Book - Contributions to Embryology Carnegie Institution No.20 part 4
Otic Capsule
The histogenesis and growth of the otic capsule and its contained periotic tissue-spaces in the human embryo
By George L. Streeter
4 text-figures and 4 plates
GROWTH AND ALTERATION OF FORM OF THE CARTILAGINOUS CANALS
In embryos .'JO mm. long the cartilaginous labyrinth has attained approximateh' the adult form. Its subsequent development is primarily an increase in size to accommodate the growing membranous labyrinth. If a cast of the superior cartilaginous canal of an 80 mm. embryo b(; compared with a similar cast of the same canal in a .30 mm. embryo, it will be seen that the general form of the canal in the older specimen is much the same as in the younger specimen But its diameter tuid length ha\e both increased, the diameter being nearly doubled and the length trebled; furthermore, its linear curvature corresi)onds to an arc with a considerably longer radius. In realitj', therefore, the developing cartilaginous labyrinth is continually undergoing changes, both in size and ff)rm. The histological evidence of these changes constitutes one of the most interesting features in the development of this region, and although it does not directly concern the development of the contained perioticular spaces, yet it may not be out of place to point out some of the elements of this process as they are seen in our material. In fact, the cartilaginous capsule of the ear is an especially favorable place for studying the general question of growth of cartilage, for two reasons: (1) there are, on account of the intricacy of form of the labyrinth, many kinds of cartilaginous changes found there that are necessary to accommodate its growth, including the deposit of new tissue and the removal of old tissues ; (2) the topography is so well marked bj^ known landmarks that all of these changes as well as the location and direction of growth can be easily followed.
Growth of cartilage is usuallj^ considered to be of two kinds, which are distinguished from each other by being either interstitial or perichondria!. Interstitial growth is described as consisting of an increase in the amount of hyaline matrix and the growth and proliferation of the encapsulated cartilage cells. The new cells form new capsules to a certain extent, but a point is finall}' reached beyond which the newly prohferated cells continue to occupy their parent capsule . From this variety of growth there results a uniform intumescence of the tissue without producing any marked change in its form. This manner of growth forms a large element in the increase in size of some parts of the capsule of the ear. In those parts, however, where a change in form is involved the growth is more Uke that described under perichondrial growth and consists of a new deposit of cartilage along the borders of the older cartilage, the consituent cells passing through a precartilage stage. In the otic capsule this latter type of growth is actively going on even before a definite perichondrium is established. The deposit of new cartilage along the margin of older cartilage and the removal of old cartilage bj' dedifferentiation are indeed the main factors in the process through which the form of the ear-capsule is modeled.
The excavation of established cartilage can be studied by comparing sections through the semicircular canals at difTerent stages, such as appear in figures 11, 12, 14, and 15. These are all sections through the same canal (lateral), taken in about the same position, and are enlarged the same number of diameters. It is, of course, possible that they were shrunken in different degrees in the process of embedding; this discrepancy, however, is probably not enough to interfere with their showing the approximate increase in size of the cartilaginous canal at the respective ages. A crude measurement of the perimeters of the canals as seen in the original photographs (100 diameters) yields the following circumferences: 30-mm. embryo, 115 mm. circumference; 37-mm. embryo, 132 nam. circumference; 43-mm. embryo, 152 mm. circumference; 50-mm. embryo, 192 mm. circumference. It is evident that we are dealing with an enlarging space and that a study of its receding edge must give the histological picture of the replacement of true cartilage by other tissue, either by dedifferentiation or by direct metaplasia.
If, with this process in mind, one makes an examination of the specimen shown in figure 11 (Carnegie Collection, No. 86) it will be seen under higher magnification that a rather definite border can be made out separating the general mass of true cartilage from the inner zone of temporarj'^ precartilage surrounding the semicircular ducts. The true cartilage has develojjed a consideral)le amount of matrix separating the encapsulated nuclei or cartilage cells. The margins of the capsules stand out as sharp refractive lines. The matrix lying between the capsules is slight I}- opaque and is beginning to take a differential stain. A narrow intermediate or transition zone separates the true cartilage from the precartilage; this zone is characterized by the presence of flattened and i)artially collapsed capsules between which there is very little or no matrix. The refractive margins of these overlai)iMng, incomplete capsules give the appearance of wavy lines that run parallel with the margin of the canal. The same appearance is not seen in other regions of the otic capsule in younger stages, where precartilage is differentiating into cartilage.
