pigk Mutation underlies macho behavior and affects Rohon-Beard cell excitability.

The study of touch-evoked behavior allows investigation of both the cells and circuits that generate a response to tactile stimulation. We investigate a touch-insensitive zebrafish mutant, macho (maco), previously shown to have reduced sodium current amplitude and lack of action potential firing in sensory neurons. In the genomes of mutant but not wild-type embryos, we identify a mutation in the pigk gene. The encoded protein, PigK, functions in attachment of glycophosphatidylinositol anchors to precursor proteins. In wild-type embryos, pigk mRNA is present at times when mutant embryos display behavioral phenotypes. Consistent with the predicted loss of function induced by the mutation, knock-down of PigK phenocopies maco touch insensitivity and leads to reduced sodium current (INa) amplitudes in sensory neurons. We further test whether the genetic defect in pigk underlies the maco phenotype by overexpressing wild-type pigk in mutant embryos. We find that ubiquitous expression of wild-type pigk rescues the touch response in maco mutants. In addition, for maco mutants, expression of wild-type pigk restricted to sensory neurons rescues sodium current amplitudes and action potential firing in sensory neurons. However, expression of wild-type pigk limited to sensory cells of mutant embryos does not allow rescue of the behavioral touch response. Our results demonstrate an essential role for pigk in generation of the touch response beyond that required for maintenance of proper INa density and action potential firing in sensory neurons.

Acoustic cavitation, bubble dynamics and sonoluminescence.

Basic facts on the dynamics of bubbles in water are presented. Measurements on the free and forced radial oscillations of single spherical bubbles and their acoustic (shock waves) and optic (luminescence) emissions are given in photographic series and diagrams. Bubble cloud patterns and their dynamics and light emission in standing acoustic fields are discussed.

The zebrafish neckless mutation reveals a requirement for raldh2 in mesodermal signals that pattern the hindbrain.

We describe a new zebrafish mutation, neckless, and present evidence that it inactivates retinaldehyde dehydrogenase type 2, an enzyme involved in retinoic acid biosynthesis. neckless embryos are characterised by a truncation of the anteroposterior axis anterior to the somites, defects in midline mesendodermal tissues and absence of pectoral fins. At a similar anteroposterior level within the nervous system, expression of the retinoic acid receptor a and hoxb4 genes is delayed and significantly reduced. Consistent with a primary defect in retinoic acid signalling, some of these defects in neckless mutants can be rescued by application of exogenous retinoic acid. We use mosaic analysis to show that the reduction in hoxb4 expression in the nervous system is a non-cell autonomous effect, reflecting a requirement for retinoic acid signalling from adjacent paraxial mesoderm. Together, our results demonstrate a conserved role for retinaldehyde dehydrogenase type 2 in patterning the posterior cranial mesoderm of the vertebrate embryo and provide definitive evidence for an involvement of endogenous retinoic acid in signalling between the paraxial mesoderm and neural tube.

Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates.

Waardenburg-Shah syndrome combines the reduced enteric nervous system characteristic of Hirschsprung's disease with reduced pigment cell number, although the cell biological basis of the disease is unclear. We have analysed a zebrafish Waardenburg-Shah syndrome model. We show that the colourless gene encodes a sox10 homologue, identify sox10 lesions in mutant alleles and rescue the mutant phenotype by ectopic sox10 expression. Using iontophoretic labelling of neural crest cells, we demonstrate that colourless mutant neural crest cells form ectomesenchymal fates. By contrast, neural crest cells which in wild types form non-ectomesenchymal fates generally fail to migrate and do not overtly differentiate. These cells die by apoptosis between 35 and 45 hours post fertilisation. We provide evidence that melanophore defects in colourless mutants can be largely explained by disruption of nacre/mitf expression. We propose that all defects of affected crest derivatives are consistent with a primary role for colourless/sox10 in specification of non-ectomesenchymal crest derivatives. This suggests a novel mechanism for the aetiology of Waardenburg-Shah syndrome in which affected neural crest derivatives fail to be generated from the neural crest.

A mutation in the Gsk3-binding domain of zebrafish Masterblind/Axin1 leads to a fate transformation of telencephalon and eyes to diencephalon.

Zebrafish embryos homozygous for the masterblind (mbl) mutation exhibit a striking phenotype in which the eyes and telencephalon are reduced or absent and diencephalic fates expand to the front of the brain. Here we show that mbl(-/-) embryos carry an amino-acid change at a conserved site in the Wnt pathway scaffolding protein, Axin1. The amino-acid substitution present in the mbl allele abolishes the binding of Axin to Gsk3 and affects Tcf-dependent transcription. Therefore, Gsk3 activity may be decreased in mbl(-/-) embryos and in support of this possibility, overexpression of either wild-type Axin1 or Gsk3beta can restore eye and telencephalic fates to mbl(-/-) embryos. Our data reveal a crucial role for Axin1-dependent inhibition of the Wnt pathway in the early regional subdivision of the anterior neural plate into telencephalic, diencephalic, and eye-forming territories.

