Time-lapse Live Imaging Research Articles

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36 hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

Timing of neurogenesis is a determinant of olfactory circuitry

An odorant receptor map in mammals, that is constructed by the glomerular coalescence of sensory neuron axons in the olfactory bulb, is essential for proper odor information processing. However, how this map is linked with olfactory cortex is unknown. Here, we use a battery of methods, including various markers of cell division in combination with tracers of neuronal connections and time-lapse live imaging, to show that early- and late-generated mouse mitral cells become differentially distributed within the dorsal and ventral subdivisions of the odorant receptor map. In addition, we demonstrate that the late-generated mitral cells extend significantly stronger projections to the olfactory tubercle than the early-generated. Together, these data indicate that the odorant receptor map is developmentally linked to the olfactory cortices in part by the birthdate of mitral cells. This endows different olfactory cortical regions a role to process information from distinct regions of odorant receptor map.

Trafficking and Surface Expression of Hyperpolarization-activated Cyclic Nucleotide-gated Channels in Hippocampal Neurons

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate the hyperpolarization-activated current I(h) and thus play important roles in the regulation of brain excitability. The subcellular distribution pattern of the HCN channels influences the effects that they exert on the properties and activity of neurons. However, little is known about the mechanisms that control HCN channel trafficking to subcellular compartments or that regulate their surface expression. Here we studied the dynamics of HCN channel trafficking in hippocampal neurons using dissociated cultures coupled with time lapse imaging of fluorophore-fused HCN channels. HCN1-green fluorescence protein (HCN1-GFP) channels resided in vesicle-like organelles that moved in distinct patterns along neuronal dendrites, and these properties were isoform-specific. HCN1 trafficking required intact actin and tubulin and was rapidly inhibited by activation of either NMDA or AMPA-type ionotropic glutamate receptors in a calcium-dependent manner. Glutamate-induced inhibition of the movement of HCN1-GFP-expressing puncta was associated with increased surface expression of both native and transfected HCN1 channels, and this surface expression was accompanied by augmented I(h). Taken together, the results reveal the highly dynamic nature of HCN1 channel trafficking in hippocampal neurons and provide a novel potential mechanism for rapid regulation of I(h), and hence of neuronal properties, via alterations of HCN1 trafficking and surface expression.

Time‐Lapse Live Imaging of Stem Cells inDrosophilaTestis

This unit describes a protocol for time-lapse live-imaging of stem cells in Drosophila testis. Testis tips are dissected from Drosophila, sliced, and transferred to glass-bottom chambers where the stem cells residing in their native microenvironment can be monitored in real time. This protocol, facilitated with various fluorescence-labeled markers, allows dynamic cellular processes in stem cells to be characterized throughout the cell cycle.

Phosphatidylinositol-phosphates mediate cytoskeletal reorganization during phagocytosis via a unique modular protein consisting of RhoGEF/DH and FYVE domains in the parasitic protozoonEntamoeba histolytica

To understand the roles of phosphoinositides [PtdIns] in phagocytosis of parasitic eukaryotes, we examined the interaction of phosphatidylinositol-3-phosphate [PtdIns(3)P] and putative PtdIns-P-binding proteins during phagocytosis in the enteric protozoan parasite Entamoeba histolytica. It was previously shown that phagocytosis in E. histolytica is indispensable for virulence and is inhibited by PtdIns 3-kinase inhibitors. We demonstrated by time-lapse live imaging that during the initiation of phagocytosis, the PtdIns(3)P biomarker GFP-Hrs-FYVE, was translocated to the phagocytic cup, phagosome, and to tunnel-like structures connecting the plasma membrane and phagosomes. E. histolytica possesses 12 FYVE domain-containing proteins (EhFP1-12), 11 of which also contain the RhoGEF/DH domain. Among them EhFP4 was shown to be recruited to the tunnel-like structures and to the proximal region of the phagosome. We further demonstrated that EhFP4 physically interacted with 4 of 10 predominant Rho/Rac small GTPases. Phosphoinositide binding assay showed that EhFP4 unexpectedly bound to PtdIns(4)P via the carboxyl-terminal domain and that the FYVE domain modulates the binding specificity of EhFP4 to PtdIns-P. Expression of the FYVE domain from EhFP4 inhibited phagocytosis while enhancement was observed when mammalian Hrs-FYVE domain was expressed. Altogether, we demonstrated that PtdIns(3)P, PtdIns(4)P and EhFP4 coordinately regulate phagocytosis and phagosome maturation in this parasitic eukaryote.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

