Book - Contributions to Embryology Carnegie Institution No.20 part 5

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DEVELOPMENT OF THE PERICHONDRIUM

In the description of the development of the periotic reticulum we have seen how it begins as a small focus along the central border of the epithelial semicircular duct and spreads at the expense of the temporary precartilage, forming as it does so a crescentic-shaped area of reticulum inclosing the duct. We have also seen how the im-asion or spread of the reticulum into the surrounding area of precartilage is brought about, at least in the later stages, by a dedifferentiation of the latter into the former.

Furthermore, along with this latter process, the inner margin of cartilage surrounding the duct is dedifferentiated into precartilage, so that a new area of precartilage becomes established as the old area disappears. The conversion of precartilage into reticulum in the later stages, however, is more rapid than the conversion of cartilage into precartilage, and consefjuently there comes a time when the precartilage has nearly all disappeared. In such specimens the reticuhnn extends practicall\' from the epithelial duct to the margin of the cartilaginous canal. The (|ualifying term "practically" is used because the inner and outer margins of the reticulum are modified in a special manner. The inner margin becomes condensed into a membrane-like coat of fibrous tissue that constitutes the membrana ])ropria of the membranous canal. The outer margin at about this time undergoes changes that result in the formation of the jjerichondrium.

In di.scussing the lu'richondrium it is important to kcej) in imnd the active alterations in the tissue along the margin of the cartilage that accomj^any the growth of the labyrinth. It has been seen how the enlargement of the cartilaginous canals and their alterations in form and position is obtained partly by excavation of cartilage and partly by the laying down of new cartilage, the excavation being accomplished by its dedifferentiation into ])recartilage and reticulum, and the new cartilage being l>uilt up through a i)recartilage stage from the periotic reticular tissue. Throughout the entire period of growth of the cartilaginous canals the elements of this continual transformation exist along their margin. The margin during this period is in a state of temporarj' eciuilibrium and is cai)able of advancing or receding as the conditions determine.

The first and relatively the major part of the hollowing-out of the cartilaginous canals is complete before the perichondrium makes its appearance. This is illustrated, for instance, by the fetus of 52 mm. crown-rump length, in figure 17, where there is as yet no indication of it shown. In fetuses between 40 and 50 mm. long the zone of precartilage surrounding the margins of the canals, as seen in figures 14 and 15. might be mistaken for perichondrium. This area, however, in fetuses sUghtly older is converted almost entirely into reticulum. Kolliker (1879), in the second edition of his text-book on embryologj', pictures a transverse section through the lateral canal of a rabbit embryo (fig. 457, page 735), in which this zone of precartilage is labeled as periosteum of the future bone.

The real perichondrium does not make its appearance until the fetus reaches a a length of about 70 mm. A specimen of this age is represented in te.xt-figure 4, which shows a segment of the posterior semicircular canal in a fetus 73 mm. crownrump length (Carnegie Collection, No. 1373). On examination of this specimen it is found that there is a distinct condensation of the reticulum along its inner margin, so that it forms a membrana propria for the epithehal duct with which it is in contact. This area has largely lost its reticular character and now resembles embryonic fibrous connective tissue. Along the outer margin of the reticulum a similar condensation of its trabeculse has taken place, forming a thin fibrous lamina or membrane near the margin of the cartilage. This is the perichondrium in its early form. It does not abut directly against the cartilage, but is separated from it by a thin layer of transition tissue that is in process of dedifferentiation from precartilage into reticulum.

Passing inward from the cartilage, the transitions are rapid from cartilage to precartilage, from precartilage to the tissue that is in transition to the reticulum and then to the perichondrium. These are found as narrow zones that merge quickly from one into the other. One should remember that the cartilaginous canal has not reached its full size yet, and that the margin of the canal is still in an unstable condition. However, as the canal becomes larger and the tissues more mature, it is found that the transitions between the different zones become more abrupt and in this process the precartilage zone becomes relatively much narrower. This can be seen by comparing text-figures 3 and 4. The width of the reticulum in these two figures can not be compared, because the.v represent diflferent canals, lateral and posterior, and no attempt was made to take them from the same relative positions. The fact that the reticulum is narrower in figure 4 has no significance in the question of growth. The wide precartilage zone in figure 3 as compared with that in figure 4, on the contrary, has a direct bearing on the relative age of the two specimens. A relatively wide zone of precartilage is characteristic of younger stages. After fetuses become 70 mm. long the precartilage zone becomes quite narrow, so that the transition from cartilage to perichondrium is relatively abrupt. In older si^ecimens one might easily obtain the impression that the perichondrium rested directly against the cartilage, as doubtless it does in the adult. In the oldest fetus examined, 130 mm. crown-rump length, there is still found a distinct though narrow precartilage-reticular transitional zone between the cartilage and the perichondrium. Presumably this indicates that the margin is still in an unstable condition.

After the perichondrium has made its first appearance it rapidly becomes thicker and more conspicuous. In a fetus 80 mmcrown-rump length (Carnegie Collection, No. 172) it is found as quite a dense fibrous coat, more than twice as thick as that shown in the 73 mm. embryo in figure 4. It is clearly separated from the cartilage and precartilage by a narrow zone of reticular tissue.

The character of the perichondrium as existing in slightly older fetuses is shown in figure 18, which represents a section through the posterior semicircular canal of a fetus 85 mm. crown-rump length (Carnegie Collection, No. 140030). Here the perichondrium consists of a relatively broad zone of enibrj'onic fibrous connective tissue, which in the photograph is about 5 mm. wide, encircling the whole canal. It can be seen on the median side (to the left) that it is sejxirated from the cartilage and adjacent transforming precartilage zone by a narrow, lighter area, which under higher magnification is found to consist of reticular tissue. The membrana propria at the inner margin of the reticulum is fairly well developed and it can be seen how it forms a supporting coat to tho epithelial duct.

\\'hen one examines the cartilaginous semicircular canals in fetuses 130 mm. long there can no longer be any ([uestion as to the identitj' of the perichondrium. A specimen showing the superior semicircular canal at this stage is represented in figure 19, which is taken from a fetus 130 mm. crown-rump length (Carnegie Collection, No. 1018). The blood-vessels are injected with India ink. The main cartilaginous mass in this specimen is (luite mature; the capsules are well defined and the cartilage cells now possess a considerable amount of granular bodj'-protoplasm.


Cartilage


Flu. 4. — Detail of the posterior canal in a human fetus 73 iniii. long (Carnegie Collection, No. 1373, slide 0, row 3. section 1)' The section is lOiU thick and is enlarged 370 diameters. It shows how the inner margin ()f< the reticulum becomes con(hm.sed into the niembnina projiria of the epithelial duct and the outer margin into I he iicricliondrium. The perichondrium does not lie in direct cdiitact with the cartilage, hut is separated by a narrow zone of tissue which consists of precartilage, into which the cartilage is still being dedifferentiated.

In many instances capsules are found containing more than one cartilage cell, showing the tendency to cell columns.

