Paper - Contribution to the structure and development of the vertebrate head 1
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Locy WA.Contribution to the structure and development of the vertebrate head. (1895) J. Morphol. 11(3): 497-595.
- Locy 1895 Contents: General Introduction | Part I - Metamerism of the Head | Part II - The Sense-Organs | Figures
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- 1 Part I - Metamerism of the Head (1895)
- 1.1 I. Basis for the Discussion
- 1.2 II. Historical Review of the Work on Neuromeres
- 1.3 III. Descriptions of Stages of Acanthias
- 1.4 IV. Supplementary Observations on Other Animals
- 1.5 V. General Considerations
- 1.5.1 1. New Aspects of the Problem of Metamerism
- 1.5.2 2. Are they Artifacts?
- 1.5.3 3. Do they afford a Clew to the Metamerism of the Brain?
- 1.5.4 4. They are formed Independently of Mesodermic Influence
- 1.5.5 5. Presence of these Segments in Embryonic Rim and Primitive Groove
- 1.5.6 6. Number of Segments represented in the Brain
- 1.5.7 7. Relation of the Neuromeres to Sense-Organs and Cranial Nerves
- 1.5.8 8. Head and Trunk
- 1.5.9 9. Summary
Part I - Metamerism of the Head (1895)
I. Basis for the Discussion
The more recent discussions on the metamerism of the head are based upon segmental divisions as shown in (1) cranial nerves and branchial clefts, (2) mesoblastic head cavities and (3) segments of the neural tube.
The first mentioned basis may now be set aside as involving too much conjecture. Mcclure has stated the objections to it as follows : «We have positive proof that the degeneration of certain branches has taken placeY This being the case, we have every reason to assume that who1e segmental nerves may have once existed, which have completely degenerated, leaving no trace whatever of their previous existence If such be the case, the segments origina1ly connected with these degenerated nerves must necessarily be overlooked, if the existing nerves are made use of as a means of determining the original number of segments.
Furthermore, the vagrant changes in the position of some of the cranial nerves must necessarily cause confusion. For examplcz take the sixth nerve, which in the frog and tadpo1e xstages is situated between the first and second roots of the ninth nerve, a position somewhat posterior to its place of origin. This remarkable shifting clearly shows not only what great changes in position the cranial nerves are capable of under—going, but it also goes to prove that we can find no reliable means of determining the primitive segments by means of their sconnection with the exit of the existing cranial nerves. Beard in taking up this problem made use of san important series of Sense-Organs for which he has proposed the name of "Branchial Sense Organs," from their development from thickenings of the epiblast over each branchial c1eft. The dorsal branchesv of certain crania1 nerves fuse with these epiblastic thickenings ; the superficial part of the thiclcening giving rise to a branchial sense-organ, while the deeper portion becomes the gang1ion of the dorsal root of the crania1 nerve. This close relation which exists between the dorsal branches of the crania1 nerves and their corresponding sense—organs is undoubtedly of segmental Character. But this line of research is beset by a great dikticu1ty, namely, that the degeneration of certain branchial sense-organs would, in time, involve the degeneration of their corresponding crania1 nerves, and such degeneration has certain1y taken place, in part. or in whole, leaving in doubt the primitive segments with which they were connected."
The second and third p0ints mentioned are more important c1ews to the metamerism of the head. Muscle and nerve are, physiologically, so fundamentally related that we should natural1y expect Some close correspondence between muscle segments and neural segments, and metamerism of the head region should be studied in light of the work done on both sets of structures.
The myotomes (or muscle segments) have received by far the most attention as they are the more conspicuous, but it is timely to ask whether they afford the most re1iable evidence as. to the primitive number of brain segmenta Comparative study shows that the neural segments are the Hrst to appear and are less subject to rnodifications than the muscle segments of the head. The large number of myotomes described in the head of se1achian embryos by Dohrn and Killian are more transitory than the neural segments. The period in which they are exhibited is a short one, and soon the seventeen or eighteen Segment-s of Killiartz and the eighteen or nineteen of Dohrn,. become reduced, by fusion, or absorption, or both, to the nine head segments of Van Wijhe
The neural segments, on the other band, begin very early, as shown in this paper, and preserve their original number and characteristics through several embryonic periods. It will bei seen as we proceed in the account of these. segments, that the assumption cannot be sustained, that the segmental divisions of the middle germ—layer (protovertebræ) are primitive.
II. Historical Review of the Work on Neuromeres
The question of Metamerism of the Head as based upon myotomes has been comp1etely reviewed by Dohrn, Ki11ian, and others; Isha11say nothing on that side of the problem, but shall limit the historical review, and confme the discussion to the side of the question that has been less cultivated.
It is a fact of comparatively recent discovery that the whole neura1 tube of vertebrates is divided by constrictions into Simi1ar segments. Each Segment is bounded, anteriorly and posterior1y, by transverse folds; and the elevated area between them constitutes the Segment to which the name metamere is given. These segments may be picturecl to the mind as a series of transverse ridges and furrows occupying each side of the neural tube and not extending across the median plane. They are exhibited in very young embryos of Vertebrates and disap— pear before what may be called the middle embryonic period. The existence of such folds in the wa1ls of the hind-brain has been known since the time of Von Baer, who in I828, first observed them in the embryonic tchiclc of the third day of development ; but it was not unti1 1889 that they were known to extend throughout the length of the neura1 tube.
Since Von Baer’s time they have been observed and commented upon by various anatomists. Bischoff1 Hgures the neural segments, but does not mention them either in the text or in the descriptions of the iigurea His figures show seven folds in the region of the fourth ventricle of a dog embryo of the twentyssiifth day of development. There are also shown three additional folds in the region of the mid-brain.
Remak in I850, made important observations, and suggested that the segments in the hind-brain are connected with the origin of the nerves in that region. He noted five or— six quadrilateral Helds on each side of the hind—brain wa11s, calling attention to the fact that they correspond c1osely in position with the viscera1 arches, and with the cranial nerves «which grow with them« According to his observations they fade away after the fifteenth day.
- I am greatly indebted to Hoffmann's historical review of the literature in Bronrks Klassen und Ordnungen des Thierreichs I have consulted nearly all the literature referred to there, but, in some few cases, where the original papers have been irr-accessible, I have depended wholly upon bis review of it.
Dursy observed them in I869, in the embryonic cow, of 6.5 mm. in 1ength. He recorded the occurrence of six folds in the regioni of the fourth ventricle Foster and Ba1four, in I874, noted the same structures in the chick, and suggested that they were of segmental importance. Dohrn, in I8y5, called attention to the occurrence of eight or nine neural segments in the fourth ventricle of bony Hshes He contrasted this early segmentation with segmental divisions in insects. Gotte figures such segments in the hind-brain of welldeveloped ernbryos of Bominator. In I877, Mihalkovics was inclined to interpret these segments as due to mechanical pressure of the mesob1ast, and, therefore, not a fundamental feature of the medullary tube.
Beraneck showed, in I884, that there is a definite connection between certain of these segments and crania1 nerves, thus giving the first real foundation for establishing their segmental re1ations. In his earlier paper, he describes five pairs of transverse folds in the hind-brain of embryos of Lacerta agilis from 3 to 4 mm. tin length He noticed that they rapidly fade away and disappear in embryos 5 or 6 mm. in length. In I887, he studied the relations of these « replis medullaires « in the chick, and, as regards their connection vsksith cranial new-es, reached similar conc1usions.
Kupffer maintained in 1885 that these segments indicate a primary metamerism of the medullary tube. He has published several brief notices on these structures In I884, he gave a record of studies on» the brain of the trout, in which he found five pairs of neural segments in the Und-brain. In sagittal Sections he noted, in addition to these, three pairs in the mid— brain. , He found no segments further forwards, and concluded that the foresbrain is not to be included in the segmented region.
In the following year ('85) he gave the results of his studies on embryos of Salamandra atra. In embryos of that form, showing as yet no traces of protovertebrae, eight pairs of neural segments in the median part of the hindbrain. It is to be carefu1ly noted that these segments observed by Kupffer were in embryos with a wide open neural groove, and occupied the median part of the cephalic p1ate, thus giving a ssmediane Gliederung des Hirnes.« In I893, in his « Vergleichende Entwicklungsgeschichte des Kopfes der Kranioten,« he gives figures (20xx and 20 F) of the forms described in I885. These iigures of Salamandra atra show segmental folds only in the median part of the neura1 plate, and none in the neural ridges. This is interesting when compared with my observations on amphibian eggs (see p. 529).
In addition to the eight segments in the brain region he (:ounted thirteen or fourteen in the cord, extending baclcwards to a point a litt1e in front of the blastopore.
Rabl ('85) spealcs of unmistakable segmentation in the hindbrain of chick embryos of from fifty to ninety hours« incubation. He found seven or eight segments in the region of the fourth ventricle-——-not being able to determine deHnitely whether there were seven or eight. Again, in 1892, Rabl has most ably discussed the question of the metamerism of the head, but as his paper deals almost exclusively with segmentation in the mesoblast, it does not come in for attention in the present connection.
