The Cell in Development and Inheritance

At the left an entire animal, showing planes of section. The middle piece, containing two nuclei, regenerates a perfect animal. The enucleated pieces, shown at the right, swim about for a time, but finally perish. stome. Lillie ('96) has recently found that Steiitor may by shaking be broken into fragments of all sizes, and that nucleated fragments as small as 2V the volume of the entire animal are still capable of complete regeneration. All non-nucleated fragments perish.

These studies of Nussbaum and Gruber formed a prelude to more extended investigations in the same direction by Gruber, Balbiani, Hofer, and especially Verworn Verworn {''^^) proved that in /V/r- stomclla, one of the Foraminifera, nucleated fragments are able to Fig. 159. — Regeneration in the unicellular animal Stentor. [From GRUBER after BALBl.\Nr.] A. Animal divided into three pieces, each containing a fragment of the nucleus. B. The three fragments shortly afterward. C. The three fragments after twenty-four hours, each regenerated to a perfect animal.

repair the shell, while non-nucleated fragments lack this power. Balbiani ('89) showed that although non-nucleated fragments of Infusoria had no power of regeneration, they might nevertheless continue to live and swim actively about for many days after the operation, the contractile vacuole pulsating as usual. Hofer ('89), experimenting on Auuvba, found that non-nucleated fragments might live as long as fourteen days after the operation (Fig. 160). Their movements continued, but were somewhat modified, and little by little ceased, but the pulsations of the contractile vacuole were but slightly affected; they lost more or less completely the capacity to

digest food, and the power of adhering to the substratum. Nearly at the same time Verworn ('89) pubUshed the results of an extended comparative investigation of various Protozoa that placed the whole matter in a very clear light. His experiments, while fully confirming the accounts of his predecessors in regard to regeneration, added many extremely important and significant results. Non-nucleated fragments both of Infusoria {e.g. Lachrymaria) and rhizopods {Poly^

Fig. 160. — Nucleated and non-nucleated fragments of A?na;ba. [HOFER.] A. B. An Amceba divided into nucleated and non-nucleated halves, five minutes after the opera- tion. C. D. The two halves after eight davs, each containing a contractile vacuole. stomcUa, TJialassicolla) not only live for a considerable period, but perform perfectly normal and characteristic movements, show the same susceptibility to stimulus, and have the same power of ingulf- ing food, as the nucleated fragments. TJicy lack, hoivever, the power of digestion and secretion. Ingested food-matters may be slightly altered, but are never completely digested. The non-nucleated frag- ments are unable to secrete the material for a new shell {Polysto-

mella) or the slime by which the animals adhere to the substratum {^Amoeba, Difflugia, Polystomclla). Beside these results should be placed the well-known fact that dissevered nerve-fibres in the higher animals are only regenerated from that end which remains in con- nection with the nerve-cell, while the remaining portion invariably degenerates. Fig. i6i. — Formation of membranes by protoplasmic fragments of plasmolyzed cells. [Town- send.] A. Plasmolvzed cell, leaf-hair of Cucitrbita, showing protoplasmic balls connected by strands.

B. Calyx-hair of Gaillardia ; nucleated fragment with membrane, non-nucleated one naked. C. Root-hair of Marchantia ; all the fragments, connected by protoplasmic strands, have formed membranes. D. Leaf-hair of Cijcurbita ; non-nucleated fragment, with membrane, connected with nucleated fragment of adjoining cell. These beautiful observations prove that destructive metabolism, as manifested by coordinated forms of protoplasmic contractility, may go on for some time undisturbed in a mass of cytoplasm deprived of a nucleus. On the other hand, the building up of new chemical or morphological products by the cytoplasm is only initiated in the presence of a nucleus and soon ceases in its absence. These facts form a complete demonstration that the nucleus plays an essential

