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.</li>
	<li>Spin the tube for 60 s at 2,000 x g via a benchtop centrifuge.</li>
	<li>Discard the supernatant by carefully pipetting out the buffer.</li>
	<li>Wash the beads again with 1 mL of MES buffer by following steps 1.2-1.4.</li>
	<li>Add 1 mL of MES buffer containing 10 mg of EDAC into the tube to activate the surface carboxyl groups. Vortex the tube to mix the beads.</li>
	<li>Rotate and incubate the tube for 15 min at room temperature.</li>
	<li>Spin down the beads for 60 s at 2,000 x g.</li>
	<li>Discard the supernatant by carefully pipetting out the buffer.</li>
	<li>To wash the beads, add 1 mL of phosphate buffered saline (PBS) into the tube. Vortex the tube to mix the beads.</li>
	<li>Spin down the tube for 60 s at 2,000 x g.</li>
	<li>Discard the supernatant by carefully pipetting out the buffer.</li>
	<li>Wash the beads again with 1 mL of PBS by following steps 1.10-1.12.</li>
	<li>The final concentration of Rhodamine used will depend on the strain being imaged. Prepare 1 mL of 1-, 10-, 100-, and 1000-fold dilution series of Rhodamine Red-X from the 0.65 mM Rhodamine Red-X stock solution.</li>
	<li>Pipette 20 &#181;L of beads into each serial dilution tube.</li>
	<li>Rotate and incubate the tube for 5 min at room temperature.</li>
	<li>Wash the beads twice with 1 mL of PBS by repeating steps 1.10-1.12.</li>
	<li>Add 1 mL of PBS into the tube and store it at 4 &#176;C for up to 6 weeks. Check the fluorescence intensity of the beads under a microscope used for live-imaging. The appropriate concentration of the Rhodamine Red-X succinimidyl ester is dependent on the imaging conditions (Figure 1). 
	NOTE: Beads without Rhodamine Red-X treatment do not adhere to cell. Rhodamine Red-X serves as fluorescence marker as well as adhesive molecule. The electro static interaction between positively charged Rhodamine Red-X and negatively charged plasma membrane is a putative cause of adhesion.</li>
</ol>
2. Assembly of mouth pipette
<ol>
	<li>Cut wide (6.35 mm inner diameter) and narrow (3.175 mm inner diameter) Tygon tubes to about 25 cm and 40 cm in length, respectively (Figure 2).</li>
	<li>Connect the Tygon tubes with a polytetrafluoroethylene (PTFE) filter (0.2 &#181;m pore size) (Figure 2).</li>
	<li>Disassemble a commercially available aspirator tube and attach its capillary holder and mouthpiece to the end of the narrow and wide Tygon tubes, respectively (Figure 2). 
	NOTE: PTFE filter was used to prevent the inhalation of fumes of hypochlorite solution via mouth pipette.</li>
</ol>
3. Isolation of embryo blastomere
NOTE: Wear gloves and lab coat to avoid cut and contact with the bleaching solution.
<ol>
	<li>Hold each end of a microcapillary (capacity; 10 &#181;L) with right and left hand.</li>
	<li>Pull the microcapillary towards both ends to apply tension and bring the center of the capillary over a burner to make two hand-pulled capillaries (Figure 3A).</li>
	<li>Trim the tips of the hand-pulled capillaries with forceps under the dissecting microscope and attach the pulled capillary into a mouth pipetting apparatus (Figure 2). Prepare two types of pipettes. The tip opening sizes for the pipettes should be approximately 2x and 1x the short axis length of C. elegans embryos (30 &#181;m) for the embryo transfer and eggshell removal, respectively Figure 3B-D).</li>
	<li>Pipette 45 &#956;L of egg salt solution onto a well of a multiwell slide (Figure 4A; bottom).</li>
	<li>Place 5-10 adult C. elegans onto a well containing egg salt solution.</li>
	<li>To obtain early C. elegans embryos, cut adults into pieces by positioning two needles to the right and left of C. elegans body and sliding the needles past each other (Figure 4A; upper schematics).</li>
	<li>Pipette 45 &#956;L of hypochlorite solution onto a well next to the well containing egg salt solution (Figure 4B).</li>
	<li>Pipette 45 &#956;L of Shelton&#39;s growth medium onto the subsequent three wells next to the well containing hypochlorite solution (Figure 4B).</li>
	<li>Transfer 1-cell stage and early 2-cell stage embryos into the hypochlorite solution by mouth pipetting with the hand-drawn capillary for embryo transfer (Figure 4B).</li>
	<li>Wait for 40&#8211;55 s.</li>
	<li>Wash the embryos by transferring the embryos from hypochlorite solution into Shelton&#39;s growth medium by mouth pipetting with the hand-drawn capillary for embryo transfer (Figure 4B).</li>
