Circadian rhythms in the pineal organ persist in zebrafish larvae that lack ventral brain

<p>Abstract</p> <p>Background</p> <p>The mammalian suprachiasmatic nucleus (SCN), located in the ventral hypothalamus, is a major regulator of circadian rhythms in mammals and birds. However, the role of the SCN in lower vertebrates remains poorly understood. Zebrafish <it>cyclops </it>(<it>cyc</it>) mutants lack ventral brain, including the region that gives rise to the SCN. We have used <it>cyc </it>embryos to define the function of the zebrafish SCN in regulating circadian rhythms in the developing pineal organ. The pineal organ is the major source of the circadian hormone melatonin, which regulates rhythms such as daily rest/activity cycles. Mammalian pineal rhythms are controlled almost exclusively by the SCN. In zebrafish and many other lower vertebrates, the pineal has an endogenous clock that is responsible in part for cyclic melatonin biosynthesis and gene expression.</p> <p>Results</p> <p>We find that pineal rhythms are present in <it>cyc </it>mutants despite the absence of an SCN. The arginine vasopressin-like protein (Avpl, formerly called Vasotocin) is a peptide hormone expressed in and around the SCN. We find <it>avpl </it>mRNA is absent in <it>cyc </it>mutants, supporting previous work suggesting the SCN is missing. In contrast, expression of the putative circadian clock genes, <it>cryptochrome 1b (cry1b) </it>and <it>cryptochrome 3 (cry3)</it>, in the brain of the developing fish is unaltered. Expression of two pineal rhythmic genes, <it>exo-rhodopsin </it>(<it>exorh) </it>and <it>serotonin-N-acetyltransferase </it>(<it>aanat2</it>), involved in photoreception and melatonin synthesis, respectively, is also similar between <it>cyc </it>embryos and their wildtype (WT) siblings. The timing of the peaks and troughs of expression are the same, although the amplitude of expression is slightly decreased in the mutants. Cyclic gene expression persists for two days in <it>cyc </it>embryos transferred to constant light or constant dark, suggesting a circadian clock is driving the rhythms. However, the amplitude of rhythms in <it>cyc </it>mutants kept in constant conditions decreased more quickly than in their WT siblings.</p> <p>Conclusion</p> <p>Our data suggests that circadian rhythms can be initiated and maintained in the absence of SCN and other tissues in the ventral brain. However, the SCN may have a role in regulating the amplitude of rhythms when environmental cues are absent. This provides some of the first evidence that the SCN of teleosts is not essential for establishing circadian rhythms during development. Several SCN-independent circadian rhythms have also been found in mammalian species. Thus, zebrafish may serve as a model system for understanding how vertebrate embryos coordinate rhythms that are controlled by different circadian clocks.</p

Towards a Synthetic Chloroplast

The evolution of eukaryotic cells is widely agreed to have proceeded through a series of endosymbiotic events between larger cells and proteobacteria or cyanobacteria, leading to the formation of mitochondria or chloroplasts, respectively. Engineered endosymbiotic relationships between different species of cells are a valuable tool for synthetic biology, where engineered pathways based on two species could take advantage of the unique abilities of each mutualistic partner.We explored the possibility of using the photosynthetic bacterium Synechococcus elongatus PCC 7942 as a platform for studying evolutionary dynamics and for designing two-species synthetic biological systems. We observed that the cyanobacteria were relatively harmless to eukaryotic host cells compared to Escherichia coli when injected into the embryos of zebrafish, Danio rerio, or taken up by mammalian macrophages. In addition, when engineered with invasin from Yersinia pestis and listeriolysin O from Listeria monocytogenes, S. elongatus was able to invade cultured mammalian cells and divide inside macrophages.Our results show that it is possible to engineer photosynthetic bacteria to invade the cytoplasm of mammalian cells for further engineering and applications in synthetic biology. Engineered invasive but non-pathogenic or immunogenic photosynthetic bacteria have great potential as synthetic biological devices

Multibow: Digital Spectral Barcodes for Cell Tracing

We introduce a multicolor labeling strategy (Multibow) for cell tracing experiments in developmental and regenerative processes. Building on Brainbow-based approaches that produce colors by differential expression levels of different fluorescent proteins, Multibow adds a layer of label diversity by introducing a binary code in which reporters are initially OFF and then probabilistically ON or OFF following Cre recombination. We have developed a library of constructs that contains seven different colors and three different subcellular localizations. Combining constructs from this library in the presence of Cre generates cells labeled with multiple independently expressed colors based on if each construct is ON or OFF following recombination. These labels form a unique "barcode" that allows the tracking of the cell and its clonal progenies in addition to expression level differences of each color. We tested Multibow in zebrafish which validates its design concept and suggests its utility for cell tracing applications in development and regeneration

De novo phosphoinositide synthesis in zebrafish is required for triad formation but not essential for myogenesis.

