CHAPTER VII.

GENERAL DEVELOPMENT OF THE TRUNK FROM STAGE H TO THE CLOSE OF EMBRYONIC LIFE.

_External Epiblast._

The change already alluded to in the previous chapter (p. 317) by which the external epiblast or epidermis becomes divided into two layers, is completed before the close of stage L.

In the tail region at this stage three distinct strata may be recognized in the epidermis. (1) An outer stratum of flattened horny cells, which fuse together to form an almost continuous membrane. (2) A middle stratum of irregular partly rounded and partly flattened cells. (3) An internal stratum of columnar cells, bounded towards the mesoblast by a distinct basement membrane (Pl. 13, fig. 8), unquestionably pertaining to the epiblast. This layer is especially thickened in the terminal parts of the paired fins (Pl. 13, fig. 1). The two former of these strata together constitute the epidermic layer of the skin, and the latter the mucous layer.

In the anterior parts of the body during stage L the skin only presents two distinct strata, viz. an inner somewhat irregular layer of rounded cells, the mucous layer, and an outer layer of flattened cells (Pl. 13, fig. 8).

The remaining history of the external epiblast, consisting as it does of a record of the gradual increase in thickness of the epidermic strata, and a topographical description of its variations in structure and thickness in different parts, is of no special interest and need not detain us here.

In the late embryonic periods subsequent to stage Q the layers of the skin cease to be so distinct as at an earlier period, partly owing to the innermost layer becoming less columnar, and partly to the presence of a large number of mucous cells, which have by that stage made their appearance.

I have followed with some care the development of the placoid scales, but my observations so completely accord with those of Dr O. Hertwig[228], that it is not necessary to record them. The so-called enamel layer is a simple product of the thickening and calcification of the basement membrane, and since this membrane is derived from the mucous layer of the epidermis, the enamel is clearly to be viewed as an epidermic product. There is no indication of a gradual conversion of the bases of the columnar cells forming the mucous layer of the epidermis into enamel prisms, as is frequently stated to occur in the formation of the enamel of the teeth in higher Vertebrates.

Footnote 228: _Jenaische Zeitschrift_, Vol. VIII.

_Lateral line._

The lateral line and the nervous structures appended to it have been recently studied from an embryological point of view by Götte[229] in Amphibians and by Semper[230] in Elasmobranchii.

Footnote 229: _Entwicklungsgeschichte d. Unke._

Footnote 230: _Urogenitalsystem d. Selachier._ Semper's _Arbeiten_, Bd. II.

The most important morphological result which these two distinguished investigators believe themselves to have arrived at is the direct derivation of the lateral nerve from the ectoderm. On this point there is a complete accord between them, and Semper especially explains that it is extremely easy to establish the fact.

As will appear from the sequel, I have not been so fortunate as Semper in elucidating the origin of the lateral nerve, and my observations bear an interpretation not in the least in accordance with the views of my predecessors, though not perhaps quite conclusive against them.

It must be premised that two distinct structures have to be dealt with, viz. the _lateral line_ formed of modified epidermis, and the _lateral nerve_ whose origin is in question.

The lateral line is the first of the two to make its appearance, at a stage slightly subsequent to K, in the form of a linear thickening of the inner row of cells of the external epiblast, on each side, at the level of the notochord.

This thickening, in my youngest embryo in which it is found, has but a very small longitudinal extension, being present through about 10 thin sections in the last part of the head and first part of the trunk. The thickening, though short, is very broad, measuring about 0.28 Mm. in transverse section, and presents no signs of a commencing differentiation of nervous structures. The large intestinal branch of the vagus can be seen in all the anterior sections in close proximity to this line, and appears to me to give off to it posteriorly a small special branch which can be traced through a few sections, vide Pl. 13, fig. 2, _n.l_. But this branch is not sufficiently well marked to enable me to be certain of its real character. In any case the posterior part of the lateral line _is absolutely without any adjoining nervous structures or traces of such_.

The rudiment of the epidermic part of the lateral line is formed of specially elongated cells of the mucous layer of the epiblast, but around the bases of these certain rounder cells of a somewhat curious appearance are intercalated.

