Book - Outlines of Chordate Development 1
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Kellicott WE. Outlines of Chordate Development (1913) Henry Holt and Co., New York.
- Outlines of Chordate Development: 1. Amphioxus | 2. Early Frog | 3. Later Frog Organogeny | 4. Early Chick - Embryonic Membranes and Appendages | 5. Later Chick - Organogeny | 6. Early Mammal - Embryonic Membranes and Appendages | Figures
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- 1 Chapter 1 The Development of Amphioxus
- 1.1 The Germ Cells And Their Production
- 1.2 The Embryonic Period - A. From Fertilization To The Time Of Hatching
- 1.3 The Embryonic Period - B. From Hatching To The Formation Of The Mouth
- 1.4 The Larval Period
- 1.4.1 The Central Nervous System
- 1.4.2 The Gill Slits
- 1.4.3 The Club-shaped Gland
- 1.4.4 The Endostyle
- 1.4.5 The Mouth and Associated Structures
- 1.4.6 The Preoral Pit and Its Derivatives
- 1.4.7 The Blood-vessels
- 1.4.8 The Atrium
- 1.4.9 Larval Asymmetry
- 1.4.10 The Mesodermal Somites
- 1.4.11 The Nephridia
- 1.4.12 The Larva at the Critical Stage
- 1.5 The Adolescent Period
- 1.6 References To Literature - Chapter 1
Chapter 1 The Development of Amphioxus
"As an introduction to the study of embryology, and as an indispensable aid to a reasonable appreciation of the value of embryological facts, the life-history of Amphioxus provides an object which ... is perhaps unrivalled. It is alike useful in a text-book of human embryology, and in one of invertebrate zoology." (Willey, "Amphioxus," etc., p. 104.)
MORPHOLOGICALLY Amphioxus (Branchiostoma lanceolatum) is recognized as one of the important types of Chordata, for it illustrates in simple form the essential characteristics of this large group, most of the members of which are extremely complex. And in its mode of development, no less than in its structure, Amphioxus serves as a key to the more complicated conditions of the Craniate groups. The anatomical and embryological simplicity of this creature is commonly regarded as an indication of true primitiveness, although the morphologist recognizes, as the embryologist must also, that the simplicity of primitiveness is here obscured, in many respects, by conditions which are obviously special adaptations or secondary alterations of primary arrangements.
Many of the simple embryological characteristics of this form are correlated with the freedom of the egg from a large yolkmass. This is equivalent to saying that the accumulation of yolk in the eggs of most Chordates is a secondary character, and is, to a considerable extent, the cause of many of those modifications of the course of development which lead to unusual conditions, proving difficult of comparison with other developmental types.
The egg of Amphioxus is small, and the deutoplasm, small in amount, is scattered through its substance. In correlation with this, cleavage is total and quite regular in its course, leading to the formation of fairly typical blastula and gastrula. The organ-rudiments are all formed first as simple epithelial structures, whose origins and fates are easily followed, for each part is sharply outlined and remains clearly demarcated through the course of its development. The embryo is free swimming and externally ciliated. In all these respects Amphioxus illustrates primitive Chordate characteristics. Most of the secondary modifications of development appear comparatively late in its course, and many of them are obviously correlated with rather unusual habits and activities.
The embryological history of Amphioxus is described here, then, not only because of the morphological importance of this form, but because of its embryological primitiveness and the diagrammatic simplicity of its early stages, which will aid in understanding the complicated development of the other Chordates described. Here we have Chordate development reduced to its simplest terms. A fair knowledge of the anatomy of Amphioxus is presupposed.
The whole developmental history of Amphioxus falls quite naturally into four general periods.
- PRODUCTION OF THE GERM CELLS. This includes the formation of the germ cells, oo- and spermato-genesis, and spawning.
- EMBRYONIC PERIOD. This extends from fertilization to the opening of the mouth. This phase of development is very rapid: at normal seasonal temperatures of the water it may occupy only thirty-six hours. The entire period is conveniently subdivided into two.
- Before Hatching. This includes fertilization, cleavage, gastrulation, and the first mapping out of the embryo. The conclusion of this period is marked by escape from the egg membranes eight hours or more.
- After Hatching. Here the chief systems and organs of the embryo become definitely laid down. The embryo is free swimming (pelagic) twenty-eight hours or more. The actual duration of these and the other periods depends chiefly upon temperature; it may be nearly doubled in the cooler waters of an early season.
- LARVAL PERIOD. From the opening of the mouth to the cessation of pelagic life and the assumption of a burrowing habit. Here development is slower and consists largely in the elaboration of the parts marked out during the preceding period about three months. At the conclusion of this period the larva is about 3.5 mm. long and has reached a condition known as the "critical stage."
- ADOLESCENT PERIOD. From the critical stage to sexual maturity. This is marked by histological differentiation and the gradual appearance of adult characteristics. Germ cells are first brought to maturity in specimens about 2 cm. in length, the age of which is doubtful. Growth to full size probably involves several years and covers several spawning periods.
The Germ Cells And Their Production
We may first describe the germ cells as they are extruded from the body.
The eggs of Amphioxus are spherical and only 0.10 to 0.13 mm. in diameter. Among the Chordates smaller eggs than these are found only in some Mammals. The egg (Fig. 1) is surrounded by two membranes, a thin outer, or vitelline membrane, which at this time is usually separated by a space from the surface of the egg, and a thick inner, or perivitelline membrane, which at the time of laying is more or less fluid and closely adherent to the surface of the egg. The egg is richly supplied with deutoplasm in the form of very numerous small granules. These are not uniformly distributed through the cytoplasm for there are two definite regions comparatively free from deutoplasm, namely, a narrow superficial layer, and an irregular conical region toward one side of the egg. Strictly speaking the egg of Amphioxus is thus telolecithal, but the deutoplasmic center is riot toward the vegetal pole of the egg but toward one side, below the equator. The precise location of this yolk-free area is important for it establishes in the egg a definite bilateral symmetry which is also that of the future embryo. The animal pole is marked by the apex of this clear area and by the point at which the first polar body is given off. The first polar body has already been formed at the time the egg is laid, but since it lies outside the vitelline membrane it is ordinarily lost. At this time the animal pole is marked, however, by the position of the egg nucleus which is just in the equatorial plate of the second polar division. The arched base of the clear protoplasmic region lies about at the level of the equator, and the whole space lies eccentrically toward that side of the egg which, from later development, is known to be anterior or antero-dorsal. A diagram of a sagittal section of the egg is shown in Fig. 2, in the position usually assumed by the egg in the water.
Fig. 1. The egg of Amphioxus. C, after Cerfontaine, others after Sobotta. A. Ovarian egg showing cortical plasm. B. Cortical layer forming a membrane on the suface of the egg, within the vitelline membrane. C. Egg membrane fully formed but still attached to surface of egg, D. Extruded, fertilized egg. Membrane fully formed and beginning to leave the egg. c, Cortical layer; e, endoplasm; m, egg membrane, externally vitelline, internally a product of the exoplasm; p, perivitelline space; s, spermatozoon; v, vitelline membrane; /, first polar body; //, second polar spindle.
The spermatozoa are very small (15-20 micra in length); the rather spherical head is about 1 micron in diameter about the size of a deutoplasm granule.
The gonads are metameric organs distributed through the middle and posterior pharyngeal region. There are about twenty-six pairs (twenty-three to twenty-eight), approximately in segments ten to thirty-six. They first begin to develop in the embryo before the mouth is opened, and apparently first come to maturity when the organisms are 2 to 2.5 cm. in length. The discharge of the germinal products involves the nearly complete loss of the gonads as such, so that after each annual spawning period they redevelop from small rudiments. Details of the
Fig. 2. Diagram of a median sagittal section through the fertilized egg of Amphioxus at the stage of the fusion of the egg and sperm pronuclei. After Cerfontaine. The arrow marks the direction of the chief or polar axis. AD, Antero-dorsal region; n, egg and sperm pronuclei; p, region relatively free from deutoplasm; PV, postero-ventral region; s, remains of tail of spermatozoon; y, yolk or deutoplasmic bodies; II, second polar body.
development of the gonads will be described later. The partially grown ovary, which the testis resembles in essentials, is a large spheroid mass, projecting from the postero-ventral margin of the segment into the atrium, covered of course by atrial epithelium. Fig. 3 represents in diagrammatic form a section through the half grown ovary in the so-called medusoid or mushroom stage. An abundant vascular supply is derived from branches of the posterior cardinal veins, which divide the germinal epithelium into dorsal and ventral portions. Inside the gonad is a space known as the primary ovarian or perigonadial cavity, in the outer wall of which the definitive germ cells are developed. Outside the gonad, and separating it from the atrial epithelium and body wall proper, is a secondary ovarian cavity or gonocoel. In the wall of the gonocoel, just above and below the stalk of attachment through which the blood-vessels pass, are thickened patches in the atrial wall of the gonoccel the dorsal and ventral cicatrices. During the later stages of their formation the germ cells oogonia or spermatogonia, enlarge so consider- fe ably as nearly to oblit- / erate, by compression, these cavities and even to cause the gonads to encroach very considerably upon the atrial cavity. Toward the end of the
Fig. 3. Diagram of a section through the gonad of Amphioxus. After Cerfon taine. 6, Peribranchial (atrial) epithelium; c, cicatrix; /, true follicular epithelium; fe, growth period the nucleus, external layer of follicular epithelium; 0, , . , . , gonocoel; ge, germinal epithelium; o\, pri WniCIl Contains a large mary ovarian cavity; o 2 , secondary ovarian
nucleoluS, passes through C , avi * y; W 'Parietal layer of gonocoel; v car dinal vein; vg, visceral layer of gonoccel.
the synizesis stage and
the deutoplasm granules, whose formation began toward the inner side of the oogonia when these were only about 0.05 mm. in diameter, crowd the entire cell body except in the perinuclear region and a narrow peripheral region which is occupied by several rows of small clearer vacuoles. It is doubtful whether there is a true cellular egg follicle: a thin layer of cells forming the wall of the primary ovarian cavity forms an ovarian follicle which folds in and covers almost completely the superficial oogonia.
After the growth period is completed the process of maturation commences while the oogonia are still within the ovary. The chromatic nuclear membrane disappears and the whole nuclear apparatus moves toward the atrial (i.e., attached) surface of the cell, marking the animal pole and establishing visibly the symmetry of the egg. As it reaches the surface a typical polar spindle with asters forms, and the chromatin forms into twelve tetrads, indicating 'a somatic chromosome number of twentyfour. About this time the very thin vitelline membrane is formed on the surface from the substance of the egg proper (Fig. 1), and as the first polar body is cut off it pushes this before it, so that a portion of the membrane is cut off with the polar body which may no longer remain attached to the egg. Immediately after the first polar body is given off the second polar spindle forms, but the division halts in the mesophase (Fig. 1). Just before or during the formation of the first polar body the inner wall of the secondary ovarian cavity is ruptured by contraction of the body wall and of muscle fibers in the thin walls of the gonoco3l, and the eggs are partly forced into the gonocoel. All of these processes occur as the animals emerge from the sand and swim about preparatory to spawning.
Spawning extends through the greater part of spring and summer in the warmer waters, and always occurs at a definite time of day about sundown, or from five to seven in the evening. Strong contractions of the general body musculature rupture the cicatrices and force the eggs from the gonocoel into the atrial cavity, and thence they are carried by bodily contractions and the respiratory current out through the atriopore into the free sea water.
