Talk:Paper - The corpus ponto-bulbare - a hitherto undeseribed nuclear mass in the human hind brain (1907)

From Embryology

The Corpus Ponto-Bulbare — A Hitherto Undescribed Nuclear Mass In The Human Hind Brain

Essick CR. The corpus ponto-bulbare - a hitherto undeseribed nuclear mass in the human hind brain. (1907) Amer. J Anat. 7: 112-136.


Charles R. Essick,

Johns Hopkins Medical School.

With 12 Figubes.

In the following paper I have described a ganglionic mass which can be seen overlapping the restiform body just caudal to the dorsal cochlear nucleus and forming a direct lateral process or extension of the ganglion mass of the pons. This structure was undoubtedly seen long ago by Clarke/ but he considered it to be an aberrant strand from the striae medullares, and probably it had been seen by Arnold, who also described it as belonging to the strial system. The caudal tip, which is often prominent, has attracted the attention of various authors, and was particularly described by Retzius and Henle ' under the name " Ligula " and " Ponticulus " ; none of these observers, however, followed the structure in cross sections, and its significance was consequently overlooked. As far as could be learned, the following paper gives the first description of its whole form and for the first time demonstrates its connection with the ganglion mass of the pons. I have found that it is constantly present to a greater or less degree in all human brains and possesses definite characteristics which seem to me to warrant a name such as is herewith proposed — the corpus ponto-bulbare or ponto-bulbar body.

An adult formalin specimen, in which the structure was especially prominently developed, was photographed (Fig. 1) and after mordanting in a double chrome salt solution was imbedded in celloidin and cut in serial sections. The alternate sections were stained by the Weigert

Mr. J. L. Clarke: On the Intimate Structure of the Brain. Phil. Trans. London, 1868.

^Gustaf Retzius: Das Menschenhirn, Vol. I, Stockholm, 1896.

'J. Henle: Handbuch der Systematischen Anatomie des Menschen, Vol. HI, Part II, Braunschweig, 1879.

American Journal of Anatomy. — Vol. VII.

120 The Corpus Ponto-Bulbare

Pal method; the intermediate ones, for purpose of cell study, were stained by various cell stains, thionin having proved most satisfactory. A wax-plate reconstruction after the method of Born was made from this series showing the ponto-bulbar body and its relations to the adjacent cranial nerves, the cochlear nuclei, the trapezoid body, and the restiform body. A composite drawing of this model showing its relations to the surrounding region is represented in Fig. 12.

Through the kindness of Prof. Franklin P. Mall and Dr. Florence E. Sabin, I had the opportunity of making a macroscopical examination of fifty brains fixed in formalin, a description of which is included in

Fig. 1. Photograph of a human medulla with a well-marked corpus pontobulbare (c), transverse sections of which are represented in Figs. 3-11. Fig. 12 represents the same medulla reconstructed into a model and drawn in ventro-lateral view. X 2.

this paper. They also placed at my disposal several series of both adult and new-born medullas which were valuable as a. control over my own series. For these and many other courtesies on their part I take pleasure in using this oiDportunity to express my thanks.

Gross Appearance.

The structure, which I have designated as the corpus ponto-bulbare, constitutes a horn-shaped process of the pons which extends caudally, wrapping around the lateral side of the restiform body so as to end on its dorsal surface; forming, in part, the lateral boundary of the fourth ventricle. If one takes a brain from which the pia mater has been

Charles E. Essick 121

stripped off, the ponto-biilbar body can be seen making its appearance on the ventro-lateral surface of the pons near the emerging root bundles of the trigeminal nerve and extending backward passing between the roots of the acoustic and facial nerves. Continuing backward ventral to the restiform body the main mass passes dorsal to the emerging glossopharyngeal roots, finally, assuming the dorsal position shown in Figs. 1 and 2. So that the ponto-bulbar body is superficial throughout its extent and its development usually throws the surface into a more or less wellmarked fold which can be traced from its beginning on the ventrolateral surface of the pons to its ending at the lateral wall of the fourth ventricle. As soon as these caudally directed fibers leave the pons it becomes evident that they are accomjDanied by a cellular mass which is itself distinct from the neighboring structures. This nuclear mass fused with the pons between the facial and .acoustic nerves gains the dorsal surface of the medulla by passing around the restiform body caudal to the dorsal cochlear nucleus.