There is between this and my next youngest embryo an unfortunately large gap with reference to the lateral line, although in almost every other respect the two embryos might be regarded as belonging to the same stage. The lateral line in the older embryo extends from the hind part of the head to a point well behind the anus, and is accompanied by a nerve for at least two-thirds of its length.

In the foremost section in which it appears the intestinal branch of the vagus is situated not far from it, _and may be seen at intervals giving off branches to it_. There is no sign that these are otherwise than perfectly normal branches of the vagus. Near the level of the last visceral cleft the intestinal branch of the vagus gives off a fair-sized branch, which from the first occupies a position close to the lateral line though well within the mesoblast (Pl. 13, fig. 3_a_, _n.l_). This branch is the lateral nerve, and though somewhat larger, is otherwise much like the nerve I fancied I could see originating from the intestinal branch of the vagus during the previous stage.

It rapidly thins out posteriorly and also approaches closer and closer to the lateral line. At the front end of the trunk it is quite in contact with it, and a short way behind this region the cells of the lateral line arrange themselves in a gable-like form, in the angle of which the nerve is situated (Pl. 13, figs. 3_b_, and 3_c_). In this position the nerve though small is still very distinct in all good sections, and is formed of a rod of protoplasm, with scattered nuclei, in which I could not detect a distinct indication of cell-areas. The hinder part of the nerve becomes continually smaller and smaller, without however presenting any indication of becoming fused with the epiblast, and eventually ceases to be visible some considerable distance in front of the posterior end of the lateral line.

The lateral line itself presents some points of not inconsiderable interest. In the first place, it is very narrow anteriorly and throughout the greater part of its length, but widens out at its hinder end, and is widest of all at its termination, which is perfectly abrupt. The following measurements of it were taken from an embryo belonging to stage L, which though not quite my second youngest embryo is only slightly older. At its hinder end it was 0.17 Mm. broad. At a point not far from this it was 0.09 Mm. broad, and anteriorly it was 0.05 Mm. broad. These measurements clearly shew that the lateral line is broadest at what may be called its growing-point, a fact which explains its extraordinary breadth in the anterior part of the body at my first stage, viz. 0.28 Mm., a breadth which strangely contrasts with the breadth, viz. 0.05 Mm., which it has in the same part of the body at the present stage.

It still continues to form a linear area of modified epidermis, and has no segmental characters. Anteriorly it is formed by the cells of the mucous layer becoming more columnar (Pl. 13 fig. 3_a_). In its middle region the cells of the mucous layer in it are still simply elongated, but, as has been said above, have a gable-like arrangement, so as partially to enclose the nerve (Pl. 13, fig. 3_b_). Nearer the hind end of the trunk a space appears in it between its columnar cells and the flattened cells of the outermost layer of the skin (Pl. 13, fig. 3_c_), and this space becomes posteriorly invested by a very definite layer of cells. The space (Pl. 13, fig. 3_d_) or lumen has a slit-like section, and is not formed by the closing in of an originally open groove, but by the formation of a cavity in the midst of the cells of the lateral line. Its walls are formed by a layer of columnar cells on the inner side, and flattened cells on the outer side, both layers however appearing to be derived from the mucous layer of the epidermis. The outer layer of cells attains its greatest thickness dorsally.

During stages M, N, O, the lateral nerve gradually passes inwards into the connective tissue between the dorso-lateral and the ventro-lateral muscles, and becomes even before the close of stage N completely isolated from the lateral line.

The growth of the lateral line itself remains for some time almost stationary; anteriorly the cells retain the gable-like arrangement which characterised them at an earlier period, but cease to enclose the nerve; posteriorly the line retains its original more complicated constitution as a closed canal. In stage O the cells of the anterior part of the line, as well as those of the posterior, commence to assume a tubular arrangement, and the lateral line takes the form of a canal. The tubular form is due to a hollowing out of the lateral line itself and a rearrangement of its cells. As the lateral line becomes converted into a canal it recedes from the surface.

In stage P the first indication of segmental apertures to the exterior make their appearance, vide Pl. 13, fig. 4. The lateral line forms a canal situated completely below the skin, but at intervals (corresponding with segments) sends upwards and outwards prolongations towards the exterior. These prolongations do not during stage P acquire external openings. As is shewn in my figure, a special area of the inner border of the canal of the lateral line becomes distinguished by its structure from the remainder.

