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)}     

Culturing of avian embryos for time-lapse imaging

Monitoring morphogenetic processes, at high resolution over time, has been a long-standing goal of many developmental cell biologists. It is critical to image cells in their natural environment whenever possible; however, imaging many warm-blooded vertebrates, especially mammals, is problematic. At early stages of development, birds are ideal for imaging, since the avian body plan is very similar to that of mammals. We have devised a culturing technique that allows for the acquisition of high-resolution differential interference contrast and epifluorescence images of developing avian embryos in a 4-D (3-D + time) system. The resulting information, from intact embryos, is derived from an area encompassing several millimeters, at micrometer resolution for up to 30 h.     

Ex vivo time-lapse confocal imaging of the mouse embryo aorta

Time-lapse confocal microscopy of mouse embryo slices was developed to access and image the living aorta. In this paper, we explain how to label all hematopoietic and endothelial cells inside the intact mouse aorta with fluorescent directly labeled antibodies. Then we describe the technique to cut nonfixed labeled embryos into thick slices that are further imaged by time-lapse confocal imaging. This approach allows direct observation of the dynamic cell behavior in the living aorta, which was previously inaccessible because of its location deep inside the opaque mouse embryo. In particular, this approach is sensitive enough to allow the experimenter to witness the transition from endothelial cells into hematopoietic stem/progenitor cells in the aorta, the first site of hematopoietic stem cell generation during development. The protocol can be applied to observe other embryonic sites throughout mouse development. A complete experiment requires ～2 d of practical work.     

High-resolution 3-D imaging of living cells in suspension using confocal axial tomography

Conventional flow cytometry (FC) methods report optical signals integrated from individual cells at throughput rates as high as thousands of cells per second. This is further combined with the powerful utility to subsequently sort and/or recover the cells of interest. However, these methods cannot extract spatial information. This limitation has prompted efforts by some commercial manufacturers to produce state-of-the-art commercial flow cytometry systems allowing fluorescence images to be recorded by an imaging detector. Nonetheless, there remains an immediate and growing need for technologies facilitating spatial analysis of fluorescent signals from cells maintained in flow suspension. Here, we report a novel methodological approach to this problem that combines micro-fluidic flow, and microelectrode dielectric-field control to manipulate, immobilize and image individual cells in suspension. The method also offers unique possibilities for imaging studies on cells in suspension. In particular, we report the system's immediate utility for confocal "axial tomography" using micro-rotation imaging and show that it greatly enhances 3-D optical resolution compared with conventional light reconstruction (deconvolution) image data treatment. That the method we present here is relatively rapid and lends itself to full automation suggests its eventual utility for 3-D imaging cytometry.     

Enzyme-linked sensitive fluorometric imaging of glutamate release from cerebral neurons of chick embryos

This paper describes a method for imaging the endogenous release of glutamate from cerebral neurons. This method is based on the reactions of glutamate oxidase and peroxidase, and on the detection of hydrogen peroxide by a fluorescent substrate of peroxidase. Glutamate has been sensitively measured in vitro in the range of 20 nM to 1 microM. We used two types of Ca(2+) channel inhibitors, MK-801 and omega-Conotoxin GVIA, which act to suppress Ca(2+) transport at postsynaptic and presynaptic neurons, respectively. MK-801 did not inhibit the increase in glutamate release after KCl stimulation, while there was no increase in glutamate release after KCl stimulation when omega-Conotoxin GVIA was used, probably due to the inhibition of voltage-activated Ca(2+) channels in the presynapse. Glutamate release and Ca(2+) flow in the synaptic regions were imaged using a laser confocal fluorescence microscope. KCl-evoked glutamate release was localized around cell bodies linked to axon terminals. This procedure allows imaging that can be sensitively detected by the fluorometric enzymatic assay of endogenous glutamate release in synapses.     

Cultivation of trematodes in chick embryos

The chick embryo can be used as a substitute for adult animals in the cultivation of a variety of parasites. Bernard Fried describes its use in cultivating trematodes, and suggests that the system might be useful as a preliminary chemotherapeutic screen.     

Full developmental potential of mammalian preimplantation embryos is maintained after imaging using a spinning-disk confocal microscope

Fluorescent live imaging of cells and embryos at subcellular resolution poses significant challenges for biologists due to morbidity and mortality ensuing from phototoxicity. Here we report the use of a spinning-disk confocal microscope to image mouse and bovine preimplantation embryos without impairing their developmental potential. We also present data indicating that this imaging technique does not affect the functionality of subcellular components as assessed by reactive oxygen species (ROS) production, caspase activity, and DNA integrity. Spinning-disk confocal microscopy was also useful in determining cell number and allocation in transgenic bovine blastocysts. We conclude that this imaging method is suitable for monitoring preimplantation embryos.     

Equivalent ages in rat, mouse and chick embryos.

