Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter ¹. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury. Open in a separate windowClick here to view.^{(76M, flv)}     

Rhythms in a diurnal brain

The neural mechanisms governing circadian rhythms generate patterns of behavior and physiology that are very different in diurnal and nocturnal species. Here we review data bearing on the issue of where and how in the brain these differences might be generated. Molecular data from several species now confirm that the central circadian clock, located in the suprachiasmatic nucleus (SCN), is coupled to the light - dark cycle in the same manner in nocturnal and diurnal species, indicating that the fundamental differences arise from mechanisms coupling the clock to effector systems. Major differences in this coupling become apparent only when one steps beyond the SCN to look at brain regions that directly or indirectly receive input from it. This review focuses on our work on brain regions and cell populations to which the SCN projects in the diurnal species Arvicanthis niloticus (Nile grass rats). We have found rhythms in the numbers of cells containing cFos, or PER1, in a number of these regions, and the patterns of these rhythms are always different from those seen in nocturnal laboratory rats. In some areas these rhythms are simply inverted in the two species, but in other extra-SCN regions the phase of the rhythms in these two species differs in less extreme ways. Taken together, these data suggest that there is no single simple switch that causes some animals to be nocturnal and others to be diurnal. Rather, the differences likely emerge through a variety of mechanisms operating within and downstream of the cells to which the SCN projects.     

Ultrasonic Telemetry, Tracking and Automated Monitoring Technology for Sharks

Sharks were among the first marine animals to carry telemetry systems because of their size and the need to understand their interactions with humans. Modern telemetry systems can gather many kinds of data (limited only by imagination, funding and sensor types), indicating which animals are near telemetry receivers and what they are doing. Receivers now range from simple autonomous detector units for deployment in mid-water in large-scale grids, to sophisticated automated benthic recorders, to triangulating radio-linked buoy systems (RAP), to ship-borne transponders. In addition, archival tags can now gather and store data even while the shark is away, to be downloaded later. Older types had to be recovered, but popup tags release from sharks automatically, surface and transfer data to satellites, while CHAT tags download whenever queried by a nearby transponding acoustic receiver. Sophisticated animal-borne tags dramatically increase the information gathered about sharks and their environment. The examples provided show the parallel progression of shark biology and acoustic biotelemetry illustrating that telemetry systems are tools for gathering data, which can often be honed to facilitate biological experiments. Future visions include sensors that directly measure shark swimming power and cardiac output, compressing the data so that it can be delivered to RAP systems tracking multiple animals with meter resolution in near real time. CHAT tags as small as 22mm diameter should be able to return similar data from trips of hundreds of kilometers. Continued communication between biologists and engineers is essential to develop these technologies.     

A prominent role for Sp1 during lipopolysaccharide-mediated induction of the IL-10 promoter in macrophages

IL-10 is an antiinflammatory cytokine secreted by activated macrophages and Th2 cells. IL-10 secretion promotes the down-regulation of proinflammatory cytokine synthesis and the development of Th2 responses. In macrophages, proinflammatory cytokines appear to be induced by similar mechanisms, but the IL-10 induction mechanisms have not been examined. We have analyzed the murine IL-10 promoter in the RAW264.7 macrophage line activated with LPS. A comprehensive mutant analysis revealed only one element upstream of the core promoter that was essential for promoter induction. A refined mutant analysis localized this element to nucleotides -89 to -78, and gel shift experiments revealed that it represents a nonconsensus binding site for Sp1. The functional relevance of Sp1 was supported by the high affinity of the interaction, the close correlation between the nucleotides required for Sp1 binding and promoter function, and the ability of an Sp1 consensus sequence to substitute for the -89/-78 promoter sequence. Evidence that Sp1 may be a target of signaling pathways involved in IL-10 induction was provided by the exclusive requirement for the Sp1 binding site, by the ability of the Sp1 site to confer induction to a heterologous promoter, and by the delineation of an Sp1 domain that can mediate induction. No relevant contribution from Rel, C/EBP (CCAAT/enhancer-binding protein), or AP-1 binding sites, which regulate most proinflammatory cytokine promoters, was observed. Together, these results demonstrate that IL-10 gene regulation is distinct from the regulation of proinflammatory cytokine genes, and suggest that Sp1 may be a central mediator of IL-10 induction.     

Highly conserved tooth morphology in allopatric elasmobranch populations despite contrasting diets—a case of Triakis megalopterus in southern Africa

Environmental Biology of Fishes - The role dentition plays in shaping feeding patterns has significant consequences for understanding the evolution of shark feeding. Despite this, research has...     

Transgenic expression of Helios in B lineage cells alters B cell properties and promotes lymphomagenesis

Helios, a member of the Ikaros family of DNA-binding proteins, is expressed in multipotential lymphoid progenitors and throughout the T lineage. However, in most B lineage cells, Helios is not expressed, suggesting that its absence may be critical for B cell development and function. To test this possibility, transgenic mice were generated that express Helios under the control of an Ig mu enhancer. Commitment to the B cell lineage was unaltered in Helios transgenic mice, and numbers of surface IgM(+) B cells were normal in the bone marrow and spleen. However, both bone marrow and splenic B cells exhibited prolonged survival and enhanced proliferation. B cells in Helios transgenic mice were also hyperresponsive to Ag stimulation. These alterations were observed even though the concentration of ectopic Helios in B lineage cells, like that of endogenous Helios in thymocytes, was well below the concentration of Ikaros. Further evidence that ectopic Helios expression contributes to B cell abnormalities was provided by the observation that Helios transgenic mice developed metastatic lymphoma as they aged. Taken together, these results demonstrate that silencing of Helios is critical for normal B cell function.     