Oscar Hertwig gives the matter passing attention in the third («88) edition of « Lehrbuch der Entwicklungsgeschichte.« He is not inc1ined to attach much importance to the neura1 segments.
Gegenbaun also, does not loolc upon these particular segments as important factors in the metamerism of the head. His position on the question is shown by the following quotation so frequently met with: «So interessant und so vielversprechend diese Thatsachen sind, so wenig scheinen sie mir gegenwärtig geeignet, zur Beurtheilung der Metamerie des Kopfes selbst als Factoren in Geltung gebracht zu werden»
0rr, in I887, traced very definitely the connection between these segments in the hind-brain and cranial nerves. In describing the segments he made use of the term "neuromeres", which has been generally adopted on this side of the Atlantic.
He describes six in the hind—brain of the lizard (Anolis), giving their anatomical characteristics with great clearness He observed no neuromeres behind the point of origin of the tenth nerve, nor did he find them in the fore— and mid—brain, but he conc1uded, hypothetica11y, that they were present in the anterior brain regions. Orr found the Hfth, seventh and eighth, ninth, and tenth nerves, respectively, connected with the first, third, fifth, and sixth neuromeres of the hind-brain.
Hoffmann, in Bronn’s « Klassen und Ordnungen des Thierreichs (1888), records his observations on these segments in Lacerta and Tropidonotus He found seven in the hindbrain of these forms. In the following year he added further details in the Zoolog-STIMM- Änaezszszm He differs somewhat from 0rr as regards the relationships of the crania1 neu-es, assigning the Hfth nerve to the second neuromere of the hindbrain, the seventh and eighth to the fourth, the ninth nerve to the sixth, and the tenth nerve to the seventh neuromere. From the first Segment the fourth nerve arises, and subsequent1y shifts its Position forwards.
Mcc1ure, following 0rr’s work, demonstrated the segmentation of the neural tube throughout its who1e extent, and published a preliminary announcement of the same in 1889. He showed the presence in the spina1 cord of segments continuous with those in the brain, and histologically simi1ar to them. He examined these structures in the chicken, Amb1ystoma, and the lizard (Anolis). He fixed upon six in the chicken and lizard, and five in Amblystoma, as the number in the Und-brain of each respectively. He found two in the fore-brain, but left the number in the mid—brain undetermined, expressing the view, however, that there are two neuromeres in that brain region. Thus he identifies ten neuromeres in the entire brain region, and agrees with 0rr in the assignrnent of nerves to the neuromeres of the hind-brain.
Miss Platt’s work (1889), on «Axia1 Segmentation of the, chickenZH agrees, in so kar as neurorneric segmentation is con-cerned, with that of her predecessors, except as regards the relation of the nervedibres to their corresp0nding neuromeres. According to her observations they spring, primarily, from the concavity between two segments, and not from the crest of a neuromere. My observations on the motor roots agree with those of Miss Platt in that particular.
Zimmerman ('91) states that he finds in embryos of the rabbit and chick, shortly before the c1osure of the neural groove, the segments observed by Kupffer in Salamandra atra. He noticed at first eight of these segrnents (encephalomeres) in the brain region. The three anterior ones were much larger than the five lying behind them in the medu1la. The three front ones are the vesicles of the fore-, mid-, and hind—brains, and they straightway undergo secondary division as follows: The first divides into two, the second into three, and the third into three, making a total of eight secondary divisions arising from three primary ones. These added to the five of the medulla give a total of thirteen segments in the brain region. He also observed these structures in Acanthias and Muste1us, and found them very clearly defined In mamma1s the metameres of the nnd-brain are not so distinct. Zimmerman goes on to say that these folds cannot be accidenta1 appearancea since in all classes of vertebrates corresponding nerves arise from corresponding segments He gives a kahle, showing nerve re1ations, with too much detail to reproduce here.
Waters, whose complete paper appeared in June, I892, studied especially the mid-brain of Teleosts He confirmed and extended the observations of Orr and McC1ure. He counted eleven neuromeres in the entire brain regiom six in the hindbrain, two in the mid-brain, and three in the fore—brain. He did not lind neuromeres in the brain of the Cod earlier than the ninth day of development He assigns the olfactory and optic nerves to the anterior two neuromeres The two neuromeres of the midsbrain give origin to the third and fourth nerves, and from the six segments in the hind—brain the näh, seventh, eighth, ninth, and tenth nerves arise as designated by Meclure The sixth nerve he found to occupy its theoretical position when the neuromere exists; when fusion has taken place between the trigeminus and abducens neuromeres the sixth nerve has been shifted backwards between the seventh and eighth nerves.
Froriep gave a noteworthy contribution to the subject of neuromeric segmentation in very early stages, before the Anatomische Gesellschaft of Germany, at the June meeting in I892. He described anew the so—ca1led neuromeres that he had previously observed in mole embryos, but concluded that they are not of true morphogenetic signiiicance He further described the conditions in Triton embryos, and concluded that the so-called primary neuromeres detected by Kupffer in those animals are simply the result of underlying mesoblastic somites.
Froriep agrees with Kupffer in tinding segmental folds while the neural groove is widely open, and in locating them in the median part of the cephalic plate. But, whereas Kupffer linds eight in the brain region of Salamandra atra, he tinds only four in the corresponding region of Salamandra maculosa, and five in Triton cristatus (Compare with my observations, p. 529.) His general conclusion is that «the jointing of the vertebrate body is originally determined by the middle germlayer; when ectodermal structures exhibit segmental arrangement, it is the result of secondary adaptation.
Herriclc ('92), in a preliminary paper, gives an account of neuromeres in the Ophidian embryo, in stages after the com— plete closure of the neural groove, and after the formation of the ear vesicle. His figures show six neuromeres in the medulla. He states a proposition that will be of use later, in helping to distinguish between primary metamerism, and metameres of secondary origin which show after the closure of the neural tube: « If neuromeres once existed in the fore-brain they would be visible only at an early stage . . . The so—called fore-brain neuromeres differ from those of the medulla and cord in involving only dorsal structures."
The present writer, in I894, gave the first account connecting the earliest formed neuromeres with those of later stages He showed that in sharlcs they arise very early, and may be traced without a break through all the stages of the open neural groove into the structures that have, in later periods, been designated neuromeres. The segmental divisions extend to the anterior tip of the fore-brain, and are distinguishable in that region for a brief time after the closure of the neural groove.
He also recorded the presence of neuromeres in Amblystoma ernbryos with wide open neural groove, but differs from Kupffer in locating them in the neural ridges instead of the median neural p1ate. (See further on this point, p. 530.)
From the foregoing historical survey it appears that our knowledge regarding metamerism in the neural tube has passed through the phase of simple observation of its occurrence (Von Baer, «28, Remalg Ho, Dursy, So, and others), and has grown by successive additions to the recent conception of its segmental importance. In reaching this point, Hrst came the work of Beraneck («84) and Orr (-87), showing that the neuromeres of the hindsbrain are definitely connected with nerves; following this it was demonstrated by Kupffen partly, in I886, and by Mcclure, definitely, in 1889, that the neuromeres extend throughout the neural tube, and that those of the trunk region merge gradually into those of the head region. Lastly, the neuromeres of the mid-brain have been especially studied by Waters («92), the condition of that brain region having been left undetermined by previous observers. Remak was the Erst (I85o) to suggest the segmental relations of nerves and neural segments; Beraneck the first (I884) to demonstrate it. From this time onward the definite relation of nerves and neuromeres began to be studied, and both Orr (-87) and Hoffmann («88) are pioneers in this line of study.
It will be observed that previous to the appearance of my paper just referred to, no one but Kupffer and Froriep had claimed a very early appearance for neural segments, and these two authors had recorded their appearance only in the median part of the neural p1ate, and not in the neural ridges. They had not shown the structures of the open neural groove stage to be in any way connected with the neuromeres of later periods, which are present in the lateral walls of the neural tube.
In the case of Froriep’s observations I thinlc there is reason to doubt whether the segments observed are really the «neural segments « of other writers. The small number (4 or s) which he observed in the head region does not correspond with the number of neuromeres observed by any other author in the same region. I thinl(, also, there is a way to bring Froriep’s observations into reconciliation with my own (see p. 529).
