Paper - The development of the cerebrospinal fluid spaces and choroid plexuses in the chick (1937)

From Embryology

Cohen H. and Davies S. The development of the cerebrospinal fluid spaces and choroid plexuses in the chick. (1937) J Anat. 72: 23-53. PMID 17104678

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

Young M. Normal facial growth in children. (1937) J Anat. 71: 458-470. PMID 17104658

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This historic 1937 paper by Young describes postnatal facial growth in children.




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The Development of the Cerebrospinal Fluid Spaces and Choroid Plexuses in the Chick

By Henry Cohen and Sarah Davies

From the Department of Medicine, University of Liverpool

I. Introduction

"Tue work described in this paper is part of a study comprising the comparative morphology and development of the choroid plexuses, the meninges and the meningeal spaces. Chick embryos of accurately determined age were investigated, utilizing a similar method to that adopted by L. H. Weed in his classical studies on pig and human embryos.

II. SURVEY OF LITERATURE (a) The development of the cerebrospinal fluid spaces in the Mammalia

Two workers, L. H. Weed (1917) and J. J. Keegan (1917), have investigated the development of the cerebrospinal fluid spaces by the method of injecting a true solution into the central nervous system of the living embryo. Weed’s comprehensive monograph appeared in 1917, and very shortly after Keegan published a very brief report of his own investigations. In a personal communication to us, Dr Keegan stated that in view of Weed’s earlier publica24 Henry Cohen and Sarah Davies

tion of almost identical work, he felt it was unnecessary to make a further detailed report of his own findings. In the light of the present investigation, however, Dr Keegan’s paper, brief though it was, proved to be of great interest.

Weed (1917) investigated the condition of the cerebrospinal fluid spaces in pig embryos at different stages of development. He replaced the fluid within the central nervous system by a 1 per cent double solution of potassium ferrocyanide and iron ammonium citrate. Fixation of the embryo in an acid medium secured precipitation of Prussian blue (ferric ferrocyanide) in situ and thus revealed the path taken by the injection solution. He records that the first extra-ventricular spread of the injected solution occurred in pig embryos of 14mm. length. Further, he found that the injected fluid made its escape into the surrounding mesenchymal tissue through two differentiated areas, one anterior and the other posterior, in the roof of the fourth ventricle. Weed terms these areas the area membranacea superior and the area membranacea inferior respectively, and states that both “differentiate at a slightly earlier period than that at which they function actively ” (1917, p. 107). The cells of these areas are distinguishable by their low stature and narrow, oval or slightly elongated nuclei.

The area membranacea superior is first differentiated in embryos of 11 mm. and reaches its maximum development at the 18 mm. stage. The fully differentiated form is maintained until about the 20 mm. stage, when the area commences a gradual regression, and disappears completely in embryos of 83 mm. Its disappearance is brought about by the encroachment of the developing cerebellum on the roof of the fourth ventricle, and by the-formation of the choroid plexus.

The inferior area. shows the first signs of differentiation in embryos of 15 mm.; it develops but slowly until the 18 mm. stage is reached, then differentiates extremely rapidly. Unlike the superior area, it persists and “‘ gradually occupies the major portion of the velum choroidea inferior” (Weed, 1917, p. 107).

The first escape of fluid from the spaces of the central nervous system in the 14mm. embryos was found to be coincident with the first indication of tufting of the developing choroid plexus of the fourth ventricle. A further marked extra-ventricular extension of the injected solution occurred in embryos of 19mm., and this was associated with the development of the choroid plexuses of the third and lateral ventricles at this stage. Furthermore, these plexuses were only definitely differentiated in embryos of 23 mm., and at this stage a complete periaxial spread occurred. From these observations Weed concludes that “‘there seems to be a very definite relationship between the developing choroid plexuses and the periaxial spread of the embryonic cerebrospinal fluid” (1917, p. 98). Weed has also identified and described similar areas of differentiation in the roof of the fourth ventricle of human foetuses at different stages of development, and the sequence of events resembles that found in pig embryos. Cerebrospinal Fluid Spaces and Choroid Plexuses 25

The presence of an area membranacea superior is also recorded in the embryos of chick, rabbit, sheep and cat, but no mention is made of an inferior area in these types.

Keegan (1917) injected a 1 per cent double solution of potassium ferrocyanide and iron ammonium citrate into the brain cavities of living rabbit and chick embryos at various stages of development, and secured precipitation of Prussian blue granules by fixation in an acid medium.

He records in both types the presence of two thin oval areas, one anterior and the other posterior to the choroid plexus, in the roof of the fourth ventricle. Each area was associated with a dense collection of protein coagula on its ventricular surface. In all early embryos injected with the double solution, a condensation of precipitated Prussian blue granules was also found below these areas.

It is further reported that “injection with the double solution in rabbit embryos up to the age of 17 days showed no extra-ventricular spread of the fluid, which was rather surprising in view of the fgct that the choroid plexus is quite well developed at this age” (Keegan, 1917, p. 8379). On the other hand, when a solution of ferric ammonium citrate alone was injected, extra-ventricular spread of the fluid occurred in the region of the fourth ventricle before the development of the choroid plexus, and there was only a slight condensation of blue granules below the thin areas of this roof. “‘ After the development of the choroid plexus there was a very rapid absorption into the circulation” (Keegan, 1917, p. 380).

Similarly, in chick embryos injected with the double solution, no extraventricular spread of the fluid had occurred, even in 9 days’ embryos, although the choroid plexus was present at this stage. Again, when the citrate solution alone was injected, extra-ventricular spread of the fluid invariably occurred, “and after the development of the choroid plexus (sixth to seventh day) very rapidly entered the circulation. At the seven day stage, a considerable portion remained within the ventricle and as a very evident blue coloration in the mesenchymal tissue over the rhombencephalon and mesencephalon... .In the eight and nine day chick the escape of the citrate solution was even more rapid, practically all leaving the ventricle within 10-20 min.” (Keegan, 1917, p. 380).

Keegan infers therefore that ‘‘this membranous area of the roof of the fourth ventricle is non-permeable to the double solution in the early embryo stages while it is permeable to the citrate solution; that a slight escape of the cerebrospinal fluid occurs before the development of the choroid plexus; and that the collection of protein coagula and Prussian blue granules in contact with the inner surface of the membranous area represents a dialysis phenomenon of this semi-permeable membrane” (Keegan, 1917, p. 380).

No details are given of the histological features of the membranous areas in the roof of the fourth ventricle, nor of the exact stages at which these areas make their appearance; neither is any mention made of their ultimate fate. Moreover, Keegan in his use of the term ‘“‘choroid plexuses” does not clearly 26 Henry Cohen and Sarah Davies

distinguish between those of the lateral and third ventricles and that of the fourth ventricle—a distinction which is shown to be important in the light of the present investigation.

(b) The avian meninges

Cuvier (1809) and Owen (1868} stated that three meninges are present in birds, a pia mater, arachnoid and dura mater, essentially similar to those found in the Mammalia and bearing the same relation to each other.

Sterzi (1902), on the other hand, states that only two meninges are present in birds, an outer, thin, non-vascular ‘dra mater, and an inner, secondary meninx, these being comparable to the two meninges found in the reptilia. The secondary meninx further consists of three layers, a thin, outermost endothelial layer, a middle vascular layer of loose texture, and an inner layer immediately adjacent to the cord. This structure of the secondary meninx shows an advance on the reptilian condition, where it is a thin single layer, and foreshadows the pia and arachnoid of the Mammalia. .

Streeter (1904), investigating the condition of the meninges in the ostrich, asserts that three membranes are present, similar to those found in the Mammalia.

The first detailed description of the development of the avian meninges appears to be that given by Farrar in 1906. In his description of the development of the pia-arachnoid membrane in the chick, Farrar designates the general mesenchymal tissue surrounding the embryonic nervous system as the arachnoid sheath or mesh, and states that the pia-arachnoid sheath arises from the cells of the arachnoid mesh immediately adjacent to the nervous system. The pia-arachnoid develops as a “‘single membrane consisting of a loose reticulum, the trabeculae of which are formed by the branching and anastomosing processes of connective tissue cells. At the outer and inner borders these cells tend to arrange themselves horizontally to form limiting membranes” (Farrar, 1906, p. 297). Blood channels make their appearance within the developing pia-arachnoid and increase in number to such an extent as to form an almost uninterrupted chain of vascular spaces within this zone. These blood channels are essentially similar to those found in the general arachnoid mesh. While the pia-arachnoid is developing from the inner zone of the arachnoid mesh, a second outer non-vascular zone of condensation makes its appearance, separated from the pia-arachnoid by an intermediate region of undifferentiated mesenchyme. In this outer zone, “the connective tissue elements are assuming elongated forms and crowding together with long axes parallel, giving rise to a very close mesh with long but extremely narrow spaces, in contradistinction to the loose, irregular reticulum of the pia-arachnoid” (Farrar, 1906, p. 297). This outer zone is the primitive dura and is clearly demarcated from the piaarachnoid. According to Farrar, therefore, two meninges are present in the chick, an outer dura and an inner pia-arachnoid; ‘“‘in the thickness of the latter, Cerebrospinal Fluid Spaces and Choroid Plexuses 27

however, no separating line can be drawn subdividing it into two membranes”’ (1906, p. 297).

