Aster-free spindle poles in insect spermatocytes: evidence for chromosome-induced spindle formation?

Tipulid spermatocytes form normally functioning bipolar spindles after one of the centrosomes is experimentally dislocated from the nucleus in late diakinesis (Dietz, R., 1959, Z. Naturforsch., 14b:749-752; Dietz, R., 1963, Zool. Anz. Suppl., 23:131-138; Dietz, R., 1966, Heredity, 19:161-166). The possibility that dissociated pericentriolar material (PCM) is nevertheless responsible for the formation of the spindle in these cells cannot be ruled out based on live observation. In studying serial sections of complete cells and of lysed cells, it was found that centrosome-free spindle poles in the crane fly show neither pericentriolar-like material nor aster microtubules, whereas the displaced centrosomes appear complete, i.e., consist of a centriole pair, aster microtubules, and PCM. Exposure to a lysis buffer containing tubulin resulted in an increase of centrosomal asters due to aster microtubule polymerization. Aster-free spindle poles did not show any reaction, also indicating the absence of PCM at these poles. The results favor the hypothesis of chromosome-induced spindle pole formation at the onset of prometaphase and the dispensability of PCM in Pales.

Fish E587 glycoprotein, a member of the L1 family of cell adhesion molecules, participates in axonal fasciculation and the age-related order of ganglion cell axons in the goldfish retina.

Axons derived from young ganglion cells in the periphery of the retinae of larval and adult goldfish are known to fasciculate with one another and their immediate forerunners, creating the typical age-related order in the retinotectal pathway. Young axons express the E587 antigen, a member of the L1 family of cell adhesion molecules. Repeated injections of Fab fragments from a polyclonal E587 antiserum (E587 Fabs) into the eye of 3.4 cm goldfish disrupted the orderly fascicle pattern of RGC axons in the retina which was preserved in controls. Instead of bundling tightly, RGC axons crossed one another, grew between fascicles and arrived at the optic disk in a broadened front. When added to RGC axons growing in vitro, E587 Fabs neutralized the preference of growth cones to elongate on lanes of E587 protein, caused defasciculation of axons which normally prefer to grow along each other when explanted on polylysine, and prevented clustering of E587 antigen at axon-axon contact sites. Monoclonal E587 antibody disturbed axonal fasciculation moderately but led to a 30% reduction in growth velocities when axons tracked other axons. Therefore we conclude that E587 antigen mediates axonal recognition, selective fasciculation and the creation of the age-related order in the fish retina.

Neurolin Ig domain 2 participates in retinal axon guidance and Ig domains 1 and 3 in fasciculation.

The optic disk-directed growth of retinal ganglion cell axons is markedly disturbed in the presence of polyclonal antineurolin antibodies, which mildly affect fasciculation (Ott, H., M. Bastmeyer, and C.A.O. Stuermer, 1998. J. Neurosci. 18:3363-3372). New monoclonal antibodies (mAbs) against goldfish neurolin, an immunoglobulin (Ig) superfamily cell adhesion/recognition molecule with five Ig domains, were generated to assign function (guidance versus fasciculation) to specific Ig domains. By their ability or failure to recognize Chinese hamster ovary cells expressing recombinant neurolin with deletions of defined Ig domains, mAbs were identified as being directed against Ig domains 1, 2, or 3, respectively. Repeated intraocular injections of a mAb against Ig domain 2 disturb the disk-directed growth: axons grow in aberrant routes and fail to reach the optic disk, but remain fasciculated. mAbs against Ig domains 1 and 3 disturb the formation of tight fascicles. mAb against Ig domain 2 significantly increases the incidence of growth cone departure from the disk-oriented fascicle track, while mAbs against Ig domains 1 and 3 do not. This was demonstrated by time-lapse videorecording of labeled growth cones. Thus, Ig domain 2 of neurolin is apparently essential for growth cone guidance towards the disk, presumably by being part of a receptor (or complex) for an axon guidance component.

The retinal axon's pathfinding to the optic disk.