Out of fifty brain specimens which were examined macroscopically, it was found that nearly all showed clearly the ponto-bulbar l3ody throughout its entire extent. Aljout half showed a prominent ridge from its beginning on the pons to its ending at the lateral wall of the fourth ventricle. In all the brains examined, the ponto-bulbar body was visible macroscopically in some part of its course. The reason it is not seen throughout its whole extent in all of the specimens is not due to its absence, but to its retraction beneath the surface of the hind-brain.

At the point of its emergence on the pons its position, size, and shape vary considerably in different brains. The ponto-bull:)ar body appears first as small bundles of fibers which turn away from the tranverse pons fibers proper somewhere median to the trigeminal nerve. Gradually these bundles converge to form a more or less compact ridge which curves backward just behind the fifth nerve and runs caudally almost at right angles to the other pons fibers, to pass between the facial and acoustic nerves. The eminence thus formed is generally single as far front as the fifth nerve and measures from 3-6 mm. in widtJi ; the extent to which it is raised above the pons surface varies from a sheet-like layer of fibers to a corded ridge. The manner in which the ponto-bulbar body sweeps out of the pons resembles somewhat the converging roots of the cauda equina ; one specimen showed a distinct claw-like arrangement of fiber bundles. At times the fibers go to form two separate ridges which unite just cephalad to the facial nerve. The region from which these fibers turn

1"32 The Corpus Ponto-Bulbare

out from the pons extends from a point about 5 mm. in front of the fifth nerve to 4 mm. in front of the seventh nerve. An interesting variation to this general arrangement is found where fiber bundles encircle the trigeminal nerve or appear to come up out of the pons immediately behind it, and then take the regular course between the facial and acoustic nerves. Few specimens did not show this part of the ponto-bulbar body very plainly.

Although no reference is made to it, an admirable picture of the pontine part is given by Eetzius * in his Atlas, Plate IV, Fig. 1. On the left half of the brain (right side of figure) the eighth and seventh nerve roots can be seen just median to the flocculus. In front of these nerves a definitely corded ridge is present passing forward, median to the trigeminal nerve to be lost in the pons. The choroid plexus from the lateral recess hides the corresponding right part of the brain in the neighborhood of the seventh and eighth nerves, but the way in which the ponto-bulbar body arises from the transversely directed pons fibers, median to the root bundles of the fifth nerve, and then assumes a caudal direction, is particularly well illustrated.

By far the most constant part of the pouto-bulbar body, appearing on mere gross examination of brain, is the welt-like ridge which it forms ventral to the restiform body after it has passed between the facial and acoustic nerves. This portion is difficult to display in the whole brains preserved in formalin, because it necessitates removing the pia mater from the depression between the flocculus and the olive and, unless great care is used, this procedure takes with it the root bundles of the glosso-pharyngeal nerves. The part of the ponto-bulbar body which is then brought into view, extends about 6 mm. caudally as a prominent elevation having a width of 3 mm. in all s^DCcimens which were measured. We find it nearly always extending so as to surround the most cephalic of the glosso-pharyngeal roots. In this event, the appearance is that of a hill-shaped eminence with the stumps of the glosso-pharyngeal nerve emerging from the summit. On the left side of the brain illustrated by Eetzius,' Fig. 11, Plate XXXVIII, there appears the best illustration of this portion of the ponto-bulbar body. Here the hypoglossal nerve roots may be recognized as they emerge mesial to the olive. Lateral to the olive is a well marked ridge with five root bundles of the glossopharyn

Gustaf Retzius: Biologische Untersuchungen, Neue Folge XII, 1905. "Gustaf Retzius: Das Menschenhirn, Vol. II, Stockholm, 1896.

Charles E. Essick 123

geal nerve emerging from the summit. This ridge can be traced anteriorily as it passes between the facial and acoustic nerves and a very little part of the pontine portion may be seen just anterior to the seventh nerve as is indicated by vertically directed lines in contrast to the horizontally placed lines representing the direction of the pons fibers proper. Other figures, 9, 13, and 13, of the same plate, furnish good illustrations of this region.

