Abstract The subcommissural organ (SCO) and the floor plate (FP) secrete high molecular weight glyco-proteins that polymerize in the form of the Reissner’s fiber (RF). To study to what extent the absence of the FP affects the expression of these glycoproteins, we have investigated the brain and spinal cord of 48-h and 72-h wildtype and cyclops (cyc) mutant zebrafish lar-vae by using a polyclonal antiserum against bovine RF. Wildtype larvae showed immunoreactivity in the SCO at the dorsal forebrain-midbrain boundary. In the ven-tricle, over the SCO surface, thin immunoreactive fi-bers aggregated into an RF that ran along the third and fourth ventricles and the central canal of the spinal cord until, at its caudal end, the fiber disintegrated and formed a strongly immunoreactive massa caudalis that left the neural tube and invaded the surrounding tissues of the tail fin. The rostral end of the FP, lining the pon-tine flexure, was also strongly immunoreactive, as was the caudal third of the FP. Cyc mutants showed an im-munoreactive SCO and fibrous material in the ventri-cle, but an RF was missing. There was no label in the ventral midline of the neural tube except in some speci-mens in which the caudal FP persisted and was immu-noreactive. It is concluded that the product of the cyc gene is not required for the expression of SCO glyco-proteins but for their polymerization into an RF in the brain ventricles.
Keywords Flexural organ · Massa caudalis · Reissner’s fiber · Anti-RF antiserum · Whole-mount immunocytochemistry · Brachydanio rerio (Teleostei)
Introduction
The floor plate (FP) of the vertebrate embryonic neural tube secretes informative molecules involved in axonal growth and pathfinding (Kennedy et al. 1994; Culotti and Kolodkin 1996). In addition, it synthesizes and releases, into the lumen of the central canal, high molecular weight glycoproteins that condense in the form of the Reissner’s fiber (RF; Reissner 1860; Rodríguez et al. 1996; López-Avalos et al. 1997; Yulis et al. 1998). Later in embryonic development, the FP ceases to produce RF glycoproteins, except in a circumscribed rostral region named the flexur-al organ (FO; Olsson 1956) where this activity persists until birth. Simultaneously, the ependyma at the dorsal forebrain-midbrain boundary, beneath the fibers of the posterior commissure, starts to produce large amounts of RF glycoproteins; this region is called the subcommissu-ral organ (SCO; Dendy and Nicholls 1910). With a few exceptions (e.g., man), the secretory activity of the SCO persists for a lifetime; SCO is the only RF-forming brain gland in adult vertebrates (Oksche et al. 1993; Rodríguez et al. 1998). RF glycoproteins are high molecular weight, N-linked, sialylated glycoproteins that are immunologi-cally similar in all vertebrates and protochordates (Sterba et al. 1982; Rodríguez et al. 1984; Oksche et al. 1993; Olsson et al. 1994; Pérez et al. 1995). Sequencing of the mRNA has revealed that RF glycoproteins contain throm-bospondin-like repeats (Gobron et al. 1996, 2000; Nualart et al. 1998); hence the name SCO-spondin has been pro-posed. Some authors have reported that SCO-spondin or peptidic domains of this protein promote neurite exten-sion and neuronal aggregation (Gobron et al. 1996, 2000; Monnerie et al. 1997).