	<li>Wash the embryos again by transferring the embryos into a new well of Shelton&#39;s growth medium by mouth pipetting with the hand-drawn capillary for embryo transfer (Figure 4B).</li>
	<li>Transfer the washed embryos into a new well of Shelton&#39;s growth medium by mouth pipetting with the hand-drawn capillary for embryo transfer. Using the hand-drawn capillary for eggshell removal, carefully repeat the pipetting (Figure 4C; middle schematics). If the eggshell is successfully removed, the embryonic cells will become more spherical (Figure 4C; right).</li>
	<li>Separate the 2-cell stage embryonic blastomeres by gently and continuously pipetting with the hand-drawn capillary for eggshell removal (Figure 4D).</li>
</ol>
4. Reconstitution of contact patterns with blastomere and bead
NOTE: Work under the imaging microscope to avoid dissociation of a cell from a bead and to facilitate the timely image acquisition.
<ol>
	<li>To observe using an inverted microscope, prepare an imaging chamber as in Figure 5.
	<ol>
		<li>Place a coverslip onto a coverslip holder (Figure 5A).</li>
		<li>Tape the edges of the coverslip to stabilize it. The side with tape is the &#39;back&#39; side (Figure 5A,B).</li>
		<li>Flip the coverslip holder over to the &#39;front&#39; side and draw a circle on the coverslip with a hydrophobic pen (Figure 5C). 
		NOTE: Any glass-bottom dish also works, provided the embryos are manipulatable by the mouth pipette.</li>
	</ol>
	</li>
	<li>Add Shelton&#39;s growth medium within the circle drawn by the hydrophobic marker (Figure 5D).</li>
	<li>Transfer the isolated blastomere to the imaging chamber.</li>
	<li>Dispense a small volume of the chemically functionalized beads using the hand-drawn capillary for embryo transfer.</li>
	<li>Control the position of the polystyrene beads by blowing into the hand-drawn capillary until the bead attaches to the isolated blastomere.</li>
	<li>Mount a coverslip to avoid evaporation of medium (Figure 5E). Perform live imaging.</li>
</ol>
5. Preparation of important reagents
<ol>
	<li>Egg Salt Solution (10 mL): Combine 235 &#181;L of 5 M NaCl and 240 &#181;L of 2 M KCl with 9525 &#181;L of dH2O.</li>
	<li>Hypochlorite Solution (10 mL): Combine 7.5 mL of Clorox (containing approximately 7.5% sodium hypochlorite) with 2.5 mL of 10 N NaOH (final concentration of sodium hypochlorite is approximately 5.625%). 
	NOTE: Although many published methods have used chitinase to digest chitinous eggshells, we have adopted a method using hypochlorite solution11. By avoiding the batch-to-batch variations of chitinase activities, we believe this method is more reproducible and cost-effective approach.</li>
	<li>0.81 mM Inulin Solution (40 mL)
	<ol>
		<li>Add 0.2 g of Inulin to 40 mL of dH2O.</li>
		<li>Autoclave to dissolve. 
		NOTE: Keeps for 1 month at 4 &#176;C.</li>
	</ol>
	</li>
	<li>0.5 M MES Buffer (500 mL)
	<ol>
		<li>Dissolve 48.81 g of MES in 400 mL of distilled water (dH2O).</li>
		<li>Adjust pH to 6.0.</li>
		<li>Bring the total volume to 500 mL with dH2O.</li>
	</ol>
	</li>
	<li>PBS Solution (1 L)
	<ol>
		<li>To make 10-fold PBS solution, dissolve 1 package of PBS premix powder in 1 L of dH2O.</li>
		<li>Combine 100 mL of 10-fold PBS solution with 900 mL of dH2O.</li>
	</ol>
	</li>
	<li>5% Polyvinylpyrrolidone (PVP) Solution (4 mL)
	<ol>
		<li>Under sterile conditions, dissolve 0.2 g of PVP in 4 mL of Drosophila Schneider&#39;s Medium. 
		NOTE: Always open Schneider&#39;s Medium stock in tissue culture (TC) hood. Keep for 1 month at 4 &#176;C.</li>
	</ol>
	</li>
	<li>0.65 mM Rhodamine Red-X stock solution (2 mL)
	<ol>
		<li>Weigh 1 mg of Rhodamine Red-X.</li>
		<li>Add 2 mL of dimethyl sulfoxide (DMSO) to dissolve.</li>
		<li>Aliquot and store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Shelton&#39;s Growth Medium (SGM) (10.25 mL)
	<ol>
		<li>Under sterile conditions, combine 8 mL of Drosophila Schneider&#39;s Medium, 1 mL of PVP solution, 1 mL of Inulin solution, 1 mL of Basal Medium Eagle (BME) Vitamins, 50 &#181;L of Penicillin-Streptomycin, and 100 &#181;L of Lipid concentrate together.</li>
		<li>Aliquot 325 &#181;L of SGM into 1.5 mL microtubes. 
		NOTE: Keep for about 1 month at 4 &#176;C.</li>
		<li>On the day of blastomere isolation, add 175 &#181;L of Fetal Bovine Serum (FBS) to the SGM (for total volume of 500 &#181;L). 
		NOTE: Store FBS at -20 &#176;C and thaw it before usage. FBS does not need to be heat killed.</li>
	</ol>
	</li>
</ol>

