Ontogenetical development of the chick and duck subcommissural organ

Histochemistry (1986) 84:31 40 stochemistry �9 Springer-Verlag 1986

Ontogenetical development of the chick and duck subcommissural organ An immunocytochemical study*

K. Schoebitz I, O. Garrido 1, M. Heinriehs 2, L. Speer 1, and E.M. Rodriguez 1 ** Instituto de Histologla y Patologia, Universidad Austral de Chile, Valdivia, Chile

2 Department of Anatomy and Cytobiology, Justus Liebig University of Giessen, Giessen, Federal Republic of Germany

Accepted September 24, 1985

Summary. The ontogenetical development of the subcommissural organ (SCO) was investigated in chick embryos collected daily from the 1st to the 21st day of incubation. Some duck embryos, and adult chickens and ducks were also studied. Immunocytochemistry using an anti-Re- issner's fiber (RF) serum as the primary antibody was the principal method used.

In the chick embryos the events occurring at different days of incubation were: day 3 morphologically undifferen- tiated cells in the dorsal diencephalon displayed immunoreactive material (IRM); days 4 to 6 immunoreactive cells proliferated, formed a multilayered structure and developed processes which traversed the growing posterior commissure and ended at the brain surface; day 7 i) blood vessels penetrated the SCO, ii) scarce hypendymal cells appeared, iii) the first signs of ventricular release of IRM were noticed, iv) appearance of IRM bound to cells of the floor of the Sylvius aqueduct; day 7 to 10 the number of apical granules and amount of extracellular IRM increased progressively; day 11 RF was observed along the Sylvian aqueduct; day 12 RF was present in the lumbar spinal cord; day 13 IRM on the aqueductal floor disappeared; days 10 to 21 i) hypendymal cells proliferated, developed processes and migrated dorsally, ii) ependymal processes elongated and their endings covered the external limiting membrane. In adult specimens the ependymal cells lacked basal processes and the external membrane was contacted by hypendymal cells. The duck SCO appears to follow a similar pattern of development.

Mollgard 1975) has also been investigated. However, certain developmental aspects, such as differentiation of SCO secretory cells, initiation of secretory activity and appearance of Reissner's fiber (RF), have only been investigated in non-mammalian species such as the chicken (Wingstrand 1953; Ziegels 1977), frog (Oksche 1961), trout (Olsson 1956), shark (Altner 1963), cyclostome fishes (Sterba et al. 1967). All of these studies were performed using classical staining methods for visualization of SCO secretory material, namely Gomori and PAS. Only in a few of these publications were other histochemical methods or electron microscopic techniques applied.

The development of a highly sensitive immunocytochemical procedure (Sternberger et al. 1970) and the avail- ability of antisera reacting specifically with the secretory material of the SCO and RF (Sterba et al. 1982; Rodriguez et al. 1984b) stimulated us to reinvestigate certain developmental aspects of the SCO. The investigation was carried out principally in chick embryos. They were chosen because they are readily available throughout the year, and may become a useful model for future experimental designs. Spe- cial emphasis has been paid to the time sequence of certain events, such initiation of secretory activity in ependymal and hypendymal cells, intracellular distribution of the secretory material, appearance of secretory material on ventricular surface of the organ and time of formation of RF. Fur- thermore, the so-called "leptomeningeal route" (see Oksche 1961; Rodriguez et al. 1984b), and the vascularization of the SCO, were also investigated in the developing chick and duck SCO.

Introduction

There are several publications dealing with various aspects of the subcommissural organ (SCO) during ontogeny. Sev- eral of these studies were concerned with the human fetal SCO (Oksche 1956, 1961; Wislocki and Roth 1958; Olsson 1961 ; Mollgard 1972). The embryonic SCO of other mammalian species such as the rat (K6hl and Linderer 1973; Marcinkiewicz and Bouchard 1983) and rabbit (Kimble and

* Supported by Grant 1/60 935 from the Stiftung Volkswagen- werk, Federal Republic of Germany, and Grant RS-82-18 from the Direcci6n de Investigaciones, Universidad Austral de Chile. M.H. was recipient of a personal grant from JNO (29-5-54), which is gratefully acknowledged