Contact with the sea water brings about the formation of a thick mucilaginous second or inner membrane over the surface of the egg. The substance of this, the perivitelline membrane, seems to be derived from the material of the peripheral layers of vacuoles in the cytoplasm; these flow together forming a homogeneous superficial layer quite distinct from the underlying protoplasm (Fig. 1). The membrane is at first rather fluid but in contact with the water it soon begins to toughen. In this condition the eggs are ready for fertilization. The formation of the spermatozoa is not so fully known but it seems to occur in a manner parallel to the formation of the ova.
The Embryonic Period - A. From Fertilization To The Time Of Hatching
Eggs and sperm are discharged into the water at about the same time and immediately each egg becomes surrounded by a crowd of the spermatozoa. The perivitelline membrane has been formed first in the region of the animal pole and at this time is gradually extending thence toward the vegetal pole. In this region then it remains longest in a more fluid condition and it is at this vegetal pole that the sperm usually gain entrance to the egg, after having penetrated first the thin vitelline membrane and traversed the perivitelline space. No micropyle has been discovered and polyspermy seems to be very frequent. It is not surely known whether the entire sperm or only the head usually enters the egg substance. Immediately after the entrance of a spermatozoon the entire perivitelline membrane is formed, toughens, leaves the surface of the egg, and comes into contact with or even partially fuses with the vitelline membrane (Fig. 1, D) obliterating the original perivitelline space but leaving a wide secondary perivitelline space between itself and the egg.
Entrance of the sperm affords also the stimulus to the completion of maturation of the egg nucleus, which at this time is still in the mesophase of the second polar division. While the sperm, lying just inside the egg, is reconstituting a normal nucleus together with sperm centrosome and aster, the second polar body is formed and the mature egg nucleus established. The second polar body forms about fifteen minutes after entrance of the sperm. The division of the sperm centrosome and aster, the approach and fusion of the egg and sperm nuclei, and the constitution of the first cleavage nucleus, are in no way unusual (Fig. 4).
The first cleavage spindle takes up a position definitely related to the symmetry of the egg, lying horizontally, slightly nearer the animal pole, and at right angles to the median plane. The relation of this symmetry of the egg to that of the future embryo is shown in Figs. 2 and 7, A. The median plane of symmetry is determined by the points of polar body formation and sperm entrance, and the eccentric position of the clear protoplasmic region. This last is displaced toward the anterodorsal region of the future embryo: one result of this is the greater freedom from yolk of this region of the embryo, and this has an important bearing upon subsequent development. The egg axis is at an angle of roughly 30 to the antero-posterior axis of the embryo : that is, the animal pole of the egg becomes ant ero- ventral, the vegetal pole postero-dorsal. It is quite probable that this bilateral symmetry, and certain that the polarity of the egg, is determined while it is still within the ovary; it gains visible expression through the assumption of a peripheral position by the primary oocyte nucleus and the eccentricity of the accompanying clear protoplasmic region. The egg of Amphioxus seems to resemble that of the Echinoderm rather than those of most other forms, in that the direction of this eccentricity of the nucleus, i.e., animal pole, is toward the region of attachment in the ovarian epithelium.
Fig. 4. Prophase of first cleavage figure in Amphioxus. After Sobotta. Inner and outer membranes fused and separated from the egg by a wide space called the perivitelline space.
The cleavage of Amphioxus is total and unequal and, though subject to much variation, it is in many eggs quite regular. It is generally believed that the Amphioxus type of cleavage is primitive among Chordates; and that the cleavage modes of Craniates are to be derived from it, the chief cause of modification being the accumulation of yolk. The first division of the egg occurs about an hour after entrance of the sperm, the second about an hour after the first, and subsequent divisions about every fifteen or twenty minutes. The plane of the first cleavage as indicated by the position of the first cleavage spindle, is median (cleavage furrow meridional), dividing the egg into exactly similar right and left halves, which become the right and left halves of the embryo. The second cleavage furrow is also meridional and the plane at right angles to the first. Cleavage becomes unequal with this division for the two cells are divided into two smaller anterodorsal, and two larger post ero- ventral cells, symmetrically arranged on each side of the median plane (Fig. 5, A).
The third cleavage is at right angles to the first two and the furrow equatorial, or rather latitudinal for again the division is slightly unequal and each of the four cells forms a smallei cell toward the animal pole, and a larger toward the vegetal.
Fig. 5. Cleavage in Amphioxus. After Cerfontaine. A. Four-cell stage viewed from animal pole. The two antero-dorsal cells are the smaller. B. Eight-cell stage viewed from animal pole showing the four sizes of the cells. C. Sixteen cells viewed from the left side. D. Thirty-two* cells viewed from vegetal pole. E. Thirty- two passing into sixty-four cells, viewed from the anterodorsal region. F. Optical section of right half of young blastula. About 128 cells, a, Animal pole; ad, antero-dorsal; /, left; pv, postero-ventral ; r, right; v, vegetal pole.
Thus in the eight-cell stage (Fig. 5, B) we have four pairs of cells of four sizes; the four micromeres of the animal pole consist of two smaller anterior, and two larger ventral cells, and the four macromeres of the vegetal pole, of two smaller dorsal, and two larger posterior cells, the smaller macromeres being larger than the larger micromeres. These relative inequalities in the quadrants, as well as their bilateral symmetry, continue throughout cleavage.
Strict synchronism of cleavage is lost in passing from four to eight cells, the smaller cells commencing their division before the larger. In the early stages the intervals between successive cleavages are such that, in spite of lack of synchronism, it is still possible to speak of sixteen- and thirty-two-cell stages, but after this the numerical progression is no longer regular.
Were the cleavage strictly regular the eight cells would form sixteen by meridional cleavages passing symmetrically through the entire egg, but as a matter of fact, during their formation the spindles of the eight blastomeres change their position so that the cleavage planes of the micromeres are nearly perpendicular to the median plane, while those of the macromeres are rather parallel with this plane. As a result the sixteen-cell stage (Fig. 5, C) consists of eight micromeres arranged in two rows of four cells each, parallel with the median plane and becoming arched in the same direction, and of eight macromeres in two rows of four cells each extending across the median plane, i.e., from right to left, and arched in this direction. The relation between the groups of micro- and macromeres is much like that of the two hands when partly closed and one in the palm of the other. The thirty-twocell stage (Fig. 5, D) is formed by the horizontal (latitudinal) divisions of each of the sixteen, forming eight meridional rows of four cells each. After thirty-two cells (Fig. 5, E) the divisions become irregular in their appearance. To summarize:
Cleavage 1st 2nd 3rd 4th 5th
!4 mi r ' 2 Sma11 = 4 Sma11 = 8 Cells
\2 large = 4 large = 8 cells
f2 small = 4 small = 8 cells
[ 4macr - 12 large = 4 large = 8 cells
Total number of cells 24 8 16 32
This typical arrangement of cleavage planes is by no means invariable. Variations are indeed very frequent but the cells are usually arranged according to definite plans. In many cases the cleavage pattern is nearly radial, and in others the blastomeres may shift more or less giving the appearance of a spiral cleavage. In some eggs the micromeres of the sixteen-cell stage may not all divide similarly but four may divide vertically and four horizontally. These variations in cleavage do not affect subsequent processes.
As a consequence of the early blastomeres remaining well rounded a central space is formed among them. Virtually present in the four-cell stage, this space becomes real at eight cells, and as the cells multiply and gradually lose their rounded form they push away from the center of the mass leaving a definite space within, at first open at the poles (cleavage pores). This is the beginning of the segmentation cavity or blastocoel. By the time thirty-two cells are formed the poles close over and soon the blastoccel is entirely closed. From about sixtyfour cells on (Fig. 5, E, F) the blastomeres lose their rounded outline and become flattened and closely packed in a simple epithelial layer. This arrangement of the blastomeres may be taken as the beginning of the blastula stage, which may be considered fully established when the number of cells reaches one hundred and twenty-eight. The hollow spherical blastula of this type (Fig. 5, F) is called a cceloblastula, and it is commonly regarded as the most primitive type of blastula. In Amphioxus this is bilaterally symmetrical and the epithelial wall is of varying thickness on account of the varying sizes of the cells. The cells are relatively free from deutoplasm, and therefore smaller, in the anterior region, richer in deutoplasm, and therefore larger, in the posterior region, and uniformly graded in size and deutoplasmic content between these regions. The animal pole remains antero-ventral (Fig. 7, B). The blastocoel is very large and, on account of the varying thickness of its wall, may be said to be slightly eccentric toward the anterior side.
By the time about two hundred and fifty-six cells have been formed in the blastula, or about four hours after fertilization, the process of gastrulation is commenced, by which the single walled blastula is to be converted into a double walled gastrula. We should notice in advance that the gastrula of Amphioxus is formed by the three processes of inwufination, involution, and epiboly, and so is not typical of Chordates generally, in which the double walled or two layered condition results more extensively from delamination, that is, by tangential divisions in the wall of the blastula, together with some involution and epiboly.
The first indication of gastrulation is the flattening of the vegetal pole of the blastula (Fig. 6, A), which is soon followed by the appearance of a slight infold on the antero-dorsal aspect, at about the level of the equator (Fig. 6, B). This infolding or invagination soon extends around the sides of the blastula, and finally the whole flattened vegetal or posterodorsal region becomes folded down into the segmentation cavity (Fig. 6, C). The process of infolding continues in a more advanced stage in the antero-dorsal region where it commenced, and here the folded layer first comes into contact with the inner surface of the cells of the animal pole so as to obliterate the blastocoel there, at a time when this remains quite widely open elsewhere. We may now speak of the invaginating and non-invaginating layers as endoderm (hypoblast) and ectoderm (epiblast) respectively, and of the whole structure as the gastrula (Fig. 6, E). While the infolding of the endoderm leads to the obliteration of the blastocoel, it leads to the formation of another cavity which is lined completely with cells of one kind endoderm. This cavity, which can be recognized as beginning with the earliest infolding of the blastula wall, is the archenteron or primitive gut cavity, which is to give rise to the chief cavities of the later embryo. In many Chordates this cavity is virtual rather than real on account of being filled with yolk-cells. The gastrula of Amphioxus is often termed a ccelogastrula to indicate the fact that the archenteron is an actual widely open cavity.
Fig. 6. Gastrulation in Amphioxus. After Cerfontaine. A. Blastul a showing flattening of the vegetal pole and the rapid proliferation of cells in the posterodorsal region (germ ring). B. Flattening more pronounced; mitoses in cells of germ ring. C. Commencement of the infolding (invagi nation) of the cells of the vegetal pole. D. Continued infolding, and inflection, or involution, of ectoderm cells in the dorsal lip of the blastopore. The blastoccel becoming obliterated and the archenteron being established. E. Invagination complete. Continued involution in the dorsal lip of blastopore. Mitoses in germ ring. F. Constriction of blastopore and commencement of elongation of the gastrula. Remnants of blastoccel in ventral lip of blastopore. G. Gastrulation completed. Continued elongation, and narrowing of blastopore. H. Neurenteric canal established by overgrowth of neural folds. Continued mitosis in germ ring.
