The Cell in Development and Inheritance

The nucleus is usually bounded by a definite membrane, and often appears to be a perfectly distinct vesicular body suspended in the cvtoplasm — a conclusion sustained by the fact that it may move actively through the latter, as often occurs in both vegetable and animal cells. Careful study of the nucleus during all its phases gives, however, reason to believe that its structural basis is similar to that of the cell-body ; and that during the course of cell-division, when the nuclear membrane usually disappears, cytoplasm and karyoplasm come into direct contmuity. Even in the resting cell there is good evidence that both the intranuclear and the extranuclear material may be structurally continuous with the nuclear membrane^ and among the Protozoa there are forms (some of the flagellates) in which no nuclear membrane can at any period be seen. For these and other reasons t/ic tcrtns ''nucleus^' and '' ccU-bQ,dy'' sJioiild probably be regarded as only topographical expressions denoting tzuo differentiated areas in a common structural basis. The terms karyoplasm and cytoplasm possess, however, a specific significance owing to the fact that there is on the whole a definite chemical contrast between the nuclear substance and that of the cell-body, the former being characterized by the abundance of a substance rich in phosphorus known as nuclein, while the latter contains no true nuclein and is especially rich in albuminous substances such as nucleo-albumins, albumins, globulins, and the like, which contain little or no phosphorus.

Both morphologically and physiologically the differentiation of the active cell-substance into nucleus and cell-body must be regarded as a fundamental character of the cell because of its universal, or all but universal, occurrence, and because there is reason to believe that it is in some manner an expression of the dual aspect of the fundamental process of metabolism, constructive and destructive, that lies at the basis of cell life. The view has been widely held that a third essen- tial element is the centrosome, discovered by Flemming and Van Beneden in 1875-76, and since shown to exist in a large number of other cells (Figs. 7, 8). This is an extremely minute body which

1 Conklin ('97, i). Obst ('99), and some others have described a direct continuity in the resting cell between the intranuclear and extranuclear ineshworks. is concerned in the process of cell-division and in the fertilization of the G.gg, and has been characterized as the " dynamic centre " of the cell. Whether it has such a significance, and whether it is in point of morphological persistence comparable with the nucleus, are ques- tions still sub judicCy which will be discussed elsewhere.^

As ordinarily seen under moderate powers of the microscope, proto- plasm appears as a more or less vague granular substance which shows as a rule no definite structure organization. More precise examination under high powers, especially after treatment by suitable fixing and staining reagents, often reveals a highly complex structure in both nucleus and cytoplasm. Since the fundamental activities of protoplasm are everywhere of the same nature, investigators have naturally sought to discover a corresponding fundamental morpho- logical organization common to all forms of protoplasm and under- lying all of its special modifications. Up to the present time, however, these attempts have not resulted in any consensus of opinion as to whether such a common organization exists. In many forms of proto- plasm, both in hfe and after fixation by reagents, the basis of the structure is a more or less regular framework or niesJiwork, consisting of at least two substances. One of these forms the substance of the meshwork proper; the other, often called the ground-substance (also cell-sap, enchylema, hyaloplasma, paramitome, interfilar substance, etc.), 2 occupies the intervening spaces. To these two elements must be added minute, deeply staining granules or " microsomes " scattered along the branches of the meshwork, sometimes quite irregularly, sometimes with such regularity that the meshwork seems to be built of them. Besides the foregoing three elements, which we may pro- visionally regard as constituting the active substance, the protoplasm almost invariably contains various passive or metaplasmic substances in the form of larger granules, drops of liquid, crystalloid bodies, and the like.