Phosphoinositides (PIPs) and their regulatory enzymes are key players in many cellular processes and are required for aspects of vertebrate development. Dysregulated PIP metabolism has been implicated in several human diseases, including a subset of skeletal myopathies that feature structural defects in the triad. The role of PIPs in skeletal muscle formation, and particularly triad biogenesis, has yet to be determined. CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) catalyzes the formation of phosphatidylinositol, which is the base of all PIP species. Loss of CDIPT should, in theory, result in the failure to produce PIPs, and thus provide a strategy for establishing the requirement for PIPs during embryogenesis. In this study, we generated cdipt mutant zebrafish and determined the impact on skeletal myogenesis. Analysis of cdipt mutant muscle revealed no apparent global effect on early muscle development. However, small but significant defects were observed in triad size, with T-tubule area, inter terminal cisternae distance and gap width being smaller in cdipt mutants. This was associated with a decrease in motor performance. Overall, these data suggest that myogenesis in zebrafish does not require de novo PIP synthesis but does implicate a role for CDIPT in triad formation

Design and test of Multibow in zebrafish.

<p><b>a.</b> Modified “Brainbow [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127822#pone.0127822.ref001" target="_blank">1</a>]” cassette that allows a binary ON/OFF switch. <b>b.</b> Multibow Strategy. Each cell harbors multiple different ON/OFF cassettes to generate random color “digital” barcodes upon Cre-mediated recombination. <b>c.</b> Table of Multibow Tags and Fluorescent Proteins (FPs). <b>d.</b> Diversity of color codes. Image is a densely labeled region along the trunk of a 40hpf <i>hsp70</i>:<i>cerulean-cre</i> embryo injected with all 21 Multibow constructs and heat-shocked at 10hpf for 1 hour. The color and tag diversity generates barcodes for cell clones that appear random and diverse. Intensity differences further help distinguish cells from neighbors visually. The Composite image is made from the green, yellow (turned to blue) and red panels. 3 different clones are highlighted by α, β, γ and corresponding arrows. Scale bar: 10μm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127822#pone.0127822.s006" target="_blank">S3 Table</a>. <b>e.</b> Partial table of clones of different color codes found in <b>d.</b>. The colored square labels of the top row indicate nuclear, membrane and cytoplasmic, respectively. A black square in the table indicates this clone being positive for the corresponding color. Distinct "barcodes" form for different clones. The α, β, γ clones are indicated by arrows. The number of annotated cells labels (~30) represents a large fraction of cells found in the image in <b>d</b>, which contains ~50 cells. The fact that most of these cells have a color code distinct from any other cell (except clones that have the same color) show that Multibow label is highly random.</p

Spatial temporal coverage and stability of Multibow labeling.

<p><b>a.</b> Spatial and cell type coverage of Multibow. The embryo was injected with 6 Multibow colors (mR/mG/nR/nG/R/G) at single cell stage and heat-shocked at 1 day-post-fertilization (dpf) for 2 hours. The whole 4dpf larva was imaged in 2 channels (G/R). Positive cells can be seen distributed from head to tail throughout the larva, indicating high spatial coverage. In inserts 1 and 2, distinctly shaped skin, muscle, mesenchymal and neural cells can be observed by cytoplasmic or membrane Multibow labeling. Scale bars: 100μm. <b>b.</b> Temporal stability of labeling. The embryo was injected with 6 Multibow colors (mR/mG/nR/nG/R/G) at single cell stage and heat-shocked at 1 day-post-fertilization (dpf) for 2 hours. The same embryo was imaged once per day to 11dpf. The persistence of labeling indicates genomic insertion of Multibow cassettes. Red patches around the eye and along the gut are auto-fluorescence. Enlarged views of white boxed areas show that the area is stably fluorescent. Scale bar in enlarged views: 100μm. <b>c.</b> Label stability of color codes over time. The embryo was injected with 12 (B/G/Y/R) Multibow constructs at one cell stage. Heat-shock of this tg(<i>hsp70</i>:<i>cerulean-cre</i>) individual was at 30hpf (duration: 2 hours). Its developing larval tail fin was imaged every 24 hours starting at 54hpf using four channels (B/G/Y/R). The color codes of the cells remain unchanged despite fluorescent intensity differences at different days, allowing identification of the same cells/clones(e.g., α and β, shown in enlarged regions marked by white boxes). Color codes: α: nG/nY; β: mB. Scale bar: 100μm. See also Fig d in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127822#pone.0127822.s002" target="_blank">S2 Fig</a>.</p

Examples of Multibow Cell Tracing in Development and Regeneration.