While cell multiplication continues in all parts of the embryo during gastrulation, immediately after gastrulation begins, a center of very rapid cell division appears just anterior to the invaginating region on the dorsal side, and extending laterally around the margin of the blastopore; on the ventral side of
Fig. 7. Diagrams illustrating the relations between the adult axes and the axes of the egg and early stages. After Cerfontaine. A. Fertilized egg. B. Fully formed blastula. C. Gastrulation begun. D. Fully formed gastrula. Note posterior elongation, a-p, Antero-posterior axis of adult; d, v, dorsal and ventral surfaces of adult; x-y, chief egg axis (x, animal pole, y, vegetal pole).
the blastopore this region is less clearly marked, although distinguishable. This band of rapidly proliferating cells is to be identified as the germ ring, an embryonic region of greater importance in the other Chordates. The increase of cells at this point is so rapid that it disturbs, for a time, the simple epithelial arrangement of the cell layers (Fig. 6, A) and complicates somewhat the otherwise simple process of gastrulation. The transitional region between ectoderm and endoderm is the blastopore. The region of rapid cell multiplication, i.e., the germ ring, is, therefore, described as located in the lip of the blastopore, chiefly toward the dorsal side (Fig. 6, B-G).
a, Animal pole; ar, archenteron; 6, blastoporal opening; ch, rudiment of notochord; dl, dorsal lip of blastopore; ec, ectoderm; en, endoderm; gr, germ ring; nc, neuenteric canal; nf, neural fold; np, neural plate; s, blastoccel or segmentation cavity; v, vegetal pole; rl, ventral lip of blastopore; II, second polar body.
After the cells within the dorsal lip of the blastopore have partly folded in, the flattened plate of large endoderm cells of the vegetal pole swings gradually inward without undergoing much bending or arching, as if hinged at the lower lip of the blastopore (Fig. 6, D). This process involves a much greater extent of motion in the dorsal margin of this plate, and this is made possible by the rapid multiplication of cells in the dorsal margin of the blastopore, a sheet of which is left behind as the endoderm cells swing inward. The obliteration of the blastocoel continues gradually toward the ventral margin of the blastopore, and though finally completed, for a long time a trace of the cavity may be seen in the ventral lip. Occasionally the entire blastocoel may be obliterated completely in a very early stage but this is not a typical condition.
From this description of invagination it is evident that the endodermal or inner layer cells are really of two kinds, first, the large deutoplasmic cells of the vegetal pole of the blastula forming the ventral region of the endoderm, and second, the smaller cells from the dorsal region of the transitional zone between the animal and vegetal poles of the blastula, which come first to lie in the dorsal lip of the blastopore, and then by rapid multiplication bud off a sheet of cells forming in general the dorsal region of the endoderm. This group of more active cells really lies just outside the margin of the blastopore so that the cells contributed by it to the endoderm are really turned in or inflected after their formation and a very brief existence as ectodermal cells (Fig. 6, D, E). This inflection (involution) of the marginal cells is of importance in comparing the gastrula of Amphioxus with that of other forms.
But the cells formed by divisions in the dorsal blastoporal lip (germ ring) are by no means all added to the endoderm layer.
They contribute also to the ectoderm, and thus cause a steady extension of the dorsal lip posteriorly; that is, gastrulation is accomplished in part by epiboly, or the growth and extension backward of part of the blastoporal region. So that while at first the invaginated blastula is only little more than hemispherical in form, it soon begins to elongate, and this elongation extends chiefly posteriorly and is accomplished mainly by the rapid posterior elongation of the dorsal margin of the blastopore (Fig. 6, F-H).
When the invaginating phase of gastrulation is completed (Fig. 6, E) the archenteron has a nearly hemispherical form and is widely open in the dorsal or postero-dorsal direction. In Amphioxus the margin of the archenteric opening coincides with the blastopore, though we have seen that in general the blastopore should be regarded as that region where the ectoderm and endoderm are continuous, whether this borders an opening or not. As the dorsal margin of the blastopore extends backward the diameter of its opening decreases, that is, the archenteron becomes a more nearly enclosed cavity, considerably elongated posteriorly. As the blastopore closes gradually the direction of its opening becomes less dorsal and more posterior (Fig. 6, F, G). Toward the end of gastrulation the sides as well as the dorsal region of the blastoporal margin grow backward, and finally the ventral region shares in the process so that the last stages in the narrowing of the blastopore are accomplished by epiboly on every side. The result of this is the formation in the endoderm, just within the blastopore, of a band of cells, narrow below and widening laterally, which have been formed differently from the remainder of the ventral and lateral endoderm, and like the endoderm forming dorsally. This has an important bearing upon the development of later structures.
At the close of gastrulation the embryo has the form shown in Fig. 6, G. The gastrula is bilaterally symmetrical, quite elongated antero- posteriorly, flattened dorsally, rounded ventrally as well as anteriorly, while at the postero-dorsal aspect the archenteron opens directly to the outside by a narrow blastoporal aperture. The ectoderm forms a fairly uniform layer of superficial cells, and during gastrulation these have developed motilecilia, almost flagelliform, so that the gastrula is slowly rotated within the egg membranes which still envelop it. The endoderm cells lining the archenteron are somewhat unlike and of three distinct kinds, according to their origin. First there are the deutoplasmic cells, which are the descendants of the original vegetal pole cells of the blastula, forming the greater part of the floor of the archenteron; second, cells derived from the dorsal margin of the blastopore which have been added to the endoderm through epiboly and inflection, forming a band along the roof of the archenteron; and third, around the blastopore, a rim of cells narrow ventrally but wider dorsally, formed also through epiboly and inflection, from the ventral and lateral margins of the blastopore. Thus the gastrula of Amphioxus, although superficially resembling the simple type of invaginate gastrula, such as that of many Coelenterates, in reality is not like that, for here epiboly plays an important part in its formation. This is a leading characteristic of the gastrulas of the Chordates in general, and it is important to recognize in Amphioxus this method of gastrulation in its simplest and probably most primitive form. If one but imagines that in Fig. 6, F, the endoderm cells derived from the vegetal pole are multiplied and filled with a great mass of yolk, the result will be not widely unlike Fig. 32, E, of a section through the gastrula of the frog. Development up to this stage has been so rapid that the completion of gastrulation occurs only six to seven hours after fertilization. During the brief period between this stage and the escape of the embryo from the egg membranes, elongation continues slowly, chiefly through the rapid multiplication of cells in a sort of " growth zone" around the blastopore. And before the close of this period certain important structures are marked out: these are, the central nervous system, the notochord, and the mesoderm and ccelom.
The Formation of the Central Nervous System
Along the dorsal flattened surface of the gastrula a median strip of ectoderm cells becomes delimited from the adjacent cells and sinks slightly below the level of the general ectoderm (Fig. 8, A). This strip of cells is* the neural plate or medullary plate. It extends from near the anterior (dorsal) margin of the blastopore forward, almost to the extremity of the embryo, and backward a short distance each side of and partially surrounding the blastoporal opening. The ectoderm bordering the lateral margins of the depressed area becomes slightly elevated, forming the neural ridges or neural folds, and these gradually flow over the margins of the neural plate as this becomes still farther de pressed (Fig. 8, B). Posteriorly the neural folds extend along the sides of the blastopore, and even posteriorly and ventrally to it, completely surrounding it. The neural folds then rapidly approach medially and soon they cover over the neural plate, though separated from it by a shallow space (Fig. 8, C). These processes of depression and roofing over do not occur at once throughout the whole dorsal surface. The depression of the neural plate commences just in front of the blastopore and proceeds thence anteriorly, while the neural folds appear first somewhat further in front of the blastopore and usually, although not invariably, fuse over the neural plate first in the same region.
Fig. 8. Transverse sections through young embryos of Amphioxus, showing formation of nerve cord, notochord, and mesoderm. After Cerfontaine. A. Commencement of the growth of the superficial ectoderm (neural folds) above the neural plate (medullary plate). B. Continued growth of the ectoderm over the neural plate. Differentiation of the notochord, and first indicatibns of mesoderm and enteroccelic cavities. C. Section through middle of larva with two somites. Neural plate folding into a tube. D. Section through first pair of mesodermal somites, now completely constricted off. E. Section through middle of larva with nine pairs of somites. Neural plate folded into a tube. Notochord completely separated. In the inner cells of the somites, muscle fibrillse are forming (compare Fig. 10). ar, archenteron; c, enteroccel; ch, notochord; ec, ectoderm; en, endoderm;/, muscle fibrillae; g, gut cavity; m, unsegmented mesoderm fold; ms, mesodermal somite: nc, neuroccel; nf, neural i'old; np; neural plate; nt, neural tube.
Posteriorly the neural folds arising from the lateral and ventral margins of the blastopore, in fusing roof over this structure without closing it, so that the blastopore no longer opens directly to the outside but into the narrow space between the neural plate and the superficial ectoderm layer formed by the fused neural folds (Fig. 6, H) . In front of the point where the folds first meet, this space remains widely open upon the surface of the embryo. This opening to the cut-side is called the neuropore, and as the fusion of the neural folds extends rapidly forward the neuropore is carried along toward the anterior end (Fig. 9, A). By the time of hatching it may be found at almost any point between the middle of the embryo and the anterior margin of the first somite (see below). In this latter region the neuropore remains as a definite opening until the middle of the larval period. There is some variation in different embryos as regards the region of the first fusion of the neural folds. In some individuals, and these have usually, and erroneously, been described as the more typical, the folds meet first over the blastopore and gradually fuse thence anteriorly. The more frequent relation seems to be that just described, and it is important to recognize that this agrees with the method of closure of the neural folds in practically all of the Craniates, where they meet and fuse first in the middle or anterior to the middle of the embryo, and then fuse in each direction from that region. Amphioxus differs, however, from all other Chordates in that the margins of the neural plate do not remain connected with the neural folds for a time, and are not elevated and closed into a tube at the same time the neural folds close together: the folding of the neural plate into a tube will be described presently.
The Formation of the Notochord
The chorda develops more slowly than the nervous system and by the time of hatching has hardly more than commenced its formation. The rudiment of the notochord is a median strip of endoderm, six to nine cells wide, forming the roof of the archenteron and lying consequently just beneath the neural plate and in contact with its lower surface (Fig. 8, B). The depression of the neural plate depresses also the flattened dorsal wall of the archenteron, so that the chorda rudiment pushes down into the archenteric cavity and appears in section concavely arched. The chorda cells are those inflected at the dorsal margin of the' blastopore together with their descendants. While the gastrula or embryo is elongating the rudiment of the notochord divides posteriorly, passing a short distance around each side of the blastopore, and terminates in a growth zone similar to that concerned in the extension of the neural plate but lying just inside instead of outside the blastoporal rim (Fig. 9, B). At the time of hatching the rudiment of the chorda still remains as a flat plate of cells directly continuous, laterally and anteriorly, with the endoderm lining the remainder of the archenteron.
The Formation of Mesoderm and Enterocoels
In the gastrula the rudiments of the middle germ layer are found in a pair of longitudinal mesoderm bands of cells lying either side of the chorda in the dorso-lateral regions of the endodermal archenteric wall. Posteriorly these bands diverge and pass either side of the blastoporal opening nearly or quite to its ventral side. The extent of the mesoderm, as of the neural plate and notochord, is increased chiefly by the addition of cells from this blastoporal region (germ ring). At the close of gastrulation therefore we can distinguish two general regions of mesoderm; first, that lying either side of the anterior part of the chorda, formed from the inflected dorso-lateral margins of the blastopore and known as gastral mesoderm, and second, that formed from the lateral and ventro-lateral margins of the blastopore, known as peristomial mesoderm. The gastral mesoderm is the earlier formed and remains limited to the anterior region, while the peristomial mesoderm forms later and during a long period after gastrulation is completed, and really constitutes all the mesoderm posterior to the very limited, and anterior gastral mesoderm. At the close of gastrulation the only difference between the two kinds of mesoderm is that of time and place of origin for they are directly continuous and not visibly differentiated from one another.