No details are given of the stages at which these developmental changes occur.

Lillie (1908), in his description of the developing brain of the chick, records that up to the eighth day the roof of the fourth ventricle retains its thin epithelial character, whilst the floor and sides thicken considerably, but no statement is made regarding the existence of any thin differentiated roof areas.

Hansen-Pruss (1923) maintains that three distinct meninges are present in birds. Moreover, he demonstrated the presence of cerebrospinal fluid in the loosely trabeculated subarachnoid space and succeeded in injecting into this space India ink, and also a double solution of potassium ferrocyanide and iron ammonium citrate. Further, he found that the injected fluids ‘extended caudally, surrounding the spinal cord, to the upper pole of the sinus rhomboidalis”’ (p. 206). Cephalad, the injection fluids were traced “over both hemispheres and optic lobes as far forward as the sinus frontalis and down on the median and lateral sides”’ (p. 206).

Ariéns-Kappers (1929), and Ariéns-Kappers et al. (1936) state that in birds two meninges are present, an outer dura and an inner meninx, also termed the secondary meninx, and in which small arachnoid spaces make their appearance.

Thus, according to Cuvier (1809), Owen (1868), Streeter (1904) and HansenPruss (1923), three meninges exist in birds. Sterzi (1902), Farrar (1906), Ariéns-Kappers (1929), and Ariéns-Kappers et al. (1986) on the other hand, maintain that only two such membranes are present.

III. MATERIAL

The present account is based on observations of six series of chick embryos, which for purposes of convenience are referred to as series A, B, C, D, E and F respectively.

The eggs of each series were incubated at a temperature of 88—40° C., care being taken that these limits were not exceeded.

IV. METHODS OF INVESTIGATION

Immediately prior to injection the embryo was removed from the egg together with a portion of the extra-embryonic yolk-sac, and placed in Pannett and Compton’s saline solution, warmed to a temperature of 38—40° C. This solution was prepared in the following way from two stock solutions:

Stock solution A:

Sodium chloride (NaCl) tee wee 12-11 g. Potassium chloride (KCl) wee wee 1:55 g. Calcium chloride (CaC],) ves wee 0-77 g. Magnesium chloride (MgCl,.6H,O) ... 1-27 g.

Distilled water to make 100 c.c. of solution. 28 Henry Cohen and Sarah Davies

Stock solution B: Prepared from the following solutions: (1) Sodium dihydrogen phosphate (NaH,PO,.12H,0): distilled water

to make 100 c.c. of solution... 0-2 g. (2) Disodium hydrogen phosphate (Na,HPO,.12H,O): distilled water to make 100 c.c. of solution... 0-52 g. These solutions are mixed in the following proportions: Solution (1)... 5 c.c. Solution (2)... 55 ¢c.¢.

and are then autoclaved.

The final solution consists of 1 c.c. of stock solution A and 1-5 c.c. of stock solution B in 22-5 c.c. of distilled water.

The embryo was exposed by careful removal of the chorion and amnion, and was kept in the warm saline solution throughout the injection.

For the purpose of injection two types of solutions were used, a double solution of potassium ferrocyanide and iron ammonium citrate, and a solution of iron ammonium citrate alone. It was found by experiment on the red blood corpuscles of the hen that a solution of 0-8 per cent concentration! was for practical purposes isotonic with the body fluids of the embryo, and this concentration was used throughout the investigation.

- During the present investigation injections were made either into the central nervous system or into the vascular system.

Two methods of injecting the fluid were employed. The first method was by means of a short glass tube of narrow bore drawn out into a fine capillary point at one end, and provided with a small rubber bulb at the other. In the case of injection into the central nervous system, a small quantity of fluid was injected under very slight pressure into the caudal end of the central canal of the spinal cord. Undue increase of intra-cerebral pressure was avoided by simultaneous withdrawal of any fluid contained within the central nervous system. This was effected by inserting a similar capillary needle into the lateral ventricle of either cerebral hemisphere. In the case of injection into the vascular system, a minute quantity of fluid was injected into the left ventricle of the heart under very slight pressure. After the dye had passed from the ventricle into the aorta, the injection was repeated and this procedure was continued until the blood vessels of the head region were seen to contain the injected dye.

The second method of injection was replacement of the fluid within the central nervous system by the injection solution, using the apparatus shown in Text-fig. 1. This consisted of a flat wooden base (A) supporting an upright rod (B), to which was affixed a graduated scale indicating the pressure in millimetres of water under which the injection was made. The upright rod also carried a rectangular brass frame (C), provided with a small pulley at each of

1 In the case of the double solution, 0-4 g. of each salt were dissolved in a 100 c.c. of solution;

in the case of the single solution 0-8 g. of iron ammonium citrate were dissolved in a 100 c.c. of solution. Cerebrospinal Fluid Spaces and Choroid Plexuses 29

its four corners. The longer bars of this frame supported two spring clamps (D, D,) fitted with rollers in order to allow of easy movement along the bars. A length of Egyptian cotton, made taut around the pulleys, was passed through the clamps, and thus their relative position could be adjusted by movement of the cotton. Each clamp held a glass reservoir (E, E,) connected to a capillary

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needle (F', F',) by a length of rubber tubing. In addition, the clamp D carried a pointer which moved up and down the graduated scale, according to the position of the clamp. The capillary needles were fixed into adjustable metal holders (G, G,) which provided free movement in all planes.

The reservoir £ was filled with the injection fluid until drops appeared at the tip of the needle. The reservoir E, was filled with 0-75 per cent saline solution 30 Henry Cohen and Sarah Davies

in the same manner. The flow of the fluid from each needle could be completely arrested by means of a clip placed on the rubber tubing at a short distance behind the needles.

The advantage of this apparatus is twofold—the reservoirs are firmly supported but can still be moved freely up and down, and the pressure under which the injection is made is automatically recorded.

The embryo to be injected was supported on a block placed on the stand 4. The reservoirs were filled with the injection fluid and saline solution to the same level. The needles were adjusted to a suitable position and then inserted into the embryo, needle F into the caudal end of the central canal of the spinal cord and needle F, into the lateral ventricle of either cerebral hemisphere. When both needles were in position the clips were removed from the rubber tubing and the reservoir E was slowly raised, while the reservoir EZ, was lowered simultaneously. As fluid was withdrawn from the central nervous system into the needle F,, it was automatically replaced by the injection solution from the needle F. It was found that complete replacement was effected at a pressure equivalent to a column of water 70-75 mm. in height.

The methods of treatment of the different series of embryos are tabulated below.

Age limit of series Series days System injected Injection fluid used A 3-9 Central nervous system Double solution of potassium ferrocyanide and iron ammonium citrate B 3-9 Central nervous system Double solution of potassium ferrocyanide and iron ammonium citrate Cc 43-9 Central nervous system Double solution of potassium ferrocyanide and iron ammonium citrate D 34-9 Central nervous system Solution of iron ammonium citrate E 5-9 Vascular system Double solution of potassium ferrocyanide and iron ammonium citrate F 5-9 Vascular system Solution of iron ammonium citrate

The embryos were injected between the age limits stated at intervals of approximately 12 hours. In each series at least two embryos were injected at each stage of development. After each injection the embryo, still in the saline solution, was kept alive in an incubator at 38-40° C. to allow of the spread of the injection fluid, the embryonic heart-beat serving as the criterion of survival. It was found that 4-3 hour in the case of the smaller embryos, and 1-14 hours in the larger, were needed for complete spread.