In the zebrafish embryo, an antiserum against Reiss-ner’s substance has been shown to label the FP and the SCO (Lichtenfeld et al. 1999). In addition, both structures This work was supported by a Grant BFI 2000-1360 from DGICYT
Madrid, Spain
P. Fernández-Llebrez (
✉
)Departamento de Biología Animal, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain
e-mail: [email protected]
Tel.: +34-952-131858, Fax: +34-952-132000
S. Hernández · J.A. Andrades
Departamento de Biología Celular, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain
DOI 10.1007/s004410100404
R E G U L A R A R T I C L E
P. Fernández-Llebrez · S. Hernández · J. A. Andrades
Immunocytochemical detection of Reissner’s fiber-like
glycoproteins in the subcommissural organ and the floor plate
of wildtype and
cyclops mutant zebrafish larvae
Received: 24 January 2001 / Accepted: 27 March 2001 / Published online: 23 May 2001 © Springer-Verlag 2001
have been shown to synthesize and release F-spondin 2, which is incorporated into the RF (Higashijima et al. 1997). Zebrafish cyclops (cyc) mutants lack an FP (Hatta et al. 1991, 1994). In order to study to what extent the se-cretory activity of the FP affects the development of the SCO-RF system, we have examined the central nervous system of 48-h and 72-h wildtype (wt) and cyc zebrafish larvae by means of an antiserum against the bovine RF.
Materials and methods
By means of immunocytochemistry, we have studied wt and cyc mutant larval zebrafish (Brachydanio rerio) at 48 h (n=10+10) and 72 h (n=10+10) after fertilization (supplied by Prof. K. Hatta, Uni-versity of Oregon, USA). Since zebrafish hatch between 48 h and 72 h, this age can be considered the first larval period or the last embryonic period. As primary antibody, we used an antiserum raised against an urea extract of bovine RF. This antiserum has been reported to bind to secretory glycoproteins of the SCO and the RF of all vertebrate species studied to date (Rodríguez et al. 1984). It also detects secretory substances in the FP of the devel-oping vertebrates including several fishes (López-Ávalos et al. 1997; Yulis et al. 1998). According to Lichtenfeld et al. (1999) who used an antiserum against Reissner’s substance and to our own previous unpublished observations in zebrafish, the SCO be-gins to be immunoreactive at 24 h, and the FP remains immunore-active until the first larval period. The aim of the present study was to compare the immunoreactivity of SCO and FP of wt and
cyc zebrafish specimens. We used larvae of 48 h and 72 h since, at
this age, wt specimens display a patent immunoreactivity in both the SCO and the FP.
All specimens were fixed by immersion in 4% paraformalde-hyde in phosphate-buffered saline (PBS) for 12 h. Whole-mount immunocytochemistry was carried out. After being washed in PBS and distilled water, the specimens were permeabilized with ace-tone at –20°C for 7 min and cleared with a mixture of benzol (20 ml), acetone (10 ml), hydrogen peroxide (5 ml), and 25% am-monium (40 µl) for 3 h at 37°C. They were then rinsed in ace-tone:water (1:1, for 10 min), distilled water (10 min), PBS (pH 7.8, 3×5 min), and subsequently exposed to bovine serum al-bumin, as saturating agent, containing 0.5% (v/v) Triton X-100 (Sigma) for 1 h. Thereafter, they were sequentially incubated in: (1) primary antibody (dilution 1:1000) for 18 h at room tempera-ture; (2) secondary antibody (anti-rabbit IgG, developed in goat in our laboratory, dilution 1:50) for 45 min at room temperature; (3) rabbit peroxidase-antiperoxidase (PAP Sigma; dilution 1:200) for 45 min at room temperature. Finally, peroxidase was visualized by incubation in 0.2% 3,3’-diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, Mo., USA) in TRIS buffer and 0.007% of H2O2for 10 min at room temperature in the dark. All antibodies were diluted in a solution composed of 100 mM TRIS-buffer, pH 7.8, containing 0.7% (w/v) lambda carrageenan as blocking agent (Sigma) and 0.5% (v/v) Triton X-100.
After the whole-mount immunocytochemistry, the specimens were viewed and photographed. They were then dehydrated and embedded in paraffin. Transverse or sagittal sections (5–10 µm thick) were mounted, deparaffinated, and viewed under a light mi-croscope. In order to control the specific binding of RF antibodies, rabbit serum (Sigma) was used as primary antibody. No immuno-reactive structures were seen in control animals.