These bodies, which usually lie in the spaces of the mesh- work, are often difficult to distinguish from the microsomes lying in the meshw^ork proper — indeed, it is by no means certain that any adequate ground of distinction exists.^ From the time of Frommann and Arnold ('65-'67) onwards, most of the earlier observers regarded the meshwork as a fibrillar structure, either forming a continuous network or reticulum somewhat like the fibrous network of a sponge ("reticular theory " of Klein, Van Bene- den, Carnoy, Heitzmann), or consisting of disconnected threads,

A. Group of epidermal cells at different foci, showing protoplasmic bridges, nuclei, and cyto- plasmic fibrillae; the central cell with nucleus in the spireme-stage. B. Connective tissue cell. C. Epidermal cell in early mitosis (segmented spireme) surrounded by protoplasmic bridges. D. Dividing cell. E.F. Cartilage-ceils with cytoplasmic fibrillas (the latter somewhat exaggerated in the plate). whether simple or branching (''filar theory" of Flcmming), and the same view is widely held at the present time. The meshwork has received various names in accordance with this conception, among which may be mentioned reticulum, thrcad-ivork, spongioplasm, mitomc, filar subslance} all of which are still in use. Under this view the " granules " described by Schultze, Virchow and still earHer observers have been variously regarded as nodes of the network, optical sec- tions of the threads, or as actual granules (" microsomes ") suspended in the network as described above.

Widely opposed to these views is the " alveolar theory " of Butschli, which has won an increasing number of adherents. Butschli regards protoplasm as having a foam-like alveolar structure ("W'aben- struktur"), nearly similar to that of an emulsion (Fig. lo), and he has shown in a series of beautiful experiments that artificial emul- sions, variously prepared, may show under the microscope a marvel- lously close resemblance to living protoplasm, and further that drops of oil-emulsion suspended in water may even exhibit amoeboid changes of form. To restate Biitschli's view, protoplasm consists of separate, closely crowded minute drops^ of a liquid alveolar substance suspended in a continuous interalvcolar substance, likewise liquid, but of different physical nature. The latter thus forms the walls of closed chambers or alveoli in which the alveolar drops lie, just as in a fine emulsion the emulsifying liquid surrounds the emulsified drops. The appear- ance of a network in protoplasm is illusory, being due to optical sec- tion of the interalvcolar walls or partitions as viewed at any given focus of the microscope. As thus seen, the walls themselves appear as fibres, while the "spaces of the network" are in like manner oi)ti- cal sections of the alveoli, the alveolar substance that fills them corresponding to the ''ground substance." As explained beyond/^ Butschli interprets in like manner the radiating systems or asters formed during cell-divison, the astral rays (usually considered as fibres) being regarded as merely the septa between radially arranged

The two (three) general views above outlined may be designated respectively as thQ fibrillar (reticular or filar) and alveolar \.\\cox\c^ of protoplasmic structure ; and each of them has been believed by some of its adherents to be universally applicable to all forms of protoplasm. Beside them may be placed, as a third general view, Xh^ granular theory especially associated with the name of Altmann, by whom it has been most fully developed, though a number of earlier writers have held similar views. According to Altmann's view, which apart from its theoretical development approaches in

2 Measuring on an average about .ooi mm. in diameter. ^ Cf. p. no. some respects that of Biitschli, protoplasm is compounded of innu- merable minute granules which alone form its essential active basis ; and while fibrillar or alveolar structures may occur, these are of only secondary importance. Fig. 10. — Alveolar or foam-structure of protoplasm, according to Biitschli. [BuTSCllLl.] A. Epidermal cell of the earthworm. B. Aster, attraction-sphere, and centrosome from sea- urchin egg. C. Intracapsular protoplasm of a radiolarian ( Thalassicolla) with vacuoles. D. Peripheral cytoplasm of sea-urchin egg. E. Artificial emulsion of olive-oil, sodium chloride, and water.

It is impossible here adequately to review the many combinations and modifications of these views which different investigators have made.^ On the whole, the present drift of opinion is toward the conclusion that none of the above interpretations has succeeded in the attempt to give a universal formula for protoplasmic structure ; and many recent observers have reached the conclusion, earlier advo- cated by Kolliker ('89), that the various types described above are connected by intermediate gradations and may be transformed one into another, in different phases of cell-activity. Unna ('95), for example, endeavours to show how an alveolar structure may pass into a sponge-like or reticular one by the breaking down of the inter-