Embryos injected with the double solution were then placed in a solution containing 10 per cent formaldehyde and 1 per cent hydrochloric acid. This caused precipitation of Prussian blue (ferric-ferrocyanide) and partial fixation of the embryo. Specimens injected with iron ammonium citrate alone were placed in a similar acid formaldehyde solution, to which some potassium ferrocyanide solution had been added. This also caused precipitation of Prussian blue and partial fixation. The time required for precipitation was, 4-1 hour, according to the size of the embryo. Cerebrospinal Fluid Spaces and Choroid Plexuses 31

The members of series A were then left in Bouin’s fluid overnight to complete fixation, but this resulted in a yellow colouration which obscured the external outline of the course of the injection fluid. Consequently, the embryos of the remaining series were fixed by allowing them to remain in the acidic formaldehyde solution overnight. Thus, the course of the injection fluid remained clearly visible, and the subsequent histological picture was in no way altered.

After fixation the embryos were dehydrated in a series of alcohol solutions, increasing from 5-100 per cent concentrations by 10 per cent gradations. They were then cleared in xylol or cedarwood oil,! embedded in paraffin wax (52 or 56° C. M.p. according to season), and serially sectioned at a thickness of 4 u, some in the longitudinal and some in the transverse plane. Ehrlich’s haematoxylin and eosin were found to be the most suitable stains, and were used throughout the investigation.

V. DESCRIPTION OF THE BRAIN AND MENINGES OF THE CHICK AT DIFFERENT STAGES OF DEVELOPMENT, WITH PARTICULAR REFERENCE TO THE STRUCTURE AND PERMEABILITY OF THE ROOF OF THE FOURTH VENTRICLE

(a) Results of injections of true solutions into the central nervous system

(i) Injections of a double solution of potassium ferrocyanide and iron ammonium citrate.

The present section is based on observations, external and histological, of the embryos of series A, B and C. In the embryos of each series injections of a double solution of potassium ferrocyanide and iron ammonium citrate were made into the central nervous system.

In embryos of 3 days’ incubation the injected solution was seen to be wholly contained within the central nervous system. It was uniformly distributed within the central canal of the spinal cord and in the ventricular cavities of the brain. The medulla was relatively very large and triangular in shape with its base directed anteriorly and its apex tapering into the spinal cord. The medullary roof was very thin and lay immediately below the integument, thus allowing the dye contained in the fourth ventricle to be more readily seen than elsewhere in the brain stem.

At 34 days the course of the injected solution was similar to that in a 8days’ embryo, but a median, deeply stained, roughly oval area was now seen in the anterior half of the medullary roof (see Text-fig. 2A). This area, in virtue of its intense blue colour, stood out in marked contrast to the more diffused colour of the remainder of the medullary roof. No spread of the injected fluid from the central nervous system was evident.

1 The transparency of the chick embryo in its earlier stages of development enabled the course of the injection fluid to be seen with ease. In older embryos the developing integument obscured

the outline of the fluid considerably, and in these cases the material was cleared overnight in cedarwood oil, to render the course of the fluid more easily distinguishable. 32


Henry Cohen and Sarah Davies



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Text-fig. 2. Cerebrospinal Fluid Spaces and Choroid Plexuses 33

Embryos of 33 and 4 days’ incubation showed a distribution of the injected fluid similar to that in the preceding stage, but the deeply staining oval area in the anterior portion of the medullary roof was relatively larger.

In 44 days’ embryos, again, the injected dye still lay wholly within the central canal of the spinal cord and ventricular system of the brain, but the median, deeply stained area was relatively much larger than previously, occupying the major portion of the anterior part of the medullary roof (see Text-fig. 2B).

At 5 days the distribution of the injected fluid was similar to that in embryos of 43 days’ incubation, but the deeply staining oval area in the medullary roof showed a slight reduction in extent when compared with that of the preceding stages (see Text-fig. 2C).

Embryos of 53 days showed that the fluid was still confined within the central nervous system, but the median oval area showed even further reduction in extent when compared with that of the preceding stage.

At 6 days the distribution of the injected fluid was similar to that of the previous stage, but the reduction in size of the deeply staining oval area in the medullary roof was even more marked.

In 6} and 63 days’ embryos the dye was still confined to the spaces of the central nervous system. The median, deeply staining area in the anterior half of the medullary roof was at this stage very small indeed, and a second similar area was now visible in the extreme apical region of the posterior half of the roof (see Text-fig. 2D and E).

Cleared specimens of 7 days’ incubation showed that a very slight spread of the dye had occurred from the roof of the fourth ventricle, but the outline of this roof was still clearly visible. The deeply stained area in the anterior part of the roof showed even greater reduction in extent than previously, whilst that in the posterior half was relatively larger (see Text-fig. 2F).

In embryos of 74 days’ incubation the outline of the medullary roof was no longer clearly visible, even in specimens cleared in cedarwood oil. This suggested tentatively that an even more extensive escape of dye had occurred from the fourth ventricle into the overlying spaces. The deeply coloured area in the anterior half of the medullary roof was now so small as to be hardly distinguishable, but that in the posterior half was relatively larger than in the preceding stage.

In cleared specimens of 8, 84 and 9 days’ incubation the outline of the medullary roof was obscured completely; the deeply coloured area in the anterior half of this roof was no longer visible, but that in the posterior half was very large and, in the 9 days’ embryos, occupied almost the whole of the apical half of the triangular medullary roof.

The embryos described above were examined histologically with a view to determining the exact nature of the deeply staining oval areas in the roof of the fourth ventricle. At the same time, an attempt was made to discover any possible correlation between the time of appearance of these areas, the spread

Anatomy LxxII 3 34 Henry Cohen and Sarah Davies

of the injected dye from the central nervous system, and the development of the choroid plexuses and meningeal spaces.

In embryos of 8 days’ incubation the wall of the brain stem consisted chiefly of ependyma only, or of ependyma associated with a narrow stratum of overlying nerve tissue. The roof of the third and fourth ventricles was in each case continuous and unfolded throughout and showed no indication of a choroid formation. The roof of the third ventricle consisted of an ependymal layer, 3-4 cells deep. The outline of the individual cells was difficult to distinguish owing to the dense granulation of the deeply staining cell cytoplasm. The nuclei were round or slightly elongated in a plane at right angles to the longitudinal axis of the body, and possessed a deeply staining nuclear membrane and 2-8 nucleoli in the chromatin mesh. The roof of the fourth ventricle was triangular in shape, the wide base being directed anteriorly, and the narrow apex posteriorly. The greater part of this roof was extremely thin, consisting of a single layer of low cuboidal cells with granular cytoplasm and round welldefined nuclei touching both the inner and outer cell walls. The nucleoli and nuclear membrane were intensely stained, in contradistinction to the pale nucleoplasm. In the anterior half of this roof a small median oval area could be distinguished, where the cells showed a slight tendency to flattening. This area was not sharply delimited from the surrounding epithelium but gradually merged into it (see Text-fig. 7).

At this stage the general mesenchymal tissue consisted of loosely arranged stellate cells enclosing large, irregular intercellular spaces. The cytoplasm of these cells assumed the form of delicate, pale, granular strands radiating from a centrally disposed nucleus. The nudlei were round or slightly elongated and possessed a deeply staining nuclear membrane, and 2-3 nucleoli in their chromatin mesh. Where the mesenchymal tissue abutted on to the embryonic brain, its cells showed a slight tendency to elongation, the cytoplasmic strands lying parallel to the brain surface.

The precipitated Prussian blue granules were seen wholly within the central nervous system, no extension of the dye into the surrounding mesenchyme being evident. The granules were distributed in a uniform manner, chiefly within the central canal of the spinal cord and in the fourth ventricle of the brain, and showed no condensation associated with any particular area in the brain stem. The distribution of the granules in the cavities of the optic lobes, thalamencephalon, and cerebral hemispheres was very sparse.

In embryos of 3} days’ incubation the slightly differentiated median area in the roof of the fourth ventricle was now abruptly demarcated from the surrounding roof epithelium. The cells of this area were extremely flat, the granular cytoplasm forming a thin investment around the elongated nucleus. Apart from a marked difference in shape, these cells resembled those of the surrounding epithelium, showing a similar degree of granulation and richness of nuclear chromatin material.

Where the mesenchyme abutted on to the brain surface its cells were not Cerebrospinal Fluid Spaces and Choroid Plexuses 35

only elongated as in the previous stage, but also showed a slight degree of condensation, and were more compactly arranged than elsewhere. The narrow ill-defined zone thus formed adhered closely to the brain surface and intimately followed its outline.

The injected fluid was again strictly confined within the central nervous

‘system but no longer exhibited the same uniformity'of distribution within the

cerebral ventricles as in the preceding stage. There was a dense accumulation of blue granules intermingled with coagulum, adhering to, and co-extensive with, the ventricular surface of the thin membranous area in the roof of the fourth ventricle. The individual cells of this area, however, were entirely free of the dye.

