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Dual function of polysialic acid during zebrafish central nervous system development

¹ Department of Biology, University of Konstanz, Fach M626, 78457 Konstanz, Germany
² Program in Cellular Biochemistry and Biophysics, Memorial Sloan-Kettering Cancer Center – Box 290, 1275 York Avenue, New York, New York 10021, USA

View larger version (82K): [in a new window]   Fig. 1. Expression pattern of PSA during zebrafish development. (A) Embryo at 17 hpf, labelled with antibodies against PSA and Tag-1. PSA labeling (red) is uniform throughout the CNS and not found on the first differentiated Tag-1-positive axons (green). (B) Single optical section through the CNS of a 27 hpf embryo demonstrating the association of PSA with membranes of almost all cell bodies. (C,D) Embryo at 27 hpf double labelled with antibodies against PSA and -tubulin as an axonal marker. (C) PSA is expressed on cell bodies in the CNS, on floorplate cells (fp) along the midline, the nuclei of cranial nerves V and VII (nV, nVII) and on their axons (aV, aVII) leaving the CNS. (D) Counterstain with -tubulin demonstrates that most axon tracts, including the medial longitudinal fascicle (mlf), the lateral longitudinal fascicle (llf) and the lateral line nerve (lln) are PSA negative. (E,F) Embryo at 30 hpf double labelled with antibodies against Tag-1 and PSA. (E) Axons in the posterior commissure (pc) express high levels of PSA, whereas the cells in the environment show lower levels of PSA. (F) The Tag-1 staining demonstrates that axons in the olfactory bulb (ob) and the epiphysis (ep) are PSA-negative. (G) Hindbrain of an embryo at 32 hpf with strong PSA expression on floorplate cells along the midline. (H) Lateral view of the tail of an embryo at 36 hpf showing PSA expression on cell bodies in spinal cord (sc), the floorplate (fp) and on secondary motor axons (mn) (Ott et al., 2001) leaving the CNS. (I) Transverse cryosection through the hindbrain of a fish larva at 13 days, double labelled for PSA (red) and Tag-1 (green). PSA staining is restricted to cells close to the ventricle and the pial surface (arrowheads), and a strong signal along the midline (arrow). ep, epiphysis; ey, eye; fb, forebrain; fp, floorplate; hb, hindbrain; mb, midbrain; mn, motor axons; ob, olfactory bulb; Oto, otocyst; sc, spinal cord; ys, yolk sack. (A-H) Confocal images; (A-F) dorsal view, rostral at the top; (G) ventral view, rostral at the top; (H) lateral view, rostral towards the left. (I) Photomicrograph of a cryosection, rostral at the top. Scale bars: 100 µm in A; 10 µm in B; 50 µm in C-F; 5 µm in G; 10 µm in H; 50 µm in I.

View larger version (48K): [in a new window]   Fig. 2. Characterisation of PSA glycoproteins in goldfish and zebrafish brain. Western blots were reacted with antibodies against PSA or against NCAM. (A-C) Proteins from goldfish brain membranes immunoprecipitated with mAb 735 against PSA. They consist of a broad high molecular weight smear between 120 and 240 kDa when reacted with mAb 735 (A). After endo N treatment, discrete bands are visible at 120 kDa and 170 kDa in a protein stain (B). The band at 170 kDa is also recognised by mAb D3 against the intracellular domain of chick NCAM (C). (D-G) Immunoblots of zebrafish brain membranes reacted with mAb 735 (D,E), a polyclonal serum against goldfish proteins immunoprecipitated with mAb 735 (F) and mAb D3 (G). mAb 735 recognised a broad band between 120 and 240 kDa (D). This labeling was completely abolished when the membranes were treated with endo N before electrophoresis (E). The polyclonal serum recognised three major bands at 170 kDa, 140 kDa and 120 kDa (F), a pattern typical of NCAM in higher vertebrates. mAb D3 recognised a band at 170 kDa (G).

View larger version (68K): [in a new window]   Fig. 3. A single injection of endo N removes PSA for at least 24 hours. (A) PSA staining in 32 hpf zebrafish embryo. (B,C) Zebrafish embryo at 32 hpf that received an injection of Endo N into the ventricle at 20 hpf, double labelled with PSA and acetylated -tubulin antibodies. (B) The specific PSA labeling has disappeared and only a faint overall fluorescence remains. (C) The tubulin label demonstrates that most longitudinal and commissural axon tracts appear normal, indicating that PSA removal does not interfere with overall development of the zebrafish nervous system. ac, anterior commissure; llf, lateral longitudinal fascicle; mlf, medial longitudinal fascicle; pc, posterior commissure; poc, postoptic commissure. (A-C) Confocal images, dorsal view, rostral at the top. Scale bar: 50 µm.

View larger version (82K): [in a new window]   Fig. 4. Removal of PSA causes a defasciculation in the posterior commissure. (A-F) The posterior commissure (pc) in zebrafish embryos at 32 hpf after injections into the ventricle at 20 hpf, labelled with acetylated -tubulin antibodies. (A,B) In buffer-injected control embryos the pc crosses the midline (dotted line) in one thick bundle. (C,D) In endo N-injected embryos, axons of the pc split into several thin bundles that cross the midline (arrowheads). (E,F) In PSA-polymer injected embryos, the fasciculated growth pattern of the pc is not affected. (A-F) All images are confocal images, dorsal view, rostral at the top. Scale bar: 10 µm.

