The Cell in Development and Inheritance

2 Flemining first called attention to the chemical difference between the true nucleoli and the chromatic reticulum ('82, pp. 138, 163) in animal-cells, and Zacharias soon afterward studied more closely the difference of staining reaction in plant-cells, showing that the former are especially coloured by alkaline carmine solutions, the latter by acid solutions. Other studies by Carnoy, Zacharias, Ogata, Rosen, Schwarz, Heidenhain, and many others show that the medullary substance (pyrenin) of true nuclei is coloured by acid tar-colours and other plasma stains, while the chromatin has a special affinity for basic dyes. Cf. p. 337.

3 For very full review of the literature of the nucleoli see Montgomery ( '98, 2). formation of chromosomes during division, and hence may play an active role in the nuclear activity. Strasburger ( '95) likewise be- lieves them to contain a store of active material which, however, has no direct relation to the chromosomes but consists of " kinoplasm " A. Permanent spireme-nucleus, salivary gland of Chirowmus larva. Chromatin in a single thread, composed of chromatin-discs (chromomeres), terminating at each end m a true nucleolus or plasmosonie. [Balkianl]

B. Permanent spireme-nuclei. intestinal epithelium of dipterous larva Ptychcptfra. [\ AN D. Polymorphic ring-nucleus, giant-cell of bone-marrow of the rabbit; c. a group of ccntro- E. Branching nucleus, spinning gland of butterfly-larva {Picris) . [KORSCHELT.J (p. 322), from which arises the achromatic part of the division- figure (p. 82). On the other hand, Hacker ( '95, '99) ^^^^^ o^^er observers regard the nucleolar material as a passive by-product of the chromatin-activity destined to be absorbed by the active sub-

Stances. This is supported by the fact that in some forms of mitosis the nucleokis is at the time of division actually cast out of the nucleus into the cytoplasm, where it degenerates without further apparent function. This seems to constitute decisive evidence in support of Hacker's view as api)lied to certain cases ; but without further evidence it must remain doubtful whether it applies to d. The groiiiid-siihstancc, nuclear sap, or kavyolympJi, a clear sub- stance occupying the interspaces of the network and left unstained by most of the dyes that colour the chromatin, the linin, or the plas- mosomes. By most observers the ground-substance is regarded as a liquid filling a more or less completely continuous space traversed by the nuclear network. By Biitschli, however, and some of his fol- lowers the nucleus is regarded as an alveolar structure, the walls of which represent the "netw^ork," while the ground-substance corre- sponds to the alveolar material. Nearly related with this is the view of Reinke ( 94) that the ground-substance consists of large pale frranules of 'Manthanin " or ** oedematin."

The configuration of the chromatic network varies greatly in dif- ferent cases. It is sometimes of a very loose and open character, as in many epithelial cells (Fig. i); sometimes extremely coarse and irregular, as in leucocytes (Fig. 49); sometimes so compact as to appear nearly or quite homogeneous, as in the nuclei of spermatozoa and in many Protozoa. In some cases the chromatin does not form a network, but appears in the form of a thread closely similar to the spireme-stage of dividing nuclei (r/. p. 65). The most striking case of this kind occurs in the salivary glands of dipterous \?.x\'^ {^Chiron 0- viHs\ where, as described by Balbiani, the chromatin has the form of a single convoluted thread, composed of transverse discs and termi- nating at each end in a large nucleolus (Fig. 14, A). Somewhat simi- lar nuclei (Fig. 14, B) occur in various epithelial cells of other insects (Van Gehuchten, Gilson), and also in the young ovarian eggs of cer- tain animals (r/. p. 273). In certain gland-cells of the marine isopod Anilocra it is arranged in regular rosettes (Vom Rath). Rabl, fol- lowed bv Van Gehuchten, Heidenhain, and others, has endeavoured to show that the nuclear network shows a distinct polarity, the nucleus having a *' pole " toward which the principal chromatin- threads converge, and near which the centrosome lies.- In many nuclei, however, no trace of such polarity can be discerned.

