Paper - Development of the connective tissues from the connective syncytium (1902)
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Mall FP. Development of the connective tissues from the connective syncytium. (1902) Amer. J Anat. 1(3): 329-366
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Development of the connective tissues from the connective syncytium
Franklin P. Mall From the Anatomical Laboratory of Johns Hopkins University.
With 18 Text Figures.
It is much disputed whether the connective-tissue fibrils arise within cells or from a substance between them. It matters little which view is entertained, the evidence in either case is unsatisfactory. The many researches upon the development of the connective tissues have not given results fully satisfactory and the reason for this is only too evident to those who have made this subject a special study. To be sure material is very abundant and at first sight the problem is a simple one to be solved easily.
The first marked step in advance regarding the histogenesis of connective-tissue fibrils was made by Flemming in 1891 to be followed by a second communication in 1897. According to Flemming, the fibrils of white fibrous tissue are formed in the protoplasm at the periphery of the cell, then gradually thrown off, after which they may still continue to grow. Simple as this is, it is extremely difficult to prove; for with this problem many others are associated to cause complications.
While there are a number of investigators who support the view of Flemming, there are also a number who oppose it. One of the most recent is Merkel, whose studies were upon the human umbilical cord. Merkel comes to the conclusion that the white fibers are formed in the intercellular substance, as taught by Kolliker. It is unnecessary to enter more into the literature of this subject, for it would only result in arranging the authorities into one group or another, or into an indefinite group. The literature has been collected recently in the article by Spuler and the reader interested in this side of the question is referred to it.
My own studies upon the development of connective-tissue fibrils began a number of years ago and my first results were published in 1891. Up to that time I could make but little headway with sections prepared in the ordinary way, and was compelled to use frozen sections and chemicals to analyze them. By using these methods alone I think all observers will also come to the conclusion I did at the time — that the connective-tissue fibrils are intercellular in their formation. Since that time, however, methods have been improved and I have gradually learned that the development of white fibrous tissue is better studied in the skin and superficial fascia of the embryo than in tendons, and that elastic tissue is better studied in the arteries and in elastic cartilage than in the ligamentum nuchas. I also have found that in their development the reticulum of the liver, the connective tissue of the cornea and cartilage are practically identical with that of white fibrous tissue. Very recently Dr. Sabin, Fellow in Anatomy at the Johns Hopkins University, has followed successfully the development of the reticulum of the lymphatic node. While ray results are now decidedly in favor of Flemming's view, the reader will soon see that if other methods and interpretations are employed (which I now consider false), it will be quite as easy to see the fibrils developing between the cells as within them.
This all brings me to a turning point, no doubt the key to the situation. The network of fibrils which forms Wharton's tissue, to employ the best known example, is composed of a mass of anastomosing cells, a syncytium, from which the connective tissues arise. Often this syncjtium is very sharply defined and differentiated, with nuclei and a little protoplasm which is less differentiated lying upon it. When differentiated to so great an extent it is very easy to designate the main portion of the syncytium as intercellular in position as well as in origin ; and since the connective-tissue fibrils arise directly from it they are of course intercellular in origin. When studying these structures in frozen sections it is quite easy to remove the nuclei, leaving only the fibrils of the syncytium. With improved methods, however, it can be shown that in later stages of the development of the syncytium the nuclei lie upon it and are therefore easily removed. In the earlier stages the nuclei lie within the syncytium, but at this time it is too delicate to isolate by the freezing method.
In very early embryos the mesenchyme is composed of individual cells which increase rapidly in protoplasm and then unite to form a dense syncytium. The protoplasm of the syncytium grows more rapidly than the nuclei divide, so that in a short time we have an extensive syncytium with a relatively small number of nuclei. In its form the syncytium appears as large bands of protoplasm with spaces between them filled at times with cells and at other times with fluid. The second condition we have in the umbilical cord of young human embryos. About this time the protoplasm of the syncytium differentiates into a fibrillar part, which forms ihe main portion of- the syncytium— the exoplasm — and a granular part, which surrounds the nucleus — the endoplasm. The fibrils of the exoplasm are very delicate and anastomose freely. When cartilage develops the exoplasm of the syncytium becomes denser and denser; the nuclei and endoplasm wander into the spaces of the exoplasm, which finally becomes semi-hyaline and takes the characteristic stain of the ground substance of cartilage. Eeticulum of the lymph node is easier studied, for here we have the least differentiated form, although the pictures are not so^ striking as they are in the development of cartilage. The development of the cornea is intermediate between reticulum and white fibrous tissue. In the development of membranous bone the process is similar to that of cartilage, only that the nuclei and endoplasm form the characteristic osteoblasts a little earlier and the ground substance deposited in the exoplasm does not stain with hgematoxylin. In the development of white fibrous tissue the nucleus and endoplasm lie upon the bundles of anastomosing exoplasm and in the course of time the anastomoses break and the exoplasm splits to give rise to the individual fibrils of white fibrous tissue.
The study of the development of elastic tissue is less satisfactory, usually, however, it develops in the middle of the exoplasm, the fibrils being extremely delicate at first, and anastomose from the beginning. Elastic tissue never develops by itself, but always in conjunction with some collagenous tissue, embryonic or mature.
Development of the Connective-Tissue Syncytium in the Tadpole
Through the kindness of Professor Harrison I am enabled to follow the formation of the connective-tissue syncytium from the mesenchyme in a set of perfect serial sections of the tadpole. The sections had all been stained in hrematoxylin and congo red and were cut 7i n thick.
In a tadpole 3 millimeters long the mesenchyme is well defined around the spinal cord and brain, on the ventral side of the head and around the chorda dorsalis. The individual cells are made up of large irregular clumps of protoplasm filled with yolk discs and pigment granules, which almost hide the nucleus. At times the cells are arranged in rows and two cells in apposition often appear to be Joined. This, however, is not frequent, and when they are united in this way they form only a syncytial rod, for they are never united with cells on all sides to form a syncytial spherule. The best place to study the mesenchyme in this stage as well as in its further development is around the anterior end of the chorda, for here it is quite transparent and a distinct group of cells can be easily followed from stage to stage. Here the cells are large and irregular, as shown in Fig. 1. When these cells are examined carefully with a 2 mm. oil immersion lens it is seen that the nucleus is almost obscured by yolk discs and pigment granules. The cell body itself is irregular in shape, running out into small elevations, or points, from which fine threads of protoplasm without pigment radiate. Occasionally one of these radiations reaches to and blends with a protoplasmic process from a neighboring cell. There is every indication of the beginning of an extensive syncytium formed by cells of the mesenchyme.
Fig. 1. Mesenchyme around the anterior end of the chorda of a tadpole 3 mm. long. Zeiss ob. 3 oc. 4 ( x 500 diameters). Hsematoxylin and con^o red.
Fig. 2. Mesenchyme around the anterior end of the chorda of a tadpole 4 mm. long (X 500 diameters). The mesenchyme forms an extensive syncytium.
That the cells of the mesenchyme unite is a well known fact and can easily be demonstrated in frozen sections, and in teased specimens of the umbilical cord. In addition I need only to refer to the description and illustrations of Flemming and of Spuler. Die mit einander vielfach in netzartigem Zuzammenhang stehenden Zellen des jSTabelstranges sind ueberwiegend spindelformig odor 3-4 zipfelig und sind an den Enden in feinsten Fibrillen aufgefasert, bald dicht an der Zelle, bald erst nachdem ein Fortsatz sich ueber eine langere Strecke bin kompact erstreckt hat." '
The syncytium as seen in tadpoles 3 mm. long progresses rapidly to form a definite tissue from which only connective tissues arise. The mesenchyme has already divided into at least two groups of tissues in the embryo, one destined, to form muscle and the other connective tissue. The syncytium destined to form the connective tissue, which I shall term the connective-tissue syncytium, begins to have its characteristic form at this time, and in its further growth it either remains as it is or gives rise to the connective tissues as we ordinarily understand them.
The point I wish to leave open is whether or not the mesenchyme was ever composed of individual cells. Was it not a syncytium throughout its development ? The most valuable and suggestive studies of His will have to answer this question for the present. At any rate, it is quite evident that the earlier syncytium, if it exists, is a very incomplete one, with very loose protoplasma bridges, easily broken and easity united to allow the cells to wander in all directions during the earliest stages of development. So it may be that the syncytium as seen in the tadpole 3 mm. long has existed ever since the appearance of the mesenchyme.
In a tadpole 4 mm. long the amount of mesenchyme, or connectivetissue syncytium, has increased a great deal around the brain, myotomes, etc. Around the anterior end of the chorda it is again very definite and can be studied better than elsewhere on account of its transparency. The main body of the cell mass has become decidedly multipolar in character and if anything, smaller than that in the embryo 3 mm. long. The yolk discs have largely disappeared while those remaining have become more transparent. On account of this change the nuclei are more distinct than in the earlier stage. The main cell body still contains many pigment granules. From each of the many poles of the cell fine threads of protoplasm arise, which divide once or twice, and anastomose into the same kind of threads from neighboring cells. In other words the multipolar cells form a complete syncytium. What now forms the main cell body gradually becomes a nodal point in an older stage of the syncytium. In this embryo we have mesenchyme pure and simple in the tail and a complete syncytium formed by the mesenchyme around the anterior end of the chorda. Between these there exist of course all intermediate stages.