** To whom offprint requests should be sent

Materials and methods

Chick embryos. SPF (specific pathogenic free) eggs of white leghorn (Gallus domesticus) were obtained from the Instituto de Patologia Aviar, Universidad Austral de Chile. The eggs were incubated at 37.5 ~ C. One or two embryos were fixed every day from the 1st (stage 6 of Hamilton 1965) to the 21st day of incubation. From the 1st to the 12th day of incubation the embryos were fixed by immersion after exposing the brain tissue with a cut along each side of the head. The remaining embryos were fixed by vascular perfusion through a cannula inserted in the left ventricle of the heart. After perfusion, the brain was exposed as above and the embryo immersed in the fixative. In all cases, fixation was in Bouin's fluid and lasted for 48 h. From day 5 on, three blocks of tissue were obtained from each embryo. One contained the brain and brain stem, another included a few lumbar segments of the spinal cord, and the third contained the caudal end of the spinal cord.

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Adult chickens. The brain of two adult chickens (1 and 11/z year old) were fixed by vascular perfusion with Bouin's fluid. A block of tissue containing the epithalamus and a portion of the brain stem with the Silvian aqueduct was dissected out and immersed in the fixative for 48 h.

Duck embryos. Fertilized eggs of Pekin ducks (Anas platyrhynchos L.) were purchased from a commercial breeder (L. M6hlenpage, Lemforde, West Germany). After shipping they were stored at room temperature in the dark, for 48 h. Eggs were incubated according to the schedule of Vogt (1979). After 5, 6, 7, 8 and 9 days of incubation the embryos were fixed by immersion in Bouin's fluid. 15, 20 and 25 day old embryos were fixed by vascular perfusion. For comparison, a duckling (5 day post-hatching) and three adult ducks were fixed by vascular perfusion and included in the series.

After fixation and washes in distilled water, all tissue blocks were dehydrated in ascending concentrations of alcohols and em- bedded in paraffin or Paraplast. The blocks were oriented in order to obtain sagittal sections of the SCO and cross sections of the spinal cord. Each block containing the SCO was serially cut and then series of sections prepared by collecting one every tenth section in each series. The blocks containing the spinal cord were serially cut and every tenth section mounted.

The series of sections of each SCO were stained by one of the following methods : 1) periodic acid-silver methenamine (Rodri- guez et al. 1984 a); 2) immunostaining using the primary antiserum (AFRU) at a dilution of 1:4,000; 3) immunostaining using the primary antiserum at a dilution of 1:150,000; 4) a procedure similar to immunoperoxidase staining but in which incubation in the primary antiserum was omitted; 5) some series of sections was kept unstained. In some specimens all sections containing the brain were mounted and immunostained using AFRU at a dilution of 1 : 4,000.

The sections of the spinal cord were stained with paraldehyde- fuchsin according to Gabe (1968) or periodic acid-silver methenamine. These methods were used to visualize RF in the central canal. At this level RF was not immunoreactive with the antisera used.

Immunostaining: The unlabeled peroxidase method of Sternberger et al. (1970) was used. An antiserum raised against bovine RF dissolved in a medium containing ethylene diamine tetraacetic acid, DL-dithiothreitol and urea (AFRU, A = antiserum, FR = Fiber of Reissner, U= Urea; see Rodriguez et al. 1984b) was used as primary antibody. AFRU was used at two dilutions, 1:4,000 for a strong immunostaining of the SCO and 1:150,000 for the demonstration of mature secretory granules of the SCO (cf. Rodriguez et al. 1986). Incubation time was 18 h. The second antibody was used at a dilution of 1 : 50. PAP (Bioproducts, Brussels, Belgium) was diluted to 1 : 75. All antibodies were diluted in TRIS buffer, pH 7.8, containing 0.7% non-gelling gelatin, lambda carrageenan (Sigma) and 0.5% Triton X-100 (Sigma) (Sofroniew et al. 1979). Coplin jars were used for incubation with the first and second antibody, whereas PAP incubation was performed in a moist chamber. All incubation were carried out at 22 ~ C.

Resu l t s

Chick embryos

Immunostaining using AFR U at a dilution o f 1:4,000. Dur- ing the first two days of incubat ion (Hamil ton stages 6 and 12) no immunoreact ive mater ial ( IRM) was found in the developing brain. The embryos collected during the third day of incubat ion (ca. 30 somites, Hami l ton stage 17; Ham- burger-Saunders stage 3) showed in the dorsal wall of the poster ior diencephalon a group o f cells containing I R M (Figs. 1 and 2). The area occupied by the immunoreact ive cells did not show any special structural feature to distin- guish it as par t of the developing SCO. When adjacent sections were stained with other methods this area could not be distinguished from the neighboring region of the dorsal wall of the diencephalon (Fig. 21). The immunoreactive cells were elongated with the I R M distr ibuted in the apical cytoplasm (Fig. 2). They did not display basal pro- cess.

