Name: in vitro reconstitution of spatial cell contact patterns with isolated caenorhabditis elegans embryo blastomeres and adhesive polystyrene beads
Uploaded: 2019-11-26T13:30:00
Description: Watch this Scientific Journal Video about In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads at JoVE.com

We thank James Priess and Bruce Bowerman for advice and providing C. elegans strains, Don Moerman, Kota Mizumoto, and Life Sciences Institute Imaging Core Facility for sharing equipment and reagents, Aoi Hiroyasu, Lisa Fernando, Min Jee Kim for the maintenance of C. elegans and critical reading of our manuscript. Our work is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), (RGPIN-2019-04442).

1. Preparation of adhesive polystyrene bead
NOTE: This protocol does not require aseptic technique.
<ol>
	<li>Weigh 10 mg of carboxylate modified polystyrene beads in a 1.5 mL microcentrifuge tube.</li>
	<li>To wash the beads, add 1 mL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer into the tube. Since MES buffer does not contain phosphate and acetate, which can reduce the reactivity of carbodiimide, it is suitable to use in protein coupling reaction. Vortex the tube to mix the beads.</...

<ol>
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Reconstitution of simplified cell contact patterns will let researchers to test the roles of specific cell contact patterns in different aspects of morphogenesis. We have used this technique to show that cell division axis is controlled by the physical contact with adhesive beads10. As division axis specification is crucial for multicellular development by contributing to morphogenesis14, stem cell division15,16, an...

During multicellular development, the cellular behaviors (e.g., division axis) of individual cells are specified by various chemical and physical cues. To understand how individual cell interprets this information, and how they regulate multicellular assembly as an emergent property is one of the ultimate goals of morphogenesis studies. The model organism C. elegans has contributed significantly to the understanding of cellular-level regulation of morphogenesis such as cell polarity1, cell division patterning1, cell fate decision2, and tissue-scale regulations such as neuronal wiring3 and organogenesis4,5. Although there are various genetic tools available, tissue engineering methods are limited.
The most successful tissue engineering method in C. elegans study is the classical blastomere isolation6; as C. elegans embryo is surrounded by an eggshell and a permeability barrier7, their removal is one of the main procedures of this method. While this blastomere isolation method enables reconstitution of cell-cell contact in a simplified manner, it does not allow for the elimination of unwanted cues; cell contact still poses both mechanical (e.g., adhesion) and chemical cues, thereby limiting our ability to fully analyze the causal relationship between the cue and cellular behavior.
The method presented in this paper uses carboxylate modified polystyrene beads that can covalently bind to any amine-reactive molecules including proteins as ligands. Particularly, we used an amine-reactive form of Rhodamine Red-X as a ligand to make beads both visually trackable and adhesive to the cell. The carboxyl groups of bead surface and primary amine groups of ligand molecule are coupled by water-soluble carbodiimide 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDAC)8,9. Obtained adhesive beads allow for the effects of the mechanical cue on cellular dynamics10. We have used this technique to identify mechanical cues required for cell division orientation10.