The depression of the notochordal plate, consequent upon the formation of the nerve cord, results in a rather sharp folding longitudinally of the dorso-lateral mesoderm bands, and the formation from the archenteric cavity there of a pair of longitudinal grooves (Fig. 8, B). These grooves are the first indications of the enteroccelic cavities and their walls are to be spoken of as the mesoderm folds. The enteroccelic grooves extend nearly the entire length of the archenteron and, though at first shallow, rapidly deepen, particularly in the anterior region. The mesoderm folds soon become sharply differentiated from the adjoining chorda and endoderm (Fig. 8, (7), and very early their continuity becomes interrupted by the appearance of paired transverse folds dropping down from their dorsal walls. The first pair of the transverse folds appears a short distance back from the anterior ends of the mesoderm folds and soon cuts off this region, forming thus the first pair of mesodermal somites, as they are called. The somites are at first nearly spherical or cubical, box-like divisions, each containing a portion of the -original archenteric space now to be known as the enterocoelic cavity of the somite or enterocod, which in these early stages remains in direct though narrowed continuity with the archenteron; later this connection becomes entirely lost (Fig. 8, D). This transverse cutting up of the continuous mesoderm folds and their cavities into somites and enteroccels proceeds, in somewhat modified form, from the anterior region posteriorly, as the embryo elongates, and by the time of hatching two pairs of somites have been constricted off (Fig. 9, A). The gastral mesoderm is approximately limited to the region of these first two pairs of somites, all of the posterior remainder forming from peristomial mesoderm.
Fig. 9. Optical sections of young embryos of Amphioxus. After Hatschek. The cilia are omitted. A. Two-somite stage, approximately at the time of hatching, showing relation of neuropore and neurenteric canal. B. Ninesomite stage, showing origin of anterior gut diverticula. C. Fifteen-somite stage. End of the embryonic period, ap, Anterior process of first somite ; c, neurenteric canal; ch, notochord (or its rudiment, in A); eg, club-shaped gland (or its rudiment, in J5); ego, external opening of club-shaped gland; co, coelomic cavity of somite; cv, cerebral vesicle; g, gut cavity (enteron, mesenteron); firs, rudiment of first gill slit; i, intestine; Id, left anterior gut diverticulum (preoral pit in C) ; m, mouth; mes, unsegmented mesoderm; n, nerve cord (or its rudiment, in A); np, neuropore; p, pigment spot in nerve cord; rd, right anterior gut diverticulum (preoral head cavity in C); si, sz, first and second mesodermal somites; spc, splanchnoccel (body cavity).
As compared with the higher Chordates the formation of the somites in Amphioxus begins very early, for in those forms the mesoderm first separates from the endoderm as a pair of longitudinal bands which later and only in part become divided into segments or somites. The formation of the somites here, as actual enterocoelic diverticula from the archenteron, has been considered of great theoretic importance on account of its supposed primitiveness, for in all of the Craniates the mesoderm bands are at first solid and later develop a cavity never directly continuous with the archenteron. It is quite possible, however, that the formation of solid peristomial mesoderm bands is the primary arrangement and that the connection between the mesoderm cavity and the archenteron, which we shall see is limited to the somites of gastral mesoderm, is in reality a secondary condition.
The first phase of embryonic development is terminated about eight to fifteen hours after fertilization by the escape of the embryo from the egg membranes, within which it has been enclosed. During the remainder of the embryonic period it swims freely near the surface by means of the covering of long ectodermal cilia. We may conveniently summarize the characters of the embryo at the time of hatching as follows (Fig. 9, A). The embryo is in general cylindrical with a flattened dorsal surface; length nearly twice the diameter, and the diameter about equal to that of the egg. External surface completely ciliated. Blastopore completely roofed, so that the only external opening is the neuropore, opposite the anterior margin of the first somite. Mesoderm folds are formed throughout and two pah's of somites are constricted off anteriorly. Notochord delimited but not developed. Archenteron open to the outside only by way of the roofed over blastopore, through the space between neural plate and superficial ectoderm, to neuropore. All organs and parts are still formed of a single layer of epithelially arranged cells.
During the remaining twenty-eight to sixty hours of the embryonic period the chief developmental processes consist in the elaboration of the rudiments already established rather than the mapping out of new organs.
The Embryonic Period - B. From Hatching To The Formation Of The Mouth
The Central Nervous System
If not already accomplished, the neural plate is now rapidly roofed over and the neuropore is carried to the anterior margin of the first somite, where it remains throughout the larval period. Next the neural plate is converted into a tubular structure, the neural tube, through the depression of its median region and the accompanying rolling up and together of its margins (Fig. 8). As in other Chordates this process begins in the region of the first somite and extends thence posteriorly and anteriorly. The original space between the neural plate and the covering ectoderm becomes the cavity of the neural tube or neurocoel (Fig. 9, B). The cilia of these originally superficial cells now line the neurocoel and continue to beat toward the posterior end of the tube. In the region of the blastopore the neural plate remains in connection with the continuous ectoderm and endoderm of that region, so that the neurocoel does not close posteriorly, but leads directly into the opening of the blastopore and so to the archenteron. This passage from the neurocoel to the archenteric cavity by way of the blastoporal opening, is the neurenteric canal. This canal remains open throughout the embryonic period, until after the mouth opening is formed, so that this, in connection with the neurocoel and neuropore, forms the Only path by which the archenteron is in connection with the exterior (Fig. 9, A, B, C). The beating of the cilia lining the neurocoel probably keeps up an interchange of fluids between enteron and the outside.
Opposite the anterior margin of the first somite the medullary tube is somewhat enlarged (Fig. 9, (7), while opposite its posterior margin, and, indeed, throughout the remainder of the tube, it becomes somewhat narrowed by the elongation of that part of the embryo, and a so-called brain region is thus marked out. About the close of this period, pigment spots begin to appear in the neural tube opposite the fifth pair of somites (Fig. 9, C) : these are the first indications of the development of sense organs and are doubtless photo-receptors. The large cranial pigment spot in the anterior wall of the brain appears about the close of the embryonic period.
The establishment of the chorda from the plate of endoderm cells lying between the mesoderm folds, commences in. the region between the first and second somites (Fig. 8). Here the plate becomes arched, its lateral halves folding together ventrally. The folding extends posteriorly and anteriorly from this region, and soon the folded plate is formed into a solid strand lying below the neural tube, between the mesodermal somites (Fig. 8, E). Posteriorly the chorda extends to the anterior wall of the neurenteric canal, and anteriorly it slowly forms to the very extremity of the embryo, and therefore in advance of the somites and neural tube (Fig. 9, B, C). The extension of the chorda in front of the brain is a peculiarity of Amphioxus, for in all other Chordates it forms only to the region of the mid-brain. The chorda cells from the two sides grow across the mid-line, interlocking and later extending from each side completely across the entire chorda. By the time nine or ten somites are formed the notochord becomes completely cut off from the endoderm layer, and the two margins of the endoderm which were originally along the outer margins of the mesoderm folds, come together in the mid-line beneath the notochord, enclosing what remains of the archenteron, as the definitive gut cavity or enteron (mesenteron) (Fig. 8, E). Finally vacuoles appear in the notochord and, increasing in size, lead to the obliteration of the cellular structure. The nuclei are displaced dorsally and ventrally and a typical "notochordal tissue" is formed before the close of the embryonic period (Fig. 9, C).
The Mesodermal Somites and Coelom
After hatching, mesodermal somites posterior to the two then present continue to form successively in the elongating mesoderm folds. The cavities of the first two pairs do not become completely separated from the archenteron until six to eight pairs have been formed and constricted off, and their enteroccels remain as definite spaces throughout their development. In the somites just posterior to the first two pairs the enteroccels close for a time just after the somites are formed, and in the more posterior ones, even before the mesoderm is quite cut off from the endoderm. In these somites cavities reappear later, and the enterocoels may be said to be virtually if not actually present throughout, for the cells composing their walls remain definitely arranged about a central point in the regions of the enteroccels (Fig. 10). Still farther posteriorly the mesoderm folds develop as solid masses without any cavities whatever at any time during their formation, but the condition in the anterior region demonstrates that Amphioxus is a true enteroccelomate organism. By the time fourteen pairs of somites have been formed the mesoderm folds cut off completely from the archenteric wall, and the remainder of the mesoderm is formed directly from the proliferating cell region around the neurenteric canal (blastopore), without ever having been included as a part of the endodermal wall of the archenteron. At the close of the embryonic period (Fig. 9, C) fourteen or fifteen pairs of somites have been formed, and posterior to these remain short undivided regions of mesoderm from which additional somites will be formed during the larval period. These somites are of course not all in the same stage of development, but passing along the series in the anterior direction they represent successively more advanced conditions. Advance is indicated by increase in the size of the cavity of the somite, thinning of the wall, and the ventral and somewhat posterior extension of the somite between the wall of the gut and the superficial ectoderm (Fig. 8, E). By the end of the embryonic period the more anterior pairs are already bent into the < -form characteristic of the adult. The first pair of somites send forward unusual extensions from the antero-dorsal region along each side of the notochord (Fig. 9, B) : it is possible ' thou hardly likely that these larva with six pairs of indicate the former presence of an additional pair of somites in this region
mesodermal somites, at the level of the notochord : it seems more likely that the condition is
After Cer- fontaine. a, Archenteron;
e, enteroccei;w, notochord; the result of the anterior prolongation of
d '/If r s l a ite S . SiXth meS - the notochord beyond the proper region
of the first somite. The walls of these
extensions later go through the same developmental history as those of a typical somite.
By the time five or six pairs of somites are formed they begin to show that bilateral alternation which is so characteristic of the adult (Fig. 10). Only the first and the upper part of the second pairs of somites lie exactly opposite. In the other pairs the left member comes to lie in advance of the right, and farther posteriorly the segments of the two sides exactly alternate. It is also noticeable that the left side develops slightly in advance of the right, e.g., when there are eight somites on the left there are but seven on the right.
The enlargement and ventral elongation of the somites mentioned above is accompanied by a differentiation of their walls. That part of the somite lying just beneath and in contact with the ectoderm becomes thinner and forms what is known as the parietal wall of the somite, or the somatic mesoderm (Fig. 8, E) ; the region in contact with the wall of the gut or enteron also becomes thinner and forms the visceral wall or splanchnic mesoderm: while the more restricted portion bordering the chorda thickens by the horizontal flattening and antero-posterior elongation of the cells through the extent of the somite. This last region of the somite is called the myotome, while the somatic and splanchnic regions together are termed the lateral plate. In the myotome the cells begin, as early as the nine-somite stage, to differentiate as muscle cells (Fig. 8, E) forming a muscular epithelium. At the close of the embryonic period the more anterior my o tomes are capable of muscular contractions.
The thinning of the parietal and visceral walls of the somite and their downward extension, considerably enlarge the enclosed cavity and carry it down around the sides of the enteron. The cavities of the somites now become recognizable as the beginnings of the coelomic space. The enlargement of the myotome partially obliterates the cavity of the somite in the dorsal region, the small cavity remaining being termed the myoccel. The larger cavity of the lateral plate is then distinguished from the myocoel by the term splanchnoccel. Before the close of the embryonic period the ventral walls of the more anterior somites push completely around the enteron and meet in the midventral region, separating the enteron from contact with the ectoderm. Presently the median walls of the splanchnoccels for the most part disappear, and the splanchnoccels of each pair of somites fuse more or less completely. Remnants of these median partitions below the enteron appear to remain as the rudiments of the subintestinal vein and branchial artery. Finally the somite becomes divided by a horizontal partition between the lateral plate and myotome, and the corresponding cavities are completely separated. By the close of the embryonic period the anterior and posterior walls of the somites in the region of the splanchnoccels also break through, leaving these cavities continuous antero-posteriorly as well as transversely. The unsegmented cavity thus formed is the coelomic cavity of the embryo and larva which is seen to be a true synccelom (Fig. 19, A).