Dur ing the 4th day of incubat ion the number of immunoreactive cells increased and a distinct knob corresponding to the developing pineal gland appeared. In the 5-day embryos the first bundles of the poster ior commissure became apparent , thus producing a local thickening of the dience- phalic wall. Parallel to this, the immunoreact ive cells developed basal processes which ended on the external basal membrane. Many of the processes and endings, but not all of them, contained IRM. By the 6th day of incubat ion the poster ior commissure had thickened and the basal processes of ependymal cells elongated (Figs. 3 and 4). Some of these processes were still devoid of I R M (Fig. 4). The number of immunoreact ive cells had increased enormously and become arranged in a stratified layer. U p to this stage no I R M was seen on the free ("vent r icu lar" ) surface of the SCO.

The SCO of 7-day embryos displayed several impor tan t fea tures : / ) The poster ior commissure had thickened consid- erably and the layer of stratified ependymal cells become taller. Thus, poster ior commissure and SCO appeared as a curved strUcture pro t ruding into the brain cavity (Fig. 5); i/) the ependymal basal processes not only became longer but, at a fixed distance from the cell body, divided into two or more slender processes that, through a terminal dila- tation, ended on the external basal membrane (Fig. 8). All processes and endings were strongly immunoreact ive (Figs. 6 and 8); iii) A thin layer of I R M lying on the free surface of the ependymal cells was first noticed (Fig. 6); iv) A few immunoreact ive hypendymal cells were seen in the poster ior commissure (Figs. 6 and 8); v) One or two

Figs. 1 and 2. 3-day chick embryo. Immunostaining, AFRU 1:4,000. The SCO (arrow) is the only positive structure. Tc telencephalon, Dc diencephalon, Me mesencephalon, Mt metencephalon, Ist isthmus, v t velum transversum, or optic recess. Fig. 1: x 35; Fig. 2: x 170

Figs. 3 and 4. 6-day chick embryo. Immunostaining, AFRU 1:4,000. Asterisk posterior commissure, arrows processes of ependymal cells, arrowhead ependymal endings on external limiting membrane. Fig. 3: x 30 ; Fig. 4: x 170

Figs. 5--8. 7-day chick embryo. Immunostaining, AFRU 1:4,000. Fig. 5 immunoreactive material appears in the SCO and on floor (arrows) of Sylvius aqueduct (SA). OL optic lobe, P pineal, x 30. Fig. 6 enlarged detail of previous figure. Arrows ependymal endings, double arrows hypendymal cell, arrowheads thin layer of immunoreactive material on free surface of SCO. x 170. Fig. 7. Enlarged detail of Fig. 5 showing the immunoreactive material on floor of Sylvius aqueduct (arrowheads) x 170. Fig. 8. Parasaggital section of SCO showing the branching of ependymal processes (arrowheads). Double arrows hypendymal cells, arrow layer of ependymal processes, Enon-secretory ependyma, x 170

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Figs. 9 and 10. 10-day chick embryo. Immunostaining, AFRU 1:4,000. Fig. 9. large arrow: fibrous immunoreactive structure, small arrows immunoreactive material on floor of Sylvius aqueduct, C cerebellum, per plica encephali ventralis, x 30. Fig. 10. Enlarged detail of previous figure. Small arrows: thick layer of immunoreactive material on free surface of SCO, large arrow fibrous structure, asterisk material on floor of Sylvius aqueduct, x 95

Figs. 1i and 12. ll-day chick embryo. Immunostaining, AFRU 1:4,000. Fig. 11. Reissner's fiber (arrows) in Sylvius aqueduct (SA) and fourth ventricle (IV). Double arrows immunoreactive material on floor on Sylvius aqueduct, C cerebellum, x 30. Fig. 12. Enlarged detail of previous figure. Arrows Reissner's fiber, arrowhead fibrous positive structure, asterisk positive material on floor of Sylvius aqueduct, x 95

blood vessels penetrated the posterior commissure and reached the SCO. So, by day 7 of incubation the SCO had acquired all of the structural features characterizing the organ in later stages of development.