part not only in the operations of synthetic metaboUsm or chemical synthesis, but also in the viorpJiological determinatioji of these opera- tions, i.e. the morphological synthesis of Bernard — a point of capital importance for the theory of inheritance, as will appear beyond. Convincing experiments of the same character and leading to the same result have been made on the cells of plants. Francis Darwin (^Ty) observed more than twenty years ago that movements actively continued in protoplasmic filaments, extruded from the leaf-hairs of Dipsaciis, that were completely severed from the body of the cell. Conversely, Klebs ('79) soon afterward showed that naked proto- plasmic fragments of Vaucheria and other algae were incapable of forming a new cellulose membrane if devoid of a nucleus ; and he afterward showed i^^y^ that the same is true of Zygnema and CEdo- gonium. By plasmolysis the cells of these forms may be broken up into fragments, both nucleated and non-nucleated. The former sur- round themselves with a new wall, grow, and develop into complete plants ; the latter, while able to form starch by means of the chloro- phyll they contain, are incapable of utilizing it, and are devoid of the power of forming a new membrane, and of growth and regeneration. A beautiful confirmation of this is given by Townsend ('97), who finds in the case of root-hairs and pollen-tubes, that when the protoplasm is thus broken up, a membrane may be formed by both nucleated and non-nucleated fragments, by the latter however only ivJien they remaiji cofinected with the nucleated masses by protoplasmic strands, however fine. If these strands be broken, the membrane-forming power is lost. Of very great interest is the further observation (made on leaf- hairs in Qicnrbita) that the influence of the nucleus may thus extend from cell to cell, an enucleated fragment of one cell having the power to form a membrane if connected by intercellular bridges with a nucleated fragment of an adjoining cell (Fig. 161).

Many observers have approached the same problem from a dif- ferent direction by considering the position, movements, and changes of form in the nucleus with regard to the formative activities in the cytoplasm. To review these researches in full would be impossible, and we must be content to consider only the well-known researches of Haberlandt i^'J'j) and Korschelt ('89), both of whom have given extensive reviews of the entire subject in this regard. Haberlandt's studies related to the position of the nucleus in plant-cells with especial regard to the growth of the cellulose membrane. He deter- mined the very significant fact that local growth of the cell-wall is always preceded by a movement of the nucleus to the point of growth. Thus, in the formation of epidermal cells, the nucleus lies at first near

the centre, but as the outer wall thickens, the nucleus moves toward it, and remains closely applied to it throughout its growth, after which the nucleus often moves into another part of the cell (Fig. 162, A, B). That this is not due simply to a movement of the nucleus toward the air and light is beautifully shown in the coats of certain seeds, where the nucleus moves, not to the outer, but to the inner wall of the cell and here the thickening takes place (Fig. 162, C). The same position

Fig. 162. — Position of the nuclei in growing plant-cells. [Haherlandt.] A. Young epidermal cell oi Luzula with central nucleus, before thickening of the membrane. B. Three epidermal cells of Monstera, during the thickening of the outer wall. C. Cell from the seed-coat of Scopulina, during the thickening of the inner wall. D. E. Position of the nuclei dur- of the nucleus is shown in the thickening of the walls of the guard- cells of stomata, in the formation of the peristome of mosses, and in many other cases. In the formation of root-hairs in the pea. the pri- mary outgrowth always takes place from the immediate neighbourhood of the nucleus, which is carried outward and remains near the tip of the growing hair (Fig. 162, D, E). The same is true of the rhizoids of fern-prothallia and liverworts. In the hairs of aerial plants this

rule is reversed, the nucleus lying near the base of the hair ; but this apparent exception proves the rule, for both Hunter and Haberlandt show that in this case growth of the hair is not apical, but proceeds from the base ! V^ery interesting is Haberlandt's observation that in the regeneration of fragments of VaiicJieria the growing region, where a new membrane is formed, contains no chlorophyll, but numerous nuclei. The general result, based on the study of a large number of cases, is, in Haberlandt's words, that **the nucleus is in most cases placed in the neighbourhood, more or less immediate, of the points at which growth is most active and continues longest." This fact points to the conclusion that '' its function is especially connected with the developmental processes of the cell," ^ and that "in the growth of the cell, more especially in the growth of the cell-wall, the nucleus plays a definite part."

