Vertebrate Embryology - A Text-book for Students and Practitioners (1893) 2

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A personal message from Dr Mark Hill (May 2020)  
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I have decided to take early retirement in September 2020. During the many years online I have received wonderful feedback from many readers, researchers and students interested in human embryology. I especially thank my research collaborators and contributors to the site. The good news is Embryology will remain online and I will continue my association with UNSW Australia. I look forward to updating and including the many exciting new discoveries in Embryology!

Marshall AM. Vertebrate Embryology: A Text-book for Students and Practitioners. (1893) Elder Smith & Co., London.

Marshall (1893): 1 Introduction | 2 Amphioxus | 3 Frog | 4 Chick | 5 The Rabbit | 6 Human Embryo | Illustrations
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Chapter II. The Development of Amphioxus

I. General Account

1. Structure of Amphioxus.

Amphioxus is a small, semi-transparent, fish-like animal, about a couple of inches in length, found in shallow parts of the Mediterranean and other seas. It is of sluggish habits, and usually remains buried in the sand, either completely or with the anterior end alone protruding ; but if disturbed it swims actively, by rapid lateral movements of the body.

In the general plan of its organisation Amphioxus agrees with the more familiar members of the group of Vertebrates, but in a large number of important respects it is far simpler than any of these.

The external appearance of Amphioxus is shown in Fig. 1 1 . The body is elongated, laterally compressed, and pointed at both ends. There is no distinct head, and no trace of limbs.

A low dorsal fin runs along the midclorsal line from end to end of the animal, becoming more prominent at the hinder end as the upper lobe of the caudal fin. The ventral surface bears a median fin along its posterior third, but in front of this is flattened, so that the body is triangular in section. The sides of this flattened ventral surface are bordered by the lateral fins or metapleural folds. (Of. Figs. 11, 12, 13.)

The skeleton is in an extremely simple condition. Neither cartilage nor bone is present, and the principal skeletal structure is an elongated elastic rod, the notochord (Fig. 11, K), which extends the entire length of the animal, lying dorsal to the alimentary canal and between this and the spinal cord. The notochord is surrounded by a thick sheath of dense connective tissue (Fig. 12, D), which is prolonged dorsalwards to form a tubular investment around the spinal cord. From these sheaths to the notochord and spinal cord, connective tissue partitions or septa arise, which, running outwards to the skin, divide the great lateral muscles of the bodv into muscle-segments or myotomes

(Fig.ll,K, and Fig. 12, x.) The attachments of these septa to the skin are indicated by a series of >shaped markings, very clearly seen on the sides of the animal along its whole length (Fig. 11).

The only other skeletal structures of importance are a series of elastic chitinous rods, supporting the side walls of the pharynx ; and an oval hoop, surrounding the mouth.

The great lateral muscles, noticed above, are the most important part of the muscular system. They form the side walls of the body along its whole length (cf. Figs. 12 and 13), and are divided, as already described, into muscle segments or myotomes by the connective tissue septa. pq . g The muscle fibres of each x! myotome run longitudinally, i.e. parallel to the axis of the body, the fibres taking origin from the connective tissue septa. The myotomes have been found to be sixty-one on each side of the body in a considerable number of specimens, and it seems probable that this number is constant. The myotomes of the two sides of the body are not arranged in pairs, but alternate with one another along the whole length of the animal ; and this lateral asymmetry, one of the most marked features of the adult Amphioxus, affects the nerves, blood-vessels, and other structui-es as well. The ventral surface of the bodv in the anterior two


FIG. 12. Amphioxus lanceolatus. Transverse section through the anterior part of the pharynx of an adult specimen. The boundary of the atrial cavity is indicated by a thick black line. The section is taken at about the level of the reference line B, in Fig. 11. (From Marshall and Hurst.)

A, skeleton of dorsal fin. B, spinal cord. C, notochord. D, connective-tissue sheath surrounding notochord. E, cavity of pharynx. F, epibranchial groove of pharynx. Gr, endostyle, which in this anterior part is flattened out or even convex. TT, atrial cavity, j, transverse muscles in floor of atrial cavity. M, dorsal coelomic canal. P, nietapleural canal. R, left dorsal aorta. S, cardiac aorta. X, myotoine. Y, suspensorv fold of pharynx, separating the dorsal ccelomic canal from the atrial cavity. Z, gill-arch or branchial bar ; the white triangular spot represents the cut surface of the skeletal rod of the arch.

thirds of the animal is covered by a thin sheet of muscle (Fig. 12, j), the fibres of which run transversely from side to side.

The alimentary canal is a nearly straight tube, the anterior part of which is modified for respiration, as in fish.

The buccal orifice (Fig. 11) is a large oval opening, on the ventral surface of the anterior end of the body ; it is fringed on each side by a series of ciliated tentacles, but there are no jaws. The buccal orifice opens into a buccal cavity (Fig. 11, A), which is bounded laterally by the buccal hood, and posteriorly by a muscular diaphragm, the velum ; a small perforation in the velum, a little way below its middle, is the true mouth and leads into the pharynx.

The pharynx (Fig. 11, c) is a wide sac, forming about half the length of the alimentary canal, and attached along its middorsal line to the under surface of the sheath of the notochord, (Fig. 12). The sides of the pharynx are perforated by a large number of slit-like apertures, the gill-slits, which run obliquely downwards and backwards, and of which in the adult animal there may be one hundred or more on each side. The parts of the pharyngeal wall left between successive slits are narrow bars, the gill-arches, each of which is strengthened by an axial rod of a chitinous substance. These arches are of two kinds, arranged alternately ; the axial rods of the second, fourth, &c., or primary arches, being forked at their ventral ends, while the rods of the alternate, or secondary arches, are unsplit. Each double gill-slit is originally a single one, but becomes divided in the course of development (vide p. 78), by the downgrowth of the unsplit bar, or tongue-bar as it is termed, from its dorsal end. The successive gill-arches are connected by horizontal bars, of which there are usually three or more crossing each slit, so that the pharynx has the character of an open meshwork.

Along the mid-dorsal line of the pharynx is a deep epibranchial groove (Fig. 12, F), lined by a single layer of long columnar ciliated cells. A band of similar cells, the endostyle (Fig. 12, G), runs along the mid-ventral wall of the pharynx ; it is folded longitudinally in its hinder part to form a groove (Fig. 13, G).

The intestine (Fig. 11) commences at the hinder end of the pharynx, close to the dorsal surface ; and runs straight back to the anus, which is on the ventral surface, some little distance from the hinder end of the body, and slightly to the left of the median plane. The intestine is extremely narrow at its commencement ; further back it dilates to form an expanded part or stomach, from which a large pouch-like outgrowth, the liver (Fig. 11, D), extends forwards some distance along the right side of the pharynx, ending blindly in front.

During life a stream of water passes through the mouth into the pharynx, and then out through the gill-slits in the sides of the pharynx, the stream being kept up by the action of columnar flagellate cells which clothe the gill-arches, and the water serving to aerate the blood in the vessels of the arches as it swills over them.

The water that has passed through the gill-slits escapes into a large space, the atrial or epipleural cavity (Fig. 12, H) : this lies between the pharynx and the body wall, and into it the pharynx hangs freely, slung up to the body walls by suspensory folds (Fig. 12, Y). The atrial cavity extends back some distance behind the pharynx, and along it the water passes, escaping finally by the atrial pore (Fig. 1 1 , i), an aperture on the ventral surface of the body, bordered by prominent lips, and about onethird the length of the animal from its hinder end. The atrial cavity 'of Amphioxus is a very characteristic feature in its anatomy, and is apparently unrepresented in the higher Vertebrates.

The coelom or body cavity is quite distinct from the atrial cavity, though its boundaries are not easy to follow. In the posterior part of the body, behind the atrial pore, the ccelom is a cavity of some width, surrounding the intestine and separating this from the body wall ; in front of the atrial pore it becomes greatly reduced owing to the increased size of the atrial cavity ; it is, however, readily recognisable as a narrow space immediately surrounding the intestine and the liver. Further forwards, in the region of the pharynx, the ccelom becomes much subdivided, and more difficult to trace ; its principal divisions are a pair of dorsal ccelomic canals (Figs. 12 and 13, M), lying at the sides of the dorsal part of the pharynx, between the body walls and the suspensory folds of the pharynx. From the dorsal ccelomic canals a series of tubular diverticula extend down the outer sides of the primary gill-arches, as far as their ventral ends. A series of spaces surrounding the reproductive organs (Fig. 13, ov) are also parts of the ccelom.

The large spaces, P, in the metapleural folds do not belong to the 003! om, but are apparently lymphatic in nature.

In the circulatory system the more important features are the following. There is no heart, but the general course of the circulation is the same as in a fish. A median longitudinal vessel, the cardiac aorta or endostylar artery (Figs. 12 and 13, s), receives venous blood from the body at its hinder end, and carries it forwards along the floor of the pharynx : from the cardiac aorta the blood passes along a series of vessels- in the gill-arches, becom ing aerated on the way, to the dorsal aortae, a pair of longitudinal vessels (Figs. 12 and 13, R), lying just beneath the notochord :



FIG. 13. Amphioxus lanceolatus. Transverse section through the hinder part of the pharynx of an adult female, passing through the liver and the ovaries. The boundary of the atrial cavity is indicated by a thick black line. The section is taken at about the level of the reference line K in Fig. 11. (From Marshall and Hurst.)

A, skeleton of dorsal fin. B. spinal cord. C, rtotocliord. D, connective-tissue sheath of notochord. E, cavity of pharynx. J 1 , epibranHiial groove. G, endostyle. TT, atrial cavity. L, liver. M, dorsal ccelomic canal. M", branchial coelomic canal. O, ccelomic space surrounding liver. OV, ovary, P, metapleural canal. R, left dorsal aorta. S, cardiac aorta. T, hepatic veins.

these unite behind the pharynx to form a single dorsal aorta, from which branches supply the various parts of the body.

The nervous system consists of a tube of nervous matter, the spinal cord, which lies immediately above the notochord, and extends almost the entire length of the body. It tapers slightly at its anterior end, and more markedly behind. The central canal of this tube is very small along the whole length of the cord, except at the extreme anterior end, where it expands to form a thin-walled chamber, or ventricle. This dilatation of the central canal constitutes the only indication, if indeed it can be regarded as such, of anything corresponding to the brain of higher Vertebrates. The nerves arise either by single roots from the dorsal surface of the cord, or by multiple roots from its ventral surface : the two sets of nerves, which are quite independent of each other, appear to correspond with the dorsal and ventral roots of the spinal nerves of other Vertebrates, although the dorsal roots have no ganglia and are both sensory and motor in function. Excepting the anterior three or four of each side, the nerves arise, not in pairs, but alternately from the right and left sides of the cord.

The sense organs are in a very simple condition, and can only doubtfully be compared with those of higher Vertebrates. From the anterior end of the ventricle of the central nervous system, a hollow outgrowth arises, which is in close relation with a ciliated pit on the dorsal surface and left side of the anterior end of the animal. This pit is commonly regarded as an olfactory organ.

The ' eye ' is a rounded pigment-spot in the anterior wall of the ventricle ; i.e. at the anterior end of the central nervous system (Fig. 11, M). There is no trace of an ear.

The sexes are distinct, but the male and female are similar, except as regards the microscopic structure of the reproductive organs. There are no genital ducts.