In addition, in the 7-day embryos IRM appeared in the mesencephalon. It was located on the "ventricular" aspect of the ventral mesencephalon, along the midline, and extending from the cranial flexure, at the level of the plica

�9 encephali ventralis (Hamilton 1965), to the fourth ventricle. This area correspond, roughly, to the floor of the developing aqueduct of Sylvius (Figs. 5, 9, 11). The IRM appeared as a thin layer on top of ependymal cells. It was not possible to determine whether it was exclusively extracellular or partly intracellular (Figs. 7, 10, 12). This material did not stain when incubation in the first antibody was omitted.

At days 8 and 9 of incubation no important changes, other than an increase in the number of hypendymal cells, occurred.

In 10-day embryos a thick layer of IRM on the free surface of the SCO was observed (Fig. 10). Lying free in the ventricle, but principally in the vicinity of the flexure and floor of the Sylvian aqueduct, IRM arranged as a loose fibrous structure was observed (Figs. 9 and 10). This struc-

ture was occasionally seen extending between the SCO and the immunoreactive surface of the flexure.

The 1 l-day embryos were characterized by the appearance of a typical RF, namely, a homogeneously dense and immunoreactive structure, 7-8 gm in diameter. RF coexisted with the fibrous immunoreactive formation, described for the 10-day embryos, that lay in the vicinity of the floor of the Sylvian aqueduct (Figs. 11 and 12).

From the 12th to 21th day of incubation the principal changes undergone by components of the SCO or related to it were: i) disappearance of the IRM on the floor of the Sylvian aqueduct, starting on the 13th day of incubation; i0 a progressive thickening of the caudal half of the posterior commissure, which, as incubation proceeded, became progressively devoid of ependymal immunoreactive processes (Figs. 13-15). At variance, cephalically, the posterior commissure became thinner and elongated and numerous ependymal processes traversed it to end on the external limiting membrane where, by the 21st day, they formed an almost continuous layer (Fig. 15). Thus, the SCO changed from a U-shaped structure to a J-shaped formation (compare Figs. 11 and 15); iii) hypendymal cells increased in number, developed processes oriented perpendicularly

Figs. 13-15. SCO of chick embryo at the 16th (Fig. 13), 19th (Fig. 14) and 21st days (Fig. 15) of incubation. Immunostaining, AFRU 1:4,000. Star ependymal processes. • 36

Fig. 16. SCO of a chicken 1 year old. Ependymal processes traversing the posterior commissure are absent (star). • 36

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to the external brain surface, and progressively migrated laterally and toward the external limiting membrane (Figs. 17-19; iv) from the 15th day on the blood vessels irrigating the posterior commissure and SCO became more numerous.

The SCO of adult specimens showed two major differ- ences in comparison to that of 21-day embryos: /) only few ependymal cells had basal processes. These were short and did not reach the external basal membrane, but instead ended on blood vessels located in the interface between the posterior commissure and the SCO (Figs. 16, 20); it') the hypendymal cells were concentrated in the thinnest portion of the posterior commissure (cephalic end) and estab- lished close contact with the external limiting membrane (Figs. 16 and 20).

Immunostaining using AFR U at a dilution of l ." 150,000. This high dilution of AFRU did not reveal any IRM in the SCO of 3-day embryos (compare Figs. 2 and 21). The SCO of 4-day embryos showed a weak and homogeneous immunostaining. From the 5th day on the basal and the most superficial layers of ependymal cells displayed a weak reaction (Fig. 22). The 7-, 8- and 9-day embryos had a few apical granules which were strongly immunostained. By day 10, these apical, strongly immunoreactive granules, were numerous (Fig. 23) and in older embryos they formed an almost continuous layer in the most apical cytoplasm (Fig. 24). In each hypendymal cell a strongly immunostained area was observed (Fig. 24).

The secretory material on the free surface of the SCO (Fig. 23), the layer of IRM on the floor of Sylvius aqueduct, the fibrous structure lying in its vicinity, and RF were all deeply stained with this high dilution of AFRU.

Spinal cord

RF was first found in the central canal of the lumbar spinal cord in 12-day embryos. RF in the central canal was not immunoreactive and was visualized with the periodic acid- silver methenamine or aldehyde-fuchsin methods (Figs. 25 and 26).

