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A new somite compartment, called the endotome, that contributes to the formation of the embryonic dorsal aorta by providing endothelial progenitors is identified here; endotome-derived endothelial progenitors, whose formation is regulated by the activity of the meox1 gene, induce haematopoietic stem cell formation upon colonization of the nascent dorsal aorta. Haematopoietic stem cells in the embryo Two papers published in this issue of Nature demonstrate the involvement of somites — paired masses of mesoderm cells that form along the anterior–posterior axis of the embryo — in the generation of haematopoietic stem cells (HSCs) during vertebrate development. Phong Dang Nguyen et al . identify a previously unknown somite compartment, called the endotome, which contributes to the formation of the embryonic dorsal aorta by providing endothelial progenitors. The formation of the endotome is regulated by the activity of meox1 , a homeobox-containing transcription factor. Isao Kobayashi et al . report that the precursors of HSCs make direct contact with the somite during their embryonic migration and that the interaction is needed to receive the necessary Notch signal. They identify two adhesion molecules that mediate the contact: Jam1a, which is expressed by HSC precursors, and Jam2a, which is expressed by the somite. Haematopoietic stem cells (HSCs) are self-renewing stem cells capable of replenishing all blood lineages. In all vertebrate embryos that have been studied, definitive HSCs are generated initially within the dorsal aorta (DA) of the embryonic vasculature by a series of poorly understood inductive events 1 , 2 , 3 . Previous studies have identified that signalling relayed from adjacent somites coordinates HSC induction, but the nature of this signal has remained elusive 4 . Here we reveal that somite specification of HSCs occurs via the deployment of a specific endothelial precursor population, which arises within a sub-compartment of the zebrafish somite that we have defined as the endotome. Endothelial cells of the endotome are specified within the nascent somite by the activity of the homeobox gene meox1 . Specified endotomal cells consequently migrate and colonize the DA, where they induce HSC formation through the deployment of chemokine signalling activated in these cells during endotome formation. Loss of meox1 activity expands the endotome at the expense of a second somitic cell type, the muscle precursors of the dermomyotomal equivalent in zebrafish, the external cell layer. The resulting increase in endotome-derived cells that migrate to colonize the DA generates a dramatic increase in chemokine-dependent HSC induction. This study reveals the molecular basis for a novel somite lineage restriction mechanism and defines a new paradigm in induction of definitive HSCs.

Most animals possess multiple opsins which sense light for visual and non-visual functions. Here, we show spectral characteristics of non-visual opsins, vertebrate Opn3s, which are widely distributed among vertebrates. We successfully expressed zebrafish Opn3 in mammalian cultured cells and measured its absorption spectrum spectroscopically. When incubated with 11-cis retinal, zebrafish Opn3 formed a blue-sensitive photopigment with an absorption maximum around 465 nm. The Opn3 converts to an all-trans retinal-bearing photoproduct with an absorption spectrum similar to the dark state following brief blue-light irradiation. The photoproduct experienced a remarkable blue-shift, with changes in position of the isosbestic point, during further irradiation. We then used a cAMP-dependent luciferase reporter assay to investigate light-dependent cAMP responses in cultured cells expressing zebrafish, pufferfish, anole and chicken Opn3. The wild type opsins did not produce responses, but cells expressing chimera mutants (WT Opn3s in which the third intracellular loops were replaced with the third intracellular loop of a Gs-coupled jellyfish opsin) displayed light-dependent changes in cAMP. The results suggest that Opn3 is capable of activating G protein(s) in a light-dependent manner. Finally, we used this assay to measure the relative wavelength-dependent response of cells expressing Opn3 chimeras to multiple quantally-matched stimuli. The inferred spectral sensitivity curve of zebrafish Opn3 accurately matched the measured absorption spectrum. We were unable to estimate the spectral sensitivity curve of mouse or anole Opn3, but, like zebrafish Opn3, the chicken and pufferfish Opn3-JiL3 chimeras also formed blue-sensitive pigments. These findings suggest that vertebrate Opn3s may form blue-sensitive G protein-coupled pigments. Further, we suggest that the method described here, combining a cAMP-dependent luciferase reporter assay with chimeric opsins possessing the third intracellular loop of jellyfish opsin, is a versatile approach for estimating absorption spectra of opsins with unknown signaling cascades or for which absorption spectra are difficult to obtain.