In the female, the ovaries (Fig. 13, ov) are a series of saccular organs, arranged in a row along the inner surface of the body wall, on each side of the pharynx, in the segments from the tenth to the thirty-sixth. They lie in cavities, which are specialised portions of the ccelom, and the true relations of which will be described when the development of the reproductive organs is considered.

The ova, when ripe, are discharged into the atrial cavity by dehiscence of the proper wall of the ovary and of the atrial membrane. The discharged ova, together with the ovaries, form a bulky mass, which causes great distension of the atrial cavity, and distortion, through pressure, of the pharynx and other organs.


The ova, which measure O105 mm. in diameter, appear to escape from the atrial cavity, as a rule, through the atrial pore ; but in some cases they have been seen to pass through the gillslits into the pharynx, and to make their exit through the mouth.

In the male, the testes are similar in form and position to the ovaries of the female ; and the spermatozoa when ripe are discharged, like the ova, into the atrial cavity, from which they escape by the atrial pore.

2. Morphological Importance of Amphioxus

It will be seen from the preceding account of its anatomy that Amphioxus, while clearly and undoubtedly a Vertebrate, yet differs from all ordinary Vertebrates, whether fish, amphibians, reptiles, birds, or mammals, in a number of points which are of great importance and affect almost every part of its body.

A closer examination shows that these points of difference between Amphioxus and the higher Vertebrates may be grouped under two chief heads.

(1) The atrial cavity, the large number of the gill-slits, the regular alternation of gill-arches of two kinds, the azygos character of the sense organs, the extension of the notochord to the extreme anterior end of the animal, and the curious lateral asymmetry shown by the rnyotomes, nerves, and other organs, are examples of a group of characters in which Amphioxus differs from the higher Vertebrates, not only in their adult condition, but at all stages of their existence.

(2) There is another and even more striking series of characters, in which Amphioxus differs from the adult forms of the higher Vertebrates, but resembles these in their early developmental stages. Thus in all the higher Vertebrates there is a stage in development when the notochord is the only skeletal structure present, neither cartilage nor bone having yet appeared ; a stage in which the limbs are absent ; and a stage in which the muscles of the body have the simple and definite segmental arrangement seen in Amphioxus throughout life. In all higher Vertebrates the heart is at first straight, like the cardiac aorta of Amphioxus ; the liver arises as one or more outgrowths of the intestine ; and the dorsal and ventral roots of the spinal nerves are at first independent of each other. In these and in


MORPHOLOGICAL IMPORTANCE. 45 many other points, Amphioxus remains throughout life in a condition characteristic of the early developmental phases of the higher Vertebrates. Amphioxus halts permanently at a stagethrough which all the higher Vertebrates pass during their development.

The Recapitulation Theory explains this as indicating that in these respects Amphioxus represents, more or less exactly, a phase through which the higher Vertebrates have passed in the history of their evolution ; that, as regards the organs in question, Amphioxus may be viewed as figuring, with more or less exactness, an ancestral form from which the higher types of Vertebrates are descended.

From this standpoint Amphioxus is an animal of very special importance to morphologists ; and the development of' Amphioxus acquires peculiar interest from the consideration that, if the adult animal is far more primitive than any other existing Vertebrate, then the earlier stages in its life history may reasonably be expected, in accordance with the law of Recapitulation, to yield valuable evidence as to the relations of Vertebrates with the simpler groups of Metazoa.

The above considerations do not imply that Amphioxus itself stands in the direct line of ancestry of any of the higher Vertebrates, but that it is a surviving representative of a type of animals which preceded the higher Vertebrates in point of time, and from which type, though not necessarily from Amphioxus itself, the higher Vertebrates have arisen.

Amphioxus shows us that, in attempting to reconstruct the characters of the ancestors of Vertebrates, we are almost certainly justified in omitting such features as paired limbs, a cartilaginous or bony skeleton, jaws, a twisted or chambered heart, a highly specialised brain, and paired sense organs ; characters which Amphioxus shows us are not necessary to an adult Vertebrate, and in the absence of which the embryos of higher Vertebrates agree with Amphioxus.

A different explanation of the peculiarities of Amphioxus has been offered by many zoologists, who consider that the simplicity that characterises so many of its organs, as the brain, heart, liver, &c., is not primitive, but due to degeneration ; that the immediate ancestors of Amphioxus were, in fact, animals higher in the zoological scale than itself. No distinct evidence of such degeneration has, however, been brought forward ; and the theory of degeneration would leave altogether unexplained what is after all the most important fact, namely, the resemblance so often referred to above, and seen not in one organ only, but in almost every part of its structure, between the adult Amphioxus and the embryonic stages of development of the higher Vertebrates.

3. General Account of the Development of Amphioxus

The development of Amphioxus has, as yet, been studied by a very limited number of investigators ; and many points, especially in the later stages, are still only imperfectly understood.

Our actual knowledge is due in the first instance to Kowalevsky, who published in 1867 an account of observations made by him at Naples in 1864. His descriptions, though brief, are exceedingly precise and well illustrated, and deal with both the earlier and later stages of development ; they were supplemented by further papers in 1870 and 1876.

In 1881, Hatschek published a detailed and admirably illustrated account of the earlier stages of development, from the laying of the eggs up to the formation of the mouth and first gill-cleft. His observations were made near Messina, the specimens being obtained from a small salt lake, communicating with the sea by a narrow channel two or three hundred yards in length.

The later stages of development, and more especially the mode of formation of the gill-clefts, the endostyle, and the atrial cavity were described very fully by Mr. Willey and Professor Lankester in 1890 and 1891, from observations on specimens obtained by Mr. Willey from the same locality as Hatschek. Quite recently, 1892, Boveri has described the mode of formation of the reproductive organs.

The spawning period, in the Mediterranean, begins early in spring, towards the end of March, and continues throughout the summer, up to September ; June being apparently the month of greatest activity. The eggs are laid about sunset, usually between 7 and 8 P.M. ; they are very small, O'lOo mm. in diameter, and consequently contain but little food-yolk. They are fertilised at once, by spermatozoa shed over them by the male, and begin to develop almost directly. The early stages are passed through with great rapidity, and early on the following morning, about eight hours after the eggs were laid, the little embryos work their way out of the egg-membranes and swim freely. Their condition at hatching is shown in Figs. 25 and 26, p. 59.

After hatching, the embryos continue to develop rapidly, and in about thirty-six hours from the time of spawning they reach the stage shown in Fig. 34, p. 74. The mouth is not formed until the end of this period, and development up to this stage is apparently effected at the expense of the small amount of food-yolk contained in the egg.

After the formation of the mouth, the embryo continues its pelagic life, but from this time develops slowly, increasing in length, and gradually acquiring the shape and characters of the adult. During this period the anterior part of the body presents an extraordinary asymmetry, by which the mode of formation of the gill-clefts, which appear in order from before backwards, is profoundly modified. The mouth is a large oval opening (Fig. 3G) placed, not on the ventral surface, but on the left side of the pharynx. The gill-slits of the two sides appear, not simultaneously, but successively ; those of the left side, which may be termed primary slits, being formed before those of the right side, or secondary slits. The primary slits, of which there are as a rule fourteen, are not at first on the left side, but in the midventral wall of the pharynx, and shift upwards so as to actually lie for a time on the right side of the pharynx. The secondary slits, usually eight in number, appear along the right side of the pharynx dorsal to the primary slits (Fig. 38) ; while between the two series of gill-slits, primary and secondary, the endostyle is formed at the anterior end of the pharynx.

During the later stages of pelagic life, the total duration of which is about three months, this curious asymmetry is gradually rectified.. The mouth assumes its median position, the primary gill-slits shift across the median line, and take up their permanent position on the left side of the pharynx ; the endostyle shifts from the right side to the mid- ventral wall ; and, by disappearance of some of the primary gill-slits, the number of primary and of secondary gill-slits becomes equalised, eight being present on each side of the pharynx.

At the close of the pelagic period, which may be called the critical stage, the young Amphioxus, now about 3'5 mm. in length, adopts the mode of life of the adult, burrowing in the sand, and gradually, by increase in the number of the gill-slits und in other ways, acquires the structure and size of the fully formed animal.

The whole developmental history of Amphioxus may, in accordance with the above account, be conveniently divided into periods, which will be dealt with in succession in the remainder of this chapter.

I. The Embryonic Period : including the stages from the commencement of development to the formation of the mouth. This lasts about thirty-six hours, and is characterised by the extreme rapidity with which the stages, especially the earlier ones, are passed through ; and by the fact that throughout the period the embryo is dependent for nutrition on the yolk granules contained within the egg. The actual rate of development varies to a certain extent with the temperature. The times here given are those recorded by Willey during the summer months ; in spring, Hatschek found the rate of development to be slower. The period may be subdivided into two parts.

1. Before hatching : from the commencement of development up te> the hatching of the embryo ; a period of about eight hours.

2. After hatching : from the hatching of the embryo to the formation of the mouth ; a period of about twenty-eight hours, during which the embryo leads a pelagic life.

II. The Larval Period : from the formation of the mouth to the critical stage. This lasts about three months, and during it the larva is pelagic. Development takes place slowly ; and the most notable events are the formation of the gill-slits and the atrial cavity ; and the curious series of changes by which the symmetrical condition of the larva is re-established.

III. The Adolescent Period : during which the young Amphioxus, having adopted the mode of life of the adult, gradually acquires its full structure, by increase in the number of the gillslits, ripening of the reproductive organs, &c.

I. The Embryonic Period

From the laying of the eggs to the formation of the mouth. Duration of the period, from thirty-two to thirty-six hours.

Part I. From the laying of the eggs to the hatching of the embryo : a period of about eight hours.


1. The Egg.

The ripe egg of Amphioxus is a spherical mass of protoplasm, 0'105 mm. in diameter on the average, and inclosed in an elastic vitelline membrane. The protoplasm is studded with numerous yolk granules, which are sufficiently opaque to hide the nucleus. At one pole, which will be spoken of as the upper pole, there is a slightly flattened patch of protoplasm comparatively free from yolk granules ; and on the top of this patch is a sharply denned polar body (Fig. 14, I, PB). A second polar body has not been seen.

The vitelline membrane, prior to fertilisation, adheres closely to the egg.

2. Fertilisation.

The male Amphioxus, as described above (p. 46), sheds spermatozoa over the eggs as these are laid by the female ; and they may be seen adhering in numbers to the vitelline membranes. The details of fertilisation have not been studied, but shortly after the spermatozoa gain access to the egg the vitelline membrane, which previously invested the egg closely, swells up rapidly by imbibition of water, and becomes separated from the egg by a considerable space ; the egg ultimately lying in the centre of a capsule three or four times its own diameter. The purpose of this swelling up of the vitelline membrane, and its separation from the egg, is probably to prevent the entrance ol other spermatozoa after the egg has been fertilised.

3. Segmentation.

The process of segmentation commences at dusk, usually about 8 P.M., and is completed in about three hours.

The first cleft appears about an hour after the eggs are laid and fertilised. It commences as a depression at the upper pole of the egg, close to the polar body, extends rapidly across the upper pole, and then spreads quickly round the egg as a groove (Fig. 14, u). The groove deepens rapidly, being always more prominent at the upper than the lower pole ; and in about five minutes from its first appearance the cleft is completed, the egg being divided by it into two halves or blastomeres of equal size, and similar in -all respects save for the presence of the polar body on the apex of one of them.


Fig. 14. Segmentation of the egg of Amphioxus. x 220. (After Hatscbek.)