In the process of cartilage differentiation in most parts of the otic capsule there is considerable intercapsular material at the time the margins of the capsules become conspicuous. The capsules are separated by the matrLx-forming syncytium. Thus, there are not the conspicuous wavy lines due to overlajJijing capsules, such as characterize the intermediate zone. The transition between this zone and the true cartilage on one hand and the temporary precartilage on the other is quite abrupt in both instances. On entering the zone of j)recartilage there is found between the nuclei, instead of the wavy refractive capsular lines, a framework having more the character of a granular syncj'tium, with only here and there the suggestion of a Ix'gimiing capsule. This, it will be remembered, is a condition the true cartilage exhibited in its earlier i)eriod. It is the intermediate zone to which we should address our especial attention, and it is this zone that moves outward as the cartilaginous canal widens.
Text-figure 1 .shows a section througli the.sc zones in a fetus of about the same age as the one just described. This section is taken through the lateral length (Carnegie Collection, No. 145).
Fig. 1. — Detail of a section through tlie lateral .wmicircular oaiial in a human fetus 33 mm. long (Carnegie
Collection, No. 14.'j, slide 7, row 1, section 3). The
section is .")(V thick and is enlarge*! 37() diameters.
It shows the epithelial wall of the seniieireular duct
and the tissue zones that intervene between it and
the cartilaginous capsule. In the outer part of llie
J (re-cartilage zone is an intermediate area which is a
transitional form between cartilage an<l precartilage.
It is characterizetl by the scant .amount of matrix and
the incomplete and flattened c(in<lilion of the cap.sule.
canal of a fetus 33 mm. crown-rump
The intermediate zone stands out conspicuously at the junction of the cartilage with the precartilage. Its wavy refractive lines are so compact that under low powers the whole zone appears as a dark rim outlining the cartilaginous margin of the canal. The compactness of these lines varies in different embryos of about the same age and even varies in the two borders of a given canal.
This latter condition can be seen in figure 14, which represents the lateral semicircular canal of a fetus 43 mm. crown-rump length (Carnegie Collection, No. 886). It will be noted that the peripheral two-thirds of the intermediate zone (toward the right hand) forms a dark, heavy margin between the true cartilage and the encircled precartilage, whereas the central one-third (toward the left hand) is wider and much less distinct. It can also be seen that the place at which this intermediate zone is well marked corresponds to the direction of the excavation necessary to allow for the growth of the canal and to make room for the elongating semicircular ducts of the contained membranous labyrinth. In this case the expansion must be toward the periphery of the cartilaginous capsule, i.e., toward the right side of the photograph. From studying various fetuses it seems to be true that where excavation of cartilage is actively going on at such a place there is found a prominent intermediate zone along the inner margin of the cartilage. Sometimes the line is uniform around the entire rim, but usually it is more marked on one side of the canal than on the other, and in such cases it is always toward the direction of the excavation of the cartilage, as can be judged from the topography of the labyrinth.
If an older specimen is examined, such as the one represented in figure 15, the character and relative position of the cartilage and precartilage are found to be the same as in the 30-mm. stage just described. They have, however, undergone an alteration to allow for the enlargement of the cartilaginous canal. Figure 15 shows the lateral canal in a human fetus about 50 mm. crown-rump length (Carnegie Collection, No. 95). The fetus is catalogued as being 46 mm. long, but this is apparently the slide-measurement. In its development it corresponds to fetuses 50 mm. long, formalin measurement, and this measurement is used so that it will accord with the other fetuses. Since figures 11 and 15 represent sections through the same canal taken at about the same place and under the same enlargement, one can superimpose them, one upon the other, and thus determine the change that has occurred between the two stages. If this is done it will be seen that the area that was precartilage in the 30-mm. stage is replaced by reticulum in the 50-mm. stage. There is just as much or more precartilage in the latter, but it ha.s moved outward into the area that was previously true cartilage. In other words, the enlargement of the cartilaginous canal has been obtained by a process of excavation based on the dedifferentiation of true cartilage into precartilage and the latter in turn into reticulum. This is shown under higher magnifications in text-figures 2 and 3, which show sections of these same canals under the same enlargement and placed side by side for the purpose of better comparison. It can be .seen in these two figures how the cartilage of 30-mm. stage becomes dedifferentiated into the precartilage of the 50-mm. stage and the border along which this process is in active operation forms the intermediate zone, which is characterized by its wavy, refractile lines. The precartilage in tuin it^ gradualh' dedifferentiated into the periotic reticukim. In this way the margin of the true cartilage graduall.y recedes from tlie epithelial duct, and the last of the precartilage is eventually dedifferentiated into a reticulum. Along with the process of excavation of cartilage there must go the laying-down of new cartilage. For instance, as the lateral cartilalaginous canal enlarges it also moves laterally, so that the distance between it and the cartilaginous vestibule increases, producing relatively a lateral migration of the si^ace as a whole. Such a migration involves the excavation of the established cartilage on its lateral margin and the formation of new cartilage on its median margin. On its laveral margin true cartilage is being dedifferentiated into precartilage and on its median margin precartilage is differentiating into cartilage. It is this phenomenon that determines the conditions shown in figure 14. On the right can be seen the prominent intermediate zone, indicating an active excavation of cartilage, and on the left the line of transition between cartilage and precartilage presents the same picture as that seen in the stage of differentiation of the latter into the former. One is forced to conclude that the cartilaginous tissue of the otic capsule is callable of differentiation and dedifferentiation in its earlier stages, at least up to the time of the completion of the encapsulation of the cartilage cells. This progressive and retrogressive adaptability of the cartilaginous tissue makes possible the changes that are necessary in the growth and alteration in form of the labyrinth.