Korschelt's work deals especially with the correlation between form and position of the nucleus and the nutrition of the cell, and since it bears more directly on chemical than on morphological synthesis, may be only briefly reviewed at this point. His general conclusion is that there is a definite correlation, on the one hand, between the position of the nucleus and the source of food-supply, on the other hand, between the size of the nucleus and the extent of its surface and the elabora- tion of material by the cell. In support of the latter conclusion many cases are brought forward of secreting cells in which the nucleus is of enormous size and has a complex branching form. Such nuclei occur, for example, in the silk-glands of various lepidopterous larvae (Meckel, Zaddach, etc.), which are characterized by an intense secretory activity concentrated into a very short period. Here the nucleus forms a labyrinthine network (Fig. 14, E), by which its surface is brought to a maximum, pointing to an active exchange of material between nucleus and cytoplasm. The same type of nucleus occurs in the Malpighian tubules of insects (Leydig, R. Hertwig), in the spinning-glands of amphipods (Mayer), and especially in the nutritive cells of the insect ovary already referred to at page 151. Here the developing ovum is accompanied and surrounded by cells, w^hich there is good reason to believe are concerned with the elaboration of food for the egg-cell. In the earwig Forficiila each ^gg is accompanied by a single large nutritive cell (Fig. 163), which has a very large nucleus rich in chro- matin (Korschelt). This cell increases in size as the ovum grows, and its nucleus assumes the complex branching form shown in the figure. In the butterfly Vanessa there is a group of such cells at one pole of the ^g,g, from which the latter is believed to draw its nutriment (Fig. jf).

A very interesting case is that of the annelid OpJuyotrocJia, referred to at page 151. Here, as described by Korschelt, the ^gg floats in the perivisceral fluid, accompanied by a nurse-cell having a very large chromatic nucleus, while that of the ^gg is smaller and poorer in chromatin. Astheecro: completes its growth, the nurse-cell dwindles away and finally perishes (Fig. jG). In all these cases it is scarcely possible to doubt that the ^gg is in a measure relieved of the task of elaborating cyto- plasmic products by the nurse-cell, and that the great development of the nucleus in the latter is correlated with this function.

Regarding the posi- tion and movements of the nucleus, Korschelt reviews many facts pointing toward the same conclusion. Per- haps the most sugges- tive of these relate to the nucleus of the e^s: during its ovarian his- tory. In many of the insects, as in both the cases referred to above, the egg-nucleus at first occupies a central posi- Fig. 163. — Upper portion of the ovary in the earw ig /l?/-- Jiciila, showing eggs and nurse-cells. [KoRSCHELT.]

Below, a portion of the nearly ripe egg {e), showing deuto- plasm-spheres and germinal vesicle {g.v.). Above it lies the nurse-cell («) with its enormous branching nucleus. Two suc- cessively younger stages of egg and nurse are shown above. tion, but as the Qgg be- gins to grow, it moves to the periphery on the side turned toward the nutritive cells. The same is true in the ovarian eggs of some other animals, good examples of which are afforded by various coelenterates, ^.^. in medusae (Claus, Hertwig) and actinians ( Korschelt, Hertwig), where the germinal vesicle is always near the point of attachment of the ^gg. Most suggestive of all is the case of the water-beetle Dytis- ciis, in which Korschelt was able to observe the movements and changes of form in the living object. The eggs here lie in a single series alter- nating with chambers of nutritive cells. The latter contain granules which are believed by Korschelt to pass into the ^gg, perhaps bodily, perhaps by dissolving and entering in a liquid form. At all events,

the tg^ contains accumulations of similar granules, which extend inward in dense masses from the nutritive cells to the germinal vesi- cle, which they may more or less completely surround. The latter meanwhile becomes amoeboid, sending out long pseudopodia, which are always directed toward the principal mass of granules (Fig. jj). The granules could not be traced into the nucleus, but the latter grows rapidly during these changes, proving that matter must be absorbed by it, probably in a liquid form.^