<p><b>a.</b> Cranial facial development mapped by Multibow. The embryo was heat-shocked at 6hpf. 4 channels (B/G/Y/R) were used. The left face of the larva was imaged. Red boxes: regions highlighted in <b>b.</b> and <b>c.</b>. Scale bars: 50μm. <b>b.</b> Lineage relationship between neuromast hair cells. Dashed line circle indicates the hair bundle. Multibow labeled hair cell color codes: 1(mB/nY/R), 2(mB/mG/nR), 3(mB), 4(nG), 5(mB/nR), 6(R). The same pattern was already observed at 30hpf. Scale bars: 10μm. <b>c.</b> Identification of cells that undergo remarkable morphological changes during semicircular canal formation. Arrows: initial locations of the two mesenchymal cells that span the projection later. Grey circle: posterior otolith. Scale bars: 50μm. <b>d.</b> Clonal expansion near the eye over long time periods. The embryo was injected with 12 constructs (B/G/Y/R) and heat-shocked at 10hpf. Arrows indicate locations of identified clones α (nG), β (nG/R), γ (nY/mR). These clones can be seen amplified in number at 54hpf or 129hpf (α: 2 to 4; β: 2 to 4; γ: 2 to 3). Scale bar: 100μm. <b>e.</b> Multibow analysis of regeneration in the larval tail. Heat-shock labeling (1 hour), amputation and imaging were performed as labeled in the timeline. Immediately after amputation, the tissue shrank and cells near the wound converged (the images overlay may appear to be slightly out of register due to the changes of the live tissue during the acquisition of different channels, cell identification is not affected as these changes are small and predictable). By 2 days after amputation, most cells that had converged at the frontier of the wound were gone (their unique color codes disappeared, red arrowheads). The regenerated tissue came from clonal expansion of cells away from the frontier (highlighted examples in enlarged view from the white boxes 1 and 2). These clones show lineage restriction to the original cell type (the morphology of cells in the same clone remains similar, e.g., the blue cells in box 1 increased in number while size and shape do not have major changes.). Scale bars: 50μm.</p

Robust correspondence of automated membrane segmentations with automated nuclear segmentations.

<p>Detection and error rates of the automated algorithm was compared with standard nuclear segmentation algorithms. The assumption was that perfect segmentations of both algorithms should theoretically establish a one-to-one correspondence between nuclei and membranes detected. <b>Matched</b> refers to cells with membrane and nuclei in exact correspondence. <b>Unmatched Cells</b> refer to membranes that did not contain a unique nucleus. <b>Unmatched Nuclei</b> refer to nuclei that did not correspond to a cell membrane.</p

Algorithm-enabled quantification of cell dynamics during somite formation.

<p>Retrospective cell tracing of epithelial (yellow) and mesenchymal (red) cells from formed somites at (B) 5ss back to the presomitic mesoderm at (A) 3ss. (C) Corresponding decrease in somite tissue surface area during the formation of somites 3, 4, and 5. (D) Epithelial and mesenchymal cell numbers in respective somites at 5ss. (E,F) Three-dimensional cell shape quantified by the length of their principal axes at 3ss and 5ss. (G,H) Scatter plots of elongation () and cell volumes at 3ss and 5ss. The two cell populations show different behavior. Statistical analysis of the two distributions show that mesenchymal cells (red) tend to cluster, round-up, and shrink in size on average.</p

Accurate and highly-sensitive algorithm performance on synthesized 3D membrane images.

<p>(A–C) Synthesized cell structures in along , and sections with image noise added (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002780#pcbi-1002780-t002" target="_blank">Table 2</a>). As in the case of real-world images, the lateral resolution significantly differs from the axial resolution. (D–F) Segmentations overlaid on the raw image with a 50% opacity function. (G) An example of under-segmentation (brown cells, black arrows) and over-segmentation (interstitial fragments, white arrows) in the image. The errors could be filtered out by size criteria.</p