At all events, it has been understood from the work of previous observers that the neural segments arise after the neural groove is closed, or while it is in process of closing. Waters («92) carries the idea throughout his paper that the metameric segmentation arises relatively late, especially in the Teleosts, where he was unab1e to lind any traces of this segmentation earlier than the ninth day of deve1opment, after the auditory pit is formed. 0rr and Mcclure do not in every case state ages, but from their flgures and the text I understand that they have not detected this segmentation in very young stages. Miss Platt mentions the fact that these segments sometimes occur in, the chick while the groove is open. Rabl mentions them as being especially clear from the iiftieth to the ninetieth hour of incubation in the chicken
I have been fortunate enough to lind these neural segments in a number of animal forms in extremely young stages, and in Squalus acanthias, to trace them coherently onward into the latet stages. In this form, the division of the embryo into segments takes place before the neural groove is formed, and, before any protovertebræ have made their appearance, the metameres not only extend the whole length of the embryo, but they are continued for Some distance into the embryonic rim. They occur under such conditions in this animal that they cannot be interpreted as depending on mesoblastic segmentation. In Amblystoma and the newt (Diemycty1us) the metameric segmentation is present in the rudiments of the neural fo1ds, just after their Erst appearance and during their period of broadest expansion. In living chick embryos of about the twentieth hour of incubation they can be made out with clearness along the walls of the beginning neural folds. It is only in Squalus acanthias, however, that I have traced the complete history of these neuromeric segments. NO. 3.] THE PEJETEBÆÄTE HEFT-D. 513
III. Descriptions of Stages of Acanthias
The ear1iest stage in which I have detected this rnetarneric segmentation is represented in Pl. XXVII, Fig. 25. This is an age somewhere between Balfour’s stages B and C. It is, in reality, the youngest embryo of Squa1us to which I have had access since I began to observe especially the metameric segments in that animal. Whether or not they occur in still younger embryos I do not know, but they are already clearly defmed in the stage referred to, and it is reasonable to suppose that they may be seen in still earlier stages.
The axial embryo (Fig. 25) is just fair1y established, and has reached a length of 1kIzmm. The headsend is already wider than the rest of the embryo. It has begun to show that tendency to broaden that is characteristic of the head-end of the embryo. The gastrular cavity is broad, and extends to the extreme anterior end of the embryo. In the iigure it is seen even protruding beyond the head-p1ate. The primitive furrowz that has often been confused with the neural groove in these Elasmobranchs, is broadened at its anterior end. Fig. 63, Pl. XXIX, is a sketch of a horizontal Section of this embryo to show the general appearance of the metameres in Section.
The segmental divisions in this embryo extend from the anterior end backwards along the margins of the axial part of the embryo, and out into the norkaxial part or embryonic rim. There are seven or eight pairs of these segments in the embryo, and as many more, directly continuous with them, in the embryonic rim. The latter is segmented to the points where it is broken from the rest of the blastodernr Whether or not these segments extend further into the blastodermic rim, I am unable to say. The segments are most clearly defmed a1ong the inner margin of the embryonic rim, and extend more faintly across it.
In the axial part of the embryo they are not in such a favor ab1e position to be observed from above—-—-they are on the rounded mai-ging; but if the embryo be rolled into such a position that the margin is brought into view, its division into segments is more plain1y seen. Near the middle part of the embryo the 1ines of segmentation are faintly traceable from the margins towards the median furrow. The two lines of segments are joined in front by a single median piece or segment. This unsegmented anterior tip becomes more prorni— nent in the irnmediately following stages There is no evidence to show whether this represents the primitive anterior Segment or several aggregated anterior segmenta These segments, once estab1ished in this very early stage, may be traced onward in an unbrolcen continuity until they become the neuromeres of other observers, and sustain deiinite relations to the spinal and crania1nerves. Ryder, in I88I, observed segmental divi sions extending into the embryonic rim of Elacate, one of the Teleosts. In I885, he Hgures such structures in a stage in which the neural groove is c1osed and the eye vesicles are well established. Although the iigure shows a considerab1y later stage than we are now dealing with, and he does not speak of their earliest origin, nevertheless, the feature of their extending beyond the embryonic axis into the blastoi derrnic rim agrees with my observations on Acanthias and I thinlc it not improbable, that Rydefs segments correspond with those I have described. These segrnents, observed by Ryder under such unusual conditions, have generally been interpreted by morphologists as due to precocious segmentation in the non——axial mesoderm. The segmentation I have just described is not capable of such interpretation, for sections show that the mesoderm is not yet divided into protovertebræ at this stage, and that the epiblast is the seat of the tsegmental divisions. The mesodermic somites of Squa1us are formed later in the usual way, and the first ones appear in the trunk or neck region at a later period.
In Fig. 26 the embryo is relative1y more slender in the trunk region, and there is coming to be an observable distinction between the broadly expanded cephalic plate and the narrower bodzn Upon the anterior end there is being forrned a pro— truding unsegmented median tip, which is much better seen in Figs. 27 and s. The median furrow ends in front in a broadly expanded depression. The gastrular cavity has become narrowed in fr0nt by folding in of the sides, so that the embryo, when viewed directly from in freut, seems mounted on a kee1; the kee1, however, is not so1id, but contains the anterior part of the gastrular cavity, which still reaches to the anteri0r 1imit of the head. For the purpose of fixing the stages as defmitely as possible, I give anatomical characteristics not immediately connected with the metameric segmentatioIL The embryo from which the ftgure was Inade measured ITBZ mal« CUT I.—-— Embryo and blastoderm of Acanthias
in length but there is so just before the kormation ok the neun! Leids. d( about 8 diameters much individual variati0n in the size of embryos of apparently the same age, that the length is not very signiöcant There are, in that part of the embryo
Cin- 2. —The same ernbryo x about 40 diameters Meeatneres no: ehe-sen. The travsverse lines and number-s indicate the Plane of the Sections shown in the succeeding out.
behind the cephalic region, three or four mesodermic somites rather imperfectly differentiated The metameric segmentation is very clearly exhibited al0ng the lateral margins of the neural plate, extending from the unsegmented tip backwards, and, as in Fig. 25, is continued in the embryonic rim to the points on either side of the latter, where it is broken from the rest of the blastoderm While it is the lateral margins that are most clearly divided into segments, in the trank region the lines of division may be traced inwards towards the median furrow. This is probably due to the appearance of the mesodermic
ctrr z. ——Twelve traust-erst: Sections ok the embryo Figur-ed. in the pteeeciing sent. d( about Zo die-merkte. The number-s reier to the positions ok the. Sections in the set-ice. ·
sornites in that region. Fig. 64 represents a horizontal Section of this embryo showing metameres in the ectob1ast.
Fig. 27 is in many respects similar to Fig. 26 ; it is slightly older and has reached a length of 2 mm. and shows about five mesodermic somites. Diverging furrows have appeared upon the cephalic plate that include between them a wedge—shaped Central piece which terminates in the anterior unsegmented tip before mentioned. The cephalic plate is thus separated into a median and two lateral parts. lt will also be noted in this iigure that the lateral margins are marked off from the rest of the medullary plate by two furrows running lengthwise of the ern— bryo, so that the plate is bordered, as it were, by marginal bands. The furrows are most disti11ct in the head region, but they extend also, with 1ess distinctness, into the trunjc and fade away without reaching the hinder extrernity The furrows do not show so distinct1y in cross—section as one would at first suppose, and they are in part an optical effect, arising from the way in which the neural folds are formed This will be understood on reference to cut s. There is, neverthe1ess, a distinct notch to be seen in the crosssectioris of many specimens, while in others it is laclcing. I am inc1ined, with my present Iight,
CUT 4. — Embryo of Äcanthias just after the formation of the neural fo1ds. d( about 40 Manier-ers. Metameres not shown. The traust-esse lines indicate the plane of the Sections in cut s.
to consider these furrows as pure1y mechanical effects Sections show (cut z) that the marginal hands, at a stage just younger than this one, are composed of an accurnulation of cells forming thickened cords running along the margins of the embryo. These bundles of cells and their immediate derivatives are the rnaterial out of which the neuraI ridges and a large part of the medullary fo1ds are straightway produced.
As noted in the preceding embryo, the tnetarneres are most isclearly seen from below, but the reason for this is not far to 518 Lock. kvon XI.
see1(. The condition of the neura1 folds in this animal is very unusual: when Hrst formed they are 1ateral, wing-1ike expansions, extending along each side of the embryo, overhanging the yolk (cut 5). No sooner are they formed than they bec0me ventrally cum-ed, and, in this way the most clearly segmented
Co? s. -—«I’welve ttansverse Sections of the emhryo of the pkecckling out. z( about zo diameteka The neural folds are expandecl laterally beyonkl the body and veuttally curved.
parts of the embryo are brought ventra1wards, and this accounts for the metameres being most distinct when viewed from the ventral surface
Figs. 28 and 29 represent two views of the same embryoz it is an older stage than that represented in Fig. 27. The neural fo1ds are now fully formed and their ventral curvature is very marked. In Fig. 28, the optic vesicles OF) are seen on each side of the Central tongueslike Process to which attention has already been ca11ed.
In Fig. 29,1 the view is taken from below ; the embryo has been removed from the b1astoderm and placed upon its dorsal surface and the recurved edges of the neural folds are thus brought prominently into view. This is of course the most favorable position for making observations The dish contain— ing the embryo should be placed over a black, non—reflecting background, and the embryo rotated into the most favorable Position with a, fme artist’s brush.