Retinal ganglion cell (RGC) axons travel in radial routes unerringly toward the optic disk, their first intermediate target in the center of the eye. The path of the RGC growth cone is restricted to a narrow zone subjacent to the endfeet of Müller glial cells and the vitreal basal lamina. The present survey indicates that RGC growth cones are guided by many molecular cues along their pathway which are recognized by receptors on their surface. Growth-promoting molecules on Müller glial endfeet and in the basal lamina assist growth cones in maintaining contact with these elements. The repellant character of deeper retinal laminae discourages them from escaping the RGC axon layer. Cell adhesion/recognition proteins enable growth cones to fasciculate with preformed axons in their vicinity. It is still unclear whether the optic disk emits long range guidance components which enable the growth cones to steer toward it. Recent evidence in fish indicates the existence of an axonal receptor (neurolin) for a guidance component of unknown identity. Receptor blockade causes RGC axons to course in aberrant routes before they reach the disk. At the disk, axons receive signals to exit the retina. Contact with netrin-1 at the optic disk/nerve head encourages growth cones to turn into the nerve. This response requires the axonal netrin receptor DCC, laminin-1, beta-integrin and most likely the UNC5H netrin receptors which convert the growth encouraging signal into a repulsive one which drives growth cones into the nerve.

Immunostaining of spindle components in tipulid spermatocytes using a serum against pericentriolar material.

First meiotic division of tipulid (Pales ferruginea) spermatocytes was investigated by double immunostaining with anti-tubulin IgG and scleroderma 5051 serum against pericentriolar material (PCM). PCM-like material became visible in late diakinesis in centrosomal areas as well as in kinetochores. Anti-PCM fluorescence was most pronounced in metaphase and diminished again in anaphase. Displacement of one of the centrosomes from the nucleus at diakinesis in Pales spermatocytes leads to the formation of a bipolar, normally functioning spindle possessing one aster and centriole-free spindle pole (AFP). In about 80% of the AFPs observed there were no traces of anti-PCM staining detectable. This finding supports the assumption based on previous studies that polar PCM is not obligatory for spindle pole formation. The chromosomes seem to be able to induce the organization of a half-spindle. The strong anti-PCM fluorescence of the kinetochores observed here may be taken as further indication of tipulid chromosome autonomy regarding spindle formation.

Cytology of Lepidoptera. V. The microtubule cytoskeleton in eupyrene spermatocytes of Ephestia kuehniella (Pyralidae), Inachis io (Nymphalidae), and Orgyia antiqua (Lymantriidae).

The structural transformations of the microtubule cytoskeleton during eupyrene meiosis were characterized in the Mediterranean mealmoth, Ephestia kuehniella. Anti-tubulin immunofluorescence was used in two strains, L and Sbr, of this species. In addition, living primary eupyrene spermatocytes from strain L were observed with phase-contrast light microscopy, and metaphase I spermatocytes were studied with the electron microscope. The analysis did not reveal strain-specific differences in the behavior of the chromatin and the microtubule cytoskeleton in eupyrene meiosis. The general pattern of the two subsequent meiotic divisions in Ephestia kuehniella did not deviate from that observed during nuclear division in other species. A bipolar spindle developed during prophase, chromatin migrated towards the poles in anaphase, and spindle elongation occurred in telophase. Spindle development and behavior were qualitatively almost identical in primary and secondary spermatocytes. The assembly of the second meiotic spindle was initiated during the depolymerization of interzone microtubules of the first division. The relationship between spindle microtubules and centrosomes, however, showed some particularities in the moth. While microtubules radiated out from the spindle poles in late prophase, most microtubules ended about half-way between the equatorial plane and the spindle poles from late prometaphase to early anaphase. The termination of most microtubules distant from the poles was confirmed using electron microscopy in Ephestia kuehniella and using anti-tubulin immunofluorescence in two other Lepidoptera species, Inachis io and Orgyia antiqua. The microtubules were oriented parallel to the pole-to-pole axis of the spindle and were confined to an area laterally sheath by membrane layers. The spindle portions between the pole-proximal termini of the microtubules and the spindle poles contained irregularly shaped membrane elements. In late anaphase, microtubules reform between the migrating chromatin plates and the spindle poles. Concomitantly with the migration of the chromosomes towards the spindle poles, interzone microtubules are assembled. The origin of most of them appears to be at the pole-distal face of the chromatin masses.

