Zebrafish vimentin: molecular characterization, assembly properties and developmental expression

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European Journal of Cell Biology 77, 175-187 (1998, November) . © Gustav Fischer Verlag· Jena

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Zebrafish vimentin: molecular characterization, assembly properties and developmental expression Joan Cerdaa, Matthias Conradb , Jtirgen Markl b , Michael Brandc , Harald Herrmann1)a a b C

Division of Cell Biology, German Cancer Research Center, Heidelberg/Germany Institute of Zoology, Johannes Gutenberg University, Mainz/Germany Department of Neurobiology, University of Heidelberg, Heidelberg/Germany

Received June 30, 1998 Received in revised version August 17,1998 Accepted August 19, 1998

Vimentin - zebrafish - brain development - retina intermediate filaments To provide a basis for the investigation of the intermediate filament (IF) protein vimentin in one of the most promising experimental vertebrate systems, the zebrafish (Danio rerio), we have isolated a eDNA clone of high sequence identity to and with the characteristic features of human vimentin. Using this clone we produced recombinant zebrafish vimentin and studied its assembly behaviour. Unlike other vimentins, zebrafish vimentin formed unusually thick fIlaments when assembled at temperatures below 21°C. At 37°C few fIlaments were observed, which often also terminated in aggregated masses, indicating that its assembly was severely disturbed at this temperature. Between 21 and 34 °C apparently normal IFs were generated. By viscometry, the temperature optimum of assembly was determined to be around 28 °C. At this temperature, zebrafish vimentin partially rescued, in mixing experiments, the temperature-dependent assembly defect of trout vimentin. Therefore it is apparently able to "instruct" the misorganized trout vimentin such that it can enter normallFs. This feature, that assembly is best at the normal body temperature of various species, puts more weight on the assumption that vimentin is vital for some aspects of generating functional adult tissues. Remarkably, like in most other vertebrates, zebrafish vimentin appears to be an abundant factor in the lens and the retina as well as transiently, during development, in various parts of the central and peripheral nervous system. Therefore, promising cell biological investigations may now be performed with cells involved in the generation of the vertebrate eye and brain, and, in particular, the retina. Moreover, the power of genetics of the zebrafish system may be employed to investigate functional properties of vimentin in vivo.

Abbreviations: Eel Enhanced chemiluminiscence. -IF(s) Intermediate filament(s). - SDS-PAGE Sodium-dodecyl sulfate polyacrylamide gel electrophoresis.

I) Dr. Harald Herrmann, Division of Cell Biology, German Cancer Research Center, 1m Neuenheimer Feld 280, D-69120 Heidelberg/Germany, e-mail: [email protected], Fax: ++ 6221423404.

Introduction Underwater the evolution of vertebrates had proceeded to a very high level of complexity long before the fish/tetrapodtransition some 365 million years ago [50]. Correspondingly, the main structural components of the cytoskeleton, microfilaments, microtubules and intermediate filaments (IFs), were already expanded in fish to differentially expressed, tissue type-specific multi-gene families, since several organizational principles are shared by present-day fishes and higher terrestrial vertebrates ([60]; for an overview see [56, 65]). Of particular complexity is the IF protein family as exemplified for type I and type II class members, i.e. cytokeratins, in trout [57] and zebrafish [10]. Moreover, several other IF protein cDNAs have been recently cloned in various fish species and shown to be very similar to their mammalian counterparts underlining their very early emergence during vertebrate evolution (cf. [35,56]). IFs differ from the other two cytoplasmic filament systems in several respects that may be a prerequisite for their specific function: they are unpolar structures and are highly insoluble under most buffer conditions. IF proteins form extended dimeric, coiled-coil molecules that assemble further into higher oligomeric complexes and, under appropriate ionic conditions, form long and smooth filaments of apparent diameter of 8-12 nm [22, 30, 62, 69]. The assembly of intermediate filaments procedes in several decisive steps that are critically dependent on temperature [30, 34-36]. Moreover, the optimal temperature for assembly of vimentin of various vertebrate classes coincides with their body temperature or their preferred ambient temperature, respectively, indicating that IF proteins indeed exert their function in a filamentous form. For example, very abrupt, heavy motions, as are typical for most fishes, may need the stabilizing forces of IFs to maintain tissue integrity. Most importantly, IFs exhibit a property named "strain hardening", i.e. the viscoelasticity of filament solutions increases dramatically when stressed mechanically, and this may be of central importance for IF function in general [39, 40]. In line with this, inherited point mutations in cytokeratins

176 J. Cerda, M. Conrad, J. Markl et al.

have been demonstrated to be the cause for human blistering diseases, indicating that only a functional cytoskeleton is able to withstand the various types of mechanical forces that skin and other tissues are routinely challenged with (for reviews see [21, 23]). The importance of IFs for cellular physiology and organismal integrity has also been established by gene targeting experiments involving various cytokeratin genes (for review see [54]), the muscle-specific desmin [52, 59] and the neurofilament triplet protein NF-M [17]. With cytokeratin and desmin "knock out" mice, the resulting phenotypes differed with respect to the specific gene targeted and the mouse strain used, but they could be quite drastic, especially in the case of desmin, since the integrity of the cardiac muscle was severely impeded. In contrast, the targeted inactivation of the single vimentin gene in mice appeared to be without obvious effect on their embryonic development and adult life functions [9]. However, natural selection does not only occur at the level of embryonic development but indeed acts on the performance of individual members of a population in the competition with others. Thus, the "survival of the fittest" may be decided through properties not evident during short experimental breeding periods, such as reactivity or topological memory, being both essentially determined by the complexity of the central nervous system. In order to set the stage for working with one of the most promising model systems for genetic investigations in vertebrate development, we isolated and characterized the vimentin cDNA in the zebrafish (Dania reria). Here we report on the biochemical and immunological properties of zebrafish vimentin, its unique assembly characteristics, as well as its expression pattern during embryogenesis and in the adult fish.

