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Results for “Zebrafish development”

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PubMed2004

Germ cell migration in zebrafish is dependent on HMGCoA reductase activity and prenylation.

Hydroxymethylglutaryl coenzyme A reductase (HMGCoAR) is required for isoprenoid and cholesterol biosynthesis. In Drosophila, reduced HMGCoAR activity results in germ cell migration defects. We show that pharmacological HMGCoAR inhibition alters zebrafish development and germ cell migration. Embryos treated with atorvastatin (Lipitor) exhibited germ cell migration defects and mild morphologic abnormalities. The effects induced by atorvastatin were completely rescued by prior injection of mevalonate, the product of HMGCoAR activity, or the prenylation precursors farnesol and geranylgeraniol. In contrast, squalene, a cholesterol intermediate further down the pathway, failed to rescue statin-induced defects. Moreover, pharmacologic inhibition of geranylgeranyl transferase 1 (GGT1) protein prenylation activity also resulted in abnormal germ cell migration. Thus, our pharmacological inhibition-and-rescue approach provided detailed information about the elements of isoprenoid biosynthesis that contribute to germ cell migration. Together with data from Drosophila (Santos and Lehmann, this issue), our results highlight a conserved role for protein geranylgeranylation in this context.

Animals↗

PubMed2006

Fgf signaling negatively regulates Nodal-dependent endoderm induction in zebrafish.

In zebrafish development, Nodal signaling is critical for the induction of endoderm and mesoderm. Three transcription factors downstream of Nodal, Bonnie and Clyde (Bon), Faust (Fau)/Gata5 and Casanova (Cas), are required for endoderm induction. However, it is not yet fully understood how the Nodal signaling pathway regulates the decision process of endoderm and mesoderm induction. In this study, we focused on Fgf signaling, downstream of Nodal signaling, during endoderm induction. We found that activation of Fgf signaling decreases the number of cas-expressing endodermal cells. Conversely, inhibition of this signaling increases the number of endodermal cells without affecting the expression of Nodal, Nodal antagonists, bon or fau/gata5. Inhibition of Fgf signaling in endoderm mutants suggests that this signaling negatively regulates cas expression by a pathway parallel to Bon and Fau/Gata5 in the molecular cascade leading to endoderm. Furthermore, activation of Fgf signaling can overcome Cas-mediated abrogation of mesodermal gene expression. Altogether, these results suggest that Fgf signaling negatively regulates endoderm induction, possibly through repression of cas expression and down-regulation of Cas function.

Animals↗

PubMed1990

Origin and organization of the zebrafish fate map.

We have analyzed lineages of cells labeled by intracellular injection of tracer dye during early zebrafish development to learn when cells become allocated to particular fates during development, and how the fate map is organized. The earliest lineage restriction was described previously, and segregates the yolk cell from the blastoderm in the midblastula. After one or two more cell divisions, the lineages of epithelial enveloping layer (EVL) cells become restricted to generate exclusively periderm. Following an additional division in the late blastula, deep layer (DEL) cells generate clones that are restricted to single deep embryonic tissues. The appearance of both the EVL and DEL restrictions could be causally linked to blastoderm morphogenesis during epiboly. A fate map emerges as the DEL cell lineages become restricted in the late blastula. It is similar in organization to that of an amphibian embryo. DEL cells located near the animal pole of the early gastrula give rise to ectodermal fates (including the definitive epidermis). Cells located near the blastoderm margin give rise to mesodermal and endodermal fates. Dorsal cells in the gastrula form dorsal and anterior structures in the embryo, and ventral cells in the gastrula form dorsal, ventral and posterior structures. The exact locations of progenitors of single cell types and of local regions of the embryo cannot be mapped at the stages we examined, because of variable cell rearrangements during gastrulation.

Animals↗

PubMed2002

Myosin heavy chain expression in cranial, pectoral fin, and tail muscle regions of zebrafish embryos.