In the actual specimen from which the figures were made the segments showed most beautifully. They appear like a row of beads running along the ventrally recurved margin, and extend with great distinctness the entire 1ength of the embryo. Those in the trunk region are continuous with those in the head and pass into the 1atter without any transition forms. There is, however, some individual variation in size of the neuromeres, and they are not abso1utely symmetrical on the right and left sides, but the signif1cant thing is, there is uniform1y the same number on each side in a given region, such as the hind—brain, or the brain region as a who1e. Fig. 29 shows the Central unsegmented piece from below with three segments on either side of it, occupying a part of the headfolds that is directed forwards Following the beaded edge, from the head into the trunk region we find it disappeap ing from view beneath the expanded walls of the gastrular cavity. viewing the same embryo from above (Fig. 28), the metameric segmentation is seen to extend the entire length of the embryo and, as in the earlier stages, lateral1y into its expanded parts. The segments are so plain that they may be easily counted There seems now to be a natural landmark sseparating the cephalic plate from the rest of the embryoz this is an abrupt downward bending (j) in the medullary folds which, as I have determined, lies just in front of the future origin of the vagus nerve. There are eleven metameres in the lateral margins of the cephalic plate, including the ones embraced inthis fold.
- I ln the process of transferring and shading, the outlines in this ögure have been rendered too symmetricah giving to it a semidiagrammatic characters.
Fig. 30 represents an older stage, in which the medullary folds have unrolled from their ventrally curved Position and are in the process of growing upwards Those of the head are at this stage nearly in the horizontal plane (cut 6). The outer margins of the folds are p1ain1y divided into segments, and the segrnentation extends backwards, also, into the trunk region, but not so clearly deftned. The optic vesicles are clearly seen on the head-plate, but the accexsory optic vesicles (see p. 57) have not yet made their appearance Beginning at the front end and counting baclcwards eleven segments on either side, we come to the point where the broadly expanded cephalic plate passes into the narrower neclc and trunk. This, as before indicated, is the point of future origin of the vagus nerve. It seems to me to be a natural line of division which may be of Service in determining the limits of the embryonic head. The question will be returned to on p. 543.
CUT H. — six transverse Sections ok an embrzso older than the preceding one. X about so diameters. The numbers below the Sections reker to their Position in the series. The neural folds are growing upwards ancl in Section 24 liave reachecl the horizontal Plane. The depressions in Sections 3 and 12 are the optic vesicles The embryo from which the Sections are made is shown on Pl. XXVL Fig. J.
It should be borne in mind that all the stages so far described are very young ; the ear1iest ones are before the formation of the embryonic medullary folds, and the oldest one is just when the Inedullary fo1ds are arching tipwarcls to form, for the first time, a medu11ary groove The mesenchymic somites have, in the interim, appeared in the trunk region, and have produced faint surface indications in the median parts of the medullary plate.
Taking a considerable step forwards in the history of these segments, we come to the condition represented in Fig. II. This iigure showS a stage in which the medullary folds have attained a nearly vertical Position; they are about to bend towards each other and meet in the median plane, but, as yet, they have not become approximated in any part of their course, and, therefore, we have an open neura1 groove (see cut 9, p. 553) extending the who1e length of the embryo. In this Hgure the embryo is viewed obliquely from the right side. The rudiments of several organs have now appeared upon-the head ; the most anterior of these organs is the primary optic vesiclez just back of this, near the margin of the head—folds, is seen a similar e1evation that represents the combined vesic1e of the mid—brain and the first act-array- optic vesicle (see p. 556). Still further back in the same 1ine, is another simi1ar but smaller elevation, which, I think, represents the second accessory optic vesicle. Behind the latter structure the niargin of the medullary fold is bent abruptly downwards; this is a normal condition in this stage, and is of course found in the earlier stages. Baclc of the primary optic vesic1e and somewhat between it and the midbrain vesic1e, is a rounded eminence which is the external indicati0n of the mandibular cavity, and behind this is the branchial pouch from which the branchiae are subsequently formed. The front end of the gastrular cavity is being cut oft· a1ong with the increase in the head flexure.
Directing our attention to the margin of the right medullary fold, we note that it is clearly segmented through the head region, and backwards into the trunk region, where, in the Hgure, it disappears behind the yo1l(. The metameres extend in reality to the posterior part of the embryo. There has been a slight change in position of the foremost segments with reference to the rest of the head-p1ate. The three anterior ones are no 1onger, as in Fig. 29, on a part of the margin that looks forwards, but they have been shifted backwards, and that part of the margin that was anterior, now constitutes a part of the lateral border. Of course this shifting of position is brought about by changes in the medullary fo1ds. The first three metameres are, at this stage, in front of the eye, the fourth, f1fth, and part of the sixth, in front of the accessory optic and midssbrain vesic1e. The following iive segments (seven to e1even) occupy the refiected part of the neural fo1d. The eleventh, as has been indicated, lies in front of the vagus nerve.
The next stage to be considered (Pl. XXVII, Fig. 32) is just after the c1osure of the neural groove, which is, however, still open in front by a small neuropore. The original segments are still visible throughout the head region; those in the fore and mid-brains still show from the outside. The surfacemarkings on the head are similar to those in Fig. II. In front is the conspicuous optic vesic1e, and behind it is another similar rounded eminence, the mid-brain vesic1e. Further back there is an area, circular in out1ine, resembling the midbrain, except it is not so prominent. It is, however, merely a pad of mesoderm applied to the walls of the cerebellum There is another surface indication of considerable interest, via, a line of mesob1ast running over the branchial region; it connects in front with the mandibular cavity and behind with the body protovertebraen
Fig. 33 shows a slightly older stage than Fig. 32. The neura1 groove is complete1y c1osed. The extreme anterior end of the gastrular cavity has been obliterated The cranial flexure is quite marked. This is the last stage in which the metameres are visible throughout the length of the embryo; those in the fore- and mid-brain have become indistinguishab1e in surface views; they are, however, still to be detected in longitudinal Sections. We possess now a particular advantage in dealing with these segments, because anatomical landmarks of the head regions have become established, and these enable us to say with defmiteness what are the relations of the segments to the rest of the headJ This is just prior to the appearance of the auditory vesicle; when first established its center occupies the space of the Segment marked Io. Some— times, in its earliest stages, the circu1ar area spreads over the space of the three segments marked 9, to, and II, but I should say from my observations that, more frequent1y, it is not so widely expanded It always settles down in squalus acanthias, to occupy the position first indicated, and, subscquent1y, it is shifted backwards The topography of the head region is similar to what it was in Fig. 32. The Chief differences to note are the further differentiation of the branchial arches and clefts; we may now distinguish the position of the future first visceral cleft, and, faintly outlined, the boundaries of branchial arches.
The Segment marked 8 serves as an important landmark in all subsequent changes that affect the segments It is seated above a depressed region in which the first visceral cleft subsequently appears, and, during all the time the segments are distinguishable from the outside, it has no nerve root.
Only a few words of description will be needed to enable us to follow the history of the metameres through the later stages. In Fig. 34, the auditory vesicle (x:2-) has been formed and the first visceral cleft has broken through. The anterior metameres 1ying in front of the one marked 6 are no longer distinguishable from surface observation. The lines of neuromeres have been brought into contact in the median plane by the closing of the neural groove, but they are soon forced apart by the growth of the dorsal wall of the neural tube. We have now reached the stages, approximate1y, in which these neuromeres have been described by previous writers, —-—that is, the stage just after the appearance of the auditory vesicle, but it is to be remembered that Kupffer and Froriep have noted a form of segmentation in very early stages of Amphibia affecting the neural plate, but not the neural ridges (see p. 529).
In Fig. 35 Some characteristic changes are to be noted ; the auditory vesicle has shifted backwards till it occupies-a position opposite the eleventh metamere ; the metameres are being forced apart laterally by the growth of the dorsal wall of the hind-brain. The eighth metamere still serves as a landmark ; there are now two clearly marked metameres in front of it and three behind it. The only metameres discernible from surface view are those belonging to the hind-brain. r The mid-brain is considerably increased in expanse, and the first accessory vesicle has been crowded forwards into the region of the thalamencephalon This may be seen after the removal of the overlying layers of mesoderm, etc. (Pl. XXVIIL Fig. 44). The kifth nerve is already well begun, and nerve iibres are also given off from the segments numbered 9 and I0.
Fig. 36 shows an embryo slightly older than that in Fig. 3 s. The line of neuromeres have been forced further apart by the 1ateral growth of the dorsal wa1l of the hind-brain. The ear— vesicle is no longer circular in outline, but is fast becoming a closed pouch. The eye shows the beginning of the lens and the choroid f1ssure. The Anlagen of the Iifth, seventh, eighth, ninth, and tenth nerves are distinctly visible from surface observation. The branchial arches are all clearly outlined and the first two gill—clefts have broken through. The specimen shows about forty-f1ve mesodermic somites. A line of surface elevations over the hinder branchiae mark the beginning of the lateral line.