<table>
	<tbody>
		<tr>
			<td>1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride</td>
			<td>Alfa Aesar</td>
			<td>AAA1080703</td>
			<td>For the bead preparation</td>
		</tr>
		<tr>
			<td>Aspirator Tube Assembly</td>
			<td>Drummond</td>
			<td>21-180-13</td>
			<td>For the blastomere isolation.</td>
		</tr>
		<tr>
			<td>Caenorhabditis elegans strain: N2, wild-type</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>N2</td>
			<td>Strain used in this study</td>
		</tr>
		<tr>
			<td>Caenorhabditis elegans strain: KSG5, genotype: zuIs45; itIs37</td>
			<td>in house</td>
			<td>KSG5</td>
			<td>Strain used in this study</td>
		</tr>
		<tr>
			<td>Calibrated Mircopipets, 10 &#181;L</td>
			<td>Drummond</td>
			<td>21-180-13</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>Carboxylate-modified polystyrene beads (30 &#181;m diameter)</td>
			<td>KISKER Biotech</td>
			<td>PPS-30.0COOHP</td>
			<td>For the bead preparation</td>
		</tr>
		<tr>
			<td>CD Lipid Concentrate</td>
			<td>Life Technologies</td>
			<td>11905031</td>
			<td>For the blastomere isolation. Work in the tissue culture hood.</td>
		</tr>
		<tr>
			<td>Clorox</td>
			<td>Clorox</td>
			<td>N. A.</td>
			<td>For the blastomere isolation. Open a new bottle when the hypochlorite treatment does not work well.</td>
		</tr>
		<tr>
			<td>Coverslip holder</td>
			<td>In house</td>
			<td>N.A.</td>
			<td>For the blastomere isolation.</td>
		</tr>
		<tr>
			<td>Dissecting microscope: Zeiss Stemi 508 with M stand. Source of light is built-in LED. Magnification of eye piece is 10X.</td>
			<td>Carl Zeiss</td>
			<td>Stemi 508</td>
			<td>For the blastomere isolation.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Qualified One Shot, Canada origin</td>
			<td>Gibco</td>
			<td>A3160701</td>
			<td>For the blastomere isolation. Work in the tissue culture hood.</td>
		</tr>
		<tr>
			<td>General Use and Precision Glide Hypodermic Needles, 25 gauge</td>
			<td>BD</td>
			<td>14-826AA</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>Inulin</td>
			<td>Alfa Aesar</td>
			<td>AAA1842509</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>MEM Vitamin Solution (100x)</td>
			<td>Gibco</td>
			<td>11120052</td>
			<td>For the blastomere isolation.</td>
		</tr>
		<tr>
			<td>MES (Fine White Crystals)</td>
			<td>Fisher BioReagents</td>
			<td>BP300-100</td>
			<td>For the bead preparation</td>
		</tr>
		<tr>
			<td>Multitest Slide 10 Well</td>
			<td>MP Biomedicals</td>
			<td>ICN6041805</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>PBS, Phosphate Buffered Saline, 10 x Powder</td>
			<td>Fisher BioReagents</td>
			<td>BP665-1</td>
			<td>For the bead preparation</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140148</td>
			<td>For the blastomere isolation.</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone</td>
			<td>Fisher BioReagents</td>
			<td>BP431-100</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Bioshop</td>
			<td>POC888</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>Rhodamine Red-X, Succinimidyl Ester, 5-isomer</td>
			<td>Molecular Probes</td>
			<td>R6160</td>
			<td>For the bead preparation</td>
		</tr>
		<tr>
			<td>Schneider's Drosophila Sterile Medium</td>
			<td>Gibco</td>
			<td>21720024</td>
			<td>For the blastomere isolation. Work in the tissue culture hood.</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Bioshop</td>
			<td>SOD001</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide Solution, 10 N</td>
			<td>Fisher Chemical</td>
			<td>SS255-1</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>Spinning disk confocal microscope: Yokogawa CSU-X1, Zeiss Axiovert inverted scope, Quant EM 512 camera, 63X NA 1.4 Plan apochromat objective lens. System was controlled by Slidebook 6.0.</td>
			<td>Intelligent Imaging Innovation</td>
			<td>N.A.</td>
			<td>For live-imaging</td>
		</tr>
		<tr>
			<td>Syringe Filters, PTFE, Non-Sterile</td>
			<td>Basix</td>
			<td>13100115</td>
			<td>For the blastomere isolation.</td>
		</tr>
		<tr>
			<td>Tygon S3 Laboratory Tubing,, Formulation E-3603, Inner diameter 3.175 mm</td>
			<td>Saint Gobain Performance Plastics</td>
			<td>89403-862</td>
			<td>For the blastomere isolation.</td>
		</tr>
		<tr>
			<td>Tygon S3 Laboratory Tubing,, Formulation E-3603, Inner diameter 6.35 mm</td>
			<td>Saint Gobain Performance Plastics</td>
			<td>89403-854</td>
			<td>For the blastomere isolation.</td>
		</tr>
	</tbody>
</table>

in vitro reconstitution of spatial cell contact patterns with isolated caenorhabditis elegans embryo blastomeres and adhesive polystyrene beads

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. This tissue complexity confounds the identification of causal link between extrinsic cues and cellular dynamics. A synthetically reconstituted multicellular system overcomes this problem by enabling researchers to test for a specific cue while eliminating others. Here, we present a method to reconstitute cell contact patterns with isolated Caenorhabditis elegans blastomere and adhesive polystyrene beads. The procedures involve eggshell removal, blastomere isolation by disrupting cell-cell adhesion, preparation of adhesive polystyrene beads, and reconstitution of cell-cell or cell-bead contact. Finally, we present the application of this method to investigate the orientation of cellular division axes that contributes to the regulation of spatial cellular patterning and cell fate specification in developing embryos. This robust, reproducible, and versatile in vitro method enables the study of direct relationships between spatial cell contact patterns and cellular responses.

For beads preparation, we determined the optimal amount of Rhodamine Red-X succinimidyl ester for the transgenic strain expressing GFP-myosin II and mCherry-histone (Figure 1A-D). We used mCherry tagged histone as a marker of cell cycle progression. Because both Rhodamine Red-X and mCherry will be illuminated by a 561 nm laser, the optimal intensity of Rhodamine Red-X signal is comparable to that of histone to allow simultaneous imaging of cell and bead. For example, the flu...

Watch this Scientific Journal Video about In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads at JoVE.com

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

Tissue complexities of multicellular systems confound the identification of causal relationship between extracellular cues and individual cellular behaviors. Here, we present a method to study the direct link between contact-dependent cues and division axes using C. elegans embryo blastomeres and adhesive polystyrene beads.

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. ...

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