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

Summary

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.

Abstract

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.

Introduction

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 organogenesis^4,5. 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.

Protocol

1. Preparation of adhesive polystyrene bead

NOTE: This protocol does not require aseptic technique.

1. Weigh 10 mg of carboxylate modified polystyrene beads in a 1.5 mL microcentrifuge tube.
2. 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.
3. Spin the tube for 60 s at 2,000 x g via a benchtop centrifuge.
4. Discard the supernatant by carefully pipetting out the buffer.
5. Wash the beads again with 1 mL of MES buffer by following steps 1.2-1.4.
6. 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.
7. Rotate and incubate the tube for 15 min at room temperature.
8. Spin down the beads for 60 s at 2,000 x g.
9. Discard the supernatant by carefully pipetting out the buffer.
10. To wash the beads, add 1 mL of phosphate buffered saline (PBS) into the tube. Vortex the tube to mix the beads.
11. Spin down the tube for 60 s at 2,000 x g.
12. Discard the supernatant by carefully pipetting out the buffer.
13. Wash the beads again with 1 mL of PBS by following steps 1.10-1.12.
14. 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.
15. Pipette 20 µL of beads into each serial dilution tube.
16. Rotate and incubate the tube for 5 min at room temperature.
17. Wash the beads twice with 1 mL of PBS by repeating steps 1.10-1.12.
18. Add 1 mL of PBS into the tube and store it at 4 °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.

2. Assembly of mouth pipette

1. 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).
2. Connect the Tygon tubes with a polytetrafluoroethylene (PTFE) filter (0.2 µm pore size) (Figure 2).
3. 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.

3. Isolation of embryo blastomere

NOTE: Wear gloves and lab coat to avoid cut and contact with the bleaching solution.

1. Hold each end of a microcapillary (capacity; 10 µL) with right and left hand.
2. 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).
3. 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 µm) for the embryo transfer and eggshell removal, respectively Figure 3B-D).
4. Pipette 45 μL of egg salt solution onto a well of a multiwell slide (Figure 4A; bottom).
5. Place 5-10 adult C. elegans onto a well containing egg salt solution.
6. 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).
7. Pipette 45 μL of hypochlorite solution onto a well next to the well containing egg salt solution (Figure 4B).
8. Pipette 45 μL of Shelton's growth medium onto the subsequent three wells next to the well containing hypochlorite solution (Figure 4B).
9. 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).
10. Wait for 40–55 s.
11. Wash the embryos by transferring the embryos from hypochlorite solution into Shelton's growth medium by mouth pipetting with the hand-drawn capillary for embryo transfer (Figure 4B).
12. Wash the embryos again by transferring the embryos into a new well of Shelton's growth medium by mouth pipetting with the hand-drawn capillary for embryo transfer (Figure 4B).
13. Transfer the washed embryos into a new well of Shelton'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).
14. Separate the 2-cell stage embryonic blastomeres by gently and continuously pipetting with the hand-drawn capillary for eggshell removal (Figure 4D).

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.

1. To observe using an inverted microscope, prepare an imaging chamber as in Figure 5.
   1. Place a coverslip onto a coverslip holder (Figure 5A).
   2. Tape the edges of the coverslip to stabilize it. The side with tape is the 'back' side (Figure 5A,B).
   3. Flip the coverslip holder over to the 'front' 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.
2. Add Shelton's growth medium within the circle drawn by the hydrophobic marker (Figure 5D).
3. Transfer the isolated blastomere to the imaging chamber.
4. Dispense a small volume of the chemically functionalized beads using the hand-drawn capillary for embryo transfer.
5. Control the position of the polystyrene beads by blowing into the hand-drawn capillary until the bead attaches to the isolated blastomere.
6. Mount a coverslip to avoid evaporation of medium (Figure 5E). Perform live imaging.

5. Preparation of important reagents

1. Egg Salt Solution (10 mL): Combine 235 µL of 5 M NaCl and 240 µL of 2 M KCl with 9525 µL of dH₂O.
2. 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.
3. 0.81 mM Inulin Solution (40 mL)
   1. Add 0.2 g of Inulin to 40 mL of dH₂O.
   2. Autoclave to dissolve.
      NOTE: Keeps for 1 month at 4 °C.
4. 0.5 M MES Buffer (500 mL)
   1. Dissolve 48.81 g of MES in 400 mL of distilled water (dH₂O).
   2. Adjust pH to 6.0.
   3. Bring the total volume to 500 mL with dH₂O.
5. PBS Solution (1 L)
   1. To make 10-fold PBS solution, dissolve 1 package of PBS premix powder in 1 L of dH₂O.
   2. Combine 100 mL of 10-fold PBS solution with 900 mL of dH₂O.
6. 5% Polyvinylpyrrolidone (PVP) Solution (4 mL)
   1. Under sterile conditions, dissolve 0.2 g of PVP in 4 mL of Drosophila Schneider's Medium.
      NOTE: Always open Schneider's Medium stock in tissue culture (TC) hood. Keep for 1 month at 4 °C.
7. 0.65 mM Rhodamine Red-X stock solution (2 mL)
   1. Weigh 1 mg of Rhodamine Red-X.
   2. Add 2 mL of dimethyl sulfoxide (DMSO) to dissolve.
   3. Aliquot and store at -20 °C.
8. Shelton's Growth Medium (SGM) (10.25 mL)
   1. Under sterile conditions, combine 8 mL of Drosophila Schneider's Medium, 1 mL of PVP solution, 1 mL of Inulin solution, 1 mL of Basal Medium Eagle (BME) Vitamins, 50 µL of Penicillin-Streptomycin, and 100 µL of Lipid concentrate together.
   2. Aliquot 325 µL of SGM into 1.5 mL microtubes.
      NOTE: Keep for about 1 month at 4 °C.
   3. On the day of blastomere isolation, add 175 µL of Fetal Bovine Serum (FBS) to the SGM (for total volume of 500 µL).
      NOTE: Store FBS at -20 °C and thaw it before usage. FBS does not need to be heat killed.

Results

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...

Discussion

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 division^15,16, an...

Materials

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