Cytology of lepidoptera. VI. Immunolocalization of microtubules in detergent-extracted apyrene spermatocytes of Ephestia kuehniella Z.

Apyrene meiosis was studied in two wild-type strains, L and Sbr, of the Mediterranean mealmoth, Ephestia kuehniella, using anti-tubulin immunofluorescence. The observations were supplemented by phase-contrast light microscopy of living spermatocytes from strain L. The study revealed that nuclear envelope breakdown, centrosome separation, migration of chromatin elements towards the poles, and spindle elongation also occur in apyrene spermatocytes. However, a conventional metaphase plate is never formed, and chromatin segregation is irregular and delayed. Chromosome laggards are frequent. As a rule, apyrene spindles have a low microtubule content. The two strains, L and Sbr, differ regarding the chromatin behavior during meiosis. In strain L, the developing spindles contain numerous small chromatin clumps which segregate asynchronously. The resulting daughter cells possess about the same amount of chromatin. In contrast, large chromatin clumps exist in strain Sbr at the onset of spindle formation. The chromatin blocks transiently occupy an equatorial position and elongate subsequently parallel to the spindle axis. These elongated chromatin bodies often divide highly unequally. As a consequence, secondary spermatocytes in strain Sbr differ greatly in chromatin content. Subjective assessment shows that the size of the microtubular cytoskeleton is positively correlated with the chromatin content of the cell. Hence, it is hypothesized that the chromatin content determines spindle size. This possibly comes about the number of available kinetochores which are exposed and able to stabilize microtubules of centrosomal origin attached with the kinetochores. However, a direct bearing of chromatin on spindle size is similarly conceivable. Other Lepidoptera species examined so far are compatible with a 'type L' or a 'type Sbr' pattern of apyrene meiosis.

Collateral branch formation related to cellular structures in the axon tract during corticopontine target recognition.

The corticopontine projection develops exclusively by collateral branches that form along the length of corticospinal axons days after they have passed their hindbrain target, the basilar pons. In vitro evidence suggests that the basilar pons releases a diffusible activity that initiates and directs the growth of collateral branches. This study investigates whether contact-dependent mechanisms may also influence the formation of collateral branches. By using immunocytochemistry, electron microscopy, and neuronal tracing techniques, we examined the region of the axon tract, the cerebral peduncle, overlying the basilar pons for cellular structures that correlate spatially and temporally with collateral branch formation. We found that radial glia are excluded from the tract. Oligodendrocyte precursors are found only at low density. Although mature astrocytes are absent, immature astrocytes are present throughout the tract. However, our evidence does not suggest a direct role for glial cell types in collateral branch formation. In contrast, dendrites of basilar pontine neurons are transiently present in the tract during the time of collateral branch formation. Although collateral branches are observed in regions of the tract devoid of dendrites, the orientation and location of most collateral branches correlates at the light microscopic level with dendrites. Electron microscopy reveals sites of increased collateral branch formation near neuronal cell bodies or dendrites. However, cell processes, whether dendritic or otherwise, are rarely found in direct contact with collateral branch points. A common and unexpected feature is the bundles of corticopontine collateral branches, oriented transversely to their parent corticospinal axons and directed across the tract to the basilar pons. Dendrites were often apposed to or embedded within the transverse bundles. These findings suggest that dendrites are not essential for collateral branch formation but that they may enhance this process and define discrete preferred locations for collateral branch initiation and elongation within the cerebral peduncle.

Optic nerve glia secrete a low-molecular-weight factor that stimulates retinal ganglion cells to regenerate axons in goldfish.