In Fig. 37 the neural segments are undergoing sorne changes in outline that are lilcely to lead to confusion in identifying them in later stages. If, for example, we loolc along the lower margin of the segmented border, we shall see that the elevations and constrictions are substantially as they have been in all the previous stages, but those along the upper margin no longer correspond with them. In all the preceding Hgures the boundaries of the segments correspond on both upper and lower margins. In Fig. 37, however, the upper margin shows e1evations just above the constrictions on the lower margin. These newckormed elevations become very quickly prominenh while the segments along the lower margin lose their individuality, and the segmented area becomes more and more an irregular sinuous band with crests upon its upper margin. The entire line of segments finally becomes indistinguishab1e, but if they be studied in stages irnmediately following that represented in Fig. 37 it will be the crests along the upper margin that first catch the eye. If the observations are made from above, these crests are seen to be transverse folds on each side of the medulla, and when counted will, of course, be one less than the original segments. It is only by viewing them from the side, and comparing them with ear1ier stages, that we shall be able to identify the boundaries of the original segments.
Just what is taking place during the appearance of the crests is not now sclear to me. I have heretofore assumed that it signified a union of the original sog-meinte, the anterior half of one with the posterior half of the Segment lying just in front of it, but at present I am inclinecl to question that interpretation The crests on the upper margin are between two neuromeres as designated by 0rr, and they correspond in Position to his inner ridge. The point of origin (motor Hbres) of the Hfth, seventh, and eighth nerves (so far as it may be determined by surface view) is now c1ear; they arise, as Miss Platt has described them in the chicken, from the concavity (on the lower margin) between two neuromeres. This will receive fuller consideration under the headingz The Nerves. lt will be interesting to note incidenta1ly, in this Hgure, the very large development of mid-brain over that in Figs. 33 and 34, and the consequent crowding forwards of the first accessory optic vesicle. The latter structure is also much reduced in size, and with its fellow is in the region of the thalamencephalon.
None of these Hgures have shown the condition of the entire neural tube. The segmentation so clearly seen in Figs. 34, 35, and 36, represents only a part of the actual segmenta-· tion, sei-Ja, that in the uppermost part of the neural tube. The rest of the tube is too much covered by mesoblast to be seen without dissection The upper part of the tube has——-—in the region of the hind-brain-—-two thiclcened lateral bands of cells, which form a border on either side of the neural tube; these are the segmented parts that are visib1e from surface observation. I have found it necessary to remove the overlying layer of mesoblast and the outer epidermic stratum, and completely expose the walls of the brain. When thus laid bare, the walls of the neural tube show in a most satisfactory manner. The ten or twelve ögures following those just described show dissections of this kind.
Pl. XXVIIL Fig. 4I, shows the surface view of an embryo slightly older than that represented in Fig. 33, and Fig. 42 shows the same embryo with the overlying tissues removed from the brain wal1s. It is clear from a comparison of these two figures that the line of metameres seen from the surface view are those occupying the upper part of the neural tube, and that below them the entire neural tube is divided into corresponcling segments. The segments (lo not reach acrosS the median plane, but they are alike in number and position on both sides of the tube.
Fig. 43 represents an embryo in which the auditory vesicle is just forming. The embryo was intermediate in age between those shown in Figs. 41 and 34.
Fig. 44 shows a dissection of an embryo of the same age as the one represented in surface view in Fig. 34.
Fig. 45 is talcen from an embryo just younger than that represented in Fig. 35. By the growth of the dorsal wall of the hind—brain the line of metameres have been forced apart. This expanded dorsal wall, being a new growth, is not divided into segments.
Fig. 46 represents a slightly older embryo with the optic vesicle and also the auditory vesicle removed. There are in this figure eight segments in the hind-brain that show plain1y, and faint indications of a ninth.
Fig. 47 represents a dissection of the embryo from which Fig. 35 was made, and therefore a direct comparison can be made between the two figures
Fig. 48 shows the condition of the brain walls in an enibryo just older than that represented in Fig. 37. From the continued growth of the dorsal wall themetameres on each side have become widely separated. The eapcapsule has not been removed. i Fig. 49 represents a slightly older embryo than the for-egoing one. The auditory and the optic vesicle have been removed. There are now distinctly nine segments in the hindbrain region. The U-shaped Segment, No. I2, in the hind brain lies opposite the ear-capsule. This figure shows well the condition of the neuromeres described in Fig. 37, in which there is no longer (as in earlier stages) a correspondence between the ridges on the upper margin and those on thelower margin of thesegmented lateral bands of the neural tube.
Up to this point the figures described have all been magni— Hed uniformly 45 diameters; but on account of the increase in size of the embryos it will be better to carry forward the history of these segments with ftgures drawn on a smal1er scale. Accordingly Figs 50-60, inclusive, are magnified only ten diameters.
Fig. 50 represents an embryo of the same age as that shown in Fig. 32.
Fig. 51 shows the entire embryo, partly dissectecL of which Fig. 43 is a portion more highly magniiied Behind the figure is seen the 1ine of fusion of the 1ips of the blastopore soon after the age represented in Fig. 55 is reached, the neural segrnents fade away.
Fig. 52 is a sketch of the embryo of which Fig. 47 is the enlarged view of a partia1 dissection. They all show well the segmented condition of the walls of the hind—brain.
Figs. 57 and 60 (PI. XXIX), represent the head region of older embryos in whichthe segments are no longer visible.
Taken togethekz the iigures now described give a comparatively full view of the neura1 segments in different ages. They show them from their first appearance to the time they fade away. We learn from this examination that the neural segments are established before any embryonic organs appear, and that in the early stages they extend not only throughout the length of the embryo, but into the embryonic rim. In the earliest stages the segments are a1ike, and there is no Structura1 distinction to be made between those in the head and those in the trunk, or even those in the embryonic rim.
In sagittal Sections the neuromeres are well exhibited Fig. 72 shows a Section of a specimen just after the closure of the neura1 groove in which the five neuromeres belonging to the fore- and mid—brain are exhibited The second neuromere coincides in the median plane with the neuropore. This is also to be seen in stirface view in Fig. 32. Very soon the anterior brain regions become so much modified and expanded that the original segmentaI divisions are no longer visible front are seen three of the brain vesicles, —-—those of the fore—» brain, the mid-brain, and the cerebellum. The thalamenc:epha1on and the prosencephalon do not show as separate parte. In the region of the hind-brain are seen three neuromeres espe— cial1y well developed They present the appearance of three bars ; they are the seventh, eighth, and ninth neuromeres respectivelzn The other neuromeres are present, but they do not stand out with such distinctness as the three mentioned. When the ear vesicle first arises it makes its appearance opposite the ninth neuromere.
Figs. 66—7I (Pl. XXIX), show six successive Sections of a specimen slightly younger than the one just described.
Figs. 73, 74, and 75 are Sections of a somewhat older embryo after the ear vesicle is estab1ished. In these figures eight neuromeres of the hind—brain are visib1e. The ear vesicle is opposite the tenth neuromere. Just in front of it, in Fig-s. 73 and 74, are the roots of the eighth and seventh nerves, respectively, those from the former nerve being connec:ted with the tenth neuromere, and those of the latter with the ninth neurowere.
In Fig. H, the iifth nerve is seen to have connections with the first and second neuromeres of the hind—brain, ins» the sixth and seventh neuromeres respectivelzn
As already indicated, the eighth neuromere bears no nerve, and Hoffmann remarks, « This seems to be the case in all Vertebrates."
IV. Supplementary Observations on Other Animals
I have also made some supplementary observations on these neural segments in Amblystoma, Diemyctylus, Rana palustris, Torpedo ocellata, and the chiclc In all of these forms the metameric divisions are to be found in very early stages before any of the embryonic organs have been formedz and they are in all essential features like those I have described for squalus acanthias.
Figs. 113 and 114 (Pl. XXX) show camera sketches of an Amblystoma egg, with broadly expanded neural plate and widely open neura1 groove. "The neural folds or ridges are divided throughout their length into a series of segments with no especial distinguishing features between those of the head and those of the body region. The median plate included between the neural ridges is smooth at this stage; at a slightly later peri0d, however, while the groove is still widely open, the median plate exhibits very faint transverse markings
The contrast between this condition in Amblystoma and that in a closely related egg (Rana palustris) is szvery instrucs tive. In the 1atter(Fig. III) the cephalic plate is very obviously segrnented, while the folds in the neural ridges are extremely difftcult to see. We thus have in these two closely related eggs strikingly different conditions In the one it isthe median plate material that is thrown into obvious folds, while those in the neural ridges are well—nigh indiscerniblez and in the other the conditions are reversed. This is not, however, to be taken as indicating profound differencesz for a little (:arefu1 observation shows that the median divisions do not correspond to those in the neural ridges, and therefore the median fo1ds in Rana palustris are not to be cornpared to the primitive segmental fo1ds in the neural ridges of Amblystoma. careful observation shows, also, that there are seg— mental divisions in the neural ridges of Rana palustris that do correspond, in number and general charaoteristics, with those in the neural ridges of Amblystoma. These latter segs mental divisions are extremely diflicult to see in Rana palustris. There are four or Ave median transverse divisions in the cephalic plate of that form, while there are ten or eleven segments in the neural ridges of the same region.