FlGURK 2.
Flo. '2.--Detailof the lateral semicircular duct in a human fetus 30 mm. long (Carnegie Collection, No. 86), being the same as that shown in figure 11. The section is .'50^ thick and is enlarged 470 diameters. It shows the epithelial wall of the duct and the <'hiir;icter of the surrouiiiliii); tissues thai lie between it and the cartilage.
FlO. 3.— Detail of the lateral canal iti a human felus Iti niiii. lorifl (Carnegie ( 'oUeil ion, No. !).">, slide 72, section 1). This i.s the same canal as that shown in tiguie I."). The .section is l(K)/u thick and is enlarged 470 diameters. By comparing it with figure 2 one can see how the cartilaginous canal is enlarged by the tledilTerciitiation of cartilage into precartilage and the precartilage in turn into the periotic reticulum.
DEVELOPMENT OF PERIOTIC RETICULAR CONNECTIVE TISSUE
The formation of the connective-tissue reticulum surrounding the semicircular ducts is first indicated by a cluster of darkly stained nuclei that lie along the central edge of the ducts in embryos soon a^ter the ducts are formed and before the differentiation of the cartilage is completed. In figure 9 such a cluster is seen just under the posterior duct in the upper part of the photograph. In figure 10, which shows the lateral semicircular duct of an embryo 27 mm. long (Carnegie Collection, No. 756a), a similar cluster of nuclei can be seen just under the duct, in reaUty just median to it. These foci mark the points at which the formation of the reticulum begins. It is not, however, until we come to embryos about 30 nmi. long that we find a definite reticulum. At that time, as is shown in figure 11, a narrow fighter area can be made out, situated between the epithelial wall of the duct and the temporary precartilage. It is the development of this area at the expense of the temporary precartilage that results in the reticulum in which the periotic spaces are subsequently formed. This area consists of a mesenchymal syncytium containing irregularly shaped clear tissue spaces and is characterized by the presence of numerous blood-vessels and connecting capillaries. The larger vessels are found resting against the inner margin of the temporary precartilage. They sometimes indent it, but never penetrate it to any extent. Such vessels can be seen in figures 11 and 12. The presence of these blood-vessels is coincident with the appearance of the reticular tissue.
In describing younger stages the statement has been made that the temporary precartilage abuts directly against the epithelial wall of the semicircular duct. This statement is based only on the gross appearance. On careful scrutiny of the tissue that immediately surrounds the ducts in embryos between 14 mm. and 20 mm. long a few mesenchymal cells can be found which possibly do not belong to the tenijjorary precartilage. These cells may very well represent some of the indifferent mesenchyme, and possibly also some angioblasts. It is conceivable that these surround the otic vesicle in its earUest stages and are inclosed along with the Otic vesicle by the condensed tissue of the otic capsule, where they remain in contact with the epithelial labyrinth in a resting condition until the embryo approaches 20 mm. in length. They then show activity and by the time the embryo is 30 mm. long we find them converted into a vascularized reticulum which forms a definite area surrounding each semicircular duct and completely separating it from the receding precartilage. The area of reticulum advances as the precartilage becomes hollowed out. This can be seen by comparing figur(>s 11, 12, 14, 15, and 16, all of which are reproduced on the same scale of enlargement.
From the histological api)earance one could maintain that the reticuhmi is derived from a few predestined mesenchymatous cells which, after a latent period, undergo proliferation and occupy the space that is vacated by the receding precartilage in the manner described above, the growth of the reticulum perhaps being the cause of the recession of the cartilage. But one could eciually well maintain that the reticulum is derived entirely from the precartilage; that it is not a predetermined tissue, but simply precartilage that has undergone dedifferentiation. It is entirely possible that the isolated cells included with the epithelial labyrinth are angioblasts only, everything else being indifferent mesenchyme. In the early stages, where only a few cells are concerned, this matter can not be determined, the histological difference between early precartilage and other embryonic cells not being sufficiently great for their certain recognition. In the later stages, however, it is quite evident that precartilage tissue is actually converted into a reticulum; that the replacement of the temporary precartilage by a reticular connective tissue is accompUshed by a process of dedifferentiation, or direct metaplasia, just as we have previously seen in the case of the dedifferentiation of cartilage into precartilage.