The ability of lower vertebrates to regenerate an injured optic nerve has been widely studied as a model for understanding neural development and plasticity. We have recently shown that, in goldfish, the optic nerve contains two molecules that stimulate retinal ganglion cells to regenerate their axons in culture: a low-molecular-weight factor that is active even at low concentrations (axogenesis factor-1) and a somewhat less active polypeptide of molecular weight 10,000-15,000 (axogenesis factor-2). Both are distinct from other molecules described previously in this system. The present study pursues the biological source and functional significance of axogenesis factor-1. Earlier studies have shown that cultured goldfish glia provide a highly favorable environment for fish or rat retinal ganglion cells to extend axons. We report that the glia in these cultures secrete high levels of a factor that is identical to axogenesis factor-1 in its chromatographic properties and biological activity, along with a larger molecule that may coincide with axogenesis factor-2. Axogenesis factor-1 derived from either goldfish glial cultures or optic nerve fragments is a hydrophilic molecule with an estimated molecular weight of 700-800. Prior studies have reported that goldfish retinal fragments, when explanted in organ culture, only extend axons if the ganglion cells had been "primed" to begin regenerating in vivo for one to two weeks. However, axogenesis factor-1 caused the same degree of outgrowth irrespective of whether ganglion cells had been induced to regenerate new axons in vivo. Moreover, ganglion cells primed to begin regenerating in vivo continued to extend axons in culture only when axogenesis factor-1 was present. In summary, this study shows that glial cells of the goldfish optic nerve secrete a low-molecular-weight factor that initiates axonal regeneration from retinal ganglion cells.

Identification of astrocyte- and oligodendrocyte-like cells of goldfish optic nerves in culture.

Fish glial cells were obtained from cultivated segments of the optic nerve and raised in vitro. Two types of cells were identified as astrocyte- and oligodendrocyte-like glia by the monoclonal antibody Mab O1 (specific for oligodendrocytes) and the rabbit serum anti-goldfish glial fibrillary acidic protein (anti-G-GFAP). Cells of compact morphology were rare, and anti-G-GFAP positive and O1 negative. Multipolar cells in 5-day-old cultures were anti-G-GFAP but rarely O1 positive. In 5-week-old cultures, however, roughly 75% of the multipolar cells were double-labeled with both anti-G-GFAP and O1; 10% were single labeled with Mab O1 and 15% with anti-G-GFAP, respectively.

The distribution of intermicrotubular bridges in meiotic spindles of the crane fly.

The distribution of intermicrotubular bridges in spindles of tipulid spermatocytes (Pales ferruginea, first meiotic division) was analyzed using serial sections of pre-selected cells. Bridges were found in all spindle regions, including kinetochore microtubules and free microtubules in the chromosome fiber. The dimensions of bridges were variable, ranging between 60 and 300 A in length and 40 and 190 A in thickness. Bridges seem to be randomly distributed. No accumulation in or absence from particular spindle regions was detected. Quantitative analysis revealed a linear, positive correlation between the number of microtubules and the number of microtubule pairs capable of forming bridges and, on the other hand, between microtubule pairs and intermicrotubular bridges. The possible composition and significance of bridges are discussed.

A model for chromosome movement based on lateral interaction of spindle microtubules.

Quantitative morphological studies of meiotic spindles in the crane fly Pales ferruginea (Fuge, 1980, 1984, 1985) were the basis for the development of a model explaining anaphase chromosome transport in higher eukaryotes. Two main features of chromosome fibres were important for the model: (1) the existence of microtubules oriented obliquely with respect to kinetochore microtubules, and (2) a higher degree of disorder in fibres exerting a pulling force. It is postulated that microtubules of the same polarity being inclined to each other at a certain angle are able to slide past each other by means of mechano-chemically active side-arms working in alternate succession. Sliding is suggested to lead to a displacement of microtubules and chromosomes in direction towards the poles. Furthermore, it is suggested that the chromosome fibre in anaphase becomes progressively disintegrated by fragmentation and disassembly of microtubules. Fragmentation may be induced by bending stress within the dynamic system.

Behavior of fish retinal growth cones encountering chick caudal tectal membranes: a time-lapse study on growth cone collapse.