Among other facts pointing in the same direction may be mentioned Miss Huie's ('97) observations on the gland-cells of Drosera, and those of Mathews ('99) on the changes of the pancreas-cell in Nect7irus. Stimulus of the gland-cells in the leaf of Drosera causes a rapid exhaustion and change of staining-capacity in the cytoplasm. During the ensuing repose the cytoplasm is rebuilt out of material laid down immediately around the nucleus, and agreeing closely in appearance and staining-reaction with the achromatic nuclear constituents. The chromatin increases in bulk during a period preceding the constructive phase, but decreases (while the nucleolar material increases) as the cytoplasm is restored. In the pancreas-cell, as has long been known, the "loaded" cell (before secretion) is filled with metaplasmic zymo- gen-granules, which disappear during secretion, the cell meanwhile becoming filled with protoplasmic fibrils (Fig. 18). During the ensu- ing period of "rest" the zymogen-granules are re-formed at the expense of the fibrillar material, which is finally found only at the base of the cell near the nucleus. Upon discharge of the secretion (granule-material) the fibrillse again advance from the nucleus toward the periphery. Mathews shows that many if not all of them may be traced at one end actually into the nuclear wall, and concludes that they are directly formed by the nucleus.

Beside the foregoing facts may be placed the strong evidence reviewed at pages 156-158, indicating the formation of the yolk-nu- cleus, and indirectly of the yolk-material, by the nucleus. All of these and a lars^e number of other observations in the same direction lead to the conclusion that the cell-nucleus plays an active part in nutrition, and that it is especially active during the constructive phases. On the whole, therefore, the behaviour of the nucleus in this regard is in har- mony with the result reached by experiment on the one-celled forms, though it gives in itself a far less certain and convincing result. ^

1 Mention may conveniently here be made of Richard Hertwig's interesting observation that in starved individuals of Actinospluvriwu the chromatin condenses into a single mass, while in richly fed animals it is divided into fine granules scattered through the nucleus - Loeb ('98, '99) makes the interesting suggestion that the nucleus is especially con- cerned in the oxydative processes of the cell, and that this is the key to its 7-dle in the syn- thetic process. It has been shown that oxydations in the living tissues are probably

We now turn to evidence which, though less direct than the above, is scarcely less convincing. This evidence, which has been exhaus- tively discussed by Hertwig, Weismann, and Strasburger, is drawn from the history of the nucleus in mitosis, fertilization, and matura- tion. It calls for only a brief review here, since the facts have been fully described in earlier chapters. To Wilhelm Roux i^^i) we owe the first clear recognition of the fact that the transformation of the chromatic substance during mitotic division is manifestly designed to effect a precise division of all its parts, — i.e. a panmeristic division as opposed to a mere mass-division, — and their definite distribution to the daughter-cells. "The essential operation of nuclear division is the division of the mother-granules " {i.e. the individual chromatin-grains) ; "all the other phenomena are for the purpose of transporting the daughter-granules derived from the division of a mother-granule, one to the centre of one of the daughter-cells, the other. to the centre of the other." In this respect the nucleus stands in marked contrast to the cytoplasm, which under- goes on the whole a mass-division, although certain of its elements, such as the plastids and the centrosome, may separately divide, like the elements of the nucleus. From this fact Roux argued, first, that different regions of the nuclear substance must represent different qualities, and second, that the apparatus of mitosis is designed to distribute these qualities, according to a definite law, to the daughter- cells. The particular form in which Roux and Weismann developed this conception has now been generally rejected, and in any form it has some serious difficulties in its way. We cannot assume a precise localization of chromatin-elements in all parts of the nucleus ; for on the one hand a large part of the chromatin may degenerate or be cast out (as in the maturation of the ^ZZ), and on the other hand in the Protozoa a small fragment of the nucleus is able to regenerate the whole.