Duck embryos

In the study of the SCO of duck embryos the main aim was to follow its earliest stages of development. So embryos were collected daily, from the 5th to the 9th day of incubation. A few samples of older embryos (15, 20 and 25 days old) were also available. So, certain events that in the chick embryos occur between day 10 and 12 of incubation, such as the appearance of IRM on free surface of the SCO and RF formation, have not been recorded for the duck embryos.

In the 5-day embryo the SCO appeared as a strongly immunoreactive patch located in the caudal half of the dorsal diencephalon (Figs. 27 and 28). The immunoreactive cells were densely packed, lacked processes, and occupied the entire thickness of the diencephalon wall (Fig. 28). Dur- ing the 6th day of incubation the first bundles of fibers of the posterior commissure appeared, and the immunoreactive cells began to move away from the external limiting membrane and to develop basal processes. Thus, by the 7th day the secretory cells had became arranged in several layers bordering the brain cavity and had developed long

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basal processes which, after traversing the posterior commissure, ended on the brain external surface (Figs. 29 and 30). At the external limiting membrane the ependymal endings were loaded with IRM and formed an almost continuous layer (Fig. 30). The developing pineal gland appeared as a distinct dorsal outgrowth (Figs. 29 and 30). By day 9, the posterior commissure had grown thicker and the ependymal processes had become longer and coarse. Before reaching the external limiting membrane most of them divided into slender branches (Figs. 30 and 31). The ependy- real cells of the basal and most apical layers displayed a stronger immunoreaction than that of the intermediate layers. A few hypendymal cells were observed.

The main changes found in the SCO of 15, 20 and 25 day old embryos were related to size and shape, and an increase in the number of the hypendymal cells.

No IRM was seen on the floor of the Sylvian aqueduct. The newly hatched duck (see Figs. 13 and 14 in Rodriguez et al. 1984b) had a SCO with similar characteristics to that of 20 and 25 day old embryos. At variance with these SCO, the SCO of adult duck specimens were virtually devoid of ependymal processes and had only a few hypendymal cells, so that the IRM was confined to the multilayered ependyma.

Discussion

Cells containing immunoreactive material (IRM) revealed by use of an anti-RF serum (AFRU) appear, in the chick embryos, at a remarkably early stage of development. In- deed, immunocytochemistry revealed the presence of secretory cells in 3-day (52-64 h) embryos. Thus, in the chick the differentiation of the secretory cells of the SCO occurs much earlier than formerly thought. By using the Gomori method, Wingstrand (1953) stated that in 6-day embryos the SCO has a general affinity to the Gomori stain and that distinct granules could be distinguished from the 7th day of incubation. Ziegels (1977) reported that secretory material (Gomori method) first appears in 7-day old chick embryos. Thus, the initiation of the synthetic activity of the chick SCO should be recorded as occurring during the third day of incubation, as shown in the present report. Among the known secretory structures of the central ner- vous system, the SCO would appear to be one of the first to develop.

Another remarkable observation is that the youngest immunoreactive cells do not show any signs of a morphological differentiation on light microscopic grounds, such

Figs. 17-19. Parasaggital sections of the SCO of chick embryos at the 9th (Fig. 17), 13th (Fig. 18) and 20th days (Fig. 19) of incubation. Immunostaining, AFRU t : 4,000. Fig. 17. EP ependymal processes, small arrows hypendymal cells, arrowheads ependymal endings on external limiting membrane, x 165. Fig. 18. Arrows processes of hypendymal cells, star area devoid of ependymal endings. x 175. Fig. 19. Most of the external surface lacks immunoreactive endings (star), E non-secretory ependyma, x 175

Fig. 20. SCO of a t year old chicken immunostained with AFRU t :4,000. Ependymal processes end on blood vessels (arrows) but not on the external limiting membrane (star) which is contacted by ependymal cells at the cephalic end of the organ (area delimited by two arrowheads), x 175

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Figs. 25 and 26. Lumbar spinal cord of 11-day (Fig. 25) and 12-day (Fig. 26) chick embryos. Aldehyde-fuchsin stain. Arrow Reissner's fiber. • 250

as their spatial organization into a multiple layered structure or the presence of basal processes. Apart from their immunoreactivity, they can not be distinguished from neighboring cells. This suggests that the expression of the gene(s) coding for the secretory product(s) could be one of the first events in the series leading to the development of the SCO.

