The authors would like to acknowledge that this work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) grant.

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The authors have nothing to disclose.

This protocol describes the use of various types of microscopy to image cell divisions during mid embryogenesis. In particular, this protocol highlights how to image the division of neuroblasts, cells that may facilitate epidermal morphogenesis. Cell-cell communication is important for tissue formation during metazoan development and C. elegans is an excellent model to study tissue formation in vivo. One event that nicely portrays the interplay of tissues is epidermal morphogenesis, which covers the emb...

While there are protocols describing how to image cell division in the early C. elegans embryo, this protocol describes how to image cell division within a tissue during mid embryogenesis. One of the major challenges in imaging organisms during development has been their sensitivity to phototoxicity. However, increased accessibility to spinning disk confocal microscopes or swept field microscopes has permitted more widespread imaging applications. Both systems use solid state lasers and scattered light, limiting the levels of UV that the organisms are exposed to. However, widefield stands can still be used for imaging in vivo, particularly if they are outfitted with cameras with high sensitivity (e.g. EMCCD), aperture control and light control (e.g. LEDs or adjustable mercury bulbs). This protocol describes how to use either a confocal based system or a widefield system to image cell division within developing C. elegans embryos. As an example, we describe how to image neuroblast cell division. Neuroblasts may facilitate epidermal morphogenesis by providing chemical or mechanical cues to the overlying epidermal cells, and provide an excellent example of the importance of intercellular communication in the formation of tissues.
Caenorhabditis elegans is an ideal model organism for microscopy based studies due to its transparency and simple tissue organization1. Furthermore, C. elegans is amenable to genetic methods and RNAi, and since many of its genes have human homologues, it can be used to identify conserved mechanisms for tissue formation2-5. In C. elegans, formation of the epidermis occurs during mid embryogenesis, when the embryo has &#62;300 cells. Epidermal morphogenesis consists of several major phases, during which the embryo is enclosed in a layer of epidermal cells that constrict and extend to transform the embryo from an ovoid form into the elongated shape of a worm6. Ventral enclosure describes one of these morphogenetic events, when the ventral epidermal cells migrate towards the ventral midline to cover the ventral surface of the embryo (Figure 1). First, two pairs of anterior located leading edge cells migrate towards the ventral midline, where they adhere and fuse with their contralateral neighbors6. This is followed by migration of the posterior located pocket cells, which form wedge like shapes creating a ventral pocket6-7. The mechanism that closes the pocket is not well understood. One possibility is that a supracellular actin myosin contractile structure ties the pocket cells together in a purse string like fashion, similar to wound healing8. Interestingly, migration of some of the pocket cells is mediated by specific subsets of underlying neuroblasts9 (neuronal precursors that are found underneath the epidermis; Figure 1B).
Previous studies showed that the neuroblasts regulate ventral epidermal cell migration and ventral enclosure.&#160;VAB-1 (Ephrin receptor) and VAB-2 (Ephrin ligand) are highly expressed in the neuroblasts and facilitate the sorting of anterior and posterior neuroblasts from one another, and mutations in vab-1 or vab-2 cause ventral enclosure phenotypes10-13. However, promoter rescue experiments showed that vab-1 is also required in the overlying ventral epidermal cells and receptors for other guidance cues secreted by the neuroblasts are expressed in the ventral epidermal cells9. Although mutations in any of these receptors cause ventral enclosure phenotypes, it is not clear if defects arise due to problems in neuroblast positioning or due to failure of the ventral epidermal cells to respond to guidance cues14. Altering the division of neuroblasts without affecting their ability to secrete guidance cues could shed light on the role of neuroblasts and their ability to provide mechanical input during epidermal morphogenesis. Recently, it was found that a cell division gene, ani-1 (anillin) is highly expressed in neuroblasts (Figure 2A) and its depletion causes neuroblast division defects. Interestingly, these embryos display ventral enclosure phenotypes (Fotopoulos, Wernike and Piekny, unpublished observations).
Anillin is required for cell division, and particularly for cytokinesis, which describes the process where a mother cell physically divides into two daughter cells. Cytokinesis is driven by the formation of an actomyosin contractile ring, which needs to be tightly controlled in space and time to ensure that it is properly coupled with sister chromatid segregation. The master regulator of metazoan cytokinesis is RhoA (RHO-1 in C. elegans), a small GTPase that is active in its GTP-bound form. The GEF Ect2/ECT-2 activates RhoA, after which RhoA-GTP interacts with downstream effectors that form the contractile ring and mediate its ingression15. Anillin is a multi domain protein that binds to RhoA via its C-terminus and to actin and myosin via its N-terminus. Anillin is required to stabilize the position of the contractile ring in mammalian or Drosophila S2 cells16. Anillin depletion causes contractile rings to undergo lateral oscillations, and cytokinesis eventually fails forming multinucleate cells17-19. Interestingly, although C. elegans ani-1 coordinates actomyosin contractility in the early embryo, it is not essential for cytokinesis. However, as described above, ani-1 is required for neuroblast cytokinesis during mid-embryogenesis (Fotopoulos, Wernike and Piekny, unpublished observations). Cytokinesis failure would alter the number and position of neuroblasts and could affect the location of chemical guidance cues, or it could alter the mechanical properties of the tissue. Both models highlight the nonautonomous role of neuroblasts for ventral enclosure, and the importance of tissue-tissue communication during embryonic development.
This experimental protocol describes how to image cell division during C. elegans mid embryogenesis using fluorescence microscopy. The majority of experiments studying the mechanisms of cell division were performed in single cells within culture dishes (e.g. HeLa or S2 cells) or in early embryos with a limited number of cells (e.g. C. elegans one-cell embryo, Xenopus,&#160;or echinoderm embryos). However, it is important to also study cell division within tissues, as there are external cues that can influence the timing and placement of the division plane. Furthermore, cells may provide chemical or mechanical cues to influence the development of neighboring tissues, and it is important to understand how intercellular communication helps tissues to form during development.