The recruitment of leukocytes to infectious foci depends strongly on the local release of chemoattractant mediators. The human CXC chemokine receptor 3 (CXCR3) is an important node in the chemokine signaling network and is expressed by multiple leukocyte lineages, including T cells and macrophages. The ligands of this receptor originate from an ancestral CXCL11 gene in early vertebrates. Here, we used the optically accessible zebrafish embryo model to explore the function of the CXCR3-CXCL11 axis in macrophage recruitment and show that disruption of this axis increases the resistance to mycobacterial infection. In a mutant of the zebrafish ortholog of CXCR3 (cxcr3.2), macrophage chemotaxis to bacterial infections was attenuated, although migration to infection-independent stimuli was unaffected. Additionally, attenuation of macrophage recruitment to infection could be mimicked by treatment with NBI74330, a high-affinity antagonist of CXCR3. We identified two infection-inducible CXCL11-like chemokines as the functional ligands of Cxcr3.2, showing that the recombinant proteins exerted a Cxcr3.2-dependent chemoattraction when locally administrated in vivo. During infection of zebrafish embryos with Mycobacterium marinum, a well-established model for tuberculosis, we found that Cxcr3.2 deficiency limited the macrophage-mediated dissemination of mycobacteria. Furthermore, the loss of Cxcr3.2 function attenuated the formation of granulomatous lesions, the typical histopathological features of tuberculosis, and led to a reduction in the total bacterial burden. Prevention of mycobacterial dissemination by targeting the CXCR3 pathway, therefore, might represent a host-directed therapeutic strategy for treatment of tuberculosis. The demonstration of a conserved CXCR3-CXCL11 signaling axis in zebrafish extends the translational applicability of this model for studying diseases involving the innate immune system.

Amyloid precursor protein (APP) is expressed in many tissues in human, mice and in zebrafish. In zebrafish, there are two orthologues, Appa and Appb. Interestingly, some cellular processes associated with APP overlap with cilia-mediated functions. Whereas the localization of APP to primary cilia of in vitro-cultured cells has been reported, we addressed the presence of APP in motile and in non-motile sensory cilia and its potential implication for ciliogenesis using zebrafish, mouse, and human samples. We report that Appa and Appb are expressed by ciliated cells and become localized at the membrane of cilia in the olfactory epithelium, otic vesicle and in the brain ventricles of zebrafish embryos. App in ependymal cilia persisted in adult zebrafish and was also detected in mouse and human brain. Finally, we found morphologically abnormal ependymal cilia and smaller brain ventricles in appa −/− appb −/− mutant zebrafish. Our findings demonstrate an evolutionary conserved localisation of APP to cilia and suggest a role of App in ciliogenesis and cilia-related functions.

Background The transition from fertilized egg to embryo is accompanied by a multitude of changes in gene expression, and the transcriptional events that underlie these processes have not yet been fully characterized. In this study RNA-Seq is used to compare the transcription profiles of four early developmental stages in zebrafish ( Danio rerio ) on a global scale. Results An average of 79 M total reads were detected from the different stages. Out of the total number of reads 65% - 73% reads were successfully mapped and 36% - 44% out of those were uniquely mapped. The total number of detected unique gene transcripts was 11187, of which 10096 were present at 1-cell stage. The largest number of common transcripts was observed between 1-cell stage and 16-cell stage. An enrichment of gene transcripts with molecular functions of DNA binding, protein folding and processing as well as metal ion binding was observed with progression of development. The sequence data (accession number ERP000635) is available at the European Nucleotide Archive. Conclusion Clustering of expression profiles shows that a majority of the detected gene transcripts are present at steady levels, and thus a minority of the gene transcripts clusters as increasing or decreasing in expression over the four investigated developmental stages. The three earliest developmental stages were similar when comparing highly expressed genes, whereas the 50% epiboly stage differed from the other three stages in the identity of highly expressed genes, number of uniquely expressed genes and enrichment of GO molecular functions. Taken together, these observations indicate a major transition in gene regulation and transcriptional activity taking place between the 512-cell and 50% epiboly stages, in accordance with previous studies.

