Zebrafish evx1 is dynamically expressed during embryogenesis in subsets of interneurones, posterior gut and urogenital system.

The even-skipped-related homeobox genes (evx) are widely distributed through animal kingdom and are thought to play key role in posterior body patterning and neurogenesis. We have cloned and analyzed the expression of evx1 in zebrafish (see also Borday et al. (Dev. Dyn. 220 (2001) in press) which displays a dynamic and restricted expression pattern during neurogenesis. In spinal cord, rhombencephalon, and epiphysis, evx1 is expressed in several subsets of emerging interneurones prior to their axonal outgrowth, identified as primary interneurones and a subset of Pax2.1(+) commissural interneurones. In the hindbrain, evx1 is expressed in reticulospinal interneurones of rhombomeres 5 and 6 as well as in rhombomere 7 interneurones. The latest emerging evx1(+) interneurones in the hindbrain correspond to commissural interneurones. evx1 is also dynamically transcribed during the formation of the posterior gut and the uro-genital system in mesenchymal cells that border the pronephric ducts, the wall of the pronephric duct, and later in the posterior gut and the wall of the uro-genital opening. In larvae, the ano-rectal epithelium and the muscular layer that surrounds the analia-genitalia region remain stained up to 27 days. In contrast other vertebrates, evx1displays no early nor caudal expression in zebrafish.

Widespread expression of the Xenopus homeobox gene Xhox3 in zebrafish eggs causes a disruption of the anterior-posterior axis.

The widespread expression of the Xenopus homeobox gene Xhox3 in zebrafish was performed by microinjection of synthetic Xhox3 mRNA into uncleaved fertilized eggs and resulted in embryos displaying varying degrees of anterior-posterior (A-P) axis disruption. The phenotype observed was dose-dependent and showed anomalies, mainly in neural keel development, from microphthalmia to acephaly. Injection of 5 pg to 10 pg of mRNA caused a range of phenotypes in prim 5 stage embryos from normal to acephalic. Anomalies have been categorized according to an index of axis deficiency (Zf-IAD). We further characterized the axis disturbance by analyzing the expression of two genes implicated in axis formation: engrailed (eng) and brachyury (ntl). eng could not be detected in the muscle pioneer cells of embryos injected with Xhox3. The pattern distribution of brachyury (Ntl) protein is abnormal in Xhox3 injected embryos. Evidence for the conservation of the NH2 terminal region of the Xhox3 protein in frogs and fish is provided by the detection of a nuclear Xhox3-like protein in 24 h zebrafish embryos located in posterior mesoderm tissue. Previous results in Xenopus embryo research and the data presented here support the conservation of an A-P patterning mechanism involving the Xhox3 gene.

Bioactive glass stimulates in vitro osteoblast differentiation and creates a favorable template for bone tissue formation.

In this study, we have investigated the behavior of fetal rat osteoblasts cultured on bioactive glasses with 55 wt% silica content (55S) and on a bioinert glass (60S) used either in the form of granules or in the form of disks. In the presence of Bioglass granules (55 wt% silica content), phase contrast microscopy permitted step-by-step visualization of the formation of bone nodules in contact with the particles. Ultrastructural observations of undecalcified sections revealed the presence of an electron-dense layer composed of needle-shaped crystals at the periphery of the material that seemed to act as a nucleating surface for biological crystals. Furthermore, energy dispersive X-ray (EDX) analysis and electron diffraction patterns showed that this interface contains calcium (Ca) and phosphorus (P) and was highly crystalline. When rat bone cells were cultured on 55S disks, scanning electron microscopic (SEM) observations revealed that cells attached, spread to all substrata, and formed multilayered nodular structures by day 10 in culture. Furthermore, cytoenzymatic localization of alkaline phosphatase (ALP) and immunolabeling with bone sialoprotein antibody revealed a positive staining for the bone nodules formed in cultures on 55S. In addition, the specific activity of ALP determined biochemically was significantly higher in 55S cultures than in the controls. SEM observations of the material surfaces after scraping off the cell layers showed that mineralized bone nodules remained attached on 55S surfaces but not on 60S. X-ray microanalysis indicated the presence of Ca and P in this bone tissue. The 55S/bone interfaces also were analyzed on transverse sections. The interfacial analysis showed a firm bone bonding to the 55S surface through an intervening apatite layer, confirmed by the X-ray mappings. All these results indicate the importance of the surface composition in supporting differentiation of osteogenic cells and the subsequent apposition of bone matrix allowing a strong bond of the bioactive materials to bone.