Fig. II.— ('?) Protoplasm of the egg of the sea-urchin {Toxopneustes) in section showing meshwork of microsomes; {b) protoplasm from a living star-fish egg {Astcrias) showing alveolar spheres with microsomes scattered between them ; {c) the same in a dying condition atler crush- ing the egg ; alveolar spheres fusing to form larger spheres ; {d) protoplasm from a young ovarian egg of the same. (All the figures magnified 1200 diameters.) alveolar walls. Flemming, for many years the foremost and most consistent advocate of the fibrillar theory, now admits that protoplasm may be fibrillar, alveolar, granular, or sensibly homogeneous,^ and that we cannot, therefore, regard any one of these types of structure as absolutely diagnostic of the living substance. In plant-cells Strasburger^ and a number of his pupils maintain that the "kino- plasm" (p. 322) or filar plasm, from which the spindle-fibres and astral rays are formed, is fibrillar, while the " trophoplasm " or alveolar plasm forming the main body of the cell is alveolar, the former, however, assuming the fibrillar structure, as a rule, only during the mitotic activity of the cell. My own long-continued studies on various forms of protoplasm have likewise led to the con- clusion that no universal formula for protoplasmic structure can be

given. ^ In that classical object, the echinoderm-egg, for example, it is easy to satisfy oneself, both in the living cell and in sections, that the protoplasm has a beautiful alveolar structure, exactly as described by Hutschli in the same object (Fig. 1 1 ). This structure is here, however, entirely of secondary origin ; for its genesis can be traced step by step during the growth of the ovarian eggs through the deposit of minute drops in a homogeneous basis, which ultimately gives rise to the interalveolar walls. In these same eggs the astral systems formed during their subsequent division (Fig. 12) are, I

Fig. 12. — Section of sea-urchin egg (Toxopficustcs), li minutes after entrance of the sperma- tozoon, showing alveoli and microsomes, sperm-nucleus, middle piece, and aster (about 2000 diameters). believe, no less certainly fibrillar ; and thus we see the protoplasm of the same cell passing successively through homogeneous, alveolar, and fibrillar phases, at different periods of growth and in different conditions of physiological activity. There is good reason to regard this as typical of protoplasm in general. BiJtschli's conclusions, based on researches so thorough, j^rolonged, and ingenious, are entitled to great weight ; yet it is impossible to resist the evidence that fibrillar and granular as well as alveolar structures are of wide occurrence ; and while each may be characteristic of certain kinds of

cells, or of certain physiological conditions,^ none is common to all forms of protoplasm. If this position be well grounded, we must admit that the attempt to find in visible protoplasmic structure any adequate insight into its fundamental modes of physiological activity has thus far proved fruitless. We must rather seek the source of these activities in the ultramicroscopical organization, accepting the probability that apparently homogeneous protoplasm is a complex mixture of substances which may assume various forms of visible structure according to its modes of activity.

Some of the theoretical speculations regarding the essential nature of that organization are discussed in Chapter VI., but one q2iasi-X\\(to- ' retical point must be here considered. Much discussion has been given to the (question as to which of the visible elements of the proto- plasm should be regarded as the "living" substance proper; and the diversity of opinion on this subject may be judged by the fact that although many of the earlier observers identified the "reticulum " as the living element, and the ground-substance as Ufeless, others, such as Leydig and Schafer, held exactly the reverse view, while Altmann insisted that only the " granules " were alive. Later discussions have shown the futility of this discussion, which is indeed largely a verbal one, turning as it does on the sense of the word "living." In practice we continually use the word "living" to denote various degrees of vital activity. Protoplasm deprived of nuclear matter has lost, wholly or in part, one of the most characteristic vital properties, namely, the power of synthetic metaboHsm ; yet we still speak of it as " living," since it still retains for a longer or shorter period such properties as irritability and the power of coordinated movement ; and, in like manner, various special elements of protoplasm may be termed " liv- ing " in a still more restricted sense. In its fullest meaning, however, the word "living" implies the existence of a group of cooperating activities more complex than those manifested by any one substance or structural element.

