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Concurrent infection with cowpox and feline parvovirus was diagnosed in a 5-month-old male European Short Hair cat. Microscopical examination of the facial skin, ears and foot pads revealed multifocal to coalescing, ulcerative to necrotizing dermatitis and panniculitis with ballooning epidermal degeneration and eosinophilic cytoplasmic inclusion bodies. Immunohistochemistry, polymerase chain reaction testing and virus isolation confirmed infection with a strain of cowpox virus similar to that isolated from a cat in Germany 5 years previously. Lymphoid tissues were depleted and there was catarrhal enteritis caused by feline parvovirus as confirmed by immunohistochemistry and in-situ hybridization. This co-infection did not result in a more severe and rapid course of the poxvirus-associated disease.

The acrosome reaction, the first step of the fertilization, is induced by calcium influx through Canonical Transient Receptor Potential channels (TRPC). The molecular nature of TRPC involved is still a debated question. In mouse, TRPC2 plays the most important role and is responsible for the calcium plateau. However, TRPC1 and TRPC5 are also localized in the acrosomal crescent of the sperm head and may participate in calcium signaling, especially in TRPC2-deficient mice. Activation of TRPC channels is an unresolved question in germ and somatic cells as well. In particular, in sperm, little is known concerning the molecular events leading to TRPC2 activation. From the discovery of IP3R binding domains on TRPC2, it has been suggested that TRPC channel activation may be due to a conformational coupling between IP3R and TRPC channels. Moreover, recent data demonstrate that junctate, an IP3R associated protein, participates also in the gating of some TRPC. In this study, we demonstrate that junctate is expressed in sperm and co-localizes with the IP3R in the acrosomal crescent of the anterior head of rodent sperm. Consistent with its specific localization, we show by pull-down experiments that junctate interacts with TRPC2 and TRPC5 but not with TRPC1. We focused on the interaction between TRPC2 and junctate, and we show that the N-terminus of junctate interacts with the C-terminus of TRPC2, both in vitro and in a heterologous expression system. We show that junctate binds to TRPC2 independently of the calcium concentration and that the junctate binding site does not overlap with the common IP3R/calmodulin binding sites. TRPC2 gating is downstream phospholipase C activation, which is a key and necessary step during the acrosome reaction. TRPC2 may then be activated directly by diacylglycerol (DAG), as in neurons of the vomeronasal organ. In the present study, we investigated whether DAG could promote the acrosome reaction. We found that 100 microM OAG, a permeant DAG analogue, was unable to trigger the acrosome reaction. Altogether, these results provide a new hypothesis concerning sperm TRPC2 gating: TRPC2 activation may be due to modifications of its interaction with both junctate and IP3R, induced by depletion of calcium from the acrosomal vesicle.

Fertilization of oocytes incites numerous changes relying on Ca(2+) signaling. In inseminated ascidian eggs, an increase in the egg surface membrane, monitored by a change in electrical capacitance, is recorded at the onset of meiosis resumption. This membrane addition to the cell surface is controlled by calcium release through a ryanodine receptor (RyR), sensitive to cyclic ADP-ribose. Using confocal microscopy analysis of ascidian oocytes immunostained with anti-RyR antibody, we show here that this calcium channel is asymmetrically located in the vegetal cortical zone. Interestingly, the increase in cell capacitance occurring at fertilization is correlated with a fluorescent signal, imaged by the marker of vesicle trafficking FM 1-43, located close to the RyR region. Two putative partners of RyR, namely an FKBP-like protein and a calmodulin, are identified in these oocyte extracts by detection of enzyme activity and PCR amplification. Both are necessary to sustain ryanodine receptor activity in these oocytes since the membrane insertion triggered by fertilization is inhibited by the FKBP ligand rapamycin and by a calmodulin antagonist peptide. These findings suggest that exocytosis in ascidian eggs is triggered at fertilization by a functional Ca(2+) release unit operating as a complex of several proteins, including a calmodulin and an immunophilin, around the intracellular calcium channel itself., (Copyright 2000 Academic Press.)

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.

alyronz12 (aln) is a recessive lethal mutation that affects early stages of neural crest development in the zebrafish. alyron appears to be an insertional mutation as the mutation was generated following microinjection of plasmid DNA into one-cell embryos and the stably integrated transgenic sequences are closely linked to the mutation. The insertion site harbors multiple copies of the plasmid sequence that have experienced complex rearrangements. Host-insert junction fragments have been molecularly cloned and host sequences adjacent to the transgene have been used to map the mutation to the distal arm of linkage group 15. alyron function is required cell-autonomously in the neural crest lineage. alyron mutants have a severe but not complete deficit of premigratory neural crest as judged by reduced expression of several markers associated with early stages of neural crest development. Lack of premigratory neural crest is likely to account for the two most conspicuous characteristics of alyron mutants: the absence of body pigmentation and the inability to affect blood circulation. The neural crest phenotype of alyron mutants resembles that observed in mouse mutants that lack Pax-3 or both Wnt-1 and Wnt-3a function, and expression of the zebrafish homologues of these genes is greatly reduced in the dorsal neural keels of alyron mutants. In contrast, ventral neural keel identity appears unaffected. Given our findings that the mutation is unlinked to pax or wnt genes that have been described in the zebrafish, we propose that alyron is a novel gene function required for the specification and/or proliferative expansion of neural crest progenitors., (Copyright 1999 Academic Press.)