Zygotic expression of the zebrafish Sox-19, an HMG box-containing gene, suggests an involvement in central nervous system development.

The zebrafish Sox-19 belongs to the Sry subfamily of HMG (Hight Mobility Group) box genes and is closely related to the Sox sub-group B, comprising the mouse Sox-1, Sox-2 and Sox-3 genes, with respect to both HMG box homology (95.3%) and neural expression during embryogenesis. Analysis of Sox-19 expression during embryogenesis by whole-mount in-situ hybridization revealed interesting features. In early gastrula embryos, Sox-19 transcripts are detected within a circular area in the region of the presomptive central nervous system (CNS) and appears to be the earliest molecular marker of the CNS in vertebrates. In the developing brain, ZfSox-19 mRNA is distributed in the ventral region of the diencephalon, midbrain and hindbrain whereas the expression is excluded from the telencephalon. In spite of the ventral localisation of its mRNA, the expression of this ZfSox-19 gene is completely normal in cyclops embryos which implies that ZfSox-19 expression is independent of the presence of the floor plate.

Scanning electron microscopic evaluation of the effects of an air-abrasive system on dental implants: a comparative in vitro study between machined and plasma-sprayed titanium surfaces.

An in vitro comparative study was conducted in order to evaluate the effects of an air-abrasive system on dental implant surfaces. Eight new titanium dental implants, four standard machined implants (machined group), and four standard plasma-sprayed implants (plasma-sprayed group) were selected for investigation. Both neck and body surfaces of the implants were analyzed. Each pair of implants in each group was treated as follows: the spray of the air-abrasive unit was applied to each area for 5 seconds on the first implant and 15 seconds on the second implant. A total of 24 areas were observed: 16 test implants and 8 controls. Scanning electron photomicrographs were analyzed by 3 examiners using a category rating scale (kappa = 0.594). The images were also computerized for texture analysis. The results indicate that a single air-powder abrasive treatment of the dental implants selected for this study modified their exposed surfaces. After treatment, the threaded neck surface of a machined group implant was least affected, whereas the body was the most altered. In the plasma-sprayed group, comparisons between implant surfaces showed little change. In the machined group, more change was observed in both neck and body areas. In all specimens, a 5-second exposure to the air-powder abrasive did not induce deep changes in the surfaces. A 15-second exposure modified all the specimen surfaces. Further studies are needed to evaluate the effect of these changes on the biological osseointegration process.

[Scanning electron microscopy study of membrane modification of blastomeres in the first stages of ovum development in trout (Salmo irideus Gibb.)]. / Etude par microscopie électronique à balayage des modifications de la membrane des blastomères au cours des premiers stades du développement de l'aeuf de Truite (Salmo irideus Gibb.).

Our study relates to the development of the membrane relief of the blastomeres of the trout egg from the stage before their division up to gastrulation. The surface of the blastoderm shows first a more or less dense system of crests which by the time of gastrulation are replaced by microvilli distributed over the whole surface of the enveloping layer. The deep blastomeres, loosely connected to each other by lamellipodes and filopodes produce only a few projections of their membranes and these are only semispherical bodies. Possible roles of the microvillosities in the respiratory exchange of the egg and in the motility of the enveloping layer, are considered.