Embryos of 3} days’ incubation presented the same essential features as in the preceding specimen, but the membranous area showed a slight increase in size. It was, however, still sharply delimited from the surrounding epithelium (see PI. I, fig. 1 and Text-fig. 3).


Text-fig. 3.

The incipient zone of mesenchyme condensation was still narrow but a little more clearly defined from the surrounding mesenchyme than in the previous stage. Further, this zone now showed the first indication of vascularization, minute capillaries being evident in its narrow,’ intercellular meshes. These capillaries, however, in their distribution bore no relation to the differentiated membranous area in the roof of the fourth ventricle. Where the zone overlay this thin area ‘its cells were less compactly arranged than elsewhere, and showed merely a slight degree of condensation or none at all, so that over this area the zone was hardly distinguishable as a separate entity from the surrounding mesenchyme.

The injected dye still lay wholly within the central nervous system, and showed a particularly dense accumulation below the membranous area in the . roof of the fourth ventricle. The cells of this area, however, showed no impregnation by the dye.

In 4 days’ embryos the histological appearance of the brain and distribution of mesenchyme were similar to those of a 82 days’ embryo. The injected fluid was still confined to the central canal of the spinal cord and ventricular system of the brain, no invasion of the periaxial mesenchyme being apparent. A heavy condensation of the dye still adhered to the ventricular surface of the membranous area in the roof of the fourth ventricle.

3—2 36 Henry Cohen and Sarah Davies

At 44 days the wall of the brain was thicker than in preceding stages, the greater part consisting throughout of ependyma, associated with a stratum of nerve tissue of variable width. The dorso-mesial walls of the cerebral hemispheres and the roof of the third ventricle, however, were narrower than elsewhere, but still showed no indication of a choroid formation. The myelencephalic roof was still smooth and unfolded. The median membranous area in its anterior portion was slightly larger than in the preceding stages, but was still abruptly demarcated from the surrounding epithelium.

The zone of mesenchyme condensation surrounding the brain was still narrow but more clearly defined than hitherto, and showed a higher degree of vascularization. The general mesenchyme overlying the roof of the fourth ventricle was more loose-meshed than elsewhere and still showed little or no condensation where it actually abutted on to the membranous area in the anterior portion of this roof.

As in earlier embryos, the dye was still confined within the central nervous system, but had now extended anteriorly in uniform distribution to the furthermost recesses of the lateral ventricles. A particularly dense accumulation of blue granules was still apparent below the membranous area in the roof of the fourth ventricle.

In embryos of 5 and 5} days’ incubation the roof of the thalamencephalon was thinner than in the previous stages, consisting only of a narrow ependymal layer 1-2 cells deep, but it was still smooth and unfolded. The epithelial roof of the fourth ventricle was still continuous throughout and showed no indication of a choroid formation. This roof was now clearly divided into anterior and posterior halves by a slight depression in the transverse median plane, and the cells participating in this fold differed from the typical epithelial roof cells in that they tended to be columnar rather than cuboidal. The thin membranous area in the anterior portion of the roof was somewhat smaller than in the preceding stage, but was still sharply delimited from the surrounding cuboidal epithelium. The greater part of the roof posterior to the transverse median fold consisted of cells which showed an unmistakable tendency to flattening, being very low cuboidal in shape, but their nuclei were still round or a little elongated (see Text-fig. 4). This slightly differentiated posterior area differed from the anterior membranous area in that its cells were not so flat and the nuclei still retained, on the whole, their rounded shape; moreover, it was not sharply delimited from the surrounding epithelium but showed gradual mergence with it.

The zone of mesenchyme condensation was still narrow but was now clearly visible as an entity distinct from the general mesenchyme from which it had originated. It consisted of 2-8 layers of cells, more densely compacted together than hitherto. The protoplasmic strands of the cells lay parallel to the brain surface or at right angles to it, and the intercellular meshes so formed contained small capillaries. Over the thin thalamencephalic roof and over the membranous areas in the roof of the fourth ventricle this zone was so narrow and ill-defined as to be hardly distinguishable. A slight indication of a second Cerebrospinal Fluid Spaces and Choroid Plexuses 37

outer zone of condensation was apparent in the general mesenchyme in certain localized areas, chiefly in front of the cerebral hemispheres and on the dorsal and ventral surfaces of the thalamencephalon. Where this zone was in process of formation it was very irregular and ill-defined, merging imperceptibly into the general mesenchyme. It was separated from the narrow inner zone by an intermediate region of mesenchymal tissue of variable width. In this newly forming outer zone the typical mesenchyme cells were more compactly arranged than elsewhere and showed a tendency to elongation in a plane parallel to the longitudinal axis of the body. This zone showed no indication of vascularization, its narrow and elongated intercellular meshes being entirely free of blood capillaries. The intermediate zone consisted of typical loosemeshed stellate mesenchyme cells and contained minute capillaries. These

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Text-fig. 4.

were uniformly distributed throughout this zone and showed no special concentration over the differentiated areas in the roof of the fourth ventricle.

The injected dye was still confined within the central nervous system, and was particularly condensed below the membranous area in the anterior region of the roof of the fourth ventricle. No such condensation was evident below the slightly flattened area in the posterior region of this roof, but merely a fine distribution of blue granules closely adhering to its ventricular surface.

In embryos of 6 days’ incubation the thin roof of the thalamencephalon presented a slightly undulating appearance, forming 2-3 shallow irregular folds, invaded by the overlying mesenchyme. The transverse median fold separating the myelencephalic roof into anterior and posterior regions was more emphasized than previously. Further, the anterior membranous area of this same roof had diminished considerably in extent, but was still sharply 38 Henry Cohen and Sarah Davies

delimited from the surrounding cuboidal epithelium. The posterior slightly flattened area was more extensive than in the preceding stage and, on the whole, exceeded the anterior area both in length and width. It still showed no clear demarcation from the surrounding epithelium. Apart from the transverse median depression, the roof of the fourth ventricle showed no indication of folding and was continuous throughout.

The inner zone of condensation was still narrow, yet clearly defined, except over the thin thalamencephalic roof and over the membranous areas in the roof of the fourth ventricle. The mesenchyme abutting on to these surfaces still showed little or no condensation. The outer zone was more clearly marked than previously but still merged gradually into the surrounding mesenchyme (see Pl. II, fig. 6). This zone had now extended posteriorly over the optic lobes and to the lateral and ventral surfaces of the medulla. In the dorsal medullary region its identity was completely lost in the general mesenchyme. Localized centres of cartilage formation were evident within the substance of this outer zone, particularly in that part of it flanking the medulla. The cells at these points of chondrification tended to a regular disposition, and were isolated from each other by a clear homogeneous matrix. Their nuclei were rounded and well defined, and possessed only a thin investment of pale granular cytoplasm. Where the nervous system approximated closely to the integument, i.e. over the anterior surfaces of the cerebral hemispheres and the dorsal surfaces of the optic lobes, the inner and outer zones lay in such close juxtaposition that the intermediate zone of loose-meshed mesenchyme was almost completely obliterated.

The injected fluid was still strictly confined within the spaces of the central nervous system, and showed the same degree of concentration below the membranous areas of the myelencephalic roof as in the previous stage, but the accumulation of blue granules below the posterior area was slightly denser.

In embryos of 6} days the incipient folding of the thalamencephalic roof was even more pronounced than previously. The transverse median fold in the roof of the fourth ventricle was deeper than in the preceding stage and its cells were more columnar in shape. The anterior membranous area in this roof showed even further reduction in extent, but was still sharply delimited from the surrounding epithelium. The posterior area, on the other hand, was slightly larger than in the previous stage, but again merged gradually into the general roof epithelium. Its cells were still very low cuboidal in shape with round or only slightly elongated nuclei, but those at the extreme posterior region of this area were extremely flat and resembled the pavement cells of the anterior area.

At this stage the narrow inner zone of mesenchyme condensation showed well-marked intrazonal spaces. Its cells were extremely flat and elongated, and arranged in two ill-defined layers parallel to the brain surface. The very narrow spaces between the cells were traversed by delicate protoplasmic trabeculae arising from the cells of both the inner and outer layers. The numerous intercellular meshes so formed contained small capillaries and consequently this zone, particularly around the cerebral hemispheres, had the appearance of Cerebrospinal Fluid Spaces and Choroid Plexuses 39

a chain of minute vascular lumina. Where the mesenchyme cells of this zone abutted on to the thin thalamencephalic roof and membranous areas in the roof of the fourth ventricle they showed little or no condensation, so that the zone was virtually absent in these regions. The outer zone of mesenchyme condensation was more clearly defined than in previous stages and had now extended round the entire brain stem. It was still irregular, however, and was separated from the inner zone by an intermediate layer of typical loosemeshed mesenchyme of variable width. Occasionally the two condensation zones were separated by large irregular spaces containing isolated mesenchyme cells, indicating that these spaces had originated by the breaking down of small portions of the mesenchymal tissue and subsequent confluence of the intercellular spaces.

