Inactivation of the zebrafish homologue of Chx10 by antisense oligonucleotides causes eye malformations similar to the ocular retardation phenotype.

We report the cloning of a zebrafish paired-type homeobox gene, Alx, closely related to the murine Chx10 and the gold fish Vsx-I homeodomain proteins. Alx is first expressed at about 12 h post-fertilization (hpf) when optic vesicles appear. Its expression is restricted to the early retinal neuroepithelium, whereas no signal can be detected in the optic placode. Later, Alx expression follows the differentiation of the neural retina. Inhibition experiments with antisense oligonucleotides resulted in specific eye malformations which are reminiscent of the phenotype of ocular retardation (or) mice, caused by a spontaneous Chx10 mutation. The expression of other developmentally relevant genes such as pax(zf-a), pax(zf-b) and krx-20 was not affected in the antisense treated embryos.

Cloning and expression pattern of a zebrafish homolog of forkhead activin signal transducer (FAST), a transcription factor mediating Nodal-related signals.

Forkhead activin signal transducer (FAST) is a member of the winged-helix family of DNA-binding proteins that has been implicated in mesoderm induction and left-right axis specification during embryonic development in Xenopus and mouse. We have cloned and characterized a zebrafish FAST homolog. Zebrafish fast is expressed maternally and zygotically. Transcripts start regionalizing and decline in level during gastrulation. During somitogenesis, fast is expressed bilaterally in the lateral plate mesoderm, like its mouse homolog. In addition, zebrafish fast is also expressed bilaterally in the dorsal diencephalon, where the nodal-related cyclops gene is only expressed on the left side. It remains to be demonstrated whether FAST expression in the brain can mediate Nodal-induced asymmetric development.

Retinoic acid induces stage-specific antero-posterior transformation of rostral central nervous system.

We report a time-course analysis of the effect of retinoic acid (RA) on the development of the mouse central nervous system (CNS) from the beginning of gastrulation throughout induction and patterning of the neural tube. RA administration induces three different, stage-specific alterations of brain development, indicating perturbation of different morphogenetic steps during the establishment of a neural pattern. In particular, treatment at mid-late streak stage (7.2-7.4 days post coitum (d.p.c.)) results in early repression of Otx2 expression in the posterior neuroectoderm of the head fold and in the ventral mid line, including the prechordal plate and the rostralmost endoderm, followed by loss of forebrain morphological and molecular identities, as revealed by analysis of the expression of regionally-restricted brain genes (Otx2, Otx1, Emx2, Emx1 and Dlx1). In these embryos, reduction of the Otx2 expression domain correlates with hindbrain expansion marked by rostral extension of the Hoxb-1 expression domain. Our analysis indicates that RA interferes with the correct definition of both planar and vertical morphogenetic signals at specific developmental stages by affecting gene expression in the regions which are likely either to produce or to respond to these signals. We suggest that retinoids may contribute to early definition of head from trunk structures by selecting different sets of regulatory genes.

Expression patterns of zebrafish sox11A, sox11B and sox21.

We have cloned three sox genes in zebrafish (Danio rerio), one related to human and chicken SOX21, and two related to mammalian and chicken Sox-11. Zebrafish sox21, sox11A and sox11B transcripts are accumulated in the egg, are present in all cells until gastrulation and become restricted later to the developing central nervous system (CNS); expression in adults is undetectable. sox21 is expressed in the forebrain, midbrain and hindbrain, but maximally at the midbrain-hindbrain junction; sox11A,B have a widespread and dynamic expression in the CNS, but in contrast to sox21 are absent at the midbrain-hindbrain boundary.

Expression pattern of two otx genes suggests a role in specifying anterior body structures in zebrafish.

We isolated two zebrafish sequences containing a homeobox related to orthodenticle (otd), a gene expressed in the developing head of Drosophila. One of these is clearly homologous to Otx1, a homeobox gene previously reported to be expressed in the developing rostral brain of the mouse. We termed this zebrafish gene otx1. The second gene is not as closely related to Otx1 and is equally divergent from Otx2, a second homeobox gene expressed in the developing rostral brain of the mouse. We termed it otx3, even if a corresponding murine Otx3 gene has not been reported yet. Both genes are expressed in early-gastrula zebrafish embryos in the involuting presumptive anterior mesendoderm. With the extension of the body axis, the expression domain of both genes extends to neuroectodermal regions fated to become fore- and mid-brain. From this stage the expression domains of the two genes differ slightly from each other but both cover the rostral brain with a sharp posterior boundary coinciding with that between midbrain and hind-brain. This late expression closely corresponds to that of the murine Otx1 gene, whereas the earliest expression of both zebrafish otx genes is different from that of Otx1 and reminiscent of that of Otx2 in the mouse. In this light, the zebrafish otx1 and otx3 genes appear to share some expression features of both murine Otx1 and Otx2. It will be of considerable interest to study the specific role of the various genes of the otx family in the development of the zebrafish brain regions. The peculiar spatio-temporal pattern of these genes during early zebrafish gastrulation suggests a role of this gene family in interactions between anterior mesendoderm and neuroectoderm.