These facts throw light upon the apparent discrepancy between the observations of Kupffer and Froriep and those I published in a preliminary artic1e. Both the former authors observed segrnents in the median plate of amphibian embryos, and none in the neural ridges, while I have ftgured segments in the neural ridges of Amb1ystoma, and none in the median plate. But my later observations show that the appearances even in close1y related eggs of the same age are not necessarily identical.
Froriep agrees with Kupffer as to the position of the seg ments -in the early stages, but not as to their number. In H 30 « LOCK [Vo1«. XI.
Salamandra maculosa he found only four segments in the head region, while Kupffer found eight in the same region of Salamandra atra (Fig. 1 Iy). All other observers have found eight or more in the head region, and Froriep stands alone in identify— ing so small a number of segments in the head. In Rana palustris there are about four obvious divisions in the median plate of the head (but, in addition, there are other segments in the neural ridges to the number of ten in the same region). This affords a suggestion that the segments observed by Froriep (see Fig. II6) correspond to those I have observed in the median plate of Rana palustris, and not to the segments in the neural ridges, which are evidently homologous with those in the same Position in Amblystoma, Acanthias, and other forms.
Whether we Hnd the median plate smooth in Amblystoma or faintly segmented depends upon the stage at which the examination is made, and we recognize that the appearances in any one egg are not constant throughout the open groove stagez and, further, that eggs of closely related animals are by no means necessarily similar at corresponding stages. The failure to take things of this nature (which are really common enough) into consideration has, I thinlc, given origin to many differences of opinion and formed the basis of many a controversy. This should make us careful in comparing results to have the same material in precisely the same stages.
In Diemyctylus the conditions are very similar to those in Amblystoma. The neural folds show metameric divisions in the stages with wide-open neural groove. In the three amphibian forms I have examined there are about ten pairs of segments in the broadly expanded neural folds of the head.
In Torpedo ocellata I have also noted the occurrence of this metameric segmentation in several stages. The youngest embryos of that animal I have had corresponds to the stage designated « c « by the Zieglers, and beginning at that point I have traced it along through several stages with a widely open neural groove. This form is so closely related to the one I have especially studied that we would naturally expect — as is the case - marlced similarity between the neural segments.
Torpedo is not so favorable for the study of the segments as» Acanthias The folds are much fainter in the former than in the 1atter, but the significant thing f is, that the number in a given region in Torpedo corresponds to that in Acanthias.
In the chick these segments may be detected as soon as the neural folds are establishechbefore there is any trace of protovertebrae A small number is visible in th—e b1astoderm of the twe1fth hour of incubation just as the head-fo1ds are first out1ined. Their history has been carefully traced by one of my students, Mr. F. A. Hayner, and it agrees in all essentiai features with the history of the same segments in Acanthias Cut 7 shows the appearance of these segments from surface view in a chick embryo of about twentyckour hours incubation There are eleven segments in front of the firsdformed protovertebrae The cell-arrange— ment in the metameres, as shown in Sections, is the same as that described by 0rr in neurorneres of older stages of the lizard (Ano1is).
From these supp1ementary observations it is c1ear that the occurrence of primitive neural segments, as the Erst dehnite expression of rnetamep «« ism, is not an isolated case in C» ,.,,E,,,bsp» «« »die« »Hm Ho» Squa1us. They occur in the same MOSOITOTIIIIO stsmitss »« Ihm« 45 very early period in al1 the other L: forms examinech and in all of them vided throughoutthsircntitslcngtb precede any adivision of the meso— Tat« primitive metamericsegments’
» » while there are but kour mesodekmicz derm into protovertebraa spmzkcs kanns,
V. General Considerations
1. New Aspects of the Problem of Metamerism
The neural segments have now been shown to occur in extremely young stages of a number of animala The fact of their presence in these early stages once established they assume new importance They have too definite a history to admit of being set aside as mere beadings or undulations of no metameric signilicance When I Erst hegen, Some two years ago, to notice these segments in extremely young embryos, I attached no particular significance to them ; but a comprehensive study has convinced me of their segmental importance and everything taken into consideration they furnish, I think a more satisfactory basis for interpretation of metamerism of the head than we have had before. I have pointed them out to several observers, in Amblystoma and the chick, and have found no one who had ever noticed them before ; but through the kindness of others I have had my observations as to the number in the head region confirmed. Since these structures have been overlooked in the earliest stages, it will not be out of place to say a word about their general appearance, and how to see them.
It is extremely difiicult to represent them on paper just as they look. All my drawings are camera tracings, and so far as number and arrangement of the segrnents are coneerned, have been verilied and reverijied over and over again. They are a little too distinct in Some of my drawings ; in making the figures clear enough so there shall be no doubt as to what I mean, their distinctness has been somewhat exaggeratekl On Pl. XXVI I have given a few photographs from untouched negatives that show these segments more as they appear in the actual specimens. To observe them successfully is largely a question of getting shadowa 0ne can loolc directly at the crenated margins without seeing the segments at all, but by tilting and rotating the speeimens until the proper angle of illutnination is found, they may be seen with more or less distinctness, according to the general state of preparation of the material. I have never seen a shark embryo, of the proper age, of any method of preparation, in which I could not detect them. A dead—black background is, of course, the best surface for observing anything of this kind by reflected light A white surface is occasionally recommended, but it is desirab1e to cut off all reflected rays except those coming from the specimen, and one may see delicate structures on a black surface that cannot be seen at all on a white background
The care with which the specimens are prepared makes a great difference in the c1earness with which these structures may be seen. The conditions under which most material is hardened are unfavorable There is usually an albuminous fluid in contact with the embryo, and this, together with minute fragments of yo1k, coagulates when the Hxing reagent is used, and forms a coating over the embryo. The embryos should be washed by a very gentle jet of the reagent immediate1y after their immersion, and the clouded reagent should be removed and rep1aced by clear fluid. This makes the preparations beautifully clear.
2. Are they Artifacts?
There is one question that must be answered for all new structures, wie» are they artifacts —produced by the reagents used? Too great precaution cannot be taken in considering this question.
I have used the following reagents in preparing my material: picro-sulphuric acid, picro-nitric acid, Flemming’s stronger solution, Davidoff's c0rrosive-acetic, chromic acid with a trace of osmic, corrosive sublimate removed with iodine. I have observed Acanthias material prepared by all these various methods, and in every case have found the segments as I have described them. I have always taken the precaution to count the number in the head region, and uniformly have found the same number of segments there, regardless of the hardening reagent used. I have had a complete parallel series of specimens hardened with DavidofFs corrosivesacetic and Kleinenberg’s picro-sulphuric, and have compared the two, step by step, one set serving to fu11y corroborate the observations made on the other.
In Acanthias I have fol1owed the history of these segments very carefu11y, and have traced the earliest-formed ones without a break into the later stages, and identiiied them with the neuromeres. If, therefore, the segrnental f0lds of the open neural groove stage are artifacts, it may with equal force be claimed that the so-ca1led neuromeres, which are later stages of the same segments, are also artifacts.
It should also be borne in mind that simi1ar segments exist in correspondingly early stages in Arnblystoma, Rana palustris, Diemycty1us, and the chic1(. It is tru1y interesting to observe the way in which these different kinds of material bear out the interpretatiom that we are dealing with veritable anatomical structures.
The most satisfactory reply to be given to the question is based on study of fresh Material. Fortunately, the chick offers at all times a source where we can get living embryonic material at any desired age. I have repeatedly observed these segments in living chick embryos I in the eighteen to twenty-twohour stages, and have treated them with reagents while they were actually under observation. The effect of the addition of picrcksnlphuric aeid is to render immediately the walls of the neural groove opaque and more clearly dejined, but not to affect the number or arrangement of the segments camera sketches have been made before the addition of the reagent and compared with those made after the specimens were hardeneii The two correspond, as regards number and arrangement of the segments I have also noted these structures in living embryos between the twelfth and fifteenth hours of incubation Their consecutive history has been traced in my laboratory by one of my students, and the results may be expected to appear later in this Journal.
I have likewise observed them in living embryos of Amblystoma in the open neural groove stage and have compared them with hardened specimens of the same age.
- 1 The embryo was removed with a part of the blastodermto normal salt solation, which was lcept at the proper temperature, and the specimens tilted and rotated with a very fme sable brush over a black background The structures are, of course, faint, and de1icately, although definitely outlined.