The type I serine/threonine kinase receptor Alk8/Lost-a-fin is required for Bmp2b/7 signal transduction during dorsoventral patterning of the zebrafish embryo.

Ventral specification of mesoderm and ectoderm depends on signaling by members of the bone morphogenetic protein (Bmp) family. Bmp signals are transmitted by a complex of type I and type II serine/threonine kinase transmembrane receptors. Here, we show that Alk8, a novel member of the Alk1 subgroup of type I receptors, is disrupted in zebrafish lost-a-fin (laf) mutants. Two alk8/laf null alleles are described. In laf(tm110), a conserved extracellular cysteine residue is replaced by an arginine, while in laf(m100), Alk8 is prematurely terminated directly after the transmembrane domain. The zygotic effect of both mutations leads to dorsalization of intermediate strength. A much stronger dorsalization, similar to that of bmp2b/swirl and bmp7/snailhouse mutants, however, is obtained by inhibiting both maternally and zygotically supplied alk8 gene products with morpholino antisense oligonucleotides. The phenotype of laf mutants and alk8 morphants can be rescued by injected mRNA encoding Alk8 or the Bmp-regulated transcription factor Smad5, but not by mRNA encoding Bmp2b or Bmp7. Conversely, injected mRNA encoding a constitutively active version of Alk8 can rescue the strong dorsalization of bmp2b/swirl and bmp7/snailhouse mutants, whereas smad5/somitabun mutant embryos do not respond. Altogether, the data suggest that Alk8 acts as a Bmp2b/7 receptor upstream of Smad5.

Conservation of Mhc class III region synteny between zebrafish and human as determined by radiation hybrid mapping.

In the HLA, H2, and other mammalian MHC:, the class I and II loci are separated by the so-called class III region comprised of approximately 60 genes that are functionally and evolutionarily unrelated to the class I/II genes. To explore the origin of this island of unrelated loci in the middle of the MHC: 19 homologues of HLA class III genes, we identified 19 homologues of HLA class III genes as well as 21 additional non-class I/II HLA homologues in the zebrafish and mapped them by testing a panel of 94 zebrafish-hamster radiation hybrid cell lines. Six of the HLA class III and eight of the flanking homologues were found to be linked to the zebrafish class I (but not class II) loci in linkage group 19. The remaining homologous loci were found to be scattered over 14 zebrafish linkage groups. The linkage group 19 contains at least 25 genes (not counting the class I loci) that are also syntenic on human chromosome 6. This gene assembly presumably represents the pre-MHC: that existed before the class I/II genes arose. The pre-MHC: may not have contained the complement and other class III genes involved in immune response.

Mariner is defective in myosin VIIA: a zebrafish model for human hereditary deafness.

The zebrafish (Danio rerio) possesses two mechanosensory organs believed to be homologous to each other: the inner ear, which is responsible for the senses of audition and equilibrium, and the lateral line organ, which is involved in the detection of water movements. Eight zebrafish circler or auditory/vestibular mutants appear to have defects specific to sensory hair cell function. The circler genes may therefore encode components of the mechanotransduction apparatus and/or be the orthologous counterparts of the genes underlying human hereditary deafness. In this report, we show that the phenotype of the circler mutant, mariner, is due to mutations in the gene encoding Myosin VIIA, an unconventional myosin which is expressed in sensory hair cells and is responsible for various types of hearing disorder in humans, namely Usher 1B syndrome, DFNB2 and DFNA11. Our analysis of the fine structure of hair bundles in the mariner mutants suggests that a missense mutation within the C-terminal FERM domain of the tail of Myosin VIIA has the potential to dissociate the two different functions of the protein in hair bundle integrity and apical endocytosis. Notably, mariner sensory hair cells display morphological and functional defects that are similar to those present in mouse shaker-1 hair cells which are defective in Myosin VIIA. Thus, this study demonstrates the striking conservation of the function of Myosin VIIA throughout vertebrate evolution and establishes mariner as the first fish model for human hereditary deafness.

Control of her1 expression during zebrafish somitogenesis by a delta-dependent oscillator and an independent wave-front activity.