No account of the lateral line would be complete without some allusion to the similar sensory structures which have such a wide distribution on the heads of Elasmobranchii; and this is especially important in the present instance, owing to the light thrown by a study of their development on the origin of the nerves which supply the sense-organs of this class. The so-called mucous canals of the head originate in the same way as does the lateral line; they are products of the mucous layer of the epidermis. They eventually form either canals with numerous openings to the exterior, or isolated tubes with terminal ampulliform dilatations.

I have not definitely determined whether the canal-system of the head arises in connection with the lateral line, or only eventually becomes so connected. The important point to be noticed is, that at first no nervous structures are to be seen in connection with it. In stage O nerves for the mucous canals make their appearance as delicate branches of the main stems. These nerve-stems are very much ramified, and their branches have, in a large number of instances, an obvious tendency towards a particular sense-organ (Pl. 13, figs. 5 and 6).

I have not during stage O been able to detect a case of direct continuity between the two. This is, however, established in the succeeding stage P, in the case of the canals, and the facility with which it may be observed would probably render the embryo Elasmobranch a very favourable object for studying the connection between nerves and terminal sense-organs. The nerve (Pl. 13, fig. 7) dilates somewhat before uniting with the sense-organ, and the protoplasm of the nerve and the sense-organ become completely fused. The basement membrane of the skin is not continuous across their point of junction, and appears to unite with a delicate membrane-like structure, which invests the termination of the nerve. The ampullæ would seem to receive their nervous supply somewhat later than the canals, and the terminal swellings of the nerves supplying them are larger than in the case of the canals, and the connection between the ampullæ and the nerves not so clear. In the case of the head, there can for Elasmobranchii be hardly a question that the nerves which supply the mucous canals grow centrifugally from the original cranial nerve-stems, and do not originate in a peripheral manner from the integument.

This is an important point to make certain of in settling any doubtful features in the nervous supply of the lateral line. Professor Semper[231], with whom as dealing with Elasmobranchii we are more directly concerned, makes the following statement: "At the time when at the front end the lateral nerve has already completely separated itself from the ectoderm, and is situated amongst the muscles, it still lies in the middle of the body close to the ectoderm, and at the hind end of the body is not yet completely segmented off (abgegliedert) from the ectoderm." Although the last sentence of this quotation may seem to be opposed to my statements, yet it appears to me probable that Professor Semper has merely seen the lateral nerve partially enclosed in the ectoderm. This position of the nerve no doubt affords a _presumption, but only a presumption_, in favour of a direct origin of the lateral nerve from the ectoderm; but against this interpretation of it are the following facts:

Footnote 231: _Loc. cit._ p. 398.

(1) That the front part of the lateral line is undoubtedly supplied by branches which arise in the ordinary way from the intestinal branch of the vagus; and we should not expect to find part of the lateral line supplied by nerves which originate in one way, and the remainder supplied by a nerve having a completely different and abnormal mode of origin.

(2) The growth of the lateral line is quite independent of that of the lateral nerve: the latter arises subsequently to the lateral line, and, so far as is shewn by the inconclusive observation of my earliest stage, as an offshoot from the intestinal branch of the vagus; and though it grows along at first in close contact with the lateral line, yet it never presents, so far as I have seen, any indubitable indication of becoming split off from this, or of fusing with it.

(3) The fact that the cranial representatives of the lateral line are supplied with nerves which originate in the normal way[232], affords a strong argument in favour of the lateral line receiving an ordinary nerve-supply.

Footnote 232: Götte extends his statements about the lateral nerve to the nerves supplying the mucous canals in the head; but my observations appear to me, as far as Elasmobranchii are concerned, nearly conclusive against such a derivation of the nerves in the head.

Considering all these facts, I am led to the conclusion _that the lateral nerve in Elasmobranchii arises as a branch of the vagus, and not as a direct product of the external epiblast_.