Materials and methods Fish maintenance and embryo collection

Wild-type zebrafish were raised and kept under standard laboratory conditions at about 28°C, as described by Westerfield [TI] and Brand et al. [4]. The eggs were spawned synchronously in the morning, and embryos were sorted and cultured in vitro in E2 medium [TI] for up to 5 days at 28°C. To prevent pigment formation, embryos were raised in 0.2mM 1-phenyl-2-thiourea (PTU; Sigma). Embryos were staged based on morphological features [44] or in days after fertilization. Embryos were either immediately processed (whole-mount procedures) or they were frozen in liquid nitrogen and stored at -80°C until use for further biochemical analysis.

Cloning and DNA sequence analysis A post-somitogenesis zebrafish embryo cDNA library in f.-ZAP II (a gift of R. Riggleman, K. Heide and D. Grunwald, Dept. Human Genetics, Eccles Institute, University of Utah, Salt Lake City, UTI USA) was screened using the random prime 32P-Iabelled EcoRI/NcoI fragment of trout vimentin (nucleotides 2-1119) [35]. Positive clones were isolated by in vivo excision (ExAssist™ Helper Phage; Stratagene, La Jolla, CA/USA). Both strands of the selected clones were sequenced employing an ABI 373 DNA sequencer (Applied Biosysterns, Foster City, CA/USA). Sequence analysis and comparisons were carried out using the computer programs contained in the Heidelberg Unix Sequence Analysis Resources (HUSAR) package, release 3.0 (German Cancer Research Center, Heidelberg/Germany, 1993). The sequence has been deposited at GeneBank, accession number AF 069994. Restriction enzymes were purchased from Boehringer Mannheim (Mannheim/Germany), Pharmacia (Freiburg/Germany) and New England Biolabs (Schwalbach/Germany).

Whole-mount in situ hybridization Whole-mount in situ hybridization was performed with a digoxigeninlabelled RNA probe, generated from the complete zebrafish vimentin cDNA, using the DIG RNA labeling kit (Boehringer Mannheim). Detection was with an anti-digoxigenin antibody coupled to alkaline phosphatase [64]. Pictures were taken on manually dissected embryos cleared and mounted in 70 % glycerol.

Protein purification and assembly experiments For the production of the zebrafish recombinant vimentin, the Ncoll partially XhoI-digested cDNA clone encoding the full coding region was subcloned into the NcoI/Sali sites of the prokaryotic expression vector pDS5, modified to contain a NcoI site following a strong ribosome-binding site, and transformed into E. coli strain TG1 [33, 35]. The recombinant protein was isolated from inclusion bodies and column purified as previously reported [33, 37]. Assembly kinetics were monitored by viscometry and electron microscopy using temperature-controlled incubators [37]. Human recombinant vimentin was obtained as described [34].

Antibodies and immunoblotting Monoclonal antibody V9 against human vimentin was obtained from Boehringer Mannheim, and the monoclonal antibody C04, specific for cytokeratin 18 in human tissues, was purchased from Progen Biotechnik (Heidelberg/Germany). Cytoskeletal proteins were prepared from embryos and from selected tissues of the adult fish. These samples were treated with Triton X-100 and high salt buffer as reported [31]. Alternatively, small pieces of tissue were homogenized directly in Laemmli sample buffer containing 6.6M urea. The proteins in the cytoskeletal fractions or in the whole homogenates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli [48]. Two-dimensional gel electrophoresis, using isoelectric focussing in the first dimension, was done as reported [31]. Blotting was done using lOmM borate pH 8.8 as a transfer buffer, and the nitrocellulose membranes were incubated with the primary antibodies at 1: 100 to 1: 1000 dilution. For the detection of bound primary antibody, we used HRP-coupled secondary antibodies (Dianova, Hamburg/Germany) and the enhanced chemiluminiscence (ECL) system from Amersham (Braunschweig/Germany) .

Immunocytochemistry and electron microscopy

Whole-mount immunocytochemistry was performed on embryos fixed with freshly prepared 4 % formaldehyde for 15-30 min, and subsequently treated as described by Westerfield [77]; the dilution of V9 was 1: S. Pictures were taken as described for the whole-mount in situ hybridization. For immunofluorescence microscopy on cryostat sections, the embryos were fixed with 4 % formaldehyde for 1 h, embedded in 30 % sucrose, transferred to Tissue-Tek® (O.C.T. 4583, Miles Inc., Elkhart, IN/USA) and frozen in isopentane cooled in liquid nitrogen. For the adult fish, the tissues were dissected, embedded in Tissue-Tek® and immediately frozen as described above. The -S-f-lm-thick cryostat sections were further fixed with acetone at -20°C for lOmin. The incubation with V9 was done for 1 h (1: 5 dilution), and incubation with the secondary antibodies coupled to Texas red, FITC, or cyanine 3.29-0Su (Biotrend, KolnlGermany, or Dianova) was performed for 30 min. Pictures were taken with a Zeiss Axiophot (Zeiss, OberkochenlGermany) using TMY films (Eastman Kodak Co., Rochester, NY/uSA). The electron microscopy techniques employed were essentially as described elsewhere [36, 37].