Climate‐driven substitution of habitat‐forming species leads to reduced biodiversity within a temperate marine community

Aim

In marine ecosystems, habitat‐forming species (HFS) such as reef‐building corals and canopy‐forming macroalgae alter local environmental conditions and can promote biodiversity by providing biogenic living space for a vast array of associated organisms. We examined community‐level impacts of observed climate‐driven shifts in the relative abundances of two superficially similar HFS, the warm‐water kelp Laminaria ochroleuca and the cool‐water kelp Laminaria hyperborea.

Location

Western English Channel, north‐east Atlantic

Methods

We compared algal and invertebrate assemblages associated with kelp stipes and holdfasts, across multiple sites and sampling events. Significant differences were recorded in the structure of assemblages between the host kelp species at each site and event.

Results

Assemblages associated with stipes of the cool‐water HFS were, on average, >12 times more diverse and supported >3600 times more biomass compared with the warm‐water HFS. Holdfast assemblages also differed significantly between species, although to a lesser extent than those associated with stipes. Overall, assemblages associated with the warm‐water HFS were markedly impoverished and comprised far fewer rare or unique taxa.

Main conclusions

While previous research has shown how climate‐driven loss of HFS can cause biodiversity loss, our study demonstrates that climate‐driven substitutions of HFS can also lead to impoverished assemblages. The indirect effects of climate change remain poorly resolved, but shifts in the distributions and abundances of HFS may invoke widespread ecological change, especially in marine ecosystems where facilitative interactions are particularly strong.     

Carbon assimilation and transfer through kelp forests in the NE Atlantic is diminished under a warmer ocean climate

Global climate change is affecting carbon cycling by driving changes in primary productivity and rates of carbon fixation, release and storage within Earth's vegetated systems. There is, however, limited understanding of how carbon flow between donor and recipient habitats will respond to climatic changes. Macroalgal‐dominated habitats, such as kelp forests, are gaining recognition as important carbon donors within coastal carbon cycles, yet rates of carbon assimilation and transfer through these habitats are poorly resolved. Here, we investigated the likely impacts of ocean warming on coastal carbon cycling by quantifying rates of carbon assimilation and transfer in Laminaria hyperborea kelp forests—one of the most extensive coastal vegetated habitat types in the NE Atlantic—along a latitudinal temperature gradient. Kelp forests within warm climatic regimes assimilated, on average, more than three times less carbon and donated less than half the amount of particulate carbon compared to those from cold regimes. These patterns were not related to variability in other environmental parameters. Across their wider geographical distribution, plants exhibited reduced sizes toward their warm‐water equatorward range edge, further suggesting that carbon flow is reduced under warmer climates. Overall, we estimated that Laminaria hyperborea forests stored ~11.49 Tg C in living biomass and released particulate carbon at a rate of ~5.71 Tg C year^?1. This estimated flow of carbon was markedly higher than reported values for most other marine and terrestrial vegetated habitat types in Europe. Together, our observations suggest that continued warming will diminish the amount of carbon that is assimilated and transported through temperate kelp forests in NE Atlantic, with potential consequences for the coastal carbon cycle. Our findings underline the need to consider climate‐driven changes in the capacity of ecosystems to fix and donate carbon when assessing the impacts of climate change on carbon cycling.     

The lymphoid transcription factor LyF-1 is encoded by specific, alternatively spliced mRNAs derived from the Ikaros gene.

The lymphocyte-specific DNA-binding protein LyF-1 interacts with a critical control element in the terminal deoxynucleotidyltransferase (TdT) promoter as well as with the promoters for other genes expressed during early stages of B- and T-cell development. We have purified LyF-1 and have obtained a partial amino acid sequence from proteolytic peptides. The amino acid sequence suggests that LyF-1 is a zinc finger protein encoded by the Ikaros gene, which previously was implicated in T-cell development. Recombinant Ikaros expressed in Escherichia coli bound to the TdT promoter, and antisera directed against the recombinant protein specifically blocked the DNA-binding activity of LyF-1 in crude extracts. Further analysis revealed that at least six distinct mRNAs are derived from the Ikaros/LyF-1 gene by alternative splicing. Only two of the isoforms possess the N-terminal zinc finger domain that is necessary and sufficient for TdT promoter binding. Although both of these isoforms bound to similar sequences in the TdT, lambda 5, VpreB, and lck promoters, one isoform contains an additional zinc finger that resulted in altered recognition of some binding sites. At least four of the Ikaros/LyF-1 isoforms were detectable in extracts from B- and T-cell lines, with the relative amounts of the isoforms varying considerably. These data reveal that the LyF-1 protein is encoded by specific mRNAs derived from the alternatively-spliced Ikaros gene, suggesting that this gene may be important for the early stages of both B- and T-lymphocyte development.     

Role of photoperiod during pregnancy and lactation in the meadow vole, Microtus pennsylvanicus

The influence of daylength on body mass and food intake of pregnant and lactating voles was tested by comparing animals housed in long versus short daylengths. Pregnancy rates were approximately 50% in long-day females and in voles kept in short days beginning 2 weeks before mating, but were significantly lower in voles preadapted to short days for 8 weeks before mating. Body mass and food intake increased substantially during pregnancy and lactation and the magnitude of the increase was unaffected by daylength; by contrast, body weight was significantly reduced in non-impregnated voles kept in short as compared to long days. The suppressive effects of short photoperiods on body weight were completely counteracted during pregnancy and lactation. Voles that breed during the short days of winter face extreme energetic challenges but the advantages of early breeding appear to justify the costs.