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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, and tissue homeostasis15,16, this method should be able to illuminate a new mechanism of animal tissue formation.
In addition to cell division orientation, this method has a potential to be a platform to test the relationship between mechanical cues and cell fate. Previous studies reported that mechanical cue controls cell fate specification. Human mesenchymal stem cells differentiate into neuron, adipocyte, skeletal muscle cell, and osteoblast when cultured in substrate with different stiffness17. In response to substrate stiffness, the transcriptional regulators Yes-associated protein (YAP) and TAZ (transcriptional co-activator with PDZ-binding motif) will translocate to nucleus, to regulate cell fate specification18. The method presented in this paper should also be useful to test the role of mechanical cues on cell fate specification, by culturing cell long-term in contact with adhesive beads.
This method has limitations in recapitulating certain mechanical cues observed in vivo. In C. elegans, chitinous eggshell and permeability barrier serve as a physical constrain. Although the removal of eggshell does not affect normal development, removal of both eggshell and permeability barrier results in abnormal pattern formation19. In the absence of eggshell and permeability barrier, cell shape becomes more spherical, suggesting that the pressure between cells and cell-eggshell plays a critical role in cell deformation and patterning. Indeed, cell-cell squeezing forces has been proposed to regulate cell division orientation20. Without having physical constrain and putative pressure among tissues, we expect that this method also cannot recapitulate in vivo cell contact area. As cell contact area is known to affect Notch signaling21, this limitation needs to be further considered when certain mechanical or chemical cue does not work using this in vitro method.
We have so far tested the cell division orientation of isolated AB, P1, EMS, P2, ABa, ABp cells (cells up to 6 cell stages) in our hands10, but the method can be applied to later development as far as individual cell can be isolated. Recent single cell sequencing study has used pronase digestion of worm body to isolate larval cell22. Therefore, potentially one can use pronase treatment to isolate later stage embryonic cells and larval cells.
In this study, we showed the example of interaction between mechanical cue and cell division orientation, but cell-cell communication is also mediated by chemical cues such as Wnt23, Notch24, adhesion coupled receptors25 and so on. Importantly, the beads preparation method presented here can couple the beads with any proteins, thereby allowing the reconstitution of chemical cues. Previous studies have also used Sepharose bead26 and protein-A magnetic bead27 coated with Wnt protein to demonstrate Wnt-dependent developmental processes. However, these beads are not commercially available in various sizes compared to carboxylate modified polystyrene beads. Hence, future research can use the presented method as a platform to test the role of both chemical and physical cues in more tunable manners. Taken together, this method is suited for researchers, who are studying cellular behaviors and responses that result from spatial cell contact patterns and wish to eliminate other cellular cues.