I, the egg before the commencement of development. PB, polar body. II, the egg in the act of dividing, by a vertical cleft, into two equal blastomeres ; about one hour after fertilisation. Ill, stage with four equal blastomeres ; about two hours after fertilisation. IV, stage with eight blastomeres : an upper tier of four slightly smaller ones, and a lower tier of four slightly larger ones. V, stage with sixteen blastomeres, in two tiers, each of eight. VI, stage with thirty two-blastomeres, in four tiers, each of eight ; about three hours after fertilisation : the embryo is represented bisected vertically to show the segmentation cavity or blastoccel, B. VII, later stage : the blastomeres have increased in number by further division. VIII, blastula stage : bisected to show die bhistocot'l, B ; about four hours after fertilisation.


A pause of about an hour now ensues, and then the second cleft is formed. This also is vertical, but in a plane at right angles to the first ; it bisects each of the two first blastomeres, and so gives rise to four equal and similar blastomeres (Fig. 14, in) ; these are ovoid in shape, with their apposed surfaces slightly flattened by mutual pressure.

The third cleft, which appears about a quarter of an hour later, is a horizontal one, dividing each of the four blastomeres of the previous stage into two (Fig. 14, iv). The cleft is a little above the equator, so that the four blastomeres of the upper tier are a little smaller than those of the lower tier. The blastomeres are in contact with one another laterally, but do not quite meet along the axis of the embryo. Hence the embryo is at this stage in the form of a ring, or short tube, with a central cavity, the segmentation cavity or blastocoel, which at present is open at both the upper and lower poles.

About a quarter of an hour later, the number of the blastomeres is again doubled by two new vertical clefts, which appear simultaneously, in planes at right angles to each other, and at angles of 45 with the first two clefts. The embryo now consists (Fig. 14, v) of sixteen blastomeres, arranged in an upper tier of eight rather smaller ones, and a lower tier of eight rather larger ones.

A little later, about three hours from the time of fertilisation, two more horizontal clefts appear simultaneously, dividing each of the tiers into two, and again doubling the number of the blastomeres. The embryo now (Fig. 14, vi) consists of four tiers, each of eight cells ; the cells of the lowest tier, as. shown in the figure, being decidedly larger than those of the other tiers. The blastocoel (Fig. 14, vi, B) still opens to the exterior at both poles, although the apertures are considerably narrowed by approximation of the cells of the upper and lower tiers respectively.

In the next stage (Fig. 14, vn)the lowest tier of blastomeres of the preceding stage has divided horizontally, giving five tiers in all ; and each of the blastomeres of the four upper tiers has divided vertically into two. The embryo now, as shown in the figure, consists of five tiers of blastomeres, the four upper of which consist each of sixteen blastomeres, while the lowest tier consists of eight much larger blastomeres. The larva is nearly spherical in shape, and by approximation of the blastomeres of the top and bottom tiers the blastocoel is no\v completely closed.

From this time the blastomeres continue to increase in number by division, but in less regular fashion than before, so that the arrangement in tiers soon becomes lost : the blastomeres at the lower pole, however, remain thi'oughout of larger size than those in other parts of the embryo. The polar body is often visible, resting on the upper pole of the egg, but it has sometimes disappeared by this stage. The blastomeres, which have hitherto been of somewhat irregular shape, rounded at their outer and inner ends, and flattened through mutual pressure at their sides, now begin to assume more definite form ; and from this stage, which marks the close of segmentation, they may be more appropriately spoken of as cells.

4. The Blastula.

The embryo has now reached the stage to which the name hlastula is given ; a stage which occurs at corresponding periods in the development of a number of the lower animals, and which is therefore of interest as possibly representing a very early ancestral form of animal life. Pandorina and Volvox are examples of organisms in which the blastula stage forms the adult condition.

The blastula (Fig. 14, vui) is a spherical or ovoid embryo, consisting of a single layer of cells, inclosing a central segmentation cavity or blastoccel, filled with fluid. In the blastula of Amphioxus the cells are not all of equal size, those of the lower half, and especially those at the lower pole, being distinctly larger than those of the upper half; the greater size and more opaque appearance of these lower cells are due to the greater quantity of yolk granules which they contain. At first, the cells of the blastula, though flattened laterally where they press against one another, remain rounded at their ends, both inner and outer. These ends, however, soon become flattened ; and the cells, in which the nuclei are now clearly visible, acquire the characters of columnar epithelial cells. These changes appear first at the upper pole of the embryo, and gradually extend to the lower pole. The blastula stage (Fig. 14, vui) is reached by the Amphioxus embryo at about the end of the fourth hour from the time of fertilisation of the eggs.


THE GASTRULA STAGE.


5. The Gastrula.


On the completion of the blastula, as described above, the multiplication of the cells ceases for a time, and the embryo undergoes a great change in shape, whereby it becomes converted into the form to which the name gastrula is given. This change is brought about as follows.

The lower surface of the blastula, consisting of the larger cells, becomes flattened (Fig. 15, H), and then invaginated within the upper surface (Fig. 16). The embryo thus becomes cupshaped, its walls consisting of two layers : an outer layer, E, formed from the original upper part of the blastula; and an inner layer, H, consisting of the invaginated cells, which originally formed the lower pole of the blastula.



FIG. 15.


FIG. 16.


The embryos are


FIGS. 15 and 16. Formation of the gastrula of Amphioxus. bisected vertically, one half alone being represented. x 220. (After Hatschek.)

Fig. 15. Flattening of the lower pole of the blastula prior to invagination. Fig. 16. Commencing invagination of the lower pole to form the gastrula.

B, blastocoel or segmentation cavity. E, epiblast. Q, archenteron or gastrocoeL H, hypoblast .

As the invagination proceeds, the blastoccel becomes gradually diminished in size, and is ultimately completely obliterated, the inner and outer layers of the gastrula coming in contact with each other (Fig. 17, H, E).

The two layers of cells of which the wall of the gastrula consists are the two primary germinal layers. The outer layer is spoken of as the epiblast, E, and the cells of which it consists are called epiblast cells : the inner layer is the hypoblast, H, and its cells, which originally were those forming the lower half of the blastula, are called hypoblast cells.

The cavity of the cup, formed by invagination of the hypoblast, is called the archenteron or gastrocoel, G : it gives rise to the greater part of the alimentary canal of the larva and adult. The mouth of the cup is called the blastopore ; it is at first (Fig. 17) a very large aperture, but in the later stages becomes greatly reduced in size (Figs. 18 and 19).

Like the blastula, the gastrula is a very widely spread embryonic form, occurring not only in Vertebrates, but in a simple or modified condition in certain members of each of the great groups of Invertebrates as well. It has therefore, like the blastula, claims to be regarded as an ancestral form ; claims which are greatly strengthened by the fact that some of the simpler sponges, and some of the Ccelenterates, such as Hydra, are closely comparable even in their adult condition to gastrulae.


FIGS. 17 and 18. Completion of the gastrula of Amphioxus. The embryos are bisected vertically, and one half only of each is represented. x 220. (After Hatschek.)

Fig. 17. Completion of the process of invagination, and consequent obliteration of the blastocffil. Fig. 18. Narrowing of the blastopore, through growth backwards of its dorsal lip. E, epiblast. G, archeuteron or gastrocrel. H, hypoblast. PC, polar ruesoblast cell.

The mechanical causes that lead to invagination, i.e. that actually occasion the change from the blastula to the gastrula condition, are not easy to determine. The epiblast cells appear to take no part in the process, and to undergo no appreciable change or alteration during it ; the active cells in the change are the hypoblast cells. By comparison of Figs. 15, 16, and 17, it will be seen that during invagination there is an increase in the number of the hypoblast cells ; and there is also, which is not so clearly brought out in the figures, an increase in the actual size of the individual cells. This increase in size is perhaps due to the cells absorbing the fluid of the blastoccel ; and this absorption of fluid may perhaps be one of the factors that determine or aid the process of invagination. It seems more probable, however, that invagination is due rather to inequality in the rates of growth of the cells at different parts, than to direct pressure from any cause on the surface of the embryo.

The later stages in the development of the gastrula show some features of importance. At its earliest formation, as shown in Fig. 16, the axis of the gastrula coincides with that of the blastula; and the blastopore or gastrula mouth is circular in outline. Later on, as shown by the careful observations of Hatschek, owing to unequal rates of growth in different directions, the blastopore becomes oval instead of circular in outline, and the shape of the embryo changes (Fig. 18) in such way that the axis of the gastrula no longer coincides with the original axis of the blastula, but forms a considerable angle with this.

At the stage shown in Fig. 18 there may be seen at the lower lip of the blastopore, and placed one on each side of the median plane, a pair of cells, PC, which differ from the other hypoblast cells in their larger size and more rounded form, and in having very large nuclei. These two cells give rise at a later stage to important portions of the middle germinal layer or mesoblast: they may be named the polar mesoblast cells.

The further stages in the completion of the gastrula will be understood from a comparison of Figs. 18, 19, and 20. The embryo elongates, becoming ovoid or egg-shaped : at the same time the blastopore becomes still further reduced in size ; the narrowing being effected, according to Hatschek, entirely by growth backwards of its anterior lip, the posterior lip, indicated by the pair of polar mesoblast cells, remaining quiescent throughout the process.

In the fully formed gastrula (Figs. 19 and 20), the ends and surfaces of the larva may be clearly recognised. The polar mesoblast cells, P C, mark the posterior end of the embryo ; the blastopore, B P, now reduced to a small circular aperture, lies at the hinder end of the embryo, and slightly on the dorsal surfaceThe anterior end of the embryo is rounded and imperforate. The dorsal surface is flattened, and is further indicated by the blastopore ; while the ventral surface is strongly convex.

If Hatschek is right in stating that the narrowing of the blastopore is effected entirely by growth backwards of its. anterior lip, then it is evident from a comparison of Figs. 17, 18, and 19 that the blastopore originally occupies almost the whole of what will afterwards be the dorsal surface of the larva ;. while the outer or convex surface of the young gastrula (Fig. 17) corresponds to the ventral surface, and perhaps also to the anterior end of the larva. If these determinations are correct.. Figs. 15, 16, and 17 show that the lower pole of the blastula corresponds to the dorsal surface of the larva, and the upper pole to its ventral surface.

Before leaving the gastrula the cells of the two layers, epiblast and hypoblast, should be noticed more fully. The epi blast


FIGS. 19 and 20. The fully formed gastrula of Amphioxus. x 220. (After Hatschek.)

Fig. 19. The gastrula bisected vertically : the left half is represented, as seen from, the inner surface.

Fig. 20. The gastrula bisected horizontally : the ventral half is represented, as seen, from above.

BP, blastopore. G, archenteron. PC, polar mesoblast cell.

(Figs. 19 and 20) is a single layer of very short columnar or almost cubical cells ; at about the stage represented by Fig. 18 these cells develop on their outer surfaces flagella or lashlike processes, one from each cell, by which the embryo is caused to rotate within the vitelline membrane. These flagella persist during the greater part of the pelagic existence of the embryo, but are not represented in the figures given here.

The hypoblast is a single layer of elongated columnar cells, with nuclei near their inner ends. At the lip of the blastopore the epiblast and hypoblast cells are necessarily continuous with one another ; in the mid-ventral line the two polar mesoblast cells render the transition an abrupt one ; but all round the rest of the lip, and especiall} r at its dorsal or anterior border, the two layers pass gradually into each other. In the figures this transition has, for diagrammatic purposes, been represented as an abrupt one.