<table><tbody><tr><td>Agar</td><td>BioShop Canada Inc.</td><td>#AGR001.1</td><td>For making &lt;em&gt;C. elegans&lt;/em&gt; NGM and RNAi plates</td></tr><tr><td>Agar</td><td>Bio Basic Inc.</td><td>#9002-18-0</td><td>For making bacteria LB agar plates</td></tr><tr><td>Agarose</td><td>BioShop Canada Inc.</td><td>#AGA001.500</td><td></td></tr><tr><td>Anti-mouse Alexa Fluor 488 antibody</td><td>Life Technologies Corporation (Invitrogen)</td><td>#A11029</td><td></td></tr><tr><td>Anti-mouse anti-GFP antibody</td><td>Roche Applied Science</td><td>#11814460001</td><td></td></tr><tr><td>Anti-rabbit Alexa Fluor 568 antibody</td><td>Life Technologies Corporation (Invitrogen)</td><td>#A11011</td><td></td></tr><tr><td>Ampicillin</td><td>BioShop Canada Inc.</td><td>#AMP201.5</td><td>Store powder at 4 &amp;deg;C and dissolved ampicillin at -20 &amp;deg;C</td></tr><tr><td>Bactopetone&amp;nbsp; (peptone-A)</td><td>Bio Basic Inc.</td><td>#G213</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (calcium chloride)</td><td>BioShop Canada Inc.</td><td>#C302.1</td><td></td></tr><tr><td>Cholesterol</td><td>BioShop Canada Inc.</td><td>#CHL380.25</td><td>Dissolve in ethanol</td></tr><tr><td>DAPI&amp;nbsp;</td><td>Sigma-Aldrich</td><td>#D9542</td><td>Use to stain nucleic acids (DNA)</td></tr><tr><td>Glycerol</td><td>BioShop Canada Inc.</td><td>#GLY001.1</td><td></td></tr><tr><td>IPTG (isopropylthio-&amp;beta;-galactoside)</td><td>Bio Basic Inc.</td><td>#367-93-1</td><td>Store powder and dissolved IPTG at -20 &amp;deg;C</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (potassium phosphate, monobasic)</td><td>BioShop Canada Inc.</td><td>#PPM666.1</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (potassium phosphate, dibasic)</td><td>BioShop Canada Inc.</td><td>#PPD303.1</td><td></td></tr><tr><td>L4440&amp;nbsp; (feeding vector)</td><td>Addgene</td><td>#1654</td><td>Keep as glycerol stock at -80 &amp;deg;C</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;nbsp; (magnesium sulfate)</td><td>BioShop Canada Inc.</td><td>#MAG511.500</td><td></td></tr><tr><td>NaCl (sodium chloride)</td><td>Bio Basic Inc.</td><td>#7647-14-5</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (sodium phosphate, dibasic)</td><td>Bio Basic Inc.</td><td>#7558-79-4</td><td></td></tr><tr><td>Normal Donkey Serum (NDS)</td><td>Wisent Bioproducts</td><td>#035-110</td><td></td></tr><tr><td>n-Propyl-3,4,5-trihydroxybenzoate (propyl gallate)</td><td>Alfa Aesar</td><td>#A10877</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>#P8920</td><td>For optimal results coat microscope slides three times&amp;nbsp;</td></tr><tr><td>Streptomycin</td><td>BioShop Canada Inc.</td><td>#STP101.50</td><td>Store powder at 4 &amp;deg;C and dissolved streptomycin at -20 &amp;deg;C</td></tr><tr><td>Tetracyclin</td><td>BioShop Canada Inc.</td><td>#TET701.10</td><td>Store powder at 4 &amp;deg;C and dissolved tetracycline at -20 &amp;deg;C</td></tr><tr><td>Tween-20</td><td>Bio Basic Inc.</td><td>CAS#9005-64-5</td><td></td></tr><tr><td>Tryptone</td><td>BioShop Canada Inc.</td><td>#TRP402.500</td><td></td></tr><tr><td>Yeast Extract</td><td>Bio Basic Inc.</td><td>#8013-01-2</td><td></td></tr></tbody></table>

visualizing neuroblast cytokinesis during c. elegans embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

This experimental protocol describes how to image cell division in C. elegans embryos during mid embryogenesis. In particular, it describes how to image neuroblasts, which may facilitate epidermal morphogenesis. Epidermal morphogenesis occurs due to a combination of epidermal cell shape changes, migration and adhesion, but also relies on chemical or mechanical cues from the underlying neuroblasts (Figure 1B). The neuroblasts secrete guidance cues that are received by receptors on the surface of ...

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Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

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11.8K Views.  Concordia University. This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

Video: Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Developmental Biology

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans ...

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Biology

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Tracking Calcium in the Early Developmental Phases of C. elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 &#176;C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter &#34;CaFE&#34; for Calcium during Fertilization in C. elegans. The CaFE reporter was&#160;engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.

Here, we present protocols and tools for visualizing calcium during fertilization and early embryogenesis using a genetically encoded calcium reporter expressed in the germline of the model nematode C. elegans.

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

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Chapters in this video

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila