detail of the cleavage, and know- ing the fate of the cells, can explain all the irregularities and pecuH- arities exhibited."^ Of the justice of this conclusion I think any one must be thoroughly convinced who carefully examines the recent literature of cell-lineage. It gives no real explanation of the phenom- ena, and is hardly more than a restatement of fact. Neither does it in any way lessen the importance of studying fully the mechanical conditions of cell-division. It does, however, show how inadequate have been most of the attempts thus far to formulate the " laws " of cell-division, and how^ superficially the subject has been considered by some of those who have sought for such "laws."

We now pass naturally to the second or promorphological aspect of cleavage, to a study of which we are driven by the foregoing con- siderations. 1 Conklin ('99) believes that many of the pecuHarities of cleavage may he explained by the assumption of protoplasmic currents which " carry the centrosomes where they will, and control the direction of division and the relative size and quality of the daughter-cells,"' I.e., p. 90. He suggests that such currents are of a chemotropic character, but recognizes that their causation and direction remain unexplained.

The cleavage of the ovum has thus far been considered in the main as a problem of cell-division. We have now to regard it in an even more interesting and suggestive aspect ; namely, in its morpho- logical relations to the body to which it gives rise. From what has been said above it is evident that cleavage is not merely a process by which the ^^g simply splits up into indifferent cells which, to use the phrase of Pfliiger, have no more definite relation to the structure of the adult body than have snowflakes to the avalanche to which they contribute. 1 It is a remarkable fact that in a very large number of cases a precise relation exists between the cleavage-products and the adult parts to which they give rise ; and this relation may often be traced back to the beginning of development, so that from the first division onward we are able to predict the exact future of every indi- vidual cell. In this regard the cleavage of the ovum often goes for- ward with a wonderful clocklike precision, giving the impression of a strictly ordered series in which every division plays a definite role and has a fixed relation to all that precedes and follows it.

But more than this, the apparent predetermination of the embryo may often be traced still farther back to the regions of the undivided and even unfertilized ovum. The Qgg, therefore, may exhibit a distinct promorphology ; and the morphological aspect of cleavage must be considered in relation to the promorphology of the ovum of which it is an expression. {a) Polarity and the Egg-axis. — It was long ago recognized by von Baer ('34) that the unsegmented ^gg of the frog has a definite egg-axis connecting two differentiated poles, and that the position of the embryo is definitely related to it. The great embryo] ogist pointed out, further, that the early cleavage-planes also are definitely related to it, the first two passing through it in two meridians inter- secting each other at a right angle, while the third is transverse to it, and is hence equatorial.^ Remak afterward recognized the fact ^ that the larger cells of the lower hemisphere represent, broadly speaking, the ''vegetative layer" of von Baer, i.e. the inner germ-layer or ento- blast, from which the ahmentary organs arise ; while the smaller cells

2 The third plane is in this case not precisely at the equator, but considerably above it, forming a " parallel " cleavage. of the upper hemisphere represent the 'f animal layer," outer germ- layer or ectoblast from which arise the epidermis, the nervous s)^tcm, and the sense-organs. This fact, afterward confirmed in a very large number of animals, led to the designation of the two poles as aniimil and vegetative, formative and nutritive, or protoplasmic and dcuto- plasmic, the latter terms referring to the fact that the nutritive deuto- plasm is mainly stored in the lower hemisphere, and that development is therefore more active in the upper. The polarity of the ovum is accentuated by other correlated phenomena. In every case where an egg-axis can be determined by the accumulation of deutoplasm in the lower hemisphere the egg-nucleus sooner or later lies eccentri- cally in the upper hemisphere, and the polar bodies are formed at the upper pole. Even in cases where the deutoplasm is equally distrib- uted or is wanting — if there really be such cases — an egg-axis is still determined by the eccentricity of the nucleus and the corre- sponding point at which the polar bodies are formed.