Formation of the three embryonic germ layers is a fundamental developmental process that initiates differentiation. How the zebrafish pluripotency factor Pou5f3 (homologous to mammalian Oct4) drives lineage commitment is unclear. Here, we introduce fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to assess the formation of Pou5f3 complexes with other transcription factors in real-time in gastrulating zebrafish embryos. We show, at single-cell resolution in vivo, that Pou5f3 complexes with Nanog to pattern mesendoderm differentiation at the blastula stage. Later, during gastrulation, Sox32 restricts Pou5f3–Nanog complexes to the ventrolateral mesendoderm by binding Pou5f3 or Nanog in prospective dorsal endoderm. In the ventrolateral endoderm, the Elabela / Aplnr pathway limits Sox32 levels, allowing the formation of Pou5f3–Nanog complexes and the activation of downstream BMP signaling. This quantitative model shows that a balance in the spatiotemporal distribution of Pou5f3–Nanog complexes, modulated by Sox32, regulates mesendoderm specification along the dorsoventral axis. As an animal embryo develops, cells divide and establish three distinct layers called the ectoderm, mesoderm and endoderm. Proteins called transcription factors control this process by regulating the activity of particular genes. Two or more transcription factors may interact to modulate each other’s activity. Zebrafish embryos provide an ideal model system for monitoring how these embryonic layers form and the interactions between transcription factors in real-time because they are transparent and develop outside their parents. Pou5f3 and Nanog are two key transcription factors involved in this process in zebrafish. However, it is not clear how Pou5f3 and Nanog instruct cells to become ectoderm, mesoderm or endoderm. Perez Camps et al. used imaging techniques to study Pou5f3 and Nanog. The experiments show that Pou5f3 and Nanog bind together to form complexes that instruct cells to form the temporary layer that later gives rise to both the mesoderm and endoderm. The cells in which there are less Pou5f3 and Nanog complexes form the ectoderm layer. To develop the body shape of adult zebrafish, the embryos need to give individual cells information about their location in the body. For example, a signal protein called bone morphogenetic protein (BMP) accumulates on the side of the embryo that will become the underside of the fish. Perez Camps et al. show that once the endoderm, mesoderm and ectoderm have formed, Pou5f3–Nanog complexes regulate BMP signalling to specify the underside of the fish. Meanwhile, in the endoderm on the opposite side, another transcription factor called Sox32 binds to individual Pou5f3 and Nanog proteins. This prevents Pou5f3 and Nanog from forming complexes and determines which side of the embryo will make the topside of the fish. A future challenge is to explore other transcription factors that may prevent Pou5f1 and Nanog from binding in the mesoderm and ectoderm of the topside of the fish.

This immunohistochemical study in zebrafish aims to extend the neurochemical characterization of enteric neuronal subpopulations and to validate a marker for identification of interstitial cells of Cajal (ICC). The expression of neuropeptides and anoctamin 1 (Ano1), a selective ICC marker in mammals, was analyzed in both embryonic and adult intestine. Neuropeptides were present from 3 days postfertilization (dpf). At 3 dpf, galanin-positive nerve fibers were found in the proximal intestine, while calcitonin gene-related peptide (CGRP)- and substance P-expressing fibers appeared in the distal intestine. At 5 dpf, immunoreactive fibers were present along the entire intestinal length, indicating a well-developed peptidergic innervation at the onset of feeding. In the adult intestine, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), galanin, CGRP and substance P were detected in nerve fibers. Colchicine pretreatment enhanced only VIP and PACAP immunoreactivity. VIP and PACAP were coexpressed in enteric neurons. Colocalization stainings revealed three neuronal subpopulations expressing VIP and PACAP: a nitrergic noncholinergic subpopulation, a serotonergic subpopulation and a subpopulation expressing no other markers. Ano1-immunostaining revealed a 3-dimensional network in the adult intestine containing multipolar cells at the myenteric plexus and bipolar cells interspersed between circular smooth muscle cells. Ano1 immunoreactivity first appeared at 3 dpf, indicative of the onset of proliferation of ICC-like cells. It is shown that the Ano1 antiserum is a selective marker of ICC-like cells in the zebrafish intestine. Finally, it is hypothesized that ICC-like cells mediate the spontaneous regular activity of the embryonic intestine.