The fully formed gastrula stage, seen in Figs. 19 and 20, is reached, in the summer, in from seven to eight hours from the time of fertilisation of the eggs. In the spring, according to Hatschek's observations, the time taken to reach the same stage is about fourteen hours. A comparison of Figs. 14 and 19 will show that the gastrula, though of different shape, is approximately the same size as the egg itself.

6. Development of the Embryo from the Completion of the Gastrula to the Time of Hatching.

The completion of the gastrula stage is followed by a short but well-marked and important period during which the rudiments of the nervous system, of the body cavity, and of the notochord are established, and at the close of which the embryo works its way out of the egg membrane, swims to the surface of the water by means of the flagella of the epiblast cells, and becomes a free living pelagic animal.

During this period it increases slightly in length but diminishes in breadth, so that at the time of hatching (Fig. 26) it is about twice as long as it is wide. Its bulk remains practically the same as before, for the mouth is not yet formed, and the embryo consequently cannot obtain food from without, but is still dependent for nourishment on the yolk granules contained in the cells, more especially in the hypoblast cells.

The nervous system is formed in the following manner. At the time of completion of the gastrula the epiblast is slightly flattened along the dorsal surface, as shown in Fig. 19, and still better in the transverse section, Fig. 21.

This flattened band of epiblast now becomes slightly depressed, and at the same time becomes marked off along its sides from the lateral epiblast (Fig. 22, NP). The lines of demarcation are at first indicated by slight modifications in the shape and arrangement of the cells, but soon become more pronounced, the edges of the lateral plates of epiblast overlapping the central depressed plate (Fig. 23), and ultimately meeting


58


AMPHIOXUS.


each other in the median plane so as to completely cover over the central plate (Fig. 24).

The central plate of epiblast, which thus becomes roofed over, is spoken of as the neural plate (Figs. 22-24, NP), and becomes converted, later on, into the central nervous system. By longitudinal folding of the neural plate a groove is formed


FIG. 21.



FIG. 23.


FIG. 24.


FIGS. 21 to 24. Transverse sections across the bodies of Amphioxus embryos, showing the mode of formation of the nervous system and of the rnesoblastic somites, x 350. (After Hatschek.)

Pig. 21. Transverse section across the middle of the back of an embryo of the same age as those shown in Figs. 19 and 20. Fig. 22. Transverse section across a slightly older embryo, with one pair of mesoblastic somites, and commencing nervous system. Fig. 23. Transverse section across the same embryo as Fig. 22, but taken rather further back, the section passing through the middle of the first pair of somites. Fig. 24. Transverse section through an embryo at the time of hatching ( cf. Figs. 23 and 24) : the section passes through the middle of the first pair of mesoblastic somites, and shows also the mode of formation of the neural tube. CE, enterocoel or mesoblastic somite. E, epiblast. Q, archenteron. H, hypoblast. NF, neural fold. HTP, neural plate.

along its upper surface, and this groove, when roofed over by the

lateral plates or neural folds, becomes the neural canal (Fig. 24).

The neural plate extends back to the blastopore, which, as

already described, is situated on the dorsal surface of the hinder


THE EMBKYO AT THE TIME OF HATCHING.


59


end of the embryo (Fig. 19, BP). The lateral plates, or neural folds, of the epiblast extend not merely along the edges of the neural plate, but round the sides and posterior lip of the blastopore as well ; and by their fusion in the median plane the blastopore becomes roofed over, so that it no longer opens directly to the exterior, but into the hinder end of the neural canal (cf. Figs. 19 and 25). The blastopore thus forms a short tubular channel of communication between the neural canal and the archenteron, and to this channel the name neurenteric canal is given (Fig. 25, NT).

It is a curious fact, and one the full meaning of which is not yet determined, that for a time the sole communication between the archenteron, or primitive alimentary canal, and the



FIG. 25.


FIGS. 25 and 26. Amphioxus embryos at the time of hatching. (After Hatschek.)


x220.


Fig. 25. The embryo bisected vertically : the left half is represented, as seen from ie inner surface. Fig. 26. The embryo bisected horizontally ; the ventral half is presented, as seen from above. CE, euteroccel or rnesoblastic somite. 7 71 *"--* K archenteron. BE, hypoblast. N"F, neural fold. NT, neurenteric canal.


G

mesoblast cell.


E, epiblast. PC, polar


exterior should be through the central canal of the nervous system. Kowalevsky, who discovered the neurenteric canal in Amphioxus and in the Ascidians, suggested that these relations may possibly be ancestral, and that animals may have existed, or may still exist, in which the nerve- tube fulfilled a non-nervous function, and possibly acted as part of the alimentary canal. Comparative anatomy has not at present, however, given any support to this suggestion.

The closure of the neural tube, by meeting and fusion of the neural folds, proceeds from behind forwards, so that a section


60 AMPIIIOXUS.

through the posterior part of an embryo (Fig. 23) will show a more advanced stage in the formation of the nervous system than one taken through the same embryo nearer its anterior end (Fig. 22).

At the time of hatching (Fig. 25), the closure of the neural tube is completed along about a third of the length of the embryo ; the anterior opening of the tube, just in front of the reference line NF in the figure, is spoken of as the neuropore.

The mesoblastic somites. During the formation of the neural canal important changes take place in the hypoblast. Theflattening of the dorsal surface of the embryo at the completion of the gastrula stage affects the hypoblast as well as the epiblast (cf. Figs. 19 and 21). As the neural plate becomes marked off and depressed, a pair of longitudinal folds of the wall of the archenteron are formed, one along each side, in the angle between its dorsal and lateral walls (Figs. 22 and 23, CE). These folds are at first very inconspicuous, but rapidly become more prominent, and especially so about the time of closure of the neural canal (Fig. 24, CE).

By the formation of these folds the archenteron becomes divided into three portions : a central division (Fig. 24, G), which is the alimentary canal itself, and a pair of lateral slit-like diverticula (Fig. 24, CE), which may be termed enteroccelic cavities, and which later on give rise to the body cavity or coelom of the adult.

The cells composing the walls of these folds are clearly of hypoblastic origin. In the later stages (cf. Figs. 27, 28, and 29), they separate completely from the wall of the alimentary canal, and are then spoken of as forming a third germinal layer, or mesoblast, situated between the two primary layers, epiblast and hypoblast.

The mesoblastic folds extend the whole length of the embryo ; they are most prominent near its anterior end, and gradually diminish posteriorly, becoming continuous at their hinder ends with the two large polar mesoblast cells (Figs. 25 and 26, PE), which have already been described as present in the posterior lip of the gastrula from an early stage in its formation (Fig. 18).

Soon after their appearance, the mesoblastic folds become divided by transverse constrictions into segments or compartments, the mesoblastic somites, arranged in pairs along the sides of the embryo. The anterior pair of somites, which are the first


THE EMBRYO AT THE TIME OF HATCHING. 61

to be formed, lie a little way behind the anterior end of the embryo, and the remaining ones are formed in succession from before backwards as the embryo increases in length ; at the time of hatching, two pairs of mesoblastic somites are usually present (Figs. 24 and 26, CE).

The notochord. The roof of the archenteron, between the mesoblastic folds, is formed by a band of hypoblast cells lying immediately below the neural plate, and in close contact with this (Figs. 21 to 24). The cells composing this band, up to the time of hatching, differ little if at all from the hypoblast cells of the sides or floor of the archenteron ; but shortly after the time of hatching, they undergo changes and become converted into the notochord, the most important part of the skeleton of Amphioxus.

Condition of the embryo at hatching. At the time of hatching, which occurs about eight hours after fertilisation of the egg, the embryo (Figs. 25 and 26) is ovoid in form, about twice as long as it is wide, and in biilk about equal to the egg from which it was developed (cf. Fig. 14, i). The epiblast is a single layer of short, almost cubical cells, each of which bears a single flagellum, by which the swimming of the embryo is effected. The neural canal is roofed in for about the hinder third of its length ; in front it opens to the exterior by a rather wide aperture, the neuropore ; posteriorly, the neural canal communicates with the archenteron through the neurenteric canal, the former blastopore. The mesoblastic folds are present, and two pairs of mesoblastic somites are already constricted off from their anterior ends.

Immediately after working its way out of the egg membrane the embryo swims to the surface of the water, and enters on the second part of the embryonic period.

Part II. From the hatching of the embryo to the formation of the mouth : a period lasting from about twenty -four to twentyeight hours (cf. p. 48).

The later stages of embryonic development consist chiefly in further elaboration of the organs which are already established at the time of hatching. The nervous system becomes more complex; the mesoblastic somites increase considerably in number, and undergo important changes whereby the muscular and other systems are formed ; the notochord is definitely established ; and at the close of the period the mouth and first


62 AMPHIOXUS.

gill-slit are formed. The embryo elongates very rapidly, and becoming much narrower and more slender, gradually acquires a shape and proportions resembling those of the adult. During the whole period the embryo is pelagic : swimming is effected at first by the flagella clothing the surface, but towards the close of the period the muscles of the body-walls become definitely established, and the young Amphioxus swims by means of muscular contractions, like the adult.

Although there is a great increase in length during the period, there is little if any change in bulk, and it is doubtful whether the embryo obtains any food from without until the formation of the mouth at the close of the period.



FIG. 27. Amphioxus embryo shortly after hatching, with five pairs of mesoblastic somites ; seen in optical section from the right side, x 224 (After Hatschek.)

E, epiblast. H, hypoblast. M"C, neural canal. M"F, neural fold. NR, neuropore. NT, neurenteric canal. PC, polar mesoblast cell. SI, first mesoblastic somite of riglit side. T, archenteron.

In dealing with this period in the developmental history it will be convenient to describe the several systems one by one.

] . The Nervous System.

After hatching of the embryo, the closure of the neural canal, by fusion of the neural folds, proceeds rapidly forwards (Figs. 25 and 27), and soon reaches the anterior border of the first somite, beyond which level the nervous system does not extend.

From the mode of its formation (Figs. 23, 24, 26, and 27), the neural canal is, in its early stages, merely the space between the neural plate and the overlapping lateral plates of epiblast, and has at first no independent roof of its own. The canal is at first wide from side to side, but shallow dorso-ventrally.


THE LATER EMBRYONIC DEVELOPMENT. 63

In the later stages the neural canal deepens, owing to longitudinal folding of the neural plate ; at the same time the cells at the free margins of the plate grow in towards one another from the two sides, and meeting in the median plane complete the wall of the neural canal (Fig. 32).

The nervous system is now a tube (Figs. 30 and 33), with proper walls of its own, extending along the dorsal surface of the embryo. It opens in front to the exterior, at the neuropore, opposite the anterior border of the first somite; and it communicates posteriorly with the archenteron, through the neurenteric canal. The wall of the tube consists of a single layer of cells, which bear flagella at their inner ends.

The anterior end of the neural tube, close to the neuropore, has, almost from the first, thicker walls than the rest of the tube. This thickening, which affects especially the ventral wall of the tube (Fig. 33), becomes much more marked in the later stages ; partly owing to actual increase in the thickness of the wall itself; and partly to a great diminution in the diameter of the hinder part of the tube, as the embryo becomes drawn out into the elongated form characteristic of the later larval condition.