View larger version (114K): [in a new window]   Fig. 5. Removal of PSA interferes with the development of hindbrain commissures. (A,B) Hindbrain of a buffer-injected control embryo at 32 hpf labelled with antibodies against PSA and Tag-1. (A) PSA is expressed by the motor nuclei of cranial nerves (nV and nVII), by their motor axons leaving the CNS (arrowheads) and on cells of the floorplate (fp). (B) Tag-1 labeling shows the VII cranial nerve and commissural axons crossing the midline (B, arrow). The boxed regions rostral to the otocyst (oto) in A and B represent the position chosen for the analysis of commissural axon growth. (C) In control embryos, PSA is expressed on cells of the floorplate (fp) but not on commissural axons. (D) Tubulin labeling of the same embryo shows commissural axons crossing the medial longitudinal fascicle (mlf) in thick bundles (asterisks). These axons defasciculate (arrowheads in D) upon arrival at the PSA-positive floorplate cells. (E,F) The growth pattern of commissural axons is markedly disturbed in endo N-injected embryos. Fewer axons cross the floorplate (E) or bundles of commissural axons appear to stop at the PSA-negative floorplate cells (arrowheads in F). All images are confocal images, ventral views, rostral at the top. Scale bars: in B, 20 µm in A,B; in F, 5 µm in C-F.

View larger version (86K): [in a new window]   Fig. 6. Removal of PSA prevents commissural axons in the hindbrain from crossing the midline. Schematic drawings illustrate the projection pattern of commissural axons in the hindbrain in cross section (A) and in a ventral view (B). Cell bodies of commissural interneurones occupy a dorsolateral position in each hindbrain segment. Their axons extend ventrally, cross the medial longitudinal fascicle (mlf) and the floorplate (FP) and terminate in contralateral mid-dorsal positions in the same hindbrain segment (A). Axons of reticulospinal neurones (B, dark green) cross the floorplate in a curved pathway and project into the contralateral mlf. Axons of commissural interneurones (B, light green) cross the mlf in a straight pathway and defasciculate upon contact with PSA-positive floorplate cells (yellow). (C,G) Hindbrain of zebrafish embryos at 36 hpf in an overlay of interference contrast and DiI-fluorescence. To analyse the growth pattern of commissural axons in more detail, DiI was inserted close to the cell bodies of commissural interneurones (asterisks) rostral to the otocyst (oto). (C-F) In buffer-injected control embryos, all DiI-labelled axons cross the midline (dotted line) and terminate on the contralateral side. (G-K) In endo N-injected embryos, either all (H,I) or subsets (K) of DiI-labelled axons stop at the midline. (C-K) Dorsal views, rostral at the top. Scale bar: 50 µm.

View larger version (41K): [in a new window]   Fig. 7. Injection of soluble PSA causes the same defects in the growth pattern of commissural axons in the hindbrain as does removal of PSA. (A,B) Hindbrain of zebrafish embryos at 36 hpf in an overlay of interference contrast and DiI fluorescence (asterisks mark DiI injection sites). (A) In sialic acid trimer-injected embryos, DiI-labelled commissural axons cross the midline and reach the contralateral side. (B) Hindbrain of a soluble-PSA injected embryo. The commissural axons fail to reach the other side and stop at the midline. oto, otocyst. Scale bar: 50 µm. (C) Quantification of the effects on the growth pattern of hindbrain commissural axons caused by the injection of control-buffer, endo N, PSA-polymer and PSA-trimer.

View larger version (37K): [in a new window]   Fig. 8. Effects of PSA on cell interactions and axon fasciculation. (A) When PSA (yellow) is attached to the cell surface via NCAM, its steric properties hinder the apposition of cell membranes and therefore reduce the interaction of receptors, both NCAM (orange) and non-NCAM (blue), on apposing cells. When PSA is removed, receptors can form enough cell-cell bonds for stable adhesion to occur. This steric effect cannot be blocked by soluble PSA. (B) Removal of PSA from the axons and/or their environment can have opposite effects on PSA-positive axon tracts, depending on the nature of the environment. Top: if the environment has powerful and stable adhesive attractants, such as potential sites for junctions (Seki and Rutishauser, 1998), loss of PSA can induce axons to follow more independent, less fasciculated pathways. Bottom: if the environment does not support such stable interactions, as in the matrix-rich plexus region of the chick hindlimb (Tang et al., 1994), the intrinsic interactions among the axons will prevail and loss of PSA will tend to promote growth of axons along other axons to form larger fascicles. In the present study, the PSA-positive posterior commissure, which traverses an environment that contains cells that are also PSA-positive, is shown to defasciculate partially when PSA is removed; this effect is not mimicked by soluble PSA. (C) Two possible binding-based modes for PSA action during midline crossing of commissural axons in the hindbrain. Top: PSA (yellow circles) binds a secreted component (blue circles) necessary for midline crossing, thereby keeping this factor at a high concentration on the surface of floorplate cells. Bottom: a dual binding mechanism in which PSA and a second molecule (blue) on the floorplate cell combine to bind to and activate an axonal receptor. Unlike steric models for PSA action (A,B), the binding-based mechanisms are predicted to be competitively inhibited by soluble PSA and blocked by endo N treatment.

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