Modulation of microtubule shape in vitro by high molecular weight microtubule associated proteins MAP1A, MAP1B, and MAP2.

The effect of microtubule associated proteins on microtubule shape has been investigated in reconstitution experiments using purified tubulin and purified MAP1A, MAP1B, and MAP2. Microtubules assembled in the presence of these MAPs were fixed with 0.1% glutaraldehyde and, after negative staining, were examined by electron microscopy. The results show that MAP1A microtubules were generally short and "straight' while those assembled with MAP1B were longer and "bendy'. MAP2 microtubules showed both types of morphologies even though straight microtubules were more abundant. These data suggest that MAPs may modulate not only microtubule dynamics but also microtubule shape which may be important in their spatial distribution and/or role in specific neuronal areas.

Oocyte development and egg envelope formation in Oreochromis niloticus, a mouth-brooding cichlid fish.

The development of the oocyte and of its associated follicle cells in the Nile tilapia, Oreochromis niloticus, has been examined by optical and transmission electron microscopy. During oocyte development the female gamete of Orochromis niloticus increases in size because of the accumulation of yolk in its cytoplasm. As the accumulation of yolk proceeds, the organization of cortex of the oocyte becomes very complex; all of the cytoplasmic organelles and several populations of vesicles can be found. On the other hand follicle cells also undergo a series of modifications: they first become cuboidal then cylindrical and their cytoplasm become densely populated with organelles. The mature egg of Oreochromis niloticus is surrounded by a thin acellular envelope (chorion) assembled during oocyte development. Biochemical analysis of isolated and purified chorions from mature females was also performed. SDS-PAGE under reducing conditions showed a reproducible pattern of three major polypeptides (121, 66 and 50 kD), most of which being glycosylated. The pattern of synthesis and assembly of the egg envelope in Oreochromis niloticus, a mouth-brooding cichlid fish, is also discussed.

Cornelia de Lange Syndrome: NIPBL haploinsufficiency downregulates canonical Wnt pathway in zebrafish embryos and patients fibroblasts.

Cornelia de Lange Syndrome is a severe genetic disorder characterized by malformations affecting multiple systems, with a common feature of severe mental retardation. Genetic variants within four genes (NIPBL (Nipped-B-like), SMC1A, SMC3, and HDAC8) are believed to be responsible for the majority of cases; all these genes encode proteins that are part of the 'cohesin complex'. Cohesins exhibit two temporally separated major roles in cells: one controlling the cell cycle and the other involved in regulating the gene expression. The present study focuses on the role of the zebrafish nipblb paralog during neural development, examining its expression in the central nervous system, and analyzing the consequences of nipblb loss of function. Neural development was impaired by the knockdown of nipblb in zebrafish. nipblb-loss-of-function embryos presented with increased apoptosis in the developing neural tissues, downregulation of canonical Wnt pathway genes, and subsequent decreased Cyclin D1 (Ccnd1) levels. Importantly, the same pattern of canonical WNT pathway and CCND1 downregulation was observed in NIPBL-mutated patient-specific fibroblasts. Finally, chemical activation of the pathway in nipblb-loss-of-function embryos rescued the adverse phenotype and restored the physiological levels of cell death.

Intermediate filament proteins immunologically related to cytokeratins in the oocyte of the fish Cyprinus carpio.

We have used monoclonal antibodies specific for different sets of human cytokeratins and the anti-IFA (Intermediate Filament Antigen) antibody to investigate the expression of intermediate filament proteins in the mature oocyte of the teleost Cyprinus carpio. Several polypeptides have been identified, showing molecular weights ranging from 43 to 65 kDa. Two-dimensional analysis of the immunoreactive species revealed the presence of at least six major protein spots and a series of minor components, grouped in quite a narrow pI range from 5.52 to 6.28. The general complexity of the carp oocyte cytokeratin-related cytoskeleton appears to be higher than those described for oocytes of other vertebrate species.

Fine structure and cytochemistry of the morphogenesis of round-headed human sperm.

Ejaculates and testicular biopsies of two infertile men were examined at ultrastructural and cytochemical levels. The two patients presented spermiograms in which all the spermatozoa had globular heads. Wide Golgi areas and large masses of annulatae lamellae were evident during spermatogenesis. Abnormal acrosomal vesicles were evident during early spermatid stage. Among the late spermatids, there was a small group characterized by a fibrous sheath showing considerable malformation, and "spindle shaped body." In Sertoli cells, detached acrosomes undergoing degeneration were noted. Low TPPase activity was found in Golgi complex and in abnormal acrosomal vesicles of early spermatids. As regards acid phosphatase, was localized in spermatocytes and in early spermatids, but not in late spermatids and mature spermatozoa. Leydig cells had high phosphatase activity.