3. Do they afford a Clew to the Metamerism of the Brain?
If we grant that these are tru1y segmenta1 structures, the question still remains : Do they afford the best or even a good clew to the number of segments in the primitive brain ? If so, they must be shown to be equal1y important in this direction with myotomes, branchiæ and cranial nerves.
In estimating the claims of these various forms of segmental divisions, to rank as primitive, the question of the time of their appearance in developmental history will be signiiicant On this point I wish to observe, that in all forms I have studied — embracing representatives of Birds, Amphibietz and Selachians — the neura1 segments are among the first anatomica1 Structures to be establishedz before the vestiges of any organs have appeared, the embryo is divided throughout its length into similar segments These metameric divisions, therefore, antedate myotomes, branchiæ, crania1 nerves, or any other Structures that exhibit metamerism. They persist through the early stages of developmenh and become deiinitely related to segrnental nerves and segmental Sense-organs. 1n the light of their very early appearance and their history, I think we are justiiied in saying that they are the most satisfactory traces of primitive metamerism that is presented in the group of Verte— brates. They may be loolced upon as a survival of that genera1 segmentatiom which all agree in assuming for the (not too remote) ancestra1 form.
1t should also be observed that the entire embryo is segmented, and the term «Metamerism of the Head « should be understood to signify merely regional metamerism and not a different kind of segmental division from that occurring in the rest of the embryo.
The study of Sections shows that the neural segments are distinctly differentiated groups of cel1s, and not merely a series of undulations. As has been already pointed out the arrange ment of ce1ls is like that described by Ort« for the neuromeres in Anolis.
4. They are formed Independently of Mesodermic Influence
The next point I wish to maintain with regard to these segments is that they are formed independently of mesodermic influence.
In a preliminary account, I went so far as to state that the segmentation is epiblastic, but that statement should be qualiHed. It is certain1y most clearly expressed in the epiblast, but the other germdayers apparently feel the same iniluence, and partake of this segmenta1 division in a modified degree. It is least c1ear in the rnesoderm — in Sections it may be made out in this layer, and also in the endoderm. The so—called neural segments are developed throughout the entire 1ength of the embryo before any protovertebræ are formed and, therefore, they must be independent of any formative influence of the latter.
There is also a notable difference in the two forms of metameric division ; the formation of protovertebræ, as is well known, proceeds from a certain point forwards and baclcwards, and if the segmentation I have described were rnoulded over the former, we should expect the neural segments to appear gradually, keeping pace with the formation of protovertebrae.
Neverthe1ess, the majority of authors who have touched upon the question have talcen the position that segmental division ok the mesoderm is primary and that the neural segments are moulded over it, but all this time the early history of the neural segments has not been known. Kupffer announced, in I885, that segments appear upon the median neural plate of Salamandra atra while the groove is widely open and before the appearance of any protovertebrae He designated this primary metamerism Seven years later, Froriep («92) studied the question of metameric segmentation upon closely related forms (Salamandra maculosa and Triton cristatus), but reached different conclusions as regards the nature of the segments.
Froriep’s conclusions are based on both surface study and Sections. He shows in very young Triton embryos a seg mented condition in the neural plate included between the incipient neural fo1ds. He expressly states that the segmental divisions do not extend beyond the plate into the neural ridges. The latter, he says, become segmented laterz only, when in the process of c1osing of the neural groove, they are brought directly over the originally segmented plate, and are made to feel the effect of the under1ying segmented mesoblast. The number of segments which he detects in the neural plate at this period is very small. He fmds only four in the greatly expanded anterior part.
My observations (already given on p. 53o), differ from his in a fundamental way: showing, in Amblystoma, Rana palustris and the newt, the presence of unmistakable segments, in the ear1iest formed neural ridges, and not less than ten pairs in the broadly expanded head—p1ate. · In Squalus acanthias, also, it is the incipient neural folds that are the most plainly segmented, and in this animal they are so situated that they cannot possibly come under the influence of the mesoblast in any mechanica1 way. It will be remembered from descriptions of Figs. 27 to 3o and from Sections of the same (cuts 3, s, 6) that the neural folds are formed as wing—like expansions, extending laterally beyond the body. The mesoblast does not accompany these outfo1dings of the epiblast, and when they best exhibit the segmenta1 divisions no mesoblast enters into them.
As for the rest, Froriep gives figures of Sections to show that the mesoblast is at this very early period actually divided into somites that correspond to the external segments His figures certainly show segmental divisions, but being wood—cuts they are not entirely satisfactory when the question arises whether they are real1y protovertebrae Froriep interprets them as such and closes his article as follows: "Die Gliederung des Wirbeltierkörpers ist ursprünglich an das mittlere Keimblatt gebunden; wo sich als Produkte des äusseren Keimblattes segmentale Anordnungen finden, sind dieselben durch Anpassung an die Metamerie des Mesoblastes sekundär entstanden."
The internal condition Hgured by Froriep is interesting when compared with that in Squalus acanthias at corresponding stages. 1 have found in that animal an undulated condition of the mesoblast, but it is not clear in my mind that we are justified in looking upon the divisions as primitive mesoblastic somites ; before seeing Froriep’s paper I had considered them as undulations, probably due to the same influence that has thrown the epiblast so clearly into segments. In many embryos of Squalus, showing epiblastic segmentations, they are, so far as I can determine from Sections, entirely lacking It seems to me that they are rather to be looked upon as a consequence of primitive segmentation and not as protoverte— brave. This view is substantiated by the fact that the formation of the latter takes place in the usual manner at a later period. Considering the late stages in which the neuromeres have genera1ly been described, it was a natura1 inference that they arise after the neural tube is established Minot, in expressing his conception of the formation of the neural segments, based on the descriptions of 0rr and McClure, says : « Their appearance seems to depend on the development of the primitive segments of the mesothelium. When the segments are fully formed, and before their inner wall has changed into mesenchymal tissue, they presst against the medullary tube and oppose its enlargement; at 1east one Sees that the tube becomes s1ight1y constricted between each pair of segments and slightly enlarged opposite each intepsegmental space.« But the neural segments appear so much earlier than the primitive segments of the mesothelium, that this interpretation is no 1onger tenab1e. i i · The facts made known will tend to materially modify the current theory of metamerism, which assumes as fundamental that metameric divisions begin in and depend on the mesoblast. As Adam sedgwick says, in a recent paper: « The Segmentation which obviously persists in the trunk region, and which begins with segmentation of the mesoderm, and is moulded upon it in the manner characteristic of all metamerically seg-— mented animals."« It seems to me a strained inference, that the middle 1ayer—the last germxlayer to be formed—should be the bearer of this primitive metameric division, but, of course, the final appeal must be made to Sections and my sectioned material has given evidence entirely corroborative of the surface observationsz Sections of the embryo i11ustrated in Fig. 25 show that the mesob1ast is nowhere divided into segments, while the epiblastic somites extend throughout its entire length When the stage represented in Fig. 26 is reached, the first mesoblastic somites (three in number) are to be detected, in the trunlc regionz the rest of the mesob1ast remains undivided, but the epib1astic segmentation is as before. I have also made a carefu1 study of the Sections of the chicken embryo illustrated in cut J, and can Hnd no segmenta1 divisions of the mesoblast outside the protovertebræ (iive in number), but the metameric divisions of the epib1ast extend from the extreme anterior tip back to the point where the neura1 fo1ds fade out.
5. Presence of these Segments in Embryonic Rim and Primitive Groove
Another point to be noted regarding these primitive segments is their range. They do not end at the posterior 1imit of the axial embryo, but extend on either side into the embryonic rim. As the embryo increases in 1ength, those in the embryonic rim are apparently drawn into the axial embryoz at any rate, I do not find evidence of extension by budding in the axial embryo, and, in the early stages, the number of neural segments therein increases inidirect ratio to the lengthening of the embrya This would indicate that at least some of the materia1 of the embryonic rim contributes to the e1ongation of the embryo.
In the chick the walls of the primitive groove are also divided into segments that are similar to those that appear in the neural fo1ds.
6. Number of Segments represented in the Brain
There is considerable variance in the conclusion of different observers as to the number of neura1 segments represented in the brain. 0rr, McC1ure, Waters, agree in identifying six in kheihindbkain ok the— chick and ward, and Ave in the hindbrain of Amb1ystoma. In estimating the entire number in the brain, there is to be added, according to Mcclures count, four for the c0mbined fore— and mid-brain, and, according to Waterss five for these regions. Thus, Mcclure considers the brain to represent a total of ten neuromeric segments, and Waters makes a total of eleven segments.