In a cross species in vitro assay, growth cones from fish temporal retina elongating on laminin lanes were observed with time-lapse videomicroscopy as they encountered lanes and territories that carried membrane fragments from the chick caudal tectum. Caudal tectal membranes of adult fish and embryonic chick are known to possess a repellent guiding component for temporal retinal axons. The caudal membranes of chick exert a particularly strong influence on fish temporal axons. Contacts with chick caudal membranes by just a few filopodia and parts of the lamellipodia evoked a turning response away from the membrane lane of the entire growth cone. Contacts by filo- and lamellipodia over the entire circumference of the growth cone, however, caused invariably growth cone collapse and retraction. During growth cone turning and collapse and retraction, filopodia remained in contact with the tectal membrane fragments, suggesting strong membrane-filopodia adhesion simultaneous to growth cone repulsion by the repellent guiding component.

Dual function of polysialic acid during zebrafish central nervous system development.

Polysialic acid (PSA), a carbohydrate epitope attached to the neural cell adhesion molecule, serves as a modulator of axonal interactions during vertebrate nervous system development. We have used PSA-specific antibodies and whole-mount immunocytochemistry to describe the spatiotemporal expression pattern of PSA during zebrafish central nervous system development. PSA is transiently expressed on all cell bodies and, except for the posterior commissure, it is not found on axons. Floorplate cells in the spinal cord and hindbrain strongly express PSA throughout development. Enzymatic removal of PSA leads to a defasciculated growth pattern of the posterior commissure and also affects distinct subsets of commissural axons in the hindbrain, which fail to cross the midline. Whereas the disordered growth pattern of hindbrain commissures produced by PSA-removal could be mimicked by injections of soluble PSA, the growth of axons in the posterior commissure was unaffected by such treatment. These results suggest that there are distinct mechanisms for PSA action during axon growth and pathfinding in the developing zebrafish CNS.

Characterization of Pales spermatocyte spindles, with reference to an MTOC-associated protein.

Pales ferruginea spermatocytes, because of their high proportion of dividing cells, offer many advantages in the study of the meiotic spindle. In the present study we have combined ultrastructural and immunofluorescence techniques with biochemical analysis of constituent spindle components. Using a centrosome-reactive serum, which recognizes an antigen in the centrosomal and midbody regions of dividing cells, we have identified an MTOC-associated polypeptide of 112,000 Mr with a pI of 5.6. The relative abundance of this protein in cells and isolated cytoskeletons indicates that it is present in other regions of the cell and is either more abundant, or is only recognized by the antibody in association with MTOCs.

Dynamics of target recognition by interstitial axon branching along developing cortical axons.

Corticospinal axons innervate their midbrain, hindbrain, and spinal targets by extending collateral branches interstitially along their length. To establish that the axon shaft rather than the axonal growth cone is responsible for target recognition in this system, and to characterize the dynamics of interstitial branch formation, we have studied this process in an in vivo-like setting using slice cultures from neonatal mice containing the entire pathway of corticospinal axons. Corticospinal axons labeled with the dye 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (or Dil) were imaged using time-lapse video microscopy of their pathway overlying the basilar pons, their major hindbrain target. The axon shaft millimeters behind the growth cone exhibits several dynamic behaviors, including the de novo formation of varicosities and filopodia-like extensions, and a behavior that we term "pulsation," which is characterized by a variable thickening and thining of short segments of the axon. An individual axon can have multiple sites of branching activity, with many of the branches being transient. These dynamic behaviors occur along the portion of the axon shaft overlying the basilar pons, but not just caudal to it. Once the collaterals extend into the pontine neuropil, they branch further in the neuropil, while the parent axon becomes quiescent. Thus, the branching activity is spatially restricted to specific portions of the axon, as well as temporally restricted to a relatively brief time window. These findings provide definitive evidence that collateral branches form de novo along corticospinal axons and establish that the process of target recognition in this system is a property of the axon shaft rather than the leading growth cone.

Astrocytes from adult rat optic nerves are nonpermissive for regenerating retinal ganglion cell axons.