In this connection it is instructive to compare again figures 11 and 15, and also fig\ires 2 and 3, which are details of the same under higher magnification. They .show under the same enlargement a section through the lateral canal made in about the same position and cut at the same thickness. It will be noticed that the space occupied by precartilage in the younger stage is entirely filled in by reticulum in the older stage. There is in the older stage, however, more precartilage than before, but it now occupies a more peripheral j^osition. With the change in the position of the precartilage area there is a corresponding enlargement of the lumen of the true cartilage, i. e., the cartilaginous canal. It is clear that we are dealing here with a dedifferentiation of true cartilage into precartilage on the one hand and a dedifferentiation of precartilage into reticulum on the other. These factors, as we already have seen, are of great imjiortance in the alteration in form and size of the cartilaginous canals.
In younger stages, as in figure 10, the epithelial semicircular duct lies near the center of the area of temjiorary pre(;artilage. ^^'hen the reticulum de\elops it makes its first appearance, and its growth continues more marked along the concave side of the duct than on the convex side— that is, on the side toward the utricle rather than toward the ix'rii)hery of the capsule. On this account the epithelial duct lo.ses its central position and gradually comes to lie along the peripheral border of the cartilaginous canal, where it eventuallj' becomes attached to the periosteum.
This eccentric position gives the canal the largest arc that is possible in the space in which it lies. It marks the point of thrust of the elongating duct against the cartilaginous chamber that confines it and it is in this direction that the cartilage must be excavated to make room for the further growth of the duct.
The spread of the reticulum into the surrounding precartilage is rather slow at first. There is very Uttle advance made in fetuses between 30 mm. and 43 mm. long, as can be seen by comparing figures 11 to 14. In figure 14 the reticulum can be recognized as a crescentic-shaped area on the central side (toward the left) and partially surrounding the epithelial duct. In the figure it is about 0.8 cm. wide at its widest point. The surrounding precartilage is also of about the same \\ddth, but it is uniformly wide around the whole circumference of the cartilaginous canal. In fetuses about 50 mm. long the dedifferentiation of precartilage into reticulum makes more rapid progress. The change is quite abrupt at this time. Figures 14, 15, 16, and 17 form a series in which is shown the alteration from a small amount of reticulum to an almost complete reticularization of the cartilaginous canal. These changes are found in fetuses varying from 43 mm. to 52 mm. long. In comparing these figures one would e.xpect that the membranous duct would be found progressive!}^ larger in the series of photographs if they were correctly arranged in the order of their age. But it should be remembered that the tissues show different degrees of response to the fixing reagents. This is particularly so in respect to the epithelial duct; in figures 14 and 17 it is distended, as can be seen by its thin wall, while in 15 and 16 it is contracted. The order in which they are arranged corresponds to their relative age, as far as could be determined by the records of the fetuses and general appearances of the sections.
In figure 15 there is a zone of precartilage, about 0.8 cm. wide in the photograph, which in reality is true cartilage that has been dedifferentiated into precartilage. The reticulum extends from the inner border of this to the membranous duct. In figure 16, which is a section through the posterior canal of a fetus 50 mm. long (Carnegie Collection, No. 184), the dedifTerentiation of precartilage into reticulum has occurred faster than that of cartilage into precartilage. There is practically none of the latter to be seen; the whole of the space between the margin on the cartilage and membranous semicircular duct is filled in by reticulum. Along the central margin of the duct there are still seen thick clusters of proliferating nuclei which are associated in part with the development of the blood-vessels and in part with the modification of the reticulum that takes place around the wall of the membranous duct.
It has been noted that precartilage is free of blood-vessels, whereas the reticulum is vascularized from the very first. Part of the dedifferentiation of precartilage into reticulum consists of the invasion of blood-vessels into the precartilage region. In the early stages of the reticulum the larger vessels hug closely against the precartilage and continue to do so as the latter recedes from the epithelial duct, as can be seen in figures 11, 12, and 14. Later, with the abrupt dedifferentiation of the remaining precartilage into reticulum, the larger vessels do not -follow the receding margin of the cartilaginous canal, but form vascular arches that are suspeiulod in the reticulum, as can be seen in figures 15, 1(), and 17, and from these a network of small vessels branches toward the membranous duct on the one hand and the cartilaginous wall on the other.
In figure 17, which is a section through the posterior semicircular canal in a fetus 52 mm. crown-rumi> length (Carnegie Cf)llection, No. 96), the reticulum is more mature in its appearance than any that have thus far been described. There is practically no precartilage to be seen. The reticulum now only lacks the membrane-like thickening of its inner and outer margins to render it complete. At the inner margin the cells arrange themselves into a fibrous coat that constitutes the memlirana proi^ria of the membranous duct. At the outer margin is formed the perichondrium, the development of which will now be considered.