To investigate whether different myosin heavy chain (MHC) isoforms may constitute myofibrils in the trunk and tail musculature and if their respective expression may be regulated by spadetail (spt) and no tail (brachyury), we identified and characterized mRNA expression patterns of an embryonic- and tail muscle-specific MHC gene (named myhz2) during zebrafish development in wild type, spt, and ntl mutant embryos. The identified myhz2 MHC gene encodes a polypeptide containing 1,935 amino acids. Deduced amino acid comparisons showed that myhz2 MHC shared 92.6% sequence identity with that of carp fast skeletal MHC. Temporal and spatial myhz2 MHC mRNA expression patterns were analyzed by quantitative RT-PCR and whole-mount in situ hybridization using primer pairs and probes designed from the 3'-untranslated region (UTR). Temporally myhz2 MHC mRNA appears in pharyngula embryos and peaks in protruding-mouth larvae. The expression level decreased in 7-day-old hatching larvae, and mRNA expression was not detectable in adult fish. Spatially in pharyngula embryos, mRNA was localized only in the tail somite region, while in long-pec embryos, transcripts were also expressed in the two cranial muscle elements of the adductor mandibulae and medial rectus, as well as in pectoral fin muscles and the tail muscle region. Myhz2 MHC mRNA was expressed in most cranial muscle elements, pectoral fin muscles, and the tail muscle region of 3-day-old hatching larvae. In contrast, no expression of myhz2 MHC mRNA could be observed in spt prim-15 mutant embryos. In spt long-pec mutant embryos, transcripts were expressed in two cranial muscle elements and the tail muscle region, but not in pectoral fin muscles, while only trace amounts of myhz2 MHC mRNA were expressed in the remaining tail muscle region of 38 hpf and long-pec ntl mutant embryos.

Amino Acid Sequence↗

PubMed1998

The shaping of pharyngeal cartilages during early development of the zebrafish.

In zebrafish the cartilages of the pharynx develop during late embryogenesis and grow extensively in the larva before eventually being replaced by bone. Here we examine chondrocyte arrangements, shapes, numbers, and divisions in the young hyoid cartilages. We observe two distinct developmental phases, morphogenesis and growth. The first phase generates stereotypically oriented chondrocyte stacks that might form by intercalations among cells within the precartilage condensations. In mutants that have deformed cartilages the orientation of the stacks is changed, and we propose that their correct formation underlies the correct initial shaping of the organ. The following period of rapid, nearly isometric cartilage growth occurs by divisions of chondrocytes that are largely located near the joints, and appears to be under quite separate regulation.

Animals↗

PubMed2027

Genomic Profiling of Chromatin State Using CUT&Tag.

Alterations in chromatin state, mediated through histone modifications and the incorporation of histone variants, are fundamental to establishing transcriptional networks and cell identity. Recent advances in low-input epigenome profiling methods, such as CUT&Tag and CUT&RUN, have enabled the study of chromatin states from very limited starting materials. In this chapter, we describe procedures for generating CUT&Tag libraries to profile histone modifications and histone variants in early-developing zebrafish embryos.

Animals↗

PubMed1995

Lateral specification of cell fate during vertebrate development.

Cells within equivalence groups interact via lateral specification to determine cell fates during development in Caenorhabditis elegans and other invertebrates. Populations of cells within the developing zebrafish have features similar to those of invertebrate equivalence groups. In a simple example, two identified zebrafish motoneurons behave as an equivalence pair in which one cell adopts a primary fate and interactions between the cells assign the other cell to a secondary fate. A more complicated situation exists for two initially equivalent populations of zebrafish neural crest cells. We consider whether mechanisms similar to those involved in fate specification within invertebrate equivalence groups also function furing fate specification in vertebrates.

Animals↗

PubMed2000

Computer-assisted visualizations of neural networks: expanding the field of view using seamless confocal montaging.

Microscopic analysis of anatomic relationships within the neural networks of adult and developing tissues often requires sampling large spatial regions of neuronal architecture. To accomplish this, there are two common imaging approaches: (1) image the entire area at once with low spatial resolution; or (2) image small sections at higher magnification/resolution and then join the sections back together by mosaic reconstruction (photomontaging). Low magnification imaging is relatively rapid to perform, resulting in a visualization that encompasses a large field of view with an extended depth of field. However, for fluorescence microscopy, low magnification visualizations are often plagued by poor spatial resolution. High magnification imaging possesses superior spatial resolution, but it produces an image with limited depth of field. When creating a larger field of view, the final image is also fragmented at the boundaries where multiple images are stitched together. Using confocal microscopy as well as features of common image processing programs, we outline a new method to transform individual, spatially contiguous z-series into a montage with a seamless field of view and an extended depth of field. In addition, we show that the manual alignment of images our method requires does not introduce significant errors into the final image. We illustrate our method for visualizing neural networks using tissues from the adult gastropod mollusc, Tritonia diomedea, and the developing zebrafish, Danio rerio.