In an embryo 6 mm. long no very great change has taken place in the development of the sj^ncytium (Fig. 3). The cells in the tissue around the anterior end of the chorda appear much as in the earlier stage, with the exception that the protoplasmic bridges between the cell bodies are somewhat thicker and have a slight fibrillar structure, forming the first exoplasm. There are also some vacuoles in each process which indicate that an individual bridge is widening and breaking up into a number of bundles. The yolk discs and pigment granules are about as numerous and as definite as in the embryo 4 mm. long. The mesenchyme of the tail is now in the form of a complete syncytium on both its dorsal and ventral sides.
In another embryo slightly larger than the one just described and just before the mouth breaks through, the connective-tissue syncytium is of different arrangement in different portions of the body. Around the anterior end of the chorda the protoplasmic filaments of exoplasm form a dense network of fibrils a little more advanced than in the embryo 6 mm. long. They are now arranged as bundles between which there are numerous spaces. The endoplasm around the nucleus, including its transparent yolk discs and pigment, is spreading over the fibrillar network of exoplasm. It appears as if the main mass of endoplasm around the nucleus is being drawn upon to form more of the fibrillar exoplasm of the syncytium in its further growth. The nuclei can now be plainly seen lying upon or within the dense masses of fibrillar exoplasm of the syncytium.
In front of the brain the cells of the mesenchyme are spindle-shaped and run out into fibrils of thicker bands of protoplasm which form a coarse network. In the mandibular arch all stages of embryonal connective tissue are seen, from single cells of mesenchyme, closely crowded together immediately below the ectoderm, to a complete syncytium lying deeper in the tissue. The single cells which are closely crowded undoubtedly form a growing point from which the syncytium arises. In the tail there is a very dense connective-tissue syncytium, more so than around the anterior end of the chorda. The nuclei are encircled with endoplasm which radiates over the exoplasm in all planes. Within the endoplasm there are imbedded numerous yolk discs and pigment granules; there are also some single yolk discs scattered throughout the exoplasm, especially in the neighborhood of the yolk of the embryo. This condition occurs before the circulation of blood is well established, and indicates that the nutrition of the syncytium of the tail is carried on in part by the inwandering of cells from Uie yolk of the ernbryo.
Fig. 3. Mesenchyme around the anterior end of the chorda in a tadpole 6 mm. long ( X 500 diameters).
Fig. 4. The same in a tadpole 9 mm. long.
In a stage somewhat older than the one just described, just after the mouth has broken through, the connective-tissue syncytium around the anterior end of the chorda is practically completed. Most of the nuclei, with a small amount of endoplasm around them, lie upon the exoplasm of the syncytium at its nodal points. Within the head in front of the brain the exoplasm has increased markedly in quantity, by an addition to it from the mesenchyme cells at the growing point as well as by a multiplication of the cells of the finished syncytium. The same is true of the syncytium on the ventral side of the head. In the tail the connective-tissue syncytium forms a very dense network of exoplasm with nuclei and a small amount of endoplasm lying upon, or imbedded within, some of the nodal points. The endoplasm about the nuclei form stellate bodies with their points running out into the general mass of exoplasm. Minute pigment granules, often in rows, are distributed throughout the syncytium.
In general we have here a stage similar to that described by Flemming and by Spuler, and what I have stated above confirms' the work of these investigators, though it formulates it somewhat differently.
The connective-tissue syncytium is practically complete in embryos 6 mm. long. In its further development it spreads and enlarges to form the general framework of the body. From now on there differentiate from it, with the exception of the chorda, the permanent connective tissues of the body, i. e., the skeleton, ligaments, tendons, true skin, etc.
Before discussing these tissues I shall describe the general arrangement of the syncytium in a tadpole 9 mm. long after the cartilages are beginning to form. In this embryo the syncytium around the anterior end of the chorda is again fully developed, with a difference, however, in the shape of the nuclei and endoplasm around them (Fig. 4). They are now spindle-shaped, lie upon and are connected with the exoplasm of the syncytium. In the course of time the nucleus and its endoplasm separates itself from the exoplasm of the syncytium, which is gradually converted into connective-tissue fibrils. The syncytium in front of the brain of the embryo is formed of bundles of anastomosing exoplasm with nuclei at some of the nodal points (Fig. 5). Each nucleus has a small quantity of endoplasm around it, forming a spindle-shaped mass which runs out into points to be lost in the exoplasm of the syncytium. In specimens of this kind it is easy to view these cells with their endoplasm as the connective-tissue cells and the exoplasm of the syncytium as the intercellular substance were not the development of the syncytium taken into consideration. Within the syncytium certain of the fibrils are more sharply defined than the rest, which indicates that in addition to the shifting of the nucleus and its envelope of endoplasm there is already some differentiation Avithin the exoplasm. The syncytium in the ventral side of the head is much like that just described. As this is followed towards the tail there is a gradual transformation of the arrangement of the bundles of exoplasm into an extremely dense network. In the tail the endoplasm around the nucleus forms a stellate mass with fibrils from the points running over into the fibrillar exo])lasm of the main body of the syncytium (Fig. (3). Within the exoplasm there are some fibrils more sliarply defined than the rest, which often appear to be composed of rows of extremely minute granules.
When the connective-tissue syncytium is fully developed in the tadpole it shows practically all of the characteristics found in mammalian embryos. I have made numerous chemical tests with the syncytium in the embryo pig, as an abundance of this material is constantly at my disposal. The tests were made with various stains, and digestive ferments npon sections which had been cut in paraffin. Frozen sections were also used a great deal, with more or less satisfactory results, to control the above, and to test with acetic acid, caustic potach, pancreatin, and pepsin.
Fig. 5. Connective-tissue syncytium just below the ectoderm in tbe anterior part of tlie head of a tadpole 9 mm. long ( x 500 diameters.)
Fig. 6. From the tail of the tadpole from which Fig. 5 was drawn.
The Connective-Tissue Syncytium in the Pig
The connective-tissue syncytium is fully developed in the embryo pig from 9 to 12 mm. long. At this time it corresponds wdth that of the tadpole 6 mm. long. In the greater portion of the embryo, however, the syncytium is pretty well obscured by its numerous nuclei with the exception of that in the skin, around the brain and on the dorsal side of the heart and lung. In these regions it is formed of an extensive network of exoplasm with nuclei at the nodal points. In other words, there are multipolar cells with anastomoses of their prolongations. At this time the nuclei are oval in shape, without the surrounding endoplasm as in the tadpole. At certain points there are indications of a beginning of a differentiation into cartilage and into white fibrous tissue, but beyond this there is the simple syncytium.
All the above may be seen in ordinary thin sections stained with acid fuchsin, but it is better shown in specimens stained with haematoxylin and Congo red. My best specimens Avere obtained by staining the sections with Mallory's connective-tissue stain, which tinges the nuclei and surrounding endoplasm, if present, slightly red and the exoplasm of the syncytium intensely blue. We have modified this stain somewhat by omitting the water and intensifying the bhie. The method now employed in our laboratory, for which we are indebted to Dr. Sabin, is as follows :
Specimens hardened in Zenker's fluid are cut in paraffin and fixed on the glass slide by the water method. They are then stained with fuchsin, yV psr cent, until they take up a proper amount of color and then without washing are fixed for a few minutes in a saturated aqueous solution of phosphomolybdic acid diluted ten times. They are next washed in alcohol, 95 per cent, and stained a very short time in the following solution: Aniline blue soluble in water, 1; orange G,, 2; oxalic acid, 2; boiling water, 100. Next they are washed in alcohol, 95 per cent, blotted, cleared in xylol, and mounted in Canada balsam.
With this modification there seems to be no difficulty in obtaining an excellent double stain in nearly all cases, which is not so with the ordinary Mallory stain. Washing the sections with water has a tendency to remove the red and this is obviated to a great extent by substituting alcohol. The blue in this modification is strengthened in order that the section need not remain in the aqueous blue stain long enough to remove the red. Successful specimens are especially valuable to trace out the exoplasm of the syncytium which is somewhat matted together when stained with hfematoxylin and congo red.
Digestion of the Syncytium in Pancreatin and Pepsin
It is extremely difficult to obtain satisfactory results by digesting sections of embryos in either pancreatin or pepsin. If the test is made with frozen sections the pancreatin causes them to swell into a transparent slimy mass, dii'Ecult to handle or to stain in any very satisfactory way. In pepsin the section becomes firmer, opaque, brittle, and it must be crushed to separate the nuclei in order to study the syncytium, in case it is not digested. Quite satisfactory results are obtained by digesting sections of embryos, which have been attached to glass slides by the water method. It is of course necessary to use sections from embryos which have been hardened in alcohol, in order to obtain results similar to those obtained from frozen sections, Not only does this statement apply to embryos but to tissues in general. With pancreatin, however, the digestion upon the glass slide is very unsatisfactory, for the alkali in the solution nearly always detaches the sections, probably on account of the great amount of mucin in them. The younger the embryo the more difficult it is to retain the sections upon the glass slides.
Not only is it difficult to obtain fairly good sections which have been digested, but there is in addition the complication of unequal as well as contradictory results. When one point appeared to be worked out in a satisfactory manner, later tests contradicted it, and so on. It is therefore with some hesitancy that I give the tests with digestive ferments upon the connective-tissue syncytium.