A casual glance at a section under lower powers might indicate that the inner maigin of the cartilage is in direct contact with the perichondrium. Examination under higher magnification, however, shows that between the thick perichondrium and the cartilage there is a narrow zone of dedifferentiated cartilage. In it the matrix has largely disappeared and the capsules have collapsed and are flattened out, allowing the elongated endoplasm of adjacent cartilage cells to come in contact, separated only by the remnants of the capsular margins. Dyes that stain endoplasm red cause this zone to appear as a deep-red line. This zone represents a state of transition between cartilage and precartilage and its presence doubtless indicates that the margin of the cartilage is still in an unstable condition. The narrowness of the zone and the abruptness of the transition are characteristic of later stages, where the process is more gradual and relatively small in amount. The transition from this zone to the perichondrium is likewise abrupt. The perichondrium consists of a dense protoplasmic stratum thickly studded with nuclei, and has all the appearance of late embryonic fibrous connective tissue. It is of about the same tliickness around the whole margin of the canal. At the outer margin (toward the right) it fuses wdth the membrana propria of the epithelial duct, therebj' forming an attachment which is regarded as a suspensory ligament for the sujjport of the membranous labyrmth. The trabeculae of the reticulum extending between the membrana propria and the perichondrium are just beginning to break apart, allowing the adjacent spaces of the reticulum, as they are seen in section, to coalesce in the formation of larger spaces.

Having completed the review of the early history of the reticulum and its formative relations to the adjacent tissues, we are now in a position to consider the development and the fate of these larger spaces in the reticulum, which ha\'e hitherto been generally known by the misleading term "perilymphatic spaces."

DEVELOPMENT OF PERIOTIC TISSUE— SPACES

In the }3receding pages of this article the main features of the development of the cartilaginous capsule that incloses the membranous labyrinth have been described. We have traced the process step by step from the first condensation of the mesenchjone around the otic vesicle, through its differentiation into a precartilaginous mass and the maturation of the latter into true cartilage, with the formation through dedifferentiation of cartilaginous chambers in wliich the membranous labyrinth is suspended. It has been shown how these spaces within the cartilaginous capsule are modified in adaptation to the continued growth of the membranous labyrinth and how they finally come to be fiUed with an open-meshed reticulum which everjT\ here bridges the space existing between the membranous labyrinth and the surrounding cartilage. It has further been shown that the membrana propria supporting the epithehal part of the labyrinth on the one hand and the perichondrium on the other are derived from and serve as the hmiting membranes of this reticulum. It is a modification in the meshes of this same reticulum that results in the formation of the so-called perilymphatic si)aces, or periotic spaces as they will be referred to in this paper, the development of which will now he outlined.

Thus far attention has been directed primarily to regions included in typical transverse sections through the semicircular canals. This was done for the purpose of uniformity and simplicity and because of the- ease with which successive stages could be compared with one another. For studying the periotic spaces, however, the region of the canals is not so favorable, because the spaces are late in developing there, and even in their completed form they are not so well defined and highly differentiated as those in the region of the vestibule and cochlea.

The earliest evidence of a periotic space makes its appearance opposite the stapes. It is developed in the reticulum that fills the interval situated between the saccule, utricle, and the cartilaginous stapes. Even before the general periotic reticulum becomes very extensive, in embryos between 30 and 40 mm. long, it can be seen that its meshes are more irregular and more open in this region than elsewhere. This is the rudimentary form of the periotic vestibular cistern, which is the first space to become established.

DEVELOPMENT OF THE PERIOTIC CISTERN OF THE VESTIBULE

Aside from the scala vestibuli and the scala tympani, the largest of the periotic spaces is the large reservoir situated between the tympanic wall of the bony vestibule with its articulated stapes and the vestibular chambers of the membranous labyrinth. This is the spatium perilymphaticum vestibuli (BNA) or the cisterna perilymphatica (Retzius). In order to eliminate the word lymphatic from the terminology it will be designated here as the cisterna periotica vestibuli, or less formally the j^eriotic cistern. In this manner the descriptive term introduced by Retzius is retained.

Before there is any trace of the scalse the initial steps in the formation of the cistern can be seen. This is well illustrated in an embryo 35 mm. long (Carnegie Collection, No. 199). This particular embryo is cut in a sagittal series and the sections on slides 53 and 54 show the periotic cistern in its most rudimentary form. It consists of an area of reticulum bounded by the utricle, saccule, ductus reuniens, the proximal end of the cochlear duct, and the ampulla of the jiosterior semicircular duct. The greater part of the periotic reticulum at this time (35-mm embryo) is characterized by a narrow and uniform mesh that is interrupted only by numerous cajjillaries branching through it; in the area mentioned, however, the spaces are larger and are more irregular both in shape and in size. They i)resent the appearance seen along the semicircular ducts in considerably older embryos, for instance, in the 52-mm. embryo, as is shown in figure 17. From the very first the increase in the size of the mesh seems to be attained by the detachment and retraction of its constituent i)rotoplasmic bridges, thereby allowing adjacent spaces to unite in the formation of (•om])osite large spaces. Thus in the above section a few irregular protopla.smic free-ends are seen still jjrojecting into the newly enlarged spaces. This interesting histogenetic process will l)e taken up again later in connection with the development of the two scalae. The area of this rudimentary periotic cistern is as yet very small and merges indefiniteh' into the adjoining reticulum. It is not until we come to fetuses about 40 mm. long that it develops spaces of any considerable size, and it is not until we come to fetuses about 50 mm. long that we find a single large space with walls that are definitely outlined, so that it can be satisfactorilj' modeled.

In a fetus 43 mm. long (Carnegie Collection, No. 886), which is cut in a coronal series, the spaces forming the rudimentary cistern stand out much more definitely than is the case in the 3o-mm. embryo that has just been referred to. There is now just opposite the stapes one space which is much larger than the adjoining spaces. On part of its margin the protoplasmic bridges are stretched along so as to form a smoothly curved continuous boundarj-, which is defective in some portions, and at such places the space merges with the adjoining secondary spaces. Within the space are some fainth' refractive branching threads of coagulated plasma. The scala vestibuli is not yet laid down and the scala tympani is only represented b}' a moderate widening of the meshes of the reticulum in the neighborliood of the fenestra cochleae (rotunda), along the basal border of the first turn of the cochlear duct.

In fetuses 50 mm. long the outlines of the cistern become very distinct, due to the marked increase in the size of its main cavity and to the more definite membrane at its junction with the rest of the reticulum. Its form and relations are shown in figures 26 and 27. Thej- represent a median and a lateral view of a wax-plate reconstruction of this region in a human fetus 50 mm. long (Carnegie Collection, Xo. 84). Onlj^ the main cavity is shown in the model. At certain places around its borders the meshes of the reticulum are uniting into larger spaces and these in turn are taken up by the main cavity as it advances into the new territory. These smaller incomplete spaces were omitted in constructing the plates of the model. The rule was adopted that only the spaces that were outlined by a membrane-like border should be traced on the plates and included in the model. This rule was adhered to in all the models of this series.

Figures 26 and 27 show that the periotic cistern in 50-mm. embryos consists of a flattened, rounded, bursa-like cavity intervening between the stapes and the lateral surface of the saccule and adjoining utricle. It extends forward to the ijmpuUa of the lateral canal and upward to the beginning of the crus commune. Posteriorly it crowds backward against the ductus reuniens, filling in the space between the utricle, saccule, and the proximal end of the cochlear duct. Both on its median and lateral surfaces there is no further opportunity for expansion except as the vestibule itself enlarges. The deUcate membrane-like wall of the cistern hugs closely against the parts of the membranous labyrinth on the one side and the tympanic wall of the cartilaginous vestibule on the other, being separated from them only by a thin layer of the original reticulum. Along the dorsal margin of the cistern, however, there is room for expansion, and the reticulum in this region shows enlarging spaces in the process of uniting with the main cavity. On its ventral margin, near the cochlea and extending along the apical surface of the latter, there is a definite row of reticular spaces actively coalescing and constituting the beginning of the scala vestibuli. These are shown in figure 21, which is a section of a fetus of about the same age. The spaces of the scala vestibuli lie between the cochlear duct and the cistern. This section also shows veiy well the relation of the stapes to the cistern. The scala tymi)ani is already well started at this time, but its development is quite independent of the cistern. Within the cistern can be seen scattered clumi:)s of faintly refractive granular threads of what seems to be a coagulated constituent of the plasma.

