Satoshi Shimozono

Visualization of an endogenous retinoic acid gradient across embryonic development

In vertebrate development, the body plan is determined by primordial morphogen gradients that suffuse the embryo. Retinoic acid (RA) is an important morphogen involved in patterning the anterior–posterior axis of structures, including the hindbrain and paraxial mesoderm. RA diffuses over long distances, and its activity is spatially restricted by synthesizing and degrading enzymes. However, gradients of endogenous morphogens in live embryos have not been directly observed; indeed, their existence, distribution and requirement for correct patterning remain controversial. Here we report a family of genetically encoded indicators for RA that we have termed GEPRAs (genetically encoded probes for RA). Using the principle of fluorescence resonance energy transfer we engineered the ligand-binding domains of RA receptors to incorporate cyan-emitting and yellow-emitting fluorescent proteins as fluorescence resonance energy transfer donor and acceptor, respectively, for the reliable detection of ambient free RA. We created three GEPRAs with different affinities for RA, enabling the quantitative measurement of physiological RA concentrations. Live imaging of zebrafish embryos at the gastrula and somitogenesis stages revealed a linear concentration gradient of endogenous RA in a two-tailed source–sink arrangement across the embryo. Modelling of the observed linear RA gradient suggests that the rate of RA diffusion exceeds the spatiotemporal dynamics of embryogenesis, resulting in stability to perturbation. Furthermore, we used GEPRAs in combination with genetic and pharmacological perturbations to resolve competing hypotheses on the structure of the RA gradient during hindbrain formation and somitogenesis. Live imaging of endogenous concentration gradients across embryonic development will allow the precise assignment of molecular mechanisms to developmental dynamics and will accelerate the application of approaches based on morphogen gradients to tissue engineering and regenerative medicine.

Engineering FRET constructs using CFP and YFP.

Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Here we discuss how to engineer constructs for FRET between the cyan- and yellow-emitting variants of green fluorescent protein (GFP) from Aequorea victoria (CFP and YFP, respectively). Throughout this chapter, we stress the fact that FRET is highly sensitive to the relative orientation and distance between the donor and the acceptor. The chapter consists of two parts. First, we discuss FRET-based indicators encoded by single genes, which were developed in our laboratory. In this approach, a number of different constructs can be made for a comparative assessment of their FRET efficiencies. For example, the length and sequence of the linker between the fluorescent protein and the host protein should be optimized for each specific application. In the second part, we describe the use of long and flexible linkers for engineering FRET constructs, including an introduction to a general and efficient tool for making successful fusion proteins with long and flexible linkers. When CFP and YFP are fused through floppy linkers to two protein domains that interact with each other, the two fluorescent proteins will associate due to the weak dimerization propensity of Aequorea GFP, which results in moderate FRET. This approach has become even more powerful due to the construction of a new pair of fluorescent proteins for FRET: CyPet and YPet.

Confocal Imaging of Subcellular Ca2+ Concentrations Using a Dual-Excitation Ratiometric Indicator Based on Green Fluorescent Protein

Dual-excitation ratiometric dyes are excited alternately at two different wavelengths, but the emission is collected at a single fixed wavelength. Therefore, the pair of intensity measurements must be collected sequentially. Ratiometric-pericam is a fluorescent Ca2+ indicator based on a chimeric fusion protein of circularly permuted green fluorescent protein and calmodulin. Upon binding to calcium, its excitation peak shifts from 415 nm to 494 nm. Ca2+ imaging using ratiometric-pericam was thought to be inadequate to follow very fast Ca2+ dynamics or Ca2+ changes in highly motile cell samples; however, we describe a technique that allows high spatial and time resolution of images acquired with ratiometric-pericam. To obtain confocal images of Ca2+ using ratiometric-pericam, we established a system in which two laser beams (excitation 408 nm and 488 nm) are alternated on every scanning line under the control of two acousto-optic tunable filters. This system increases the rate at which ratio measurements are done to 200 Hz, and provides confocal images at 1 to 10 Hz depending on the image size. The ratio images are free from noise caused by the fluctuation of laser power, because the system is equipped with a violet laser diode (408 nm) and a diode-pumped solid-state laser (488 nm), both of which are stable. We visualized the dynamic propagation of Ca2+ waves from the cytosol to the nucleus and changes in Ca2+ concentrations in motile mitochondria of HeLa cells. We demonstrate that this new confocal imaging system expands the range of potential applications of ratiometric-pericam and other dual-excitation ratiometric indicators.