In the ventral wall of the neural tube, opposite the fifth pair of somites, a black pigment spot, possibly a sense organ, appears at about the stage represented in Fig. 33 ; and much later, towards the end of the embryonic period, another pigment spot, the eye, is formed in the anterior wall of the brain swelling (cf. Fig. 36).

2. The Notochord.

The notochord is developed, as already noticed, from the band of hypoblast cells which forms the dorsal wall of the archenteron, and lies between the two lateral mesoblast folds.

Its earliest appearance as a distinct structure is seen in a larva with three pairs of somites, i.e. immediately after the time of hatching ; and the successive stages in its formation are shown in Figs. 28, 29, and 32, CH.

The median plate of hypoblast cells, fonning the roof of the archenteron, first becomes marked off, by a difference in mode of arrangement of the cells, from the lateral mesoblast folds, and


64 AMPHIOXUS.

then grooved ventrally along the median plane (Fig. 28). The ventral groove deepens, and at a stage with five pairs of mesoblastic somites the plate is completely folded on itself, so that its two halves are in contact with each other. The cells of the two halves now begin to grow across the median plane, interdigitating with one another (Fig. 29, CH), and forming a solid ridge of cells along the mid-dorsal surface of the archenteron. At a slightly later stage, with eight or nine pairs of mesoblastic somites, this ridge begins to separate from the gut wall as a cylindrical rod of cells, the notochord (Fig. 32, CH).

Behind the first somite, i.e. along the greater part of its



FIGS. 28 and 29. Transverse sections through Amphioxus embryos shortly after the time of hatching ; showing stages in the formation of the notochord and mesoblastic somites, x 435. (After Hatschek.)

Fig. 28. Embryo with five pairs of somites : transverse section through the middle of the first pair. Fig. 29. Embryo with six pairs of somites : transverse section through the hinder end of the first pair. CE, enterocoelic pouch or mesoblastic somite. CH, iiotochord. G, archenteron. MS, mesoblastic somite. NGr, neural canal. T, mesenteron.

length, the notochord develops from before backwards. Opposite the first somite the notochord forms more slowly, and is always a little behind the stage reached in the second somite. In front of the first somite the notochord is developed from behind forwards, but otherwise in the same manner as in the hinder part, though much more slowly ; towards the close of the embryonic period, at the time when the pointed anterior end of the animal is forming, it grows much more rapidly (Fig. 33). This late development of the anterior end of the notochord will be referred to again further on.,


THE LATEE EMBRYONIC DEVELOPMENT. 65

Opposite the neuropore, and corresponding to the marked thickening in the ventral wall of the neural tube already described, there is a distinct bending of the notochord (Fig. 33), traces of which persist even in the adult animal.

The histological development of the notochord presents some features of interest. The interdigitation of the cells of the two sides, the commencement of which is shown in Fig. 29, proceeds rapidly ; and, at the time of its separation from the gut, the notochord consists (Fig. 32) of four or five rows of cells, arranged horizontally one above another, each cell extending across the whole of its width. Within the notochordal cells numerous small vacuoles now appear ; these vacuoles are, from the first, most abundant in the two middle rows of cells, and in these they increase greatly in size ; so that in its later stages, as in the adult, the notochord consists of a middle series of cells, enormously distended by vacuoles, and covered on its dorsal and ventral surfaces by rows of smaller and comparatively little modified cells.

3. The Mesoblastic Somites.

The mesoblastic ridges, as described above, are a pair of longitudinal folds of the dorso-lateral walls of the archenteron, inclosing slit-like diverticula of the archenteric cavity (Figs. 26, 28). By transverse constrictions these ridges become divided into somites, which, though separated from one another by the constrictions, still retain for a time their communications with the archenteron (Figs. 27, 28).

At the time of hatching, two pairs of these somites are present; and, as the embryo elongates, other pairs are added in succession from before backwards, the number of pairs of somites present affording a convenient basis for estimating the age of an embryo (Figs. 27, 30, 33).

The anterior somites, which are the first formed, are also the largest, and the remainder decrease in size towards the hinder end of the embryo (Figs. 27, 30) ; the hindmost pair passing into the, as yet, unsegmented mesoblast folds, which end posteriorly in the two polar mesoblast cells (Figs. 30, 31, PC).

At a stage when six pairs of somites are present, the cavities of the anterior ones become constricted off from the

F


66


AMPIIIOXUS.


archenteron. and separate completely from this (Fig. 29). This separation rapidly extends backwards, involving the hinder somites in succession; and the somites now form (Figs. 27, 29) a series of squarish hollow bodies, arranged in a row along each side of the embryo, at the level of the notochord.

The somites are at first small, and lie above or dorsal to the alimentary canal (Fig. 29) ; but they rapidly increase in size, and, extending ventral wards (Figs. 30 and 32), make their way


MR



NT


FIG. 30.


FIG. 31.


FlGS. 30 and 31. Amphioxus embryos with nine pairs of ruesoblastic somites, x 224. (After Hatschek.)

Fig. 30. Embryo seen in optical section from the right side. Pig. 31. Embryo seen in horizontal section, at the level of the notochord. CH, uotochord. DL, left anterior gut diverticulum. DR, right anterior gut diverticulum. M"R, ueuroi>ore. NT, ueurenteric canal. PC, polar rnesoblast cell. SI, first mesoblastic somite of the right side. S9, ninth mesoblastic somite of the right side. T> mesenteron.

round the sides of the embryo, between the gut wall and the external epiblast, ultimately reaching the mid-ventral line, where the somites of the right and left sides of the body become continuous with one another.

During their earlier stages (Figs. 27, 30), the long axes of the somites lie transversely, or slightly obliquely to the axis of the embryo ; but towards the close of the embryonic period


THE LATER EMBEYONIC DEVELOPMENT.


67


ML



(Fig. 33) they acquire the >-like shape so characteristic of the adult (Fig. 11. x).

The walls of the somites soon undergo important changes. At the time of separation from the archenteron (Fig. 29, MS), the wall of each somite consists of a single layer of cells, somewhat irregular in shape and size, but showing no marked differences in different parts. As the somites extend down the sides of the body they become somewhat triangular in transverse section. In each somite there may now be distinguished (Fig. 32) an outer or parietal wall, next the external epiblast ; a visceral wall, in contact with the hypoblast of the archenteron ; and a notochordal wall, forming the base of the triangle, and in contact with the notochord and the nerve cord. The cells of the parietal and visceral walls are slightly flattened, but show no special peculiarities ; those of the notochordal wall, on the other hand, show marked changes. Each cell (Fig. 32, ML) is much flattened dorso-ventrally, and elongated in a direction parallel to the axis of the embryo (Fig. 31); and is undergoing changes by which it becomes converted into a muscle cell or fibre. This differentiation of muscle cells begins at a stage with about nine pair of somites, and proceeds rapidly ; the muscles, at a stage with eleven pairs of somites, beginning to contract and cause lateral undulations of the body. The mass of muscle cells, formed in this way by modification of the notochordal wall of a somite, is called a myotome : the myotomes, being formed from the somites, are, like these, arranged segmentally from their first appearance ; they increase rapidly in size, and become the great lateral muscles or myotomes of the adult Amphioxus (Fig. 12, x). Each muscle cell extends the whole length of the somite to which it belongs.

In the higher Vertebrates it will be found that the earliest

F 2


FIG. 82. Transverse section through the middle of an Amphioxus embryo with nine pairs of mesoblastic somites, x 435. (After Hatschek.)

CH, uotochord. I, spinal cord. ML, muscle layer. MS, cavity of mci-oblastic somite. T, mesenterou.


68 AMPHIOXUS.

muscles to appear in the development of the embryo correspond in mode of formation, and in relations, to the myotomes of Amphioxus. The formation of muscles, as indeed of all other tissues, by direct modification of epithelial cells, is a further point of very great and general interest, indicating that the epithelial cell is a more primitive type of structure than muscle, connective tissue, nerve tissue, or any of the other histological elements of which the body of an adult animal is composed.

The cavities of the somites give rise to the coelom or body cavity of the adult. After their separation from the archenteron they are completely closed, and remain so for some time ; theanterior and posterior walls of adjacent somites becoming closely applied to one another, and forming septa which separate the cavities of successive somites from one another (Fig. 31). Towards the close of the embryonic period, the ventral portions of these septa disappear, so that the somites open into one another ; and the body cavity, which up to this time has been represented by a series of isolated chambers, now becomes continuous from end to end of the animal. The dorsal portions of tbe somites, however, remain separate from one another throughout life.

The first somite (Fig. 27, s i) is a little distance from the anterior end of the body : from its anterior and dorsal border, at a stage with about nine pairs of somites, a hollow conical process is given off towards the anterior end of the embryo (Figs. 30, 31) ; the walls of this process undergo changes similar to those described above as occurring in the body of the somite itself.

At the time of their first appearance the somites are paired ; the two somites of each pair being exactly opposite each other, and the whole embryo being bilaterally symmetrical. At a stage with nine pairs of somites this symmetry becomes disturbed (Fig. 31), the somites of the right side becoming situated a little behind the corresponding ones of the left side, and ultimately alternating with these. This curious lateral asymmetry is preserved in all the later stages, and in the adult animal as well. The fact that the somites are at first symmetrically arranged shows that it is a secondary and not a primitive feature, and the further fact that it appears just at the time when the great lateral muscles are being formed, and are coming into use for swimming, suggests that the explanation of the asymmetry is to be found in some mechanical advantage gained by the alternating arrangement of


THE LATEK EMBRYONIC DEVELOPMENT.


69


the muscles in an animal in which the skeleton is represented merely by an elastic notochord.

The development of new somites during the later stages of embryonic life occurs very slowly ; and at the time of the formation of the mouth, marking the close of the period, there are not more than fourteen or fifteen pairs. The elongation of the body, which is so marked a feature of the later embryonic stages, is due, not so much to addition of new segments, as to lengthening of those already present ; and this lengthening, as shown in Figs. 33 and 34, principally concerns the anterior or oldest somites.

A. The Alimentary Canal.

After separation of the somites and the notochord, the archenteron, or, as it is usually termed from this time, the mesenteron, forms a straight tube (Figs. 30 and 33, T), dilated at its anterior end, but narrow and cylindrical along the greater part of its length. It is closed in front, but at its hinder end it communicates through the neurenteric canal with the neural tube, and so, indirectly, through the neuropore, with the exterior. It is ciliated along its entire length, but no food particles have as yet been observed in it prior to the formation of the mouth.



70 AMPHIOXUS.

a. The anterior gut diverticula. At a stage with seven pairs of somites, a pair of lateral diverticula arise from the dilated anterior end of the mesenteron. These are situated (Figs. 30, 31, DL, DK), near the dorsal surface of the mesenteron, just in front of the first pair of somites, and ventral to the anterior prolongations of these somites.

The two diverticula soon separate from the mesenteron, which then shrinks back from the anterior end of the body. They are , at first of equal size, but from a stage with about ten pairs of somites, onwards, they develop very unequally.

The right anterior gut diverticulum (Fig. 33, DR) forms a thinwalled sac, which extends forwards so as to occupy a large space at the anterior end of the body, below the notochord ; its walls become flattened epithelial cells, and the space which they inclose may be spoken of as the head-cavity.

The left anterior gut diverticulum (Fig. 33, DL) remains ot small size, and forms a spherical thick-walled sac, lying on the left side of the head, just in front of the mesenteron and a little way behind the level of the neuropore ; its wall consists of a single layer of columnar ciliated epithelial cells. Towards the close of the embryonic period it opens to the surface by a small pore on the left side of the head (Fig. 34, DL), and from this time is spoken of as the praeoral pit.