The subsequent growth of the cistern is shown in figures 28 to 31. Figures 28 and 29 show respectively a median and lateral view of a wax-plate reconstruction of the membranous labyrinth and its periotic spaces in a human fetus 85 mm. long (Carnegie Collection, No. 1400-30). The growth of the cistern here has kept pace with the increase in size of the lab3'rinth and maintains the same general relations as regards the stapes and the parts of the membranous labyrinth. The view of the cistern in figure 28 is an oblique one which would tend to mislead one as to its width. In reality it is relatively a little wider. It has also extended upward on the dorsal surface of the utricle and is beginning to creep along the inner side of the jjostcrior end of the lateral semicircular duct. Ventrally it communicates freely with the scala vestibuli, which now extends well down along the cochlear duct.

The oldest stage studied is shown in figures 30 and 31. These show two views of a wax-plate reconstruction of these structures in a human fetus 130 mm. long (Carnegie Collection, No. 1018). At this time the periotic cistern has spread over the vestibular part of the meml>ranous labyrinth, covering it nearly eveiywhere excepting at the macular jiortions where the nerves terminate. In figure 31 it can be seen that the mesial surface of the saccule is not covered ; this lies close against the wall of the cartilaginous vestibule. The uppermost division of the cistern, situated between the crus commune and the ampulla of the posterior semicircular duct, does not yet open into the general cavit3^ It has formed separately and owing to the i)osition in which it lies its coalescence with the other parts of the cistern is retarded ; otherwise, free communication exists between all divisions of the cistern.

DEVELOPMENT OF THE PERIOTIC SPACES OF THE SEMICIRCULAR DUCTS

From the descriptions given of the adult the reticulum along the ducts never develops a single continuous wide periotic space like that of the cistern and the two scala?. There always remain a few trabecular, such as are seen in the cistern and scala? in their earlier stages, and these constitute partitions which traverse the space and give it in sections the a])pearance of a series of sej^arate spaces extending along the inner margins of the semicircular ducts. Although these spaces along the ducts are inc()mi)lete as compaicd with the cistern and scahc, they are, however, entirely analogous with them in their formation.

The space along the lateral semicircular duct is the largest. Its posterior end exists as a continuation of the cistern. This can be seen in the lateral view of the model shown in figure 30, where the cistern extends for a considerable distance along the inner border of the lateral duct. Along the other two ducts of the same specimen (130 mm. crown-rump length) the reticulum has commenced the process of space-formation, but complete channels are not yet established. A typical section through one of the semicircular ducts in a fetus of this size, and this is the oldest fetus studied, is shown in figure 23. As compared with the scalse in the same fetus, as shown in figure 20, the space-formation along the ducts is very much retarded.

DEVELOPMENT OF SCALA TYMPANI AND SCALA VESTIBULI

The scala vestibuli may be regarded as an extension of the cistern downward into the region of the cochlea and as such its growth starts from a focus opposite the fenestra vestibuli (ovahs) . The scala tympani in a similar way makes its first appearance opposite the fenestra cochleae. From these two foci the scalae extend gradually downward along the cochlear duct as two separate spaces which do not communicate with each other until they reach the tip of the duct, where there is finallj^ developed a free opening between them known as the helicotrema.

In their formation they go through a series of histogenetic changes essentially in the same manner that has been followed in the case of the formation of the cistern ; this (as we shall see) consists of the enlargement of the spaces of the periotic reticulum that originally occupied this region, the enlargement being a result of the disappearance of the protoplasmic bridges of the reticulum, whereby adjacent spaces unite in the formation of composite larger spaces. This process continues until there is a single continuous space extending down along the cochlear duct representing each scala and at the margins of each of them there is developed a membranous arrangement of the reticular cells which completely walls ofif the space from the surrounding tissue. In these alterations in the reticular mesh and in the formation of the surrounding membrane there is an active change in the form of the reticular cells, which repeatedly adapt themselves to the new conditions. There is no evidence to indicate that smy other cells take part in the formation of the scalae.

The first evidence of the formation, of scalae is found in fetuses about 40 mm. long, which stage is a little later than the first appearance of the cistern. In a fetus 43 nmi. crown-rump length (Cargnegie Collection, No. 886), along the proximal part of the cochlear duct on its basal surface there is a distinct widening of the meshes of the periotic reticulum. This is the beginning of the scala tj^mpani. On the opposite side of the cochlear duct, where one would look for the scala vestibuh, the periotic reticulum retains its primitive appearance characterized by a narrow and rather uniform mesh. Thus the scala tjTnpani makes its appearance slightly in advance of the scala yestibuli — that is, if we regard the latter as distinct from the cistern.

In fetuses 50 mm. long both the scala tympani and the scala vestibuli can be plainly identified, although they are still very incomplete. A wax-plate reconstruction of them, rejiresenting their form and their relation to the membranous labyrinth in a human fetus 50 mm. crown-rump length (Carnegie Collection, No. 84), is shown in figures 26 and 27, being a median and a lateral view respectively.

In preparing this and tlie models shown in figures 28 to 31, it is to be remembered that only those periotic spaces are included that were outlined by a membranelike margin. In the adjacent reticulum there are spaces that are actively coalescing and gradually uniting with the main cavity. No attempt, however, was made to show such spaces in the models. From figures 26 and 27 it will be seen that the scala tympani is larger and more advanced in its development than the scala vestibuli. The hitter is in its earliest stage and consists of hardly more than a row of enlarged reticular spaces which extend downward from the cistern along the dorsal and apical surface of the cochlear duct. A section through the scala vestibuli in another fetus of about the same age (Carnegie Collection, No. 448) is roughly shown in figure 21, the spaces of the scala being situated between the cistern and the cochlear duct.

The scala tympani consists of an elongated oval space lying along the basal surface of the proximal jiart of the cochlear duct, about corresponding to the proximal half of the first turn of the duct. In the main part it is a single space with a distinct margin separating it from the general periotic reticulum. In the more apical portion it tapers off into multiple incompletely united smaller spaces which actively coalesce as the process advances into the new territory along the duct. It is of interest to note that the most mature and the largest part of this scala, representing the focus at which it first appeared, is opposite the fenestra cochleae (rotunda), just as the cistern forms opposite the stapes and the fenestra vestibuli. The scala tympani alw-aj's begins at the same place and extends downward along the cochlear duct, at first a Uttle in advance of the scala vestibuli, but subsequenth^ the latter catches up with it and the two reach the tip of the duct at about the same time.

It is well known that the proximal portions of the cochlear duct mature sooner than the distal portions. One might expect that the accompanying periotic spaces would correspond in their development to the maturity of the duct and therefore the proximal parts of the scalse would differentiate first. In other words, the maturation of the cochlea proceeds as a wave from the proximal end to its tip, involving all of its constituent structures as it passes along, including mesenchymal j)arts as well as epithelial.