I am therefore entirely in accord with the view urged by Sachs, Kolliker, Verworn, and other recent writers, that life can 'only be properly regarded as a property of the cell- system as a whole ; and the separate elements of the system would, with Sachs, better be designated as "active" or "passive," rather than as "living" or "lifeless." Thus regarded, the distinction 1 Thus the alveolar structure seems to be characteristic of Protozoa in general, and of the protoplasm of plant-cells when in the vegetative state, the fibrillar of nerve-cells and muscle-cells. The granular type is characteristic of some forms of leucocytes and gland- cells; but many of the granules in these cells are no doubt metaplasmic, and it is further very doubtful whether such a granular or "pseudo-alveolar" structure can be logically dis- tinguished from an alveolar (c/. Wilson, '99). In the pancreas-cell granular and hbr.llar structures alternate with the varying phases of secretory activity {r/. Mathews, '99).

between "protoplasmic" and " nictai:)lasmic " substances, while a real and necessary one, becomes after all one of degree. I believe that we are probably justified in regarding the continuous substance as the most constant and active element, and that which forms the fundamental basis of the system, transforming itself into granules, drops, fibrilla?, or networks in accordance with varying physiological needs. ^ Thus stated, the question as to the relative activity of the various elements becomes a real and important one. It now seems probable that the substance of the meshwork (fibrillar or interalveolar structure) is most active in the processes of cell-division, in contractile organs such as cilia and muscle-fibres, and in nerve-cells ; but the ground- substance, while apparently the most frequent seat of metaplasmic deposits, is certainly also the seat of active chemical changes. This subject has, however, not yet been sufficiently investigated.

A fragment of a cell deprived of its nucleus may live for a consid- erable time and manifest the power of coordinated movement without perceptible impairment. Such a mass of protoplasm is, however, devoid of the powers of assimilation, growth, and repair, and sooner or later dies. In other words, those functions that involve destructive metabolism may continue for a time in the absence of the nucleus ; those that involve constructive metabolism cease with its removal. There is, therefore, strong reason to believe that the nucleus plays an essential part in the constructive metabolism of the cell, and through this is especially concerned with the formative processes involved in growth and development. For these and many other reasons, to be discussed hereafter, the nucleus is generally regarded as a controlling

1 Wilson, '99. Cf. Sachs ('92. '95), Kulliker ('97), Meyer ('96), and Kupffcr ('96) on energids. Sachs sharply distinguishes between the energid {r^\x(^^\x% and protoplasm), which forms a living unit, and the passive ^x^^ix^xA-prodticts, placing in the former the nucleus, nucleolus, general cytoplasm, centrosome and plastids (chloroplasts and leucoplasts), and in the latter the starch-grains, aleurone-crystals, and membrane. Meyer carries the analysis further, classifying the active energid-elemcnts m\.o protoplasmatic and alloplasmatic organs, the former (nucleus cytoplasm, chromatophores, and perhaps the centrosomes) arising only by division, the latter (cilia, and according to Kolliker, also the muscle- and nerve-fibriiiee) formed by differentiation from the protoplasmatic elements. The passive energid-products {ergastic structures or " formed material " of Beale) are formed as enclosures (starch-grains, etc.), or excretions (membranes). These general views arc accejited by Kolliker; but none of these writers has undertaken to show how " alloplasmatic "' structures are to be distinguished from metaplasmic or ergastic. I believe Sachs' view to be in principle not only true but of high utility. Practically, however, it involves us in considerable difficulty, unless the terminology adopted above — itself directly suggested by and nearly agreeing with the usage of Sachs and Kolliker — be employed.

centre of cell-activity, and hence a primary factor in growth, develop- ment, and the transmission of specific qualities from cell to cell, and so from one generation to another. The cell-nucleus passes through two widely different phases, one of which is characteristic of cells in their ordinary or vegetative condi- tion, while the other only occurs during the complicated changes involved in cell-division. In the first phase, falsely characterized as the *' resting state," the nucleus usually appears as a rounded sac-like body surrounded by a distinct membrane and containing a conspicu- ous irregular network (Figs. 6, 7, 13), which is in some cases plainly visible in the living cell (Fig. 9). The form of the nucleus, though subject to variation, is on the whole singularly constant, and as a rule shows no very definite relation to that of the cell-body, though in elon- gated cells such as muscle-cells, in certain forms of parenchyma, and in epithelial cells (Fig. 49), the nucleus is itself often elongated. Typically spherical, it may, in certain cases, assume an irregular or amoeboid form, may break up into a group of more or less completely separated lobes (polymorphic nuclei. Fig. 49), sometimes forming an irregular ring (" ring-nuclei " of leucocytes, giant-cells, etc., Fig. 14. J)). It is usually very large in gland-cells and others that show a very active metabolism, and in such cases its surface is sometimes increased by the formation of complex branches ramifying through the cell

