Cell Grafting and Labeling in Postimplantation Mouse Embryos

Gabriel A. Quinlan, Paul A. Trainor, and Patrick P. L. Tam 1. Introduction

Fate mapping experiments provide direct information on the differentiation pathways normally taken by cells or tissues during embryogenesis. Systematic analyses of the developmental fate of cell populations localized in different parts of the embryo enables the construction of fate maps. A comparison of the expression pattern of lineage-specific genes and the fate map allows the identification of precursor tissue for cell lineages well before definitive histogenesis takes place. The ability to trace the early lineage history of cells greatly facilitates the elucidation of the forces and processes which lead to the specification of cell lineages and the determination (or commitment) of cell fate. The knowledge of cell fate may also assist the interpretation of the phenotype of mutant embryos produced either by spontaneous mutation or by gene knockout experiments.

This chapter describes the technical aspect of fate mapping the mouse embryo during gastrulation (6.5 d post-coitum [p. c.]) (1-3) and organogenesis (8.5 d p.c.) (4-8). Two experimental strategies are used to study the developmental fate of cells. First, a specific population of cells can be marked by labeling with vital carbocyanine dyes in situ, and second, the same population of cells can be isolated from a transgenic embryo followed by trasnplantation (grafting) to a host embryo. The pattern of tissue colonization and differentiation of the descendants of these marked or transplanted cells is then analyzed after a period of in vitro development to assay their development fate.

Cell labeling and grafting procedures have their special advantages. When grafting a genetically identifiable population of cells, there is no dilution of the label owing to cell proliferation, so the contribution of transgenic cells to every

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

available lineages can be assessed. Cell transplantation techniques can also be applied to the study of the developmental potency of a population of cells, by confronting the cells with novel tissue environment or inductive signals (9). The usefulness of the cell grafting approach depends critically on the ability to isolate a defined cell population for transplantation and to place these cells at the appropriate site in the host. By contrast to cell transplantations, in situ labeling experiments do not require tedious dissection of tissue fragments. Fate mapping studies can be carried out directly after in situ labeling with minimal disruption of existing tissue architecture. However, the label must be noncytotoxic and should remain only among the descendants of the labeled cells.

2. Materials

2.1. Culturing 6.5-8.5 D Embryos

1. Roller/rotator bottle culture apparatus (B.T.C. Engineering, Milton, Cambridge, UK).

2. Water-jacketed CO2 incubator (Forma Scientific, Marietta, OH, Model 3336).

3. Four-well chamber slides (NUNC, Naperville, IL).

4. Glass culture bottles, thin-walled,15-mL, 30-mL capacity (B.T.C. Engineering).

5. Refrigerated bench top centrifuge, (CENTRA-7R, International Equipment Company, Needingham Heights, MA).

6. 15-mL sterile centrifuge tubes (Corning, Cambridge, MA, cat. no. 25310-15).

7. Penicillin/streptomycin, 5000 |g/mL (Trace Biosciences, Sydney, NSW, Australia).

8. Glutamine 200 |M (Trace Bioscience).

9. Dulbecco's Modified Eagle's Medium (DMEM) (Gibco-BRL, Grand Island, NY, cat. no. 12100-103, glucose 4 g/L).

11. Human cord serum (HCS) (see Subheading 3., step 1).

2.2. Isolation of Tissue Fragments for Grafting

Alloy metal needles are made by electrolytically sharpening orthodontic wire (Rocky Mountain Orthodontics, Denver, CO) with the a wire polishing unit (Dental Corporation of America, Hagerstown, MD).

Glass needles are made from thick-walled glass capillaries (Leitz, Rockleigh, NJ, #520-119). The capillaries are heated over a small flame to fuse a segment of the glass. This fused segment is then pulled with a vertical pipet puller (David Kopf Instruments, Tujunga, CA, Model 720) to give two needles. These needles are coated with Repelcote (BDH Chemicals, Poole, UK) to prevent the tissue from adhering.

Dissecting microscope (Wild M3Z or MDG 17).

Tissue culture dishes (60-mm, Corning).