In general it is quite certain that when the main mass of the syncytium is formed of exoplasm it is digestible in pancreatin and bicarbonate of soda. This treatment causes the section, if fresh, to become a swollen and slimy mass in which the delicate fibrils can be seen after it is treated with picric acid. The ground substance of the cartilage, if present, is well isolated and the more developed fibrils of the perichondrium can also be seen. It appears that the more the syncytium is differentiated the more it resists pancreatic digestion. In case a section of an older embryo is digested upon the glass slide it will be found that at the end of 3-4 hours all of the nuclei are dissolved, while all of the fibrils of the exoplasm of the S5^ncytium, the white fibrils and the ground substance of the cartilage remain. In a section of this kind, which includes the umbilical cord, all stages of the syncytium can be studied; that in the cord is not differentiated, while that toward the back is changed into white fibrous tissue and cartilage. In the aorta there are at this time numerous elastic fibers. With Mallory's stain it is shown that a beautifuL network of fibers alone remains, the nuclei having begn digested in the pancreatin. Furthermore, it is shown by staining with Weigert's elastic tissue stain that the elastic fibrils have also been dissolved. The above-named tests were made many times on embryos from 7 mm. to 20 cm. in length. The older the tissue the easier it is to isolate fibrils by digesting in pancreatin and bicarbonate of soda.
The action of pepsin and hydrochloric acid upon the connective-tissue syncytium appears to be just the opposite of that of pancreatin. The younger the syncytium the more ditticult it is to digest it in pepsin. A section of a young embryo becomes opaque and shrinks a little when placed in dilute hydrochloric acid and pepsin. It is shown by examining it with the microscope at this time that the nuclei are opaque, and the white fibrous tissue, if present, has become transparent. After the sections have been kept in the digestive fluid for a few hours at 37° C. they still remain opaque and are somewhat elastic, for pressing a section under a covergiass only spreads it but does not break it. When stained with magenta the delicate fibrils of the syncytium are easily recognized. When the sections are digested for 34 hours or longer they usually fall into granules, showing that the syncytium is fully broken up. This is especially the case if the sections are from embryos 10 cm. long or longer. It is only in the smaller embryos that the syncytium is well developed and in them we are to make the valuable tests. The sections of an embryo 5 cm. long were still opaque at the end of 48 hours, with considerable elasticity, indicating that the ' syncytium must be present in considerable quantity. Furthermore, some fibrils could be seen from time to time in bits of crushed sections. The cartilage and white fibrous tissue of the perichordium resisted the action of the pepsin for 24 hours, but at the end of 48 hours they were fully dissolved.
Sections of embryos hardened in alcohol can be easily digested upon the glass slide and then stained with Mallory's connective-tissue stain or with Weigert's elastic-tissue stain. If the digestion is continued 24 or 48 hours usually all of the connective tissue and syncytium are dissolved, leaving only broken cells to outline the structures of the body. When the digestion is not complete it is found that usually the white fibrous tissue is dissolved first, then the cartilage and then the syncytium. A section through the body of an embryo, including the umbilical cord, has in it from the ventral to the dorsal side all stages of the syncytium in the process of differentiation. When such sections are digested to a proper degree, usually from 24 to 36 hours, it is often found that the white fibrous tissue of the body wall and back are dissolved while the syncytium of the cord is almost entirely intact; the cartilages are destroyed only in part.
It appears then that the connective-tissue syncytium is more resistant towards the action of pepsin than is white fibrous tissue. The more mucoid, that is, the younger, the syncytium is the more resistant it is toward the action of pepsin. The action of pancreatin is in a measure the opposite.
Cartilage appears as a band of condensed tissue on either side of the head of tadpoles just before the mouth breaks through. In the region destined to become cartilage the nuclei of the connective-tissue syncytium become first slightly enlarged, for nuclear figures are here more numerous. The eudoplasm around the nuclei extends rapidly, and due to the multiplication of nuclei now fills the entire space and partly obscures the exoplasm of the syncytium. Where the endoplasm passes over into the exoplasm there is now a sharp line of demarcation making it appear as if the capsule of the cartilage cell were forming.
In tadpoles about 6 mm. long, immediately after the mouth has broken through, the nuclei of the precartilage are surrounded by a solid mass of endoplasm, thus filling up all the space between them. Where the nuclei are more separated the exoplasm is at the periphery of the endoplasm, but where the nuclei are packed together the exoplasm is wholly obscured. The endoplasm stains quite intensely with Congo red, not more so, however, than the protoplasm of other cells.
A tadpole 9 mm. long has in it slender bands of cartilage fully developed from which the ground substance is directly continued into the exoplasm of the surrounding syncytium (Fig. 7). The best pictures are found at the tips of growing cartilage which are being added to by a transformation of the neighboring syncytium. Where the border of the cartilage is sharply defined the transition into the surrounding tissue is not marked, for its boundary line is obscured by a layer of flat cells. In a suitable specimen it is seen in passing from the syncytium over into the cartilage that the nuclei gradually become more and more crowded and the bundles of exoplasm become smaller and smaller. The nuclei gradually come to lie in the meshes formed by this exoplasm, that is, they have been extruded from the syncytium. With this change of relations between the nuclei and the exoplasm there is an increase of the endoplasm which now fills the meshes, encroaches upon the exoplasm, and partly obscures it. At this time the endoplasm of the syncytium stains with congo red, but as the finished cartilage is approached, the nuclei and surrounding endoplasm are separated by delicate lines or fibrils of exoplasm, which stain with hgematoxylin. These lines now widen, stain more intensely with hgematoxylin, and form the ground substance of the cartilage. The endoplasm becomes clearer and clearer, separates from the ground substance, and finally encircles the nucleus only, leaving a space between it and the ground substance. In other words, we have a thickening and transformation of the exoplasm of the syncytium to form the ground substance of the cartilage, while the nuclei and endoplasm of the syncytium become the cartilage cells. This statement is found to be true in following the development of cartilage from embryo to embryo as well as in the transformation of the connective-tissue syncytium when cartilage grows into it.
The very early change in the syncytium preceding the formation of cartilage is much more easily followed in tadpoles than in mammals, the pig, for instance. On the other hand, when the cartilage is once formed its further growth is better studied in pig's embryo.
Fig. 7. Transition between cartilage and syncytium in a tadpole 9 mm. long. ( X 350 diameters). Hsematoxylin and eosin.
Fig. 8. Beginning of the occipital cartilage in a pig's embryo 16 mm. long ( x 250 diameters). Mallory's stain.
Fig. 9. Transition between the syncytium and cartilage in an embryo pig 24 mm. long. The specimen had been macerated in Miiller's fluid 24 hours before it was hardened in alcohol ( x 250 diameters). Hsematoxylin and congo red. The central dark zone stained with hematoxylin while the zone of ground substance between it and the syncytium only took the congo red.
The beginning of the formation of cartilage can be recognized in pig's embryos from 10 to 15 mm. long, in the condensed mass of cells around the chorda. When sections which have been stained in acid fuchsin are studied the connective-tissue syncytium can be followed to the areas of precartilage, but not into them, for the numerous nuclei obscure entirely the exoplasm of the syncytium. In sections stained by Mallory's method the beautiful and definite exo^olasm of the syncytium can be followed to the precartilage, and between its numerous nuclei. As this tissue is followed from the region of the spinal cord towards the chorda it is found that exoplasm becomes denser and denser and the nuclei somewhat larger with numerous karyokinetic figures. In the neighborhood of the chorda the exoplasm of the syncytium is so dense that it appears as a granular mass between the nuclei. The sheath of the chorda is stained intensely blue. The endoplasm which is quite marked around the nuclei of the syncytium near the spinal cord gradually disappears as the chorda is approached. By most methods of hardening the nuclei of the precartilage become so packed that the exoplasm of the syncytium is entirely obscured. Especially is this true in the development of the cartilage of the arm. In an embryo pig, 12 mm. long, which had been macerated in Miiller's fluid for 24 hours, washed in water, then in alcohol, stained in hiematoxylin and congo red, the precartilage of the arm could also be analyzed. The nuclei of the precartilage are all surrounded with a continvious mass of protoplasm, stained red, which is directly continuous with the exoplasm of the neighboring connective-tissue syncytium. In specimens which have been macerated it is very difficult to separate the endoplasui from the exoplasm of the syncytium. Therefore between the nuclei there is one continuous mass of protoplasm practically of the same structure.
In pigs' embryos from 15 to 20 mm. long the cartilages of the vertebral bodies are well developed and are pretty sharply defined. In sections treated with Mallory's stain it is found that on the dorsal side of the bodies of the vertebrae the ground substance of the cartilage is directly continuous Avith the exoplasm of the connective-tissue syncytium. By all odds the best place to study the early development of the cartilage is in the occipital cartilages, which lie on either side of the dorsal middle line. At this time the exoplasm of the connective-tissue syncytium in the region of the occipital precartilage is in the form of sharpened bands encircling definite openings, some of which contain nuclei (Fig. 8).
In a pig's embryo a little over 2 cm. long the main cartilages of the body are all well developed, and in this specimen we obtain the best pictures showing the relation of cartilage to the connective-tissue syncytium. Again, the occipital cartilage shows all the transitional stages from the completed ground substance to the exoplasm of the syncytium (Fig. 10). Passing from the ectoderm of the embryo towards the cartilage it is seen that the main spaces between the exoplasm of the syncytium become gradually smaller and smaller, with the nuclei shifting into them as the cartilage is approached. This takes place before any true ground substance is deposited, as the figure shows. Next we reach a zone in which the exoplasm has ground substance deposited between its fibrils. Finally, when the cartilage is complete, the fibrils of the exoplasm are entirely obscured by the ground substance.