These forms seem in general to be fairly constant in a given species of cell, but in a large number of cases the nucleus has been seen in the living cell (cartilage-cells, leucocytes, ova) to undergo more or less active changes of form, sometimes so marked as to merit the name of amoeboid (Fig. 77). Perhaps the most remarkable deviations from the usual type of nucleus occur among the unicellular forms. In the dil- ate Infusoria the nuclei are massive bodies of two kinds, viz. a large macrormcleiis and one or more smaller viicroujiclci, both of which arc present in the same cell, the former kind being generally regarded as the active nucleus, the latter as a reserve nucleus from which at cer- tain periods new macronuclei arise (p. 224). The macronuclei show a remarkable diversity of form and structure in different species. Still more interesting are the so-called scattered or distributed nuclei, de- scribed by Butschli in flagellates and Bacteria, by Gruber in certain rhizopods and Infusoria, and by several authors in the Cyanophyccx (Figs. 15, 16). The nuclear material is here apparently scattered through the cell in the form of numerous minute, deeply stained gran- ules, which, if this identification is correct, represent the most primi-

tive known types of nucleus ; but this subject is still sub jiidice (p. 39). A transition from this condition to nuclei of the ordinary type appears to be L^iven in the nuclei of certain flagellates (e.g. CJii- Uwionas and Tracluhnotias), where the chromatin-granules are aggre- gated about a nucleolus-like body, but are not enclosed by a membrane.^ In considering the structure of the nucleus, as seen in sections, we must, as in the case of the cytoplasm, bear in mind the possibility, or

rather probability, that some of the elements described may be coagulation - products ; for the nucleus is in life comj^osed of liquid or semi-liquid substance, and Albrecht ('99) has recently shown that nuclei isolated in the fresh condition will flow together to form a single body. Most of the main features of the nucleus, both in the resting and in the dividing phases, have, however, been seen in life (Fig. 9), and the principal danger of mistaking artifacts for normal structures re- lates to the finer elements, con- sidered beyond.

In the ordinary forms of nuclei in their resting state the follow- ing structural elements may as a rule be distinguished (Figs. 6, 7, Fig. 13. — Two nuclei from the crypts of ^^ q^^e liuclcav DUmbraUC, a Lieberkiihn in the salamander. [Heidenhain.] ^^ -^ r i i t ^ n i • i (^aj/r/4r^wa//«) is accurately shown. The upper glVCS the UUClCUS a Sharp COUtOUr nucleus contains three plasmosomes or true -^^-^^ differentiates it clcarly from {oxychromattn) are seen in the upper nucleus the SUrrOUuding Cytoplasm. ThlS

running off from the chromatin-masses. The wall SOmctimCS StaiuS but VCiy clear spaces are occupied by the ground-sub- ^|i n] ^,^,| ^aU SCarCcly be dif- stance. n ./ ' j ferentiated from the outlying cytoplasm. In other and perhaps more frequent cases, it approaches in staining capacity the chromatin. b. The nuclear rcticuliiDi. This, the most essential part of the nucleus, forms an irregular branching network or reticulum which con- sists of two very different constituents. The first of these, forming the general protoplasmic basis of the nucleus, is a substance known as lini7i