In vastly the greater number of cases the polarity of the ovum has a definite promorphological significance ; for the egg-axis shows a definite and constant relation to the axes of the adult body. It is a very general rule that the upper or ectodermic pole, as marked by the position of the polar bodies, lies in the median plane at a point which is afterward found to lie at or near the anterior end. Through- out the annelids and mollusks, for example, the upper pole is the point at which the cerebral ganglia are afterward formed ; and these organs lie in the adult on the dorsal side near the anterior extremity. This relation holds true for many of the Bilateralia, though the primitive relation is often disguised by asymmetrical growth in the later stages, such as occur in echinoderms. There is, however, some reason to believe that it is not a universal rule. The recent observa- tions of Castle ('96), which are in accordance with the earlier work of Seeliger, show that in the tunicate Ciona the usual relation is reversed, the polar bodies being formed at the vegetative {i.e. deutoplasmic or entodermic) pole, which afterward becomes the dorsal side of the larva. My own observations ('95) on the echinoderm-egg indicate that here the primitive egg-axis has an entirely inconstant and casual relation to the gastrula-axis. It may, however, still be possi]:)lc to show that these exceptions are only apparent, and the principle in- volved is too important to be accepted without further jDroof.

{b) Axial Relations of the Primary Cleavage-planes. — Since the egg-axis is definitely related to the embryonic axes, and since the first two cleavage-planes pass through it, we may naturally look for a definite relation between these planes and the embryonic axes ; and if such a relation exists, then the first two or four bkistomeres must likewise have a definite prospective value in the development. Such relations have, in fact, been accurately determined in a large number of cases. The first to call attention to such a relation seems to have been Newport ('54), who discovered the remarkable fact that tJie first cleavage-plane in the frog s egg coincides with tJie niediaji plane of the adult body ; that, in other words, one of the first two blastomeres gives rise to the left side of the body, the other to the right. This discovery, though long overlooked and, indeed, forgotten, was con- firmed more than thirty years later by Pfliiger and Roux {'^7). It

A. Four-celled stage of Clavellna, viewed from the ventral side. B. Sixteen-cell stage (VAN Beneden and JULIN). C. Cross-section through the gastrula stage (Castle) ; a. anterior; p. posterior end ; /. left, /-. right side. [Orientation according to Castle.] was placed beyond all question by a remarkable experiment by Roux ('88), who succeeded in killing one of the blastomeres by puncture with a heated needle, whereupon the uninjured cell gave rise to a half-body as if the embryo had been bisected down the middle line (Fig. 182).

A similar result has been reached in a number of other animals by following out the cell-lineage ; e.g. by Van Beneden and Julin ('84) in the ^g^ of the tunicate Claveliiia (Fig. 177), and by W^atase ('91) in the eggs of cephalopods (Fig. 178). In both these cases all the early stages of cleavage show a beautiful bilateral symmetry, and not only can the right and left halves of the segmenting o^^^^ be di.'^tin- guished with the greatest clearness, but also the anterior and jjoste- rior regions, and the dorsal and ventral aspects. These discoveries seemed, at first, to justify the hope that a fundamental law of develop- ment had been discovered, and Van Beneden was thus led, as early as 1883, to express the view that the development of all bilateral animals would probably be found to agree with the frog and ascidian in respect to the relations of the first cleavage.

This cleavage was soon proved to have been premature. In one series of forms, not the first but the second cleavage-plane was found A. Eight-cell stage. B. The fifth cleavage in progress. The first cleavage {a-p) coincides with the future median plane; the second {l-r) is transverse. to coincide with the future long axis (7\^^;r2>, and some other annelids ; Crepidula, UiJibrclla, and other gasteropods). In another series (A forms neither of the first cleavages passes through the median plane, but both form an angle of about 45° to it {Clcpsinc and other leeches ; RJiynchelniis and other annelids ; Planorbis, Xassa, L 'nio, and other mollusks ; Discococlis and other platodes). In a few cases the first cleavage departs entirely from the rule, and is equatorial, as in Ascans and some other nematodes. The whole subject was finally thrown into apparent confusion, first by the discovery of Clapp ('91 ), Jordan, and Eycleshymer ('94) that in some cases there seems to be no con- stant relation whatever between the early cleavage-planes and the adult axes, even in the same species (teleosts, urodeles); and even in