Results Cloning and molecular characterization of zebrafish vimentin By screening a post-somitogenesis zebrafish embryo cDNA library with a random-primed trout vimentin cDNA probe, we

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Fig. 1. Amino acid sequence comparison of vimentin from zebrafish (Zf), trout (Tro) [35] and human (Hum) [34]. The asterisks indicate the residues identical in all three species, whereas the dots indicate the residues identical in two species, with a conserved one in the third species. The small open arrows below the sequences indicate those amino acids that are unique to zebrafish vimentin and identical in trout, human and Xenopus vimentin (the latter is not shown but see [31, 35]). The vertical arrows above the sequences indicate conserved aromatic residues of the head domain. Conserved basic amino acids of the head domain are boxed. The large box close to the amino-terminus marks a evolutionarily conserved sequence motif (see [32]). A potential cdc2 kinase phosphorylation site is indicated by a bar. Note that the position differs slightly between fishes and terrestrial vertebrates. The boxed prolines in the tail domain point to their strict positional conservation. A nonapeptide motif highly conserved in the tail domain of fish is indicated by a bar. The horizontal arrows above the sequence indicate the proposed beginning and end of the helical subdomain; PR, pre-coiled coil; C, coil; L, linker. The bracket marks the proposed region of the helical "stutter". Head and tail indicate the non-helical N-terminal and C-terminal domains, respectively.

obtained a clone of 1525 base pairs containing the open reading frame coding for a polypeptide of 455 amino acids with a molecular mass of 52597 Da, an isoelectric point of 4.93 and high sequence identity with known vertebrate vimentin (Fig. 1). Its deduced amino acid sequence shares between 80-90 % identity with that of carp [7], goldfish [27] and trout vimentins [35], and still 72 % identity with human vimentin [34]. Therefore, we conclude that we identified zebrafish vimentin. Quite remarkably, the central helical rod exhibits a very high degree of sequence identity with that of trout and human vimentin (Fig. 1). However, in the 334 amino acids of the a-helical rod (including the pre-coiled coil region PR) there are 18 positions, scattered over the whole rod domain, where the zebrafish sequence differs from the other two vertebrates and also from the amphibian Xenopus laevis (not shown here; see [31, 35] for sequence comparison), the latter three being identical at these positions (arrows in Fig. 1). Also, if conservative changes are taken into account, the non-helical tail domain has been conserved considerably during evolution. In contrast, the non-helical head domain shows less iden-

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tity at the primary sequence level, except for a nonapeptide motif near the N-terminus. Despite these extensive sequence differences, general principles have also been conserved in this domain such as the number and spacing of arginines as well as that of aromatic amino acids (boxes and upper arrows, respectively, in Fig. 1; for details see [30]). Finally, also in the zebrafish vimentin the pre-coiled coil sequence (PR in Fig. 1) is completely compatible with a-helix formation indicating that vimentin indeed is principally different from cytokeratins in this part of the molecule . Given such a high degree of homology with other vertebrate vimentins, we tested three available monoclonal antibodies for recognition of zebrafish vimentin. Western blot experiments indicated that the monoclonal antibodies XL-VIM14.13 [35], 3B4 [31] and V9 [61], raised against X. laevis, bovine and porcine vimentin, respectively, recognized zebrafish vimentin in protein extracts from several tissues (data not shown). However, with exception of the V9 antibody, these antibodies cross-reacted significantly with other unknown proteins with different molecular mass than zebrafish vimentin, and therefore, V9 was used for further experiments, although the major findings were corroborated with the other two antibodies. To test the affinity of the V9 antibody for zebrafish vimentin when compared with human vimentin, the respective recombinant proteins were produced in E. coli, and serial dilutions of the proteins were probed with the antibody in a standard immunoblot experiment. The V9 antibody indeed recognized zebrafish vimentin down to 100 ng of protein at this level of exposure, although the reaction was approximately 25-fold weaker when compared to that of human vimentin (Fig. 2a). To further investigate the specificity of the V9 antibody recognizing zebrafish vimentin, the recombinant protein, total protein extracts and cytoskeletal fractions from the adult eye were analyzed by immunoblotting after two-dimensional gel electrophoresis (Fig. 2b-d). Recombinant vimentin was separated into three isoelectric variants, the major one focussing at a pI slightly more acidic than a-actin. In the second dimension they migrated with an apparent Mr of 55000 (Fig. 2b). In the protein extracts of zebrafish eye (Fig.2c), a single, distinct spot was detected at a position corresponding to that of recombinant vimentin run in parallel. Also, a strong signal identically located as the recombinant protein was obtained in the cytoskeletal preparations. At prolonged exposure, a few weak spots less acidic than the vimentin protein were seen (Fig.2d).

Assembly of zebrafish vimentin in vitro The way vimentin from different species assembles is drastically influenced by the increase of temperature, whereas low temperatures seem to be of less influence [34-36, 70]. In contrast, zebrafish vimentin filaments, assembled at low temperatures (12,15 and 18°C), were quite thicker than standard IFs (diameter up to 30 nm, arrowheads in Fig. 3b) and appeared to be engaged in extensive lateral filament-filament contacts, sometimes fusing for more than 100 nm (arrow in Fig. 3a, b). At the higher temperatures, i.e. 21 to 34°C, zebrafish vimentin filaments were indistinguishable from typical vimentin IFs (Fig.3c, and data not shown). At 37°C, however, relatively few, short filaments were observed, most of them terminating in ball-like aggregates (data not shown) resembling those seen with trout vimentin at 28°C [35]. In order to quantitatively determine the effect of temperature on assembly, we performed viscometry using the Ostwald

178 J. Cerda, M. Conrad, J. Markl et 01.

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d Fig. 2. Characterization of zebrafish vimentin using the monoclonal antibody V9. (a) Immunoblot with different amounts of human (lanes 1-4) and zebrafish (lanes 5-8) recombinant vimentin. SOOng (lanes 1 and 5), 100ng (lanes 2 and 6), 20ng (lanes 3 and 7), 4ng (lanes 4 and 8) were analyzed in paralell by SDS-PAGE (12.5 %), probed with V9 antibody (1: 10(0) for 1 h at room temperature, and detected by the ECL system. On the right in s, the relative molecular weight of marker proteins is indicated (from top to bottom: 116000; 97000; 66000; 45(00). (b-d) Immunoblot analysis of (b) zebrafish recombinant vimentin, (c) whole protein extract from adult eye and (d) cytoskeletal preparation from adult eye. Proteins were separated by isoelectric focusing (large horizontal arrow pointing to the anode) followed by SDS-PAGE (large vertical arrow), and detected using V9 (1: 100) in combination with the ECL system. In b, the small arrow indicates the position of bovine serum albumin (67 kDa, pI 6.4), the arrowhead that of actin (42kDa, pI5.4). The left of the three vimentin-positive components most probably is vimentin without the initiating formylmethionine. The minor acidic component very likely is an acetylated form of vimentin (data not shown).