Nevertheless, the essential fact remains, as Hertwig, Kolliker, Strasburger, De Vries, and many others have insisted, that in mitotic cell-division the chromatin of the mother-cell is distributed with the most scrupulous equality to the nuclei of the daughter-cells, and that in this regard there is a most remarkable contrast between nucleus and cytoplasm. This holds true with such wonderful constancy dependent upon certain substances (oxydation ferments) that in some manner, not vet clearly understood, facilitate the process; and the work of Spitzer ('97) has shown that these substances (obtained from tissue-extracts) belong to the group of nucleo-proteids, which are characteristic nuclear substances. The view thus suggested opens a further way toward more exact inquiry into the nuclear functions, though it is not to be supposed that the nucleus is the sole oxydative centre of the cell, as is obvious from the prolonged activity of non-nucleaied protoplasmic masses.

throughout the series of living forms, from the lowest to the highest, that it must have a deep significance. And while we are not yet in a position to grasp its full meaning, this contrast points unmistakably to the conclusion that the most essential material handed on by the mother-cell to its progeny is the chromatin, and that this substance therefore has a special significance in inheritance. The foregoing argument receives an overwhelming reenforcement from the facts of fertilization. Although the ovum supplies nearly

all the cytoplasm for the embry- onic body, and the spermatozoon at most only a trace, the latter is nevertheless as potent in its effect on the offspring as the former. On the other hand, the nuclei con- tributed by the germ-cells, though apparently different, become in the end exactly equivalent in every visible respect — in structure, in staining-reactions, and in the num- ber and form of the chromosomes to which each gives rise. But furthermore the substance of the two germ-nuclei is distributed with absolute equality, certainly to the first two cells of the embrvo, and probably to all later-formed cells. The latter conclusion, which long remained a mere surmise, has been rendered nearly a certainty by the remarkable observations of Ruckert, Zoja, and Hacker, de- scribed in Chapters IV. and VI. We must therefore accept the high probability of the conclusion that the specific character of the cell is in the last analysis determined by that of the nucleus, that is by the chromatin, and that in the equal distribution of paternal and ma- egg-fragment of Sphcer echinus granular is, fertil- tcmal chromatin tO all the CClls of ized with spermatozoon of Echinus microtuber- ^j^^ offsprinP" We find the physio- culatus, ana showing purely paternal characters. . ,1 • r ■, r \

B. Normal Pluteus of Echinus microtuberculatus. loglCal explanation of the fact that Fig. 164. — Normal and dwarf larvae of the sea-urchin. [BOVERI.] every part of the latter may show the characteristics of cither or both parents. Boveri ('89, '95, i) has attempted to test this conckision by a most ingenious and beautiful experiment ; and although his conclusions do not rest on absolutely certain ground, they at least open the way to a decisive test. The Hertwig brothers showed that the eggs of sea- urchins might be broken into pieces by shaking, and that spermatozoa would enter the enucleated fragments and cause them to segment. Boveri proved that such a fragment, if fertilized by a spermatozoon, would even give rise to a dwarf larva, indistinguishable from the nor- mal in general appearance except in size. The nuclei of such larvae are considerably smaller than those of the normal larvae, and were shown by Morgan ('95, 4) to contain only half the nuDibcr of cJiromo- somes, thus demonstrating their origin from a single sperm-nucleus. Now, by fertilizing enucleated egg-fragments of one species ( SphcB- recJiiniis granulans) with the spermatozoa of another {Echinus niicro- tuberculatus), Boveri obtained in a few instances dwarf Plutei show- ing except in size the pure paternal cJiaracters {i.e. those of Echinus, Fig. 164). From this he concluded that the maternal cytoplasm has no determining effect on the offspring, but supplies only the material in which the sperm-nucleus operates. Inheritance is, therefore, ef- fected by the nucleus alone.