Sections stained by Mallory's method show that the endoplasm is almost wanting around the nuclei of the syncytium immediately below the ectoderm. Practically none is seen until the nuclei shift into the spaces between the exoplasm in the neighborhood of the cartilage. In this region the nuclei are larger than those more distant and in the region of the completed cartilage both nuclei and endoplasm are several times as large as in the surrounding syncytium. When the cartilage is fully developed the relatively large granular nuclei are encircled with vacuolated endoplasm. Each nucleus and endoplasm is encircled by a transparent space separating it from the surrounding exoplasm or ground substance. In specimens which have been macerated in Miiller's fluid for a day, then washed and hardened in alcohol, and stained with hsematoxylin and congo red, it is seen that no space exists between the endoplasm about the nucleus and the ground substance. By this method the endoplasm becomes more marked and the ground substance less marked than in the specimens hardened in Zenker's fluid and stained by Mallory's method.
Specimens of developing cartilage macerated as described above show at the jimcture of the cartilage v;-ith the connective-tissue syncytium a zone of ground substance which will noL stain with hgematoxylin, but tinges with congo red (Fig. 9). Passing from the surrounding syncytium into the developing cartilage, the nuclei become larger, the exoplasm increases, condenses and obscures the endoplasm. Gradually the fibrillated exoplasm becomes granular, making it appear as if the nuclei were imbedded in a continuous granular mass. This all seems to be due to the maceration in Mliller's fluid. On the periphery of the cartilage there is a zone of ground substance which does not stain with hematoxylin but tinges with congo red (Fig. 9). This zone is of the width of one or two nuclei which are surrounded with some endoplasm. The completed ground substance, which stains with hgematoxylin, begins quite abruptly; the nuclei are encircled with a considerable quantity of endoplasm, filling almost entirely the spaces in Avhich they lie.
The definite conclusion to be drawn from the above specimens is that the ground substance of the cartilage is deposited directly into the exoplasm of the syncytium and its nuclei and endoplasm become the cartilage cells.
Not only can the direct connection between the ground substance and the exoplasm be seen in the occipital cartilage, but also at the dorsal side of the bodies of the vertebrjB, the petrous portion of the temporal cartilage, and occasionally in other cartilages of the head as well as in the ribs. Otherwise the boundary line between the cartilage and the surrounding connective-tissue syncytium is quite sharp and is obliterated by the dense tissue and nuclei of the perichondrium. Specimens stained by Mallory's method are the best by all odds for studying the transition of syncytium into cartilage, for in them the ground substance and exoplasm are stained intensely blue, while the nuclei and endoplasm are shrunken and tinged red.
After the cartilage is once well formed its further growth is interstitial as well as peripheral. Not only do the nuclei divide, but the ground substance increases out of proportion, forcing the nuclei apart. This is beautifully illustrated in the sheath of the chorda, which is gradually incorporated with the vertebral cartilage. Often the thickened sheath appears as a great stump with its roots extending out into the cartilage.
In an embryo 3 cm. long the occipital and petrous cartilages have extended greatly, but still show beautifully the connection of the ground substance with the exoplasm of the syncytium. The transitions from the nuclei, endoplasm and exoplasm, into cartilage are again as distinct as in embryos 2 cm. long. This indicates that the syncytium at the peripher} is being changed into and added to the cartilage already formed.
In older embryos the cartilage becomes separated more and more from the surrounding syncytium, Avith the exception where the cartilage is still extending into it. In an embryo 5 cm. long this condition is still present in the occipital cartilage and in the sternum. The borders of the vertebra and most of the other cartilages are well defined and are beginning to ossify at different points.
The frontal and mandibular are the first membranous bones to appear in embryos about 2 cm. long. In smaller embryos no indication of the formation of bone can be seen. Sections through the frontal region of embryos 2 cm. long stained by Mallory's method shoAV that the bone begins by a very blue zone of hyaline deposit in the exoplasm of the connective-tissue syncytium (Fig. 11). The deposit appears to be equally distributed throughout the exoplasm within this zone. The nuclei stain somewhat more intensely than those of the surrounding syncytium, and the endoplasm around them is increased in quantity. Nuclei and endoplasm now show all the characteristics of osteoblasts and may be considered such. Sections certainly show definitely that when the syncytium turns into bone the nuclei become more sharply defined, the endoplasm increases greatly in quantity, and the bone substance is either transformed exoplasm or is deposited into it. This latter process is first marked by the fibrils of the exoplasm becoming sharper and staining more intensely blue than before. Soon, however, the substance between the fibrils of the exoplasm takes up the blue stain, making it appear as if the tissue were injected with a blue color. In fact I thought for a long time that in this region there was an extravasation which stained blue, until I recognized the osteoblasts in older stages. Furthermore, the extravasation proved to be constant and took on the characteristic bone stain when treated with hfematoxylin and eongo red. The gradations in color from hferaatoxylin to congo red are in the order, nuclei, endoplasm, exoplasm, and bone substance.
The frontal and mandibular bones have increased in size in embryos 3 cm. long, the bone deposit is beginning to radiate into the surrounding exoplasm, and the osteoblasts are larger and more numerous along these radiations. The bone radiations are still more pronounced in embryos 5 cm. long and are lost in the prefibrous tissue which is now arising from the surrounding exoplasm.
Fig. 10. Section through the occipital cartilage of au embryo pig 20 mm. long ( X 250 diameters). The ground substance is deposited in the exoplasm of the syncytium.
Fig. 11. Section through the frontal bone of a pig 30 mm. long ( x 250 diameters).
Most instructive specimens showing the - extension of membranous bone as well as of the beginning of periosteal ossification are obtained from embryos 5 cm. long, stained by Mallory's method. In such specimens it is seen that the frontal bone is well started, with bone corpuscles imbedded within it, and osteoblasts encircling it. The bone substance with its radiations stains intensely blue, and it is seen that the radiations, with the accompanying osteoblasts, extend into the membranous skull and are gradually lost in the exoplasm and prefibrous tissue. At the extreme tips of the bone radiations the scattering fibrils are surrounded with osteoblasts. At the outer border of the zone of osteoblasts, where there are transition forms between them and the nuclei of the syncytium, there are heavier fibrils of bone radiations between the nuclei. Passing from these fibrils towards the finished bone it is seen that they soon unite to form bundles which are soon stuck together to form the apparently homogeneous bone substance. After the bone is once well formed the primitive Haversian canals are filled with osteoblasts and a core of connective-tissue syncytium in which course the blood-vessels.
The difference in appearance between the beginning of bone in embryos 3 and 5 cm. long is probably due to the perfect syncytium in the former and the syncytium differentiating into prefibrous tissue in the latter. In the first the bone is deposited directly in the exoplasm, while in the second the exoplasm is first partly changed into prefibrous tissue and then into bone.
There are marked changes in the cartilages of embryos 5 cm. long, preparatory to their ossification. The shafts of the clavicle and ribs are encircled with a shell of bone and the transverse processes of the vertebra are beginning to ossify. In suitable sections of the latter it is seen that the perichondrium is thickened and filled with osteoblasts. Not only do the osteoblasts appear to be arising from the nuclei of the perichondrium, but also from the outer layer of cartilage cells. Between the osteoblasts the bone fibrils first appear; they stain intensely blue with Mallory's stain, more so than the ground substance of the cartilage, extend throughout the perichondrium and extend somewhat into the cartilage. In order to separate the bone fibrils from cartilage it is necessary to stain sections with hsematoxylin and congo red. Such sections show that the greater part of the bone is first deposited in the perichondrium, and only a small amount, if any, in the ground substance of the periphery of the cartilage, in periosteal ossification.
So it appears that in periosteal ossification the connective-tissue syncytium changes partly into cartilage and partly into white fibrous tissue before it gives rise to bone. Possibly the study of some suitable sections from embryos smaller than 5 cm. will give results identical with those obtained for the frontal bone, but so far I have been unable to obtain such sections.
White Fibfous Tissue
In the study of the development of cartilage and bone definite spots can be located and followed from stage to stage. To do the same with white fibrous tissue and the other connective-tissue fibrils is much more difficult. Finally, after trying many regions, I settled on the development of the connective tissues in the skin on the dorsal side of the body, between the two shoulder blades. Here there is the underlying broad trapezius, which marks a region in the section of the skin on one side with the epidermis on the other. Some 50 stages of this region were cut from embryos measuring from 1 to 30 cm. AIJ: important stages were hardened in Zenker's fluid, stained by Mallory's method, as well as by other methods. A parallel set of specimens was also made by macerating them in Midler's fluid for 24 hours, washing, hardening, etc., then staining in hsematoxylin and congo red. All the stages were also frozen, cut and examined fresh, then treated with dilute acetic acid and with caustic potash, after which they were stained with magenta and other aniline dyes. While the specimens stained by Mallory's method give the most definite and permanent preparations, the macerated and fresh preparations give an excellent control. Mallory's method stains pretty much all connective tissues in the embryos, while with macerations and digestions there is some differentiation.