Results
Wildtype larvae of 48 h and 72 h displayed similar fea-tures. They showed strong immunoreactivity in the dor-sal forebrain-midbrain boundary region beneath the
pos-terior commissure, i.e., in the SCO. Labeling was appar-ent in whole-mount preparations as a dark region in the head (Fig. 1A). In paraffin sections (Fig. 1B, C), immu-noreactive SCO cells showed basal processes that reached the outer surface of the brain via the posterior commissure. The apical cytoplasm of SCO cells was strongly labeled and immunoreactive fibrous material was visualized in the ventricular lumen over the apical surface of the SCO cells. This fibrous material aggregat-ed to form a thick RF that extendaggregat-ed caudally close to the ventral midline of the brain ventricles and the central ca-nal of the spica-nal cord (Fig. 1E, F). The rostral FP near the anterior end of the notochord at the level of the pon-tine flexure exhibited immunoreactivity. This region cor-responded to the FO (Fig. 1B, D). In transverse sections, the FO appeared as a monolayer of four or five secretory ependymal cells in the floor of the neural tube and dis-played immunoreactivity at its apical and basal pole (Fig. 1D). In the ventricular lumen, immunoreactive ma-terial was found on the surface of the FO cells. A distinct RF occurred in whole-mount preparations along the en-tire length of the neural tube from the SCO to the caudal tip of the spinal cord. In transverse sections, RF ap-peared as a dark dot close to the ventral surface of the neural tube (Fig. 1E). FP cells in the caudal spinal cord were also immunopositive (Fig. 1G, H); the label was visible in the cytoplasm around the cell nuclei giving a zipper-like appearance to the caudal FP in whole-mount preparations (Fig. 1G). The RF depolymerized and filled the caudal tip of the neural tube in an enlarged portion of the ventricular system called ampulla caudalis where it formed a massa caudalis (Fig. 1G). Immunoreactive ma-terial of the massa caudalis escaped from the ampulla caudalis via dorsal openings of the neural tube (Fig. 1I) and extended outside the neural tube throughout the sur-rounding tissues, mainly those ocupying a dorsal posi-tion (Fig. 1G, I).
All whole-mount preparations of cyc zebrafish larvae examined, either at 48 h and 72 h, showed strong immu-noreactivity in a region of the head corresponding to the location of the SCO (Fig. 2A, B). An immunoreactive SCO was always found in paraffin sections (Fig. 1C). The ventricular surface of the organ faced the lumen of an irregular dorsal portion of the third ventricle in which immunoreactive fibrous material could be found (Fig. 1D); however, we never observed morphological profiles suggesting aggregation of this fibrous material into an RF. An FO was never observed in any of the ex-amined specimens. We were unable to detect an RF in the spinal central canal and, in most specimens, immunoreac-tivity was missing at all levels of the ventral spinal cord (Fig. 2E, F). However, in some cyc specimens, FP cells could be identified in the caudalmost region of the spinal cord, and these FP ependymal cells were immunoreactive to the antibody (Fig. 2G, H). In these specimens, profiles resembling an ampulla caudalis and an immunoreactive massa caudalis were frequently seen (Fig. 2G). Immuno-reactive material outside the caudal neural tube, in the surrounding tissues, was however completely absent.