The injected dye was still retained within the spaces of the central nervous system. A particularly dense accumulation of blue granules and coagulum was still seen adhering to, and coextensive with, the ventricular surface of the anterior membranous area. Further, a heavy condensation was now evident below the extremely flat cells at the apex of the slightly differentiated posterior membranous area.

In embryos of 63 days the invaginations of the thalamencephalic roof were more pronounced than previously, and extended forward for a very short distance into the lateral ventricles via the foramina of Munro. The anterior membranous area of the myelencephalic roof was smaller than in the preceding stage, and its border no longer showed an abrupt transition from the surrounding epithelium but gradually merged into it. The posterior membranous area, on the other hand, was now sharply delimited from the general roof epithelium and showed considerable increase in extent. Its cells were extremely flat and possessed narrow elongated nuclei with a thin investment of cytoplasm (see Pl. I, fig. 2).

The outer zone of mesenchyme condensation was a little more clearly defined than previously. It showed a slight increase in width and a higher degree of cartilage formation within its substance.

The injected fluid was still confined within the central nervous system, and was particularly condensed below the membranous areas in the roof of the fourth ventricle.

In 7 days’ embryos the folds of the thalamencephalic roof were more pronounced than in the preceding stage and, as they extended forward into the lateral ventricles, showed a slight degree of branching, forming incipient choroid villi. In transverse section each villus consisted of an axial core of loose-meshed mesenchyme containing minute capillaries in its intercellular spaces and surrounded by an epithelium 1-2 cells deep. The cells were columnar in shape with granular cytoplasm and round well-defined nuclei. The median transverse fold in the roof of the fourth ventricle was deeper than hitherto and its cells were more markedly columnar. The anterior membranous area was smaller than previously but the posterior area, on the other hand, was larger 40 Henry Cohen and Sarah Davies

and still showed an abrupt transition from the surrounding roof epithelium: (see Text-fig. 5).

The distribution of mesenchyme was similar to that in the preceding stage, but the inner zone of condensation was more clearly defined and highly vascular. Where it overlay the thin thalamencephalic roof and the membranous areas in the roof of the fourth ventricle it showed little or no condensation and was, therefore, virtually absent over these regions. The outer zone still merged gradually into the surrounding mesenchyme; it enclosed the brain without closely following its outline and was completely non-vascular. This zone showed a higher degree of cartilage formation within its substance than in the previous stage. The intermediate zone consisted of typical loose


Text-fig. 5.

meshed mesenchyme cells with minute capillaries scattered in the intercellular meshes. These capillaries, in their distribution, bore no relation to the differentiated roof areas of the fourth ventricle.

Some of the dye, which in all previous stages had been strictly confined to the central canal of the spinal cord and ventricular system of the brain, had at this stage invaded the dorsal medullary mesenchyme (see Text-fig. 5). Apparently the site of fluid escape was the anterior membranous area in the roof of the fourth ventricle. Not only was there a dense accumulation of blue granules below this area, but its cells were actually impregnated with the dye. Further, in the mesenchyme immediately above this thin area there was a particularly dense accumulation of blue granules. Since the inner zone of mesenchyme condensation was virtually absent over the membranous areas in the roof of the fourth ventricle, the dye escaped directly into the meshes of the intermediate zone and did not invade the spaces within the inner zone of condensation. The blood capillaries of the intermediate zone were also entirely Cerebrospinal Fluid Spaces and Choroid Plexuses 41

free of the dye, although here and there a slight accumulation of blue granules was evident, adhering to the outer surface of the capillary wall. A heavy precipitation of the injected fluid was also seen below the posterior membranous area. There was, however, no evidence of escape from this region, its cells showing no invasion by the dye. Neither was there evidence of fluid escape from any other point in the ependymal roof of the fourth ventricle. The cuboidal cells of this layer were entirely free of the dye, and exhibited only a fine granular distribution on their ventricular surface.

In embryos of 74 days’ incubation the folding of the thalamencephalic roof was even more marked than in the preceding stage, the folds being slenderer and exhibiting a higher degree of branching. The median transverse invagination in the roof of the fourth ventricle was deeper and the columnar appearance of its constituent cells more pronounced. The ventro-lateral walls of this fold showed a slight degree of undulation, but no true choroid villi were apparent. The anterior membranous area of this roof was even smaller than in the preceding stage, but the posterior area was relatively larger.

The outer zone of mesenchyme condensation was wider and more clearly defined than in the previous stage. The mesenchyme of the intermediate zone immediately above the differentiated roof areas of the fourth ventricle was more loose-meshed than elsewhere and contained large irregular spaces. The capillaries in this zone now tended to be more concentrated in the dorsal medullary region.

The extra-ventricular spread of the injected fluid was slightly more marked than previously. Anteriorly it had extended as far as the cerebellar anlage, and posteriorly as far as the median transverse fold in the medullary roof. The blue granules lay in the meshes of the intermediate zone and did not invade the inner zone of condensation.

In 8 days’ embryos, as in preceding stages, the thalamencephalic roof consisted of an ependymal layer only 2-8 cells deep. The degree of folding of this roof, however, was much more marked than hitherto, and the long narrow branching choroid villi so formed extended forward into the lateral ventricles via the foramina of Munro (see Text-fig. 8). The roof of the fourth ventricle showed striking morphological changes. The median transverse fold was extremely well developed and extended almost to the floor of the ventricle. This fold was lined by the inner zone of condensation, and was supported by mesenchymal tissue continuous with that of the intermediate zone. Its walls consisted of columnar cells with granular cytoplasm and round well-defined nuclei. The undulations in the floor of this fold, first recorded in 74 days’ embryos, were here much more pronounced, forming short unbranched incipient villi. These were concentrated in two ill-defined clusters, one at each side of the floor. The anterior portion of the roof of the fourth ventricle was much shorter than in the preceding stage, owing apparently to the encroachment of the developing cerebellar anlage. A median membranous area was still evident in this anterior region but was much smaller in extent than previously. The 42 Henry Cohen and Sarah Davies

posterior membranous area, on the other hand, was relatively much larger and occupied practically the whole of the roof region posterior to the median fold (see Text-fig. 9). ,

The outer zone of mesenchyme condensation was wider and a little more clearly defined than in the preceding stage. Where the intermediate zone abutted on to the areas of chondrification in the outer zone its cells showed a slight condensation. They were slightly elongated in a plane parallel to the inner surface of the developing bands of cartilage, thus forming an extremely narrow ill-defined zone of fibrous appearance.

The extra-ventricular distribution of the injected dye was more extensive than in the previous stage. It extended for a short distance over the posterior lip of the cerebellar anlage, and even invaded the mesenchyme lying in the upper region of the transverse median fold in the roof of the fourth ventricle. The inner zone of condensation still showed no invasion by the dye. Apparently the posterior membranous area in the medullary roof was now functioning as the site of fluid escape. There was a particularly dense accumulation of blue granules adhering to its ventricular surface. Its cells were deeply impregnated with the dye, and the mesenchyme immediately above this area was more intensely stained than elsewhere. Below the anterior area there was little condensation of blue granules, its cells showed scarcely any impregnation with the dye and the overlying mesenchyme was only slightly coloured.

In embryos of 84 days’ incubation the choroid villi of the lateral ventricles exhibited a higher degree of branching than previously. The incipient villi in the floor of the median fold of the medullary roof were more pronounced than in the preceding specimen, and were slightly branched (see Pl. I, fig. 3). The anterior portion of the medullary roof was even shorter in longitudinal section, bringing the posterior lip of the cerebellum near to the transverse median fold. The membranous area in the anterior region of the roof was very small indeed and was now relegated to the upper part of the anterior wall of the median fold. This membranous area was no longer sharply delimited from the surrounding epithelium but gradually merged into it. Its constituent cells had lost their flat appearance and were now low cuboidal in shape. The posterior membranous area was very extensive, occupying practically the whole of the roof behind the transverse fold. It remained sharply delimited from the surrounding epithelium, and its cells, in contrast to those of the anterior area, were still extremely flat (see Text-fig. 6).