The network may undergo great changes both in physical con- figuration and in staining capacity at different periods in the life of the same cell, and the actual amount of chromatin fluctuates, sometimes to an enormous extent. Embryonic cells are in general characterized by the large size of the nucleus; and Zacharias has shown in the case of plants that the nuclei of meristem and other embryonic tissues are not only relatively large, but contain a larger percentage of chromatin than in later stages. The relation of these changes to the physiological activity of the nucleus is still imperfectly understood.^

A considerable number of observers have raised the question whether the nuclear structures may not be regarded as aggregates of more elementary morphological bodies, though there is still no general agreement regarding their nature and relationships. The most definite evidence in this direction relates to the chromatic network. In the stages preparatory to division this network resolves itself into a definite number of rod-shaped bodies known as chroniosoines (Fig. 21), which split lengthwise as the cell divides. These bodies arise as aggregations of minute rounded bodies or microsomes to which various names have been g\vQx\{cJiroi)io- mcres, Fol ; ids, Weismann). They are as a rule most clearly visible and most regularly arranged during cell- division, when the chromatin is ar- ranged in a thread {spireme), or in separate cJironiosoines (Figs. 8, D, 53, B)\ but in many cases they are dis- tinctly visible in the reticulum of the scatteredchromatin-graniiles. [GRUBKK.]

however, an open question whether the chromatin-granules of the reticulum are individually identical with those forming the chromo- somes or the spireme-thread. The larger masses of the reticu- Fig. 15. — An infusorian. Trachelo- cerca, with diffused nucleus consisting of 1 Both chromatin-granules and nucleoli have been seen in a considerable number of living cells (Fig. 9). Favourable objects for this purpose are according to Korschelt ('96) the silk- glands of caterpillars, where the whole nucleus may be seen to be filled with fine granules ("microsomes"), among which are scattered many larger granules (" macrosomes "). 'I he later studies of Meves ('97, i) make it probable that the latter are true nucleoli and the for- ?r chromatin-granules. Korschelt, however, regards the "macrosomes" as composed of romatin and the "microsomes" as representing the so-called "achromatic sulistance."

lum undoubtedly represent aggregations of such granules, but whether the latter completely fuse or remain always distinct is unknown. Even the chromosomes at certain stages appear perfectly homoge- neous, and the same is sometimes true of the entire nucleus, as in the spermatozoon. It is nevertheless possible that the chromatin-gran- ules have a persistent identity and are to be regarded as morpho- logical units of which the chromatin is built up.^

Heidenhain ('93, 94), whose views have been accepted by Rcinke, Waldeyer, and others, has shown that the "achromatic" nuclear net- work is likewise composed of granules, which he distinguishes as /tint/ianiu- or <u;jr//;v;;/^?//;/-granules from the has ic/iroiULi t i fi-gr3.\\\i\Q.s> of the chromatic network. Like the latter, the oxychromatin-granules are suspended in a non-staining clear substance, for which he reserves the term //;////. Both forms of granules occur in the chromatic network, while the achromatic network contains only oxychromatin. Thev are sharply differentiated by dyes, the basichromatin being coloured by the basic tar-colours (methyl-green, saffranin, etc.) and other true "nuclear stains"; while the oxychromatin-granules, like many cytoplasmic structures, and like the substance of true nucleoli (pyrenin), are coloured by acid tar-colours (rubin, eosin, etc.) and other "plasma stains." This distinction, as will appear in Chapter VI I., is possibly one of great physiological significance.

Still other forms of granules have been distinguished in the nucleus by Reinke ('94) and Schloter ('94). Of these the most important are the " oedematin-granules," which according to the first of these authors form the principal mass of the ground-substance or " nuclear sap " of Hertwig and other authors. These granules are identified by both observers with the " cyanophilous granules," which Altmann regarded as the essential elements of the nucleus. It is at present impossible to give a consistent interpretation of the morphological value and physiological relations of these various forms of granules. The most that can be said is that the basichromatin-granules are probably normal structures ; that they play a principal role in the life of the nucleus ; that the oxychromatin-granules are nearly related to them ; and that not improbably the one form may be transformed into the other in the manner suggested in Chapter VII.