Somitogenesis has been linked both to a molecular clock that controls the oscillation of gene expression in the presomitic mesoderm (PSM) and to Notch pathway signaling. The oscillator, or clock, is thought to create a prepattern of stripes of gene expression that regulates the activity of the Notch pathway that subsequently directs somite border formation. Here, we report that the zebrafish gene after eight (aei) that is required for both somitogenesis and neurogenesis encodes the Notch ligand DeltaD. Additional analysis revealed that stripes of her1 expression oscillate within the PSM and that aei/DeltaD signaling is required for this oscillation. aei/DeltaD expression does not oscillate, indicating that the activity of the Notch pathway upstream of her1 may function within the oscillator itself. Moreover, we found that her1 stripes are expressed in the anlage of consecutive somites, indicating that its expression pattern is not pair-rule. Analysis of her1 expression in aei/DeltaD, fused somites (fss), and aei;fss embryos uncovered a wave-front activity that is capable of continually inducing her1 expression de novo in the anterior PSM in the absence of the oscillation of her1. The wave-front activity, in reference to the clock and wave-front model, is defined as such because it interacts with the oscillator-derived pattern in the anterior PSM and is required for somite morphogenesis. This wave-front activity is blocked in embryos mutant for fss but not aei/DeltaD. Thus, our analysis indicates that the smooth sequence of formation, refinement, and fading of her1 stripes in the PSM is governed by two separate activities.

Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation.

Vertebrate gastrulation involves the specification and coordinated movement of large populations of cells that give rise to the ectodermal, mesodermal and endodermal germ layers. Although many of the genes involved in the specification of cell identity during this process have been identified, little is known of the genes that coordinate cell movement. Here we show that the zebrafish silberblick (slb) locus encodes Wnt11 and that Slb/Wnt11 activity is required for cells to undergo correct convergent extension movements during gastrulation. In the absence of Slb/Wnt11 function, abnormal extension of axial tissue results in cyclopia and other midline defects in the head. The requirement for Slb/Wnt11 is cell non-autonomous, and our results indicate that the correct extension of axial tissue is at least partly dependent on medio-lateral cell intercalation in paraxial tissue. We also show that the slb phenotype is rescued by a truncated form of Dishevelled that does not signal through the canonical Wnt pathway, suggesting that, as in flies, Wnt signalling might mediate morphogenetic events through a divergent signal transduction cascade. Our results provide genetic and experimental evidence that Wnt activity in lateral tissues has a crucial role in driving the convergent extension movements underlying vertebrate gastrulation.

A radiation hybrid map of the zebrafish genome.

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

Maternal and embryonic expression of zebrafish lef1.

Transcription factors of the TCF/LEF family interact with the Wnt signaling pathway to control transcription of downstream genes (Clevers, H., van de Wetering, M., 1997. TCF/LEF factor earn their wings. Trends Genet. 13, 485-489). We were interested in cloning family members which were expressed in zebrafish neural crest, because Wnt signaling modulates specification of neural crest fate (Dorsky, R.I., Moon, R.T., Raible, D.W., 1998. Control of neural crest cell fate by the Wnt signalling pathway. Nature 396, 370-373). We cloned a zebrafish homolog of lef1 and localized its chromosomal position by radiation hybrid mapping. lef1 is expressed in the neural crest as well as the tailbud and developing mesoderm, and is maternally expressed in zebrafish, unlike mouse and Xenopus homologs. In addition, we cloned two tcf3 genes and a homolog of tcf4, neither of which were strongly expressed in premigratory neural crest.

A gradient of cytoplasmic Cactus degradation establishes the nuclear localization gradient of the dorsal morphogen in Drosophila.

Dorsoventral axis formation in the Drosophila embryo is established by a signal transduction pathway that comprises the products of at least 12 maternal genes. Two of these genes, dorsal and cactus, show homology to the mammalian transcription factor NF-kappa B and its inhibitor I kappa B, respectively. As in the case for I kappa B and NF-kappa B, Cactus inhibits Dorsal by retaining it in the cytoplasm. In response to the signal produced and transmitted by the products of the other genes, Dorsal translocates to the nucleus preferentially on the ventral side of the embryo. Here, we show that Cactus forms a cytoplasmic concentration gradient inversely correlated to the nuclear translocation gradient of Dorsal. Deletions of the N-terminus and C-terminus of Cactus reveal that two modes of degradation control cactus activity: signal-induced degradation and signal-independent degradation, respectively. Genetic evidence indicates that degradation of Cactus is required, but not sufficient to translocates Dorsal completely into the nucleus.

cactus, a gene involved in dorsoventral pattern formation of Drosophila, is related to the I kappa B gene family of vertebrates.

Among the maternally active genes of Drosophila, cactus is the only one whose loss of function mutations specifically produce ventralized embryos. Its product inhibits nuclear translocation of the dorsal morphogen in the dorsal region of the embryo. Here we report the cloning of cactus and the sequencing of its maternal transcript. The identity of our clones was verified by induction of phenocopies with antisense RNA and rescue of the mutant phenotype with sense RNA. cactus is predicted to encode an acidic, cytoplasmic protein with seven ankyrin repeats. The sequence has similarity to the I kappa B proteins that inhibit the vertebrate transcription factor NF-kappa B. In analogy to results obtained with I kappa B and NF-kappa B, bacterially expressed cactus protein can inhibit DNA binding of dorsal protein in vitro.