capillary viscometer at temperatures ranging from 4°C to 37°C. At 28 °C, highest values of specific viscosity were reached followed by those obtained at 24 and 31°C, respectively (Fig. 4a, b). At 34 and 21°C medium viscosity values

were measured, which gradually decreased when the temperature was lowered from 18, 15, 12, and 8 to 4°C (Fig.4b). In contrast, between 34 and 37°C, a sudden drop of specific viscosity occurred indicating that filament elongation was severely impaired at temperatures above 34°C (Fig. 4a). We have recently shown that mutated, assemblyincompetent vimentin can be "rescued" for assembly by addition of relatively small amounts of wild-type vimentin [33]. Therefore, we asked if a temperature-sensitive phenotype such as the "null-assembly" of trout vimentin at 28°C could be rescued by addition of zebrafish vimentin, which has its assembly optimum at 28°C. We applied two experimental regimens. Firstly, trout and zebrafish tetramers were mixed in low salt/ high pH-buffer. After initiation of assembly conditions, viscosity reached quite higher values than zebrafish vimentin alone (at 0.25 mg/ml, Fig.4c; compare filled triangle with filled rhombus), although not those obtained with zebrafish vimentin at 0.5 mg/ml (filled square). This indicates that part of the trout vimentin, which on its own (at 0.5mg/ml; filled circle) developed hardly any viscosity, was in part recruited for IF formation by co-assembly with zebrafish vimentin. The same effect was observed when zebrafish and trout vimentin were mixed at a ratio of 1: 3 (i.e. end concentration 0.125 and 0.375 mg/ml, respectively). In the second series, both vimentins were mixed at the monomeric stage in 8 M urea and then dialyzed as a mixture into the low salt/high pH-buffer. This results in the formation of mixed dimers and mixed tetramers. Also in this case zebrafish vimentin appeared to be partly able to integrate trout vimentin into the assembly process as deduced from the viscosity profiles (data not shown). However, the temperature sensitivity effect of trout vimentin is still comparatively dominant, since 75 % of the dimers should, in this type of experiment, contain at least one zebrafish vimentin, and therefore most of the tetramers should also contain at least one or two zebrafish molecules. Our experiments show that this is not sufficient for a complete rescue, i.e. to obtain specific viscosity values identical to those gained with 100 % zebrafish vimentin. Since zebrafish is routinely reared at 28°C, we investigated how filament assembly proceeds with time at this "natural" temperature in order to compare the structures formed with those found for human vimentin at 37°C [36]. At 1 second after initiation of assembly, "unit-length" filaments of approximately 65 nm length and of up to 20 nm diameter, with slightly "sharpened" ends, were observed very similar to those seen with human, Xenopus and trout vimentin [36]. Also, apparently fused filaments of wide diameter and length of 150 to 210nm were seen (Fig. Sa). After 10 seconds, longer and more compact filaments were frequently encountered side by side with open forms (Fig.5b), which at 30 seconds had further increased in length. One minute after initiation of assembly, filaments were more compact and already so long that filament ends were hardly ever detected (Fig. 5c). By 5 minutes, filaments had compacted completely to diameters of around 12 nm, some filaments already performing loop-like turns indicating their high flexibility (arrows in Fig.5d). By 15 min, dense meshworks, as seen after 1 h of dialysis, were prevailing throughout the electron microscopic grids (data not shown). Corresponding kinetic experiments were also performed at 12, 15, 18, and 21°C. Also at these lower temperatures, the "early" structures seen at 10 seconds were very similar to those observed at 28 °C at this time point (data not shown). Remarkably, at the lower temperatures, filaments appeared thicker in

Characterization ond expression of zebrafish vimentin

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Fig. 3. Electron microscopic analysis of negatively stained preparations of zebrafish vimentin (0.2 mg/ml) assembled by the addition of concentrated filament buffer at (a) 12°C, (b) 15°C, (c) 21°C. Arrowheads in a and b point to thick regions of the filaments, the arrow in b

indicates apparently fused filaments. Note that at 21°C (c) filaments appear a lot more uniform and exhibit the "normal" diameter of around 10 nm. Bar, 100 nm.

accordance with the pictures obtained at 1 h of assembly (see Fig. 3). Thus, although the first phase of filament assembly, i.e. lateral aggregation of tetramers/octamers to unit-length filaments, is apparently fast at 12°C, filament elongation appears to be less efficient, as also observed in the viscometric analyses (see Fig. 4b). However, as filaments do interact with

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Fig. 4. Temperature-dependence of zebrafish vimentin assembly. (a, b) Viscometric analysis ofthe effect of temperature on the assembly of zebrafish vimentin. Soluble protein was preincubated in 5 mM TrisHCI, pH 8.4, at (a) 24°C, filled rhombus; 28 °C, filled square; 31°C, open circle; 34 °C, filled triangle; 37°C, open square; (b) 4°C, open triangle; 8 °C, open square; 12°C, filled triangle; 15 °C, open circle; 18°C, asterisk; 21 °C, filled circle; 24°C, filled rhombus. Measurements were carried out with one batch of protein using the same viscometer. (c) Mixing experiments with trout and zebrafish vimentin. Proteins were mixed in the indicated ratios at the tetrameric stage at 28°C. Assembly was initiated at 10 min by addition of concentrated filament buffer. Filled square, 0.5 mg/ml zebrafish vimentin; filled triangle, 0.25 mg/ml of both zebrafish and trout vimentin; open triangle, 0.125 mg/ml zebrafish and 0.375 mg/ml trout vimentin; filled rhombus, 0.25 mg/ml zebrafish vimentin; open rhombus, 0.125 mg/ml zebrafish vimentin; filled circles, 0.5 mg/ml trout vimentin. Note that the addition of 0.25 mg/mi trout vimentin to zebrafish vimentin (0.25 mg/ml) increases viscosity considerably (compare filled triangles with filled rhombus), much more than could be expected from 0.25 mg/ml trout vimentin alone which would give hardly any viscosity increase on is own (compare with 0.5 mg/ml trout vimentin, filled circles). Abscissa: time (minutes); ordinate: specific viscosity.