Fig. 11. Sections through young Amphioxus embryos showing the origin of the anterior gut diverticula. After Hatschek. The cilia are omitted. A. Frontal section through embryo with nine pairs of somites. (See Fig. 9, 5). The dotted line marks the course of the gut wall ventral to the level of the section. B. Optical sagittal section through anterior end of embryo with thirteen pairs of somites, showing position of right anterior gut diverticulum. C. Same in ventral view, c, coelomic cavity of somite; ch, notochord; csg, rudiment of club-shaped gland; d, rudiment of anterior gut diverticula; ec, ectoderm; en, endoderm; g, gut cavity (enteron, mesenteron); gsl, rudiment of first gill slit; Id, left anterior gut diverticulum; n, nerve cord; np, neuropore; rd, right anterior gut diverticulum; si, $2, sg, first, second, and ninth mesodermal somites.
There remain to be described, in connection with the development of the mesoderm, a pair of outgrowths from the anterior end of the enteron in front of the first pair of mesodermal somites. These cavities are known as the anterior gut diverticula and have been commonly regarded as endodermal derivatives of peculiar character, but although their exact homologies remain rather obscure it seems clear that they are essentially a pair of mesodermal somites which develop late, on account of the delayed forward extension of the whole anterior tip of the body. The anterior gut diverticula first appear in the embryo with seven pairs of somites, as a pair of narrow anterodorsal evaginations from an expanded anterior region of the enteron (Figs. 9, B; 11, A, B). These push forward much like the somites, and quickly pinch off from the enteron as a pair of closed sacs (Fig. 11, C).
Although symmetrical in their origin they differ greatly in their subsequent development. In the embryo with ten pairs of somites the right diverticulum begins to grow forward and to enlarge while its wall becomes very thin. Soon it extends across the mid-line and finally, as a median structure, it occupies the whole of the space below the chorda and from the enteron forward to the tip of the embryo. It remains wholly in front of the first somite and forms what is known as the preoral cavity or head cavity (Fig. 9, C). The left anterior gut diverticulum differs widely from its antimere. It remains quite small and unmodified throughout the embryonic period, shortly after which it acquires an opening to the surface in the left side of the head below the level of the chorda, and is then known as the preoral pit (Fig. 9, C). Its history is quite complicated and will be considered later.
The Enteron and its Appendages
After the wall of the archenteric cavity has lost successively the mesoblast folds, notochord, and anterior gut diverticula, the endodermal tube remains as the wall of the enteron or mesenteron. This gives rise to the epithelial lining of the alimentary canal and related structures. After the time of hatching the enteron elongates rapidly and, except at its anterior extremity, narrows very markedly, differentiating in this way the posterior stomach-intestine from the wide anterior pharyngeal region opposite the first two somites (Fig. 9, C). Throughout the embryonic period, as described above, the only opening to the exterior is by way of the neurenteric canal, neuroccel, and neuropore, the essential relations among which do not change during this period. We may describe briefly the chief structures arising in connection with the enteron during the embryonic period.
A. THE CLUB-SHAPED GLAND AND ENDOSTYLE
In the floor of the enteron, just back of the anterior gut diverticula, and therefore opposite the anterior margin of the first somite, a transverse groove appears about the time nine or ten somites are present (Figs. 9, B; 11). During the latter part of the embryonic period this groove constricts off as a narrow tube, separate from and below the gut, and acquires an opening at its left end to the exterior in the region below the preoral pit, while the right end dilates into a conspicuous sac, closed at this time (Fig. 9, C).
Lying just in front of the grooved rudiment of the club-shaped gland, is a strip of high ciliated cells which seems to be the first indication of the endostyle. The later development of the gland and endostyle is described in connection with the history of the larval period.
B. THE MOUTH
The mouth, first gill slit, and anus develop almost simultaneously, toward the close of the embryonic period. At the age of fourteen somites a large patch of ectoderm on the left side of the head, along the margin of the first somite, thickens considerably and with this thickened area the adjacent wall of the enteron fuses. This fused region then becomes perforated by a small opening (Fig. 9, C), which rapidly enlarges and becomes the relatively enormous mouth opening bordered with elongated cilia (Fig. 12).
C. THE FIRST GILL SLIT
At the same time the mouth is forming, the endoderm pushes out ventrally, opposite the second somite, and fuses with another thickened patch of ectoderm (Fig. 9, C). Perforation follows and forms the first gill slit which rapidly enlarges and moves up on the right side of the head, nearly opposite the mouth (Fig. 12, A). It remains smaller than the mouth but is similarly bordered with long cilia.
Fig. 12. Anterior ends of Amphioxus larvae, in optical section. A. One gill slit stage, from right side. After Hatschek. B. Four gill slit stage, from left side. After Lankester and Willey (mouth added). 6, Brain region; c, coelpm (preoral head cavity); ch, notochord; csg, club-shaped gland; d, dorsal portion of preoral pit (later forming the groove of Hatschek and Hatschek's nephridium) ; e, rudiment of endostyle; gsl, first gill slit; Hn, Hatschek's nephridium; i, intestine; ra, mouth; n, nerve cord; np, neuropore; o, external opening of clubshaped gland; p, pigment; ro, Raderorgan; v, cerebral vesicle.
D. THE ANUS
Very shortly after the mouth and first gill slit are formed the wall of the narrow enteron, immediately below the neurenteric canal, fuses with the ectoderm and the region is perforated as the anus. At first this lies in the mid-line but later is displaced toward the left, by the development of the provisional caudal fin. Upon the formation of the anus the intestine is separated from the neurenteric canal and this structure closes and graduually moves posteriorly, away from the anal region, as the tail region grows out; but for some time the tip of the nerve cord remains curved around toward the region of its original connection with the enteron.
About the time the mouth and anus open, the alimentary tract becomes ciliated and a small diverticulum forms in the anterior part of the stomach-intestine; this is the rudiment of the caecum or liver.
At the close of the embryonic period, which is arbitrarily marked by the opening of the mouth, the embryo is about 1.0 mm. long and very slender (Fig. 9, C). Probably its actual bulk is little greater than that of the egg. The embryo is almost perfectly transparent, and swims about with the elongated ectodermal cilia, accompanied by irregular muscular contractions when strongly stimulated. The anterior end is dilated and prolonged forward into a rostrum, containing the preoral head cavity and supported by the elongated notochord. The external openings are, the mouth and first gill slit on opposite sides of the head, above these the small median neuropore, and near the posterior end the small ventral anus. Posteriorly the tail is expanded into a provisional caudal fin formed of elongated ectodermal cells. There are about fifteen pairs of somites in every stage of development, from the completely undifferentiated condition of the posterior pairs of the series, to the quite completely differentiated anterior pairs. Anteriorly the nervous system shows the rudiment of the brain; posteriorly it is no longer connected with the enteron on account of the closure of the neurenteric canal. The notochord is completely established. In addition to the mouth and first gill slit the enteron has formed the anterior gut diverticula and clubshaped gland, all of which have become separate structures and commenced their differentiation. In spite of its rather complex structure all parts of the embryo are still of epithelial character, though the epithelia now show some degree of elementary cellular differentiation.
The Larval Period
The duration of this period is roughly about three months, during which the larva is free swimming but descends to deep water. Development is very slow and consists largely in the elaboration and modification or multiplication of structures indicated at its beginning. Although up to this time development has been simple and typical (primitive) in general, now the anterior portion of the larva becomes highly modified through the development of characters not found in the Craniata. These are, chiefly, the extensive asymmetry of the pharyngeal and associated structures, and the development of the atrium or peribranchial cavity both to be regarded as special adaptations to the habits of the larva. The close of the larval period is marked by an extensive metamorphosis which consists largely in a " symmetrization" of the anterior end. At the close of this period the larva is said to be at the critical stage. 7 '
The Central Nervous System
Very soon after hatching the walls of the spinal cord thicken, constricting the neuroccel, and becoming differentiated into three regions. These are (a), a thin epithelial layer the ependyma, lining the neurocoel, (&), dorso-lateral and lateral columns of nerve-cell bodies (gray substance] connecting respectively with the dorsal and ventral spinal nerves, and (c) ventral columns composed of nerve fibers (white substance).
In the brain region the walls become comparatively thin and the cavity dilates considerably (Fig. 13). At first the brain lies beneath the epidermis, and around the neuropore its wall is directly continuous with the superficial ectoderm. When the dorsal fin appears in the mid-line the neuropore is pushed to one side, usually the left. Gradually the brain sinks away from the epidermis, drawing down the neuropore into a funnel-shaped depression, and at the same time drawing out the antero-dorsal wall of the brain into a conical elevation, the two remaining connected by a very small tube. Not until after metamorphosis does this opening become entirely closed and the neuropore then remains as a ciliated funnel known as the olfactory pit, which retains an innervation from the elevated region of the brain and becomes sensory in character, the assumption being that it functions as an olfactory organ. This antero-dorsal outgrowth of the brain may correspond with the lobus olfactorius impar which marks the morphologically anterior end of the brain of the Craniates. Just in front of and below this is the large cranial pigment spot (Fig. 13) in the general region from which the olfactory lobes grow out in higher forms. Postero-dorsally the brain wall is thickened by an accumulation of ganglion cells, and in its posterior part two important structures appear. Just in front of the opening of the cavity of the cord the ependymal cells elongate forming a small median pad which is regarded as the infundibulum (Fig. 13). Posterior to the cerebral vesicle proper is an extensive dorsal diverticulum of the neuroccel : this has been compared with the IV ventricle of Craniates.
Fig. 13. Median, sagittal sections through the brain of Amphioxus. After Boeke. A. Of a larva with seven primary gill slits. B. Diagram of a section through the brain of a larva of 2.25 mm., with five pairs of primary gill slits, X 933. C. Same of a young specimen of 10 mm. X 233; c, Central canal of cord; i, infundibulum; n, neuropore; p, cerebral pigment; op, olfactory pit; v, cerebral vesicle; vd, postero-dorsal extension of cerebral vesicle.
The simple character of the brain of Amphioxus is obviously correlated with the general lack of special sense organs, particularly with the absence of the important optic and auditory organs, and the feebly developed olfactory apparatus.
The Gill Slits
We may describe first the general morphological arrangements of the developing gill slits: the development of the first gill slit has already been mentioned.
Although finally symmetrical and paired organs the gill slits of the right and left sides develop independently, those of the left side first. These appear successively in the midventral line, posterior to the first gill slit, and as they form they shift upward, on the right side of the pharynx (Fig. 14). These are called the primary gill slits, and when twelve to fifteen, typically fourteen, have appeared, their formation ceases. At this time the more anterior slits are large ovoid openings lying well up on the right side; posteriorly they diminish in size and lie toward the mid-line. In all these early stages they are metameric and correspond with the somites, a correspondence which is entirely lost later on. When the full number of fourteen primary gill slits has been formed, the secondary gill slits, or those of the proper right side, appear, also on the right side of the pharynx and dorsal to the primary series. Their first indication is a longitudinal ridge, in which appear six thickenings alternating with the primary gill slits in the region between the third and ninth of these (Fig: 14,* B).
The relations of these parts of the pharynx will become clear if it be imagined that the formative centers located in the most anterior region of the pharynx have been twisted out of their normal positions, so that the morphological mid-ventral line becomes displaced to the topographical right side, and structures morphologically of the right and left sides appear, respectively, dorsally and ventrally on the right side alone.