An interesting feature about the lateral line and the similar cephalic structures, is the fact of these being the only sense-organs in Elasmobranchii which originate entirely from the mucous layer of the epiblast. This, coupled with the well-known facts about the Amphibian epiblast, and the fact that the mucous canals are the only sense-organs which originate subsequently to the distinct differentiation of the epiblast into mucous and horny layers, goes far to prove[233] that the mucous layer is to be regarded as the active layer of the epiblast, and that after this has become differentiated, an organ formed from the epiblast is always a product of it.

Footnote 233: I believe that Götte, amongst his very numerous valuable remarks in the _Entwicklungsgeschichte der Unke_, has put forward a view similar to this, though I cannot put my hand on the reference.

_Muscle-plates._

The muscle-plates at the close of stage K were flattened angular bodies with the apex directed forwards, their ventral edge being opposite the segmental duct, and their dorsal edge on a level with the middle of the spinal cord. They were composed of two layers, formed for the most part of columnar cells, but a small part of their splanchnic layer opposite the notochord had already become differentiated into longitudinal muscles.

During stage L the growth of these plates is very rapid, and their upper ends extend to the summit of the neural canal, and their lower ones nearly meet in the median ventral line. The original band of muscles (Pl. 11, fig. 8, _m.p´_), whose growth was so slow during stages I and K, now increases with great rapidity, and forms the nucleus of the whole voluntary muscular system. It extends upwards and downwards by the continuous conversion of fresh cells of the splanchnic layer into muscle-cells. At the same time it grows rapidly in thickness, but it requires some little patience and care to unravel the details of this growth; and it will be necessary to enter on a slight digression as to the relations of the muscle-plates to the surrounding connective tissue.

As the muscle-plates grow dorsalwards and ventralwards their ends dive into the general connective tissue, whose origin has already been described (Pl. 13, fig. 1). At the same time the connective-tissue cells, which by this process become situated between the ends of the muscle-plates and the skin, grow upwards and downwards, and gradually form a complete layer separating the muscle-plates from the skin. The cells forming the ends of the muscle-plates retain unaltered their primitive undifferentiated character, and the separation between them and the surrounding connective-tissue cells is very marked. This however ceases to be the case in the parts of the muscle-plates on a level with the notochord and lower part of the medullary canal; the thinnest sections and most careful examination are needed to elucidate the changes taking place in this region. The cells which form the somatic layer of the muscle-plates then begin to elongate and become converted into muscle-cells, at the same time that they are increasing in number to meet the rapid demands upon them. One result of these changes is the loss of the original clearness in the external boundary between the muscle-plates and the adjoining connective-tissue cells, which is only in exceptional cases to be seen so distinctly as it may be in Pl. 13, figs. 1 and 8. Longitudinal horizontal sections are the most instructive for studying the growth of the muscles, but transverse sections are also needed. The interpretation of the transverse ones is however rendered difficult, both by rapid alterations in the thickness of the connective-tissue layer between the skin and the muscle-plates (shewn in Pl. 13, fig. 8), and by the angular shape of the muscle-plates themselves.

A careful study of both longitudinal and transverse sections has enabled me to satisfy myself of the fact that the cells of the somatic layer of the protovertebræ, equally with the cells of the splanchnic layer, are converted into muscle-cells, and some of these are represented in the act of undergoing this conversion in Pl. 13, fig. 8; but the difficulty of distinguishing the outline of the somatic layer of the muscle-plates, at the time its cells become converted into muscle-cells, renders it very difficult to determine whether any cells of this layer join the surrounding connective tissue. General considerations certainly lead me to think that they do not; but my observations do not definitely settle the point.

From these facts it is clear, as was briefly stated in the last chapter, _that both layers of the muscle-plate are concerned in forming the great lateral muscle, though the splanchnic layer is converted into muscles very much sooner than the somatic_[234].

Footnote 234: The difference between Dr Götte's account of the development of the muscles and my own consists mainly in my attributing to the somatic layer of the muscle-plates a share in the formation of the great lateral muscles, which he denies to it. In an earlier section of this Monograph, pp. 333, 334, too much stress was unintentionally laid on the divergence of our views; a divergence which appears to have, in part at least, arisen, not from our observations being opposed, but from Dr Götte's having taken the highly differentiated Bombinator as his type instead of the less differentiated Elasmobranch.

The remainder of the history of the muscle-plates presents no points of special interest.