Cortisol was reported to downregulate body-fluid Ca(2+) levels in mammals but was proposed to show hypercalcemic effects in teleostean fish. Fish, unlike terrestrial vertebrates, obtain Ca(2+) from the environment mainly via the gills and skin rather than by dietary means, and have to regulate the Ca(2+) uptake functions to cope with fluctuating Ca(2+) levels in aquatic environments. Cortisol was previously found to regulate Ca(2+) uptake in fish; however, the molecular mechanism behind this is largely unclear. Zebrafish were used as a model to explore this issue. Acclimation to low-Ca(2+) fresh water stimulated Ca(2+) influx and expression of epithelial calcium channel (ecac), 11β-hydroxylase and the glucocorticoid receptor (gr). Exogenous cortisol increased Ca(2+) influx and the expressions of ecac and hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2), but downregulated 11β-hydroxylase and the gr with no effects on other Ca(2+) transporters or the mineralocorticoid receptor (mr). Morpholino knockdown of the GR, but not the MR, was found to impair zebrafish Ca(2+) uptake function by inhibiting the ecac expression. To further explore the regulatory mechanism of cortisol in Ca(2+) uptake, the involvement of vitamin D(3) was analyzed. Cortisol stimulated expressions of vitamin D-25hydroxylase (cyp27a1), cyp27a1 like (cyp27a1l), 1α-OHase (cyp27b1) at 3 dpf through GR, the first time to demonstrate the relationship between cortisol and vitamin D(3) in fish. In conclusion, cortisol stimulates ecac expression to enhance Ca(2+) uptake functions, and this control pathway is suggested to be mediated by the GR. Lastly, cortisol also could mediate vitamin D(3) signaling to stimulate Ca(2+) uptake in zebrafish.

Transient receptor potential melastatin 7 (TRPM7) is a broadly expressed, non-selective cation channel. Studies in cultured cells implicate TRPM7 in regulation of cell growth, spreading, and survival. However, zebrafish trpm7 homozygous mutants display death of melanophores and temporary paralysis, but no gross morphological defects during embryonic stages. This phenotype implies that melanophores are unusually sensitive to decreases in Trpm7 levels, a hypothesis we investigate here. We find that pharmacological inhibition of caspases does not rescue melanophore viability in trpm7 mutants, implying that melanophores die by a mechanism other than apoptosis. Consistent with this possibility, ultrastructural analysis of dying melanophores in trpm7 mutants reveals abnormal melanosomes and evidence of a ruptured plasma membrane, indicating that cell death occurs by necrosis. Interestingly, inhibition of melanin synthesis largely prevents melanophore cell death in trpm7 mutants. These results suggest that melanophores require Trpm7 in order to detoxify intermediates of melanin synthesis. We find that unlike TRPM1, TRPM7 is expressed in human melanoma cell lines, indicating that these cells may also be sensitized to reduction of TRPM7 levels.

During development of the vertebrate neuroepithelium, the nucleus in neural progenitor cells (NPCs) moves from the apex toward the base and returns to the apex (called interkinetic nuclear migration) at which point the cell divides. The fate of the resulting daughter cells is thought to depend on the sampling by the moving nucleus of a spatial concentration profile of the cytoplasmic Notch intracellular domain (NICD). However, the nucleus executes complex stochastic motions including random waiting and back and forth motions, which can expose the nucleus to randomly varying levels of cytoplasmic NICD. How nuclear position can determine daughter cell fate despite the stochastic nature of nuclear migration is not clear. Here we derived a mathematical model for reaction, diffusion, and nuclear accumulation of NICD in NPCs during interkinetic nuclear migration (INM). Using experimentally measured trajectory-dependent probabilities of nuclear turning, nuclear waiting times and average nuclear speeds in NPCs in the developing zebrafish retina, we performed stochastic simulations to compute the nuclear trajectory-dependent probabilities of NPC differentiation. Comparison with experimentally measured nuclear NICD concentrations and trajectory-dependent probabilities of differentiation allowed estimation of the NICD cytoplasmic gradient. Spatially polarized production of NICD, rapid NICD cytoplasmic consumption and the time-averaging effect of nuclear import/export kinetics are sufficient to explain the experimentally observed differentiation probabilities. Our computational studies lend quantitative support to the feasibility of the nuclear concentration-sensing mechanism for NPC fate determination in zebrafish retina.