Discussion
The presence of Reissner’s material in zebrafish embryos has previously been reported by means of immunocyto-chemistry (Lichtenfeld et al. 1999). According to Lich-tenfeld et al. (1999), immunoreactivity was first detected in early embryos (15–20 h) along the whole FP. In 48-h embryos, immunoreactivity was present in the caudal one-third of the FP, the rostral FP in a region correspond-ing to the FO, and the SCO. Later on, an RF originatcorrespond-ing in the SCO was described. The results of the present work with a different antiserum agree with those of Lich-tenfeld et al. (1999). Moreover, the same sequence of
events, but at different times, have been reported for em-bryos of other fishes, such as Sparus aurata, Onco-rhynchus kisutch (Yulis et al. 1998), and Scyliorhinus canicula (López-Avalos et al. 1997). Virtually the same regional and temporal pattern of labeling has been de-scribed in embryos of other vertebrate classes (Olsson 1993; Fernández-Llebrez et al. 1996; Rodríguez et al. 1996; Yulis et al. 1998). Thus, during the embryonic de-velopment of the vertebrate central nervous system, the FP appears to be the first RF glycoprotein secreting struc-ture. This activity then gradually disappears as the neuro-epithelial cells of the roof of the forebrain-midbrain boundary begin to synthesize RF glycoproteins and
dif-Fig. 1A–I
Immunocytochemi-cal staining of wildtype zebra-fish embryos of 48 h by using an antiserum against bovine Reiss-ner’s fiber. A Whole-mount im-munostaining of the head region showing obvious labeling of the SCO in the rostral brain (arrow).
Bar 400 µm. B Sagittal section
through the head showing the localization of the SCO (boxed) and the flexural organ (FO). Bar 200 µm. C Detail of the region
boxed in B. Note
immunoreac-tivity in the subcommissural or-gan (SCO) around the posterior commissure (pc) and immunore-active material in the ventricle forming Reissner’s fiber (RF).
Bar 50 µm. D Transverse section
through the flexural organ (FO) in the floor of the third ventricle (v). Bar 25 µm. E Detail of transversely sectioned spinal cord (outlined) over the noto-chord (not). The sectioned Reissner’s fiber (RF) appears as a dark dot in the lumen of the central canal. Bar 25 µm. F Sag-ittal section through the spinal cord (outlined) showing an im-munoreactive Reissner’s fiber (RF). Bar 50 µm. G Whole-mount immunostaining of the caudal region of the spinal cord showing an obvious Reissner’s fiber (RF) along the central ca-nal at the end of which it depo-lymerizes and forms a massa caudalis (mc). Immunoreactive material escapes and invades the surrounding tissues. Floor-plate (FP) cells are labeled (not noto-chord). Bar 60 µm. H Sagittal section through the immunore-active caudal floor plate; note Reissner’s fiber (RF) over FP cells (not notochord). Bar 30 µm. I Transverse section at the level indicated by the dotted
line in G; note an opening of the
neural tube (nt, arrow) through which immunoreactive material escapes. Bar 30 µm
ferentiate into an SCO, whose activity persists for the lifetime of the animal in most of the vertebrate species studied.
At least in lower vertebrates, RF glycoproteins re-leased from the FP seem to be able to polymerize into an RF, since an RF is visible before the SCO is immunologi-cally detectable (López-Avalos et al. 1997; Yulis et al. 1998). It is, however, not known to what extent RF-form-ing glycoproteins from the FP and the embryonic SCO are identical compounds. An immunocytochemical study
of dogfish embryos with antisera against different elec-trophoretic bands of adult dogfish SCO extracts suggests that the secretions are similar (López-Avalos et al. 1997).
The results obtained in zebrafish by immunocyto-chemistry with an antiserum against RF glycoproteins (Lichtenfeld et al. 1999; present results) parallel those reported in zebrafish for F-spondin 2 following the use of in situ hybridization and immunocytochemistry by Hi-gashijima et al. (1997). These authors have detected ex-pression of F-spondin 2 in the FP from 15 h and in the
Fig. 2A–H
Immunocytochemi-cal staining of cyc zebrafish embryos of 72 h by using an antiserum against bovine Reiss-ner’s fiber. A Whole-mount im-munostaining of a cyc mutant; only the SCO (arrow) is la-beled. Bar 450 µm. B Detail of the head region showing an ob-vious SCO. Bar 150 µm. C Transverse section through the level indicated by the dotted
line in B showing a strongly
la-beled subcommissural organ (SCO). Bar 50 µm. D Trans-verse section through a level caudal to the subcommissural organ. Note immunoreactive material (arrows) in the wall of the ventricle (v). Bar 75 µm. E Transverse section through the neural tube (nt) over the noto-chord (not). No immunoreac-tive structure is visible. Bar 50 µm. F Whole-mount immu-nostaining of the tail region
boxed in A; no immunoreactive
structure is visible (nt neural tube, not notochord). Bar 100 µm. G Whole-mount im-munostaining of the tail region of a specimen showing immu-noreactive floor plate cells (FP) and a massa caudalis (mc), but no Reissner’s fiber (not notochord). Bar 100 µm.