(Schwarz), invisible until after treatment by reagents, which in sections shows a finely granular structure and stains like the cytoplasmic sub- stance, to which it is nearly related chemically (Figs. 7, 49). The second constituent, a deeply staining substance known as chromatiji (Flemming), is the nuclear substance /(^r excellence, for in many cases it appears to be the only element of the nucleus that is directly handed on by division from cell to cell, and it seems to have the power to pro- duce all the other elements. The chromatin often appears in the form of scattered granules and masses of differing size and form, which are embedded in and supported by the linin-substance (Figs. 7, 19). In some cases the entire chromatin-content of the nucleus appears to be condensed into a single mass which simulates a nucleolus ; for exam- ple, in Spirogyra and in various flagellates and rhizopods (e.g. Acti- jiospJicBviuni, Ai'cella) ; or there may be several such chromatin-masses, as in some of the Foraminifera and in Noctihica. More commonly the chromatin forms a more or less regular network intermingled with and more or less embedded in the linin, from which it is often hardly dis- tinguishable until the approach of mitosis, when a condensation of the chromatin-substance occurs.

In contradistinction to the other nuclear elements, chromatin is nut acted upon, or is but slowly affected, by peptic digestion. It may thus be easily isolated for chemical analysis, which shows it to consist mainly of luiclein, i.e. a compound in varying proportions of a complex phosphorus-containing acid known as inicleinic acid, with albumi- nous bodies such as histon, protamin, or in some cases albumin itself.' Upon this, as will be show^n in Chapter VL, probably depends the pro- nounced staining capacity when treated with the so-called *' nuclear stains " {e.g. hsematoxylin, methyl-green, and the basic tar-colours gen- erally) from which chromatin takes its name. This capacity always increases as the nucleus prepares for division, reaching a climax in the spireme- and chromosome-stages, and it is also very marked in con- densed nuclei such as those of spermatozoa. These variations are almost certainly due to varying proportions in the constituents of the nuclein, the staining capacity standing in direct ratio to the amount of nucleinic acid.

c. The nucleoli, one or more larger rounded or irregular bodies, suspended in the network, and staining intensely with many dyes. In some nuclei they are entirely absent. When present the nucleoli vary in number from one to five or more; and the number is otten inconstant in the same species of cell, and even varies in the same cell with varying physiological conditions. In the eggs of some animals, at certain periods of growth {e.g. lower vertebrates), the nucleus may contain hundreds of nucleoli. An interesting case is

that of the subcutaneous gland-cells of Pisciola, the nuclei of which contain in early phases of secretion but a single nucleolus. During growth of the cell the nucleolus fragments, finally giving rise to several hundred nucleoli which then appear to migrate out into the cytoplasm, leaving but a single nucleolus to repeat the cycle. ^ The bodies known as nucleoli are of at least two different kinds. The first of these, the so-called true nucleoli or phuinosomcs (Figs. 6, 8, B, 13), are of spherical form, and are shown by the staining reactions to differ widely from chromatin, being in general sharply stained by dyes which, like eosin, orange or acid fuchsin, stain the linin and the general cytoplasm. The plasmosomes sometimes seem to have no envelope, but in many cases {e.g. in leucocytes) are surrounded by a thin layer that stains Hke chromatin. Nucleoli of a quite different type are the *' net-knots " (Netzknoten), chromatin- nucleoli, or karyosojucs, which agree in staining reaction with chro- matin and are doubtless to be regarded as merely a portion of the chromatin-network (Figs. 8, 49). These are sometimes spherical, more often irregular (Fig. 8), and often are hardly to be distinguished, except in size, from nodes of the chromatin-reticulum.''^ The relations between these two forms of nucleoli are far from certain, and the variations in staining reaction shown by true nucleoli render it not improbable that intermediate forms exist which may represent an actual transition from one to the other.-*^ In many of the Protozoa, as described beyond, the ''nucleolus" is shown by its behaviour during mitosis to be comparable with an attraction-sphere or centro- some ('Muicleolo-centrosome," Keuten); and even in higher forms there are some cells in which the centrosome is intranuclear

There is good reason to believe that the chromatin-nucleoli are merely more condensed portions of the chromatin-network. since during cell-division they have the same history as the remaining portion of the chromatin-substance.* The nature of the true nucleoli is still imperfectly known. By some observers, including Flemming, O. and R. Hertwig, and Carnoy, they have been regarded as store- houses of material (para-nuclein, plastin) which contributes to the