Animals↗

PubMed2000

Comparative immunocytochemical study of FMRFamide neuronal system in the brain of Danio rerio and Acipenser ruthenus during development.

The distribution of FMRFamide-like immunoreactive (ir) neurons and fibers was investigated in the central nervous system of developing zebrafish and juvenile sturgeon (sterlet). Adult zebrafish was also studied. In zebrafish embryos FMRFamide-ir elements first appeared 30 h post-fertilization (PF). Ir somata were located in the olfactory placode and in the ventral diencephalon. FMRFamide-ir fibers originating from diencephalic neurons were found in the ventral telencephalon and in ventral portions of the brainstem. At 48 h PF, the ir perikarya in the olfactory placode displayed increased immunoreactivity and stained fibers emerged from the somata. At 60 h PF, bilaterally, clusters of FMRFamide-ir neurons were found along the rostro-caudal axis of the brain, from the olfactory placode to rostral regions of the ventro-lateral telencephalon. At 60 h PF, numerous ir fibers appeared in the dorsal telencephalon, optic lobes, optic nerves, and retina. Except for ir fibers in the hypophysis at the age of 72 h PF, and a few ir cells in the nucleus olfacto-retinalis (NOR) at the age of 2 months PF, no major re-organization was noted in subsequent ontogenetic stages. The number of stained NOR neurons increased markedly in sexually mature zebrafish. In adult zebrafish, other ir neurons were located in the dorsal zones of the periventricular hypothalamus and in components of the nervus terminalis. We are inclined to believe that neurons expressing FMRFamide originate in the olfactory placode and in the ventricular ependyma in the hypothalamus. On the same grounds, a dual origin of FMRFamide-ir neurons is inferred in the sturgeon, an ancestral bony fish: prior to the observation of ir cells in the nasal area and in the telencephalon stained neurons were noted in circumventricular hypothalamic regions.

Age Factors↗

PubMed2006

Embryonic and tumorigenic pathways converge via Nodal signaling: role in melanoma aggressiveness.

Bidirectional cellular communication is integral to both cancer progression and embryological development. In addition, aggressive tumor cells are phenotypically plastic, sharing many properties with embryonic cells. Owing to the similarities between these two types of cells, the developing zebrafish can be used as a biosensor for tumor-derived signals. Using this system, we show that aggressive melanoma cells secrete Nodal (a potent embryonic morphogen) and consequently can induce ectopic formation of the embryonic axis. We further show that Nodal is present in human metastatic tumors, but not in normal skin, and thus may be involved in melanoma pathogenesis. Inhibition of Nodal signaling reduces melanoma cell invasiveness, colony formation and tumorigenicity. Nodal inhibition also promotes the reversion of melanoma cells toward a melanocytic phenotype. These data suggest that Nodal signaling has a key role in melanoma cell plasticity and tumorigenicity, thereby providing a previously unknown molecular target for regulating tumor progression.

Animals↗

PubMed2005

The 'lipid raft' microdomain proteins reggie-1 and reggie-2 (flotillins) are scaffolds for protein interaction and signalling.

Reggie-1 and reggie-2 are two evolutionarily highly conserved proteins which are up-regulated in retinal ganglion cells during regeneration of lesioned axons in the goldfish optic nerve. They are located at the cytoplasmic face of the plasma membrane and are considered to be 'lipid raft' constituents due to their insolubility in Triton X-100 and presence in the 'floating fractions'; hence they were independently named flotillins. According to our current view, the reggies subserve functions as protein scaffolds which form microdomains in neurons, lymphocytes and many other cell types across species as distant as flies and humans. These microdomains are of a surprisingly constant size of less than or equal to 0.1 mm in all cell types, whereas the distance between them is variable. The microdomains co-ordinate signal transduction of specific cell-surface proteins and especially of GPI (glycosylphosphatidylinositol)-anchored proteins into the cell, as is demonstrated for PrP(c) (cellular prion protein) in T-lymphocytes. These cells possess a pre-formed reggie cap scaffold consisting of densely packed reggie microdomains. PrP(c) is targeted to the lymphocyte reggie cap when activated by antibody cross-linking, and induces a distinct Ca(2+) signal. In developing zebrafish, reggies become concentrated in neurons and axon tracts, and their absence, after morpholino antisense RNA-knockdown, results in deformed embryos with reduced brains. Likewise, defects in Drosophila eye morphogenesis occur upon reggie overexpression in mutant flies. The defects observed in the organism, as well as in single cells in culture, indicate a morphogenetic function of the reggies, with emphasis on the nervous system. This complies with their role as scaffolds for the formation of multiprotein complexes involved in signalling across the plasma membrane.