The nuclear membrane is not yet thoroughly understood, and much discussion has been devoted to the question of its origin and structure. The most probable view is that long since advocated by Klein i^yd)) and Van Beneden ('83) that the membrane arises as a condensation of the general protoplasmic substance, and is part of the same structure as the linin-network and the cytoplasmic mesh- w^ork. Like these, it is in some cases "achromatic," but in other cases

it shows the same staining reactions as chromatin, or may be double, consisting of an outer achromatic and an inner chromatic layer. Ac- cording to Reinke, it consists of oxychromatin-granules like those of the linin-network. Interesting questions are raised by a comparison of these facts with the conditions observed in some of the lowest organisms, such as the flagellates and lower rhizopods among animals and the Fig i6.- Forms of Cyanophyce^, Bacteria, and Flagellates showing the so-called scattered

A. Oscillaria. B. Chromafmrn, C. Bacterium lineola. D. Achrowatiuw. E. The same m H. Aggregation of the granules. /. Division of the sphere. J. Fission of the cell. Cyanophyce^ and Bacteria among plants. In many ol these forms (Fig. i6) no distinct nucleus can be demonstrated, the cell consistmg of a mass of protoplasm in which are scattered numerous deeply staining granules. Many of these granules stam mtensely with hematoxylin and other " nuclear" dyes; like chromatm, they resist the action of peptic digestion, and in at least one case (the bacterium- like AckromatiHm, according to Schewiakoff, '93) they have the power of division like the chromatin-granules of higher forms. For these

reasons most observers (Biitschli, Gruber, Schewiakoff, Nadson, etc.) recrard them as true chromatin-f(ranules which rein-esent a scattered or distributed nucleus not differentiated as a definite morphological body. If this identification is correct, such forms probably give us the most primitive condition of the nuclear substance, which only in higher forms is collected into a distinct mass enclosed by a membrane; and the scattered granules are comparable to those forming the chro- matin-reticulum and chromosomes in the higher types. The identi- fication is, however, difficult, owing to the impossibility of actual chemical analysis; and Fischer (97) has shown in the case of the Bacteria and Cyanophycene that we cannot safely trust either the staining reactions or the digestion test, since the former are variable, while the latter does not differentiate the granules from some other cytoplasmic constituents.^ It is, however, certain that the staining power of chromatin in the higher forms varies with different condi- tions, and furthermore there is reason to believe that these granules may divide by fission. Besides these observations of Schewiakoff on AcJiromatiuin (see above), we have those of several authors on Infusoria, and more recently those of Calkins on flagellates, both pointing to the same conclusion. Balbiani, Gruber, Maupas, and others have described various Infusoria (^/r^i-Zj/Z^', Trac/icloccrca, HolosticJia, Urolcptns), as well as some rhizopods {Pcloniyxa), in which the body contains very numerous minute chromatin-granules of "nuclei" (Fig. 15), which Gruber {"^'J) showed to multiply by division.

Balbiani ('61) long since showed that in Urostyla these bodies become concentrated toward the centre of the cell at the time of division, and Bergh ('89) demonstrated that they then fuse to form a macronucleus of the usual type, that elongates, assumes a fibrillar structure, and divides by fission. After division of the cell-body the macronucleus again fragments into minute scattered granules, which in this case certainly represent a distributed nucleus. In the flagellate Tctramitus Calkins ('98, i) likewise finds numerous scat- tered chromatin-granules, which at the time of division become aggre- gated into a single dividing mass (p. 92); while in other forms the mass (nucleus) persists as such without (yTracJiclomouas, Lagcnclla, CJiiloDionas) or with {Euglcna, Synura) a surrounding membrane.

Taken together, the foregoing facts, while certainly not conclusive, give good ground for the provisional acceptance of Biitschli's con- ception of the distributed nucleus, and indicate that nucleus and cytoplasm have arisen through the differentiation of a common protoplasmic mass. The nucleus, as Carnoy has well said,- is like a house built to contain the chromatic elements, and its achromatic ele- ments (Unin, etc.) were originally a part of the general cell-substance. Moreover, as Carnoy points out, the house periodically goes to pieces in the process of mitotic division, the chromatin afterward "buildin"- for itself a new dwelling."

The chemical nature of the various nuclear elements will be considered in Chapter VII., and a brief statement will here suffice. The following classification of the nuclear substances, proposed by Schwarz in 1887, has been widely accepted, thou'di open to criticism on various grounds. 1. Chromatin. The chromatic substance (basichromatin) of the network and of 2. Linin. The achromatic network and the spindle fibres arising from it. 4. Pyrcniii or Parachroniatiii. The inner mass of true nucleoli.

Chromatin is probably identical with imclein (p. 332). which is a compound of nucleiiiic acid (a complex organic acid, rich in phosphorus) and albuminous sub- stances. In certain cases (nuclei of spermatozoa, and probably also the chromo- somes at the time of mitosis) the percentage of nucleinic acid is very large (p. 'Ji'}^'},). The /////;/ is supposed to be composed of "plastin'' — a substance identified -by Reinke and Rodewald ('81) and probably a nucleo-albumin or a related substance. '• Pyrenin " is related to plastin ; and Carnoy and Zacharias apply the latter word to the nucleolar substance, while O. Hertw'ig calls it paranuclein. " Amphipyrenin"" has no very definite meaning ; for the nuclear membrane sometimes appears to be of the same nature as the linin, w'hile in other cases it stains like chromatin. For cri- tique of the staining reactions see page 334.

It has long been recognized that in the unicellular forms the cytoplasmic substance is often differentiated into two well-marked zones : viz. an inner medullary substance or cjuioplasui in which the nucleus lies, and an outer cortical substance or exoplasm (ectoplasm) from which the more differentiated products of the cytoplasm, such as cilia, trichocysts, and membrane, take their origin. Indications of a similar differentiation are often shown in the tissue-cells of higher plants and animals, ^ though it may take the form of a polar differen- tiation of the cell-substance, or may be wholly wanting. Whether the distinction is of fundamental importance remains to be seen ; but it appears to be a general rule that the nucleus is surrounded by

1 This fact was first pointed out in the tissue-cells of animals liy Kupffer ('75). and its importance has since been urged by Waldeyer, Reinke, and others. The cortical layer is by Kupffer termed paraplasm, by p'feffer hyaloplasm, by Tringsheim the Haiitschiclit. The medullary zone is termed by Y.^x^^'^^x protoplasm, sensii strictu; by Strasburger, K'dmer- plasma ; by ^'■k<g€(\, polioplasm. protoplasm of relatively slight differentiation, while the more highly differentiated products of cell-activity are laid down in the more peripheral region of the cell, either in the cortical zone or at one end of the cell.^ This fact is full of meaning, not only because it is an expression of the adaptation of the cell to its external environment, but also because of its bearing on the problems of nutrition. ^ For if, as we shall see reason to conclude in Chapter VI I., the nucleus be immediately concerned with synthetic metabolism, we should expect to find the immediate and less differentiated products of its action in its neighbourhood, and on the whole the facts bear out this view.

The most pressing of all questions regarding the cytoplasmic structure is whether the sponge-like, fibrillar, or alveolar appearance is a normal condition existing during life. There are many cases, especially among plant-cells, in which the most careful examination has thus far failed to reveal the presence of a reticulum, the cyto- plasm appearing, even under the highest powers and after the most careful treatment, merely as a finely granular substance. This and the additional fact that the cytoplasm may show active streaming and flowing movements, has led some authors, especially among bota- nists, to regard the reticulum as non-essential and as being, when present, either a secondary differentiation of the cytoplasmic sub- stance specially developed for the performance of particular functions or a mere coagulation-product due to the action of fixatives. It has been shown that structureless proteids, such as egg-albumin and other substances, when coagulated by various reagents, often show a structure closely similar to that of protoplasm as observed in micro- scopical sections. Flemming ('82) long since called attention to the danger of mistaking such coagulation-products for normal structures as seen in fixed and stained material, and his warning has been emphasized by the later experiments of Berthold {"^6\ Schwarz i^^jX and especially of Butschli ('92, '98), Fischer ('94, '95, '99), and Hardy ('99). Butschli's extensive studies of such coagulation-phe- nomena show that coagulated or dried albumin, starch-solutions, gela- tin, gum arable, and other substances show a fine alveolar structure scarcely to be distinguished from that which he believes to be the normal and typical structure of protoplasm.