Ethanol consumption and serum lipid profiles in Sinclair(S-1) miniature swine.

Ethanol consumption was correlated with changes in acyl group profiles of phosphatidylcholine and triacylglycerols in serum of Sinclair(S-1) miniature boars. Serum triacylglycerols in the control pigs were high in linoleate (18:2) (48%) and low in stearate (18:0 (3%). Upon feeding with 10% (w/v) ethanol ad lib for two weeks, the proportion of 18:2 in serum triacylglycerols decreased to 12-15% with a concomitant increase in 16:0, 18:0 and 18:1. Similar, but less extensive, acyl group changes were observed in the serum phosphatidylcholine. In addition, there was a decrease in the proportion of 20:3(n-6), but a biphasic change was shown in 20:4(n-6) with respect to ethanol consumption. In general, the high ethanol consumers (7.0 g/kg/day) indicated a more rapid rate of acyl group change than the low consumers (3.8 g/kg/day). Upon withdrawal of ethanol, acyl groups of triacylglycerols rapidly returned towards the control values, whereas only small changes were observed for the recovery in phospholipids. In this situation, the low-consumer group indicated a more rapid recovery than the high-consumer group. Results indicate that with the swine model, serum lipid changes can be a useful parameter for correlating biological changes upon ethanol consumption.

The route of migration of lymphocytes from blood to spleen and mesenteric lymph nodes in the pig.

Porcine peripheral blood lymphocytes were labelled with tritiated uridine in vitro and transfused into normal young pigs. Serial biopsies were taken from the spleen and mesenteric lymph nodes, and the localization of labelled lymphocytes determined on autoradiographs. Four to five hours after cell infusion samples of Peyer's patches, tonsils and small intestine were taken and the labelled lymphocytes classified by their localization in different organ compartments. In spleens most labelled cells were found in the red pulp and about 30% in the marginal zone. Within 4 to 5 h there was a constant increase in labelled lymphocytes in the compartment of the white pulp with more cells localized in the periarteriolar lymphatic sheath than in the corona. In lymph nodes about two thirds of all labelled cells were seen in the wall of high endothelial venules 15 min after cell infusion. Labelled cells then decreased in the vessel wall, but, at the same time, increased in the neighbourhood of these vessels and other parts of the paracortex. After 5 h about 10% of labelled lymphocytes had already migrated into the corona. In tonsils and Peyer's patches a comparable distribution of labelled cells was found in the organ compartments. The kinetics and the route of migration in the spleen and lymph nodes are therefore comparable to data for other species, despite the peculiar structure of pig lymph nodes and the paucity of lymphocytes in efferent lymphatics in pigs.

The effects of insulin on the turnover of glucose-6-phosphate dehydrogenase in epididymal adipose tissue of the rat.

The effects of insulin on the turnover of glucose-6-phosphate dehydrogenase in rat epididymal adipose tissue were studied by immunochemical technique in in vitro incubations. Insulin increased the relative rate of synthesis of glucose-6-phosphate dehydrogenase by two-fold in tissue obtained from normal rats. Insulin also had an effect on the rate of degradation of this enzyme. In the absence of insulin in the incubation medium the rate constant of degradation was 0.11 h-1 (half-life, 6.3 h). When insulin was added to the medium degradation of this enzyme was slowed. The new rate constant of degradation was 0.04 h-1 (half-life, 17 h). In the presence of insulin, the rate constant of degradation of total protein in adipose tissue was unchanged; therefore the effects of insulin on the degradation of glucose-6-phosphate dehydrogenase are specific to that protein and perhaps to a few other specific proteins.

Ethanol dependence in the rat: temporal changes in neuroexcitability following withdrawal.

Electrodes were chronically implanted in the dorsal hippocampus of rats. Pretreatment levels of neural excitability were assessed by determining the duration of direct hippocampal electrical stimulation sufficient to induce forelimb clonus in each rat. Following baseline measurements the rats were administered an alcohol or sucrose-containing liquid diet and 19--22 days later were withdrawan. Two postwithdrawal stimulation sessions were conducted on each animal. Separate groups of ethanol-dependent and control rats were given primary stimulation sessions at 8 h, 24 h, 72 h, or 1 week postwithdrawal. Secondary stimulation sessions were conducted 1 week or 2 weeks postwithdrawal. The results from the primary stimulations indicated that ethanol-dependent animals exhibited significant neural hyperexcitability for at least 72 h, but not 1 week, postwithdrawal. Results from the secondary stimulations demonstrated the presence of a relative neural hypoexcitability in ethanol-dependent groups as compared to controls. The pattern of results suggests, however, that the observed relative neural hypoexcitability was not the direct result of ethanol withdrawal.