Till the close of stage L, the muscle-plates are not distinctly divided into dorsal and ventral segments, but this division, which is so characteristic of the adult, commences to manifest itself during stage M, and is quite completed in the succeeding stage. It is effected by the appearance, nearly opposite the lateral line, of a layer of connective tissue which divides the muscles on each side into a dorso-lateral and ventro-lateral section. Even during stage O the ends of the muscle-plates are formed of undifferentiated columnar cells. The peculiar outlines of the intermuscular septa gradually appear during the later stages of development, causing the well-known appearances of the muscles in transverse sections, but require no special notice here.

With reference to the histological features of the development of the muscle-fibres, I have not pushed my investigations very far. The primitive cells present the ordinary division, well known since Remak, into a striated portion and a non-striated portion, and in the latter a nucleus is to be seen which soon undergoes division and gives rise to several nuclei in the non-striated part, while the striated part of each cell becomes divided up into a number of fibrillæ. I have not however determined what exact relation the original cells hold to the eventual primitive bundles, or anything with reference to the development of the sarcolemma.

_The Muscles of the Limbs._--These are formed during stage O coincidently with the cartilaginous skeleton, in the form of two bands of longitudinal fibres on the dorsal and ventral surfaces of the limbs. Dr Kleinenberg first called my attention to the fact that he had proved the limb-muscles in _Lacerta_ to be derived from the muscle-plates. This I at first believed did not hold good for Elasmobranchii, but have since determined that it does so. Between stages K and L the muscle-plates grow downwards as far as the limbs and then turn outwards and grow into them (Pl. 18, fig. 1). Small portions of several muscle-plates come in this way to be situated in the limbs, and are very soon segmented off from the remainder of the muscle-plates. The portions of muscle-plates thus introduced into the limbs soon lose their original distinctness, and can no longer be recognized in stage L. There can however be but little doubt that they supply the tissue for the muscles of the limbs. The muscle-plates themselves after giving off these buds to the limbs grow downwards, and by stage L cease to shew any trace of what has occurred (Pl. 13, fig. 1). This fact, coupled with the late development of the muscles of the limbs (stage O), caused me to fall into my original error.

_The Vertebral Column and Notochord._

In the previous chapter (p. 325) an account was given of the origin of the tissue destined to form the vertebral bodies; it merely remains to describe the changes undergone by this in becoming converted into the permanent vertebræ.

This subject has already been dealt with by a considerable number of anatomists, and my investigations coincide in the main with the results of my predecessors. Especially the researches of Gegenbaur[235] may be singled out as containing the pith of the whole subject, and my results, while agreeing in all but minor points with his, do not supplement them to any very great extent. I cannot do more than confirm Götte's[236] account of the development of the hæmal arches, and may add that Cartier[237] has given a good account of the later development of the centra. Under the circumstances it has not appeared to me to be worth while recording with great detail my investigations; but I hope to be able to give a somewhat more complete history of the whole subject than has appeared in any single previous memoir.

Footnote 235: _Das Kopfskelet d. Selachier_, p. 123.

Footnote 236: _Entwicklungsgeschichte d. Unke_, pp. 433-4.

Footnote 237: _Zeitschrift f. Wiss. Anat._ Bd. XXV., Supplement.

At their first appearance the cells destined to form the permanent vertebræ present the same segmentation as the muscle-plates. This segmentation soon disappears, and between stages K and L the tissue of the vertebral column forms a continuous investment of the notochord which cannot be distinguished from the adjoining connective tissue. Immediately surrounding the notochord a layer formed of a single row of cells may be observed, which is not however very distinctly marked[238].

Footnote 238: Vide pp. 356, 357.

During the stage L there appear four special concentrations of mesoblastic tissue adjoining the notochord, two of them dorsal and two of them ventral. They are not segmented, and form four ridges seated on the sides of the notochord. They are united with each other by a delicate layer of tissue, and constitute the rudiments of the neural and hæmal arches. In longitudinal sections of stage L special concentrated wedge-shaped masses of tissue are to be seen between the muscle-plates, which must not be confused with these rudiments. Immediately around the notochord the delicate investment of cells previously mentioned, is still present.