The later studies of Seehger ('94), Morgan ('95, 4), and Drisch ('98, 3) showed that this result is not entirely conclusive, since hybrid larvae arising by the fertilization of an entire ovum of one species bv a spermatozoon of the other show a very considerable range of varia- tion ; and while most such hybrids are intermediate in character between the two species, some individuals may nearly approximate to the characters of the father or the mother. Despite this fact Boveri ('95, i) has strongly defended his conclusion, though admitting that only further research can definitely decide the question. It is to be hoped that this highly ingenious experiment may be repeated on other forms which may afford a decisive result.

Scarcely less convincing, finally, is the contrast between nucleus and cytoplasm in the maturation of the germ-cells. It is scarcely an exaggeration to say that the whole process of maturation, in its broadest sense, renders the cytoplasm of the germ-cells as unlike, the nuclei as like, as possible. The latter undergo a scries of com- plicated changes which result in a perfect equivalence between them at the time of their union, and, more remotely, a perfect equality of distribution to the embryonic cells. The cytoplasm, on the other

hand, undergoes a special differentiation in each to effect a second- ary division of labour between the germ-cells. When this is corre- lated with the fact that the germ-cells, on the whole, have an equal effect on the specific character of the embryo, we are again forced to the conclusion that this effect must primarily be sought in the nucleus, and that the cytoplasm is in a sense only its agent. Existing views regarding the functions of the centrosome may con- veniently be arranged in two general groups, the first including those which regard this structure as a relatively passive body, the second those which assume it to be an active organ. To the first belongs the hypothesis of Heidenhain ('94), accepted by Kostanecki ('97, i) and some others, that the centrosome serves essentiall}^ as an insertion- point for the astral rays (''organic radii"), and plays a relatively passive part in the phenomena of mitosis, the active functions being mainly performed by the surrounding structures. To the same category belongs the view of Miss Foot that the formation of the centrosome is, as it were, incidental to that of the aster — ''the expression, rather than the cause, of cell-activity " ('97, p. 810). To the second group belong the views of Van Beneden, Boveri, Biitschli, Carnoy, and others who regard the centrosome as playing a more active role in the life of the cell. Both of the former authors have assumed the centrosomes to be active centres by the action of which the astral systems are organized ; and they are thus led to the conclu- sion that the centrosome is essentially an organ for cell-division and fertilization (Boveri), and in this sense is the "dynamic centre" of the cell.^ To Carnoy and Biitschli is due the interesting suggestion ^ that the centrosomes are to be regarded further as centres of cJiemical action to which their remarkable effect on the cytoplasm is due. That the centrosome is an active centre, rather than a passive body or one created by the aster-formation, is strongly indicated by its behaviour both in mitosis and in fertilization.

Griffin ('96, '99) points out that at the close of division in TJialassema the daughter-centro- somes migrate away from the old astral centre and incite about themselves in a different region the new astral systems for the ensuing mitosis (Figs. 99, 155); and similar conditions are described by Coe in Cerebratnlns ('98). In fertilization the aster-formation can- not be regarded as a general action of the cytoplasm, but as a local one due to a local stimulus given by something in the spermatozoon ; for in polyspermy a sperm-aster is formed for every spermatozoon (p. 198). This stimulus is given by something in the middle-

piece (p. 212), which is itself genetically related to the centrosome of the last cell-generation (p. 170). These facts seem explicable only under the assumption that in these cases the centrosome, or a sub- stance which it carries, gives an active stimulus to the cytoplasm which incites the aster-formation about itself, and in the words of Grifhn '' disengages the forces at work in mitosis " ('96, p. 174). For these reasons I incline to the view that in the artificial aster-formation described by Morgan ^ the centrosomes there observed should not be regarded as the creations of the asters, but rather as local deposits of material which incite the aster-formation around them. That the centrosomes or astral centres are centres of division (whether active or passive) is beautifully shown by Boveri's interesting observations on ''partial fertilization" referred to at page 194.