Animals↗

PubMed2005

Diversity and functional plasticity of eukaryotic selenoproteins: identification and characterization of the SelJ family.

Selenoproteins are a diverse group of proteins that contain selenocysteine (Sec), the 21st amino acid. In the genetic code, UGA serves as a termination signal and a Sec codon. This dual role has precluded the automatic annotation of selenoproteins. Recent advances in the computational identification of selenoprotein genes have provided a first glimpse of the size, functions, and phylogenetic diversity of eukaryotic selenoproteomes. Here, we describe the identification of a selenoprotein family named SelJ. In contrast to known selenoproteins, SelJ appears to be restricted to actinopterygian fishes and sea urchin, with Cys homologues only found in cnidarians. SelJ shows significant similarity to the jellyfish J1-crystallins and with them constitutes a distinct subfamily within the large family of ADP-ribosylation enzymes. Consistent with its potential role as a structural crystallin, SelJ has preferential and homogeneous expression in the eye lens in early stages of zebrafish development. A structural role for SelJ would be in contrast to the majority of known selenoenzymes. The unusually highly restricted phylogenetic distribution of SelJ, its specialization, and the comparative analysis of eukaryotic selenoproteomes reveal the diversity and functional plasticity of selenoproteins and point to a mosaic evolution of the use of Sec in proteins.

Adenosine Diphosphate Ribose↗

PubMed1996

Magnetic resonance microscopy and spectroscopy reveal kinetics of cryoprotectant permeation in a multicompartmental biological system.

Successful cryopreservation of most multicompartmental biological systems has not been achieved. One prerequisite for success is quantitative information on cryoprotectant permeation into and amongst the compartments. This report describes direct measurements of cryoprotectant permeation into a multicompartmental system using chemical shift selective magnetic resonance (MR) microscopy and MR spectroscopy. We used the developing zebrafish embryo as a model for studying these complex systems because these embryos are composed of two membrane-limited compartments: (i) a large yolk (surrounded by the yolk syncytial layer) and (ii) differentiating blastoderm cells (each surrounded by a plasma membrane). MR images of the spatial distribution of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that methanol permeated the entire embryo within 15 min. In contrast, the other cryoprotectants exhibited little or no permeation over 2.5 h. MR spectroscopy and microinjections of cryoprotectants into the yolk inferred that the yolk syncytial layer plays a critical role in limiting the permeation of some cryoprotectants throughout the embryo. This study demonstrates the power of MR technology combined with micromanipulation for elucidating key physiological factors in cryobiology.

Animals↗

PubMed2005

The hedgehog-PKA pathway regulates two distinct steps of the differentiation of retinal ganglion cells: the cell-cycle exit of retinoblasts and their neuronal maturation.

In the developing zebrafish retina, neurogenesis is initiated in cells adjacent to the optic stalk and progresses to the entire neural retina. It has been reported that hedgehog (Hh) signalling mediates the progression of the differentiation of retinal ganglion cells (RGCs) in zebrafish. However, the progression of neurogenesis seems to be only mildly delayed by genetic or chemical blockade of the Hh signalling pathway. Here, we show that cAMP-dependent protein kinase (PKA) effectively inhibits the progression of retinal neurogenesis in zebrafish. Almost all retinal cells continue to proliferate when PKA is activated, suggesting that PKA inhibits the cell-cycle exit of retinoblasts. A cyclin-dependent kinase (cdk) inhibitor p27 inhibits the PKA-induced proliferation, suggesting that PKA functions upstream of cyclins and cdk inhibitors. Activation of the Wnt signalling pathway induces the hyperproliferation of retinal cells in zebrafish. The blockade of Wnt signalling inhibits the PKA-induced proliferation, but the activation of Wnt signalling promotes proliferation even in the absence of PKA activity. These observations suggest that PKA inhibits exit from the Wnt-mediated cell cycle rather than stimulates Wnt-mediated cell-cycle progression. PKA is an inhibitor of Hh signalling, and Hh signalling molecule morphants show severe defects in cell-cycle exit of retinoblasts. Together, these data suggest that Hh acts as a short-range signal to induce the cell-cycle exit of retinoblasts. The pulse inhibition of Hh signalling revealed that Hh signalling regulates at least two distinct steps of RGC differentiation: the cell-cycle exit of retinoblasts and RGC maturation. This dual requirement of Hh signalling in RGC differentiation implies that the regulation of a neurogenic wave is more complex in the zebrafish retina than in the Drosophila eye.