This conception might exi)lain the direction of development of the scalae, but can hardly be ai:)plied to the cistern, the vestibular repres(>ntative of the scala vestibuli. One can not say that those portions of the membranous labyrinth lying opi^osite the focus of development of the cistern (that is, the lateral walls of the saccule and utricle) mature in advance of the rest of the labyrinth. There is no indication that a wave of differentiation passes through the epithelial elements of the labyrinth in the same direction and sj'nchronously with the extension of the cistern as it advances from its j)rimary focus u])()n the roof of the utricle and over on its median surface. In the case of the ci.stern it seems much more likely that the point at which it first apjiears is determined by the position of the stai)es, which is doubtless an expression of the physical relation that subsequently exists between the two. Hy analogy this would yield additional significance to the relation existing between the fenestra cochlea; and the point of beginning development of the scala tympani.

In dealing with the cistern and also with the scalse one should not consider them as insignificant accessories that merely fill in the waste intervals between the membranous labyrinth and the surrounding cartilage. From stud3dng their development it becomes apparent that they have a morphological individuality in many respects as definite as that of the ossicles themselves. They make their appearance at a definite time and at definite places, they spread in a definite manner, and eventually they attain a form and structure that are adapted to a definite function. This becomes more and more evident as we examine older stages.

The form and relations of the scalie in fetuses between 12 and 13 weeks old are shown in figures 28 and 29. These figures show median and lateral views of a waxplate reconstruction of the membranous labyrinth and the surrounding periotic spaces in a human fetus 85 mm. crown-rump length (Carnegie Collection, Xo. 1400-30). Attention has already been directed to these figures in the description previously given of the cistern. The scala vestibuli can be seen in figure 28. Above, it opens freely into the cistern and extends downward along the apical side of the duct as a single main space, possessing a rather uniform diameter. It extends along the first two turns of the duct, gradually tapering off and showing a less mature character in its distal portions. Along the second turn of the duct the spaces are incompletely fused and the contour becomes correspondingly irregular. As a rule the peripheral margin of the scala is less mature and more irregular than the central margin. The scala, vestibuli does not connect with the scala tjTiipani at any point as yet. The two are separated in the first place by the cochlear duct and then more centrally b}- a framework of connective tissue in which are the radiating bundles of the cochlear nerve with the nodes of ganglion cells that form the spinal gangUon. These latter structures are not shown in the model; they occupy, however, the V-shaped groove seen between the two scalae.

The scala tj^mpani, as can be seen in figure 29, extends downward on the basal side of the cochlear duct along its first two turns. This corresponds to about the same linear dimension as that of the scala vestibuli. In its proximal portion it shows a greater area in cross-section than the latter, but further toward the apical region it is of about the same size and in some places it is even smaller. The peripheral margin of the scala tympani is distinctly more irregular than the central margin. The irregularity is due to spaces along this margin that are actively coalescing with the main sjiace, but in which the fusion is not yet complete. The irregularity of this margin is thus an indication of the direction of the expansion of the scala. As the diameter of the whole cochlear mass increases, it is evident that the main growth of the scala must radiate outward in a peripheral direction. This is accomplished by the continual assimilation of new reticular spaces along this margin. At the proximal end of the scala tympani can be seen an oval depression which corresponds to the fenestra cochleie (rotunda) and with which it stands in intimate relation.

In fetuses about 16 weeks old the form and relations of the scalae have nearly attained the adult conditions, and this represents the oldest stage studied in connection with the present paper. The conditions found at this time are shown in figures 30 and 31, which present nuMhan and lateral views of a wax-plate model of a human fetus 130 mm. crown-rump length (Carnegie Collection, No. 1018). On comparing the scala tympani and scala vestibuli as seen in these figures with those in figures 28 and 29, it will be seen that they are larger in cross-section and more nearlj- cover the cochlear duct. Furthermore, they now extend to the extreme tip of the duct and communicate with each other across its central margin, thus forming a helicotrema. A section through this point can be seen in figure 25, in which these structures are shown as seen under low magnification. It will be noted that now, even as far as the tip of the cochlea, each of the scalse consists of a continuous principal space, though both are more mature and larger in their proximal portions. Along the first turn of the cochlear duct they are walled off by a smooth membranous margin which separates them from the adjacent reticular tissue. The spaces of the latter do not seem to be taking anj^ further part in the I)rocess of enlargement of the scala". Along the second turn of the cochlear duct, a section of which is shown in figure 20, the coalescence of reticular spaces w'ith each other and with the scalse is still in active operation. This produces a greater irregularity of the scalar than is shown in the model. The subsidiary spaces are shown as a solid mass; the slender clefts separating them are not represented. The nearer one a})proaches the tip of the duct the more immature are the scalse, until the condition is reached that is shown in figure 25, where the membrane-like margin is {[uite incomplete and the spaces merge irregularly with the surrounding reticulum. Thus a single specimen, if studied in its different parts, shows several stages in this interesting process of the formation and growth of the scalaj.

The figures grouped on plate 3 illustrate some of the histological features of this process. An early stage in space-formation is shown in figure 23. This is a section through the canal region where the changes in the reticulum are late in making their appearance. In fact, the periotic spaces never reach the same degree of differentiation here that occurs in the case of the cistern and scalse. The initial steps, how-evcr, are the same, and this figure presents very w^ell the appearance of the periotic reticulum as it begins to open up into larger spaces. Unmodified reticulum is characterized by a rather uniform narrow mesh. The essential change in space-formation consists in the disappearance of some of the trabeculse of the mesh, with the consequent coalescence of the corresponding adjacent spaces. The trabecuhe consist of the protoplasmic processes of the constituent cells of the reticulum and their disappearance is to be explained in either of two ways: It is I)ossiblc that owing to some property of the fiuid element of the tissue the protoplasmic strands are dissolved or liquefied; this would account for their complete disajipearance. On the other hand, the same result could be accomj)lished by an alt(.'ration in the form of the cell processes. A given trabecula could separate at either end, or at some jioint along its line, and the free ends of protoplasm could then retract and reshape themselves and become a part of the remaining framework. Whether we are dealing with a licjuefaction of tissue or with active motility of the cell, protoplasm involviiig detachment and retraction of the trabeculae can not be definitel}^ determined by observations of fixed tissue; but the appearance of sections where the process is in active operation seems to the writer to indicate the latter.

In the above paragraph and elsewhere in this paper reference is made to trabeculse ser^dng as "partitions" between "spaces" and the disappearance of trabeculse resulting in the "coalescence of adjacent spaces." In making this use of the term "space" it should be explained that it is done in a descriptive sense, in application to the appearance of the tissue as seen in sections in which form human embryological material is mainly available. In thin sections of a reticular tissue one sees trabecule as partitions separating adjacent spaces. The same tissue in a mass would show that the spaces everywhere communicate freely with each other, hke the spaces in a sponge, and that the trabeculse are thread-like strands which at the best are very incomplete partitions. Instead of a meshwork containing manj' small spaces, one could perhaps equalh' well describe reticular tissue as a single large space traversed by mam^ trabeculse. If the latter practice were adopted, one would describe the development of the tissue-spaces with which we are concerned as a process of gradual decrease in the number of traversing trabeculse, with the result that the mesh thereby becomes coarser. For descriptive purposes, however, it is convenient to refer to the intervals between the strands of the mesh as spaces, at the same time not granting them the significance that is attached to such membrane-Uned tissue-spaces as are represented by the vestibular cistern and the two scalse, though the latter are in reaUty derived from them.

In figure 23 the free detached ends of the trabeculse will be noted everywhere, as is characteristic of this stage of development. It is a necessarj^ step in the coalescence of adjacent spaces. The detached trabeculse seem to be gradually retracting and adapting themselves to the formation of larger spaces. Their constituent protoplasm reshapes itself as a smooth border or as a part of other trabeculse. Larger spaces necessitate longer trabeculse, and as trabecuUe become longer they also tend to become heavier. These phenomena are all in evidence in the spreading and enlargement of the scalse.

Figure 20 shows a characteristic view of the scalse as seen under low magnification. It will be noted that the scala vestibuli is relatively mature at this point; the scala tympani, however, is in the act of spreading peripherally, so as to underUe, as it eventually will do, the future basilar membrane. The scala tympani finally reaches the peripheral margin of the cochlear duct, and it does this by the coalescence of the enlarging reticular spaces, which become incorporated with the main cavity of the scala. This can be observed better in figure 22, which shows a detail of the same section as seen under higher magnification. By comparing this figure with figure 20 the exact location can be readily made out. A portion of the main cavity of the scala is indicated and to the right of this are a few enlarged reticular spaces that are uniting with each other and will in the end become part of the main space. In adcUtion to the enlarged reticular spaces there is a certain amount of residual undifferentiated reticulum. It is this tissue that will play the part of an adventitial coat to the completed scala. The trabecula? that separate the enlarged spaces seem to be under tension and about ready to snap apart. In fact, in most sections one can see the fragmentary ends of trabeculse where this interruption of continuity has apparently occurred.

The differentiation of the margin of the scalse constitutes the final feature in their maturation. During the i)eriod in which the enlargement of an individual scala is being brought about by the coalescence f)f enlarging reticular spaces, the margins of the main cavity can be seen to consist of smooth, delicate strands of nucleated protoplasm that resembles in all essentials that of the trabeculae between the large reticular spaces. These linear margins are interrupted here and there by openings into adjacent spaces, but they tend to form a continuous line that definitely marks off the space from the adjacent reticulum. An early stage in the formation of such a margin is shown in figure 25, where the margin is indicated at a few places, but for the most i)art the space abuts against the surrounding ragged reticulum. The margin of the space is more complete in the scala tympani shown in figure 22, but it is still thin and delicate and can be easil}^ opened up to allow the taking in of new spaces. If we examine the borders of more mature spaces we find them inclosed by a firmer membrane, which finally reaches a state that will probably not admit of any further oi)ening up for the coalesence of additional spaces. Any further growth must thereafter be limited to simple distention of the wall of the space with the consequent adjustment of its constituent cells. Such a condition is represented in figure 24. This shows a more mature section of the wall of the scala vestibuli, being a detail of the same section shown in figure 20. The only difference between such a membrane, as we must now call it, and the corresponding structure in younger stages is its density; it is wider and its protoplasm perhaps more opaque, or in other words, more protoplasm is accumulated there.

If figures 24, 22, 25, and 23 are comi^aied and followed in that order, it will be seen that the lining m('ml)rane of the scala* can be traced backward, step by step, to the ordinary trabeculie of the periotic reticulum. There is no histological evidence that any new cells enter into its formation. It seems to be simply a product of the proliferation and adaptive reshaping of the cells already there. In its final form the margin of the space r(\seml)les an endothelial membrane. One could describe, as inunediately lining the s])ace, a thin membrane with flattened nuclei, which is sui)i)orted underneath by a thin coat of nucleated protoplasm thai, has the form of fibrous connective tissue. The former, judging only from its final aj)pearance, one might designate as endothelium and thus make a distinction between it and the underl^ying tissue. In its histogenesis, however, it differs in no way from the rest of the wall and the difference that exists later seems to be merely the result of its adaptation to the existing physical conditions. Its early l)ehavior is entirely different from that of vasculiu- endothcliuiu. Thus if its final ajipearance is stres.sed and the term endothelium is used for its designation, il must be done with a considerable amount of reservation. It is preeminently a i)lace where the term mesothelium could be used with great advantage.

COMMUNICATION OF PERIOTIC SPACES WITH ARACHNOID SPACES

The relation of the scala tj^mpani and scala vestibuli to the subarachnoid spaces surrounding the hind-brain is of considerable interest, both on account of the possibiUty of their functional relationship and on account of the similarity that exists in their development. For a satisfactory investigation of the establishment and the character of the communications that are formed between these two allied systems of tissue-spaces, one should resort to other methods than those used in the present study, and, furthermore, one should examine older fetuses than those described here. In fact, a problem lies here that would be well worth careful study.

Certain observations, however, were made in the course of the above investigation that bear relation to these matters, and they will be briefly outlined here. In the first place, the histological picture of the periotic reticulum is essentially the same as that of the early stages of the pia-arachnoidal tissue investing the central nervous system. The enlargement of the meshes of the latter and the formation of the subarachnoid spaces and the arachnoid cistern, as has been recently described by Weed (1917), correspond exactly with the appearance seen in the histogenesis of the periotic spaces in the ear. The periotic spaces are not, however, extensions of the arachnoid spaces that have invaded the cavity of the cartilaginous labyrinth. If this were so we should find them first appearing among the rootlets of the vestibular and cochlear nerves, along which the .subarachnoid space extends for some little distance. Instead, they begin at points where there can be no connection with the arachnoid tissue and their direction of growth is quite independent of it. The periotic spaces maj' be analogous to the arachnoid spaces, but they are not identical with them, nor are they an extension of them.

According to the descriptions of the adult anatomy of the ear, a communication becomes established between the scala tympani and the subarachnoid space near the fenestra cochleae, the so-called aquaeductus cochleae. Vague and conflicting statements are also made concerning a communication through the internal auditory meatus connecting the arachnoid spaces with the scalae. Such communication must be estabhshed quite late. In the oldest fetus examined, 130 mm. crown-rump length, they did not yet exist. As to the latter communication, it can be seen that the arachnoid spaces extend peri])herally through the internal auditory meatus along the trunk of the acoustic nerve-comjilex, and slender pockets and clefts from them extend along the larger bundles of the cochlear nerve; they terminate, however, before reaching the margins of the scalae, and there is no evidence at this stage that there is ever to be a conununication between them and the scalae. As to the aquaeductus cochleae, in the 130 mm. fetus it can be plainly seen that it is already forming as a deri\'ative of the arachnoid spaces, although the communication with the scala tympani is not yet established. The arachnoid spaces invest the glossojiharyngeal nerve and extend down along its trunk and pa.ss directly V)y the region of the fenestra cochleae (rotunda). A thin-walled tubular pouch projects from these spaces, leaving the nerve trunk and extending obliquely toward the scala tympani in a direction that would meet it just distal to the fenestral impression on its basal surface. This fundament of the aqua?ductus cochleae is present in fetuses 85 mm. crown-rump length, but is longer in the 130 mm. fetus, where it nearly reaches the scala tympani. The communication must be established soon after this.


SUMMARY

The changes in size and form which the cartilaginous capsule of the ear undergoes during its development in the human embryo are accomplished in part by a progressive and in part by a retrogressive differentiation of its constituent tissues. Throughout the entire period of growth, as far as material was available for study, it was foimd that the margins of the cartilaginous cavities undergo a process of continual transformation. They exhibit a state of unstable equilibrium in respect to the opposing tendencies toward a deposit of new cartilage on the one hand and toward the excavation of the old on the other. The margins therebj^ are always either advancing or receding, and it is in this way that the progressive alterations in the size, shape, and position of the cavities are produced, due to which a suitable suite of chambers is always provided for the enlarging membranous labyrinth.