Expression pa"ern of vimentin during embryonic development The expression pattern of vimentin was examined by in situ hybridization with a digoxigenin-Iabelled antisense RNA probe in whole zebrafish embryos fixed at regular intervals between the onset of the two-cell stage and the prim-15 stage (see [44] for details and terminology of staging). The expression of vimentin mRNA was first detected at the 2-somites stage (approximately 10.5 hpf), increasing further at the 4-somites stage (approximately 11 hpf) in presumptive lateral ganglia adjacent to the developing hindbrain and midbrain (Fig. 6a). By the 18-somites stage (17.5 hpf), the level of expression became higher in ventral areas of the developing forebrain, midbrain and hindbrain, and in the pial surface of the neural plate along the neural tube (Fig. 6b-f). In prim-5stage embryos (24 hpf), the expression in the posterior forebrain, the ventral midbrain and the anterior hindbrain was extended (Fig. 6g), and a new subset of cells in the anterior hindbrain started to express vimentin (Fig. 6h). By this stage, the vimentin transcripts also increased in the latero-dorsal cells of the neural tube, and its ventricular cells also appeared to express low levels of vimentin (data not shown). By the 15prim stage (30 hpf), the expression of vimentin in the embryonic brain was increased (Fig. 6i), and differently located subsets of vimentin-expressing cells were distinguished along the neural tube (Fig. 6j, k). The developmental patterns of expression of the vimentin protein, together with that of cytokeratins, were then investigated by Western blot experiments on cytoskeletal fractions of embryos at different embryonic stages. For the immunodetection of cytokeratin we used the monoclonal antibody C04, which recognizes zebrafish cytokeratin 18 [10]. Low but detectable levels of vimentin were present at prim-5 stage, and the intensity of the reaction became gradually stronger in long-pec-stage embryos (48hpf) and larvae (5 days pf; data not shown). In contrast, cytokeratins were detected earlier,

180 J. Cerda, M. Conrad, J. Markl et 01.

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Fig. 5. Time course of the assembly of zebrafish vimentin at 28°C. Soluble vimentin, in 5 mM Tris-HCI, pH 8.4, was induced to assemble by addition of an equal volume of 2 x concentrated filament buffer, and assembly terminated by addition of an equal volume of filament buffer containing 0.2 % glutaraldehyde at (a) 1 second; (b) 10 seconds;

(c) 1 minute; (d) 5 minutes. Note that from 1 minute, but especially clear from 5 minutes on, filaments appear to be much more compact. The arrows in d point to sharp turns in the filament's direction indicating that the filaments are already of high flexibility at this time point. Bar,100nm.

since a strong reaction was already detected at the bud stage (lOhpf). Similar to the situation observed for vimentin, the expression of cytokeratins seemed to increase in later developmental stages. To study the localization of vimentin during embryonic development, whole-mount immunocytochemistry and immunofluorescence microscopy on cryostat sections were carried out (Fig. 7). Under the conditions employed, vimentin protein was first detected in 15-somites-stage embryos, i.e. approximately 4 hours after the respective mRNA, in ventral parts of the forebrain, midbrain and hindbrain (Fig. 7a). By the prim-5 stage, vimentin synthesis had increased further in the embryonic brain and became also prominent in the trigeminal ganglia (Fig. 7b) and in areas of the anterior hindbrain surrounding the otic vesicle. Along the neural tube, high levels of synthesis were seen in dorsal and ventral cells, presumably being motor neurons extending from the anterior hindbrain (Fig.7c). From both populations of cells, decorated axons extending to a more ventral position were observed (Fig.7d,e). In long-pec-stage embryos (48 hpf) , vimentin started to be synthesized in the pigmented epithelium of the embryonic retina (Fig. 7f), in some cells of the diencephalon, and in numerous cells within the developing spinal cord. By the protruding-mouth stage (72hpf) and in larva (5 days pf), the signal in the pigmented epithelium of the retina and in other retinal cell types (Fig. 7g), as well as in axonal tracts possibly from the trigeminal ganglia increased markedly. In larva, cells from the anterior hindbrain and putative glial cells along the spinal cord were also strongly decorated (Fig. 7h).

copy on cryostat sections of all major tissues. Prominent staining was obtained in dermal cells within the dermis (Fig. 8a, a'), dispersed fibroblasts in the connective tissue of the basal cell layer of the intestine (Fig. 8b, b'), dermal cells from the bulbus arteriosus of the heart (Fig. 8c, c'), glial cells of the spinal cord (Fig. 8d, d'), nerves (Fig. 8f, f' and i, i') and brain (not shown), epithelial cells of the ocular lens (Fig. 8g, g') and a number of retinal cell types (Fig. 8h, h'). These results indicated that the expression of vimentin in the adult zebrafish was in fact restricted to few tissues and cell types.

Localization of vimentin in the adult fish The expression of the vimentin protein in the adult fish was initially studied by Western blotting of total protein extracts from different organs. Substantial amounts of vimentin were detected only in skin and eye, whereas no signal was seen in extracts from heart, intestine, swimbladder, muscle, liver, brain, and ovary (data not shown). Thus, vimentin expression appeared to be highly restricted in the adult zebrafish. To study more precisely the localization of vimentin in the adult zebrafish, we performed immunofluorescence micros-

Discussion The high degree of sequence conservation, and especially the notion that zebrafish vimentin is nearly as related to the respective protein in man as it is in the rainbow trout, indicates that it is engaged in cellular processes of general importance. The assembly properties of zebrafish vimentin are best in the temperature range in which this particular organism lives. Beside these biophysical properties, the expression pattern of vimentin in various species demonstrates indeed that vimentin is principally present, irrespective of the species looked at, in certain specific tissues like retina. The synthesis in other cells or tissues, such as the nucleated red blood cells of amphibia and birds or the first mesodermal cells of the mouse embryo, appears to be related to more species-specific needs. This evolutionary conservation of a biophysical property, together with that of distinct expression principles, leads to the idea that the existence of vimentin in a filamentous form is necessary for the optimal establishment or performance of the corresponding tissue. In particular, lens and retina express vimentin strongly, and therefore it may be involved in the establishment of visual acuity - and vision truly is a decisive property to survive competition during evolution. Furthermore, the intricate expression pattern of vimentin in the embryonic brain points to the fact that its presence in early differentiating neurons and mature glia may be related with the generation of the complexity of the neuronal network.

