1. A pathologic phenomenon consisting of complex folding of the brain-wall has been obtained in living chick embryos after operations upon the rhombencephalon in early stages. The operation consisted of the extirpation of the tip of the notochord and the overlying neuromere. Its results mainly appear in the hemispheres and tectum opticum region, but sometimes also extend to other evaginated brain parts, viz. the optic évaginations, the epiphyseal region, and possibly also the caudal parts of the hypothalamus.

  2. The pathologic phenomenon is identical with certain abnormal foldings, described in human embryonic brains by Patten (1952) and called by him ‘overgrowth’. It may be combined with encephaloschisis in the mesencephalic roof.

Patten (1952) described ‘a curious distortion of the central nervous system’ in human embryos measuring 5,7,12·5,20, and 30 mm. in length, as well as in some pig embryos. The malformation was called‘overgrowthof the neural tube’. Instead of the indecisive word ‘overgrowth’ the present writer suggests the more exact term ‘hypermorphosis’ should be used for this malformation. Patten described it in the following way: ‘the neural tube epithelium had started to grow wildly so that it became folded, and refolded on itself, as if it was crowded into a cranial space fairly normal in size and shape’. The phenomenon was most distinctly developed in the rostral part of the neural tube. In some cases the cranial cavity was expanded by the process, giving rise to a high-crowned skull. In other cases an encephalocoel was formed. In later papers (1953,1957) Patten discussed this phenomenon further.

A malformation in the chick brain, similar to that described by Patten, was obtained by Källén (1955) as the result of operations in the rostral part of the rhombencephalon.

Other papers have been published on similar malformations. The literature is reviewed by Kappers (1956, 1957), who described overgrowth in a human embryo 7 weeks of age which had been damaged by a rubeola infection of its mother. The folding process was especially marked in the telencephalon, and similar abnormalities were seen in the dorsal parts of the diencephalon, mesencephalon, and rhombencephalon. The optic vesicles and the ventral parts of the brain were only slightly affected. A cranioschisis and encephaloschisis were present. In the chorioid plexus Kappers described abnormalities consisting of rosette-like cell formations. He is of the opinion that the encephalocoel was secondary to the strongly marked folding of the brain-wall.

Van Limborgh (1956) found pronounced overgrowth in the hemispheres, the mesencephalic tectum, and the myelencephalon in a 9-mm. human embryo. An encephaloschisis was present, interpreted as the result of incomplete closure of the neural folds.

Sjodin (1957) studied post-mortem changes in chick embryos. He demonstrated that a low oxygen tension for 2-3 days resulted in strong folding of the neural epithelium, comparable with Patten’s overgrowth. Similar results were obtained after injection of 2,3-dimercaptopropanol (BAL), KCN, HgCb, or indolacetic acid in high dilutions. After in vitro cultivation of embryos similar phenomena also appeared. Sjodin found no indications of an increased mitotic activity in the neural epithelium. A still more pronounced folding could be obtained by addition of growth inhibitors. After studies of Feulgen-stained material, Sjodin came to the conclusion that cell swelling and lysis was the basis of the folding. By studying embryos in solutions of different tonicity it could be shown that hypotonic solutions increased the folding processes, while hypertonic solutions had a tendency to inhibit them, though the results of the latter experiments were less marked than those of the former. Summing up, Sjodin remarks that the folding obtained in his experiments was no ‘case of teratogeny in the usual sense of the word. It is, rather, a side effect in the morbid process.’ He thinks there is a similar basis for the formation of Patten’s overgrowth malformations.

Operations were performed on chick embryos, incubated for 36–45 hours, at stage 11-14 (Hamburger & Hamilton, 1951). After opening of the egg-shells and vital staining with neutral red of the embryos, an operation was performed with glass needles of the Spemann type or with vibrating steel needles (Drury, 1941). After operation the shell openings were closed and the eggs were incubated for another 2–3 days. The age at fixation was thus days. The normal and operated embryos were fixed in Bouin’s fluid, sectioned transversely at 10 μ. and stained with Delafield’s haematoxylin and eosin. A total of 28 wax-plate recon-structions and 48 graphic reconstructions have been made.