The general tissue mass of the otic capsule, during the period represented by embryos from 4 to 30 mm. long, passes through three consecutive histogenetic periods, nameh', the stage of mesenchymal syncj'tium, the stage of precartilage, and the stage of true cartilage. In the subsequent growth of the capsule it is found that in areas where new cartilage is being deposited the tissues of the areas concerned follow a definite and progressive order of development. In areas, however, where excavation occurs, where cartilage previously laid down is being removed, it is found that the process is reversed. The tissue in such areas returns to an earlier embryonic state — that is, it undergoes dedifferentiation. Tissue that has accjuired all the histological characteristics of true cartilage can thus be traced in its reversion to precartilage and from jirecartilage in turn to a mesenchymal syncytium. In the latter form it redifferentiates into a more specialized tissue, in this case for the most part into a vascular reticulum.

The formation of the periotic reticulum is first indicated by a cluster of deeply staining nuclei that can be seen along the central edge of the semicircular ducts in embryos soon after the ducts are formed, and at about the time the otic capsule begins to change from condensed mesenchyme into precartilage. These nuclei constitute a focus at which the development of the reticulum and its blood-vessels takes origin. Here the tissue of the otic capsule takes on an appearance that is less like that of a cartilage-forming tissue and more like that of an embryonic connective tissue. Spreading from this focus, a narrow area is established which soon encircles the semicircular ducts and becomes the open-meshed vascular reticulum which, in embryos 30 mm. long, everywhere bridges the space existing lietween the epithelial lal)yrinth and the surrounding cartilage. In the earlier stages it could not be definitely shown that the primordium of the i^eriotic reticular tissue is not derived from a few ])rcdestined mesenchyme cells which become inclosed, along with the otic vesicle, by the condensed tissue of the capsule and after a certain latent period undergo proliferation and occup}' the space vacated by the receding precartilage. In the later stages, however, it is cjuite evident that precartilage tissue is actually' converted into a reticulum, and that the replacement of precartilage by a reticular connective tissue is brought about through a process of dedifferentiation.

The perichondrium is a derivative of the periotic reticulum and forms an outei limiting membrane along its cartilaginous margin. During the fetal period the perichondrium does not rest directly against the true cartilage, but is separated from it by a zone of transitional tissue consisting partly of precartilage and partly of reticulum. This transitional zone intervening between the perichondrium and the surrounding cartilage was ob-served in all of the specimens that were studied, which includes fetuses up to 130 mm. crown-rump length. Owing to the fact that the perichondrium is late in making its appearance, being first seen in fetuses about 70 mm. long, it can take no part in the early changes in the cartilaginous capsule, either as regards the deposit of new cartilage or the excavation of cartilage that had been previously laid down.

The periotic tissue-spaces are formed by a modification of the meshes of the periotic reticulum. The latter consists originally of a rather uniform narrow mesh. The essential change which it undergoes in the process of space-formation consists in the gradual disappearance of the traversing trabeculse. The trabeculae consist of the protoplasmic processes of the constituent cells of the reticulum, and their disappearance is apparently due, not to a dissolution or Uquefaction of these cellprocesses, but to an alteration in their form. It apparently is the result of an active motility of the cell protoplasm involving the successive detachment and retraction of the trabeculse. Wlien a trabecula becomes detached it retracts and adapts itself to the formation of the enlarging space, reshaping itself either as a smooth border or as a constituent part of another trabecula.

The differentiation of the margin of the periotic spaces constitutes the final feature in their maturation. During the period in which the enlargement of an individual space is activeh' going on,. the margins of the main cavity consist of smooth, delicate strands of nucleated protoplasm that resemble the trabeculse between the large reticular spaces. These linear margins are interrupted here and there by openings into adjacent spaces. They tend, however, to form a continuous hne that definitely marks ofT the space from the adjacent reticulum. As the space becomes more mature, the membrane-like border becomes thicker until it reaches a state that will probably not admit of any further opening-up for the coalescence of additional spaces. Any further growth is thereafter limited to a simple distention of the wall of the space, with the consequent adjustment of its constituent cells. In its final form the margin of the space constitutes a mesothehal membrane. Immediately lining the space is a thin membrane with flattened nuclei which is supported underneath by a thin coat of nucleated protoplasm having the form of fibrous connective tissue. The former in its histogenesis differs in no way from the rest of the wall and the difference that exists later seems to be merely the result of its adaptation to the existing physical conditions.

'J'hc oarlie.st histological evidence of the formation of the periotic spaces occurs near the stapes, in the reticulum that bridges the interval l)etween the sacculus and the fenestra vestibuh. In embrj'os between 30 mm. and 40 mm. long, it can be seen that the meshes in this region are becoming irregular and larger, due to the disapjiearance of some of the trabeculae and a consequent coalescence of the intertrabecular si)aces. The widening of the mesh at this point constitutes the primordium of the vestibular cistern. It makes its appearance before there is any trace of the scalse, but it is not until the fetus reaches a length of about 50 mm. that the cistern becomes definitely outlined and clearly differentiated from the adjoining reticulum.

Following the appearance of the cistern, the scala tympani is the next space to become established. It can be recognized as a moderate widening of the meshes of the reticulum in the region of the fenestra cochleae in fetuses 43 mm. long, along the basal border of the first turn of the cochlear duct. The scala vestibuli, as can be seen in fetuses 50 mm. long, develops as an extension downward of the cistern along the apical border of the cochlear duct. Starting from these definite foci, these three spaces spread into their destined territory, absorbing as they go the enlarging reticular spaces of the invaded region by a process of space-coalescence, or, in other words, the progressive formation of areas that are free of trabeculse. In fetuses 85 mm. long the two scalse extend downward along the cochlear duct to its last turn, as two separate spaces which do not communicate with each other. When they reach the tip of the duct, which occurs in fetuses about 130 mm. crown-rump length, a free opening is developed between them which represents the hehcotrema. After being completely established along the whole length of the cochlear duct, the scalae continue to enlarge by further coalescence of tissue along their peripheral border, in which the trabecular disappear.

The periotic sjxices are analcjgous in their development to the pia-arachnoidal spaces; they are not, however, extensions of them that have invaded the cavity of the cartilaginous labyrinth. They begin at points where there can be no connection with the arachnoidal tissue and their direction of growth is quite independent of it. The communication that is found in the adult between the scala tympani and the subarachnoid sjjace in the neighborhood of the fenestra cochleae, the socalled af|ua'ductus cochleae, is established (juite late. In fetuses 85 mm. crownrump length it exists as a tubular pouch projecting from the subarachnoid s])aces along the glossopharyngeal nerve toward the scala tj^mpani. In the 13()-mm. fetus, the (jldest examined, this j^ouch is longer and nearly reaches the scala. The communication must be established soon after this.

Similar iirojections from the subarachnoid spaces at the internal auditory meatus extend as perineural clefts along the trunk and branches of the acoustic nerve. No actual cf)mnnuiications, however, were seen between these spaces and the two scalae.


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EXPLANATION OF PLATES

Plate 1.

The figures on Plates I aiui II represent a series of photographs of the ear region inhuman embryos varying from 4 mm. to i;{0 mm. long. The photographs were taken at a magnification of 1()0 diameters and as far as possible at similar positions, so that a eomparison of them would indicate the actual increase in size and the relative amount and form of the individual tissue-masses. In the reproduction tliey were reduced to about 90 diameters. The different figures include the principal stages in the development of the cartilaginous capsule of the car and show the gross features of the histogenesis of the periotic reticulum. Figures 5 to 7 cover the period during which the mesenchyme becomes condensed around the otic vesicle. Figures 8 to 10 show the otic capsule in its precartilage stage and the manner in which the precartilage becomes difTerentiated into relatively i)ermanent and temporary zones. The latter encircle the epithelial ducts and correspond to the future cartilaginous canals. In figures 1 1 to 13 the main capsular mass has become true cartilage, whereius the temporary zone of precartilage surrounding the canal is on the point of dedifferentiating into periotic reticulum. A focal area of vascularized reticulum is already established at the inner margin of the epitheUal duct.