[Histochemical localization of lactate dehydrogenase (LDH) during cartilaginous cell differentiation in pleurodeles (Pleurodeles waltlii Michah]. / Localisation histochimique de la lacticodéshydrogénase (LDH) au cours de la différenciation des cellules cartilagineuses chez le Pleurodèle (Pleurodeles waltlii Michah.).

As revealed in chondrogenetic cells the LDH activity that is very low during cellular migration, suddenly increases at the time of cytodifferentiation and reaches a peak at the onset of the first chondrocyte differentiation. A relationship is likely to exist between this increase in LDH-activity, chondroitin-sulfate synthesis and NAD metabolism.

Cytochalasin D induces changes in cell shape and promotes in vitro chondrogenesis: a morphological study.

One of the initial events required for the expression of cartilage-specific macromolecules in monolayer cultures is the reversion to the initial round shape of chondrocytes. Thus, considerable research efforts have focused on developing reliable procedures to maintain a round morphology of cultured chondrocytes. Our study focuses on evaluating the response of dedifferentiated fetal rat chondrocytes to cytochalasin D, an actin-disrupting agent, with special emphasis on the morphological events. Immediately after exposure to the drug, cells round up but flatten again after removing the agent. However, immunocytochemical procedures revealed a disorganization of microfilaments and intermediate filaments. Phase-contrast and scanning electron microscopic observations revealed that on day 6 of culture, cells located at the top of the cell layer adopted a spherical morphology. Prominent differences were noted in control cultures where cells had to aggregate prior to overt chondrogenesis. Transmission electron microscopy confirmed the round morphology of the cells situated at the top layer but also revealed the presence of cell contacts between the cells. In addition, cells located at the central part of the cell layer displayed a typical morphology of mature chondrocytes, separated by an extensive extracellular matrix. These morphological changes occurred parallel to the expression of type II collagen and chondroitin sulfate, both hallmarks of the chondrocyte phenotype strong in experimental cultures, relatively weak in control cultures, and only restricted on areas of polygonal cellular aggregates. Furthermore, [35S]-sulfate incorporation into sulfated glycosaminoglycans increased rapidly with the period of culture to a maximum after 7 days and was then two-fold in treated cultures. Taken together, these findings indicated that cytochalasin D stimulates chondrogenesis in response to modification of cytoskeleton architecture and the subsequent rounding up of the cells.

[Histochemical localization of lactate dehydrogenase in blastomeres during early development of the rainbow trout embryo (Salmo irideus Gibb)]. / Localisation histochimique de la lacticodéshydrogénase dans les blastomères au cours du développement précoce de l'embryon de la truite arc-en-ciel (Salmo irideus Gill)

During the early development of the trout egg, some blastomers display a certain activity of the L.D.H., variable according to their position in the embryo. At the morula stage, the histochemical ltion of those blastomeres. On the contrary, during the gastrula stage, the blastomeres which have a high L.D.H. activity concentrate in the deep layer of the germ, in the "embryonic button", and in the germinal ring.

[Histochemical localization of lactate dehydrogenase in the trout embryo (Salmo irideus, Gibb) during early organogenesis of the cord and the neural tube]. / Localisation histochimique de la lacticodéshydrogénase dans l'embryon de la truite (Salmo iredeus, Gibb), au cours de l'organogenése précoce de la corde et du tube neural

The histochemical study of the LDH in the Trout embryo during the early organogenesis shows a specific localization in notochord cells, in mesodermic cells of the terminal knob and in some prosencephalic neuroblasts. The role of the LDH in the metabolism of NAD as well as in the energetic metabolism of embryonic cells is discussed.

Prevention of gastrulation but not neurulation by antibodies to fibronectin in amphibian embryos.