It is shown by means of digestion in pancreatin that the earliest definite fibrils in the syncytial exoplasm are resistant in fresh specimens as well as in hardened specimens which have been fixed upon the glass slide. In acetic acid and in caustic potash the exoplasm and its collagenous derivatives become transparent in embryos 25 mm. long. In larger embryos there is a residual syncytium which resists acetic acid, even when boiled in it for hours, and probably is related in some way to yellow elastic tissue. This will be considered later.
In general the connective-tissue syncytium is fully developed in embryos 15 mm. long. It is practically of equal density throughout the skin. The nuclei are mostly round or somewhat oval, usually quite naked or with only a small amount of endoplasm around them. The exoplasm is very delicate, with a slight amount of fibrillation.
In an embryo 2 cm. long the connective-tissue syncytium has begun to differentiate; many nuclei are oval in shape and they are enveloped with an increased quantity of endoplasm, which runs out on either side of the nucleus, forming two poles— the well-known connective " tissue cells " of the embryo. The nuclei lying just below the ectoderm have the least quantity of endoplasm around them. As the muscle lying under the skin is approached the nuclei increase in number, forming quite a dense layer over it. Analyzing this layer by means of Mallory's stain shows that it is formed of syncytium which is drawn out parallel with the long axis of the body. The fibrillated exoplasm tends to form parallel bundles which anastomose quite frequently with one another. In this region the nuclei are markedly spindle-shaped, Avith the surrounding endoplasm running out into two poles to be lost in the exoplasm.
Digesting the syncytium and the prefibrous tissue in pancreatin shows that they resist its action to a marked extent. In order to obtain any satisfactory result the digestion must be mild, i. e., for a short timeat room temperature. Great quantities of resistant white fibers cannot be obtained from the skin by means of digestion in pancreatin until the embryo is about 15 cm. long. Although no elastic fibers can be demonstrated in the skin of embryos 15 cm. long, frozen sections of it will resist boiling acetic acid for a very long time. In embryos 25 mm. long the first prefibrous tissue of the perimysium resists pancreatin more than the remaining syncytium. The fibrils of the prefibrous tissue also swell in dilute actic acid. These reactions, together with the position of the tissue in question, make it very definite that the changes in the syncytium immediately over the muscle mark the beginning of white fibrous tissue.
The prefibrous tissue of an embryo 2 cm: long is formed of anastomosing fibrils which are in direct connection with the exoplasm lying between the perimysium and the ectoderm. Immediately below the ectoderm the meshes of the exoplasm are smaller, the syncytium thus forming a more compact felt upon which the epithelium rests.
In embryos 3 cm. long the perimysium is a little more compact and sends reflections between the muscle fasciculi. The layer immediately below the ectoderm is a little more extensive than before, while between it and the perimysium the syncytium is quite typical. The two zones of altered exoplasm have approached each other in an embryo 4 cm, long, leaving a narrow zone of typical syncytium between them, within which the first lymph channels have appeared.
In the next stage, 5 cm. long, all of the exoplasm of the syncytium of the skin has changed considerably, being more fibrillated, with some of the fibrils staining more intensely than the rest (Fig. 13). The prefibrous perimysium is advanced one step more now, being composed of layers of fibrillated exoplasm, the layers anastomosing between themselves, and the fibrils within a given layer forming a dense network. True white fibrous tissue is not yet present.
The prefibrous tissue has extended still more in an embryo 7 cm. long. Its development is most advanced in the perimysium, where the individual fibrils are beginning to become wavy. In a transverse section of the body it is seen that the development is most advanced in the neighborhood of the vertebral column and least in the umbilical cord. From without inward the permysium is most developed, diminishing in the intermuscular septa, ligaments, superficial fascia, and cutis, the least development being immediately helow the epidermis. Even here it is no longer typical syncytium, but partly differentiated. The process continues in embryos 9, 10 and 12 cm. long; most of the prefibrous tissue is still within the syncytium. Immediately over the muscle a further differentiation of the prefibrous tissue of the perimysium has taken place. In this narrow zone the fibrils are arranged in parallel bundles apparently communicating with one another as well as being continuous with the neighboring exoplasm. In this region the individual fibrils anastomose with one another. Prefibrous tissue is changing into fibrous tissue very rapidly in an embryo 16 cm. long. The perimysium, composed of parallel fibers, sends processes of wavy fibers into the superficial fascia, and from them fibers enter the cutis. All of the tissue between the epidermis and the underlying muscle is composed of these wavy fibers, either isolated or connected with the exoplasm, which is very fibrillated. The process is quite complete in embryos from 20 to 30 cm. long. In the older embryos, however, the density of the fibers is greater in the skin than in the underlying superficial fascia.
Fig. 12. Section through the skin of a pig 5 cm. long ( x 250 diameters). The first white fibers are just forming in the exoplasm.
Fig. 13. Section through the skin of a pig 16 cm. long ( x 250 diameters). The nuclei and endoplasm on the left are immediately below the root of a hair.
The individual fibrils after once well formed are of unequal size, often appearing in bands and frequently anastomosing. The anastomoses are finally broken and the bands and thicker fibers split into the individual fibrils.
I have now followed the development of the white fibers from the exoplasm of the sj'ncytium without considering the nuclei and the endoplasm. What follows relates to them.
In smaller embryos (2 cm.), which have been stained by Mallory's method, the nuclei are round or oval with a small amount of endoplasm around them. When the tissue is macerated in Miiller's fluid for 24 hours before cutting, the amount of endoplasm around the nucleus is greatly increased, showing that by Mallory's method it becomes , shrunken. x\s the embryo grows the endojDlasm increases in quantity until in specimens 5 cm. long it forms a spindle-shaped mass around the nucleus, the tips of which run out into the exoplasm and are lost. Hajid in hand with the expansion of the exoplasm the endoplasm continues to grow until in embr^^os 10 to 13 cm. long it is ditl'erentiated to correspond with that of the exoplasm. In the neighborhood of the first white fibers the nuclei and endoplasm are arranged in rows, while, where the exoplasm is changing into prefibrous tissue, they are as before. More towards the epidermis the spindle-shaped endoplasm is much larger, indicating that it is also active in the conversion of exoplasm into prefibrous tissue. At this time the hairs are beginning to develop and below their roots the nuclei are multiplying and accumulating, apparently preparing much new endoplasm and exoplasm at these points (Fig. 13). At any rate, in larger embryos (15-20 cm.), there are islands of new syncytium at the roots of the embryonic hairs, making it appear as if the soft syncytium is present at these points to enable the hairs, in their further growth, to sink into the skin with greater ease. In the rest of the skin the embryonic white fibers, the prefibrous tissue, and fibrillated exoplasm are accompanied with nuclei surrounded with a spindle-shaped mass of endoplasm, Not only are all of these stages seen in the skin, but also between the radiations of embryonic white fibers from the perimysium into the superficial fascia.
After the activity of the nuclei and endoplasm has produced enough exoplasm to give rise to all the white fibers of the skin, which is the case in embryos from 20 to 30 cm. long, they cease to be so prominent and sink back into the form of irregular cells. Around the roots of the hairs there are still the islands of quite typical syncytium. Probably in both the scattered cells (nuclei and endoplasm) as well as in the islands of syncytium we have forces which can develop new white fibers, should circumstances so demand. The syncytium at the roots of the luiirs undergoes a further differentiation in the development of elastic tissue, which I shall take up presently.
It appears then that the connective-tissue syncytium grows rapidly before it gives rise to white fibrous tissue. The nuclei multiply, the endoplasm becomes larger, the exoplasm increases absolutely and relatively in quantity. The nuclei and endoplasm form the well-known bipolar cells, the tips of which rvm into and are lost in the exoplasm, » making it appear as if the exoplasm were spinning its fibrils from the granular endoplasm. Soon the fibrillated exoplasm is drawn out intobundles, the bands between them beginning to break, thus forming the prefibrous tissue. The process of drawing out continues and the prefibrous tissue is changed into embryonic white fibers, which at first are irregular in size and anastomose occasionally. In the further development the bridges break and the thicker fibers split into the individual fibrils of white fibrous tissue.
The first white fibers appear in the perimysium, then they, grow as radiations into the superficial fascia and cutis. Not only do the nuclei of the syncytium multiply, but the exoplasm increases much out of proportion. This continues in the prefibrous and embryonic fibrous tissues by stretching, widening, and splitting the individual fibrils.
The reticulum of the lymph node is developed directly from the connective-tissue syncytium, and is probably the least differentiated of the connective tissues. This view has been advanced by writers, most recently by Waldeyer. In its differentiation it begins much like white fibrous tissue and when fully developed is about as far advanced as the tissue I have termed prefibrous. When white fibrous tissue and reticulum develop side by side it is impossible to separate them in their early stages, but when the early development of the perimysium is compared with the develojjment of reticulum of a lymph node it is noticed that the arrangement of the fibrils is different, although their development is parallel. In the liver the reticulum develops from Kupffer's cells.
The development of rhe reticulum of the lymph node is now under investigation by Dr. Sabin who has given me the following resume with the permission to publish it. " The lymph node has just appeared as a plexiform mass of lymph ducts in embryo pigs 4 cm. long. These duets can be injected from more distant lymph channels and within the node they are relatively large and are separated from one another by bridges of tissue, or primitive lymph cords. The lymph cords are composed of a syncytium of delicate bands of exoplasm, with oval nuclei surrounded by spindle-shaped endoplasm. In addition there are many round cells which lie in the meshes — the first lymph cells. By the time the embryo is 10 cm. long the lymph node is one millimeter in diameter.
