International audienceWe implemented two-photon scanned light-sheet microscopy, combining nonlinear excitation with orthogonal illumination of light-sheet microscopy, and showed its excellent performance for in vivo, cellular-resolution, three-dimensional imaging of large biological samples. Live imaging of fruit fly and zebrafish embryos confirmed that the technique can be used to image up to twice deeper than with one-photon light-sheet microscopy and more than ten times faster than with point-scanning two-photon microscopy without compromising normal biology. Cop. 2011 Nature America, Inc. All rights reserved

SUMMARY Patterning of the dorsal-ventral axis in the early Drosophila embryo depends on the nuclear distribution of the Dorsal transcription factor. Using live two-photon light-sheet microscopy, we quantified the nuclear Dorsal gradient in space and time and found that its amplitude and basal levels display oscillations throughout early embryonic development. These dynamics raise questions regarding how cells can reproducibly establish patterns of gene expression from a rapidly varying signal. We therefore quantified domains of Dorsal target genes, discovering their expression patterns are also dynamic. Computational modeling of this system reveals a correlation between Dorsal gradient dynamics and changes in target gene expression and suggests that these dynamics, together with time averaging of noise, results in the formation of graded gene expression borders in regions where the gradient is nearly flat. We propose that mRNA levels remain plastic during transient signaling events, allowing tissues to refine patterns in the face of genetic or environmental variation.

Spatially confined green-to-red photoconversion of fluorescent proteins with high-power, pulsed laser illumination is negligible, thus precluding optical selection of single cells in vivo. We report primed conversion, in which low-power, dual-wavelength, continuous-wave illumination results in pronounced photoconversion. With a straightforward addition to a conventional confocal microscope, we show confined primed conversion in living zebrafish and reveal the complex anatomy of individual neurons packed between neighboring cells.

Telomeres are essential in maintaining chromosome integrity and in controlling cellular replication. Attrition of telomere length in peripheral blood mononuclear cells (PBMCs) with age is well documented from cross-sectional studies. But the actual in vivo changes in telomere lengths and its relationship with the contributing factors within the individuals with age have not been fully addressed. In the present paper, we report a longitudinal analysis of telomere length in the PBMCs, lymphocytes and monocytes of 216 human subjects aged from 20–90 years assessed at 0-, 5- and 12-year follow-up. For the 5- and 12-year follow-up, telomere length in the PBMCs decreased in 34 % and 46 %, exhibited no detectable change in 56 % and 47 % and increased in 10 % and 7 % of the subjects respectively. The rate of telomere change was distinct for T-cells, B-cells and monocytes for any given subject. Telomerase activity declined with age in the resting T-cells and B-cells and the activated T-cells. Finally, a significant portion of telomere attrition in T-cells with age was explained by a decline in the telomerase activity, decreased naïve cells and the change in physiological conditions such as elevated blood glucose and interleukin (IL)-6 levels. These findings show that changes in the telomere length of the PBMCs with age in vivo occur at different rates in different individuals and cell types and reveal that changes in the telomere length in the T-cells with age is influenced by the telomerase activity, naïve T-cell percentage and changes in health conditions.

Sleep is an essential and phylogenetically conserved behavioral state, but it remains unclear to what extent genes identified in invertebrates also regulate vertebrate sleep. RFamide-related neuropeptides have been shown to promote invertebrate sleep, and here we report that the vertebrate hypothalamic RFamide neuropeptide VF (NPVF) regulates sleep in the zebrafish, a diurnal vertebrate. We found that NPVF signaling and npvf-expressing neurons are both necessary and sufficient to promote sleep, that mature peptides derived from the NPVF preproprotein promote sleep in a synergistic manner, and that stimulation of npvf-expressing neurons induces neuronal activity levels consistent with normal sleep. These results identify NPVF signaling and npvf-expressing neurons as a novel vertebrate sleep-promoting system and suggest that RFamide neuropeptides participate in an ancient and central aspect of sleep control.