The rudiments of the arches increase in size and distinctness in the succeeding stages, and by stage N have unquestionably assumed the constitution of embryonic cartilage. In the meantime there has appeared surrounding the sheath of the notochord a well-marked layer of tissue which stains deeply with hæmatoxylin, and with the highest power may be observed to contain flattened nuclei. It is barely thicker than the adjoining sheath, but is nevertheless the rudiment of the vertebral bodies. Pl. 13, fig. 9, _vb_. Whence does this layer arise? To this question I cannot give a quite satisfactory answer. It is natural to conclude that it is derived from the previously existing mesoblastic investment of the notochord, but in the case of the vertebral column I have not been able to prove this. Observations on the base of the brain afford fairly conclusive evidence that the homologous tissue present there has this origin. Gegenbaur apparently answers the question of the origin of this layer in the way suggested above, and gives a figure in support of his conclusion (Pl. XXII. fig. 3)[239].

Footnote 239: None of my specimens resembles this figure, and the layer when first formed is in my embryos much thinner than represented by Gegenbaur, and the histological structure of the embryonic cartilage is very different from that of the cartilage in the figures alluded to. Götte's very valuable researches with reference to the origin of this layer in Amphibians tend to confirm the view advocated in the text.

The layer of tissue which forms the vertebral bodies rapidly increases in thickness, and very soon, at a somewhat earlier period than represented in Gegenbaur's Pl. XXII. fig. 4, a distinct membrane (Kölliker's Membrana Elastica Externa) may easily be recognized surrounding it and separating it from the adjoining tissue of the arches. Gegenbaur's figure gives an excellent representation of the appearance of this layer at the period under consideration. It is formed of a homogeneous basis containing elongated concentrically arranged nuclei, and constitutes a uniform unsegmented investment for the notochord (vide Pl. 13, fig. 10).

The neural and hæmal arches now either cease altogether to be united with each other by a layer of embryonic cartilage, or else the layer uniting them is so delicate that it cannot be recognized as true cartilage. They have moreover by stage P undergone a series of important changes. The tissue of the neural arches does not any longer form a continuous sheet, but is divided into (1) a series of arches encircling the spinal cord, and (2) a basal portion resting on the cartilaginous sheath of the notochord. There are two arches to each muscle-plate, one continuous with the basal portion of the arch-tissue and forming the true arch, which springs opposite the centre of a vertebral body, and the second not so continuous, which forms what is usually known as the intercalated piece. Between every pair of true arches the two roots of a single spinal nerve pass out. The anterior root passes out in front of an intercalated piece and the posterior behind it[240].

Footnote 240: In the adult Scyllium it is well known that the posterior root pierces the intercalated cartilage and the anterior root the true neural arch. This however does not seem to be the case in the embryo at stage P.

The basal portion of the arch-tissue likewise undergoes differentiation into a vertebral part continuous with the true arch and formed of hyaline cartilage, and an intervertebral segment formed of a more fibrous tissue.

The hæmal arches, like the neural arches, become divided into a layer of tissue adjoining the cartilaginous sheath of the notochord, and processes springing out from this opposite the centres of the vertebræ. These processes throughout the region of the trunk in front of the anus pass into the space between the dorsal and ventral muscles, and are to be regarded as rudiments of ribs. The tissue with which they are continuous, which is exactly equivalent to the tissue from which the neural arches originate, is not truly a part of the rib. In the tail, behind the anus and kidneys, the cardinal veins fuse to form an unpaired caudal vein below the aorta, and in this part a fresh series of processes originates on each side from the hæmal tissue adjoining the cartilaginous sheath of the notochord, and eventually, by the junction of the processes of the two sides, a canal which contains the aorta and caudal vein is formed below the notochord. These processes for a few segments coexist with small ribs (vide Pl. 13, fig. 10), a fact which shews (1) that they cannot be regarded as modified ribs, and (2) that the tissue from which they spring is to be viewed as a kind of general basis for all the hæmal processes which may arise, and is not specially connected with any one set of processes.

While these changes (all of which are effected during stage P) are taking place in the arches, the tissue of the vertebral bodies or cartilaginous investment of the notochord, though much thicker than before, still remains as a continuous tube whose wall exhibits no segmental differentiations.