Fetal calf serum (FCS) (Trace Biosciences): The FCS is thawed and inactivated by heating at 56°C for 30 min immediately before use.

Polyvinylpyrrolidone (PVP, Sigma, St. Louis, MO). Dialyze a 0.5% aqueous PVP solution against water at 4°C overnight followed by lyophilization.

Enzymatic solution contains 0.5% trypsin (Trace Biosciences), 2.5% pancre-atin (Boehringer Mannheim, Indianapolis, IN), 0.2% glucose (Sigma), and 0.1% PVP dissolved in calcium-magnesium-free phosphate-buffered saline (PBS) (Flow Laboratory, Costa Mesa, CA).

NaCl (BDH Chemicals).

Na2HPO4 (Ajax Chemicals, Sydney, NSW, Australia).

KH2PO4 (BDH Chemicals).

CaCl22H2O (BDH Chemicals).

MgCl26H2O (Ajax Chemicals).

Sodium pyruvate (Sigma): Dissolve 85 mg of sodium pyruvate in 10 mL of 0.9% NaCl. Dilute 1:50 in 0.9% (w/v) NaCl before use. It can keep for 2 wk at 4°C. Phenol red (Sigma): Add 13 mg of phenol red and 129 mg of NaHCO3 (BDH Chemicals) to 10 mL of dH2O. It can be stored for 2 wk at 4°C.

Penicillin (Sigma): Add 599 mg to 1 mL of 0.9% (w/v) NaCl. Dilute 1:100 before use. Store at -20°C.

PB1 is prepared according to the formulation in Table 1. After preparation, the solution is equilibrated with 5% CO2 in air for 5-10 min, 130 mg of glucose and 520 mg of bovine serum albumin (Fraction V, Sigma) are added, and the solution is sterilized with a 0.22-|m Millipore filter. The solution is stored in 10- or 40-mL aliquot at 4°C.

2.3. Labeling and Grafting of Cell in 6.5-Day Embryos

Embryo culture requirement as in Subheading 1.

Vertical pipet puller (David Kopf Instruments, Model 720).

Microforge (Narishige Scientific Instrument Laboratory, Greenvale, NY, MF79).

Holding, injection, and grafting pipets (Fig. 1A-D). Holding pipets are made from thick-walled glass capillaries (Leica, cat. no. 520-119). Injection and grafting pipets are made from glass capillaries (outer diameter: 1 mm, inner diameter: 0.75 mm, Thomas, Swedesboro, NJ).

Transfer pipets made from Pasteur pipets.

Diamond glass cutter (Thomas).

Micromanipulation apparatus: base plate with fixing elements for both manipulators (Leitz, cat. no. 335 520 139); manipulators (Leitz, cat. no. 335 520 137 and cat. no. 335 520 138); instrument holders (Leitz, cat. no. 335 520 142 and cat. no. 335 520 143), and instrument sleeves.

Laborlux S microscope with fixed mechanical stage (Leitz).

Dissecting microscope (Wild, MDG 17).

de Fonbrune syringe (Alcatel, Malakoff, France).

Micrometer syringe (Wellcome, London, UK).

Manipulation chamber (Fig. 5).

Tissue-culture dishes (60-mm, Corning).

Table 1

The Composition of PB1 Medium for Handling Embryos and Tissue Fragments

Stock solution (g/500 mL)

Volume, mL, to add to make 100 mL solution

NaCl (4.5)

65.8

KCl (5.75)

1.8

Na2HPO4 (10.93)

5.2

KH2PO4 (10.5)

0.9

CaCL2 2H2O (8.1)

0.8

MgCl2 6H2O (16.55)

0.3

Na pyruvatea

21.4

Phenol reda

0.8

Penicillina

0.3

Distilled H2O

0.27

aSee text for method of preparation.

aSee text for method of preparation.