At the point where it emerges below the lateral recess, we see it turning around the restiform body just caudal to the dorsal cochlear nucleus from which it is separated by a shallow sulcus. In some specimens it might easily be mistaken for the dorsal cochlear nucleus ; but if one will carefully follow the cochlear nerve, the real dorsal cochlear nucleus can be made out in front of the attachment of the tela choroidea inferior, i. e., the dorsal cochlear nucleus lies in the lateral recess and is covered with ependymal cells; while the ponto-bulbar body, which often approaches the dorsal cochlear nucleus in elevation and extent, is situated more caudal, and makes a curve of greater radius around the restiform body to gain the dorsal surface of the medulla.

On the dorsal surface of the medulla, the ponto-bulbar body undergoes its greatest variation in different brains and, in fact, on the two sides of the same brain. Instead of the welt-like ridge which it presents in the more cephalic part of its course, it here spreads out into a flattened leaf-like structure overlapping the restiform body and the lateral part of the floor of the fourth ventricle. In its exact form it shows considerable variation. Of the fifty brains which were examined five of them presented the prominent mitten-like form 4.5 — 6.0 mm. wide with a free tip, projecting, as it were, out into the tela choroidea, as is seen in the Fig. 1. These would be called markedly well developed. On the other hand, in about 40% of the specimens this part of the ponto-bulbar body forms such a thin lamella that it is not noticeable macroscopically on at least one side of the brain. Between these two extremes there are all gradations. It may take the form of a single ridge usually 2 — 3 mm. in width; or may be divided into two finger-like ridges. In well developed cases the caudal tip may help to form the roof of the fourth ventricle, forming a free end to the edge of which is attached the tela choroidea. In the specimen photographed. Fig. 1, there was a tip of this kind 1.5 mm. long. The fact that the tela choroidea is attached to it, makes it a thing easily damaged in removal of the pia. A drawing of an average human medulla is represented in Fig. 2 and this gives the usual position and size which the ponto-bulbar body presents in a dorsal


The Corpus Ponto-Biilbare

view of the fourth ventricle. It may be seen turning around the restiform body caudal to the line of attachment of the tela choroidea along the lateral recess and projecting as a lamella into the roof of the fourtli ventricle.

There are many excellent photogravures of this region in Ketzius," in which the relations of the ponto-bulbar body to the restiform body and the fourth ventricle are brought out. In ]iis PJate XXXV, Fig. 9 shows



im 1


w ^

!,■ ^



^^^^^^^^Nu. cock. dors.

^MwB^^^ Taerviai

^^^^^^•' Corpus ponto


^x ,


^^^HF bulbars ^^^W Corpus restlforme

^» Funiculus cuneatus

Fig. 2. Drawing of an average human medulla giving the usual appearance of the corpus ponto-bulbare caudal to the dorsal cochlear nucleus. X 2.

a doubled ridge, above described as " finger-like," on the right side extending antero-lateral from the base of the detached taenia (labeled " t "). A similar though not so well marked arrangement is shown in Plate XXXVI, Fig. 2. Figure 4 of Plate XXXVII gives the best illustration of the whole dorsal extent of the ponto-bulbar body because the pia has been completely removed from the specimen. It may be recognized as the first transversely placed ridge which encircles the restiform body in front of the clava. The way in which it turns around the restiform body

" Loc. cit.

Charles E. Essick 135

and ends at the attachment of the tela choroidea is particularl}^ well shown. It compares with the right half of my figure 1 except that the free edge is apparently lacking, possibly liaving been torn away with the pia. Plate XXXVIII, Figs. 5 and 6, also give admirable pictures of this region; attention might be called to the free edges of the ponto-bulbar body on both sides of the former figure.

Histological Appearance.

On microscopical examination it is foujid that the ponto-bulbar body consists of a mass of multipolar ganglion cells supported in a dense neuroglia network closely resembling the nuclear masses of the pons. The lateral or free surface of tliis ganglionic body is entirely covered by a layer of medullated nerve fibers which in general follow the direction of the long axis of this structure and continually give off collaterals which branch among the underlying ganglion cells. On its median surface throughout its greater extent there is no such layer of fibers present and consequently the ganglion mass lies directly adherent to the adjacent structures and fuses more or less completely with them. The ponto-bulbar body presents a uniformity of histological structure throughout, so that in passing from section to section the same characteristics can be made out everywhere. The cells lie very close to one another and in large areas are separated from each other by less than the width of a cell. The narrow space between the cells is found to be filled by a dense framework of neuroglia and an intricately interwoven network of very fine nerve fibers, which in their complexity and number resemble the hypoglossal nucleus. On the other hand this rich felt-work stands out in contrast to the ala cinerea, which in Weigert sections gives such a clear picture.

The cells are of the multipolar type and show several different forms ; many are spindle-shaped with their long axes parallel to the fibers; wliile most have the characteristic multipolar form. The former are found in greater numbers in the part of the ponto-bulbar body which lies lateral to the restiform body, the latter in the rest of the body. The size is fairly uniform, so that, of cells which were measured in sections stained in thionin, the average was 23 x 14.5 fx, some cells being as large as 29 x 17 ^, others only 17x10 fi. This corresponds exactly to measurements taken of the ganglion cells scattered through the pons. To afford a means of comparison, cells from the hypoglossal nucleus were measured in the same sections and were found to be 10 x 23 /j.. These measurements

126 The Corpus Ponto-Biilbare

were made on celloidin sections of tissue which had been fixed in formalin and mordanted in a double chrome salt.

A section in any part of the ponto-bulbar body shows the free border of the nucleus outlined by a layer of nerve fibers cut either transversely as in Figs. 3, 4, 5, 6, and 7, or longitudinally as in Figs. 8, 9, 10, and 11; in no case was there a stratum of cellular material on the surface. The diameter of these fibers, including their medullary sheath, ranges from 0.8 — 2 ix; those making up the larger strands 1 — 2 ju, thick, while those between the cells generally 0.8 in thickness. This small size makes the fibers easily distinguishable from the surrounding structures with which they come into close relation. The fibers and cells together form a structure whose shape varies in section but the relation of cells to fibers are always such that masses of nuclear material lie between the fiber bundles and the rest of the brain. Occasionally only do we find strata of cells between the fiber bundles as in Figs. 9 and 10.

For the sake of simplicity it might be well in describing its finer structure to arbitrarily divide the ponto-bulbar body into a pontine part which fuses with the ventro-lateral surface of the pons, a middle part which extends median to the ventral cochlear nucleus and is perforated by the glosso-pharyngeal roots, and lastly a broad caudal part which overlaps the restiform body. With this iii mind each part will be treated separately as was done in the gross description.

In the region of the trigeminal nerve small strands of fine fibers separate from the transverse fibers of the pons and turn caudally, forming rather compact small bundles whicli lie on the ventro-lateral surface of the pons just behind the root bundles of the fifth cranial nerve. The addition of fibers from the lateral side or middle penduncle is .comparatively small, while the strands from the pons proper continue to turn in almost as far caudal as the emerging facial nerve. The nerve fibers gradually unite to form more or less rounded bundles which are spread out on the ventro-lateral border of the pons. Although there is this gradual increase of fibers from both sides, the cross section does not increase proportionately as it runs caudally. The reason for this is found to be due to rather large bundles of fibers which separate from the other fibers and run into the middle peduncle of the cerebellum.

In .the region of its most cephalic part, the ganglionic mass adjacent to the fibers is very small ; but as it approaches the caudal border of the pons, it increases in amount, fusing so completely with the pontine nuclei that no sharp line of demarcation can be drawn. This is well illustrated in Figs. 3 and 4, where the fibers, cut transversely, can be seen on the

Charles E. Essick 127

surface of the pons between the facial and acoustic nerves. The nuclear mass which lies adjacent to the fibers can be traced serially back into the caudal tip of the process, but it will be noticed in these two sections that the nuclear mass is directly continuous with that scattered throughout the pons. Fig. 4 shows the nervus intermedins passing tlirough the ponto-bulbar body on its way to join the facial nerve in the same manner as the giosso-pharyngeal nerve does farther caudally.

We may say its middle division begins at the point where the pontobulbar body leaves the pons between the facial and acoustic nerves. It lies ventral to the restiform body and all the fibers in our sections, as in Fig. 5, are cut across transversely — that is they are running caudocephalically. Here again it will be noted that the fibers are arranged along the surface. Throughout the length of the ventral portion numerous small strands of obliquely cut fibers are found scattered in the nuclear mass. By carefully following the sections serially it becomes evident that these small bundles are passing between the restiform body and the ponto-bulbar body. These nerve fibers leave the restiform body and travel obliquely out across the nucleus in a caudal direction and finally take their position on the surface of the nucleus, thereby swelling the number of fibers. As we pass back and forth in serial sections, a variability in the number of fibers cut transversely, calls attention to the fact that there are not only fibers arising in the nucleus itself and running along the edge, but that there are also fibers which are passing into other structures. These obliquely cut fiber bundles can be seen in Figs. 5 and 6, between the superficial fringe of transversely cut fibers and the restiform body. The exchange of fibers between the ponto-bulbar body and the restiform body takes place even in the regions where the nucleus has fused with the pontine nuclei and although the obliquely cut fibers with the same directions persist, the cross fibers from the pons and the trapezoid body make it impossible to trace bundles from the ponto-bulbar body to their junction with the restiform body. The ponto-bulbar body is separated from the restiform body by the trapezoid body as soon as the level of the ventral cochlear nucleus is reached. The separation is partial in Figs. 5 and 6, and complete in Figs. 3 and 4, where the trapezoid body can be traced to the superior olive. Attention might be called to Fig. 7, where the coarse fibers of the most cephalic of the giosso-pharyngeal nerve roots may be followed from the fasciculus solitarius through the descending spinal root of the fifth nerve and the restiform body and out through the most ventral part of the ponto-bulbar body. Fig. 7 also shows the dorsal cochlear nucleus in its relation to the ponto-bulbar body which, a

128 The Corpus Ponto-Bulbare

few sections farther caudally (c.p. Fig. 8), occupies a simihir position with regard to the restiform Ijody. Sections like Fig. 8 might easily be misinterpreted as containing a dorsal cochlear nucleus.

The caudal part embraces that portion of the ponto-bulbar body which sweeps around the restiform body to gain the dorsal surface of the medulla. As its most cephalic border is reached, the fibers are cut a little more obliquely indicating that they are turning out of their caudocephalic course, see Fig. 8 and Fig. 9. A little farther backward the fibers run around the restiform body in a rather heavy layer (Fig. 10) ; the nuclear mass, on the other hand^, does not follow them immediately, so that in sections where the front part appears, fibers alone are seen encircling the restiform body — c.p. Fig. 8. When the body reaches its greatest development, the fibers can be traced for some distance as they bend around the restiform body; always separated from it by a mass of nuclear material. It is here that the fiber bundles which skirt along the edge may be interspersed with thin strata of ganglion cells as in Figs. 9 and 10. From the part of the nucleus which adjoins the median vestibular nucleus a few small strands of fine fibers could be traced as they ran cephalically toward one of the striae acusticte, which had a caudal direction. On reaching this stria, the fibers made a sharp turn and joined it. N'umerous other smaller groups of fibers had a similar direction and gave the impression of joining the striae but only one such bundle could be followed from the ponto-bulbar body to the striee acusticje. It is probably this connection which led Clarke to interjjret tliis structure, which he undoubtedly saw, as belonging to the group of stris aciisticas.

The caudal tip extends out as a tongue-like structure which projects to a greater or less extent into the roof of the fourth ventricle in the edge of the tela choroidea inferior, see Figs. 9, 10, and 11. The ventral side of the free process is lined Ijy a layer of ependyma which is continuous with the ependymal cells lining the rest of the ventricular cavity, while the dorsal side is covered by pia mater — in every respect then it appears to be an enlarged portion of the secondary " Eautenlippe " of His. An illustration not unlike my Fig. 9 is given by Spalteholz ' in his figure 727. Here it appears to be disregarded as an individual structure and has no label referring to it; the characteristic superficial fiber layer, the fusion of the cell mass with the restiform body, and even the

'Werner Spalteholz: Hand Atlas of Human Anatomy, Vol. HI, Leipsig, 1906.

Charles R. Essick 129

tongue-like process are well illustrated. His figure 728 has almost the same level as Fig. o, aud here he has called it "pons (Varoli)." This latter is very natural when we consider that a little farther front the ponto-bulbar body is actually fused with the pons.

In the tongue-like projection the cells occupy a central position, being surrounded on all sides by the fiber bundles which may then be regarded as forming a capsule; Figs. 9, 10, and 11 show this very well. Single fibers may be traced for some distance in a section showing that here in the dorsal portion the fibers take the same direction as the body that is toward the median line. TJiese fiber strands run along the border of the nucleus and converge as they turn around to gain the lateral side of the restiform body forming a bundle which is gathered together in wisp-like fashion. The eft'erent fibers from the abducens, facial, and other nuclei have this same confluence into a root bundle. Toward the median end of this projection appears a rather large bundle cut transversely which gives off collaterals to the nuclear m.aterial in a very irregular manner.

The above description applies to adult material, but I have also had the opportunity of examining a series of the new-born babe, the one which was prepared by Dr. John Hewetson and belongs to the collection of this laboratory. In this series the ponto-bulbar body could be distinctly made out, and in its relations to the surrounding structures showed the same features as described in the adult. However none of its fibers are as yet myelinated, the non-myelinization made it particularly easy to identify it in the region of the cochlear nerve, ventral cochlear nucleus, and trapezoid body, all of which have at this age acquired their myeline sheaths. Further caudally where it lies against the side of the restiform body it comes into contact with the cerebello-olivary fibers. Although these unmyelinated fibers fuse with the ponto-bulbar body, the closely arranged ganglion cells of the latter are easily distinguished from the coarse nerve fibers. The pons with which this body fuses cephalically also contains no myelinated fibers. It may also be added that it is present in the early stages of both human and pig embryos. It can be seen in the reconstructions of this region in a 50 mm. human embryo which are about to be published by Dr. Streeter, and it was also noted by him in dissections of pig embryos.*

'G. L. Streeter: Development of the Membranous Labyrinth and the Accoustic and Facial Nerves in the Human Embryo. Amer. Jour, of Anat., Vol. VI, p. 163.

130 The Corpus Ponto-BiTlbare

In conclusion, I wish to acknowledge my indebtedness to Dr. George L. Streeter, at whose suggestion and under whose supervision the study of this ganglion mass was carried on. I also desire to express my thanks to Prof. Max Brodel for his assistance in preparing Figs. 2 and 12.


Brach. pont.^ Brachiiim pontis. Corp. Rest. =^ Corpus restiforme. Corp. Trap. = Corpus trapezoideum. Fibr. Cereb. OZty. = Fibrfe cerebello-olivares. N. Coch. = Nervus cochlearis. N. Vest. = Nervus vestibularis. N. Glossoph.^ Nervus glossopharyngeus. Nu. Coch. Dors.^ Nucleus cochlearis dorsalis. Nu. Coch. Yent. = Nucleus cochlearis ventralis.


Fig. 3. Transverse section through the pons immediately in front of the emerging facial nerve. X 4.5.

Fig. 4. Transverse section just caudad to the preceding, through the nervus intermedins, showing the fibers of the corpus ponto-bulbare in cross section. X 4.5.

Fig. 5. Transverse section through the medulla at the border of the pons. X 4.5.

Fig". 6. Transverse section through the cephalic end of the olive. X 4.5.

Fig. 7. Transverse section through the caudal- border of the dorsal cochlear nucleus. X 4.5.

Fig. 8. Transverse section through the cephalic border of the corpus ponto-bulbare where it encircles the restiform body. X 4.5.

Fig. 9. Transverse section immediately caudad to the preceding, showing the longitudinally cut fiber strands turning around the restiform body. X 4.5.

Fig. 10. Transverse section through the thickest part of the projecting caudal tip of the corpus ponto-bulbare. X 4.5.

Fig. 11. Transverse section through the middle of the olive, showing the caudal end of the corpus ponto-bulbare. X 4.5.






















^^l^r .jf








Fig. 12 represents a somewhat schematic drawing of a model reconstructed from sections of the specimen shown in Fig. 1, in a ventro-lateral view of the medulla and pons. Part of the nucleus cochlearis dorsalis (nu. coch. dors.) has been removed to show the relations of the corpus restiforme (c. r.) Numerals V-X refer to the cranial nerves. I refers to the nervus intermedius. X 3.75.




From the Anatomical Laboratory, Johns Hopkins University.

With 5 Figures.

Eoux ' first pointed out that the material which forms the embryo of the frog is laid down in the black-white ring around the equator of the egg, and that the embr^^o is formed b}^ a process of concurrence in that this material grows over the white hemisphere. If this is prevented, as in his case of asyntaxia medullaris, a half embryo develops on either side of the equator of the egg. Eoux's observations and experiments were confirmed by Morgan ^ in a somewhat similar series of experiments. Hertwig* was able to produce the bilateral half embryos by allowing the eggs to develop in salt solutions. I have seen many similar forms from eggs of rana sylvatica and rana palustris which were kept on ice for a prolonged period of time. In such abnormal forms the lips of the blastopore which fail to grow over the large yolk plug differentiate into these modified half embryos with a central nervous system, muscle, the chorda, and the roof of the archenteron.

More recently Morgan * has investigated the question of the location of the embryo-forming substances and concludes that the material is first in the upper hemisphere of the developing frog's egg and is later carried downward into the germ ring.

'■ Ueber die Lagerung des Materials des Medullarrohes in gefurchten Froschei. Anat. Anz., Vol. 3, 1888.

- The formation of the embryo of the frog. Anat. Anz., Vol. 9, 1894.

= Urmund und Spina bifida. Arch. f. mikr. Anat., Vol. 39, 1892.

•* The relation between normal and abnormal development of the embryo of the frog, X. A re-examination of the early stages of the normal development from the point of view of the results of abnormal development. Arch. f. Entwickelungsmech. XIX, 1905.

The origin of the organ-forming materials in the frog's embryo. Biol. Bui., XI, 1906.

American Journal or Anatomy. — Vol. VII.

138 TransjDlantation of the Lips of the Blastopore

The following experiments were made, partly with the view of determining what organ-forming stuffs are present in the lips of the blastopore; but more especially to determine the extent of independent selfdifferentiation possessed by this structure when small pieces were isolated and transferred into strange environments. In the examples of asyntaxia medullaris there develops from the lips of the blastopore on either side a half embryo with spinal cord, notochord, and myotomes, so that evidently the fusion of the lips is not necessary for their differentiation, but it might be, of course, that some of the other relations are essential, such as the anterior, posterior, or the lateral connections with the embryo. But by cutting out and transplanting small sections of the lips these factors were eliminated.

The first series of experiments were made on rana palustris at a time when the dorsal and lateral lips are well marked, the ventral lip is just beginning to appear. Small blocks of tissue were cut from the dorsal and lateral lips (1, 2, 3, and 4, Fig. 1), so as to include the entire thickness of the lip with both ectoderm and endoderm. These pieces were transplanted beneath the ectoderm of older embryos of the same species in the region of the otic vesicle. The -embryos into which they were transplanted show the beginnings of the tail bud. The pieces transplanted are soft and delicate, and of course very liable to injury during the operation by distortion, tearing, or loss of a portion.

In the first experiment (taj, piece (1), Fig. 1, was transplanted into an older embryo which was killed 7 days after the operation. The sections (Fig. 2) show in the region of the otic vesicle a large notochord, perfectly normal in its differentiation and extending for many sections in a direction parallel to the long axis of the central nervous system. Portions of the chorda are irregular in outline and it is not quite as large as the normal one in cross diameter. Near the chorda and ventral to the otic vesicle is a large mass of muscle, irregularly arranged, normal in its differentiation, showing striation and other characters of myotomic muscles. I am unable to find any nerves going to this muscle mass and it lies in a position where normally there is no muscle.

Ventral to the notochord and medial to the muscle mass is a piece of central nervous system having both white and gray substance and a central canal. In places the nerve cells seem to be degenerating. The central canal is closed at either end.

I am unable to determine if any of the transplanted tissue differentiated into endoderm, though some degenerating cells in this region may

Warren Harmon Lewis 139

possibly be endoderm. It is evident from this experiment that tissue from the dorsal lip of the blastopore possesses great power of self -differentiation, is already predetermined, and does not need the usual normal relations with the rest of the embryo for its differentiation. At how early a period these tissues are capable of independent self-differentiation is, of course, only possible to determine by further experiments along perhaps somewhat similar lines of transplantation.

The transplanted piece in another similar experiment (ta,) of this series, taken from the region (2) (see Fig. 1), differentiated into notochord, striated muscle, and nervous tissue. The latter, however, is only imperfectly differentiated and shows many degenerating cells. This embryo was killed 7 days after the operation.

In another experiment of the same series (tag), piece (3) (see Fig. 1), was transplanted and the embryo killed after 7 days. There differentiated from it a large mass of striated muscle without nervous connection and a small piece of the neural tube consisting of a single layer of cells (see Fig. 3). ISTo traces of notochord were found so it seems probable that the cells of the transplanted piece destined to form chorda were lost during the operation. The more lateral position of piece (3) does not explain its absence as from piece (4) (see Fig. 1, experiment ta^), a large normal appearing notochord and neural tube tissue differentiated, the latter contains many degenerating cells. No muscle tissue was found and its rudiment was probably lost in transplantation. Both of the above embryos were killed 7 days after the operation.