Fig. 5. Frontal section through the region of the ear in a human embryo 4 mm. long (Carnegie Collection, No. 588, slide 6, row 6, section 6). The section is 1.5^ thick and is enlarged 90 diameters. It shows part of the brain-wall and t he ot ic vesicle with the surrounding mesenchyme. The nuclei of the latter are more numerous in the neighborhood of the vesicle, indicating the beginning of the capsular condensation.

Fig. 6. Horizontal section through the region of the ear in a human embryo 9 mm. long (Carnegie Collection, No. 721, slide ,"), row 2, section 1). The section is l.'i/i thick and is enlarged 90 diameters. It shows a distinct condensation of the mesenchyme around the otic vesicle, particularly on its lateral surface (above) where it extends frotn the surface of the vesicle to about half the distance from the vesicle to the ectoderm.

Fig. 7. Frontal section through the labyrinth in a human embryo 11 mm. long (Carnegie Collection, No. 353, shde 16, row 3, section 4). The section is 10ft thick and is enlarged 90 diameters. It shows the vestibular part of the labyrinth with the appendage opening out of it and passes transversely through the pouches whose margins are to form the superior and lateral semicircular ducts. There is now a very complete capsule of condensed mesenchyme surrounding every part of the labyrinth, with the exception of the appendage and the regions of the interna! auditory meatus and the fenestra cochle«.

Fig. 8. Horizontal sec'tioii through the otic capsule in a human embryo 15 mm. long (Carnegie Collection, No. 719, slide 3, row 2, section 3). The .section is 40^ thick and is enlarged 90 diameters. It shows a portion of the utricle below and the superior semicircular duct above. Surrounding these is a definite capsule of precartilage tissue.

Fig. 9. Sagittal section through the otic capsule in a human embryo 18 mm. long (Carnegie Collection, No. 144, slide 4, row 1, section 3). The section is 40fi thick and is enlarged 90 diameters. Above is the posterior semicircular duct, and just below the center is the lateral semicircular duct. The otic capsule is now differentiated into relatively permanent are:is of prccarlilago and other are;is that are more temporary. The latter surround the epithelial ducts and indicate the future cartilaginous canals.

Fig. 10. Frontal .section through the otic capsule in a human embryo 27 mm. crown-rump length (Carnegie Collection, No. 756a, slide 47, section 2). The section is .")()/i thi<-k and is enlarged 90 diameters. It passes transversely through the lateral .semicircular canal. The epithelial duct is surrounded by a zone of temporary precartilage corresponding to the future cartilaginous canal. Just median to the duct (below it in the photograph) is a group of nuclei that forms the focus of the future fp-owth of reticulum.

Fio. 1 1 . Section through the lateral semicircular canal in a human fetus 30 mm. crown-rump (Carnegie Collection, No. 86, slide 46, section 2). The section is HOit thick and is enlargetl 90 diameters. The main capsular mass is now differentiated into true cartilage. The zoik- of temporary precartilage is beginning to recede from the epithelial <luct, leaving a reticular area in the interval, which is more pronounced on the median side of the duct (below it in the photograph).

Fig. 12. .Section through the lateral semicircular canal in a human fetus 37 mm. crown-rump length (Carnegie Collection, No. 972, slide 20, section 1). The section is 50m thick and is enlarged 90 diameters. The nuclei of the zone of temporary precartilage form a dark field that corresponds to the future cartiliiginous canal. Along the inner margin of this zone are seen large blood-vessels that belong to the periotic reticulum.

Fig. 13. Section through the lateral semicircular canal in a human fetus 35 mm. crown-rump length (Carnegie Collection, No. 199, .slide .58, section 2). The section is 50;u thick and is enlarged 90 diameters. It is stained deeply with hematoxylin, showing the matrix of the cartilage but not the zone of precartilage that is to become the cartilaginous canal.

Plate 2.

The figures on Plate II are in continuation of those on Plate I and .show the final establishment of the periotic reticular tissue. They also show, on being comp.arerl with younger stages, the manner in which the cartilage becomes excavated in order lo yield room for the enlarging duct and also to allow for its changing position. The excavation is brought about by the dedilTerentiation of cartilage into reticuku- tissue. Throughout this period the margin of the cartilaginous canal continues in an unstable condition and is gradually either

52


EXPLANATION OF PLATES. 53

receding or advancing, through the processes of dedifferentiation, into precartilage or differentiation from precartilage respectively. The periotic reticulum in its later stages develops fibrous membranes at its inner and outer borders. The one at the inner border forms the membrana propria for the epithelial duct, and the one at the outer border becomes the perichondrium.

Fig. 14. Section through the lateral semicircular canal in a human fetu.s 43 mm. crown-rump length (Carnegie Collection, No. 886, slide 42, section 3). The section is 100m thick and is enlarged 90 diameters. The zone of precartilage is expaniling around its peripheral margin by dedifferentiation of the surrounding cartilage and on its central margin the precartilage is giving way before the advancing reticulum. A crescentic area of periotic reticulum is established on the median side (to the left) of the epithelial duct, about 8 mm. deep in the photograph.

Fig, 1.5. Section through the lateral semicircular canal in a human fetus 46 mm. crown-rump length (Carnegie Collection, No. 9.5, slide 72, section 1). The section i.s 100m thick and is enlarged 90 diameters. The original area of precartilage is now all dedifferentiated into reticulum, and a new area of precartilage has formed outside of this at the expense of the smioundiiig cartilage. The new area of precartilage is about OS cm. deep in the photograph. Everj-thing between this and the epithelium is reticulum, the peripheral part of which is not yet completely vascularized.

Fig. 16. Section thiough the posterior semicircular canal in a human fetus 50 mm. crown-rump length (Carnegie Collection, No. 184, shde 23). The .section is 50^ thick and is enlarged 90 diameters. The dedifferentiation of precartilage into reticulum is nearly complete, there being left ordy a narrow hne of it along the margin of the cartilage. The vascularization of the reticulum is not yet completed. The small diameter and the thick wall of the epithelial duct in this figure and in figure 15 result from contraction. If they were distended in the process of fixation they would doubtless be as large as those in figures 14 and 17.

Fig. 17. Section through the posterior semicircular canal in a human fetus 52 mm. crown-rump length (Carnegie Collection, No. 96, shde 12, section 2). The section is 100m thick and is enlarged 90 diameters. It differs from figui'e 16 in having a more mature periotic reticulum.

Fig. is. Section through the posterior semicircular canal in a human fetus, So mm. crown-rump length (Carnegie Collection, No. 1400-30, slide 43, section 2). The section is 100m thick and is enlarged 90 diameters. At the inner margin of the reticulum can now be seen the membrana propria .supporting the semicircular duct and at the outer margin is the thick peri.'hondrium, between which and the cartilage there is a narrow open space that is better seen on the left part of the photograph. The sharp dark line along the margin of the cartilage on the right is an appearance due to the excavation of cartilage at that point. It consists of an intermediate zone in which the cartilage is being dedifferentiated into precartilage and that in turn into reticular tissue.