Although in the early stages of development no distinc- tion can be made between ependyma and hypendyma, it is clear that all primitive immunoreactive cells become dif- ferentiated into ependymal cells, since hypendymal cells dif- ferentiate at later stages. The development of the basal processes of the ependymal cells is time-related to the development of the posterior commissure resulting in a continuous contact between the ependymal cells and the external limiting membrane.

Although synthesis of the secretory material would be- gin during the third day of incubation, the first sign of ventricular release was noted at day 7 as a thin layer of IRM on the free surface of the SCO. This is in agreement with Wingstrand's (1953) statement that " in 7-day embryos there is a delicate membrane of precipitated matter (Go- m o i l + ) from the liquor which rests on the tops of the flagellae".

There is evidence that in the SCO the secretory material is synthetized in the form of precursor molecules which, on their way from the perinuclear region to the apical cell pole, undergo processing resulting in mature secretory granules located in the vicinity of the ventricular cavity (Rodri- guez et al. 1986). These mature granules have a high affinity for A F R U , clearly demonstrated by a positive reaction at a high dilution of the antiserum (1:200,000), in contrast with the immature material which displays low or non immunoreactivity at this dilution (Rodriguez et al. 1986).

The use o f A F R U at a dilution of 1 : 150,000 for the immunostaining of the SCO of chick embryos showed a lack of staining of the SCO at day 3 and a homogeneous

Figs. 21-24. SCO of chick embryos at the 3rd (Fig. 21), 5th (Fig. 22), 10th (Fig. 23) and 21st days (Fig. 24) of incubation. Im- munostaining, AFRU 1:150,000. Fig. 21. Lack of immunoreactive material (compare with Fig. 2). x 175. Fig. 22 A weak immunoreaction is seen in cells of the apical (double arrows) and basal (arrows) layers. Star posterior commissure, x 175. Fig. 23. A strong reaction is seen in cells of basal layer (star), apical secretory granules (arrows) and pre-Reissner's fiber material (PRF) x 175. Fig. 24. Strong immunostaining of apical granules of ependymal cells (arrows) and in granules of hypendymal cells (double arrows). x 120

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Figs. 27 and 28. 5-day duck embryo. Immunostaining, AFRU 1:4,000. Fig. 27. The SCO (arrow) is the only reactive structure, tc

telencephalon, dc diencephalon, m c mesencephalon, vt velum transversum, or optic recess, x 25. Fig. 28. Enlarged detail of previous figure, x 110

Figs. 29 and 30. 7-day duck embryo. Immunostaining, AFRU 1:4,000. Fig. 29. p pineal, m c mesencephalon, m t metencephalon, I s t

istmo, x 25. Fig. 30. High magnification of SCO shown in Fig. 29. A r r o w s endings of slender ependymal processes, s tar bundles of posterior commissure, p pineal, x 110

Figs. 31 and 32. 9-day duck embryo, hnmunostaining, AFRU 1:4,000. P pineal. Fig. 31 : x 25, Fig. 32: x 110

weak reaction from day 4 to 6. The 7-day embryos showed, in addition to the weak background staining, strongly immunoreactive apical granules. It is suggested that the material synthetized by the SCO from the 3rd to the 6th day of incubation does not undergo processing, thus remaining as immature material. Maturation would operate from the 7th day of incubation, leading to the appearance of the

first apical secretory granules. As incubation proceeds, the apical granules start to accumulate at the apical cell pole so that by day 10, and thereafter, these granules appear as a dotted line underneath the apical plasma membrane. The apical mature granules are strongly stained by the Go- mori stain (Rodriguez et al. 1986). This is in agreement with Wingstrand's (1953) observation that the SCO of

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7-day embryos contains, in addition to the general Gomori staining shade of the cytoplasm, distinct granules located in the apical cytoplasm.

So, the appearance of i) an extracellular layer of IRM; and i0 apical secretory granules, are time-related events (both occur at the 7th day of incubation), supporting the suggestion that ventricular release of secretory material first occurs during the 7th day of incubation.

During days 8, 9 and 10 not only do the apical secretory granules increase in number, but also the layer of IRM attached to the free surface of the SCO becomes thicker. In the SCO of adult mammals this layer covering the surface of the organ has been shown to have different immunohis- tochemical reactivity than the RF proper, and has been regarded as a pre-RF material (Rodriguez et al. 1986). For the embryonic SCO we shall also refer to this layer as pre- RF. Although pre-RF was present from the 7th day of incubation, a RF proper was seen within the Sylvius aqueduct only from the I l th day of incubation. So, factors other than the ventricular release of IRM, must be required for the formation of RF.