In the main experimental series of 55 specimens (4 of them from the material published by Källén, 1955) neuromere d, together with the underlying notochord tip, was extirpated (see Text-fig. 1; for the terminology of the neuromeres, see Bergquist, 1956). In the control series of embryos other neuromeres were extirpated: neuromere a in 5 specimens, neuromere b in 1 specimen; neuromere c in 7 specimens; neuromere e with underlying notochord in 9 specimens (including 1 of Källen’s); neuromere f with underlying notochord in 6 specimens (including 1 of Källén’s). Studies have also been made on specimens prepared by Hugosson (1957) after operations on the mesencephalon and on the most rostral part of the spinal cord; and on various normal embryos of Hamburger & Hamilton stages 21–29 belonging to the collection of the Tornblad Institute.

TEXT-FIG. 1.

Schematic drawing of the neural tube of a chick embryo in dorsal view, roughly corresponding to Hamburger & Hamilton’s stage 12 (incubation age approx. 43 hours); also cf. Bergquist (1956). The levels . of operation are shown.

TEXT-FIG. 1.

Schematic drawing of the neural tube of a chick embryo in dorsal view, roughly corresponding to Hamburger & Hamilton’s stage 12 (incubation age approx. 43 hours); also cf. Bergquist (1956). The levels . of operation are shown.

Normal embryos

In the normal chick brain no foldings exist which could be misinterpreted as overgrowth. The surface of the hemispheres is smooth and so is that of the mesencephalon. In the epiphysis, the rest of the diencephalic roof, the hypothalamus, and the rhombencephalon no signs of foldings similar to those described as overgrowth phenomena can be seen.

Experimental material

After extirpation of neuromere d, the gap in the brains of the surviving embryos was often bridged by fusion of the remnants of the brain-stem. Usually there was a thin string of nervous tissue, but in other cases the two ends fused directly. In a few cases no fusion occurred and the two ends remained separated. The location of the operation can always be seen clearly in the sections. Abnormal foldings appeared both in the hemispheres and in the mesencephalon: in 38 specimens these overgrowths were strongly marked (4 specimens having encephaloschisis in the optic tectum, and 6 having overgrowths in the optic stalks), in 5 specimens they were moderate, in 7 specimens they were faint, and in 5 specimens there were none. In some cases the roof of the diencephalon with the epiphysis was folded, and in a few cases signs of abnormal folds could be seen in the caudal parts of the hypothalamus. The morphology of these abnor-malities are very similar to those described as overgrowth by Patten in human embryos.

The intention in extirpating neuromere d was to remove the notochordal tip. That this was done successfully was seen in the serial sections, which showed that a smaller or larger portion of the tip of the notochord was absent. In other cases the rostral end of the notochord was split into two or was club-shaped. A combination of a split and a club ending has also been seen. In one case the end was connected with the rest of the notochord by only a thin string. A complete separation of the tip from the rest of the notochord has also been observed.

In most cases the overgrowth was strongly marked in the hemispheres (Plate, fig. A). In some cases the two hemispheres were not distinctly separated, and in some extreme cases they merely formed (in transverse section) a compressed tubular structure (Plate, fig. B). The wax-plate reconstructions show, however, that in these cases there is really a vesicle folded and compressed in all directions. A characteristic feature of the hemisphere overgrowth was the presence of a more or less distinct protrusion or pouch of the lateral wall of the hemisphere (Plate, figs. A, C), which was either unilateral or bilateral. There was always a marked asymmetry in form between the two halves, and in some cases asymmetry in size.

There was usually a strong folding of the mesencephalic roof (Plate, figs. A, B). The separation of the tectum opticum vesicles was sometimes incomplete. In some embryos there was an encephaloschisis with strongly everted lateral parts, bending ventral wards. As with the hemispheres, there was always asymmetry with regard to size and shape of the two brain halves in the mesencephalon. In the material operated upon by Hugosson, parts of neuromere c were also removed—i.e. the future mesencephalic anlage; hence these embryos showed only a partial development of the tectum opticum. In these parts the overgrowth phenomenon was strongly marked.

The degree of overgrowth in the mesencephalic roof was highly variable and was sometimes practically absent. There is no relation between the degree of the overgrowth in the hemispheres and in the tectum opticum vesicles.