It is in stage Q that these differentiations first appear in the vertebral regions opposite the origin of the neural arches. The outermost part of the cartilage at these points becomes hyaline and almost undistinguishable in structure from the tissue of the arches[241]. These patches of hyaline cartilage grow larger and cause the vertebral parts of the column to constrict the notochord, whilst the intervertebral parts remain more passive, but become composed of cells with very little intercellular substance. Coincidently also with these changes, part of the layer internal to the hyaline cartilage becomes modified to form a somewhat peculiar tissue, the intercellular substance of which does not stain, and in which calcification eventually arises (Pl. 13, fig. 11). The innermost layer adjoining the notochord retains its primitive fibrous character, and is distinguishable as a separate layer through both the vertebral and the intervertebral regions. As a result of these changes a transverse section through the centre of the vertebral regions now exhibits three successive rings (vide Pl. 13, fig. 11), an external ring of hyaline cartilage invested by "the membrana elastica externa" (_m.el_), followed by a ring of calcifying cartilage, and internal to this a ring of fibrous cartilage, which adjoins the now slightly constricted notochord. A transverse section of an intervertebral region shews only a thick outer and thin inner ring of fibrous cartilage, the latter in contact with the sheath of the unconstricted notochord.

Footnote 241: A good representation of a longitudinal section at this stage is given by Cartier (_Zeitschrift f. Wiss. Zoologie_, Bd. XXV., Supplement Pl. IV. fig. 1), who also gives a fair description of the succeeding changes of the vertebral column.

The constriction of the notochord proceeds till in the centre of the vertebræ it merely forms a fibrous band. The tissue internal to the calcifying cartilage then becomes hyaline, so that there is formed in the centre of each vertebral body a ring of hyaline cartilage immediately surrounding the fibrous band which connects the two unconstricted segments of the notochord. The intervertebral tissue becomes more and more fibrous. In Cartier's paper before quoted there is a figure (fig. 3) which represents the appearance presented by a longitudinal section of the vertebral column at this stage.

The relation of the vertebral bodies to the arches requires a short notice. The vertebral hyaline cartilage becomes almost precisely similar to the tissue of the arches, and the result is, that were it not for the "membrana elastica externa" it would be hardly possible to distinguish the limits of the two tissues. This membrane however persists till the hyaline cartilage has become a very thick layer (Pl. 13, fig. 11), but I have failed to detect it in the adult, so that I cannot there clearly distinguish the arches from the body of the vertebræ. From a comparison however of the adult with the embryo, it is clear that the arches at most form but a small part of what is usually spoken of as the body of the vertebræ.

The changes in the notochord itself during the stages subsequent to K are not of great importance. The central part retains for some time its previous structure, being formed of large vacuolated cells with an occasional triangular patch of protoplasm containing the starved nucleus and invested by indurated layers of protoplasm. These indurated layers are all fused, and are probably rightly regarded by Gegenbaur and Götte as representing a sparse intercellular matter. The external protoplasmic layer of the notochord ceases shortly after stage K to exhibit any traces of a division into separate cells, but forms a continuous layer with irregular prominences and numerous nuclei (Pl. 13, fig. 9). In the stages subsequent to P further changes take place in the notochord: the remains of the cells become more scanty and the intercellular tissue assumes a radiating arrangement, giving to sections of the notochord the appearance of a number of lines radiating from the centre to the periphery (Pl. 13, fig. 11).

The sheath of the notochord at first grows in thickness, and during stage L there is no difficulty in seeing in it the fine radial markings already noticed by Müller[242] and Gegenbaur[243], and regarded by them as indicating pores. Closely investing the sheath of the notochord there is to be seen a distinct membrane, which, though as a rule closely adherent to the sheath, in some examples separates itself from it. It is perhaps the membrane identified by W. Müller[244] (though not by Gegenbaur) as Kölliker's "membrana elastica interna." After the formation of the cartilaginous investment of the notochord, this membrane becomes more difficult to see than in the earlier stage, though I still fancy that I have been able to detect it. The sheath of notochord also appears to me to become thinner, and its radial striation is certainly less easy to detect[245].

Footnote 242: _Jenaische Zeitschrift_, Vol. VI.