Gastrulation and formation of the neural plate are major steps in early vertebrate embryogenesis. Although morphogenetic movements leading to the formation of the primary germ layers have been extensively described, the mechanisms governing migration of mesodermal cells and their interactions with ectoderm remain ill-defined. A large body of evidence indicates that fibronectin (FN), a high molecular weight cell-surface-associated glycoprotein, promotes cell adhesion and cell migration throughout embryogenesis. FN has been detected at an early blastula stage in Pleurodeles waltlii. We now show that FN is a component of a dense fibrillar matrix underlying the blastocoel roof; in contrast, the exterior surface of the embryo is devoid of FN. Microsurgical inversion of part of the blastocoel roof does not prevent mesodermal cell migration except at the site of inversion where no FN matrix is available. Perturbation experiments using antibodies to FN demonstrate that the invagination of presumptive mesodermal cells does not occur when the monovalent antibodies are injected before or at the onset of gastrulation; on the other hand, the formation of a neural plate is not prevented when late gastrula stage embryos are treated with antibodies to FN. We conclude that the presence of FN is required for cell migration during gastrulation.

Effects of 1,2-propanediol on the cytoskeletal organization of the mouse oocyte.

The effects of 1,2-propanediol, a cryoprotectant used for the freezing of embryos, were tested on the organization of microtubules and microfilaments in mouse oocytes arrested in metaphase II. At low doses (less than or equal to 1.0 M), 1,2-propanediol induced disorganization of the meiotic spindle but at 1.5 M and higher, it stabilized the spindle. Cytoplasmic asters were observed at all doses tested. An extensive network of free microtubules was observed at 1.0 M and 2.0 M 1,2-propanediol, the former having the stronger disruptive effect on the spindle. Higher doses of 1,2-propanediol (greater than or equal to 1.5 M) caused the oocytes to form cytoplasmic blebs. These blebs lacked detectable cortical microfilaments. In contrast, the microfilament-rich area of the cell cortex overlying the meiotic spindle was not modified.

Role of catecholamines in the control of newborn kidney adenine nucleotide content.

During the 1st h of extrauterine life, the adenine nucleotide content of the rat kidney is modified: the ATP level increases (+30%) while ADP and AMP are lowered (-30 and -50%, respectively). This leads to a high value of energy charge in the newborn kidney (0.89 vs. 0.80 in the fetus). It was possible to obtain in utero a similar modification of ATP, ADP, AMP concentrations by injections to the fetuses of cAMP, dibutyryl cAMP, or isoprenaline. Conversely, the postnatal changes in adenine nucleotide content could be prevented by administration to the fetus, just before birth, of beta- or beta 1-adrenoreceptor antagonists. Therefore the rise of kidney energy charge following parturition appears to be under hormonal control. Glucagon had no effect on kidney adenine nucleotide content. It is strongly suggested that the catecholamines released at the time of parturition are the triggering factor of this evolution.

The ventral and posterior expression of the zebrafish homeobox gene eve1 is perturbed in dorsalized and mutant embryos.

We have identified and characterized zebrafish eve1, a novel member of the Drosophila even-skipped (eve) gene family. eve1 RNAs are expressed initially in late blastulae with a peak during the gastrula stage, at which time expression is confined to ventral and lateral cells of the marginal zone of the zebrafish embryo. Later, eve1 transcripts are located in the most posterior part of the extending tail tip. We show that LiCl, known to dorsalize Xenopus embryos, has the same effect in zebrafish, resulting in embryos with exaggerated dorsoanterior structures. In LiCl-treated embryos, eve1 transcripts are completely absent. eve1 is therefore a marker of ventral and posterior cells. In the light of its ventroposterior expression domain, the localization of eve1 transcripts was analysed in spadetail (spt) and no tail (ntl), two mutants with abnormal caudal development. In sptb140 homozygous mutants, there is an accumulation of cells in the tail region, resulting from inadequate migratory behaviour of precursors to the trunk somites. These cells, in their abnormal environment, express eve1, emphasizing the correlation between ventroposterior position and eve1 expression. In homozygous mutant embryos for the gene ntl (the homologue of mouse Brachyury, originally called Zf-T), posterior structures are missing (M. E. Halpern, C. B. Kimmel, R. K. Ho and C. Walker, 1993; Cell In press). While mutant and wild-type embryos do not differ in their eve1 transcript distribution during gastrulation, eve1 expression is absent in the caudal region of mutant ntl embryos during early somitogenesis, indicating a requirement for ntl in the maintenance of eve1 expression during tail extension. Our findings suggest that eve1 expression is correlated with a ventral and posterior cell fate, and provide first insights into its regulation.

Perinatal changes in adenine nucleotide content of developing rat kidney.

Adenine nucleotides have been measured in fetal and newborn kidneys of rat using the luciferine-luciferase method. In fetuses, between days 18 and 21 of gestation there is a drop of the relative amount of ATP in the renal nucleotide pool. Consequently, the kidneys of 21 days-old fetuses have lowered ATP/ADP ratio (3.6) and energy charge (0.80) compared with values found on day 18 (6.9 and 0.91, respectively); this relative energy deficit is heightened in progesterone induced postmaturity. One hour after delivery whether the gestational stage is 21, 22 or 23 days, there is a rise in ATP and a decrease in AMP content which restore a high energy level in kidney of the newborn and a 30% increase in the total adenine nucleotide pool.

Mineralization in vitro of matrix formed by osteoblasts isolated by collagenase digestion.

Osteoblasts from calvaria of 18-day-old fetal Sprague-Dawley rats were isolated using a dissecting procedure followed by collagenase digestion. Freshly isolated or previously frozen cells were cultured for up to 4 weeks in a Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 micrograms/ml ascorbic acid, with or without 10 mM beta-glycerophosphate. Most of the cells were alkaline phosphatase positive throughout the culture period and expressed a type-I collagen as assessed by immunofluorescence. Cells cultured in the presence of beta-glycerophosphate formed a matrix with type-I collagen in 7 days. The matrix underwent mineralization in less than 2 weeks. In the absence of beta-glycerophosphate, only the formation of a nonmineralized matrix was observed. Electron-microscopic examination revealed osteoblasts embedded in a dense network of collagen fibers, with a well-defined mineralization process in association with matrix vesicles. Scanning electron-microscopy showed that the matrix composed of layers of irregularly shaped spread cells with smooth surfaces trapped in a fiber matrix. No mineralization process was observed when rat skin fibroblasts were cultured under similar conditions. These data demonstrate the ability of enzymatically isolated osteoblasts cultured in the presence of beta-glycerophosphate to form bone in vitro, and that this process is similar to bone formation in vivo.

Behavior of fetal rat chondrocytes cultured on a bioactive glass-ceramic.

We examined the behavior of fetal rat chondrocytes cultured on a bioactive glass-ceramic containing apatite and wollastonite (A.W.G.C.). Biomaterial surface topography and profiles were evaluated by bidimensional profilometry and revealed a rough surface for the glass-ceramic compared to the plastic coverslips used as controls. Chondrocyte attachment was evaluated by measuring the number of attached cells after one day of culture and by morphological observations. Chondrocytes attached in great numbers to the material surface by means of focal contacts containing vinculin and beta1-integrin. Fluorescent labeling of actin and vimentin revealed a poor spreading of chondrocytes on the bioactive glass-ceramic compared to the plastic coverslips, where the cells appeared to adhere intimately to the surface and exhibited polygonal arrays of stress fibers. During the following days of culture, chondrocytes proliferated, colonized the surface of the material, and, finally, on day 10, formed nodular structures composed of round cells separated by a dense extracellular matrix. Furthermore, these clusters of round cells were positive for type II collagen and chondroitin sulfate, both hard markers of the chondrocyte phenotype. In addition, protein synthesis, alkaline phosphatase activity, and proteoglycan production were found to increase gradually during the culture period with a pattern similar to that observed on control cultures. These results demonstrate that the bioactive glass-ceramic tested in this study appears to be a suitable substrate for in vitro chondrocyte attachment, differentiation, and matrix production.

Cartilage formation by fetal rat chondrocytes cultured in alginate beads: a proposed model for investigating tissue-biomaterial interactions.

Chondrocytes from 21-day-old rat fetal nasal cartilage were cultured in alginate beads for up to 20 days. It was found that chondrocytes retained their spherical shape and typical chondrocytic appearance. During the culture time, chondrocytes underwent differentiation, as demonstrated by the alkaline phosphatase-specific activity and rate of proteoglycan synthesis. Morphological data confirmed chondrocyte differentiation with the appearance of hypertrophic chondrocytes scattered in the alginate gel and a dense extracellular matrix containing filamentous structures and matrix vesicles. In addition, Northern blot analysis performed on day 8 of culture showed that chondrocytes cultured in alginate beads expressed type II collagen mRNA. The alginate bead method also appeared to be suitable for testing biomaterials, and the ready dissolution of the alginate beads by chelating agents provided a simple means for the rapid recovery of encapsulated chondrocytes. Powdered glass-ceramic particles entrapped in the alginate gel were colonized by chondrocytes, which then proliferated and formed a tissue similar to a true calcified cartilaginous structure. These results indicate that the alginate system represents a relevant model for studies of chondrogenesis and endochondral ossification. Furthermore, the encapsulation method could prove useful for studies of tissue-biomaterial interactions in an in vitro environment which more closely mirrors the cartilage matrix than other culture methods.

Widespread expression of the eve1 gene in zebrafish embryos affects the anterior-posterior axis pattern.

The zygotic expression of the eve1 gene is restricted to the ventral and lateral cells of the marginal zone. At later stages, the mRNAs are localized in the most posterior part of the extending tail tip. An eve1 clone (pcZf14), containing a poly-A tail, has been isolated. In order to address eve1 gene function, pcZf14 transcript injections into zebrafish embryos have been performed. The injection into uncleaved eggs of a synthetic eve1 mRNA (12 pg), which encodes a protein of approximately 28 kd, produces embryos with anterior-posterior (A-P) axis defects and the formation of additional axial structures. The first category of 24 h phenotypes (87%) mainly displays a gradual decrease in anterior structures. This is comparable to previous phenotypes observed following Xhox3 messenger injection either in Xenopus or in zebrafish that have been classified according to the index of axis deficiency (zf-IAD). These phenotypes result in anomalies of the development of the neural keel, from microphthalmia to acephaly. The second category (13%) corresponds to the phenotypes described above together with truncal or caudal supernumerary structures. Additional truncal structures are the most prominent of these duplicated phenotypes, displaying a "zipper" shape of axial structures including neural keels and notochords. Caudal duplication presents no evident axis supernumerary structures. The observation of these phenotypes suggests an important role for the eve1 gene in mesodermal cell specification and in the development of the posterior region, and more particularly of the most posterior tail tip where endogenous eve1 messengers are found.

[Ventral and posterior expression of the homeo box gene eve1 in zebrafish (Brachydanio rerio) is repressed in dorsalized embryos]. / L'expression ventrale et postérieure du gène à homéoboîte eve1 de poisson-zèbre (Brachydanio rerio) est réprimée dans les embryons dorsalisés.

We have identified and characterized the zebrafish eve1 homeo box gene, a member of the Drosophila even-skipped (eve) gene family. eve1 is expressed in the most posterior part of the tail bud during somitogenesis. During gastrulation, transcripts are confined to ventral and lateral cells of the marginal zone of the embryo. We show that LiCl, known to dorsalize Xenopus embryos, has the same effect in zebrafish embryos. In LiCl-treated embryos, eve1 transcripts are completely absent, suggesting that eve1 marks the ventral specification of mesoderm in zebrafish gastrulae.