Fig. 14. Anterior ends of Amphioxus larvae. A. Larva with eleven primary gill slits, from left side. After Goodrich. B. Larva with eleven primary gill slits, from right side showing metapleural fold and secondary gill slit rudiments. After Willey. C. Larva with fourteen primary gill slits, from left side. After Lankester and Willey. ao, Ventral aorta (branchial artery); at, atrial cavity; ch, notochord; en, cranial nerve; csg, club-shaped gland; dn, dorsal spinal nerve; e, end ostyle;gH, groove of Hatschek; gsl, gsll, gs!4, first, eleventh, and fourteenth primary gill slits; H0s, rudiments of secondary gill slits; i, internal opening of club-shaped gland; Im, lower margin of mouth; m, mouth; my, myotomes; n, nerve cord; ne, nephridium; o, external opening of club-shaped gland; p, pigment in nerve cord; r, renal cells in floor of atrial cavity; rm, edge of right metapleural fold; ro, Raderorgan.
The rudiments of the secondary gill slits enlarge and become perforated, and then an additional perforation appears at each end of the series making eight in all. As these enlarge, chiefly in the ventral direction, the primary gill slits below them are moved down to the ventral side, and then over on the left side of the pharynx to their proper position (Figs. 14, C; 15). As a result of this migration the dorsal and ventral margins of the primary gill slits are reversed, and it is evident that the morphological ventral margins of these actually appeared first to lie dorsally. As this migration is going on, first the secondary, then the primary gill slits become divided by a downward extension of a tongue-like process from the dorsal wall of the slit which finally reaches the ventral side, divides the originally simple opening into two, and forms the tongue bar of the completed structure of the adult (Fig. 15). These tongue bars appear in all the secondary slits except the first, and in all of the primary slits except the first two and a variable number of the last. As the primary gill slits approach their final position the most anterior disappears completely (Fig. 15) and soon after, the last five also disappear, reducing the number in the primary series to eight, the same as the number originally appearing in the secondary series. This is the arrangement of the gill slits at the critical stage, the symmetrization of these structures being one of the important phases of metamorphosis. After the reduction of the gill slits to eight symmetrical pairs, arranged metamerically, additional pairs form slowly, posterior to the primary and secondary. These are the tertiary gill slits. They are not metameric in their appearance and they displace anteriorly the primary and secondary slits, so that the whole gill slit series loses its metameric arrangement.
Regarding the actual details of the formation of the gill slits little is known except in certain of the secondary series, or those of the right side proper. In these, and presumably in most of the primary series also, true gill pouches seem to be formed first, purely of endodermal cells. These are drawn out ventro-laterally into small tubes closed by a very thin superficial layer of transparent ectoderm cells. The tongue bar grows down like a stalactite from the roof of the gill pouch, and just before it reaches the ventral side of the pouch the ectoderm is perforated opposite the tongue bar. But by this time the atrial cavity has formed in this region, as will be described later, so that the gill slits never open directly to the
Fig. 15. Anterior ends of Amphioxus larvae showing the migration of the mouth, formation of tongue bars, reduction in primary gill slits, etc. After Willey. A. Larva with fourteen primary gill slits nearly in their definitive position on the left side. Oral hood in process of formation. B. Larva with nine primary, and eight secondary gill slits. Mouth nearly in its definitive anterior position and oral hood nearly completed. This larva has nearly reached the "critical stage." at, Atrial cavity; 6, buccal (oral) cirri; be, buccal cavity (cavity of oral hood); c, coelom; cv, cerebral vesicle; df, dorsal fin chambers; dgsl, dgs8, first and eighth definitive gill slits (second and ninth of the primary series) ; dph, dorsal wall of pharynx (region of epipharyngeal groove); e, endostyle; gsl, gs9, gsl4, first, ninth, and fourteenth primary gill slits; h, hepatic caecum; i, intestine; Im, lower margin of left metapleural fold; ra, mouth; nH, Hatschek's nephridium; pb, peripharyngeal band; r, renal papilla in floor of atrial cavity; ro, Raderorgan. tb, tongue bar; uh, upper margin of oral hood; v, velum.
outside; the more anterior primary gill slits apparently form exceptions to the usual arrangement in opening directly for a time (Fig. 17). As the external openings of the pouches enlarge, the tongue bars move to the surface and if it is true that they are completely covered with endoderm, the atrial cavity on which they border must therefore to this extent be lined with endoderm (Legros).
The Club-shaped Gland
The club-shaped gland lies far forward in the pharyngeal region, and does not share in the shifting of the gill slits and other derivatives of the pharynx with which it is at this time connected only at a -single point. During the early larval period its dilated right end narrows and acquires an opening into the pharynx (Fig. 14, A). This " gland" now is in the form of a narrow tube, opening at the right end into the pharynx and at the left to the outside. It is of doubtful significance, both functional and morphological; it has been compared with a gill slit and may represent the antimere of the first primary, which is otherwise entirely lacking. It soon atrophies like this gill slit and disappears without leaving any trace (Figs. 14, 15).
This appeared as a transverse, ciliated thickening, in the floor of the pharynx in front of the club-shaped gland. As a thickening of the pharyngeal wall the endostyle is involved in the general torsion of this region and passes over to the right side (Fig. 12). There its middle region draws back above the primary gill slits, i.e., in the morphological mid-ventral line, converting the transverse >and m t o a > -shaped structure with the apex directed posteriorly (Fig. 14). It continues to extend backward, between the rows of primary and secondary gill slits, while its limbs draw together coming into contact and forming a double strip of cells, which in part become differentiated as glandular cells. The endostyle is finally carried back into a median ventral position by the time of the critical stage (Fig. 15, B).
From the anterior end of the endostyle two narrow ciliated bands the rudiments of the peripharyngeal bands (Fig. 15, B), pass around the pharyngeal wall to the dorsal side, and there extend backward as a pair of ciliated bands which later fuse into a single median structure the epipharyngeal or hyperbranchial groove.
The Mouth and Associated Structures
This opening on the left side of the head continues to enlarge during the early part of the larval period, and finally extends from a point considerably in front of the first gill slit to the region of the fifth gill slit (Fig. 14). When the secondary slits begin to develop and the pharynx rotates back to a normal position, the mouth gradually shifts its position in a horizontal plane, moving around to the anterior end: it finally reaches an anterior median position at about the critical stage (Fig. 15). As the mouth migrates its margin extends inward as the velum, like that of Ammocoetes, finally reducing the mouth opening to a small aperture. Later this becomes fringed with outgrowths from the free margin of the velum the velar tentacles (Figs. 15, 16). From the base of the velum the oral cirri grow out. These are first indicated about the time the secondary gill slits appear, and by the time of the critical stage they are well developed and their cartilaginous supports have become differentiated. At the same time the velum begins to form, folds of the integument, first above and then below the mouth, grow out, the latter passing forward toward the region of the snout. These folds are the rudiments of the oral hood, and as the mouth passes forward they enlarge and shift so as to form the left and right sides respectively of the oral hood. The right fold becomes continuous with the extremity of the dorsal fin as this turns the anterior end of the larva (Figs. 15, 16).
The Preoral Pit and Its Derivatives
The preoral pit, the formation of which from the left anterior gut diverticulum has been described, was left as a small sac (Fig. 9, C), opening upon the surface of the left side of the head, in front of the mouth. When the rudimentary folds of the oral hood appear, the dorsal fold develops just above the opening of the preoral pit, which thereafter remains as a structure chiefly related with the buccal cavity. Early in the larval period the preoral pit enlarges and then divides into a dorsal and a ventral portion. The ventral or stomodceal portion, as it is called, enlarges very considerably, moves to the surface, and spreads out over what is by that time the partly formed oral hood. It forms here the irregularly lobed ciliated patch of cells called the "Rdderorgan" (Figs. 12, 15, 16). The dorsal portion of the preoral pit again divides into two parts, one lying anteriorly and toward the right, the other posteriorly and toward the left. The first of these remains on the right side, in the roof of the buccal cavity, and forms there the glandular "groove of Hatschek" leading to the oral aperture (Figs. 14, 16). The second part remains toward the left side and draws out into a long fine tube, extending back to the pharynx, into which, by the time the larva has six gill slits, it acquires an opening above the mouth, and loses its external opening (Figs. 12, 15, 16). This part is sometimes called " Hatschek' s nephridium": its final structure is that of a greatly enlarged nephridium (Fig. 16). There seems to be little doubt that the dorsal division of the preoral pit is homologous with the hypophysis of the Craniates, and with the neural gland and its duct of the Tunicates. Some would restrict this homology to the anterior portion (groove of Hatschek) alone, others to the posterior part alone (Hatschek's nephridium).
Fig. 16. Ventral view of anterior end of adult Amphioxus. The buccal cavity has been cut open along the mid- ventral line and spread out. After Goodrich, b, Buccal skeleton; ch, notochord; cr, cirri; gH, groove of Hatschek; h, oral hood, Hn, Hatschek's nephridium; m\ t mz, first and third myotomes; n, nerve coming from left side; o, pharyngeal opening of Hatschek's nephridium. TO, Raderorgan; s, solenocytes; v, velum; vt, velar tentacles.
The ventral blood-vessel which was formed from cells of somewhat uncertain origin between the lower ends of the lateral plates as they approached the ventral side, is formed throughout the pharyngeal region during the larval period, lying between the primary and secondary gill slits (Fig 14, B). It first begins to show contractions about the time the first gill slit is perforated.
The presence of the atrium is one of the two chief characteristics wherein Amphioxus is essentially unlike other Chordates, for a similar structure is found only among the Urochorda. It is to be recognized in Amphioxus as a special adaptation to the burrowing habit, serving to protect the delicate and complicated pharynx. The enclosure of the branchial region by the atrium occurs early during the larval period. About the time six or seven primary gill slits are present, a pair of low folds appears in the middle of the body region, along the ventral surface. These are the so called metapleural folds, at first merely thickenings of the ectoderm, but later hollow ridges of considerable size; their primary cavities are to be regarded as coelomic in origin.
The metapleural folds lie close together and gradually extend forward, the right fold proceeding rapidly in advance of the left and passing far up on the right side of the pharynx, above the primary gill slits, which it reaches about the time ten are formed (Fig. 17, A ) . Later the left fold reaches the gill-slit region, diverges widely from the right fold, and
FIG. 17. Diagrams of three larvae of
passes tO the Opposite Side Ot Amphioxus, viewed ventrally, showing
the Secondary gill slits, lying the , Rations of the metapleural folds
J . and the method of their closure. After
at first in the topographical Lankester and Willey. A. Folds still
i / i i i completely separate. B. Folds fused,
Ventral region (morphological and P atria l cavity thereby established!
left side) (FigS. 14 B; 17). from atriopore to posterior end of
.. ' , , , , ! , pharynx. C. Folds fused throughout,
The metapleural folds enlarge except in the region of the first two
and new cavities of lymphatic
character appear within them, left metapleural fold; m, mouth; rm, , . , T . , right metapleural fold.
which encroach upon the
original coelomic space and lead to their obliteration. From the inner face of each metapleural fold a horizontal ridge or shelf appears, just below the level of the body wall. These are the subatrial ridges (Fig. 18, A); they grow toward one another and fuse across the mid-line, enclosing between themselves and the ventral body wall a small space lined with ectoderm, which is the beginning of the atrial cavity (Fig. 18, B) . The formation and fusion of the subatrial ridges occur first posterior to the gill-slit region, and proceed thence forward, and by the time the secondary gill-slit series is completed the branchial region is completely covered. Anteriorly the subatrial ridges merge with the body wall closing the cavity at this end, but posteriorly the atrial cavity remains open to the outside as the atriopore (Fig. 17). The cavity, at first very small, gradually enlarges (Fig. 18, C) pushing 'upward each side of the pharynx, ultimately surrounding it on all sides except the dorsal, where alone the pharynx remains connected with the body wall. The space occupied by the atrium was previously the region of the general ccelom. The development of the atrium therefore reduces the ccelom to a number of greatly reduced and somewhat scattered spaces. The chief of these are (a) the pair of dorsal coelomic canals along the dorso-lateral regions of the pharynx, from which the atrium remains separated by the suspensory folds or denticulate ligament; (b) the endostylar ccelom in the ventral region below the endostyle; and (c) the branchial coelomic canals connecting the two preceding regions by way of very small canals in the primary branchial arches. Posterior to the atriopore the ccelom remains for a time in its normal relation, but later the atrial cavity extends into this region on the right side nearly to the anus, leaving the ccelom widely open only on the left side of the intestine.
