Name: visualizing neuroblast cytokinesis during c. elegans embryogenesis
Uploaded: 2014-03-12T18:05:00
Description: Watch this Scientific Journal Video about Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis at JoVE.com

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

1.&#160;Preparation of Plates for Maintaining Worm Strains and Performing RNAi
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	<li>Nematode Growth Media Plates
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		<li>Plates
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			<li>Prepare Nematode Growth Media (NGM) plates to maintain worm strains and to perform genetic crosses. Combine 3 g NaCl, 17 g Agar and 2.5 g BactoPeptone with 1 L of distilled water in a 2 L flask and add a metal stirring bar.</li>
			<li>Autoclave the flask to dissolve the agar and to sterilize the media. Then place the flask on a stir plate and allow the media to cool whi...

<ol>
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Genome sequence of the nematode C. elegans: a platform for investigating biology.</a> Science. 282, 2012-2018 (1998).</li><li>Pennisi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worming+secrets+from+the+C.+elegans+genome.">Worming secrets from the C. elegans genome.</a> Science. 282, 1972-1974 (1998).</li><li>Tabara, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rde-1+gene,+RNA+interference,+and+transposon+silencing+in+C.+elegans.">The rde-1 gene, RNA interference, and transposon silencing in C. elegans.</a> Cell. 99, 123-132 (1999).</li><li>Chisholm, A. D., Hardin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Epidermal morphogenesis. WormBook. The C. elegans Research Community. , 1-22 (2005).</li><li>Zhang, H., Gally, C., Labouesse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+morphogenesis:+how+multiple+cells+cooperate+to+generate+a+tissue.">Tissue morphogenesis: how multiple cells cooperate to generate a tissue.</a> Curr. Opin. Cell Biol. 22, 575-582 (2010).</li><li>Kiehart, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing:+the+power+of+the+purse+string.">Wound healing: the power of the purse string.</a> Curr. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+divergent+C.+elegans+ephrin+EFN-4+functions+in+embryonic+morphogenesis+in+a+pathway+independent+of+the+VAB-1+Eph+receptor.">The divergent C. elegans ephrin EFN-4 functions in embryonic morphogenesis in a pathway independent of the VAB-1 Eph receptor.</a> Development. 129, 5499-5510 (2002).</li><li>Ghenea, S., Boudreau, J. R., Lague, N. P., Chin-Sang, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+VAB-1+Eph+receptor+tyrosine+kinase+and+SAX-3/Robo+neuronal+receptors+function+together+during+C.+elegans+embryonic+morphogenesis.">The VAB-1 Eph receptor tyrosine kinase and SAX-3/Robo neuronal receptors function together during C. elegans embryonic morphogenesis.</a> Development. 132, 3679-3690 (2005).</li><li>Bernadskaya, Y. Y., Wallace, A., Nguyen, J., Mohler, W. A., Soto, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UNC-40/DCC,+SAX-3/Robo,+and+VAB-1/Eph+polarize+F-actin+during+embryonic+morphogenesis+by+regulating+the+WAVE/SCAR+actin+nucleation+complex.">UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph polarize F-actin during embryonic morphogenesis by regulating the WAVE/SCAR actin nucleation complex.</a> PLoS Genet. 8, (2012).</li><li>Piekny, A. J., Werner, M., Glotzer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokinesis:+welcome+to+the+Rho+zone.">Cytokinesis: welcome to the Rho zone.</a> Trends Cell Biol. 15, 651-658 (2005).</li><li>Piekny, A. J., Maddox, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myriad+roles+of+Anillin+during+cytokinesis.">The myriad roles of Anillin during cytokinesis.</a> Sem. Cell Dev. Biol. 21, 881-891 (2010).</li><li>Zhao, W. M., Fang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anillin+is+a+substrate+of+anaphase-promoting+complex/cyclosome+(APC/C)+that+controls+spatial+contractility+of+myosin+during+late+cytokinesis.">Anillin is a substrate of anaphase-promoting complex/cyclosome (APC/C) that controls spatial contractility of myosin during late cytokinesis.</a> J. Biol. Chem. 280, 33516-33524 (2005).</li><li>Piekny, A. J., Glotzer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anillin+is+a+scaffold+protein+that+links+RhoA,+actin+and+myosin+during+cytokinesis.">Anillin is a scaffold protein that links RhoA, actin and myosin during cytokinesis.</a> Curr. Biol. 18, 30-36 (2008).</li><li>Hickson, G. R., O&#8217;Farrell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-dependent+control+of+anillin+behavior+during+cytokinesis.">Rho-dependent control of anillin behavior during cytokinesis.</a> J. Cell Biol. 180, 285-294 (2008).</li><li>Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+analysis+of+C.+elegans+chromosome+I+by+systematic+RNA+interference.">Functional genomic analysis of C. elegans chromosome I by systematic RNA interference.</a> Nature. 408, 325-330 (2000).</li><li>Kamath, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+functional+analysis+of+the+Caenorhabditis+elegans+genome+using+RNAi.">Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.</a> Nature. 421, 231-237 (2003).</li><li>Duerr, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunohistochemistry. WormBook, The C. elegans Research Community. , 1-61 (2006).</li><li>Maddox, A. S., Habermann, B., Desai, A., Oegema, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+two+C.+elegans+anillins+in+the+gonad+and+early+embryo.">Distinct roles for two C. elegans anillins in the gonad and early embryo.</a> Development. 132, 2837-2848 (2005).</li><li>Sulston, J. E., Schierenberg, E., White, J. G., Thomson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+cell+lineage+of+the+nematode+Caenorhabditis+elegans.">The embryonic cell lineage of the nematode Caenorhabditis elegans.</a> Dev. Biol. 100, 64-119 (1983).</li><li>Chin-Sang, I. D., Chisholm, A. 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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.

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			<td height="21" style="height:21px;width:291px;">Agar</td>
			<td style="width:247px;">BioShop Canada Inc.</td>
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			<td style="width:335px;">For making C. elegans NGM and RNAi plates</td>
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			<td height="21" style="height:21px;">Agar</td>
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			<td>Sigma-Aldrich&#160;</td>
			<td>#P8920</td>
			<td>For optimal results coat microscope slides three times&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin</td>
			<td>BioShop Canada Inc.</td>
			<td>#STP101.50</td>
			<td>Store powder at 4 &#176;C and dissolved streptomycin at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracyclin</td>
			<td>BioShop Canada Inc.</td>
			<td>#TET701.10</td>
			<td>Store powder at 4 &#176;C and dissolved tetracycline at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Bio Basic Inc.</td>
			<td>CAS#9005-64-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>BioShop Canada Inc.</td>
			<td>#TRP402.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ...

Watch this Scientific Journal Video about Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis at JoVE.com

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 ...

Research

JoVE Journal

Neuroscience

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

In vivo Laser Axotomy in C. elegans

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

C. elegans Tracking and Behavioral Measurement

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...