In another series (tb) four pieces from the dorsal lip of the blastopore of a gastrula, the same age as in the preceding series, were transplanted into the otic region of similar older embryos, with beginning tail buds. These embryos were killed 6 days after the operation. As in the preceding series muscle and chorda develop into quite normal tissue, the former without any nervous connection. The neural tube is always present but shows more or less extensive degenerative changes.

In one of these experiments (tbg) the chorda is in contact with the ectoderm, the latter shows here a modification of its staining reaction with hematoxylin and Congo red. Here, also, the cells of the inner layer of the ectoderm are elongated in an axis perpendicular to the surface. These modifications were very likely brought about in some way through the contact influence of the notochord.

In another similar series (te) an embryo (tCo) was killed 9 days after the operation. The sections show ventro-lateral to the otic vesicle

140 Transjilantation of the Lips of the Blastopore

a well-formed chorda, a group of striated muscle fibers which seem to be spreading out in the form of a muscle (m, m, Fig. 5), and a small and imperfect neural tube. The other experiments in this series show that the dorsal lip has differentiated into chorda, muscle, and nervous system, but all three tissues are not always present, however, owing to loss, probably in transplantation, of their rudiments. The mutual relation of these three tissues varies in such a manner that there is evidently no interdependence, as regards differentiation. The muscle develops perfectly normally without any nervous connection for 9 days at least after the transplantation, as in none of the experiments can nerves be traced to the muscles, either from the host or the transplanted nervous tissue.

These tissues do not seem to influence the configuration or arrangement of the connective tissue about them in any especial manner nor with the exception of the one instance where the chorda has modified the ectoderm do they influence other tissues in the region in which they are developing. In one instance (te,) the chorda rudiment was evidently transplanted near the normal chorda and has differentiated into a chorda, lying parallel to the normal one, both are encased in cartilage of this region, and in some places there seems to be a slight excess of cartilage about the abnormally placed chorda.

It is possible that by the transplantation of small pieces or even groups of cells from younger and younger embryos that the localization of the primary organ or tissue-forming substances can be traced back, step by step, to their more primitive locations in the egg. It may be possible, also, to determine in these early stages correlations necessary for the formation of secondary tissues or for the differentiation of these.

In almost all of these experiments the tissues which have developed from the transplanted piece are much greater in bulk, very much greater in the case of the chorda and muscle than such a piece would have produced in the same tihie had it remained in the normal position in the embryo from which it was taken. This is an indication of how the neighboring parts in a normal embryo must interact upon each other, regulating the size or extent of growth for each such part. It is possible that when such pieces are freed from this influence of the whole on the part, that cell division can take place more rapidly and so produce a larger piece from the same number of cells than under normal conditions.

Although at this early gastrula stage the dorsal and lateral lips of the blastopore are already determined as regards there subsequent differentiation to give rise to chorda, muscle, and nervous tissue, there is evidently considerable difference in the power of self-differentiation in that

Warren Harmon Lewis 1-il

the chorda and muscle develop into much more normal-appearing tissue than the rudiment of the nervous system. The latter, though predetermined at this time, does not seem to be able to' differentiate into perfectly normal tissue after its transplantation. It is thus j)robably dependent on certain, as yet unknown, relations in its normal environment for its more perfect differentiation. Just what these relational factors are or for how long a period they must act is not clear, but a somewhat later stage after the first faint indications of the medullary plate appear the nervous tissue or its rudiment possesses great power of self-differentiation.

142 Transplantation of the Lips of the Bhistopore

Fig. 1. Outline of blastopore region showing pieces transplanted from the dorsal and lateral lip.

Fig. 2. Experiment taj . Piece of the dorsal lip of the blastopore (see (1) Fig. 1) transplanted into the otic region of an older embryo, the tail of which is beginning to show. Embryo killed 7 days after the operation. Section through otic region showing piece of nervous system (n) with central canal and marginal layer, chorda (c) and large muscle fiber mass (m). X 100 diameters.

Fig. 3. Experiment tag . Section through abortive neural tube, from piece (3) Fig. 1. X 200 diameters.

Fig. 4. Experiment tai . Section through chorda and brain from transplanted piece (4) Fig. 1. X 100 diameters.

Fig. 5. Experiment te„. c, chorda; m, m, muscle; n, neural tube. X 50 diameters.