Live Imaging and Genetic Analysis of Mouse Notochord Formation Reveals Regional Morphogenetic Mechanisms

The node and notochord have been extensively studied as signaling centers in the vertebrate embryo. The morphogenesis of these tissues, particularly in mouse, is not well understood. Using time-lapse live imaging and cell lineage tracking, we show the notochord has distinct morphogenetic origins along the anterior-posterior axis. The anterior head process notochord arises independently of the node by condensation of dispersed cells. The trunk notochord is derived from the node and forms by convergent extension. The tail notochord forms by node-derived progenitors that actively migrate toward the posterior. We also reveal distinct genetic regulation within these different regions. We show that Foxa2 compensates for and genetically interacts with Noto in the trunk notochord, and that Noto has an evolutionarily conserved role in regulating axial versus paraxial cell fate. Therefore, we propose three distinct regions within the mouse notochord, each with unique morphogenetic origins.

Fission yeast MO25 protein is localized at SPB and septum and is essential for cell morphogenesis

Cell morphogenesis is of fundamental significance in all eukaryotes for development, differentiation, and cell proliferation. In fission yeast, Drosophila Furry-like Mor2 plays an essential role in cell morphogenesis in concert with the NDR/Tricornered kinase Orb6. Mutations of these genes result in the loss of cell polarity. Here we show that the conserved proteins, MO25-like Pmo25, GC kinase Nak1, Mor2, and Orb6, constitute a morphogenesis network that is important for polarity control and cell separation. Intriguingly, Pmo25 was localized at the mitotic spindle pole bodies (SPBs) and then underwent translocation to the dividing medial region upon cytokinesis. Pmo25 formed a complex with Nak1 and was required for both the localization and kinase activity of Nak1. Pmo25 and Nak1 in turn were essential for Orb6 kinase activity. Further, the Pmo25 localization at the SPBs and the Nak1-Orb6 kinase activities during interphase were under the control of the Cdc7 and Sid1 kinases in the septation initiation network (SIN), suggesting a functional linkage between SIN and the network for cell morphogenesis/separation following cytokinesis.

Fission yeast ch-TOG/XMAP215 homologue Alp14 connects mitotic spindles with the kinetochore and is a component of the Mad2-dependent spindle checkpoint.

The TOG/XMAP215-related proteins play a role in microtubule dynamics at its plus end. Fission yeast Alp14, a newly identified TOG/XMAP215 family protein, is essential for proper chromosome segregation in concert with a second homologue Dis1. We show that the alp14 mutant fails to progress towards normal bipolar spindle formation. Intriguingly, Alp14 itself is a component of the Mad2-dependent spindle checkpoint cascade, as upon addition of microtubule-destabilizing drugs the alp14 mutant is incapable of maintaining high H1 kinase activity, which results in securin destruction and premature chromosome separation. Live imaging of Alp14-green fluorescent protein shows that during mitosis, Alp14 is associated with the peripheral region of the kinetochores as well as with the spindle poles. This is supported by ChIP (chromatin immunoprecipitation) and overlapping localization with the kinetochore marker Mis6. An intact spindle is required for Alp14 localization to the kinetochore periphery, but not to the poles. These results indicate that the TOG/XMAP215 family may play a central role as a bridge between the kinetochores and the plus end of pole to chromosome microtubules.