H Transverse section (not
noto-chord) through the level indi-cated by the dotted line in G showing a labeled floor plate (FP). Bar 50 µm
SCO from 28 h. They describe immunoreactivity in both structures and in the RF from 26 h; in conclusion, they propose that, in the zebrafish, F-spondin 2 is produced by the FP and subsequently deposited in the RF. Since F-spondin 2 and SCO-F-spondin (Gobron et al. 2000) share sequence homologies, one may expect cross-reactivities when using antisera against one of these two structurally related proteins. It is possible that some antibodies pres-ent in our antiserum are capable of binding to F-spon-din 2 in addition to SCO-sponF-spon-din. Unfortunately, since the work of Higashijima et al. (1997) was performed on embryos from 11 h to 40 h and our study was in embryos of 48 h and 72 h, it is open to discussion whether F-spondin 2 is indeed detectable in zebrafish embryos used in the present investigation.
The cyc mutation in zebrafish blocks the formation of FP in the spinal cord and virtually deletes or reduces the whole ventral forebrain and midbrain. The effect is more severe in rostro-ventral structures such as the ventral dien-cephalon (i.e., the hypothalamus) and diminishes follow-ing an antero-posterior gradient (Hatta et al. 1991, 1994). Thus, one may frequently find some remaining FP cells in the caudal portion of the the cyc mutant spinal cord, as re-ported here. In principle, all substances produced by FP cells, e.g., RF glycoproteins, should be absent in the ven-tral midline of the neural tube of cyc mutants including the rostral-most portion of the FP, i.e., the FO, but occasional-ly, label has been seen in the caudal FP cells that persist in some specimens. Indeed, these remaining caudal FP cells in cyc mutants have been reported to express other FP markers, such as sonic hedgehog (shh) or axial (Strähle et al. 1996). Since we have observed immunoreactivity in the ampulla caudalis of mutants, the possibility that caudal FP cells of some cyc mutants can release reactive material into the lumen of the caudal central canal must be consid-ered. Alternatively, material in the lumen could originate from the active SCO present in cyc mutants (see below) followed by transport along the central canal. In any case, the amount of immunoreactive material in the tail of mu-tants is clearly reduced in comparison with the deposit in the tail of wt specimens.
In cyc mutants, an immunoreactive SCO is always present and, judging by the presence of specifically stainable fibrous material in the brain ventricle, it releas-es glycoproteins into the ventricle. Thus, the missing product(s) in the cyc mutants does (do) not seem to be essential for the expression of immunologically detect-able RF glycoproteins in the SCO and the remaining caudal FP cells or for the discharge of these glycopro-teins into the lumen of the neural tube.