Characterization and expression of zebrafish vimentin

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Fig. 6. Vimentin mRNA expression in developing embryos detected by whole-mount in situ hybridization using a digoxigenin-labelled fulllength probe. Lateral (b, d, g, h, i,j) and dorsal (a, c, e, f, h, k) views of embryos; rostral is to the left. (a) 4-somites embryo. Vimentin expression in a presumptive peripheral ganglion adjacent to the developing hindbrain (arrowhead), and in the presumptive midbrain neural plate (arrow). (b-f) I8-somites embryo. (b, c) A more extended and intense staining appears in ventral areas of the forebrain (asterisk), midbrain (arrow) and hindbrain (arrowheads). (d) Numerous cells in the neural tube are labelled. (e, f) A subset of cells located close to the pial surface (arrows) extends along the neural tube. (g-b) Prim-5 stage. (g) General view of the extensive expression of vimentin in the

posterior forebrain, midbrain and anterior hindbrain. (h) Intense staining in developing peripheral ganglia (arrowheads), and a new subset of cells in the anterior hindbrain (arrow) start to express vimentin. (i-k) Prim-IS stage. (i) Increased staining in the posterior forebrain, ventral midbrain and hindbrain. (j) Two populations of cells in the developing spinal cord express vimentin strongly: one subset of cells close to the floor plate (arrow) and the other in a more dorsal position (arrowhead). (k) Intense vimentin expression is also seen in ventricular cells of the spinal cord of the tail (arrow). Abbreviations: CB, cerebellum E, eye; HB, hindbrain; NO, notochord; OV, otic vesicle; TE, tectum. Scale bar: I00llm (a-e), SOllm (f-k).

Molecular characterization of zebrafish vimentin

(chicken) or proline (human). A potential cdc2 phosphorylation site in fish is located between the 4th common aromatic acid (upper arrows in Fig. 1) and the 7th common arginine; in tetrapods such a sequence is found further downstream between the 5th common aromatic amino acid and the 9th common basic amino acid, a lysine (in man it is an arginine). The functional significance of these three features is not clear, but they are sufficient to indicate specific differences.

Despite the high sequence identity of zebrafish vimentin to both man and trout vimentin, zebrafish vimentin has some sequence features unique to fish. In the conserved nonapeptide motif near the amino terminus, the diarginine is replaced in fish by lysine-arginine and the diglycine is followed by a glutamic acid in zebrafish, trout and carp (see Fig. 1 and [30)), whereas in tetrapods this is an asparagine (frog), glycine

182 J. Cerda, M. Conrad, J. Markl et 01.

Fig. 7. Expression of vimentin protein in developing embryos. Whole-mount immunocytochemistry (a-e) and immunofluorescence microscopy on 5-J.Lm cryostat sections (f-b) using the V9 antibody. Lateral (a, d, e) and dorsal (b, c) views of embryos (rostral is to the left) or transversal section (g). (a) 15-somites embryo. Vimentin expression in basal areas of the forebrain (arrows) and midbrain (arrowhead). (b--e) Prim-5 stage. (b) Dorsal view of the head with labelled cells in the lateral trigeminal ganglia (arrow), and in several cells of the forebrain, midbrain, cerebellum and anterior hindbrain (arrowheads). (c, d) Dorsal cells extending from the anterior hindbrain and located along the neural tube (arrowheads) are also intensely labelled; occasionally, axons extending from these cells to a more ventral position can be seen (arrow). (e) Another population of ventral cells of the neural tube, presumably motor neurons, are positive for vimentin;

these cells (arrowheatI) are located around the floor plate and also extend processes (arrow) to a more ventral position. (I) Long-pee stage. Parasagittal section through the head with positive reaction in the pigmented epithelium of the retina (arrow), as well as in axonal tracts close to the otic vesicle (arrowheatI). (g, h) Larvae. (g) Transversal section through the head with the retina showing positive reaction in the inner plexiform layer (1), amacrine cells (2) and cone cells (3), in addition to the pigmented epithelium (4). The cells located in the integument (asterisk), precursors of the adult skin, are also decorated. (h) In a parasagittal section, some cells of the anterior hindbrain show specific staining (arrowheatI). The signal is also intense in putative glial cells (arrow) along the spinal cord. Abbreviations: CB, cerebellum; E, eye; FB, forebrain; HB, hindbrain; MB, midbrain; NO, notochord; OV, otic vesicle. Scale bar: 100 J.Lm (a-c), 75 J.Lffi (d-b).

We have provided evidence that the temperature sensitivity of assembly of Xenopus and rainbow trout vimentin lies within the a-helical rod domain, and that the tail domain has even an opposite influence, as both tailless human and Xenopus vimentin assemble better than wild-type vimentin [16, 34-36]. Recently we have shown that swapping of the non-helical head domains between Xenopus and human vimentin yields chimeric proteins that retain their assembly characteristics with regard to temperature [30], and therefore, the temperature sensitivity is indeed caused by the behavior of the helical rod. Figure 9 compares the zebrafish, human, trout, and frog rod sequences in a schematic way, highlighting those residues in the amino acid sequence of one species that are distinctly dif-

ferent from those of the other three species, i.e. the corresponding other three sequences are identical in that position. This diagram shows that the most thermotolerant protein, human vimentin, contains several (9) unique residues spread over the first half of the rod. Then, however, with regard to the unique residues of zebrafish vimentin, human vimentin shares quite a lot of identical positions with the "colder" vimentins of trout and frog. Finally, there are 22 residues, most of them in coil 2, where the two fish sequences differ significantly from the tetrapod sequences (black arrowheads). This comparison strongly suggests that not a single amino acid is responsible for the corresponding temperature-dependent behavior of assembly.