The whole node is composed of a delicate syncj'^tium which now shows all of the characteristics of a fnliy developed reticulum, with many nuclei and endoplasm lying upon it. The meshes are partly filled with lymph cells. At the surface of the node the reticulum is continuous with the syncytium of the surrounding tissue. That there is a continuous network is best seen in sections stained by Mallory's method, which also show that the meshes are smaller and the fibrils are more delicate than those of the surrounding syncytium.
" The node has grown to be 3 mm. in diameter in embryos 20 cm. long. Each node is now surrounded with a delicate capsule of prefibrous tissue, and the reticulum, prefibrous tissue and surrounding syncytium form one continuous network. Upon the reticulum there are but few spindle cells and within the meshes there are many lymph cells."
From the above description it is seen that the reticulum develops directly from the exoplasm of the syncytium, while the nuclei and endoplasm are converted into cells which lie upon the reticulum fibrils. After the node is outlined the surrounding syncytium develops into prefibrous tissue to form the capsule.
The study of sections of the pig's intestine stained by Mallory's method shows definitely that both white fibrous tissue and reticulum are developed directly from the syncytium lying between the muscle wall and the epithelium. In embryos 20 cm. long there are small villi and rudimentary crypts present, but there is no marked muscularis muscosfB to separate the submucosa from the mucosa. There is no line of demarcation between the reticulum of the mucosa and the white fibrous tissue of the submucosa, more than a few scattered muscle cells of the muscularis mucosae. The tissue around the bases of the embryonic crypts is fibrillated, wavy, and generally parallel with the muscularis mucosa3, stains more intensely and corresponds with the prefibrous tissue found elsewhere. From this layer there are gradual gradations towards reticulum in the villi on one side to a less developed white fibrous tissue in the submucosa ou the other side. The degree of development of the layer of prefibrous tissue of the intestine is about the same as that of the skin of the same embryo.
The results here given suggest very much that reticulum represents an embryonic form of white fibrous tissue. That these two tissues blend and arise from a common syncytium does not speak for their identity any more than it does for the identity of either cartilage or bone with white fibrous tissue. As the matter now stands all of these tissues, including that of the cornea, are to be classed as collagenous, but still as distinct tissues. I have recently given the reasons for classing reticulum as a separate tissue and will not enter upon the discussion " of this subject at present. At any rate, if these reasons are overcome, reticulum will remain as peculiar white fibrous tissue not fully developed, in case we can consider any tissue in the adult body as embryonic.
In examining various tissues for the development of reticulum, I found that in the liver it arises from Kupffer's endothelial cells, which here also form a beautiful syncytium.'"
Frozen sections of the liver of a pig 3 cm. long are very delicate, and can easily be crushed under a coverglass. When such preparations are stained with a little magenta it is seen that a network of fibrils lies between clumps of liver cells. It can now be determined that all of the fibrils surround the capillaries and are formed by prolongations from Kupffer's cells. The fibrils, or rather the syncytium, is delicate, can easily be stretched and broken by slight pressure upon the cover glass. Such sections are also very easily broken into granules by giving them a delicate shake in water. When digested a short time in pancreatin at room temperature the liver cells break up and fall out, leaving the delicate syncytium to which are attached many small granules. In such preparations the syncyidum is still very elastic and does not appear to swell in acetic acid.
The observations upon the development of the reticulum of the liver are entirely out of harmony with those of the development of connective tissue elsewhere. In all other places the syncytium arises from the mesenchyme but here it is from the endothelial lining of blood-vessels.
It is not difficult to obtain fresh specimens with all the capillaries surrounded with this syncytium which has the nuclei imbedded in it; the union is so complete that it is impossible to consider the nuclei and exoplasm in apposition only. The fibrils are in no way connected with the liver cells and true mesenchyme cells are not present at all.
The cornea of a pig 2 cm. long is composed of a dense syncytium. The exoplasm is fibrillated and it radiates from nodal points where are located the nuclei and endoplasm. In an embryo 3 cm. long the general direction of the fibrils of the exoplasm is parallel with the surface of the cornea, i. e., the lamellse are beginning to form. Between these primitive lamellfe the nuclei lie and are surrounded by spindle-shaped masses of endoplasm. A faint Descement's membrane is shown in specimens stained by Mallory's method; it does not stain by Weigert's method. Practically the same condition is found in the cornea of pigs 4 cm. long.
11 Mall, Zeit. f. Morpholog. u. Anthropol., ii, 9.
Kiipffer, Archiv f. mik. Anat., 54.
In an embryo 6 cm. long the cornea has grown in thickness, the quantity of exoplasm has increased and the nuclei have multiplied. The general character of the exoplasm is as before. In the cornea of pigs 9 cm. long the adult condition is present, the lamellfe of the anterior portion of the cornea being more developed than those of the posterior. The exoplasm forms definite lamellae in the cornea of pigs 14 cm. long. The fibrillated lamellae are bound together by bridges which run between them. Descement's membrane is sharply defined, stains intensely blue by Mallory's method, but does not stain by Weigert's method. It gives the same reactions in the cornea of the adult.
No elastic tissue can be demonstrated in the cornea of the adult either by Weigert's method or by treating frozen sections with boiling acetic acid and magenta. The lamellae of the cornea can be easily resolved into fibrils by forming artificial cedema or by spreading frozen sections. " In specimens made in this way the endoplasm is seen to encircle the nuclei and forms an extensive syncytium, as is well known. The tissue of the cornea contains much mucin and has often been spoken of as an embryonic connective tissue. It appears to be tlje only collagenous tissue which contains no accompanying elastic fibers. In many respects the cornea resembles the perimysium of the embryo before the white fibers have been fully formed from the exoplasm of the syncytium. At this time there are also no elastic fibers in the perimysium.
It is quite evident that in order to obtain any definite ideas regarding the development of elastic tissue it must be studied when it first appears. Studying its extension when once formed may give results which are misleading, for in older embryos the tissues which are being invaded have also undergone development.
In order to study the first appearance of elastic tissue I first tried to follow it in the skin, both human and pig's, for here I obtained the best preparations of developing white fibrous tissue. Furthermore, pieces of skin are easily cut by the freezing method and treated with the reagents usually employed in studying the connective-tissue fibrils. Although numerous tests and specimens were made the results were unsatisfactory until I had gained clearer pictures of the development of elastic tissue in the arteries and in cartilage. For this reason I shall consider the development of elastic tissue in the skin at the end of the discussion.
At first it was extremely difficult to obtain any clear pictures of young elastic fibers in the walls of the arteries by means of Weigert's method, for the surrounding tissues were also stained somewhat black. Finall}^ by staining the paraffin section upon the glass slide just long enough, complete differentiation was obtained by subsequent treatment with alcohol and hydrochloric acid, stronger than usual, and with a saturated aqueous solution of picric acid. By this method numerous sections were obtained with the elastic tissue only stained black. These were then counterstained with congo red or first with a very dilute solution of Delafield's hematoxylin to tinge the nuclei a little and then with Congo red. In this way perfect specimens were obtained with the nuclei stained with hjematoxylin, the elastic fibers stained intensely blue and the rest of the protoplasm red.
Not any elastic fibers could be demonstrated by Weigert's method in embryos less than 4 cm. long. As soon as the embryo has grown to this length a delicate network of elastic fibrils is stained intensely blue-black in the aorta and extends from the origin of the aorta into the arteries arising from it. Here they are gradually lost. The arteries of the skin do not have any elastic tissue in them. In a section of the 'carotid artery it is seen that there is a thick layer of elastic fibrils in the intima forming nearh' a complete membrane. The media is but a few cells thick with a few individual fibrils between them. There are no elastic fibrils in the adventitia.
In an embryo 5 cm. long the elastic tissue is in the walls of the subdivisions of the main branches arising from the aorta. The walls of the whole aorta and its main branches are filled with fibers which extend into the adventitia. In the intima of the aorta the fibrils have coalesced to form the Avell-known fenestrated membrane. In the carotid the individual fibrils are present in the intima, the fenestrated membrane appearing in an embryo somewhat older. The muscularis is filled with most delicate elastic fibrils which together make a network of meshes which are filled with nuclei. At the outer border of the muscularis there is a gradual transition of the elastic tissue into the connectivetissue syncytium of the adventitia. In a thin section stained with Weigert's method and counterstained with congo red the relation of the elastic fibers to the syncytium is especially well seen when examined with the 2 mm. oil immersion lens of Zeiss. The elastic fibrils lie within the exoplasm together with other fibrils and the spindle-shaped nuclei and endoplasni lie upon these bundles. The degree of development of the exoplasm is practically of the stage I have termed preiibrous above with numerous elastic fibrils, which stain with Weigert's stain, added. This process is slightly more advanced in the umbilical artery, which is especially suited for the study of early elastic fibrils, in longitudinal or oblique sections (Fig. 1-1). In such sections all grades of the development of elastic fibrils are easily found — from perfect syncytium in the cord without elastic fibers to the finished elastic tissue in the intima. At the point of juncture between the media and the adventitia it is seen that the white fibrous tissue gradually passes over into prefibrous tissue and this in turn over into typical exoplasm of the syncytium of the cord. The degree of development of the elastic tissue is exactly parallel with this. In the media the elastic network encircles the bundles of white fibers, while in the region of prefibrous tissue the network is in the periphery of the exoplasm. Farther out, in the adventitia, the network of elastic fibrils is all through the exoplasm. The fibrillated exoplasm in the walls of the arteries is composed of two kinds of fibrils, destined to become the fibrils of white fibrous and yellow elastic tissue. As this process of differentiation begins the white fibers swell in acetic acid, are not digested in pancreatin, etc. While the yellow elastic fibrils resist acids and dilute solutions of potassium hydrate and stain intensely when treated by Weigert's method. At first the elastic fibrils form a network throughout the exoplasm but they gradually shift to its outer border, leaving the prefibrous tissue within. At this time the nuclei and endoplasm lie upon the exoplasm. A further development liberates the nuclei and endoplasm more and more and the elastic fibers come to form a network which encircles bundles of white fibers, to form the characteristic and fully developed connective tissue.