Footnote 243: _Loc. cit._

Footnote 244: _Loc. cit._

Footnote 245: Gegenbaur makes the reserve statement with reference to the sheath of the notochord. For my own sections the statement in the text certainly holds good. Fortunately the point is one of no importance.

EXPLANATION OF PLATE 13.

COMPLETE LIST OF REFERENCE LETTERS.

_al._ Alimentary tract. _ao._ Aorta. _c._ Connective tissue. _cav._ Cardinal vein. _ch._ Notochord. _ep._ Epiblast. _ha._ Hæmal arch. _l._ Liver. _ll._ Lateral line. _mc._ Mucous canal of the head. _mel._ Membrana elastica externa. _mp._ Muscle-plate. _mp´._ Muscles of muscle-plate. _na._ Neural arch. _nl._ Nervus lateralis. _rp._ Rib process. _sd._ Segmental duct. _sh._ Sheath of notochord. _spc._ Spinal cord. _spg._ Spinal ganglion. _syg._ Sympathetic ganglion. _um._ Ductus choledochus. _v._ Blood-vessel. _var._ Vertebral arch. _vb._ Vertebral body. _vcau._ Caudal vein. _vin._ Intestinal branch of the vagus. _vop._ Ramus ophthalmicus of the fifth nerve. _x._ Subnotochordal rod.

Fig. 1. Section through the anterior part of an embryo of _Scyllium canicula_ during stage L.

_c._ Peculiar large cells which are found at the dorsal part of the spinal cord. Sympathetic ganglion shewn at _syg._ Zeiss A, ocul. 1.

Fig. 2. Section through the lateral line at the time of its first formation.

The cells marked _nl_ were not sufficiently distinct to make it quite certain that they really formed part of the lateral nerve. Zeiss B, ocul. 2.

Figs. 3_a_, 3_b_, 3_c_, 3_d_. Four sections of the lateral line from an embryo belonging to stage L. 3_a_ is the most anterior. In 3_a_ the lateral nerve (_nl_) is seen to lie in the mesoblast at some little distance from the lateral line. In 3_b_ and 3_c_ it lies in immediate contact with and partly enclosed by the modified epiblast cells of the lateral line. In 3_d_, the hindermost section, the lateral line is much larger than in the other sections, but no trace is present of the lateral nerve. The sections were taken from the following slides of my series of the embryo (the series commencing at the tail end) 3_d_ (46), 3_c_ (64), 3_b_ (84), 3_a_ (93). The figures all drawn on the same scale, but 3 _a_ is not from the same side of the body as the other sections.

Fig. 4. Section through lateral line of an embryo of stage P at the point where it is acquiring an opening to the exterior. The peculiar modified cells of its innermost part deserve to be noticed. Zeiss D, ocul. 2.

Fig. 5. Mucous canals of the head with branches of the ramus ophthalmicus growing towards them. Stage O. Zeiss A, ocul. 2.

Fig. 6. Mucous canals of head with branches of the ramus ophthalmicus growing towards them. Stage between O and P. Zeiss a a, ocul. 2.

Fig. 7. Junction of a nerve and mucous canal. Stage P. Zeiss D, ocul. 2.

Fig. 8. Longitudinal and horizontal section through the muscle-plates and adjoining structures at a stage intermediate between L and M. The section is intended to shew the gradual conversion of the cells of the somatic layer of muscle-plates into muscles.

Fig. 9. Longitudinal section through the notochord and adjoining parts to shew the first appearance of the cartilaginous notochordal sheath which forms the vertebral centra. Stage N.

Fig. 10. Transverse section through the tail of an embryo of stage P to shew the coexistence of the rib-process and hæmal arches in the first few sections behind the point where the latter appear. Zeiss C, ocul. 1.

Fig. 11. Transverse section through the centre of a caudal vertebra of an embryo somewhat older than Q. It shews (1) the similarity between the arch-tissue and the hyaline tissue of the outer layer of the vertebral centrum, and (2) the separation of the two by the membrana elastica externa[246] (_mel_). It shews also the differentiation of three layers in the vertebral centrum: vide p. 374.

Footnote 246: The slight difference observable between these two tissues in the arrangement of their nuclei has been much exaggerated by the engraver.