In some cases the epiphysis also developed signs of overgrowth. The dience-phalic roof, which normally forms a single dome, may be divided into two ridges, one of them carrying the epiphyseal rudiment, or it may be completely transformed into an overgrowth.

In some cases, clusters of cells grew into the ventricular lumen in locations where overgrowth was present (Plate, fig. B). These clusters resemble the cell rosettes, described in the telencephalic chorioid plexus by Kappers (1957; his fig. 9). In fig. 18 in that paper, Kappers also showed tumour-like structures resembling the formations observed in the present writer’s material. There are numerous mitoses in his structures.

There is a left-sided microphthalmia in three embryos and overgrowth was found in the optic stalks and vesicles of three others.

In no single embryo belonging to the present material have signs of overgrowth been observed in the rhombencephalon or in other parts of the brain.

In the five embryos in which no signs of overgrowth could be found, in spite of the fact that neuromere d had been extirpated, the sections and the reconstructions show that the notochordal tip had not been removed.

Control operations

After extirpations of neuromeres situated caudad and rostrad to neuromere d, no signs of overgrowth could be seen except in three embryos in which neuromere e had been extirpated. The sections show, however, that neuromere d had also been damaged and probably, too, the notochord underlying neuromere d.

In the introduction to the present paper it is pointed out that Sjodin obtained formations in chick embryos resembling the overgrowth phenomenon after either hypoxia or chemical influence. Sjodin regarded the phenomenon as a postmortem change. He supposed that the same was true for the malformations described by Patten.

The overgrowth described in the present paper occurs in embryos which are alive and apparently otherwise normal and is also of a different nature. In fig. D of the Plate part of the mesencephalic overgrowth in embryo 9 (see Plate, fig. A) is shown at high magnification. It may be compared with fig. 6 A and B, p. 600, in Sjodin’s paper. In the present material no pyknotic nuclei or autolysed cells can be seen, but numerous mitoses, showing that the tissue is strongly proliferating. It is possible that the spontaneous overgrowth phenomena described by Patten and others are of a similar nature. It cannot be shown that the terato-genetic factor, operating in spontaneous overgrowth, acts upon the same region as that damaged in the present operations, although this possibility is not unlikely. In some of Sjodin’s experiments damage to this region may also have occurred. Whether the influence of the notochordal tip is essentially inductive in character has yet to be shown experimentally; but it is striking that Eyal-Giladi (1958) has demonstrated a specific action by the notochordal tip in the formation of the orohypophysis.

The cost of this investigation was defrayed by grants from the Swedish Medical Research Council.

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KEY: 1, tectum opticum in overgrowth; 2, thalamus; 3, left hemisphere in overgrowth; 4, pouch of lateral wall of hemisphere; 5, cell clusters; 6, right hemisphere in overgrowth; 7, cells in mitosis.

FIG. A. Transverse section of embryo 9, cutting the caudal parts of the hemispheres, the thalamus, and the mesencephalon. There is a distinct overgrowth in the hemispheres and mesencephalon. See also fig. D. × 22.

FIG. B. Transverse section of embryo 9, showing the cell clusters extending from the neural epithelium into the ventricular lumen, ×22.

FIG. C. Transverse section of embryo 35, showing overgrowth in the hemispheres, x 22.

FIG. D. Detail of the mesencephalic overgrowth in embryo 9. The section shown here lies one section in front of that shown in fig. A. × 255.

KEY: 1, tectum opticum in overgrowth; 2, thalamus; 3, left hemisphere in overgrowth; 4, pouch of lateral wall of hemisphere; 5, cell clusters; 6, right hemisphere in overgrowth; 7, cells in mitosis.

FIG. A. Transverse section of embryo 9, cutting the caudal parts of the hemispheres, the thalamus, and the mesencephalon. There is a distinct overgrowth in the hemispheres and mesencephalon. See also fig. D. × 22.

FIG. B. Transverse section of embryo 9, showing the cell clusters extending from the neural epithelium into the ventricular lumen, ×22.

FIG. C. Transverse section of embryo 35, showing overgrowth in the hemispheres, x 22.

FIG. D. Detail of the mesencephalic overgrowth in embryo 9. The section shown here lies one section in front of that shown in fig. A. × 255.