Characterization and expression of zebrafish vimentin

Assembly of zebrafish vimentin The assembly mechanism of zebrafish vimentin appears to be principally identical to that of vimentin from higher vertebrates (cf. [30]). However, at low temperature thick non-IFtype fibrils are formed. This polymorphism again indicates that IF proteins have several options to interact in generating the polymer. The physiological meaning of this property, however, is not known at the moment. In contrast, actin from antarctic fishes (body temperature -1.9 0c) has similar association characteristics like actin from fishes of temperate zones or even birds and mammals, and also heat stability of actins from various species does not entirely correlate with the corresponding body temperature [71]. We have recently shown that in vitro assembly of Xenopus vimentin at a high, "non-permissive" temperature generates protein aggregates that are permanently trapped in a wrong conformation [34]. Moreover, we had shown that rather low amounts of wild-type vimentin were able to rescue assemblydefective mutant vimentins [33]. In this report, we show that the temperature sensitivity of a certain vimentin can also be partially rescued by a vimentin from a "warm" species. For instance, zebrafish vimentin can "instruct" trout vimentin to assemble at 28°C. The salt-induced lateral aggregation of soluble subunits, the first phase of assembly (see [30]), apparently allows the incorporation of several trout vimentin units into the unit-length filaments. However, if the ratio of such "temperature-misfolded" units is too high per unit-length filaments, non-productive subunits may form that cannot join the regular elongation process. Consistent with this idea, only very loose, "open" structures resembling unit-length filaments in their outline are obtained from trout vimentin assembled at 28°C for 1 to 10 sec (unpublished observations).

Expression of vimentin during embryonic development The expression and organization of the vimentin and cytokeratin filament systems during mouse and X. laevis embryonic development has been extensively documented [18-20, 31, 49]. In fish, cytokeratins are the major IF proteins being abundantly expressed in certain tissues which in mammals appear to be devoid of this class of IF proteins, such as the optic nerve, mesenchymal cells and oocytes [26, 35, 55]. These studies suggest that the integrity of the early embryonic cytokeratin system is important for normal gastrulation (reviewed in [15,45]). For vimentin, which has been detected in a number of different cell types during vertebrate embryogenesis, less information is currently available on its possible role(s) during embryonic development. In the zebrafish, however, the dynamics of the intermediate filament system, including that of vimentin, during embryonic development is largely unknown. A recent study has revealed that a zebrafish type II cytokeratin mRNA is inherited maternally and that it is present in mature oocytes, the zygote and in the cleavage stage embryo [38], thus resembling the situation in mammals [24] and amphibians (see [45]). After midblastula transition, this cytokeratin gene is expressed in all surface cells of the embryo, including those of the enveloping layer, periderm (embryonic epidermis), and in a subpopulation of deep cells presumed to be intestinal progenitors. Therefore, this gene could be a suitable epidermal marker during zebrafish development ([38]; for discussion see [10]). In the present work, by using whole-mount in situ hybridization, we have observed that zebrafish vimentin mRNA was expressed after

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the gastrulation period, approximately at the 2-somites stage (Fig. 6). Thus, its expression is turned on much later in development than that of this cytokeratin. Moreover, immunoblots of embryonic cytoskeletal extracts employing the human cytokeratin 18-specific monoclonal antibody C04, which specifically reacts with a zebrafish cytokeratin [10], indicated that substantial amounts of cytokeratin protein were present at the bud stage (end of gastrulation) towards later stages of development, whereas vimentin was detected only weakly from the prim-5 stage on. Therefore, our findings and those from Imboden et al. [38] suggest that in the zebrafish, as in amphibians and mammals, cytokeratins are expressed before vimentin, which seems, unlike cytokeratins, not to be of maternal origin. This scenario would be consistent with a possible role of cytokeratins during zebrafish gastrulation as it has been demonstrated in X.laevis [46, 47, 74]. From our expression studies in the zebrafish, it appeared that both vimentin mRNA and protein displayed a complex pattern of expression during embryonic development (Figs. 6 and 7). In developing embryos, vimentin was expressed in a subset of cells, predominantly in early differentiating neurons and putative peripheral glial cells, which were located in different regions of the forebrain, midbrain and hindbrain, and along the trunk within the developing spinal cord. At late stages of development, vimentin immunoreactivity was abundant in the brain, the spinal cord, and in different cell types of the retina. In contrast to other vertebrates (e.g. [11, 19,31]), vimentin was not detected in embryonic mesenchymal or muscle cells (nor in the adult). The anatomical pattern of expression of vimentin in the zebrafish, as shown in this study, was closely associated with the development and differentiation of the central and peripheral nervous system and strongly resembles that found in mammals [1, 6, 75], birds [73] and amphibia [14,28,31, 72]. In these species, vimentin is initially expressed following gastrulation (or at early neurula stages) in differentiating neurons and neuroepithelial cells of the embryonic brain and spinal cord. Therefore, it appears that vimentin could be a reliable marker of early neurogenesis in the zebrafish, similarly to the case in mammals and birds where it is considered to be a marker for immature glia of the developing brain (e.g. [12,58]). However, in some teleost species [35, 42, 43, 66] and apparently also in the zebrafish, vimentin expression persists in mature glia, particularly from the optical nerve and from other peripheral nerves. This has been associated with the preservation of regenerative activity of the fish and amphibian brain [8, 43] unlike its counterpart in higher vertebrates in which this feature appears more restricted.