Fig. 14. Elastic tissue just beginning in the syncytium of tlie umbilical vein of a pig 7 cm. long ( x 2.50 diameters). The specimen was first stained by Weigert's method, then tinged with hcematoxylin and couuterstained with congo red.
Fig. 15. Elastic fibers isolated from the skin of a pig 16 cm. long by means of boiling acetic acid ( x 250 diameters). Stained with gentian violet. The fibrils form baskets arouud the bundles of white fibrous tissue which are converted into a jellylike mass.
The youngest elastic fibrils which are stained by Weigert's method form a delicate network of homogeneous fibrils less than one n thick (smaller than the chromatin granule of the nucleus), and at no time are the fibrils composed of a row of granules as described by Eanvier. Eanvier's description of elastic granules in arytenoid cartilage is correct so far as it goes but does not apply to the development of elastic fibers.
I have studied carefully the development of elastic tissue in an embryo 7 cm. long, which had been hardened in alcohol, thus permitting tests with various digestion ferments. This was not possible with most of the sections I studied, for they were from embryos hardened in Zenker's fluid. Thin sections of the aorta show, when stained by Mallory's method, a beautiful syncytium composed of fibrillated exoplasm M^ithin which there is a network of sharply defined fibrils which stain intensely blue. The elastic membrane of the intima is also stained intensely blue. The arrangement of the network which stains more intensely by Mallory's method is identical with that stained by Weigert's method. If, now, a section is first digested in pancreatin for 24 or 48 hours, the network of the syncytium and the membrane of the intima are no longer present; as is shown in sections which have been stained with either Mallory's or Weigert's method. A shining mass of anastomosing fibrils of the exoplasm alone remains intact, nuclei, endoplasm, and elastic fibrils having been removed by the action of the pancreatin. From time to time specimens may be obtained by the action of pepsin in which only some elastic fibers and fragments of nuclei are left. In general, it is as difficult to isolate elastic fibers by the action of pepsin in the tissues of the embryo as it is to isolate them in the adult.
In thin sections of the embryo 7 cm. long which have been stained successfully with Weigert's elastic tissue stain, Delafield's hrematoxylin and Congo red the relation of white fibrous and yellow elastic tissue to the syncytium is beautifully shown in the adventitia. The two kinds of fibrils alternate in bundles with nuclei and endoplasm lying upon them. The individuaf elastic fibrils may appear as rows of granules, but the granules never leave the field of the microscope while focusing i. e., the granules are optical sections of fibrils of elastic tissue closely packed around the bundles of white fibers. In the adventitia of the umbilical artery, where the fibrils are cut parallel in this specimen, the fibrils are all homogeneous and continuous.
As the embryo grows the elastic tissue gradually extends along the arteries to every part of the body, reaching those of the skin in embryos about 20 cm. long. Shortly after the arteries of the skin have elastic tissue in their wall, it can also be demonstrated in the loose tissue below the hair follicles.
From the study of the development of elastic tissue in the arteries it is seen that the exoplasm of the connective-tissue syncytium forming their walls differentiates into two kinds of fibrils, which give rise to the white fibrous and elastic tissues, respectively. In other words, one cell gives rise to both tissues.
In the arteries the elastic and white fibrous tissues develop at the same time from the common exoplasm, as would be expected in a region where the elastic tissue develops so early. In cartilage, on the other hand, the exoplasm is converted completely into the ground substance before the elastic fibers develop. A condition which is parallel with that in cartilage is found in the skin, in bone and in reticulated tissue when accompanied by elastic fibers.
The arytenoid cartilage of the adult pig is partly hyaline and partly elastic. Where the two kinds of tissue come together the fibrils course in the ground substance between the fartilage cells. The hyaline cartilage near the elastic is infiltrated with granules which are sometimes in rows but more frequently in clumps around one or more cartilage cells. Generally the granules in the ground substance lie midway between the cells but where they begin to form masses they are usually around a single cartilage cell. According to Kanvier the granules form rows which coalesce to form elastic fibers. My own observations show that whenever fibers or granules are in the same neighborhood that they are separated and that one is never continuous with the other. We have here to do with a special kind of elastic tissue composed only of granules, as we have another form in the fenestrated membrane in the smaller arteries. Conclusive proof is obtained when the development of these structures is followed in the embryo pig.
The arytenoid cartilage of a pig 12 cm long is a few millimeters long and can easily be dissected out. It is then to be frozen and cut, stained by Weigert's method, and mounted as usual. Such sections show that most of the cartilage is hyaline, with some elastic fibers appearing at one end of the cartilage. The fibrils are extremely delicate and lie within the ground substance midway between tlie cells. At no point is the diameter of the fibers as great as that of the granules in the arytenoid cartilage of the adult. Furthermore, there are absolutely no granules of elastic tissue in the cartilage in which the elastic fibers have appeared and are growing. The same pictures, only more advanced, are seen in the arytenoid cartilages of pigs' embryos up to 24 cm. long. I have been unable to obtain specimens between embryos 24z cm. long and the adult, so cannot contribute anything regarding the development of the elastic granules. It is probable that they appear as minute specks and gradually grow larger and larger, for where they are in clumps granules of all sizes are seen.
Mucous Membrane of the Intestine
The reticulum of the mucosa and the prefibrous tissue of the submucosa form a single layer in the intestine of the embryo pig 24 cm. long. At this time no elastic fibers whatever can be demonstrated in any of the layers of the intestine by Weigert's method. Unfortunately the succeeding stages were not at my disposal, but from the examination of the intestine in the adult it is shown that the bundle of white fibers of the submucosa are surrounded with numerous elastic fibers which form a dense network throughout the muscularis mucosa and the stratum fibrosum. From this point a few fibrils extend between the crypts but not into the villi. Spalteholz  has followed them throughout the mucosa, showing that they accompany the muscle bundles of the villi. At any rate there is considerable reticulum in the mucosa of the intestine which has no accompanying elastic fibers, as is also the case in the ground sul)stance of cartilage.
Frozen sections of lymph nodes Avhich have been stained by Weigert's method show beautiful networks of elastic fibers throughout the trabeculae and the follicles. Within the trabecular the elastic fibrils are very numerous and from there they pass at regular intervals along tlie bands of reticulum through the sinus to the periphery of the follicle. Their course is quite direct towards the center of the follicle where they anastomose to form an irregular network. If the section is macerated for a few days in a solution of bicarbonate of soda to soften the cells, the sections can be cleared pretty well, leavin*j only the reticulum and the elastic fibers. When specimens thus obtained are stained by Weigert's method it is found that not all the reticulum fibrils are accompanied with elastic. At the periphery of the follicle about every second fibril, while more towards its center, about every fifth reticulum fibril is accompanied by an elastic fiber.
The examination of nunicrous thin sections cut in parattin and stained by Weigert's method showed that the amount of elastic tissue in the follicle is by no means constant. Occasionally no fibrils at all could be demonstrated by this method while frequently they were only at the periphery of the follicle. Care must be taken in such tests not to stain the sections too long, for the reticulum, and the wdiite fibrous tissue of the capsule, take up considerable stain and thus lead to confusion. The only definite tests are those in which the surrounding elastic tissue stains intensely, leaving the white fibrous tissue colorless. In a beautiful specimen of a Peyer's patch the elastic tissue accompanies every reticulum fibril into the follicle for two-thirds of the distance to its center and then ends quite abruptly. When not highly magnified it appears as if the reticulum itself were stained intensely, but with the 2 mm. oil immersion it is very apparent that each fibril of reticulum is encircled with several delicate elastic fibrils. At the center of the follicles there are no elastic fibrils at all. . The variation in the amount of elastic tissue in the lymph node suggests at once whether it is not due to some pathological process, for most of my sections were from human lymph nodes which had been cut for other purposes. The recent work of Melnikow-Easnednekow, Flexner, and others upon the formation of elastic tissue in cirrhosis of the liver suggests this view. The observations are sufficient, however, to show that elastic fibrils accompany some, but not all. of the reticulum fibrils in the follicle of the lymph node. Furthermore, the development of reticulum precedes that of elastic tissue.
It is extremely difficult to obtain clear pictures of the development of elastic tissue of the Template:Skin, when the youngest fibers which take Weigert's stain are studied in relation to the nuclei or to the white fibers. Practically no better results are obtained from the embryo than from the adult. In each case there are sharply defined fibers"^ and that is all. On the other hand, when the skin is macerated by boding frozen sections in 1 per cent acetic acid until the white fibers are mostly dissolved or are converted into a jelly-like mass the relations are somewhat distorted but the results are instructive, when compared with sections of the skin and of the larger arteries which have been stained by Weigert's method.