Fig. 19. Section through the superior semicircular canal in a human fetus 130 mm. crown-rump length (Carnegie Collection, No. 1018, slide 30, section 1). The section is 50m thick and is enlarged 90 diameters. It shows a rather mature perichondrium closely attached to the cartilage, separated from it, however, by a narrow intermediate zone that is not seen in the photograph. ThLs zone is connected with the further enlargement of the cartilaginous canal, the growth of which is not yet completed. In the outer part of the canal the perichondrium fuses with the membrana propria of the semicircular duct. The periotic reticulum is beginning to break up in the formation of larger spaces, which it does by the retraction of its trabecute, thereby allowing adjacent spaces to coalesce. The blood-vessels in this specimen were injected with India ink.

Plate 3.

The figures on Plate III show the histological appearance of the periotic tissue-spaces and the manner in which they are formed from the periotic reticulum. This is accomplished by the disappearance of the trabeculae and the consequent repeated coalescence of adjoining spaces.

Fig. 20. Section through the second turn of the cochlea in a human fetus 130 mm. crown-rump length (Carnegie Collection, No. 1018, slide 32, section 2), enlarged 57 diameters. This section shows the topograph}' of the cochlear duct and the general character of the periotic spaces that are developing along its inner margins. Details of this same section as seen under higher magnification are shown in figures 22 and 24.

Fig. 22. Detail of the section shown in figure 20, enlarged 278 diameters. This figure shows the part of the coclilear duct that is to form the organ of Corti with the adjacent tissue that becomes incorporated in the basilar membrane. Below is the periotic reticulum, whose spaces are in the process of enlarging. By repeated coalescence these spaces finally unite with the large space which constitutes the scala tympani. This figure shows the histological appearance of the reticulum where the formation of ti.ssue-spaces is in active operation.

Fig. 24. Detail of the section shown in figure 20, enlarged 300 diameters. It shows the character of the margin of the scala vestibuli in a fairly mature condition. The scala vestibuli is inclosed by a membrane consisting of the cells that had previously constituted the reticulum occupying this area and which have been modified in form in adaptation to the formation of this large tissue-.space, closing it off from the surrounding tissue.

Fig. 21 . Section through the vestibular portion of the labyrinth in a human fetus 52 mm. crown-iump length (Carnegie Collection, No. 448, slide 154, section 2), enlarged 31 diameters. This section shows the general character of the periotic spaces and their relation to the different parts of the membranous labyrinth and the surrounding cartilaginous capsule. The first space to develop and the largest shown in this figure is the vestibular cistern, situated between the utricle and the cartilaginous stapes. The smaller spaces, belowthe cistern and extending downward along the cochlear duct, represent the scala vestibuli in an early form. The arteries in this specimen were injected with Imlia ink and are shown in black.


54 EXPLANATION OF PLATES.

Fir,. iV Section tlirough the superior semicircular canal in a human fetus 130 mm. crown-rump length (Carnegie Collection, \o. 1018, slide 29, section 2), enlarged 90 diameters. The periotic reticulum Ls undergoing the alterations characteristic of the earlj' st^es of the formation of tissue-spaces. Along the margins of the cartilage the reticular tissue is condensed and (^oiLstitutes the fibrous pcrichoiulrium. .\round the epithelial canal there is developed a layer of supporting tissue h hicli forms the iiieinbrana propria. This layer fuses with the perichondrium along the jx-ripheral margin of thi- ciiiial :in<l thereby constitutes a ligament that attaches each membranous duct throughout its whole length to the cartilaginous .space in which it is suspended.

Fin. 2.'i. Section through the apex of the cochlea of a human fetus 130 mm. crown-rump length (Carnegie Collection, No. 1018, slide 32, section 2), enlarged 57 diameters. This section shows the tip of the cochlear duct and the character of the communication that develops between the two scate forming the helicotrenia. It will be seen that the margins of the periotic spaces are not so mature here as in the proximal parts of the cochlea of the same fetua, on comparing tliis figure with figure 20.

Plate 4.

The figures shown on this plate represent a series of median and lateral views of wax-plate reconstructions of the membranous labyrinth and the surrounding periotic tissue-spaces. They illustrate under the same scale of enlargement three typical stages in the development of these spaces. Abbreviations: C. s. 1., ductus semicircuiaris lateralis; C. s. p., ductus semicircularis posterior; C. s. s., ductus semicircularis superior; Duct, coch., ductus cochlearis; Impressio rotund., area opposite the fenestra cochleae; Impressio staped., area in contact with base of stapes; Saccus endol., saccus endolymphaticus; Scala tymp., scala tynipani; Scala vestib., scala vestibuH.

Fio. 26. Lateral view of a model reconstructed from a human fetus 50 mm. crown-rump length (Carnegie Collection, No. 84). The cistern and the scala vestibuli are shown in green and the scala tympani is shown in orange. The scala vestibuU is in the first stage of its development and consists of a row of large reticular spaces which extend from the ventral margin of the cistern downward along the apical surface of the cochlear duct. The scala tympani is more advanced and shows more complete coalescence of its constituent spaces. Enlart;eii 1 1 .4 diameters.

Fin. 27. Median view of the same model .shown in figure 26. This view shows the tojjography of the scala tympani. Its large proximal end lies opposite the fenestra cochleje (rotunda) and corresponds to the focus at which its development originates. Distally it tapers off rapidly where the spaces are smaller and their coalescence less complete. Enlarged 11.4 diameters.

Fi(i. 28. Lateral view of wax-plate reconstruction of the left membranous labyrinth and the periotic spaces in a human fetus 85 mm. crown-rump length (Carnegie Collection, No. 1400-30), enlarged 11.4 diameters. The cistern and the connecting scala vestibuli are shown in green. Although the greater jiart of the cistern abuts against the stapes, it will be noted that it is also begiiming to spread over the liorsal surface of the utricle and along the inner border of the lateral semicircular duct. The scala vestibuh communicates freely with the cistern and extends downward alotig the apical surface of the cochlear duct, throughout nearly two turns, showing the characteristic sacculated appearance near its tip, w here the coalescence of the spaces is less complete.

Fi<;. 29. Median view of same model shown in figure 28, enlarged 11.4 diameters. The scala tympani is shown in orange. The oval indentation in its proximal end corresponds to the fenestra cochlea (rotunda). This space extends along the cochlear duct about the same distance as the scala vestibuli, but the two do not comMUinicate yet at any place. The peripheral border of the scala tympani is characterized by sacculations corresponding to spaces that are coalescing with the main s|)a(c. The grow th of the scala is due to a coalescence of new spaces along its peripheral border rather than along its cential border.

Fio. 30. Lateral view of a wax-plate reconstruction of the li'ft membnmoiis labyrinth and the periotic spaces in a human fetus 130 mm. crown-rump length (Carnegie Collection, No. 1018), enlarged 11.4 diameters. The cistern and scala vestibuli are shown in green and the scala tympani is shown in orange, as in the previous figures. The cartilaginous stapes was removed from this model and the oval impression that it makes on the cistern can be plaiidy seen. The cistern has spread over the top of the utricle and part way along the lateral semicircular duct. The scala vestibuli extends to the ti|) of the cochlear duct, where it communicates with the McaLi tympani, thas forming the helicotrema.

Fki. 31 . Mcnlian view of .same model shown in figure 30, enlarginl 1 1.4 diameters. The oval impression on the proximal i-nd of the scala tympani corresponds to the fenestra cochle;c (rotunda). .\s yet there is no conmiunication at this point between the scala tympani and subarachnoid spaces, such :is is found in the adult and known as the aqua-ductus cochlete. The spaces making up the <ist<'rn cover almost the whole of th<' utricle and saccule except the places at whicli t he nervi-s enter and a small part of the medial surface near the attachment of tlie appendage.