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.</...

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The authors declare no conflict of interests.

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...

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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. ...

Multicellular tissues complex, this protocol reconstitutes cellular conduct in a simplified manner. One thing is this technique to study the direct relationships between cellular conductor protons and cellular response. This protocol uses chemically functional polystyrene beads to reconstitute the cellular conductor protons.The beads can be cut with any protons of interest, to test their effects on cellular response. We have used this method in C-Elegance on mouse embryos. Therefore, this protocol can be upright to the studies of wide organisms.As embryos are fragile after high full chlorite treatment, significant practice is required before one can successfully isolate blastomere. Visual demonstration will provide a greater understanding of how one should handle capillary during blastomere-isolation methods. To begin, weigh 10 milli-grams of carboxylate modified polystyrene beads in a 1.5 milli-liter micro centrifuge tube.Add one milli-liter of MES buffer into the tube to wash. Vortex the tube to mix the beads. Spin the tube for 60 seconds at 2000XG via bench-top centrifuge.Dicard the supernatant by carefully pipetting out the buffer. Wash the beads again with one milli-liter of MES buffer. After the wash, add one milli-liter of MES buffer containing 10 milli-grams of EDAC into the tube, to activate the surface carboxyl groups.Vortex the tube to mix the beads. Rotate and incubate the tube for 15 minutes at room temperature. Spin down the beads for 60 seconds at 2000XG.Discard the supernatant by carefully pippetting out the buffer, and wash with one milli-liter of PBS. Vortex the tube to mix the beads and repeat the PBS wash one more time. Prepare one milli-liter of one, 10, 100, and one-thousandfold dilution series of Rhodamine Red-X from the 0.65 milli-liter Rhodamine Red-X stock solution.Pippette 20 micro-liters of beads into each serial dilution tube. Rotate and incubate the tubes for five minutes at room temperature. Wash the beads twice with one milli-liter of PBS.After wash, add one milli-liter of PBS into the tubes and store them at four degrees Celsius for up to six weeks. Check the fluorescence intensity of the beads under a microscope used for live imaging. Hold each end of a micro-capillary at a capacity of 10 micro-liters with right and left hand.Pull the micro-capillary towards both ends to apply tension and bring the center of the capillary over a burner to make two hand-pulled capillaries. Under the dissecting microscope, trim the tips of the hand-pulled capillaries with forceps. Make the two tip opening sizes approximately two times, and one times the short access length of C-Elegance Embryos for the embryo transfer and eggshell removal respectively.Attach the pulled capillary into a mouth-pippetting apparatus. Pippette 45 micro-liters of egg-salts solution onto a well of a multi-welled slide. Place five to 10 adult C-Elegance onto the well.To obtain early C-Elegance Embryos cut adults into pieces by positioning two needles to the right and left of C-Elegance's body, and sliding the needles past each other. Pippette 45 micro-liters of hypochlorite solution onto a well next to the well containing egg-salt solution and pippette 45 micro-liters of Shelton's Growth Medium onto the subsequent three wells. Transfer one cell stage and early two cell stage embryos into the hypochlorite solution by the mouth pippette with the larger sized opening and wait for 40 to 55 seconds.Wash the embryos by transferring them into the next three wells with Shelton's Growth Medium. With one to two seconds in each of the first two wells. Using the hand-drawn capillary with the smaller sized opening carefully repeat pippetting the embryo for eggshell removal.After that, continuously pippette with the hand-drawn capillary to separate the two-cell stage embryonic blastomeres. After eggshell removal, minimize the force in pippetting the embryos up and down to prevent burst. Prepare an imaging chamber by placing a coverslip onto a coverslip holder and tape the edges of the coverslip to stabalize it.Flip the coverslip holder, and draw a circle on the coverslip with a hydrophobic pen. Add Shelton's Growth Medium within the circle drawn by the hydrophobic marker, and transfer the isolated blastomere to the imaging chamber within the circle. Now, dispense a small volume of the chemically functionalized beads onto the coverslip using the hand-drawn capillary with the larger opening.Control the position of the beads by blowing into the hand-drawn capillary until the beads attach to the isolated blastomere. Mount the coverslip to avoid evaporation of medium and perform live imaging using an inverted microscope. In this study, the fluorescent signal from the bead treated with 0.005 micro-grams per milli-liter Rhodamine Red-X Succinimidyl Ester for the transgenic strain expressing GFP miosin-2 and M-cherihistone was too weak.On the other hand, the fluorescent signal from the beads treated with five micro-grams per milli-liter Rhodamine Red-X Succinimidyl Ester was too strong. The concentration of Rhodamine Red-X Succinimidyl Ester at 0.5 micro-grams per milli-liter was optimal for this particular transgenic strain. During the live imaging a plane was identified where two centrosomes were vertically aligned.The plane with plus or minus 90 degree angle of this plane is spindle plane. The angle of spindle orientation relative to the cell/bead contact interface after cytokinesis shows that both daughter cells were attached to the bead in a line that passes both contact sights was used as a contact orientation. One needs to learn how embryos and eggshell react to certain external forces applied by the mouth pippette.Theoretically, any cellular response can be studied, for example, sulfate specification can be studied by culturing cells and contact with adhesive beads over longer terms. This technique has been used to understand contact dependent cell division orientation mechanism. PTF filter was used to prevent inhalation of fumes of hypochlorite solution via mouth pippette.

in-vitro-reconstitution-spatial-cell-contact-patterns-with-isolated

Research

JoVE Journal

Developmental Biology

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