Identification and characterization of the major components of the Oncorhynchus mykiss egg chorion.

The extracellular coat surrounding the fish egg, commonly called the chorion, is a primary envelope that confers biochemical and morphological identity typical of the species. Purified chorions can be easily isolated from either oocytes or ovulated eggs. The aim of this work was to analyze the macromolecular composition of the various chorion components in Oncorhynchus mykiss (Salmonids). SDS-PAGE analysis of purified chorion showed a reproducible pattern of four major components (129, 62, 54, and 47 kD), representing about 80% of total chorion proteins. The 129 and 47 kD polypeptides were periodic-acid Schiff (PAS) and concanavalin A positive. After chemical and enzymatic deglycosylation treatments only the 129 and 47 kD components proved to be glycosylated and to belong to the "asparagine-linked" glycoprotein family. Furthermore, peptide mapping performed on isolated polypeptides showed comigrating fragments on SDS-PAGE. These results suggest that the four main chorion polypeptides might share common structural features.

Identification and spatial distribution of the mRNA encoding an egg envelope component of the Cyprinid zebrafish, Danio rerio, homologous to the mammalian ZP3 (ZPC).

Using degenerate reverse transcription polymerase chain reaction (RT-PCR) techniques we have isolated a cDNA encoding a putative component of the zebrafish Danio rerio egg chorion, homologous to the mammalian ZP3 (ZPC). The predicted protein (zfZPC) has a calculated molecular mass of 58.4 kDa and contains a signal peptide (located in the N-terminal region) composed of 11 hydrophobic amino acid residues followed by a signal peptide cleavage site. The zfZPC contains the ZP domain, a characteristic amino acid sequence shared by all ZP proteins of the mammalian zona pellucida and of both amphibian and bird egg envelope components. The zfZPC also exhibits certain unique features including five N-terminal Q-rich tandem repeats presumably involved in the hardening of the chorion after the fertilization of the egg and a long C-terminal tail containing two potential sites of N-linked type glycosylation. RT-PCR and in situ hybridization revealed a restricted pattern of tissue distribution: the gene encoding zfZPC is transcribed only in the growing oocyte of sexually mature female fish.

Structure and macromolecular composition of the zebrafish egg chorion.

The chorion is the acellular envelope surrounding mature eggs of teleostean fish. The macromolecular composition of the zebrafish (Danio rerio) egg chorion, organised as a three-layered structure, has been analysed. SDS-PAGE analysis, under reducing conditions, of isolated and purified chorions revealed a reproducible pattern of four major polypeptides (116, 97, 50 and 43 kDa) and several minor bands. Lectin binding assays showed that both the 116 kDa and 50 kDa proteins were recognised by concanavalin agglutinin (Con A), Galanthus nivalis agglutinin (GNA), Sambucus nigra bark agglutinin (SNA) and Ricinus communis agglutinin (RCA 120), suggesting that these polypeptides are N-linked glycoproteins. By contrast, neither the 97 kDa nor the 43 kDa polypeptides were stained by these lectins, indicating that these polypeptides are not glycosylated. Amino acid analysis also showed significant differences in the average content of some amino acids, for example serine and proline, when compared with previous reports.

Ultrastructure of the tubular wall of the prepuberal cryptorchid human testis.

The tubular wall in the human prepuberal testis of cryptorchid patients has been examined by ultrastructural and histochemical analysis. When compared with the normolocated testes, a disorganization of the tubular wall due to cryptorchidism is apparent. Alterations regarding single elements of the tubular wall are described and a positive correlation seems to exist between the age and the amount of the damage. A possible relation between morphological changes and hormone therapy is discussed.

Nonelectrolyte fluxes across gastric mucosa in relation to gastric stimulation. Is gastric juice secreted by osmosis or exocytosis?

The effects of histamine and thiocyanate, added to the serosal bathing solution, on unidirectional fluxes of some nonelectrolytes (thiourea, methylated thiourea derivates, mannitol), and on H+, pepsinogen and mucous secretion were investigated in frog (Rana esculenta) fundic gastric mucosa. Histamine (10(-4) M) increases significantly the outfluxes (serosa to mucosa fluxes) of only thiourea and its derivates (but not mannitol) and the stimulation is the greater the more lipidsoluble the nonelectrolyte is. Influxes (mucosa to serosa fluxes) of the same molecules are not affected. In parallel histamine stimulates H+-secretion but does not modify pepsinogen and mucous secretion. SCN- (10(-2) M) inhibits the histamine effect on thiourea outfluxes and on H+-secretion, while pepsinogen and mucous secretion are not affected. Colchicine (10(-4) M) pretreatment inhibits the histamine effect on outfluxes and H+-secretion. It is concluded that: (1) histamine induces a secretion of nonelectrolytes towards the lumen; (2) such secretion is correlated with the hormone-induced secretion of HCl and fluid; (3) this process is mediated by an exocytotic mechanism.