Expression of vimentin in the adult fish After embryonic development, the expression of vimentin was confined to few tissues and cell types, including some dermal cells of the skin, mature glial cells and astrocytes, several cell types of the retina, and dispersed fibroblasts within the connective tissue (Fig. 8). This restricted expression of vimentin in the adult suggests that a number of embryonic neurons and differentiating glial cells, which initially synthesized vimentin, possibly changed their expression of cytoskeletal proteins as development proceeded to other differentiation-specific intermediate filaments, such as those containing glial fibrillary protein or neurofilament triplet proteins, as it is well known to occur in other vertebrates (e.g. [25,67,76]). The general pattern of expression of vimentin found in the adult fish is also essentially identical to that reported in other teleosts, such as

184 J. Cerda, M. Conrad, J. Markl et al.

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Characterization and expression of zebrafish vimenlin

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Fig. 9. Schematic representation of the sequence differences in the a-helical rod of vimentin between zebrafish (red balls), human (rose balls), trout (blue balls) and frog (light-blue balls). White balls indicate identical residues in the "cold" vertebrates (trout, frog) versus "warm" vertebrates (zebrafish, human). The black arrowheads below the colored bar indicate amino acid positions identical in fish versus those identical in tetrapods. The a-helical rod domain is divided into the following subdomains: PR, precoiled coil; CIA, coil 1A; Ll, linker 1; C1B, coil 18; Ll2, linker 12; C2A, coil 2A; L2, linker 2; C2B1, first

half of coil2B; S, stutter (see [62)); C2B2, second half of coil2B. The numbers above the colored bar indicate amino acid positions of zebrafish vimentin. The bold numbers below the colored bar indicate the percentage of amino acid identity of the corresponding subdomain. The dark green second half of C2B2 indicates a region with 100 % identity between all four sequences. Note that only those amino acids are highlighted where the corresponding other three sequences are identically different from the indicated one.

the rainbow trout [35], the barbel [3] or the goldfish [8, 42]. In these species, vimentin is expressed predominantly in glial cells and astrocytes of the brain, spinal cord and optical nerve, and in the case of the rainbow trout, also in white blood cells and other cells of the immune system. In the zebrafish, however, the white blood cells and other blood cell types, the epidermis, and the ovary appear to be devoid of vimentin (data not shown), unlike in the rainbow trout [35]. Remarkably, both in zebrafish (this study) and in other teleosts [13, 35, 41, 53], as in higher vertebrates (e.g. [29,51,68]), vimentin is found in several parts of the developing and mature visual pathway, and specially in a number of cell types within the retina and in the lens, particularly in Muller cells and retinal ganglion cells (and in some cases, also in horizontal cells). This indicates that the expression of this intermediate filament within the retina and the lens may follow the same pattern in both mammalian and non-mammalian species (for discussion see [63]). However, the significance of this conserved pattern of expression of vimentin in the eye of vertebrates throughout evolution, which may point to an important role of this intermediate filament in the visual system, is still unclear.

The targeted inactivation of the vimentin gene in mice yielded no obvious histological phenotype in animals lacking vimentin, including the epithelial outer layer of the lens [9]. However, in that study the evaluation of the general status of the visual perception in mutant mice was not investigated. Notably, the overexpression of vimentin or the expression of mutated forms of vimentin in the lenses of transgenic mice has been found to interfere with the terminal differentiation of lens fiber cells [2, 5]. Therefore, important functions of the vimentin filament system in the vertebrate eye are very likely to occur, although we cannot specify them at the moment. The information provided in this work regarding the pattern of expression of vimentin both in the embryo and in the adult zebrafish, which at least in brain appears to be strikingly similar to that known in higher vertebrates, should establish the basis towards the design of future genetic studies using the advantages of this new model for vertebrates in order to elucidate the role and consequences of vimentin expression during development.

Fig. 8. Immunofluorescence microscopy of different tissue cryostat sections from the adult zebrafish after reaction with V9 antibody. Phase-contrast (a-i) and epifluorescence (a'-i') optics. (a, a') Crosssection of the skin showing variable decoration in the dermis. (b, b') Longitudinal section of the intestine with staining in some cells within the connective tissue, but the mucosa epithelium (arrowhead) and lamina propia (arrow) were not stained. (c, c') Section of the heart showing positive reaction in the dermal cells from the bulbus arteriosus (arrow), whereas the muscle cells were negative. (d, d') Zebrafish spinal cord showing specific decoration of glial cells; and (e, e') the edge of the spinal cord showing staining of glial cells in a projecting spinal nerve. (f, f') Cross-section of the nervus vagus with positive reaction associated with the surrounding astrocytes and internal glial cells. (g, g') Longitudinal section of the ocular lens showing positive reaction in the epithelium, whereas the lens fibers were negative. (h, h') Crosssection of the retina with specific staining in the ganglion cell layer, bipolar cell layer, outer plexiform layer and pigmented cells. (i, i') Cross-section of the optical nerve with positive reaction in glial cells. BC, bipolar cell layer; CB, cell bodies; CT, connective tissue; D, dermis; EP, epidermis; GC, ganglion cell layer; IP, inner plexiform layer; LE, lens epithelium; LF,lens fibers; MC, muscle cells; OP, outer plexiform layer; PC, pigmented cells; RC, rods and cones. Scale bar: 100 Ilm in a-e, g and h; 200 Ilm in f; and 50 Ilm in i.

Acknowledgements. We thank A. Hunziker, S. Reidenbach and M. Brettel for excellent technical assistance, A Picker for technical demonstrations and helpful discussion, R. Zimbelmann for preparation of Figure 1, and E. Ouis for typing of the manuscript. We gratefully acknowledge Prof. Dr. W. W. Franke's interest and support. This work was supported by the Deutsche Forschungsgemeinschaft (grant HeI853 to H. Herrmann) and by the "Stiftung Rheinland-Pfalz flir Innovation" (grant 836-386261/138 to 1. Markl). 1. Cerda is a recipient of a postdoctoral grant from the European Commission (Training and Mobility of Researchers Program).

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