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 &amp;micro;L</td><td>Drummond</td><td>21-180-13</td><td>For the blastomere isolation</td></tr><tr><td>Carboxylate-modified polystyrene beads (30 &amp;micro;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&amp;rsquo;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 fluorescence signal from the bead treated with 0.005 &#181;g/mL Rhodamine Red-X succinimidyl ester was too weak to visualize the bead (Figure 1A). On the other hand, the fluorescence signal from the beads treated with 5 &#181;g/mL Rhodamine Red-X succinimidyl ester was too strong to image mCherry-histone (Figure 1D). We determined that 0.5 &#181;g/mL Rhodamine Red-X succinimidyl ester is optimal for this particular transgenic strain (Figure 1C).
Blastomere isolation was performed according to the procedures shown in Figure 4. The following points should be addressed for successful experiments. 1) The media in the well evaporates over time and becomes viscous; this causes the embryos to stick to the glass capillary and other embryos, so a new well with fresh media needs to be used. 2) In the hypochlorite solution, embryos float to the surface. Therefore, lower the magnification of the microscope and focus on the surface of the droplet to ease with the transferring of embryos. 3) The effectiveness of hypochlorite solution may differ. Hence, duration of embryos spend in the hypochlorite solution should be tested for each new batch of hypochlorite solution made. 4) Place the mouth pipette as close to the embryo as possible to minimize the strength used to blow into the pipette, but also not too close so that the embryo is sucked up as well (this is also true when attaching beads). 5) After eggshell removal, embryos become very delicate. Hence, forces exerted on the mouth pipette need to be slightly lowered to prevent embryos from bursting. 6) Prolonged usage of the mouth pipette may dampen the PTFE filter, and 7) 2-cell stage embryos should only be separated when the nucleus has been clearly formed (Figure 4C, left schematics). Compared to cells in intact embryos (Figure 4A; right), cells in embryos without eggshell look rounder (Figure 4C; right). Additionally, after blastomere isolation, the shape of blastomeres become spherical (Figure 4D; right).
To test the effects of physical contact-dependent cue on cell division orientation, we attached Rhodamine Red-X coated beads to AB blastomeres isolated from the 2-cell stage and performed live-imaging (Figure 6, Figure 7). The bead without Rhodamine Red-X treatment did not adhere to the blastomeres, suggesting that the Rhodamine Red-X serves as a both fluorescence marker and adhesive molecule. For the analysis of cell division orientation, we have 3D reconstructed the time-lapse image using the &#34;3D project&#34; function of ImageJ12 (Figure 6A; tilting around Y-axis). To determine the plane containing mitotic spindle (spindle plane), we first identified a plane where two centrosomes were vertically aligned (Figure 6B; 110&#176;): the plane with &#177; 90&#176; angle of this plane is spindle plane (Figure 6B; -20&#176;). Next, we have measured the angle of spindle (spindle orientation) relative to the cell-bead contact interface (contact orientation) after cytokinesis (Figure 6C). When both daughter cells were attached to the bead, a line that passes both contact sites was used as a contact orientation.
In our previous study, we have identified an anisotropic cell surface myosin flow during the bead-induced AB cell division orientation10. However, it is difficult to measure the intracellular myosin flow as cell moves during oriented division. To perform this, first we have selected the samples with spindle aligned to the imaging plane (xy plane) (Figure 7). Second, Z-stacks were projected using maximum projection method (Figure 7). Third, cell movements were corrected using the subpixel registration algorithm Stack reg plug-in13 of ImageJ with Rigid body option (Figure 7B). By the registration of images, the position of cell was stabled (Figure 7B). Finally, by using Manual tracking plug-in of ImageJ, myosin foci were tracked (Figure 7C). According to the coordinate information, velocities of myosin along division axis, and axis perpendicular to it were calculated within 50 s after the cytokinesis onset.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60422/60422fig01.jpg" /> 
Figure 1: Determination of Rhodamine Red-X concentration. (A-D) Embryos expressing GFP-myosin (green) and mCherry-histone (magenta) were placed in proximity to beads bound with different concentrations of Rhodamine Red-X (magenta). The signal intensities of mCherry and Rhodamine Red-X are plotted along the white dotted lines (bottom graphs). The beads and histone signals were clearly detected for beads treated with 0.5 &#181;g/mL Rhodamine Red-X. Scale bars show 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60422/60422fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60422/60422fig02.jpg" /> 
Figure 2: Assembly of mouth pipette. Mouth pipette is assembled by connecting narrow (1) and wide (2) Tygon tubes with a PTFE filter (3). At the end of the narrow and wide Tygon tube, capillary holder (4) and mouth piece (5) were attached, respectively. A hand-drawn glass capillary (6) can be inserted in the capillary holder. Scale bar is 50 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60422/60422fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60422/60422fig03.jpg" /> 