As in previous stages, the inner zone of mesenchyme condensation was still narrow, but nevertheless very clearly defined. Over the membranous areas in the roof of the fourth ventricle it was absent altogether, so that in these regions only a very thin membrane separated the fourth ventricle and its contents from the intercellular meshes of the overlying intermediate zone. The outer zone was wider than previously and showed an even higher degree of cartilage formation. Further, the fibrous layer immediately within this zone was a little more clearly defined. Cerebrospinal Fluid Spaces and Choroid Plexuses 43

The extra-ventricular spread of the injected fluid was more pronounced than hitherto.. Anteriorly the dye had extended over the cerebellar anlage, and posteriorly for a short distance along the dorsal surface of the spinal cord. The inner zone of condensation still showed no invasion by the dye. Again, the evidence pointed to the posterior membranous area in the roof of the fourth ventricle as being the site of escape. There was a dense accumulation of blue granules, intermingled with coagulum, closely adhering to and coextensive with the ventricular surface of this area. Its cells were deeply impregnated with the dye, and the mesenchyme immediately above showed a very heavy invasion of blue granules. The anterior area, on the other hand, exhibited only a fine granular distribution on its ventricular surface, its cells were entirely free of the dye, and the mesenchyme above this area showed only a slight colouration.


Text-fig. 6.

In embryos of 9 days’ incubation the branching of the choroid villi in the lateral and third ventricles was even more emphasized than previously. The developing cerebellum had encroached on almost the whole of the anterior portion of the medullary roof, with the result that the now almost vestigeal membranous area of this region was incorporated yet further into the anterior wall of the transverse median fold. The posterior membranous area, on the other hand, had developed to such an extent that it now occupied almost the whole of the posterior region of the roof, and even extended into the median fold, forming part of its posterior wall (see Pl. I, fig. 4). The walls of the fold’ below their junction with the membranous roof areas consisted of a singlelayered epithelium of cuboidal cells with granular cytoplasm and round welldefined nuclei. This gradually merged into an epithelium 2-3 cells deep, which formed the remainder of the walls and floor of the fold. The cells were columnar 44 Henry Cohen and Sarah Davies

and their cytoplasm was so densely granular that the individual cell boundaries were difficult to distinguish. Their nuclei were round or slightly elongated and possessed deeply staining nucleoli and nuclear membrane. The floor of the fold exhibited a higher degree of villus formation than in the preceding stage, and the branching of the villi was even more pronounced (see PI. I, fig. 5).

As in previous stages, the brain was surrounded by two zones of mesenchyme condensation separated by an intermediate zone of loosé meshed mesenchyme tissue. The inner zone was still narrow and closely adhered to the brain surface. As in all previous stages, the mesenchyme immediately adjacent to the membranous areas in the roof of the fourth ventricle showed little or no ‘condensation, so that above these regions this zone was virtually absent. The width of the intermediate zone varied considerably. On the dorsal surface of the optic lobes and anterior surfaces of the cerebral hemispheres it was very narrow indeed, due to close approximation of the inner and outer zones. Over


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Text-fig. 7.

the thalamencephalon, mid-brain and cerebellum it was much wider, but as it extended posteriorly over the medullary roof it then became increasingly narrow. In the mid-dorsal line above the medulla this intermediate zone contained a large irregular longitudinal sinus, extending from the anterior end of the cerebellum to the posterior end of the medulla. The outer zone of condensation was more clearly defined than in the previous stage and showed a higher degree of chondrification. The cells of the intermediate layer immediately adjacent to the cartilaginous bands of the outer zone were even more densely compacted together than previously, forming a definite but narrow zone of fibrous appearance (see Pl. II, fig. 7).

The periaxial spread of the dye was more extensive than hitherto. Anteriorly it had extended over the cerebellum, and posteriorly along the spinal cord for about half of its length. The meshes of the inner zone of condensation were still entirely free of the dye. The injected fluid had obviously escaped through the posterior membranous area in the roof of the fourth ventricle. A heavy condensation of blue granules and coagulum adhered to the ventricular surface of this area, and its cells, together with those of the overlying Cerebrospinal Fluid Spaces and Choroid Plexuses 45

mesenchyme, were deeply permeated with the dye. The anterior membranous area showed only a fine granular distribution on its ventricular surface, and its cells were entirely free of the dye. Since the inner zone of condensation was absent over the differentiated roof areas, only a thin membrane separated the fourth ventricle and its contents from the overlying mesenchyme of the intermediate zone; consequently, the escape of dye from the fourth ventricle was directly into the intercellular meshes of this zone. The capillaries and longitudinal sinus of this region showed no invasion by the dye, except at one point where the sinus actually abutted on to the posterior membranous area, and here there was only a very slight, almost negligible, infiltration of the dye through the sinus wall.


Text-fig. 8.

To summarize: the external appearance of chick embryos in which the central nervous system was injected with the double solution of potassium ferrocyanide and iron ammonium citrate suggested that two specialized areas, one anterior and the other posterior, were present in the roof of the fourth ventricle. These were roughly oval in shape and were distinguishable from the remainder of the roof by their intense colour. The anterior area was first visible in embryos of 8} days’ incubation, and rapidly increased in size until the 5th day. It then commenced a gradual regression, till at 8 days it was no longer visible externally. The first indication of a posterior area was seen in embryos of 64 days. This gradually increased in size, till at 9 days it occupied the greater part of the posterior region of the roof. 46 Henry Cohen and Sarah Davies

During the earlier stages of development there appeared to be no escape of the injected dye from the central nervous system. At the seventh day of incubation, however, a slight spread was evident from the roof of the fourth ventricle, and this became increasingly marked from the seventh day onward.

These observations of the injected embryos were verified by a detailed histological examination.

The first indication of a differentiated area on the roof of the fourth ventricle was seen in embryos of 3 days’ incubation. At this stage it exhibited only a fine distribution of blue granules on its ventricular surface. Therefore no indication of the presence of this area was given by external examination of the embryo. At 8} days this membranous area was fully differentiated. A heavy


condensation of blue granules now adhered to its ventricular surface, and therefore its position could be identified externally. Up to the 4} days’ stage this area rapidly increased in size, but at 5 days it showed a slight reduction. It gradually diminished in extent during succeeding stages, and in 9 days’ embryos it was practically non-existent. Furthermore, at 5 days the roof of the fourth ventricle was divided by a median transverse fold into anterior and posterior regions. At this stage, too, indications of the differentiation of a second membranous area were evident in the posterior region of the roof. The presence of this second area could not be ascertained externally until the 64 days’ stage, when for the first time a dense accumulation of blue granules was present on its ventricular surface. This posterior membranous area became fully differentiated at 6} days, and thereafter increased rapidly in extent till at 9 days it occupied almost the whole of the posterior region of the roof. Meanwhile, the median transverse fold in this same roof had become increasingly marked Cerebrospinal Fluid Spaces and Choroid Plexuses 47

throughout successive stages, till at 9 days it almost reached the floor of the ventricle. At the same time the cells participating in this fold gradually lost their original cuboidal appearance and became columnar.

In embryos up to and including the age of 6} days the injected dye remained entirely within the central canal of the spinal cord and ventricular system of the brain. At 7 days, however, there was a slight extension of the dye from the anterior membranous area in the roof of the fourth ventricle into the overlying mesenchyme of the intermediate zone. At 7} days this spread was slightly more marked, the injected dye still escaping through the anterior membranous area. In embryos of 8 days’ incubation the passage of the dye from the fourth ventricle was even more extensive than previously. At this stage, the injected fluid made its escape through the posterior membranous area in the medullary roof, and no longer through the anterior membranous area. At 8} days there was a very marked periaxial spread of the dye, even along the dorsal surface of the spinal cord, its passage again being through the posterior membranous area. At 9 days the spread of the dye from this same area was even more pronounced, Anteriorly it had invaded the mesenchyme overlying the whole cerebellar anlage, while posteriorly it extended along the cord for a considerable distance.

The first choroid plexuses to develop were those of the third and lateral ventricles. In embryos of 6 days’ incubation the thin thalamencephalic roof presented a slightly undulating appearance owing to the formation of 2-3 broad irregular folds. These undulations were more marked at 6} days, while in embryos of 6? days they extended anteriorly for a short distance into the lateral ventricles via the foramina of Munro, still in the form of simple unbranched villi. At 7 days, however, the folds exhibited a slight degree of branching, forming an embryonic choroid plexus. From this stage onward the formation of branching choroid villi increased rapidly, till at 9 days they occupied almost the whole of the lateral ventricles. The first indication of the development of the choroid plexus of the fourth ventricle was seen in embryos of 74 days’ incubation. At this stage the ventro-lateral walls of the transverse median fold in the roof of this ventricle showed a slight degree of undulation, but no true villi were apparent. At 8 days the undulations were much more marked, resulting in the formation of short unbranched villi concentrated in two ill-defined clusters in the floor of the fold. In 8} days’ embryos the formation of these villi was more pronounced, and they exhibited a slight tendency to branching, which became even more marked at 9 days.