The homologies of these anterior gut diverticula with organs of higher Vertebrates are very uncertain. They are probably to be regarded as parts of the body cavity or ccelom, though it must be admitted that their development differs in important respects from the rest of the ccelom. In the mode of their origin, in their asymmetry, and in the fact that the left diverticulum early acquires an opening to the exterior, they resemble the anterior ccelomic diverticula of Balanoglossus, and the enteroccelic outgrowths of Echinoderms, with which they have by some observers been held to correspond.

b. The club-shaped gland. In embryos with nine or ten pairs of somites a shallow transverse groove appears across the floor of the mesenteron, and extending up its sides, opposite the septum between the first and second pairs of somites. The first commencement of this groove is seen in Fig. 30, opposite the ventral end of the first somite, but is not indicated by a reference letter. Towards the end of embryonic life the lips of the


THE LATER EMBRYONIC DEVELOPMENT. 71

groove close to form a tube, which splits off along its whole length from the mesenteron, but remains in close contact with this. The limb of the tube which lies at the right side of the mesenteron expands slightly to form the club-shaped gland (Fig. 30, GL) ; the rest of the tube forms a slender duct, which passing across the body, under the mesenteron, to its left side (Fig. 36, GD), acquires an opening to the exterior just below the anterior border of the mouth, as soon as this latter is formed. The further development of the club-shaped gland will be described in the section dealing with the larval stages.

c. The mouth. At the close of the embryonic period, a disclike thickening of the epiblast forms on the left side of the head, opposite the first somite but ventral to its lower edge. The hypoblast of the mesenteron fuses with this patch of epiblast, and the mouth is formed as a perforation in the middle of the fused patch. The mouth is at first a minute circular aperture, but it rapidly increases in size, and at the end of the embryonic period is a large oval opening (Fig. 36. o), with a slightly thickened border, on the left side of the head.

d. The first gill-slit. Simultaneously with the formation of the mouth, a slight depression of the hypoblast of the ventral surface of the mesenteron appears, opposite the second pair of somites ; this fuses with the epiblast, and then, by perforation, an opening is formed which is the first gill-slit (Fig. 34, L). The perforation is formed from within outwards : the gill-slit is at first very small, and situated in the mid-ventral wall ; but it soon enlarges, and as it does so shifts upwards to the right side of the body (Fig. 36, HK i). Like the mouth, it is bordered by long cilia.

e. The anus. This is formed shortly after the mouth and the first gill-slit (Fig. 34, u). It is at first much nearer the hinder end of the body than in the adult, and is placed slightly to the left of the median plane.

5. The Blood-vessels.

The development of the blood-vessels in Amphioxus has been but very imperfectly studied. The first vessel to appear is said to be the ventral or cardiac aorta, which is developed in a longitudinal strip of mesoblast, formed by fusion of the ventral edges of the somites of the two sides along the mid-ventral line,


72 AMPHIOXUS.

and extends along the whole length of the under surface of the intestine. The anterior end of the aorta, on reaching the level of the second somite, turns upwards, and runs obliquely forwards along the right side of the pharynx, passing dorsal to the first gill-cleft, and ending in close relation with the club-shaped gland.

6. Structure of the Embryo at the Close of the Embryonic Period.

The general appearance of the embryo at this stage is shown in Fig. 34. The embryo has a total length of about 1'3 mm., and is of a glassy transparency in all its parts and organs, owing to the complete absorption of the yolk granules originally present in the egg. It is widest about the level of the mouth, in front of which it tapers rapidly, ending in a sharply pointed snout. The hinder part of the body tapers very gradually, and ends in a thin vertical fin of rather larger size than is shown in the figure.

The embryo swims actively, by alternating contractions of the myotomes of the two sides of the body. Of these myotomes there are fifteen pairs present ; the myotomes of the first pair are opposite each other, those of the next two or three pairs are placed more or less obliquely, and behind the fourth pair the myotomes alternate regularly along the two sides of the body. The first pair of myotomes give off anterior prolongations, which extend along the sides of the notochord to the tip of the snout, and by their contractions bend the snout freely from side to side. Each muscle fibre is formed by elongation of a single cell, and the majority of the fibres show more or less evident transverse striation. The alimentary canal is divided into an anterior, dilated, pharyngeal region, lying opposite the first two myotomes ; and a posterior, cylindrical, intestinal region which extends to the anus. In connection with the pharyngeal region are the mouth, the first gill-slit, and the club-shaped gland ; there is as yet no trace of the liver.

The nervous system consists of a neural tube, with proper walls of its own, extending the whole length of the back of the animal, just above the notochord. The neural tube opens to the exterior at its anterior end through the neuropore, immediately behind which the tube presents a slight dilatation or ' brain.' The posterior end of the neural tube (Fig. 34, NE) bends downwards


STRUCTURE AT CLOSE OF EMBRYONIC PERIOD. 73

round the end of the notochord, and still communicates, though by a very minute aperture, with the hinder end of the intestine. Sense organs are represented by pigment spots in the wall of the neural tube ; and a pair of small filaments, formed of elongated and adherent cilia, and situated on the under surface of the body behind the mouth, are very possibly taste organs.

One of the most interesting points to notice is that, up to this stage, all the various parts of the body, the epidermis, the walls of the neural tube and of the alimentary canal, the myotoraes, &c., all alike consist of single layers of cells, and cells which, at any rate in their earlier stages of development, are of epithelial origin.

II. THE LARVAL PERIOD.

This extends from the formation of the mouth to the critical stage, at which latter date the mouth assumes its median position, and the gill-slits become symmetrically arranged on the two sides of the pharynx. The duration of the period is about three months.

During the larval period, development proceeds far more slowly than in the earlier stages. An interval of about a fortnight is said to elapse between the formation of the first and the second gill- slits ; and the close of the larval period, which indicates a very definite stage in development, is also marked by a pause of considerable duration. The chief events that occur during the larval period are the formation of the gillslits of both sides of the pharynx, the formation of the endostyle, the development of the atrial cavity, the shifting of the mouth to its adult position, the establishment of the full number of myotomes, together with certain important changes in their relations to other organs, and the disappearance of the clubshaped gland.

Until recently our acquaintance with these stages was very fragmentary, and due entirely to Kowalevsky's careful, but brief and incomplete descriptions. Now, owing to Hatschek's observations on the development of the myotomes, and those of Willey and Lankester on the formation of the gill-slits, atrial cavity, and endostyle, we have far more complete and satisfactory knowledge of the actual course of events, although there are many points that still require investigation.


AMPHIOXUS.


During the larval stages the young Amphioxus leads a pelagic life, and is found most abundantly, not at the surface.



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THE LARVAL PEEIOD. 75

which the larva takes to living in the sand varies greatly in different individuals. At the close of the larval period the larva measures about 3*5 mm. in length.

1. The Gill-slits.

It has already been mentioned, in the general account of the development of Amphioxus, that the gill-slits of the two sides are not formed simultaneously ; those of the left side, which may be termed primary slits, appearing before those of the right side, or secondary slits. The primary slits, of which there are as a rule fourteen, are formed, not on the left side, but in the mid- ventral wall of the pharynx, and, after their formation, shift upwards so as actually to be for a time on the right side of the pharynx. The secondary slits, usually eight in number, are formed at a later stage, along the right side of the pharynx, dorsal to the primary slits. Towards the close of the larval period, as the mouth assumes its median position, the primary slits shift across to their permanent position on the left side : at the same time, by an actual diminution in number, through disappearance of the slits at the two ends of the series, the primary slits become reduced to eight, and the critical stage is reached, at which the primary and secondary slits are equal in number, and symmetrically arranged along the left and right sides of the pharynx respectively.

a. The primary gill-slits, or the gill-slits of the adult left side of the pharynx, are formed in succession from before backwards. Like the first gill-slit, the development of which has already been described, each of the succeeding primary gill-slits lies at first in the mid-ventral wall, but, with the exception of the hindermost two or three, shifts almost at once to the right side of the pharynx. The full number of primary gill-slits is as a rule fourteen, but varies in different specimens from twelve to fifteen. The slits are at first metarnerically arranged, corresponding, when they are fourteen in number, to the somites from the second to the fifteenth inclusive ; this metameric arrangement is, however, entirely lost in the later stages of development.

The condition with three fully developed primary gill-slits, and a fourth slit in the act of forming, is shown in Fig. 36 ; and the stage in which all fourteen primary gill-slits are present, in


76


AMPHIOXUS.


Fig. 37. The gill-slits are at first wide, window-like apertures in the wall of the pharynx ; and, until the formation of the atrial



UK 4


HK I


HK2


HK 5


FIG. 36. The anterior end of an Amphioxus Larva with four primary gill-slits, from the left side. (After Lankester and Willey.) x 200.

CH, iiotoehonl. DL, praeoral pit. ES, endostyle. GD, aperture of duct of clubshaped gland. GL, club-shaped gland. HK 1, 2, 3, 4, first, second, third, and fourth primary gill-slits. NS, spinal cord. O, margin of mouth opening. OC, eye-spot. P 6, sixth niyotome of the left side.

cavity, they open directly to the exterior. At a comparatively early stage (Fig. 37), the first primary gill-slit becomes markedly smaller than the succeeding ones.



DL


HK 14


HK 7


MD

FIG. 37. The anterior end of an Amphioxus Larva with fourteen primary gillslits, seen from the right side. (After Willey.)

CH, notochord. DL , praeoral pit. ES, endostyle. GL, club-shaped gland. GO. opening from dub-shaped gland into pharynx. HK 1, 7, 14, first, seventh, and fourteenth primary gill-slits. HP 2, 7, thickened patches in which the second and seventh secondary gill-slits will be formed at a slightly later stage. MD, free edge of right metapleural MJ. NC, neurnl canal. NO, anterior dilatation, or ventricle of neural canal. M"S, spinal cord. O, mouth. OC, eye-spot. P 13, septum between thirteenth and fourteenth myotornes.

b. The secondary gill-slits, or the gill-slits of the adult right side of the pharynx, appear later than the primary slits, and in the following manner. At a stage (Fig. 37) when fourteen


THE GILL-SLITS.


77


primary slits are present, of which the hinder three or four already open into the atrial cavity, a longitudinal ridge appears in the right wall of the pharynx, above the primary gill-slits. In this ridge six oval thickenings or enlargements appear simultaneously, formed by fusion of the hypoblastic wall of the pharynx with the external epiblast. These fused patches alternate with the primary gill-slits; the first patch (Fig. 37, HP 2) lying above and between the third and fourth primary slits, and the sixth patch, HP 7, above and between the eighth and ninth primary slits. Each patch now becomes perforated by a minute aperture, which by enlargement becomes one of the secondary gill-slits.

The most anterior of these six slits is usually formed a little later than the remaining five ; and a little later still two more


CH


NO


Gfl ES


NO


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HKI3 HP 8


FIG. 38. The anterior end of an Amphioxus Larva with thirteen primary, and eight secondary gill-slits, seen from the right side. (After Willey.)



slits are formed in similar fashion, one at each end of the series. In this manner the full number of eight secondary gill-slits is acquired (Fig. 38) ; the first, HP ], lying above and between the second and third primary slits ; and the eighth, HP 8, above and between the ninth and tenth primary slits. A ninth secondary gill-slit is sometimes developed at the hinder end of the series.

c. Further development of the primary and secondary gillslits. The secondary gill-slits are at first very small, but they rapidly increase in size, extending down the right side of the pharynx ; as they do so, the primary slits move downwards to the ventral wall of the pharynx, and then extend up its left


78


AMPHIOXUS.