Fig. 18. Diagrams of transverse sections through Amphioxus larvae, showing the formation of the atrial cavity. After Lankester and Willey. A. Section through larva with eleven or twelve primary gill slits, showing subatrial ridges on metapleural folds. B. Section through slightly older larva (Fig. 17, 5), showing first fusion of subatrial ridges to form the rudiment of the atrial cavity. C. Section through advanced larva showing enlargement of atrial cavity and the method of its encroachment upon the coelom. a, Atrial cavity; ao, dorsal aorta; c, ccelom (splanchnoccel) ; ch, notochord;/, dorsal fin cavity; i, intestine; ra, metapleural fold containing coelomic space; mt, myotome; my, myoccel; n, nerve cord; sa, subatrial ridges; sc, sclerotome; v, subintestinal vein.
The atrial cavity is formed in the branchial region just before the primary gill slits (except perhaps the more anterior) are perforated, so that from the first these open into the atrial cavity and never directly to the outside. From its mode of formation the denticulate ligament or- suspensory fold is seen to be a membrane equivalent to the body wall, and therefore covered on its atrial surface with ectoderm. Of course the remainder of the atrial cavity is lined with ectoderm, except, as we have seen, that the endodermally covered tongue bars line a small portion of it.
The nature and extent of the larval asymmetry of Amphioxus represent the most important difference between the development of this form and other Chorda tes. The asymmetry of such structures as the neuropore and anus, which have merely been displaced slightly by a later medially developing structure (in these cases the median fins), is not unusual or of particular importance. Nor is the alternation on opposite sides of the body of the mesodermal somites and structures primarily related to them, such as the spinal nerves and gonads. The asymmetry which is unique in Amphioxus concerns the formation of the mouth, gill slits, and other organs connected with the anterior end of the pharynx and with the oral hood. One of the remarkable facts in this connection is that the asymmetry is not indicated in early development and is wholly corrected later in larval life, so that it is wholly limited to a comparatively brief period during larval development. It is obviously a purely secondary or ccenogenetic character, and must be explained as in some way adaptive either to present or past conditions of development. We may mention but a single explanatory hypothesis that of Willey.
The starting point of this hypothesis is the assumption of a primitively median dorsal position of the mouth, for which there is some evidence. The second consideration is the extension of the notochord forward to the tip of the snout, far in advance of the central nervous system and enteron. So far as is known, Amphioxus is the only form in which the chorda extends in this way in all Craniates it stops in the region of the midbrain. This anterior extension of the chorda, which is obviously an adaptation to the burrowing habit assumed at the close of the larval period, occurs very early in development and necessarily prevents the mouth from appearing in the middorsal region. The net result of this is the shifting of the formative centers of the mouth, and associated structures of the oral hood, to one side, actually the left, and the correlated shifting of the formative centers of other pharyngeal organs. The whole rotation occurs in a counterclockwise direction, throwing structures of the proper left side over to the median line, or even to the right side, structures of the median line up on the right side, and structures of the right side proper, still higher up on the same side. The region of extreme torsion is around the mouth so that posteriorly the amount of displacement becomes less and less and in the posterior part of the pharynx the parts appear and remain in a normal position.
Such an asymmetry is in itself obviously not advantageous (adaptive) and during later development it becomes corrected by a compensatory rotary growth of the pharyngeal structures in the opposite direction, while the mouth and associated structures (Raderorgan, groove of Hatschek, etc.) move chiefly in the anterior direction.
This explanation of the origin and correction of the larval asymmetry may be accepted though its necessarily hypothetical character should be clearly recognized.
The Mesodermal Somites
At the commencement of the larval period there were fourteen or fifteen pairs of somites formed, and we have seen how each of these divides into a dorsal myotome, with its small myoccel and thickened muscular wall, and a ventral lateral plate with thin walls and large splanchnoccel, and further how the median, anterior, and posterior faces of the lateral plates break through, forming a continuous ccelom, walled externally by somatic and internally by splanchnic mesoderm (Fig. 19, A). By the time three primary gill slits have been formed the number of somites has more than doubled, and the full adult number (sixty-one in the common species of Amphioxus, Branchiostoma lanceolatum) is acquired by the time the series of primary gill slits is completed. All of these additional somites are formed from the rapidly elongating posterior region of unsegmented mesoderm, which has been carried backward past the neurenteric region by the outgrowth of the tail.
During the later development of the somites their myotomal region increases largely in vertical extent, and the narrowed myoccel sends downward a thin-walled extension below the myotome proper. This divides into two folds or outgrowths: one of these folds extends inward and upward, between the myotome or protovertebra as 'it is sometimes called, and the axial structures notochord and nerve cord, to the region, of the dorsal fin (Fig. 19, B). This is the sclerotome; its inner or axial layer is called the skeletogenous layer and gives rise to dense fibrous connective tissue forming the notochordal and neural sheaths; its outer or myotomal layer remains thin and forms the fascia covering the muscular protovertebra : the cavity between the two layers is soon obliterated by an accumulation of connective tissue. There is no fascia on the outer surface of the protovertebra or myotome such as is found in Craniates. The second or dermal fold (dermatome) of the myocoelic wall, extends downward and outward between the somatic layer of the lateral plate and the ectoderm (Fig. 19, B), where its inner and outer layers come into apposition and form the cutis layer of the integument. The blood cells and many of the important blood-vessels appear to be derived from the skeletogenous layer of the sclerotome.
Fig. 19. Diagrams of transverse sections through Amphioxus larvse. A. Through the body region of a larva with five gill slits, showing separation of myocoel and splanchnocoel (coelom). B. Through the region between atriopore and anus of young individual, shortly after metamorphosis, showing relations of sclerotome. After Hatschek. o, Dorsal aorta; c, ccelom (splanchnocoel); ch, notochord; d, dermatome; df, dorsal fin cavity; e, epidermis; i, intestine; me, myocoel; mp, muscle plate (myotome); n, nerve cord; s, sclerotome; , subintestinal vein; vf, ventral fin cavity.
In the adult the lateral plate forms only the lining membrane of the ccelom, its somatic layer, the body wall, its splanchnic layer, chiefly the wall of the gut outside the endodermal epithelium. The ventral musculature develops later and appears to be derived from the parietal (somatic) layer of the lateral plate.
Among the Craniates the sclerotome is formed from a mass of mesenchyme cells, a tissue which is noticeably lacking in Amphioxus. It remains an open question whether in this organism the formation of the sclerotome by a folding of the myocoelic wall, and the correlated paucity of mesenchyme cells, is a primitive or secondary character.
The development of the nephridia is very incompletely known (Legros, Goodrich). They appear in connection with each gill pouch, as these are first marked out (Fig. 14, A), as groups of mesoblast cells along the outer faces of the endodermal gill pouches, lying at the base of a narrow coelomic groove in this region. These mesoblast cells soon form a definite blind tube extending posteriorly and ventrally from the coelom toward the gill pouch. The mesodermal cells composing the small nephridial rudiments become differentiated, for the most part, as clear cubic cells, but a group of solenocytes is distinguished as a small collection of elongated granular cells in the neighborhood of the coelomic opening of the tubule. Then the lower end of the nephridial tube fuses with the dorsal wall of the gill pouch, just posterior to the region of the developing tongue bar. Finally it opens into the gill pouch, about the time this becomes perforated externally, and at the same time its coelomic opening is closed and the solenocytes elongate into the coelom (Fig. 20).
The first formed nephridia apparently become functional when four or five slits are formed. When the anterior and posterior primary gill slits atrophy the associated nephridia seem also to atrophy and later to be re-formed in connection with the tertiary gill slits although actual details in the process are unknown.
The Larva at the Critical Stage
We may now summarize briefly the most important characters of the larva at the close of this period, in what is called the critical stage, marked by the symmetrization of the mouth and pharynx, and the reduction of the gill slits to eight symmetrical pairs. The larva (Fig. 15, B) is about 3.5 mm. in length and tapered at each extremity. The only external opening added during this period is the atriopore, and the neuropore is about to close. The mouth has assumed a symmetrical anterior position, and in front of it a buccal cavity has been established by the outgrowth of the oral hood, from the base of which the buccal cirri have begun to grow out. The f u n number (61) of somites has been formed for some time, and a definite tail region has grown out posterior to the ana l opening. The provisional caudal fin has been replaced by a permanent caudal fin fold continuing anteriorly as space; n, wall of nephri- the median dorsal and ventral fins, the phridiZ former continuous with the right side of the oral hood. The ventral fin has disthe anus to the left side. The atrium is a spacious cavity receiving the external openings of the eight pairs of gill slits, of which the anterior pair remains undivided by a tongue bar, while the posterior one or two pairs are as yet in
Fig. 20. Portion of a transverse section of Amphioxus larva passhridium. After Goodrich, c, Coelom; e, branchial epithelium; g, wall placed completely divided. The liver or caecum is just beginning to develop.
At this time the larva largely gives up its free swimming habit, and assumes the adult habit of burrowing in the sand or mud of the bottom, frequently remaining buried with only the anterior end protruding. The cilia of this exposed region, and of the pharynx in general, vibrate actively, carrying into the pharynx a respiratory current containing small nutritive organisms. The age of the larvae at the critical stage varies greatly but three months may be taken as a rough approximation.
The Adolescent Period
The larva now enters upon its long period of adolescence. This is characterized by the very gradual assumption of adult characteristics, chiefly through histological differentiations and increasing complexity of many regions of the body. The development of the brain and cord has been mentioned. In the pharynx pairs of gill slits (tertiary) are slowly added and the pharyngeal wall assumes the complex structure of the adult. Apparently gill slits are added slowly throughout life, and usually number upward of one hundred pairs in mature specimens. The liver pushes forward as a simple blind sac, into the atrial cavity on the right side, carrying before it a fold of fused atrial and coelomic walls; finally it extends far forward into the pharyngeal region. The most important development of the period is the formation of the gonads.
In reality the gonads begin to develop before the end of the larval period. They appear in the ninth or tenth segment and continue to about the thirty-fifth. They are first indicated as small groups of cells in the floor of the myocoel, in the region where the skeletogenous layer passes into the cutis layer. This is the region of the nephrostome in the Craniates, and the gonadial cavities which appear later have been compared with the nephrostomes, or "gono-nephrostomes," of the higher forms, and it seems likely that Amphioxus may be primitive as regards the position and origin of the germ cells. At any rate Amphioxus is primitive in that the gonads arise and remain as metameric structures, entirely separate from the excretory system.