In 9 days’ embryos the brain was enclosed by the developing cranium and two meninges, an inner and an outer, which were separated by an intermediate layer of undifferentiated mesenchyme. The inner meninx was narrow, closely adhered to the surface of the brain, was loosely trabeculated and contained minute capillaries in its intercellular meshes. Where this layer abutted on to the membranous areas in the roof of the fourth ventricle it was so thin as to be almost absent. The outer meninx, on the other hand, lay close to the inner 48 Henry Cohen and Sarah Davies

surface of the embryonic skull; it was wider than the inner membrane, was non-vascular and presented a fibrous appearance- The intermediate zone separating the inner and outer meninges was of variable width and possessed a fairly rich blood supply. Where this zone overlay the roof of the fourth ventricle it contained large irregular spaces.

Both meninges and the cranial anlage originated in situ by a process of cellular condensation in the general mesenchyme surrounding the brain. The inner meninx arose as a single membrane from a zone of condensation immediately adjacent to the brain surface. It was first distinguishable in embryos of 34 days’ incubation, but at this stage was narrow and ill-defined. At 44 days it was still narrow but more clearly visible as a distinct layer, and now showed a slight degree of vascularization. During successive stages of development this zone became increasingly vascular and more clearly defined. The cranial anlage was first visible in embryos of 5 days’ incubation where it was represented by an extremely irregular and ill-defined zone of mesenchyme condensation situated at some distance from the inner meninx. At 6 days this zone was more clearly marked, and localized centres of cartilage formation were evident within its substance. In later stages this zone became increasingly well defined and showed a progressive degree of chondrification. Meanwhile, at 8 days the cells of the intermediate zone immediately adjacent to the developing cranium showed a slight degree of condensation, forming a very narrow ill-defined layer of fibrous appearance. At 8} days this zone was more marked, and at 9 days it was clearly visible as a definite but narrow fibrous membrane immediately within the embryonic cranium.

Owing to the absence of the inner meninx over the membranous areas in the roof of the fourth ventricle, the injected dye, in its passage from this ventricle, escaped directly into the meshes of the intermediate zone. Never at any stage was it seen to invade either the inner or the outer meninx.

(ii) Injections of a solution of iron ammonium citrate alone

The present section is based on external and histological observations of the embryos of series D. These were injected with a solution of iron ammonium citrate into the central nervous system. Since a detailed account of the development of the roof of the fourth ventricle, choroid plexuses and meninges has been given in the previous section, a description of the distribution of the injected fluid alone is given below.

In embryos up to and including the age of 64 days the injected solution was seen to be contained within the central nervous system. In cleared specimens of 7 days’ incubation a slight escape of fluid was evident from the roof of the fourth ventricle. At 7} days this spread was more marked and partly obscured the outline of the medullary roof. In embryos of 8, 84 and 9 days’ incubation the outline of this roof was no longer visible externally, suggesting that an even greater extra-ventricular spread of the injected fluid had occurred. Cerebrospinal Fluid Spaces and Choroid Plexuses 49

These observations on the external appearance of the injected embryos were confirmed by histological examination.

In embryos of this series the development of the membranous areas in the roof of the fourth ventricle was similar to that described in embryos of series A, B and C. When these areas were present a heavy condensation of dye was seen adhering to their ventricular surface (see Pl. II, fig. 8).

Until the age of 7 days the injected solution was confined within the central nervous system. At 7 days there was a slight escape of the dye through the anterior membranous area into the overlying mesenchyme between the two zones of condensation (see Pl. II, fig. 9). At 74 days this spread had increased, but was still through the anterior area. In 8 days’ embryos the extension of the dye from the fourth ventricle was even more pronounced than previously. At this stage the posterior area alone functioned as the site of fluid escape. At 84 days the injected fluid had spread to an even greater extent, its passage still being through the posterior membranous area. In 9 days’ embryos the escape of the dye through this same area was even more marked. Anteriorly it had invaded the mesenchyme overlying the optic lobes, while posteriorly it extended along the spinal cord for about half of its length.

The meninges of the brain in embryos of series D showed no invasion by the dye. The injected fluid in its passage from the fourth ventricle escaped directly into the meshes of the intermediate zone.

Thus, embryos in which the central nervous system is injected with a solution of iron ammonium citrate alone yield the same results as those injected with the double solution of potassium ferrocyanide and iron ammonium citrate.

(b) Results of injections of true solutions into the vascular system

The observations recorded in sections Va (i) and Va (ii) showed that the first escape of injected fluids from the central nervous system into the surrounding tissue occurred in embryos of 7 days’ incubation. At this stage the passage of the dye was through the anterior membranous area in the roof of the fourth ventricle, and from 8 days onwards through the posterior area.

Injections were made into the vascular system in order to ascertain whether the anterior and posterior areas at 7 and 8 days respectively allowed the passage of dye into the fourth ventricle from the surrounding tissue. For this purpose two series of embryos were injected, series E and F. In both cases the age limits of the series were 5 and 9 days, thus allowing an ample margin of time before and after the critical ages.

In the embryos of series E, injections of the double solution of potassium ferrocyanide and iron ammonium citrate were made into the left ventricle of the heart. Embryos of series F were similarly injected, but with a solution of iron ammonium citrate alone.

In embryos of both series Prussian blue granules were seen in the capillaries -of the head region and even in those of the inner meninx. There was, however,

Anatomy Lxxi1 4 50 Henry Cohen and Sarah Davies

no evidence of any passage of the dye through the capillary walls into the surrounding tissue. Therefore, it cannot be said whether the membranous areas allow the passage of dye into the fourth ventricle or not. But one fact emerged clearly—there was no passage of fluid from the vascular system into the ventricles of the brain.

VI. GENERAL SUMMARY AND DISCUSSION

The present investigation shows that at certain stages in the development of chick embryos two differentiated areas, one anterior and the other posterior, are present in the roof of the fourth ventricle. The two areas are sharply delimited from the general roof epithelium, and consist of very flat cells with narrow elongated nuclei. These areas can be identified with the area membranacea superior and the area membranacea inferior described by Weed (1917), in the roof of the fourth ventricle of pig and human embryos.

In the chick the anterior area is first differentiated in embryos of 34 days. It then increases rapidly, till at 44 days it occupies the greater part of the anterior region of the medullary roof. At 5 days this area shows a slight reduction in extent, and gradually diminishes during successive stages, till at 9 days it is almost non-existent. As in pig and in human embryos, the disappearance of this area is brought about by the encroachment of the developing cerebellum on the anterior portion of the medullary roof. The posterior area first appears in embryos of 5 days’ incubation. At 63 days it is fully differentiated, and thereafter increases rapidly, till at 9 days it occupies the greater part of the medullary roof behind the choroid plexus.

Thus the development of these membranous areas in the roof of the fourth ventricle of the chick shows the same sequence of events as that described by Weed (1917) in pig and in human embryos.

The passage of injected fluids from the central nervous system into the surrounding tissue is first seen in chick embryos of 7 days. At this stage the spread of the fluid is very slight and occurs through the anterior membranous area alone. At 74 days the escape of the solution is more marked and still takes place through the anterior area. This extra-ventricular spread is much more extensive at 8 days, but the dye now passes through the posterior area only. In embryos of 84 and 9 days the fluid escapes even more extensively through this same area, spreading over the cerebellum, optic lobes and anterior part of the spinal cord.

From the work of Weed (1917), it appears that the membranous areas in the roof of the fourth ventricle function almost as soon as they are formed. In the chick, however, these areas are fully differentiated several days before they allow of any fluid escape.

According to Keegan (1917), in chick embryos injected with the double solution of potassium ferrocyanide and iron ammonium citrate, no escape of fluid occurs from the central nervous system, even when the choroid plexuses are well developed. We found, however, that in embryos injected with the Cerebrospinal Fluid Spaces and Choroid Plexuses 51

double solution there is a definite spread of fluid from the central nervous system, correlated with the development of the choroid plexuses. The first choroid plexuses to develop are those of the third and lateral ventricles. These are fully formed at 7 days, and at this stage the first extra-ventricular spread of the injection fluid occurs. The choroid plexuses of the fourth ventricle are only fully developed between 8} and 9 days, and this was associated with a further marked escape of fluid from the fourth ventricle.