Data for estimating equivalent ages of rat, mouse and chick embryos are presented, using several sources from the literature. When the time of development of various embryonic structures is matched for the three species, the differences in time for the stages are primarily due to delays in development of the preimplantation blastulas of the rat and mouse in comparison with the incubated chick egg. Development from the blastula stage to completion of major organogenesis proceeds at approximately the same rate for all three species.     

Direct cell lineage analysis in Drosophila melanogaster by time-lapse, three-dimensional optical microscopy of living embryos

One of the first signs of cell differentiation in the Drosophila melanogaster embryo occurs 3 h after fertilization, when discrete groups of cells enter their fourteenth mitosis in a spatially and temporally patterned manner creating mitotic domains (Foe, V. E. and G. M. Odell, 1989, Am. Zool. 29:617-652). To determine whether cell residency in a mitotic domain is determined solely by cell position in this early embryo, or whether cell lineage also has a role, we have developed a technique for directly analyzing the behavior of nuclei in living embryos. By microinjecting fluorescently labeled histones into the syncytial embryo, the movements and divisions of each nucleus were recorded without perturbing development by using a microscope equipped with a high resolution, charge-coupled device. Two types of developmental maps were generated from three-dimensional time-lapse recordings: one traced the lineage history of each nucleus from nuclear cycle 11 through nuclear cycle 14 in a small region of the embryo; the other recorded nuclear fate according to the timing and pattern of the 14th nuclear division. By comparing these lineage and fate maps for two embryos, we conclude that, at least for the examined area, the pattern of mitotic domain formation in Drosophila is determined by the position of each cell, with no effect of cell lineage.     

Gene manipulation of chick embryos in vitro, early chick culture, and long survival in transplanted eggs

We introduce a revolutionary gene transfer system in chick: transfect chick embryos at early developmental stage by electroporation in vitro, Early Chick (EC) culture, and transplant to the egg to let the embryo survive until E5.5. Referring to the fate map, we could target the tissues of transfection, or transfect large areas of the embryo. We could get tissue-specific expression of a transgene by tissue-specific promoter. This method is very convenient and rapid, but allows us to get stable expression of the transgene in combination with transposon system.     

Teratogenic effects of phenytoin on chick embryos

The antiepileptic drug phenytoin was injected into the yolk sac of White Leghorn chick embryos. A dose-response study was followed by a detailed teratological study using a single dose of 3 mg. The surviving embryos were sacrificed on the 19th day of incubation. The embryos showed a generalized decrease in body weight together with a wide range of malformations. The malformations could be roughly divided into limb, craniofacial, abdominal, and ocular defects, as well as deficiencies in growth. Skeletal defects included hypoplasia of digital phalanges and nails and shortened wings.     

Expression of DLX3 in chick embryos

Higher vertebrates appear to possess six genes encoding a homeodomain of the distal-less type. We report the cloning and expression pattern of the chicken DLX3 gene, a homeobox gene highly related to the DLX5 gene with regard to both the encoded protein structure and the expression pattern. DLX3 RNA was observed during the development of the olfactory and otic placodes, in the distal portion of the first and second visceral arch mesenchyme, in the growing limb buds, and in the tail tip. No expression occurs in the central nervous system.     

Cholinesterase in the amnion of chick embryos

Cholinesterase (ChE) activity in amnion was studied in developing chick embryos and the enzyme's substrate-inhibitory characteristics were established. The enzyme activity increased until the 8th day of incubation and then gradually decreased; on day 12-15 the activity is 40% only of the maximal one. On the basis of substrate-inhibitory analysis the enzyme was referred to propionyl-cholinesterases. Relations between age changes in ChE activity and morphological structure of smooth-muscle amnion tissue, its differentiation during development and functional activity of amnion are discussed.     

Ribosome crystallization in nuclei of chick embryos

Ribosome crystallization within nuclei has been studied in chick embryos with procedures which increase its frequency by various orders of magnitude as compared to previous findings. The extrusion of ribosome microcrystals from nuclei is reported for the first time, and a model for the transfer of ribosomes from nucleus to cytoplasm is proposed.     

Propagation of Chandipura virus in chick embryos

Stocks of three Indian Chandipura virus (CHPV) isolates; one isolate from an adult febrile case in 1965 from Chandipura town, Maharashtra, and two isolates from two pediatric encephalitis cases from Andhra Pradesh, 2003 were inoculated in 10-day-old chick embryos by allantoic route. All three virus isolates replicated in chick embryos showing titre of log 10(12) to log 10(13) EID50. The results demonstrated that chick embryos are susceptible to CHPV and virus grows to high titres in this system. Therefore chick embryos can be used as an alternative host system for cultivation and isolation of CHPV as they are less expensive than laboratory animals and have several other advantages over cell cultures. Also this system can be used for the development of vaccine and diagnostic reagents.