Direct cell lineage analysis in Drosophila melanogaster by time-lapse, three-dimensional optical microscopy of living embryos (original) (raw)
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Embryonic lineage analysis using three-dimensional, time-lapse in-vivo fluorescent microscopy
Bioimaging and Two-Dimensional Spectroscopy, 1990
Drosophila melanogaster has become one of the most extensively studied organisms because of its amenability to genetic analysis. Unfortunately, the biochemistry and cell biology ofDrosophila has lagged behind. To this end we have been microinjecting fluorescently labelled proteins into the living embryo and observing the behavior of these proteins to determine their role in the cell cycle and development. Imaging of these fluorescent probes is an extremely important element to this form of analysis. We have taken advantage of the sensitivity and well behaved characteristics of the charge coupled device (CCD) camera in conjunction with digital image enhancement schemes to produce highly accurate images of these fluorescent probes in vivo. One of our major goals is to produce a detailed map of cell fate so that we can understand how fate is determined and maintained. In order produce such a detailed map, protocols for following the movements and mitotic behavior of a large number of cells in three dimensions over relatively long periods of time were developed. We will present our results using fluorescently labelled histone proteins as a marker for nuclear location1. In addition, we will also present our initial results using a photoactivatable analog of fluorescein to mark single cells so that their long range fate can be unambiguously determined.
Journal of Theoretical Biology, 1995
Time-lapse microscopy of biological systems has provided new and exciting information about the dynamics of cellular and developmental events. However, these events are often complex and difficult to analyze. This paper describes a study in which computation was indispensable for formulating and evaluating a cellular/developmental hypothesis directly from observations of time-lapse fluorescence images. Previous analyses of time-lapse microscopy sequences of Drosophila melanogaster embryonic syncytial nuclear cycles 10-13, when the nuclei form an evenly spaced monolayer at the surface of the embryo, have failed to identify any pattern in these divisions. However, computational analysis of the data has provided evidence that the direction of syncytial nuclear mitosis is not random, but is clearly influenced by the relative positions of neighboring nuclei. An approximate law governing mitotic direction that is based on a scheme that compromises among ''votes'' made by neighboring nuclei is introduced.
The onset of homologous chromosome pairing during Drosophila melanogaster embryogenesis
The Journal of cell …, 1993
We have determined the position within the nucleus of homologous sites of the histone gene cluster in Drosophila melanogaster using in situ hybridization and high-resolution, three-dimensional wide field fluorescence microscopy. A 4.8-kb biotinylated probe for the histone gene repeat, located approximately midway along the short arm of chromosome 2, was hybridized to whole-mount embryos in late syncytial and early cellular blastoderm stages. Our results show that the two homologous histone loci are distinct and separate through all stages of the cell cycle up to nuclear cycle 13. By dramatic contrast, the two homologous clusters were found to colocalize with high frequency during interphase of cycle 14. Concomitant with homolog pairing at cycle 14, both histone loci were also found to move from their position near the midline of the nucleus toward the apical side. This result suggests that coincident with the initiation of zygotic transcription, there is dramatic chromosome and nuclear reorganization between nuclear cycles 13 and 14.
Genetics, 1987
An embryonic cell marker system has been developed in Drosophila melanogaster that has enabled us to identify the genotype of cells as early as the cellular blastoderm stage of development. This system allows unambiguous detection of embryos homozygous for most X-linked lethal mutations at stages prior to when their first defects become obvious. By examining gynandromorphs at this stage, we have observed that the number of nuclei per unit area in male regions is about half that in female regions. An examination of early cleavage stage embryos whose DNA has been stained with Hoechst 33258 and whose actin has been stained with phalloidin suggests that this difference is due to a cell cycle delay in cells losing the ring-X. These experiments also demonstrate the existence of a mechanism which controls the timing of nuclear divisions in cycle 10-14 embryos.
Drosophila under the lens: imaging from chromosomes to whole embryos
Chromosome Research, 2006
Microscopy has been a very powerful tool for Drosophila research since its inception, proving to be essential for the evaluation of mutant phenotypes, the understanding of cellular and tissue physiology, and the illumination of complex biological questions. In this article we review the breadth of this field, making note of some of the seminal papers. We expand on the use of microscopy to study questions related to gene locus and nuclear architecture, presenting new data using fluorescence in-situ hybridization techniques that demonstrate the flexibility of Drosophila chromosomes. Finally, we review the burgeoning use of fluorescence in-vivo imaging methods to yield quantitative information about cellular processes.
Spatial Organization of the Drosophila Nucleus: A Three-Dimensional Cytogenetic Study
Journal of Cell Science, 1984
SUMMARY The combination of optical fluorescence microscopy with digital image processing and analysis has been used to examine the three-dimensional organization of chromosomes within intact polytene nuclei. Although the arrangement indicates a high degree of flexibility, there are many conserved features between nuclei at the same developmental state. For example, chromosome arms are loosely coiled with centromeres clustered at the opposite end of the nucleus from the telomeres. Individual chromosome arms are not interwoven but occupy different spatial domains. Chromosomal sites that contact the envelope correlate with intercalary heterochromatin. Connections are observed between actively transcribing regions.
Imaging Cell Shape Change in Living Drosophila Embryos
Journal of Visualized Experiments
The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1) 1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells. The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility 6 , but is instead fueled largely by exocytosis of membrane from internal stores 7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle. Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1) 8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques 9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.
Lineage analysis of transplanted individual cells in embryos of Drosophila melanogaster
Roux's Archives of Developmental Biology, 1987
A method is presented which allows the study of the progeny of single cells during Drosophila embryogenesis. Cells from various larval anlagen of donor embryos labelled with a lineage tracer are individually transplanted from defined positions into similar, or different, positions in unlabelled hosts. The clones produced by these cells can be seen in whole mounts or in sections of fixed material, when using a histochemical marker (i. e. HRP), and/or in living embryos, when using fluorescent lineage tracers. The characteristics of the clones disclose lineage parameters, such as division patterns, morphogenetic movements and differentiation. The method is especially useful for testing the respective roles of positional information and cell lineage on the commitment of progenitor cells by transplanting these cells into heterotopic positions or into hosts of different genotypes.
Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
Current Protocols in Cell Biology, 2001
This unit describes fluorescence-based techniques for noninvasive imaging of development in living Drosophila embryos, discussing considerations for fluorescent imaging within living embryos and providing protocols for generation of flies expressing fluorescently tagged proteins and for preparation of embryos for fluorescent imaging. The unit details time-lapse confocal imaging of live embryos and discusses optimizing image acquisition and performing three-dimensional imaging. Finally, the unit provides a variety of specific methods for optical highlighting of specific subsets of fluorescently tagged proteins and organelles in the embryo, including fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and photoactivation techniques, permitting analysis of specific movements of fluorescently tagged proteins within cells. These protocols, together with the relative ease of generating transgenic animals and the ability to express tagged proteins in specific tissues or at specific developmental times, provide powerful means for examining in vivo behavior of any tagged protein in embryos in myriad mutant backgrounds.