HK.I2


wall, finally assuming their permanent position on the left side of the pharynx. During the process of shifting, the primary and secondary slits gradually become equal in size, and of similar shape. From the dorsal border of each slit a small process, the tongue-bar, grows downwards across the slit, dividing it into anterior and posterior portions ; these tongue-bars (Fig. 38) appear rather earlier in the secondary than in the primary slits. Of the fourteen primary slits, the first and the fourteenth close up and disappear; and at slightly later stages the thirteenth, twelfth, eleventh, and tenth similarly, and in succession, close and disappear (cf. Fig. 39). In this way the primary gill


OB


HT HK 2 HKJ

FIG. 39. The anterior end of an Amphioxus Larva with twelve primary gillslits, of which the first and twelfth are disappearing, and eight secondary gill-slits ; seen from the ventral surface. (After Willey.)

CH, notochord. ES, endostyle. HK1, first primary gill-slit just before its final disappearance. HK 2, second primary gill-slit. HK 12, twelfth primary gill-slit, in the act of closing, prior to its disappearance. HP 1, 8, first and eighth secondary gillslits. HT, tongue-bar. LM, velum. OB, buccal cavity. OT, buccal tentacles.

slits become reduced to the same number, eight, as the secondary slits, the eight persisting primary slits being the second to the ninth inclusive.

The anterior persisting slits of both series, i.e. the second primary slit and the first secondary slit, differ from the others in their smaller size, and in the fact that they alone do not develop tongue-bars (Fig. 39, HK 2 ; HP i).

The gill- slits have now reached the condition characteristic of the critical stage. Eight slits are present on each side of the pharynx, alternating with one another as in the adult ; the anterior slit of the right side, i.e. the first secondary slit, HP i, being opposite the interval between the first and second slits of the left side, i.e. the second and third primary slits.


THE GILL-SLITS AND THE ENDOSTYLE. 79

2. The Endostyle.

The endostyle appears at the commencement of the larval period, or towards the close of the embryonic period, as a band of columnar ciliated cells on the right side of the anterior end of the pharynx, immediately in front of the club-shaped gland, and in close contact with this. Its condition at an early stage of the larval period is shown in Fig. 36, ES, where it is seen as a broad > -shaped band, formed by modification of the hypoblast cells of the right side of the pharynx, opposite the anterior part of the mouth opening. The apex of the > is directed backwards ; the upper arm is much shorter than the lower ; and the whole band is divided down its centre by a groove.

In the later stages (Figs. 37 and 38, ES), the endostyle extends backwards, its apex passing behind the duct of the clubshaped gland and making its way between the primary and secondary series of gill-slits. As the critical stage is approached, and the primary gill-slits shift across to the left side, the endostyle (Fig. 39) moves to its permanent position on the mid-ventral wall of the pharynx. At the same time it continues to extend backwards, and at the critical stage has reached to about the level of the fifth gill-slits. During the shifting of its position the two limbs of the >, which were originally upper and lower, become right and left respectively ; and as it extends backwards along the floor of the pharynx the two limbs become closely applied, and fused together. From the anterior ends of the limbs, a pair of ciliated ridges of epithelial cells extend up the sides of the pharynx, and grow backwards along its dorsal surface to form the epibranchial band of the adult.

3. The Club-shaped Gland.

The early stages in the formation of the club-shaped gland have been already described, p. 70. The gland reaches its maximum development about the commencement of the larval period (Fig. 36), when it consists of a dilated sac, GL, lying on the right side of the pharynx, and continuous with a narrow tubular duct, which passes round the ventral surface of the pharynx and opens to the exterior on the left side, close to the anterior border of the mouth, GD.

The dilated part of the gland soon becomes narrower, and tubular, but according to Willey acquires an opening into the


80 AMPHIOXUS.

pharynx at its dorsal end. It does not shift its position in any way ; but, about the stage represented in Fig. 38, when the secondary gill-slits are formed, and the primary slits are moving across to the left side, the club-shaped gland begins to atrophy, and by the stage shown in Fig. 39 has disappeared completely.

The function and the morphological meaning of the clubshaped gland are very doubtful. Willey has suggested that it may be the modified first gill-slit of the right side, adducing in support of the suggestion the fact that the first gill-slit of the left side is also a structure which disappears early ; indeed, about the same time as the club-shaped gland itself. It is difficult, however, to understand, if the club-shaped gland is formed from a gill-slit of the right side, why its external opening should be on the left side of the head.

4. The Mouth.

The most striking features about the mouth, at the commencement of the larval period, are its position on the left side of the head, and its enormous size. As shown in Fig. 36, the mouth, o, and the first gill-slit, HK 1, with the part of the pharyiigeal cavity between them, form a huge opening, perforating the animal from side to side like the eye of a needle.

During the formation of the primary gill-slits the mouth remains on the left side of the head, and increases considerably in length ; extending, at the close of the stage (Fig. 37, o), from the second to the seventh myotome inclusive.

From the commencement of the formation of the secondary gill-slits the mouth gradually shifts its position, growing round the anterior end of the pharynx, and eventually attaining the median position and the shape characteristic of the adult. The shifting commences with the formation of a groove on the surface of the head, leading from the prasoral pit to the upper and anterior angle of the mouth. By deepening of this groove the mouth opening becomes placed obliquely across the body, and by a continuance of the process, together with growth forwards of its posterior lip, it ultimately becomes median in position. The mouth is relatively much smaller in the adult than in the larva, but not actually so.

The margin of the mouth opening of the larva becomes the velum of the adult, from which the velar tentacles arise as out


THE MOUTH. 81

growths ; of these, there are four present at the critical stage, the remaining eight being developed later.

5. The Buccal Hood and Buccal Tentacles.

The true mouth of the adult Amphioxus, the development of which has just been described, is the small opening in the velum, or partition separating the buccal cavity from the pharynx (Fig. 11, p. 38).

The buccal cavity itself is formed by a pair of folds of integument, which appear about the time of formation of the secondaiy gill-slits. The two folds are at first upper and lower respectively ; the iipper fold commencing above the pra3oral pit, and becoming continuous posteriorly with the upper margin of the mouth ; while the lower fold arises as a ridge along the lower and hinder border of the mouth, extending in front across the ventral surface to the right side.

As the mouth assumes its median position the upper and lower folds increase in size, and form the left and right halves of the buccal hood respectively.

The buccal tentacles appear early, as papilla-like outgrowths from the buccal folds (Fig. 39, ox). They arise at first entirely from the lower, or future right fold, about the time the mouth commences to shift its position, and they do not extend into the left fold until a much later period. The median ventral tentacles are the first to be formed, and the others are added on in succession at either end of the series. Small cartilaginoid skeletal elements are present at the bases of the tentacles from their first appearance, and ultimately give rise to the buccal. skeleton.

6. The Praeoral Pit.

At the commencement of the larval period, the prasoral pit, which, it will be remembered, is formed from, the left anterior gut diverticulum (p. 70), is a small pit with thick ciliated walls, lying on the left side of the anterior part of the head, above and in front of the mouth, and opening to the exterior by a small aperture (Fig. 36, DL). When the mouth commences to shift towards the median plane, a ciliated groove is formed, connecting its upper and anterior angle with the aperture of the praaoral pit ; and as the mouth sinks further and further towards the i-ight side the prasoral pit gradually becomes flattened out (Figs. 37, 38, DL)>

G


82


AMPHIOXUS.


its walls becoming ultimately converted into the tract of columnar ciliated epithelium, which in the adult Ainphioxus lines the posterior part of the buccal cavity.

7. The Atrial Cavity.

The atrial chamber begins to form in larvae which have from nine to ten primary gill-slits, but in which the secondary gillslits have not commenced to develop. A narrow longitudinal groove appears along the ventral surface of the body of the larva,


CM



71


AF


MD


MV


FIG. 40. A diagrammatic transverse section across an Amphioxus Larva with eleven or twelve primary gill-slits, but no secondary ones. (Slightly modified from Lankester and Willey.)

A, aorta. AC, atrial cavity. AF, subatrial fold. CH, notochord, CM, myocoel. CN, diverticuluin of myocrel lying between uotochord ami myotome. CS, splanchuoccel. CTJ, cutis layer. DF, cavity of dorsal fin. US. skeletogenous layer. I, spinal cord. MD, metapleural ridge. MF, muscle-fascia layer. ML, myotomic muscle. MV, metapleural caual. TI, intestine. V, subintestinal vessel.

behind the region of the pharynx. The groove is bordered by two folds, which become later the metapleural ridges of the adult (Fig. 40, MD) : on reaching the pharyngeal region, the two metapleural ridges are deflected towards the right side of the larva, and run forwards one on each side of the row of primary gill-slits. From the inner side of each metapleural ridge a horizontal shelf-like outgrowth, the subatrial fold, arises ; and the two sub


THE ATEIAL CAVITY.


83


ntrial folds meet and fuse, converting the groove into a tube (Fig. 40, AC). This tube, of which the roof is formed by the ventral wall of the body, the sides by the metapleural folds, and the floor by the fused subatrial folds, is the atrial chamber. The formation of the floor of the chamber proceeds from behind forwards. In the larva shown in Fig. 37, in which there are fourteen primary slits, and the secondary slits are just commencing to form, the


CM


CH


ML


CN



71


AC


AF


FiG. 41. A diagrammatic transverse section through an advanced Amphioxus Larva with fully formed atrial cavity. (Slightly modified from Lankester and Willey, and from Boveri.)

A, aorta. AC, atrial cavity. A3P, floor of atrial cavity, formed by fusion of the subatrial folds. CH, notochord. CM, myocrel. CN, diverticulum of myocoel lying between notochord and myotomic muscle. CS, splanchnoco3l. CTJ, cutis layer. JDF, cavity of dorsal fin. US, skeletogenous layer. I, spinal cord. MD, metapleural ridge. MF, muscle-fascia layer. ML, myotomic muscle. MV, metapleural canal. OR, commencing reproductive organs. TI, intestine. V, subintestiual vessel.

atrial tube is completed to about the level of the ninth primary gill-slit ; and at a stage shortly before that shown in Fig. 38 the tube is completed along the whole length of the pharynx. The anterior end of the tube ends blindly, but the posterior end remains open as the atrial pore.

The atrial tube is at first very narrow, and of nearly equal

G 2


84 AMPHIOXUS.

diameter along its whole length. Later on, it enlarges very greatly, and, pushing the ventral body-wall before it, encroaches on the space hitherto occupied by the ccelom, finally extending so far dorsal wards as nearly to surround the alimentary canal (Fig. 41, AC; cf. also Figs. 12 and 13).

The primary gill-slits at first open directly to the exterior, but, as they lie between the two metapleural folds, they become boxed in on the formation of the floor of the atrial tube, and from this time open into the atrial tube or chamber. The secondary gill- slits, which also lie between the two metapleural folds, very close to the base of the right metapleural fold, are not formed until the floor of the atrial chamber is completed, and consequently open into this chamber from the first.

The metapleural folds are at first solid ridges ; large spaces soon appear in them, which become the metapleural canals of the adult (Figs. 12, 13, and 41, MV).

b. The Mesoblastic Somites.