We have directly compared the abilities of astrocytes from newborn and adult rats to support or inhibit the growth of regenerating axons in vitro. Astrocytes prepared from newborn rats were able to promote retinal ganglion cell (RGC) axon growth from embryonic and adult rat and from adult fish retinal explants. Retinal axons from E16 rat retinae grew significantly faster on astrocytes from neonatal rats than those from E18 or adult rat retinae with growth rates comparable to RGC axons from adult fish retinae. RGC regeneration from adult rat retinae was almost completely inhibited on adult rat optic nerve astrocytes. Only axons from adult fish retinae were able to extend onto monolayers from these reactive astrocytes, although their growth rates were significantly reduced. We conclude that the failure of mammalian RGC axons to regrow within the lesioned optic nerve environment is, at least in part, due to nonpermissive aspects of adult "reactive" optic nerve astrocytes. However, the cell intrinsic growth potential of RGCs also appears to influence their ability to extend axons on cellular substrates.

The spatiotemporal distribution of N-CAM in the retinotectal pathway of adult goldfish detected by the monoclonal antibody D3.

The spatiotemporal distribution of neural cell adhesion molecule (N-CAM) in the retinotectal system of adult goldfish was assessed by immunofluorescence using the monoclonal antibody (Mab) D3 against chick N-CAM. In immunoblots with extracts of cell surface membranes of fish brains, Mab D3 recognized a prominent band at 170K and a weak band at 130K (K = 10(3) Mr). N-CAM immunofluorescence on cells was restricted to the marginal growth zones of the retina and the tectum and, in normal fish, to the youngest axons from the new ganglion cells of the peripheral retinal margin. In fish with previously transected optic nerves (ONS), Mab D3 staining was found transiently on all axons from the site of the cut into the retinorecipient layers of the tectum, but disappeared from these axons 450 days after ONS. Growing retinal axons in vitro exhibited N-CAM immunofluorescence throughout their entire extent, including their growth cones. Glial cells cultured from regenerating optic nerves were, however, unlabeled. These data are consistent with the idea that N-CAM is involved in adhesive interactions of growing axons. The temporally regulated expression of N-CAM on the new retinal axons may contribute to the creation of the age-related organization of the axons in the retinotectal pathway of fish.

Similarities and differences between fish oligodendrocytes and Schwann cells in vitro.

In light of the striking differences between oligodendrocytes of the optic nerve/tract of adult goldfish and their mammalian counterparts, a further characterization of goldfish oligodendrocytes was performed. A comparison with Schwann cells was included because fish optic nerve/tract-derived oligodendrocytes bear remarkable similarities to this type of glial cell. Fish optic nerve/tract-derived oligodendrocytes that had differentiated into 04 and 6D2-positive cells and thus expressed early myelin marker molecules were found to incorporate BrdU and to divide in vitro over a period of weeks. For the induction of more advanced markers of myelinogenesis such as the CNS-specific myelin protein 36K, oligodendrocytes required extensive contact with axons. Other agents, such as fetal calf or carp serum, substrate components, or forscolin failed, however, to induce 36K expression. 04/6D2-positive oligodendrocytes could be distinguished from fish 6D2-positive Schwann cells derived from cranial nerves by their antigenic phenotype: Schwann cells but not oligodendrocytes exhibited the low affinity NGF receptor. While both cell types carry the cell adhesion molecules NCAM, E 587 antigen, and the L2/HNK-1 epitope, only Schwann cells possess a further adhesion molecule, Neurolin.

Function of Neurolin (DM-GRASP/SC-1) in guidance of motor axons during zebrafish development.

Neurolin (zf DM-GRASP), a transmembrane protein with five extracellular immunoglobulin domains, is expressed by secondary but not primary motoneurons during zebrafish development. The spatiotemporally restricted expression pattern suggests that Neurolin plays a role in motor axon growth and guidance. To test this hypothesis, we injected zebrafish embryos with function-blocking Neurolin antibodies. In injected embryos, secondary motor axons form a broadened bundle along the common path and ectopic branches leave the common path at right angles. Moreover, the formation of the ventral and the rostral projection of secondary motor axons is inhibited during the second day of development. Pathfinding errors, resulting in secondary motor axons growing through ectopic regions of the somites, occur along the common path and in the dorsal and rostral projection. Our data are compatible with the view that Neurolin is involved in the recognition of guidance cues and acts as a receptor on secondary motor axons. Consistent with this idea is the binding pattern of a soluble Neurolin-Fc construct showing that putative ligands are distributed along the common path, the ventral projection, and in the area where the rostral projection develops.