Slow Ca2+ dynamics in pharyngeal muscles in Caenorhabditis elegans during fast pumping

The pharyngeal muscles of Caenorhabditis elegans are composed of the corpus, isthmus and terminal bulb from anterior to posterior. These components are excited in a coordinated fashion to facilitate proper feeding through pumping and peristalsis. We analysed the spatiotemporal pattern of intracellular calcium dynamics in the pharyngeal muscles during feeding. We used a new ratiometric fluorescent calcium indicator and a new optical system that allows simultaneous illumination and detection at any two wavelengths. Pumping was observed with fast, repetitive and synchronous spikes in calcium concentrations in the corpus and terminal bulb, indicative of electrical coupling throughout the muscles. The posterior isthmus, however, responded to only one out of several pumping spikes to produce broad calcium transients, leading to peristalsis, the slow and gradual motion needed for efficient swallows. The excitation–calcium coupling may be uniquely modulated in this region at the level of calcium channels on the plasma membrane.

Identification and characterization of an output neuron from the oscillatory molluscan olfactory network.

Synchronous oscillations in olfactory systems have been thought to play critical roles in encoding olfactory information. However, their role in determining behavior is unknown. As a first step toward understanding the decoding process of coherent oscillation, we looked for a neuron in the terrestrial slug Limax marginatus that receives output signals from the procerebrum (PC), which is the olfactory center of Limax. We identified a neuron in the metacerebrum that extends its neurites into both the PC and the metacerebrum, and named it the metacerebro-procerebral neuron (MPN). The MPN exhibited a membrane potential oscillation that was synchronous with the local field potential oscillation in the PC. When we cut the PC off, the membrane potential oscillation of the MPN disappeared. Numerous varicosities were found on the neurites in the metacerebrum, while no varicosities were found on the neurites inside the PC. From these morphological and physiological results, we conclude that the MPN is an output neuron from the PC. The MPN also receives monosynaptic inputs from the superior and inferior tentacle nerves. The MPN thus may receive olfactory information from two pathways, one directly from the sensory organ and the other by way of the PC, possibly functioning to integrate them.

Diffusion of Large Molecules into Assembling Nuclei Revealed Using an Optical Highlighting Technique

The nuclear envelope (NE) defines the nuclear compartment, and nuclear pore complexes (NPCs) on the NE form aqueous passages through which small water-soluble molecules can passively diffuse. It is well known that proteins smaller than 50 kDa can diffuse though NPCs, whereas proteins larger than 60 kDa rarely enter by passive diffusion. Little, however, is known about how this size cutoff develops as the NE reassembles and the nucleus expands. In 1987, a well-known study identified an efficient mechanism by which large diffusing proteins (> 60 kDa) were excluded from the reassembling nucleus after mitosis. Since then, it has been generally accepted that after mitosis, newly formed nuclei completely exclude all proteins except those that are initially bound to the mitotic chromosomes and those that are selectively imported through NPCs. Here, the tetrameric complex of the photoconvertible fluorescent protein KikGR ( approximately 103 kDa) was optically highlighted in the cytoplasm and followed to examine its entry into nuclei. Remarkably, highlighted complexes efficiently entered newly assembled nuclei during an approximately 20-min period after the completion of cytokinesis. Because KikGR contains no known nuclear-localization or chromosome-binding sequences, our results indicate the diffusion barrier is less restrictive during nuclear reassembly.

Single-cell bioluminescence imaging of deep tissue in freely moving animals

Improved spy tactics for single cells Bioluminescence imaging is a tremendous asset to medical research, providing a way to monitor living cells noninvasively within their natural environments. Advances in imaging methods allow researchers to measure tumor growth, visualize developmental processes, and track cell-cell interactions. Yet technical limitations exist, and it is difficult to image deep tissues or detect low cell numbers in vivo. Iwano et al. designed a bioluminescence imaging system that produces brighter emission by up to a factor of 1000 compared with conventional technology (see the Perspective by Nasu and Campbell). Individual tumor cells were successfully visualized in the lungs of mice. Small numbers of striatal neurons were detected in the brains of naturally behaving marmosets. The ability of the substrate to cross the blood-brain barrier should provide important opportunities for neuroscience research. Science, this issue p. 935; see also p. 868 A bioengineered light source allows in vivo imaging of individual cells. Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.

Optical recording of oscillatory neural activities in the molluscan brain

The procerebrum (PC) of the terrestrial slug shows a coherent oscillatory activity. Information is encoded in the PC by neurons with synchronized oscillatory activity, and the oscillatory activity will propagate to other brain regions as the PC transmits information. Using the optical recording of membrane potentials and the correlation analysis, we showed that the metacerebrum/mesocerebrum (MC) region also shows an oscillatory activity coherent with that of the PC. The MC oscillation was either inphase or antiphase with the PC oscillation, and its amplitude was larger when it was antiphase than it was inphase. These results indicate that the MC is capable of producing an oscillatory activity, possibly driven by synaptic input from the PC.