RF was first seen in the lumbar spinal cord in the 12-day embryos. This would mean that the growth of RF from the Sylvian aqueduct to the lumbar spinal cord took place in a period of 24 h or less. This would imply a growing rate of about 70% 80% of total length/24 h. Such a growing rate of the developing RF is much higher than the esti- mated growing rate of RF in adult specimens of several species, in which the rate ranges from 1% of the total length/day in Lampetra fluviatitis (Ermisch et al. 1971) to 10%/day in the mouse (Ermisch 1973).

Hypendymal cells mainly develop during the second half of embryonic life, when they increase in number, migrate toward the brain surface, and develop cell processes through which they contact the external limiting membrane. In the chick embryos, the "leptomeningeal projection" of the SCO (cf. Oksche 1961 ; Rodriguez et al. 1984b), namely, the contact of the external limiting membrane by secretory processes, is exclusively provided by the ependymal cells during most part of embryonic life. Only during the last stages of development do hypendymal cells contribute to the leptomeningeal projection. On the other hand, in the adult chicken hypendymal cells are the only source of the leptomeningeal projection. Hence, regarding the leptomeningeal projection, the SCO of chick embryos is similar to the SCO of adult specimens of amphibian and reptilian species, whereas the SCO of the adult chicken resembles that of adult mammalian species (cf. Rodriguez et al. 1984b). The same reasoning applies to the duck SCO.

Although the synthesis of the IRM starts much earlier than the invasion of the SCO by the first blood vessels (7th day), this latter event coincides with the first signs of ventricular release. Whether these two events are interre- lated or not, can not be decided at the present stage. How- ever, the scarcity of blood vessels of the embryonic SCO and the virtual absence of secretory processes ending on these vessels would suggest that in the developing SCO of the chick and duck, the "vascular route" of secretion (cf. Rodriguez et al. 1984c) is either missing or of little impor- tance. Still, in the adult chick the ependymo-vascular path- way seems to be relatively well developed.

The location of IRM on the free surface of the cells lining the ventral mesencephalon agrees well with the de- scription given by Olsson (1956) of what he calls the "flex-

ural organ". According to Olsson the flexural organ is only present during certain developmental stages of the salmon, and during the earliest stages RF would be produced by this organ and not by the SCO. Altner (1963), who also described the flexural organ only in certain stages of the development of the shark brain, described RF connecting the SCO and the flexural organ. In the chick embryos the IRM on the floor of the ventral mesencephalon is also present only during certain stages (from day 7 to day 12). We have also seen a fibrous immunoreactive structure, different from RF, associated with the mesencephalic IRM or connecting the tip of the flexure and the SCO. So, in many respects our findings agree with those of Olsson (1956) and specially with those of Altner (1963). However, both authors regard the flexural organ as a secretory organ. We have no evidence for such a secretory activity in the cells lining the ventral mesencephalon. Instead, the IRM seems to be bound to the apical plasma membrane of these cells. Another important difference with Olsson's finding in salmon embryos or with his interpretation, is that in the chick embryos the SCO is the exclusive structure responsible for the formation of RF. In fact, for the six days in which the mesencephalic IRM was found (7th to 12th day), this material and RF coexisted only during two days (1 l th and 12th). Furthermore, the mesencephalic IRM was first observed at day 7, the same day on which the first signs of ventricular release were noticed in the SCO (see above). This, together with the presence of a fibrous structure inter- posed between the SCO and the floor of the mesencephalon and the extracellular location of the mesencephalic IRM strongly suggest that this latter material is of SCO origin. The significance of the IRM located on the mesencephalic floor is puzzling. Why should it be present only at certain stages of brain development ? Is its presence related to the onset of the release of secretory material from the SCO and the lack of operation of the factor(s) responsible for the condensation of the secreted material into RF?

Acknowledgement. The authors wish to thank the Dept. of Poulty Diseases, University of Giessen, for bacteriological examination of samples and Instituto de Patologia Aviar, Universidad Austral de Chile, for providing SPF eggs.

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Ontogenetical development of the chick and duck subcommissural organ

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