Animals↗

PubMed2004

Primitive roles for inhibitory interneurons in developing frog spinal cord.

Understanding the neuronal networks in the mammal spinal cord is hampered by the diversity of neurons and their connections. The simpler networks in developing lower vertebrates may offer insights into basic organization. To investigate the function of spinal inhibitory interneurons in Xenopus tadpoles, paired whole-cell recordings were used. We show directly that one class of interneuron, with distinctive anatomy, produces glycinergic, negative feedback inhibition that can limit firing in motoneurons and interneurons of the central pattern generator during swimming. These same neurons also produce inhibitory gating of sensory pathways during swimming. This discovery raises the possibility that some classes of interneuron, with distinct functions later in development, may differentiate from an earlier class in which these functions are shared. Preliminary evidence suggests that these inhibitory interneurons express the transcription factor engrailed, supporting a probable homology with interneurons in developing zebrafish that also express engrailed and have very similar anatomy and functions.

Action Potentials↗

PubMed2004

Semaphorin3D guides retinal axons along the dorsoventral axis of the tectum.

We examined the role of Sema3D, a semaphorin of previously unknown function, in guiding retinal ganglion cell (RGC) axons to the optic tectum in the developing zebrafish. Sema3D is expressed more strongly in the ventral versus dorsal tectum, suggesting that it may participate in guiding RGC axons along the dorsoventral axis of the tectum. Ubiquitous misexpression of Sema3D in transgenic zebrafish inhibits ventral but not dorsal RGC axon growth. In addition, ventral RGC axons avoid or stop at individual cells misexpressing Sema3D along their pathway. Sema3D ubiquitous misexpression at later stages also causes ventral RGC axon arbors to spread more widely along the dorsoventral axis of the tectum. Knock-down of Sema3D with morpholino antisense causes ventral RGC axons to extend aberrantly into the ventral tectum. These results suggest that Sema3D in the ventral tectum normally acts to inhibit ventral RGCs from extending into ventral tectum, ensuring their correct innervation of dorsal tectum.

Animals↗

PubMed1998

Real-time measurements of calcium dynamics in neurons developing in situ within zebrafish embryos.

We have developed a non-invasive technique to measure intracellular calcium ([Ca2+]i) in neurons growing within intact embryos of the zebrafish (Danio rerio). A single blastomere was injected with a calcium-sensitive fluorescent dye (Calcium Green dextran) between the 32- and 128-cell stage and the embryo imaged between 16 h and 20 h postfertilisation using laser scanning confocal microscopy. Labelled nerve cells from embryos preinjected with dye and dissociated at 16 h showed a fluorescence increase (66+/-22%; n=11) in response to depolarisation with KCl confirming that the dye remained intracellular and was sensitive to calcium. In addition, fluorescence changes in activated muscle cells of intact embryos showed that the dye was capable of responding to [Ca2+]i changes in vivo. Imaging of dye loaded cells over 30-min periods in embryos between 16 and 20 h revealed that the majority of neurons within the brain and spinal cord did not show spontaneous fluorescence changes distinguishable from noise. However, a subset of neurons within the ventral spinal cord exhibited spontaneous, repetitive [Ca2+]i oscillations which may have a functional significance during neuronal development.

Animals↗

PubMed2006

Zebrafish pax5 regulates development of the utricular macula and vestibular function.

The zebrafish otic vesicle initially forms with only two sensory epithelia, the utricular and saccular maculae, which primarily mediate vestibular and auditory function, respectively. Here, we test the role of pax5, which is preferentially expressed in the utricular macula. Morpholino knockdown of pax5 disrupts vestibular function but not hearing. Neurons of the statoacoustic ganglion (SAG) develop normally. Utricular hair cells appear to form normally but a variable number subsequently undergo apoptosis and are extruded from the otic vesicle. Dendrites of the SAG persist in the utricle but become disorganized after hair cell loss. Hair cells in the saccule develop and survive normally. Otic expression of pax5 requires pax2a and fgf3, mutations in which cause vestibular defects, albeit by distinct mechanisms. Thus, pax5 works in conjunction with fgf3 and pax2a to establish and/or maintain the utricular macula and is essential for vestibular function.

Acoustic Maculae↗