Shortly after the completion of the larval period, in specimens about 5 mm. long, these small groups of cells are found in the ant ero- ventral region of the segment, toward its inner or atrial surface, lying along the posterior face of the dissepiment separating the surrounding myocoel from that next anterior. As these cells slowly multiply they push forward into this next anterior myocoel forming, in its postero-ventral region, a small bud covered with a fold of the dissepiment. This dissepiment soon becomes sac-like and remains attached by a short stalk to the anterior face of the remainder. This solid gonadial rudiment soon develops a cavity within its mass. When the larva is about 12 mm. long, this general region of the myoccel becomes cut off by a fold the outer myotomal wall, leaving the gonad surrounded outside of its own wall by a portion of the general myocoelic space; this is the perigonadial coelom or gonocoel (Fig. 21, A). In the base of this fold is a blood-.vessel a branch of the posterior cardinal vein, which extends through this entire region.
Then the gonadial cells move toward the atrial side of the original gonadial sac, leaving the dissepiment al wall as the visceral wall of the gonocoel while its parietal wall is formed by this latter downgrowth of the myotomal wall (Fig. 21, B). The cavity of the gonad enlarges, as the primary gonadial (ovarian or testicular) cavity, toward the outer or atrial side of which the definitive germ cells are crowded and become covered by a follicular epithelium, also formed from cells of the original mass. The later history is more completely w r orked out in connection with the ovary, although it is known that the development of the testis is closely similar.
The outer (atrial) region of the gonocoel, lying between the genital cells and the atrial epithelium, enlarges above and below the region of the original stalk of attachment, which, by the general growth of the gonad, is pushed from the myotomal to the atrial surface. This cavity is now known as the secondary gonadial (ovarian or testicular) cavity (Fig. 21, C). In the outer wall of this, just above and below the stalk of attachment, two specialized thickenings develop; these are the cicatrices. As the gonadial cells multiply and enlarge they crowd upon these cavities and nearly obliterate them, particularly those toward the myotomal surface, but the essentially epithelial arrangement is not lost, and as the ova develop, as described at the beginning of this chapter, their polarity has a definite relation to their position in the epithelium, such that the animal pole is toward the free surface. When the germ cells are fully developed the inner and outer gonadial envelopes contain muscle fibers, the contractions of which appear to assist in rupturing the visceral layer of the gonocoel in the region of the secondary gonadial cavity, and force the germ cells into this cavity, where they remain for some time before extrusion. Sexual differentiation within the gonad appears in specimens about 18 mm. in length.
Fig. 21. Diagrams of sections through the gonads of Amphioxus in three stages of development. After Cerfontaine. Atrial surface toward the right. A. Early stage. B. Intermediate stage. C, Late stage showing definitive arrangement. 6, Peribranchial (atrial) epithelium; c, cicatrix; /, true follicular epithelium; fe, external layer of follicular epithelium; g, gonocoel; ge, germinal epithelium; 01, primary ovarian cavity; 02, secondary ovarian cavity; pg, parietal layer of gonocoel; v, cardinal vein; vg, visceral layer of gonocoel.
When the fully formed germ cells, ready for laying, have accumulated in the secondary gonadial cavity, strong contractions of the body wall and ventral musculature rupture the outer membrane in the region of the cicatrices, and they are forced into the atrial cavity, whence they are carried to the outside through the atriopore by the respiratory current, aided by continued muscular contraction. Individuals of the common species of Amphioxus apparently first produce mature germ cells, that is, become adult, when they reach a length of about 2 cm.; the age of such specimens is unknown.
References To Literature - Chapter 1
The literature lists here and at the end of Chapters III, V and VI, include only a few of the more important or more recent titles. References to the sources of borrowed figures are included, and a few works of historical importance, as well as articles containing extensive literature references. In each reference the author's name is followed by the title of the work and the reference to the journal in which the article appeared, or to the place of publication, in case the work appeared separately. The number of the volume (Band, tome, etc.} is printed in black-face Arabic numerals, followed by the year of its publication. References to pages, parts, etc. are omitted except in a few instances where this information is necessary.
The abbreviations of the journals more frequently referred to are as follows :
Amer. Jour. Anat. American Journal of Anatomy. Baltimore and Philadelphia.
Amer. Jour. Physiol. American Journal of Physiology. Boston.
Anat. Anz. Anatomischer Anzeiger. Jena.
Anat. Hefte. Anatomische He/te. Wiesbaden.
Anat. Record. Anatomical Record. Philadelphia.
Arch. Anat. u. Entw. Same as Arch. Anat. Physiol.
Arch. Anat. Physiol. Archiv fur Anatomic und Physiologic. Leipzig.
Arch. Biol. Archives de Biologic. Leipzig and Paris.
Arch. d'Anat. Micr. Archives d y Anatomic Microscopique. Paris.
Arch. Entw.-Mech. Archiv fur Entwickelungsmechanik der Organismen, Leipzig.
Arch. mikr. Anat. Archiv fiir mikroscopische Anatomic undEntwickelungsgeschichte. Bonn.
Arch. Naturgesch. Archiv fur Naturgeschichte. Berlin.
Arch. Zellf. Archiv fiir Zellforschung. Leipzig.
Arch. Zool. Exp. Archives de zoologie experimentale et general. Paris.
Biol. Bull. Biological Bulletin. Woods Hole, Mass.
Biol. Centr. Biologisches Centralblatt. Leipzig.
Bull. Acad. Sci. Cracovie. Bulletin de I 'Academic des sciences de Cracovie.
Bull. Mus. Comp. Zool. Harvard Coll. Bulletin of the Museum of Comparative Zoology at Harvard College. Cambridge, Mass.
C. R. Acad. Sci. Paris. Comptes rendus hebdomaires des seances de V Academic de sciences. Paris.
C. R. Soc. Biol. Paris. Comptes rendus des seances et memoires de la Societe de biologie. Paris.
Ergeb. Anat. u. Entw. Ergebnisse der Anatomic und Entwickelungsgeschichte. Wiesbaden.
Intern. Monatsschr. Anat. Phys. Internationale Monatsschrift fiir Anatomic und Physiologic. Leipzig.
Jena. Zeit. Jenaische Zeitschrift fiir Naturwissenschaft. Jena.
Jour. Anat. Physiol. Journal of Anatomy and Physiology. London.
Jour. Coll. Sci. Imp. Univ. Tokyo. Journal of the College of Science, Imperial University of Tokyo. Jour. Exp. Zool. Journal of Experimental Zoology. Baltimore and Philadelphia.
Jour. Morph. Journal of Morphology. Boston and Philadelphia. Mitt. Stat. Neapel. Mitteilungen aus der zoologischen Station zu Neapel. Berlin.
Monit. Zool. Ital. Monitore Zoologico Italiano. Firenze. Morph. Jahrb. MorphologischeJahrbuch. Leipzig. Phil. Trans. Roy. Soc. Philosophical Transactions of the Royal Society of London.
Proc. Roy. Soc. Proceedings of the Royal Society of London. Q. J. M. S. Quarterly Journal of Microscopical Science. London. Sitz.-Ber. Acad. Wiss. Wien. Sitzungsberichte der Kaiserlichen Akademie der Wissenschaften zu Wien. Mathematisch-naturwissenschaft liche Klasse. Wien.
Zeit. wiss. Zool. Zeitschrift filr wissenschaftliche Zoologie. Leipzig. Zool. Anz. Zoologischer Anzeiger. Leipzig. Zool. Jahrb. Zoologische Jahrbilcher. Abteilung filr Anatomie und Ontogenie der Tiere. Jena.
BOEKE, J., Das Infundibularorgan im Gehirne des Amphioxus. Anat. Anz. 32. 1908.
CERFONTAINE, P., Recherches sur le deVeloppement de TAmphioxus. Arch. Biol. 22. 1906.
GARBOWSKI, T., Amphioxus als Grundlage der Mesodermtheorie. Anat. Anz. 14. 1898.
GOODRICH, E. S., On the Structure of the Excretory Organs of Amphioxus. Q. J. M. S. 54. 1909.
HAMMAR, J. A., Zur Kenntnis der Leberentwickelung bei Amphioxus. Anat. Anz. 14. 1898.
HATSCHEK, B., Studien iiber Entwicklung des Amphioxus. Arbeiten a. d. Zool. Inst. Wien. 4. 1882. Ueber den Schichtenbau von Amphioxus. Verh. d. Anat. Gesell. 2. Anat. Anz. 3. 1888.
KLAATSCH, H., Bemerkung liber die Gastrula des Amphioxus. Morph. Jahrb. 25. 1897.
KOWALEWSKY, A., Entwickelungsgeschichte des Amphioxus lanceolatus. Me"m. de 1'Acad. Impe*r. de St. Petersbourg. VII. 11. 1867. Weitere Studien liber die Entwickelungsgeschichte des Amphioxus lanceolatus, nebst einem Beitrage zur Homologie des Nervensystems der Wlirmer und Wirbelthiere. Arch. mikr. Anat. 13. 1877.
LANKESTER, E. R. and WILLEY, A., The Development of the Atrial Chamber of Amphioxus. Q. J. M. S. 31. 1890.
LEGROS, R., Sur quelques cas d'asyntaxie blastoporale chez P Amphioxus. Mitt. Zool. Stat. Neapei. 18. 1907. Sur le developpement des fentes branchiales et des canalicules de Weiss-Boveri chez 1'Amphioxus. Anat. Anz. 34. 1909. Published anonymously. Sur quelques points de 1'anatomie et du developpement de PAmphioxus. Notes preliminaires. 1. Sur le nephridium de Hatschek. Anat. Anz. 36. 1910.
LWOFF, B., Ueber einige wichtige Punkte in der Entwicklung des Amphioxus. Biol. Centralbl. 12. 1892. Die Bildung der primaren Keimblatter und die Entstehung der Chorda und des Mesoderms bei den Wirbelthieren. Bull. Soc. Impe"r. de Natural, de Moscou. II. 8. 1894.
MAC BRIDE, E. W., The Early Development of Amphioxus. Q. J. M. S. 40. 1898. Further Remarks on the Development of Amphioxus. Q. J. M. S. 43. 1900. The Formation of the Layers in Amphioxus and its Bearing on the Interpretation of the Early Ontogenetic Processes in Other Vertebrates. Q. J. M. S. 54. 1909.
MAR^CHAL, J., Sur Tovogenese des Selaciens et de quelques autres Chordates. Premier memoire: morphologic de Pelement chromosomique dans Povocyte I chez les Selaciens, les Teleosteens, les Tuniciers et 1'Amphioxus. Cellule. 24. 1907.
MORGAN, T. H. and HAZEN, A. P., The Gastrulation of Amphioxus. Jour. Morph. 16. 1900.
SAMASSA, P., Studien liber den Einfluss des Dotters auf die Gastrulation und die Bildung der primaren Keimblatter der Wirbelthiere. IV. Amphioxus. Arch. Entw.-Mech. 7. 1898.
SOBOTTA, J., Die Reifung und Befruchtung des Eies von Amphioxus lanceolatus. Arch. mikr. Anat. 50. 1897.
WILLEY, A., The Later Larval Development of Amphioxus. Q. J. M. S. 32. 1891. Amphioxus and the Ancestry of the Vertebrates. Columbia Univ. Biol. Ser. II. New York. 1894.
WILSON, E. B., Amphioxus and the Mosaic Theory of Development. Jour. Morph. 8. 1893.
ZIEGLER, H. E., Die phylogenetische Entstehung des Kopfes der Wirbelthiere. Jena. Zeit. 43. 1908.
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Kellicott WE. Outlines of Chordate Development (1913) Henry Holt and Co., New York.
Outlines of Chordate Development: 1. Amphioxus | 2. Early Frog | 3. Later Frog Organogeny | 4. Early Chick - Embryonic Membranes and Appendages | 5. Later Chick - Organogeny | 6. Early Mammal - Embryonic Membranes and Appendages | Figures
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