It is noteworthy that, according to our results, despite the presence of immunoreactive RF glycoproteins in the brain ventricles and the central canal, a typical RF was never observed in cyc mutants. Moreover, in another FP-deprived zebrafish mutant one-eye pinhead, an RF is missing (Higashijima et al. 1997). It could be reasoned that mutants lack an RF because of a putative delay in development. However, as discussed above, an RF is present in fish even before the SCO displays
immunore-activity (López-Avalos et al. 1997; Yulis et al. 1998). Thus, it seems unlikely that the absence of RF in 72-h cyc zebrafish mutants, in which a well-developed and immunoreactive SCO can be seen, is attributable to a de-layed development, which has so far not been reported for any central nervous structure in these mutants. The absence of RF could depend on one of the following fac-tors. (1) A putative dysfunction in the circulation of ce-rebrospinal fluid via the modified brain ventricles and the central canal in cyc and other FP mutants may pre-vent the formation of an RF. In this respect, the polymer-ization of the RF has been considered to depend on hy-drodynamic factors related to the circulation of the cere-brospinal fluid (cf. Fernández-Llebrez et al. 1993). (2) Unknown substances essential for the polymerization of RF-glycoproteins may be absent in FP-deprived mutants. (3) The SCO of cyc mutants may secrete different, al-though immunologically recognizable, RF glycoproteins that are unable to polymerize. (4) Finally, the released RF glycoproteins may be resorbed by the ventricular walls in mutants, thus preventing the formation of RF. The causal relationship between any of these posibilities and the absence of FP cells is completely unknown.
In addition to RF glycoproteins (Lichtenfeld et al. 1999; present results) and F-spondin 2 (Higashijima et al. 1997), the SCO and FP share the expression of other FP markers. Strähle et al. (1996) have reported that both axial, a transcription factor involved in FP development, and shh, a secretory protein that directs the patterning of the ventral neural tube, are expressed in the FP and in a region of the dorsal forebrain-midbrain boundary of wt zebrafish embryos. This region corresponds precisely to the region where the developing SCO can be found. In-terestingly, in cyc mutants, the expression of both genes is absent in the FP but persists in a region described as “a small patch of cells in the dorsal aspect of the mid-diencephalic boundary” (Strähle et al. 1996). According to this description and the corresponding images shown in the paper, this zone probably represents the SCO. It is not known whether shh or axial are involved in the de-velopment or the secretory activity of the SCO. Never-theless, the product(s) absent in cyc mutants does (do) not seem to interact with the expression of RF glycopro-teins, shh, or axial (and perhaps F-spondin 2) in the SCO, but with the formation of the FP and RF.
Little is known about the function of RF glycopro-teins, although the presence of an RF in vertebrate em-bryos and adults might reflect a role in the development and homeostasis of the central nervous system. RF glyco-proteins contain thrombospondin-like repeats (Gobron et al. 1996, 2000; Nualart et al. 1998), and thus the term SCO-spondin has been proposed for glycoproteins secret-ed by the SCO (Gobron et al. 1996, 2000). The embryon-ic FP secretes several proteins containing thrombospon-din repeats, such as F-sponthrombospon-dins (Klar et al. 1992), min-dins (Higashijima et al. 1997), or semaphorins (Adams et al. 1996; Culotti and Kolodkin 1996), which are involved in axonal growth and guidance. SCO-spondin has been reported to promote in vitro neuronal aggregation and
neurite extension (Gobron et al. 1996, 2000; Monnerie et al. 1997). Thus, it is possible that SCO-spondin could play a role in axonal guidance of nerve fibers in the dor-sal diencephalic-mesencephalic boundary, where the pos-terior commissure develops, and in the ventral spinal cord, in a manner similar to that reported for the other proteins containing thrombospondin repeats.
The SCO is an ancient ependymal gland of the verte-brate brain (Oksche et al. 1993); according to its anatomi-cal loanatomi-calization, it appears to originate from the rostral roof plate of the neural tube. However, primitive vertebrates such as myxinoids possess a secretory SCO surrounding a tubular third ventricle at its dorsal, lateral, and ventral as-pects, suggesting that the SCO might share a common phy-logenetic origin from the ventral part of the neural tube (Olsson 1993). Thus, it would not be surprising to find the expression of the same genes in the FP and SCO.
Acknowledgements We are indebted to Prof. K. Hatta (Oregon, USA) who kindly provided wt and cyc zebrafish larvae, and to Prof. E. Rodríguez (Valdivia, Chile) for providing the anti-bovine-RF serum used in this study.
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