Fischer and Hardy have likewise made extensive tests of solutions of albumin, peptone, and related substances, in various degrees of concentration, fixed and stained by a great variety of the reagents ordinarily used for the demonstration of cell-structures. The result was to produce a mar- vellously close simnlacniui of the appearances observed in the cell, alveolar, reticulated, and fibrillar structures being produced that often contain granules closely similar in every respect to those described as

"microsomes " in sections of actual protoplasm. After impregnating pith with peptone-solution and then hardening, sectioning, and stain- ing, the cells may even contain a central nucleus-like mass suspended in a network of anastomosing threads that extend in every direction outward to the walls, and give a remarkable likeness of a normal cell. These facts show how cautious we must be in judging the appear- ances seen in preserved cells, and justify in some measure the hesita-

Fig. 17. — Ciliated cells, showing cytoplasmic fibriilcTe terminating in a zone of peripheral microsomes to which the cilia are attached. [Engelmann.] A. From intestinal epithelium of Anodonta. B. From gill of Anodonta. CD. Intestinal epi- thelium of Cyclas. tion with which many existing accounts of cell-structure are received. The evidence is nevertheless overwhelmingly strong, as I believe, that not only the fibrillar and alveolar formations, but also the micro- somes observed in cell-structures, are in part normal structures. This evidence is derived partly from a study of the living cell, partly from the resfular and characteristic arrangement of the thread-work and

microsomes in certain cases. In many Protozoa, for example, a fine alveolar structure may be seen in the living jirotoplasm ; and Flem- ming as well as manv later observers has clearly seen fibrillar struc- tures in the living cells of cartilage, epithelium connective-tissue, and some other animal cells ( Fig. 9). Mikosch, also, has recently described ^;v7;///A7/' threads in living plant-cells. Almost equally conclusive is the beautifully regular arrangement of the fibrillct' in ciliated cells ( Fig. 17, Fngelmann), in muscle-fibres and ncrve-hbres, and especially in the mitotic figure of dividing cells

A-C. Nectiiriis ; D. Rami. A and H represent two stages of the " loaded " cell, showing zymogen-granules in the-peripheral and fibrillar structures in tlie basal part of the cell. C shows cells after discharge of the granule-material and invasion of the entire cell by fibrillie. In D por- tions of the fibrillar material are coiled to form the mitosome (" paranucleus " or " Nebenkern "}. (Figs. 2 1, 31), where they are likewise more or less clearly visible in life. A very convincing case is afforded by the pancreas-cells of Nccturiis, which Mathews has carefully studied in my laboratory. Here the thread-work consists of long, conspicuous, defmite fibrillae, some of which may under certain conditions be wound up more or less closely in a spiral mass to form the so-called Nebcjikern. In all these cases it is impossible to regard the thread-work as an accidental coagulation-product. In the case of echinoderm eggs, I have made ('99) a critical comparison of the living structure, as seen under powers

of a thousand diameters and upwards, with the same ohject stained in thin sections after fixation by picro-acetic, subHmate-acetic, and Fig. 19. — Section through a nephridia! cell of the leech, Clepstite (drawn by Arnold Graf from The centre of the cell is occupied by a large vacuole, filled with a watery licinid. The cyto- plasm forms a very regular and distinct reticulum with scattered microsomes which become very large in the peripheral zone. The larger pale bodies, lying in the ground-substance, are cvcretory granules {i.e. metaplasm). The nucleus, at the right, is surrounded by a thick chromatic mem- brane, is traversed by a very distinct linin-network, contains numerous scattered chromatin- granules, and a single large nucleolus within which is a vacuole. Above are two isolated nuclei showing nucleoli and chromatin-granules suspended in the linin-threads.

Other reagents. The comparison leaves no doubt that the normal structures are in this case very perfectly preserved, thoui^h the sec- tions give at first sight an appearance somewhat different from that of the living object, owing to differences of staining capacity. In these eggs the microsomes, thickly scattered through the alveolar walls, stain deeply (Figs. ii. i J ), while the alveolar spheres hardly stain at all. When, therefore, the stained sections are cleared in balsam, the contours of the alveolar spheres almost disappear, and the eye is caught by the walls, which give at first sight quite the appear- ance of a granular reticulum, as it has been in fact described by many observers. Careful study of the sections shows, however, that t lie form and arraui^cjuiut of all the elements is almost idcntieally t/ie same as in life.

This result shows that careful treatment by reagents in some cases at least gives a very faithful picture of the normal structure ; and while it should never be forgotten that in sections we are viewing coagulated material, much of which is liquid or semi-liquid in life, w^ should not adopt too pessimistic a view of the results based on fixed material, as I think some of the experimenters referred to above have done. Wherever possible, the structures observed in sections should be compared with those in the living material. When this is imprac- ticable we must rely on indirect evidence; but this is in many cases hardly less convincing than the direct.