The European investigators, in general, have found a larger number of these segments in the hind-brain, and usually have not observed them in the fore—brain. Dohrn gives eight in the bony ftshes, Rabl seven or eight in the chiclg Hoffmann seven in Reptilia ; Froriep counted twelve in the entire brain of mole embry0s, and Zimmerman gives thirteen in front of the vagus The fore-part of the brain has been regarded as non—metameric, and the question of the anterior extension of segmentation is an important one. In regard to that point, the evidence furnished by the neural segments, shows that the segmentation extends throughout the fore—brain. This corresponds with the investigations of Whitman on the nervous System of C1epsine, in which he shows conclusively that the cerebra1 ganglia of that anima1 belong to the metameric series.
Recent observations on a different set of segments, sei-Jan, segmental divisions in the mesoderm of the head, have brought to light a re1atively large number of cephalic segments Dohrn, in I890, observed eighteen or nineteen mesodermic segments in the head of Torpedo marmorata of 3 mm. in length. Killian («91) in the following year describes seventeen or eighteen in Balfour stages F and J of Torpedo ocel1ata. He also makes a correction to Dohrn’s enumeration, making it correspond with his own. ln later stages of Selachians, as Van Wijhe has shown, they are reduced to nine, or to ten according to Miss P1att.
There is such a fundamental relation between muscle and nerve that we might expect myotomes and neural segments to bear a direct numerical relation. My observations show a smaller number of actual segments in the brain-walls than that tarrived at by Dohrn and Killian through studying the myotomes of the head region. In the hind-brain I have found» eight segments represented in the earlier stages, and a ninth segment which, to all appearances, belongs to those of the spinal cord, but later this ninth Segment becomes clearly associated with those of the hind-brain. I have taken this to signify that the hind-brain has encroached on the territory of the .spinal cord, and has embraced at least one Segment origina11y be1onging to the cord. It is reasonable, on theoretical grounds, to suppose that such a process has taken place and has been several times repeated.
In order to bring the segments of the brain into satisfactory evidence I have completely laid bare the brain-walls by removing the overlying layers of mesoderm and epidermis The segments after being exposed in this way stand out so clearly that I feel considerable coniidence in my count of them in Squalus acanthias. In addition to the nine segments in the hind-brain, I have found five in the combined fore- and mid-brain. In the latter particular I agree with Waters, but, of course, differ as regards the total number in the brain. Mcclure says (p. 39): «I will show that Dr. Hoffmann is probab1y wrong in considering the hind—brain as consisting of seven segments, and that the Segment considered by him as the iirst Segment of the hind-brain is rather the posterior Segment of the mid-brain; in other words, it is the second neuromere of the mid-brain." The recently adduced evidence is all in favor of Hoffmanns observation. According to my observations, there are still represented in the otogeny of squalus at least fourteen paired neural segments belonging to the brain region. There should be added to this enumeration the median unsegmented tip which terminates the line of segments in front, and, as stated above, there are reasons to suspect (a1though no direct evidence in that structure) that there may be more than one segment included in that terminal piece. If the terminal piece represents a single pair consolidated, then there are ftfteen neural segments in the earliest condition of the brain of Acanthias.
7. Relation of the Neuromeres to Sense-Organs and Cranial Nerves
The Sense-Organs and cranial nerves, undoubtedly, at Hrst sustained definite segmental relations to the neural segments. In the spinal cord there is still a pair of nerves for each neuromere, but in the brain the primitive re1ations have been greatly modified or ob1iterated. There is not sufåcient evidence to show what might have been the primitive arrangement in that region. Of course, we bear in mind that the sensory fibres grow centripeta11y, but if they appertained to a particular segment, we might make a tentative estimate of numerica1 relations as follows :
- I. First Neuromere of Fore-brain, Olfactory
- II. second N euromere of Fore-brain, Optic.
- III. Third N euromere of Fore—brain, possibly nerve to Pineal Senseorgan ?
- IV. First N euromere of Mid-brain, 0culo-motor.
- V. second Neuromere of Mid-brain, Trochlearis.
It is easier to assign relations for the segments of the hind-brain. In my assignment I agree substantia1ly with Hoffmann.
- VI. First Neuromere of Hindibraikn anterior root of Trigeminua
- VII. Main root of Trigeminus
- VIII. Third Neuromere of blind-main; no nerve root, at least in ear1y stages.
- IX. Facialis.
- X. Auditorzn The roots of the Facialis and the Auditory arise Separately in squalus acanthias.
- XI. G1osso—pharyngea1.
- XII, XIII, XIV. Roots of the vagus.
At the time of its first appearance the auditory saucer 1ies in front of the tenth neuromere, but is soon shifted backwards opposite the eleventh.
Minot has directed attention to the fact that Miss PIatt and McClure ignored the difference between ganglionic and medu1lary nerve-f1bres, and this is important ; but the ganglion ridges are divided in the same way as the neura1 ruhe, and we may spealc of the neuromeres as including the segments of the ganglion ridge.
The re1ation of the nerveckibres to the neuromeres will be considered more in detail under the heading of The Nerves.
8. Head and Trunk
It would be a great convenience to anatomists to have Some means of distinguishing between the head and trunk of very young embryos. It is genera11y regarded as impossib1e, on account of the laek of deHnite landmarks, to assign such a line of division, in early stages, before the origin of the auditory vesic1e. As Sedgwick says, in the artic1e referred to above, « The term head here must be regarded as meaning the anterior end of the body, for it is not possible in these young embryos t·o distinguish the head from the trunk." Nevertheless, in the young embryos of Squalus acanthias, there seems to be a natural line of division. The neural folds in this anima1 run forwards with the margins nearly parallel to one another, and then expand in front into the broad cephalic p1ate. The expansion takes place rather abrupt1y, and it is possib1e, in very young stages, to draw a line indicating where the expanded part of the cepha1ic pIate joins the nonexpanded part of the embryo. This line may be drawn without any reference to the number of primitive segments that it will cut off. The position of such a line is indicated by fix-i in Fig. 26. This is, in Squalus acanthias, just in front of the point where, subsequently, the vagus nerve begins. As before indicated, there are uniform1y eleven neural segments in front of this line. Their number remains the same from the ear1iest stages until the anatomical landmarks (auditory vesicle and nerves) appear that enable us to determine the limits of the head -region. In this animal, we may identify that part of the head that 1ies in front of the vagus nerve by counting the first e1even neural segments. It will be merely a question of agreeing upon the number of primitive segments be1onging to the vagus, to enable us to locate with defmiteness the hindermost limit of the head. Besides being of use in other ways, this would enable us to say, even in the earliest stages, what is head mesoblast and what is trunk-mesob1ast.
It is interesting, in this connection, to notice that there is in Amblystoma a similar greatly expanded cephalic part with an assignable line of its union with the non-expanded trunk region, and there are also in this form ten or eleven segments included in the expanded head part. In the chick there are eleven segments in front of the first formed protovertebrae.
Some of the more important facts and conclusions regarding the neural Segment may be briefly stated:
1. They are natural structures, not artifacts produced by the reagents This has been shown by their constancy in ap pearance and general characteristics when different reagents are used; their consecutive history; their similarity in different kinds of embryos; and lastly, by their presence in fresh rnaterial before any reagents have been used.
2. Early Appearance. — The so-called neuromeric segmentation is the first to appear. It arises long before there are any segmental divisions of the mesoderm, and therefore cannot depend upon segmental divisions of the middle germ-layer. Neuromeric segmentation is more primitive than mesodermic segmentation.
3. Structure. — The cells in these segments are characteristica1ly arranged even in the earliest stages, and their arrangement and their structure would indicate that they are definite differentiations of cell areas, not merely mechanical undulations.
4. Extent. — The entire embryo is divided into similar segments, and passes through an arthromeric condition simi1ar to that of arthropods and worms. i ln Squalus the neural segments extend also into the germ-ring, and in the chiclc at times into the primitive streak.
5. Number. - There are in Squa1us acanthias eleven segments in the brain region in front of the vagus nerve, and fourteen paired segments in the entire brain region, as follows: nine in the hind-brain, two in the mid-brain, and three in the fore-brain; this does not include the anterior tip, which may represent several consolidated segments.
6. Backward growth. — There is Some evidence to show that the spinal cord region is being encroached upon by backward differentiation. Early in the history of these segments there are seven that clearly belong to the hind-brain, and later there are successively added two more.
7. Nature, position. — These segrnents are clearest in the epiblastz the other layers are slightly afkected by the segmental influencez the mesob1ast least of all. The segments are serially homologous
8. XXVII-Eises. —They are directly (if not genetically) related to the cranial and spinal nerves. The neural ridges are divided in the same manner as the neural tube. The segments are also directly related to the Sense-Organs through non-es.
9. Twcxæsosweczxiows w- MociMc-2sz«2·o-2F. —The modifications are most extreme in the anterior part with the early obliterasp tion of those belonging to the foresi and mid—brains. Those in the hind—brain region are clearly deiined for some time after« the establishment of the cranial nerves, and then they fade away.
This is about all that can be said about their transformations, for the modiftcations have not yet been worked out in detaiL
- Locy 1895 Contents: General Introduction | Part I - Metamerism of the Head | Part II - The Sense-Organs | Figures
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