At the commencement of the larval period fourteen or fifteen pairs of somites are present ; during the early part of this period the number steadily increases, and, shortly before the appearance of the secondary gill-slits, the full number of somites of the adult animal, which appears to be very generally sixtyone, is attained. The somites formed during the larval period differ from those developed in the embryonic stages in not communicating with the mesenteron at any time in their formation. In the development of these hinder somites it is probable that the polar mesoblast cells take an important share.

Concerning the further development of the somites some interesting details are given by Hatschek. At the commencement of the larval period, i.e. about the time of formation of the mouth, each somite (cf. Figs. 32 and 42) becomes divided into a dorsal portion or proto vertebra, and a ventral portion or lateral plate.

The proto vertebras retain the original segmental arrangement, i.e. the cavities of successive protovertebras remain separate from one another ; but in the ventral portions of the somites, or lateral plates, the septa become absorbed, and the cavities open into one another along the whole length of the body, forming a continuous body cavity or ccelom.


THE MESOBLASHC SOMITES.


85


The cavity of the protovertebra is spoken of as a myocoel (Fig. 42, CM) ; and at a stage when five primary gill-slits are present (cf. Fig. 36) the myocoels of each pair of pro to vertebras communicate with each other above the spinal cord (Fig. 42). The outer or parietal wall of the protovertebra is very thin, and closely applied to the epidermis : it gives rise to the cutis, or connective tissue basis of the skin, and may be spoken of as the cutis layer (Fig. 42, cu). The inner or notochordal wall of the protovertebra, as already noticed (p. 67), thickens very greatly, and, though still remaining only one cell thick, becomes converted into the myotomic muscles (Fig. 42, ML). The lower or visceral wall of the protovertebra, like the parietal wall, is thin, and is in contact with the dorsal wall of the alimentary canal.

The cavity of the lateral plates, or splanchnocoel (Fig. 42, cs), is continuous from end to end of the body, through absorption of the septa between the successive somites ; it is also continuous from side to side across the mid-ventral plane. The walls of the splanchnoccel are thin ; the outer, or parietal layer, is in contact with the ventral epidermis, while the inner or splanchnic layer clothes the sides and ventral wall of the alimentary canal.

In the later stages important changes occur in these relations, and the condition immediately after the completion of the larval period is shown in Fig. 43.

The myocoels now extend ventral wards much further than before, so that the parietal layer of the splanchnocoel (Fig. 43, cs) no longer touches the epidermis. The median dorsal and ventral parts of the myocoels have separated off as the compartments, DF and VF, of the dorsal and ventral fins, which are now prominent structures.

The ventral or splanchnic wall of each myoccel is folded to



cs


FIG. 42. Diagrammatic transverse section across the intestinal region of an Amphioxus larva with five primary gill-slits : cf. Fig. 36. (After Hatschek.)

CH, notochord. CM, myocoel. CS, spliuiclmocal. CU, cutis layer. EP. epidermis. I, spinal cord. ML, myotomic muscle. TI, intestine. V", subintestinal blood-vessel.


86


AMPHIOXUS.


DF


form a pouch, which extends upwards, between the myotome on the outer side, and the notochord and spinal cord on the inner side. The outer wall of this pouch (Fig. 43, MF) becomes the fascia covering the inner^surface of the myotome ; while the inner wall of the pouch (Fig. 43, HS) gives rise to the

skeletal connective tissue, which invests the notochord and the spinal cord. The cavity of the pouch becomes ultimately obliterated by growth of the connective tissue, except in the anterior three or four segments of the body.

The splanchnocoel (Fig. 43, cs) undergoes but slight modification. It extends further dorsalwards than before, and almost completely surrounds the alimentary canal, cutting out the myocoel from its former share ; while the myoccel in its turn, owing to its ventral extension, shuts out the splanchnocoelic wall from all contact with the external epidermis. The splanchnoccel becomes the body cavity, or ccelom, of the adult. .

It is interesting to note that even at this stage, when the larval development is completed, all the parts of the body are, as in the earlier stages already noticed in this respect,* made up of epithelial



cs


VF


FlG. 43. Diagrammatic transverse section across a young Amphioxus immediately after the completion of the larval period. The section is taken at a level between the atrial pore and the anus. (After Hatschek.)


A, dorsal aorta. CH, notochord. CM, myocoel. CS, splanchnoccel. CU, cutis layer. DF. cavity of dorsal fin. EP,

epidermis. HS, skeietogenous layers, which in each case are but

layer. I, spinal cord. MF, muscle-fascia layer. ML, myotouiic muscle. V, subintestinal vessel. "VF, cavity of ventral fin. (Compare also Figs. 40 and 41.)


one cell thick ; the complications in various regions being brought about by differences in the shapes of the

cells at different places, together with foldings of the walls of

the several cavities.

The origin of the connective tissue is not determined with

certainty. Hatschek considers that it is at first of a gelatinous

nature, probably formed by excretion from, and between, the


THE ASYMMETRY OF THE LARVA. 87

several epithelial layers ; any cellular elements it may obtain being derived by migration from these epithelial layers.

0. The Asymmetry of the Larva.

The asymmetry of the larva during its early stages is one of the most striking features in the development of Amphioxus. The fact that, but for the alternation of the myotomes on the two sides of the body, the embryonic stages are symmetrical ; and the further fact that at the close of the larval period the symmetry is regained, indicate that the asymmetry of the earlier larval stages is a secondary or acquired character, and that the explanation of it is probably to be found in peculiarities of habit or environment of the larva during these stages.

The cardinal point in the asymmetry of the larva is the lateral position of the mouth, which, coupled with its huge size, is probably sufficient to explain the displacement of the gills of the left side.

Willey has suggested that the lateral position of the mouth is correlated with, or actually due to, the anterior extension of the notochord. The mode bf development of this front end of the notochord, and a comparison with other Vertebrates, strongly suggest that the prolongation forwards in front of all the other organs of the head is a secondary feature, associated not improbably with the burrowing habits of Amphioxus ; and if we assume that the ancestral mouth was, as in the Ascidian tadpoles, dorsal in position, then the forward growth of the notochord would of necessity cause lateral displacement of the mouth. The suggestion is an ingenious one, and may be accepted as at any rate a provisional explanation.

III. THE ADOLESCENT PERIOD.

At the close of the larval period, i.e. at the completion of the critical stage, the young Amphioxus abandons its pelagic habits and burrows into the sand, where it passes the rest of its life ; burying itself upright, with the tail downwards and the buccal hood alone projecting from the sand.

The further development takes place gradually. There is a steady increase in size, but no new myotomes are formed, the full number being present at the critical stage. The gill-slits,


88 AMPHIOXUS.

on the contrary, increase greatly in number, new ones being added on at the hinder end of the series, apparently throughout the life of the animal. Each new gill-slit (Fig. 35, I/) becomes divided into two, at an early stage in its development, by the growth downwards of a tongue-bar from its dorsal border, just as in the earlier formed slits. The slits further become divided transversely by the horizontal bars characteristic of the adult (Fig. 35, L). Owing to this increase in number of the gill-slits, without any increase in the number of the myotomes, the correspondence between the two sets of structures is speedily lost ; the alimentary canal, and the body generally, each acquiring a metamerism of its own.

The Reproductive Organs.

The reproductive organs are formed by proliferation of the epithelial walls of the septa which divide the successive somites from one another. Each of these septa (cf. Fig. 31) is formed by the coalescence of the posterior and anterior walls of adjacent somites, and consists in the young Amphioxus of a thin connective tissue lamella, clothed on each surface by a single layer of flattened epithelial cells, these latter being really parts of the walls of the protovertebrsB.

In young specimens of Amphioxus, of about 5 mm. length, the epithelial layer becomes modified over a very small patch at the outer and lower corner of the septum, in the angle between the parietal wall or cutis layer, and the visceral wall of the protovertebra (cf. Fig. 41, OR) : at this spot the cells become cubical or columnar in shape, while over the rest of the septum they remain flattened.

This modification does not occur along the whole length of the body, but is from the first confined to the somites in which the reproductive organs lie in the adult animal ; i.e. the patches of modified epithelium are found on the septa forming the walls of the somites from the tenth to the thirty-sixth inclusive.

The modification affects the cells of both surfaces of each septum, but the cells of the posterior surface are almost from the first of larger size than those of the anterior surface, and, growing much more rapidly than these latter, push the septum forwards, and project into the segment in front of that to which they really belong, as a small stalked knob, to which the cells of


THE ADOLESCENT PERIOD. 89

the anterior surface of the septum form a follicular epithelial investment.

These knobs, each of which is a solid mass of enlarged epithelial cells, gradually increase in size, extending forwards until they ultimately occupy the whole length of the segments. The cavities in which they lie, really parts of the myoccel (Fig. 41, CM), widen considerably to allow for this increased size, and in specimens of about 15 or 16 mm. length become shut off completely from the rest of the myocoel. Boveri has proposed the term gonotome for this portion of the somite, which is specially connected with the reproductive organs, and which is only found in the somites from the tenth to the thirty-fifth or thirty-sixth, in which these oi'gans lie ; he has further directed attention to the fact that the position at which the reproductive organs appear in Amphioxus, close to the line of separation between myocoel and splanchnoccel, corresponds very nearly to that which they hold, in the earlier stages of their development, in the higher Vertebrates.


List of the more important Publications dealing with the development of Amphioxus.

Boveri, T. : ' Ueber die Bildungsstatte der Geschlechtsdriisen und die Entste hnng der Genitalkammern beim Amphioxus.' Anatomischer Anzeiger,

vii. 1892. Hatschek, B. : ' Studien iiber Entwicklung des Amphioxus.' Arbeiten aus

dem Zoologiscben Institute der Universitat Wien, iv. 1881.

'Ueber den Schichtenbau von Amphioxus.' Anatomischer Anzeiger,

iii. 1888.

' Die Metamerie des Amphioxus und des Ammocoetes.' Verhand lungen der Anatomischen Gesellschaft, 1892. Kowalevsky, A. : ' Entwickelungsgeschichte des Amphioxus lanceolatus.'

Memoires de I'Academie Imperiale des Sciences de Saint-Petersbourg,

vii e serie, tome xi. No. 4. 1867.

' Weitere Studien iiber die Entwicklungsgescbichte des Amphioxns

lanceolatus, nebst einem Beitrage zur Homologie des Nervensystems

der Wurmer und Wirbelthiere.' Archiv fur mikroskopische Anatomie,

xiii. 1876.

Lankester, E. Ray : ' Contributions to the Knowledge of Amphioxus lanceolatus.' Quarterly Journal of Microscopical Science, New Series, xxix.

1889. Lankester, E. Ray, and Willey, A. : ' The Development of the Atrial Chamber

of Amphioxus.' Quarterly Journal of Microscopical Science, New

Series, xxxi. 1890. Willey, A. : ' The Later Larval Development of Amphioxus.' Quarterly Journal

of Microscopical Science, New Series, xxxii. 1891. Wilson, E. B. : ' On Multiple and Partial Development in Amphioxus.'

Anatomischer Anzeiger, vii. 1892.


Marshall (1893): 1 Introduction | 2 Amphioxus | 3 Frog | 4 Chick | 5 The Rabbit | 6 Human Embryo | Illustrations

Marshall AM. Vertebrate Embryology: A Text-book for Students and Practitioners. (1893) Elder Smith & Co., London.

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