Classical embryology experiments have indicated the existence of dorsal-type and ventral-type mesoderms that arise as a consequence of mesoderm induction during vertebrate development. Here we report that the zebrafish tbx6 gene, a member of the Brachyury-related T-box family of genes, is exclusively expressed by ventral mesendoderm. Three observations link the expression of tbx6 to ventral mesoderm specification. First, the gene is initially expressed at the onset of gastrulation within a ventrolateral subpopulation of cells that express the pan-mesodermal gene, no tail (Brachyury). Second, the mesoderm-inducing factors activin and bFGF activate tbx6 expression in animal caps. Third, dorsalization of the mesendodermal precursor population following exposure of embryos to lithium ions causes down-regulation of tbx6 transcription. tbx6 is expressed transiently in the involuting derivatives of the ventral mesendoderm, which give rise to nonaxial mesodermal tissues; its expression is extinguished as tissue differentiation progresses. Transcription of tbx6 commences about an hour after initiation of expression of the pan-mesendodermal gene no tail and the organizer gene goosecoid. The dependence of tbx6 expression on no tail activity was examined in no tail mutant embryos. The activation of tbx6 transcription in ventral mesoderm does not depend on no tail gene activity. However, no tail appears to contribute to the maintenance of normal levels of tbx6 transcription and may be required for tbx6 transcription in the developing tail.

Using whole cell patch clamp recordings on unfertilized eggs of the ascidian Ciona intestinalis, we are able to detect ryanodine receptors within the oocytes. Our approach is based on measurements of the voltage-activated inward calcium currents. Two types of Ca2+ currents have been described on the oocyte membrane of Ciona: a low threshold slowly activating current, and a high threshold faster one. We show here that caffeine induces a decrease in the intensity of the Ca2+ currents, when applied either externally or internally from the mouth of a patch pipette. Caffeine application mimics fertilization which transiently decreases the high threshold Ca2+ current density during density during the first meiotic cycle. Ryanodine (> 1 nM) has an effect similar to caffeine. This partial decrease in Ca2+ current density elicited by caffeine or ryanodine is prevented by intracellular application of the calcium chelator BAPTA, then imputable to calcium release. In summary, the depolarization-induced Ca2+ current intensity allows monitoring of an intracellular calcium store which is sensitive to low concentrations of ryanodine in Ciona oocytes. Further identification of a ryanodine receptor was obtained by immunological staining with antibodies against mammalian skeletal muscle ryanodine receptor. Ryanodine receptors were asymmetrically localized in the cortex of Ciona eggs. We discuss the methodological relevance of our patch-clamp approach, in connection with the possible biological role of such a ryanodine receptor in the early stages of development.

Zebrafish holds great potential as a model system for studying inner ear development because genetic techniques are highly efficient and inner ear development is a conspicuous and manipulable feature of zebrafish embryogenesis. Here we describe analysis of a semilethal dominant mutation, termed monolith (mnl), that specifically perturbs formation of the anterior (utricular) otolith in the developing ear. Other than the utricular otolith deficiency, all structures in the ear appear morphologically normal in mutant embryos, including posterior otoliths and all sensory epithelia. Expression patterns of several ear marker genes (msxC, msxD, and dlx3) also appear normal in the mutant. To identify the cell type(s) affected by the mnl mutation, chimeras were generated by transplanting dye-labeled +/+ cells into unlabeled mnl/mnl host embryos. Roughly half of such chimeras formed utricular otoliths normally, indicating that the transplanted wild-type cells rescued their mutant hosts. Detailed analysis of +/+ cell fates revealed that virtually all chimeras in which +/+ cells formed support cells in the utricular sensory epithelium were rescued. In contrast, wild-type cells forming other cell types (such as hair cells) or colonizing other regions of the host were not sufficient to facilitate rescue. These data indicate that support cells are required for normal otolith formation, providing the first experimentally established role for support cells in vertebrate sensory epithelia. The data also provide the first clear indication that otolith formation is controlled independently in different regions of the ear by localized cellular functions.