The elastic tissue of the arteries of the skin stains by Weigert's method in embryos from 20-25 cm. long. There are no elastic fibers within the skin itself. The clear areas at the roots of the hairs are filled with nuclei encircled with endoplasm lying upon a delicate network of exoplasm. This is beautifully showai in specimens stained by Mallory's method, and also to a certain extent by Weigert's method, provided the stain is pushed until the surrounding white fibrous tissue stains also. In embryos a little over 25 cm. long the elastic tissue of the arteries of the skin has increased in quantity, and the exoplasm of the syncytium below the roots of the hairs undoubtedly is stained more readily by Weigert's method than before.
11 Melnikow-Rasnednekow, Ziegler's Beitrage, 26, 1899.
'5Flexner, Univ. Med. Mag., 1900.
When frozen sections of the skin (which show no elastic tissue by Weigert's method) are boiled in dilute acetic acid until the white fibrous tissue is either dissolved or converted into a jelly mass, a network of sharp fibers can still be demonstrated. by staining the swollen section with magenta or with very dilute gentian violet (Fig. 15). In case the sections are not boiled very long the gelatinous exoplasm of the syncytium has imbedded within it sharp fibrils upon which lie oval nuclei surrounded with a plate of endoplasm. When the boiling is pushed still further, until the section falls nearly into pieces, it can still be coaxed upon the glass slide and stained with magenta under the coverglass. The main bands of syncytium are now practically all dissolved, leaving a network of delicate and sharply defined fibrils which appear to be directly continuous with the endoplasm around the nuclei. Often some of the anastomosing fibrils are quite free and upon them lie the nuclei and surrounding endoplasm (Fig. 18). These specimens, which are extremely instructive, show definitely that the nuclei and endoplasm lie upon the fibers. Furthermore, when frozen sections are treated a short time in boiling dilute caustic potash only a network — the elastic fibers — remains, all the rest, including the nuclei, having been dissolved. These tests show that an elastic network is present in the skin in young embryos before it can be stained by Weigert's method. Elastic tissue can be demonstrated by Weigert's method in the skin of the embryo pig about 25 cm. long, and by maceration in boiling acetic acid, and staining with magenta, the fibrils can easily be isolated (Fig. 16). It is therefore seen that elastic tissue is present in the skin long before it can be stained by Weigert's method.
In a section of the skin in Avhich the elastic fibers just begin to take the Weigert's stain it is seen that the bundles of white fibrous tissue are accompanied by one or two elastic fibers. In the region of the roots of the hairs, where the development is not so far advanced, the fibers are continued into the exoplasm of the syncytium and are related to the nuclei and endoplasm as described above. Frozen sections boiled in acetic acid (1 per cent) until very soft, then coaxed upon the glass slide and stained with magenta, show that the elastic fibers are related to the exoplasm of the syncytium much as they are to the reticulum of the lymph follicle.
It is much more difficult to obtain specimens of the human skin in which the elastic tissue is just beginning to appear. Fresh specimens are not always at hand and preserved specimens are often unsuited to cut into frozen sections which can be boiled or macerated.
In the skin of a human foetus, measuring 22 cm. from head to breech, practically no elastic fibers are stained by Weigert's method — much as in pig's embryos of the same length. Sections which have been boiled in dilute acetic acid for 4 hours had the white fibrous tissue destroyed completely, leaving only a delicate network of fibers upon which the nuclei lie (Fig. 17). In this specimen it really seemed at first as if there is a complete network formed by the anastomoses of the ends of numerous multipolar cells, but crushing the section and pulling it apart, showed that a delicate network of fibrils remains, which stain with magenta, is jDartly buried in the gelatinous remnant of the white fibrous tissue, and is partly covered with nuclei and endoplasm. The elastic network can be further isolated by boiling the section in a dilute solution of caustic potash; the delicate elastic fibers alone remain, the white fibers and nuclei having been removed completely.
Fig. 16. Elastic fibers isolated from the skin of a pig 24 cm. long ( x 250 diameters). Magenta. The skin was frozen and cut, then boiled in acetic acid (1^) for one hour. The fibrils form baskets around swollen bundles of white fibers. To them cling nuclei and endoplasm.
Fig. 17. Elastic network obtained from the skin of a human foetus 22 cm. long ( X 250 diameters). Stained with magenta. The specimen had been hardened in alcohol, was washed in water, frozen, and cut. Sections were then boiled in acetic acid (\<fo) for 4 hours. Further treatment showed that the nuclei and endoplasm could be removed by means of dilute caustic potash, leaving only the delicate elastic fibers.
Fig. 18. Elastic fibers from the skin of a human foetus 26 cm. long ( x 250 diameters). The fresh tissue was cut by the freezing method and boiled in acetic acid (1^) for an hour. It was then coaxed upon a slide and stained with magenta. AH of the fibers have large nuclei clinging to them.
The skin of a foetus 7 months old (36 cm. long) has wdthin it many delicate elastic fibers which are stained by Weigert's method. The individual fibrils are in general parallel with the bundles of white fibrils, are not composed of rows of individual granules, but are homogeneous. When the sections are boiled to remove the white fibers in part and then stained by Weigert's method, a beautiful network remains, one or two fibrils accompanying each swollen bundle of white fibrils. Frozen sections boiled in dilute acetic acid and stained with magenta give the same picture. The oval nuclei with the surrounding endoplasm lie upon the elastic fibrils, surround them, but are not continuous with them (Fig. 18). Similar results have been obtained by Jores, who studied the formation of elastic fibers in a myxoma.'"
The elastic fibers have increased greatly in number in the skin of a fcetus 8 months old. The fibers are closely packed to form baskets encircling the individual bundles of Avhite fibers. Specimens made by the aid of boiling acetic acid are again most instructive, for in such specimens the fibers are isolated with nuclei and endoplasm clinging to them. Thick sections made in this way appear as a felt in which there are numerous holes, where the bundles of white fibers lay, with nuclei and endoplasm clinging to the elastic fibers. In the skin at birth the elastic fibers have become a little larger and denser, and therefore more numerous as the skin has expanded and become thicker. Frozen sections which have been treated with boiling actic acid and stained in magenta show nuclei and endoplasm attached to the individual fibers. Sometimes they are spindle-shaped but usually they form plates which are easily separated from the elastic fibrils after the white fibers have been dissolved.
In the skin of an infant two months old the elastic and white fibrous tissues are about equal in quantity. The elastic fiber baskets encircle and frequently sink into the bundles of the white fibers, as is easily shown in sections which have been stained by Weigert's method. The same picture is seen in the skin of infants from two to six months old.
While the elastic fibers are present in relatively small number in the skin of a foetus 22 cm. long, and gradually increase in size and quantity as the fretus grows older and after birth, the study of their development in this region gives unsatisfactory results. It is definite, however, that they always appear around the bundles of white fibers^ being covered, especially at their points of anastomosis, with nuclei and endoplasm. If the early formation of elastic tissue in the syncytium of the walls of the umbilical artery is considered the type we must interpret what has been found in the skin as a secondary differentiation of the exoplasm, which is already collagenous, into elastic tissue, as is also the case in the ground substance of the cartilage. In the cartilage, however, the fibers develop in the middle of the ground substance, as far away from the nuclei as possible, while in the skin the elastic fibers appear at the periphery of the bundles of white fibers, close to the nuclei." The same is true regarding the elastic fibers which are formed in the lymph follicle along some of the reticulum fibrils.
'6 Jores, Ziegler's Beitrii^e, xxvii, Fig. 3.
From this histogenetic study it must be concluded that elastic tissue is a more highly differentiated tissue accolnpanying to a greater or less extent all collagenous tissues (reticulum, cartilage, bone, and white fibrous tissue) with the exception of the cornea.
The study of the growth of all connective tissues is difficult, for after they are once differentiated and quite sharply separated from the nuclei and endoplasm they have then power of further growth and expansion without a continuous transformation of endoplasm into exoplasm.
'^ See also Jores, Ziegler's Beitrage, xxvii, Fig. 4.
- Flemming:, Virchow's Festschrift, Berlin, 1891.
- Flemming, Archiv fṻr Anatomic, 1897.
- Merkel, Verhandl. d. Anatom. Gesellscliaft, 189.5.
- Spuler, Anatom. Hefte, Bd. 7, 1896.
- Mall, Abhandl. d. K. S. Gesellscli. d. Wiss , Bd. 17, 1891.
- Flemming, His' Archiv, 1897, S. 183.
- Spuler, Anat. Hefte, viii, 133.
- His, Zellen uud Syncytialbildung; Protoplasmastudien ; Lecitboblast und Angioblast. Abhandl. d. K. S. Gesellschaft d. Wiss., Bd. 34, 2.5 u. 26, 1898-1900.
- Mallory, Jour, of Exp. Med., Vol. 5, 1901.
- Reddinghaus (Ziegler's Beitrage, 29, 1901) has shown that in inflammation of the omentum the fixed cells become active and form a syncytium which is in every respect identical with the connective tissue syncytium of the embryo. His pictures are in every respect like the normal specimens I obtained with Mallory's method.
- Spalteholz, Arch. f. Auat., Supplement Band, 1897.
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