Figure 3: Blastomere isolation. (A) Hand pulling of glass capillary. (B) Hand-drawn glass capillary for embryo transfer. (C) Hand-drawn glass capillary for eggshell removal. (D) Schematics showing the appropriate size of capillary opening for the eggshell removal. Arrows indicate embryos. Scale bars show 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60422/60422fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60422/60422fig4a.jpg" /> 
Figure 4: Blastomere isolation workflow. (A) Dissection of adult C. elegans in egg salt buffer to obtain embryos. Photographs show 2-cell and 4-cell stage embryos before eggshell removal. (B) Hypochlorite treatment and washing. (C) Schematics depict the appropriate timing for eggshell removal. Photograph shows a 4-cell stage embryo after eggshell removal. (D) Blastomere separation. Photograph shows a separated 2-cell stage embryo. Sizes of the arrows in C and D indicate the relative forces required during pipetting. Scale bars show 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60422/60422fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60422/60422fig05.jpg" /> 
Figure 5: Assembly of imaging chamber. (A) Attaching coverslip to the coverslip holder. (B) Images of a coverslip holder. Coverslip holder before and after assembly is indicated on the left and right, respectively. (C) A circle drawn via a hydrophobic pen. (D) Addition of Shelton&#39;s growth medium and sample within the circle. (E) Mounting a coverslip to avoid evaporation. Scale bars show 50 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60422/60422fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60422/60422fig06.jpg" /> 
Figure 6: Analysis of cell division orientation. (A) A diagram of cell division orientation analysis. (B) Determination of spindle plane. Left images show an example of a sample. 3D reconstructed 4-D movies were rotated around Y-axis to determine the plane wherein two centrosomes align vertically (right image; right upper schematics). In this example, spindle plane is &#177; 90&#176; of 110&#176; (middle image; right bottom schematics). (C) Measurement of spindle orientation relative to the cell-bead contact. Using images of the spindle plane, spindle orientation after cytokinesis was determined based on angle between lines connecting two centrosomes (orange dotted lines in the right schematics) and cell contact (blue dotted lines). When both daughter cells were attached to the beads, cell-bead contact orientation was the line that passes both contact sites. Green is myosin and centrosome, magenta is histone and beads. Scale bars are 10 &#181;m. Times are minutes and seconds. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60422/60422fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60422/60422fig07.jpg" /> 
Figure 7: Analysis of myosin flow. (A) A diagram of myosin flow analysis. (B) Correction of cell division orientation. To quantify intracellular myosin flow dynamics, the rotation of dividing cell was corrected using the ImageJ plugin Stack reg (Rigid body option). Upper and bottom images are before and after the Stack reg processing. Right most images show temporal color code of time series. (C) Tracking of myosin foci. Using the images processed by Stack reg, individual myosin foci movements were tracked by ImageJ Manual tracking plugin. Division axis and the axis perpendicular to it were defined as x and y, respectively (right schematics). Myosin flow velocities in x and y axis (Vx and Vy) were measured according to the coordinate information. Scale bars are 10 &#181;m. Times are minutes and seconds. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60422/60422fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.1K Views.  The University of British Columbia. Meercellige weefsels complex, dit protocol reconstrueert cellulair gedrag op een vereenvoudigde manier. Een ding is deze techniek om de directe relaties tussen cellulaire dirigent protonen en cellulaire respons te bestuderen. Dit protocol maakt gebruik van chemisch functionele polystyreen kralen om de cellulaire geleider protonen te reconstrueren.De kralen kunnen worden gesneden met eventuele protonen van belang, om hun effecten op cellulaire respons te testen. We hebben deze methode g...

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

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/&#946;-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol to generate aggregates of mouse embryonic stem cells that display self-organization, symmetry breaking and elongation paralleling axial development. This technique allows the study of axial developmental processes and the generation of cell types that are otherwise difficult to perform in monolayer culture.

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation ...

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Biology

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Bioengineering

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 ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

Neuroscience

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and ...

Single Cell Dissociation of Caenorhabditis elegans: A Method to Isolate Live Cells

Source: Germany, E. M., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60145/isolation-specific-neuron-populations-from-roundworm-caenorhabditis">Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans</a>. J. Vis. Exp. (2019).
This video describes a method to dissociate whole worms into a single-cell suspension suitable for FACS or immunoprecipitation of intact cells.

Source: Germany, E. M., et al. Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans. J. Vis. Exp. (2019).
This video describes a ...

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

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