Functional significance of intermediate filament meshwork in annelid helical muscles.

A desmin-like protein of mol wt 54 kDa was identified in the body wall muscles of some Polychaeta, Oligochaeta, and Hirudinea utilizing SDS-PAGE followed by blot and screening with a vertebrate anti-desmin antibody. The pattern in immunofluorescence is compared to electron micrographs where several bundles of filamentous structures are clearly identifiable. These bundles are unevenly arranged in round or flattened circomyarian fibers and sometimes clearly connect Z elements with hemidesmosomes. The mechanism of intermediate filaments as a functional integration in muscle fibers is analyzed and a possible role as a block to superelongation typical of helical muscles is discussed.

Immunocytochemical localization of actin in the sperm head of Talpa europaea (Insectivora).

Actin in the sperm head of Talpa europaea was observed by immunofluorescence and immunoelectron microscopy. The indirect immunofluorescence technique, using both anti-actin and DNase anti-DNase methods, showed a shining fluorescent band around the sperm head in some spermatozoa, whereas in others the fluorescence was found in the postacrosomal region. Since no labeling was detected in sperms treated with NBD-phallacidin, it is likely that mature mole sperms contain G-actin but not F-actin. The results of electron microscopy indicated the deposition of the anti-actin antibodies in two places in mole spermatozoa: the postacrosomal region and the nuclear segment of the acrosome. In the first case, the actin was localized in the space between the outer surface of the postacrosomal sheath and the plasma membrane; in the second one, the actin was localized in the space between the outer acrosomal membrane and the plasma membrane. The significance of the presence of actin and its role(s) during fertilization are discussed.

Localization and distribution of actin in mammalian sperm heads.

Actin was identified in boar and mole spermatozoa by utilizing indirect immunofluorescence, immunoelectron microscopy, and SDS-PAGE, followed by blot and screening with an anti-actin monoclonal antibody. Actin was detected in two places in the sperm head: the equatorial segment of the acrosome and the postacrosomal region. The protein was present in a nonfilamentous form and was localized under the plasma membrane. A small amount of actin was also detected in the sperm tail. The function of actin in the sperm head is discussed.

Seasonal fluctuations of selected physiological characteristics of elite alpine skiers.

The effects of heavy resistance training and jumping exercise were examined during the 1989-1990 season in 12 international level alpine skiers. The athletes were tested before, during, immediately after training and during the period off training (June, July, October 1989, April 1990). Their mechanical behaviour was investigated using firstly squat jumps performed without (SJ) or with low extra loads (20 kg, SJ20kg) and high extra loads (equivalent to body mass on the shoulders, SJbm) and secondly 15-30 s continuous jumping. These tests allowed the assessment of explosive dynamic strength production (SJ and SJ20kg), slow dynamic strength (SJbm) and maximal mechanical power (continuous jumping). The training adopted resulted in specific changes in neuromuscular performance; in fact all the variables studied showed a significant improvement (P < 0.01) from the beginning compared to the end of training. The range of improvement was between 55.4% (SJbm) and 12.5% (average power during 15-s continuous jumping). The enhancement of SJ had become significant by July. Surprisingly, even when no strength or jumping training was performed during the competition period (November-April), no deterioration in the neuromuscular performance was observed, there being no significant difference between the test values obtained in October 1989 and April 1990. It was concluded that the demanding competition programme of alpine skiers may provide a training stimulus adequate to maintain the neuromuscular improvement induced by training throughout the competition season.

Heart rate profiles and energy cost of locomotion during cross-country skiing races.

The purpose of this study was to compare heart rate responses and speed in two cross-country skiing races, which were run by seven male and seven female subjects by using classic and free style. Heart rates and skiing velocities were analyzed over flat, uphill and downhill sections, which were run from one to three times. Heart rates were higher in uphill sections than in flat sections; a steady-state heart rate was never reached in the downhill section. When the same uphill section was repeated, the heart rate tended to increase but the speed to decrease. Oxygen uptake (VO2) was calculated from heart rate:VO2 ratio, measured during uphill walking with the aid of poles. The mean (SD) energy cost of locomotion (i.e., the ratio between net VO2 and speed) was 162.1 (9.4) ml.km(-1).kg(-1) and 147.7 (7.1) ml.km(-1).kg(-1) when male subjects ran the flat section after first downhill by using classic and free style, respectively. Females had lower values for VO2 and speed, but similar energy costs. In general, the variability of the energy cost of locomotion in skiers of a similar competitive level is of the same order as that found in uphill walking on a treadmill.