BackgroundAlterations in the number and composition of lymphocytes and their subsets in blood are considered a hallmark of immune system aging. However, it is unknown whether the rates of change of lymphocytes are stable or change with age, or whether the inter-individual variations of lymphocyte composition are stable over time or undergo different rates of change at different ages. Here, we report a longitudinal analysis of T- and B-cells and their subsets, and NK cells in the blood of 165 subjects aged from 24 to 90 years, with each subject assessed at baseline and an average of 5.6 years follow-up.ResultsThe rates of change of T-(CD4+ and CD8+) and B-cells, and NK cells were relative stable throughout the adult life. A great degree of individual variations in numbers of lymphocytes and their subsets and in the rates of their changes with age was observed. Among them, CD4+ T cells exhibited the highest degree of individual variation followed by NK cells, CD8+ T cells, and B cells. Different types of lymphocytes had distinct trends in their rates of change which did not appear to be influenced by CMV infection. Finally, the rates of CD4+, CD8+ T cells, naive CD4+ and naïve CD8+ T cells were closely positively correlated.ConclusionOur findings provide evidence that the age-associated changes in circulating lymphocytes were at relative stable rates in vivo in a highly individualized manner and the levels of selected cytokines/cytokine receptors in serum might influence these age-associated changes of lymphocytes in circulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12979-016-0079-7) contains supplementary material, which is available to authorized users.

Schlemm's canal is an important structure of the conventional aqueous humor outflow pathway and is critically involved in regulating the intraocular pressure. In this study, we report a novel finding that prospero homeobox protein 1 (Prox-1), the master control gene for lymphatic development, is expressed in Schlemm's canal. Moreover, we provide a novel in vivo method of visualizing Schlemm's canal using a transgenic mouse model of Prox-1-green fluorescent protein (GFP). The anatomical location of Prox-1+ Schlemm's canal was further confirmed by in vivo gonioscopic examination and ex vivo immunohistochemical analysis. Additionally, we show that the Schlemm's canal is distinguishable from typical lymphatic vessels by lack of lymphatic vessel endothelial hyaluronan receptor (LYVE-1) expression and absence of apparent sprouting reaction when inflammatory lymphangiogenesis occurred in the cornea. Taken together, our findings offer new insights into Schlemm's canal and provide a new experimental model for live imaging of this critical structure to help further our understanding of the aqueous humor outflow. This may lead to new avenues toward the development of novel therapeutic intervention for relevant diseases, most notably glaucoma.

The aggregation stage of the life cycle of Dictyostelium discoideum is governed by the chemotactic response of individual amoebae to excitable waves of cAMP. We modeled this process through a recently introduced hybrid automata-continuum scheme and used computer simulation to unravel the role of specific components of this complex developmental process. Our results indicated an essential role for positive feedback between the cAMP signaling and the expression of the genes encoding the signal transduction and response machinery.Upon starvation, Dictyostelium amoebae turn on a genetic program, leading to the formation of aggregates, migrating slugs, and eventually fruiting bodies (1). Aggregation is controlled by nonlinear waves of cAMP that propagate through the colony and "inform" individual cells as to the direction to move toward the nearest aggregation center (2). Often, these waves take the form of rotating spirals that are familiar from the study of excitable chemical media such as the BelusovZhabotinsky reaction (3) or the catalysis of carbon monoxide on a platinum surface (4). These cAMP waves can either be seen by direct methods (5) or inferred based on cell response visualized by darkfield microscopy (6).Many authors have published darkfield images from the early aggregation stage of Dictyostelium (for example, see refs.2 and 6). These wave patterns, believed to be initiated by occasional random cAMP emission, start out with a rather disordered spatial structure. At some point, the systems makes a transition to a state with large wavelength spirals. As time progresses, the spiral wavelength shrinks, leaving the system with large domains, each of which is controlled by a coherent spiral wave. Of particular note is that these patterns have on average a large domain size as compared with the most natural scale available, that of the spiral core (see below). After some number of hours, the cells begin to move and the dynamics becomes more complex. The biochemistry of the signal transduction underlying the cAMP waves has been partially elucidated (7). It is known that the CAR1 receptor detects an above-threshold concentration of cAMP and causes the activation of adenyl cyclase so as to produce (and secrete) additional cAMP. This emission phase is followed by a refractory period during which the receptor is desensitized (8) and the cell cannot be excited. cAMP is degraded by a phosphodiesterase that is itself secreted by the aggregating cells (9). Of crucial importance for our study is the fact that the rate of expression of many critical aggregationstage genes (including carA, adenyl cyclase, etc.) is not constant over the time scale over which the wave patterns develop. Instead, there is a positive feedback loop in which gene expression is stimulated by the detection of cAMP pulses (10). As we shall see, this dynamical mechanism is essential for the generation of the observed coherent spiral patterns.To model cAMP waves and resultant chemotaxis, we use a recently introduced (11, 12) a hybrid methodo...