The conductance change after fertilization in the oocyte of the ascidian Ciona intestinalis has been followed by the whole cell patch-clamp technique. Two new inward currents, which are absent in unfertilized eggs, are elicited by hyperpolarization from a holding potential of +20 mV, which is the resting potential soon after fertilization. These currents reach their maximum level during the first meiotic division cycle, and then decrease in intensity, becoming almost undetectable at the 2-cell stage. These currents are most easily seen at high concentrations of barium. At least one, and likely both, of these currents appears to be carried by Ca ions. One of the currents is blocked by low concentrations of gadolinium; the other one is blocked by higher concentrations, although gadolinium at these levels does not block fertilization and the associated early depolarizing jump of the eggs. Thus these currents are not carried by channels that mediate the fertilization current. However, gadolinium blocks normal transition to 2-cell stage and blocks current oscillations synchronous to free calcium oscillations that occur normally in eggs around meiosis II. The electrical signature of calcium-release activated currents, taken together with these findings, suggests that these inward currents ensure a calcium entry pathway throughout meiosis. A plausible function of these currents may be to refill the Ca stores that are depleted after fertilization and that are required to progress into mitotic cell division. This interpretation is reinforced by experiments on unfertilized eggs with intracellular Ca stores depleted by thapsigargin, where both the newly described currents are observed.

Flow cytometry (FCM) allows the simultaneous measurement of different parameters in a population of cell or sub-cellular particles, which are individually analysed at a rate of 500 to 5,000 objects/second. Results are displayed as univariate or bivariate histograms, giving the distribution of the population in relation to the studied parameters. Moreover, FCM offers the possibility to physically sort the sub populations defined by the analysis.

It is not known how region- or tissue-specific differences are generated in the zebrafish embryo. To look at the potential role of maternal transcripts in generating cell diversity, we have isolated and characterized the zebrafish homologue of Xenopus DVR-1 (Vg1), a maternally supplied RNA that encodes a member of the transforming growth factor-beta superfamily. The zebrafish DVR-1 RNA is maternally supplied and its protein product shares a high degree of sequence identity with Xenopus DVR-1. These conserved features indicate that DVR-1 is likely to have an essential function in early embryogenesis. However, unlike the frog transcript, which is restricted to vegetal cells, DVR-1 RNA is distributed equally among all zebrafish blastomeres. We suggest that the ubiquitous distribution of DVR-1 RNA reflects a significant aspect of the developmental strategy of the zebrafish in which each blastomere retains an equivalent developmental potential throughout the cleavage period.

We identified a B2 repetitive element approximately 1.9kb down stream from mouse p53 coding gene. This element was then used as a probe to investigate the expression of B2 containing RNA during the induced differentiation of murine erythroleukemia (MEL) cells. This probe revealed two nuclear and one cytoplasmic RNA species. Nuclear small RNAs had a biphasic variation: a decrease followed by a reaccumulation. The cytoplasmic species was essentially non polysomal, and disappeared after the induced differentiation. The presented results suggest that the regulation of these RNAs is associated to cell proliferation and differentiation respectively.

We describe an embryonic lethal mutation in the zebrafish Brachydanio rerio that specifically affects the viability of most cells in the embryonic central nervous system (CNS). The mutation ned-1 (b39rl) was induced with gamma-irradiation and segregates as a single recessive allele closely linked to its centromere. It produces massive cell death in the CNS but a small set of specific neurons, including Rohon-Beard sensory neurons, large hindbrain interneurons, and primary motoneurons, survive embryogenesis and are functional. Synaptic connections between embryonic motoneurons and muscle cells appear physiologically normal, and the normally observed spontaneous flexions are present. Correlated with the presence of sensory neurons and interneurons, mutant embryos display reflexive movements in response to mechanical stimulation. Together, the surviving neurons, called primary neurons, form a class of cells that are prominent in size and arise early during development. Thus, this mutation may define a function that is differentially required by developmentally distinguishable sets of cells in the embryonic CNS.

Mosaic analysis has been used to study the clonal basis of the development of the pigmented retina of the zebrafish, Brachydanio rerio. Zebrafish embryos heterozygous for a recessive mutation at the gol-1 locus were exposed to gamma-irradiation at various developmental stages to create mosaic individuals consisting of wild-type pigmented cells and a clone of pigmentless (golden) cells in the eye. The contribution of individual embryonic cells to the pigmented retina was measured and the total number of cells in the embryo that contributed descendants to this tissue was determined. Until the 32-cell stage, almost every blastomere has some descendants that participate in the formation of the pigmented retina of the zebrafish. During subsequent cell divisions, up to the several thousand-cell stage, the number of ancestral cells is constant: approximately 40 cells are present that will give rise to progeny in the pigmented retina. Analysis of the size of clones in the pigmented retina indicates that the cells of this tissue do not arise through a rigid series of cell divisions originating in the early embryo. The findings that each cleavage stage cell contributes to the pigmented retina and yet the contribution of such cells is highly variable are consistent with the interpretation that clonal descendants of different blastomeres normally intermix extensively prior to formation of the pigmented retina.