In the chick, therefore, the first spread of fluid is coincident with the development of the choroid plexuses of the third and lateral ventricles. A similar correlation between the development of the choroid plexuses and escape of injection solution is recorded by Weed (1917) in pig embryos. Here the first spread corresponds with the formation of the choroid plexus of the fourth ventricle, this plexus being the first to develop.

Observations on chick embryos in which the central nervous system was injected with a solution of iron ammonium citrate alone do not agree with the findings of Keegan (1917), who made the same investigation. He states that the citrate solution invariably leaves the fourth ventricle even when no choroid plexuses are present. The present investigation shows a definite correlation between the escape of the dye and the formation of the choroid plexuses. An extra-ventricular spread was never observed before the appearance of a choroid plexus.

The observations herein recorded show that only two meninges are present in the chick, an inner and an outer, separated by a region of undifferentiated mesenchyme. The outer meninx, lying immediately within the embryonic skull, is narrow, non-vascular and presents a fibrous appearance. The inner meninx, closely adhering to the surface of the brain, is narrow, richly vascular and presents a loosely trabeculated appearance, but cannot be divided into two separate membranes.

The outer meninx is obviously the dura mater. From its appearance the inner meninx can be identified with the secondary meninx described in birds by Sterzi (1902), Ariéns-Kappers (1929), and Ariéns-Kappers e¢ al. (1936). The development of this inner meninx also shows that it corresponds to the piaarachnoid of the chick, described by Farrar in 1906.

Never at any stage of development was the inner meninx invaded by solutions injected into the central nervous system. These fluids in their passage from the fourth ventricle escape directly into the meshes of the intermediate zone. Therefore, the present investigation clearly shows that, contrary to the statement of Hansen-Pruss (1928), the inner meninx functions as a single membrane and its spaces are not comparable physiologically to the subarachnoid spaces of the Mammalia. 52 Henry Cohen and Sarah Davies

VII. CONCLUSIONS

1. In chick embryos the cerebrospinal fluid, as indicated by the distribution of injected solutions, first escapes from the central nervous system at the seventh day of incubation. In 7 and 7} days’ embryos the passage of the fluid is through a membranous area in the anterior portion of the medullary roof. At 8, 84 and 9 days the fluid escapes through a similar area in the posterior region of the roof.

2. The first choroid plexuses to develop are those of the third and lateral ventricles. These are fully formed at 7 days, and at this stage the first extraventricular spread of fluid occurs. The choroid plexus of the fourth ventricle is fully developed between 8} and 9 days, and this is correlated with a further marked escape of fluid from the fourth ventricle. Therefore, the spread of cerebrospinal fluid from the central nervous system is coincident with the development of the choroid plexuses of the brain.

8. Two meninges are present in the chick, an outer dura mater and an inner secondary meninx. The inner meninx, though loosely trabeculated, develops and functions as a single membrane. Its spaces contain no cerebrospinal fluid, and are therefore not comparable with the subarachnoid spaces of the Mammalia.

We wish to thank Mr K. M. Wilson for constructing the apparatus shown in Text-fig. 1, and Mr F. Beckwith for taking the photographs.

REFERENCES

Anritns-Kapprrs, C. U. (1929). The Evolution of the Nervous System, p. 195. Haarlem.

Axnrins-Kaprers, C. U., Huser, G. C. & Crossy, E. C. (1936). The Comparative Anatomy of the Nervous System of Vertebrates including Man, vol. 1, p. 63. New York.

Cuvigr, G. L. (1809). Legons d’ Anatomie Comparée. Paris.

Farrag, C. B. (1906). “The embryonic pia.” Amer. J. Insanity, vol. Lx1u, p. 296.

Hansen-Pruvss, O. C. (1923). ‘“‘Meninges of birds with a consideration of the sinus rhomboidalis.” J. comp. Neurol. vol. xxxvt, p. 193.

Kegean, J. J. (1917). “‘A comparative study of the roof of the fourth ventricle.” Anat. Rec. vol. x1, No. 6, p. 379.

Liz, F. R. (1908). The Development of the Chick, p. 252. New York.

Owen, R. (1868). Comparative Anatomy and Physiology of Vertebrates, vol. m1. London.

Sterzi, G. (1902). “Recherches sur l’Anatomie Comparée et sur l’Ontogenése des Méninges.” Arch. ital. Biol. t. xxxvu, p. 287.

Streeter, G. L. (1904). “The structure of the spinal cord of the ostrich.” Amer. J. Anat. vol. 111, p. 1.

Wexp, L. H. (1917). “The development of the cerebrospinal spaces in pig and in man.” Contr. Embryol. Carneg. Instn, vol. v, No. 14. Journal of Anatomy, Vol. LX XII, Part 1 Plate I |


Fig. 3 Fig. 4


Fig. 5

COHEN anp DAVIES—CEREBROsSPINAL FLUID Spaces AND CHOROID PLEXUSES Journal of Anatomy, Vol. LX XII, Part 1 Plate II


Fig. 6 Fig. 7



Fig. 8 Fig. 9

COHEN anp DAVIES—CrEREBROSPINAL FLUID Spaces AND CHOROID PLEXUSES Fig

Fig. Fig. Fig.

Fig.

Fig. Fig.

‘Fig.

Fig.

Cerebrospinal Fluid Spaces and Choroid Plexuses 53

EXPLANATION OF PLATES I AND II Prats I

. 1. Photomicrograph of a longitudinal section of the roof of the fourth ventricle in a 3} days’ embryo. In this specimen an injection of a double solution of potassium ferrocyanide and iron ammonium citrate was made into the central nervous system. (Acid formaldehyde : Ehrlich’s haematoxylin and eosin.) x31 approx. .

2. Photomicrograph of a longitudinal section of the roof of the fourth ventricle in a 6} days’ embryo, injected as in specimen illustrated in fig. 1. (Acid formaldehyde : Ehrlich’s haematoxylin and eosin.) x24 approx.

3. Photomicrograph of a longitudinal section of the roof of the fourth ventricle, showing the development of the choroid villi in an 8} days’ embryo, injected as in specimen illustrated in fig. 1. (Acid formaldehyde : Ehrlich’s haematoxylin and eosin.) x 38 approx.

4. Photomicrograph of a longitudinal section of the roof of the fourth ventricle in a 9 days’ embryo, injected as in specimen illustrated in fig. 1. (Acid formaldehyde : Ehrlich’s haematoxylin and eosin.) x28 approx.

5. Photomicrograph of a longitudinal section of the roof of the fourth ventricle showing the choroid villi in a 9 days’ embryo, injected as in specimen illustrated in fig. 1. (Acid formaldehyde : Ehrlich’s haematoxylin and eosin.) x36 approx.

Prats IT

6. Photomicrograph of a transverse section of the cerebral hemispheres, showing the inner and outer zones of mesenchyme condensation in a 6 days’ embryo. (Bouin’s Fluid : Ehrlich’s haematoxylin and eosin.) x37 approx.

7. Photomicrograph of part of a longitudinal section of the floor of the medulla, showing the dura mater in a 9 days’ embryo. (Acid formaldehyde: Ehrlich’s haematoxylin and eosin.) x 37 approx.

8. Photomicrograph of a longitudinal section of the roof of the fourth ventricle in a 43 days’ embryo. In this specimen the central nervous system was injected with a solution of iron ammonium citrate. (Acid formaldehyde and potassium ferrocyanide solution: Ehrlich’s haematoxylin and eosin.) x30 approx.

9. Photomicrograph of a longitudinal section of the roof of the fourth ventricle in a 7 day’s embryo, injected as in specimen illustrated in fig. 8. (Acid formaldehyde and potassium ferrocyanide solution : Ehrlich’s haematoxylin and eosin.) x24 approx.

ABBREVIATIONS

A.M. Anterior membranous area. LV. Lateral ventricles. C.B. Cerebellum. M.T.F. Median transverse fold. CR. Cranium. 0.2. Outer zone of mesenchyme C.V. Choroid villi. condensation. Dz Dura mater. ; P.M. Posterior membranous area. D.A.G. Dense accumulation of blue granules. 8. Extra-ventricular spread of injected F.M. Floor of medulla. fluid. 1.Z. Inner zone of mesenchyme condensation.


Cite this page: Hill, M.A. (2020, July 15) Embryology Paper - The development of the cerebrospinal fluid spaces and choroid plexuses in the chick (1937). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_The_development_of_the_cerebrospinal_fluid_spaces_and_choroid_plexuses_in_the_chick_(1937)

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