Fig. 1. Pipets used for micromanipulation. (A) The holding pipet, (B) the injection pipet used for labeling embryos, (C) the pipet used for grafting cell clumps, and (D) a beveled pipet that can also be used for grafting tissue. The pipets in (A-D) are used for manipulating 6.5-d embryos in hanging drops. (E) An angled holding pipet that is used for manipulating 7.5-8.5-d embryos in drops on a Petri dish. Angled grafting or labeling pipets can be made by introducing similar bends in pipets shown in (B-D).

Fig. 1. Pipets used for micromanipulation. (A) The holding pipet, (B) the injection pipet used for labeling embryos, (C) the pipet used for grafting cell clumps, and (D) a beveled pipet that can also be used for grafting tissue. The pipets in (A-D) are used for manipulating 6.5-d embryos in hanging drops. (E) An angled holding pipet that is used for manipulating 7.5-8.5-d embryos in drops on a Petri dish. Angled grafting or labeling pipets can be made by introducing similar bends in pipets shown in (B-D).

Coverslips (24 x 50-mm, Mediglass, Sydney, NSW, Australia).

Light and heavy paraffin oil (BDH Chemicals).

1,1-Dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine percholate (Dil) (Molecular Probes, Eugene, OR) and 3,3'-dioctadecyloxacarbobyanide percholate (DiO) (Molecular probes): Stock dye solutions (0.5% w/v) were prepared by dissolving the crystals in 100% ethanol. Dilute 1:10 (by volume) for Dil and 1:5 (by volume) for DiO in 0.3 M sucrose (BDH Chemicals) immediately before use for cell labeling.

2.4. Labeling and Grafting Cells in 7.5-D Embryos

The materials are the same as outline in Subheadings 2.1. and 2.3.

Fluovert FS (fixed-stage) inverted microscope (Leitz).

Holding, injection, and grafting pipets (Fig. 1).

Tissue-culture dishes (60 mm, Corning).

2.5. Transplanting and Labeling Cells in the Cranial Region of 8.5-D Embryos

The materials are the same as outlined in Subheadings 2.1. and 2.4. 3. Methods

3.1. Culturing 6.5-8.5 D Embryos

3.1.1. Preparation of Culture Media

3.1.1.1. Preparation of DMEM

1. Dissolve the contents of one packet of powdered formula in 4.75 L (less 5% of the final volume required).

2. Stir gently at room temperature until the contents dissolve.

4. Bring the volume up to 5 L by adding reverse osmosis (RO) water.

5. Adjust the pH to 7.2-7.4 using 1 N NaOH or 1 N HCl.

6. Sterilize immediately by membrane filtration (pH value usually rises 0.1-0.3 on filtration).

7. Test sterility of a 50-|L aliquot from each bottle by incubating at 37°C for 3-5 d.

8. Before DMEM is used for culture, add 10 mL each of glutamine and penicillin/ streptomycin solution to 1 L of DMEM. Fresh glutamine and penicillin/streptomycin should be added to the DMEM working solution after 2 wk. The working solution is good for 4 wk after preparation when kept at 4°C.

3.1.1.2. Collection of Rat Serum (RS)

1. Anesthetize the rat using 5% Halothane in 1-1.5 L of O2.

2. Blood is collected from the anesthetized rat by drawing blood from the aorta into a nonheparinized syringe using a G20 hypodermic needle.

3. Dispense the freshly collected blood in 15-mL centrifuge tubes and centrifuge at 3000 rpm for 10 min (see Notes 3-5).

4. Grasp the fibrin clot with a flame-sterilized forceps, and spool it around the shaft of the forceps to squeeze out the serum trapped in the clot.

5. Transfer the serum to a new centrifuge tube using an autoclaved Pasteur pipet, and spin again at 3000 rpm for 10 min.

6. Collect the serum from the second spin aseptically and store at -20°C.

3.1.1.3. Collection of Human Cord Serum (HCS)

1. Collect cord blood from the placenta obtained following Caesarean delivery, and keep the blood on ice.

2. Centrifuge the blood as soon as possible in 15-mL centrifuge tubes at 3000 rpm for 10 min at 4°C.

3. Removed the serum and store at -20°C (see Note 3-5).

3.1.2. Static Culture in 4-Well Chamber Slides at 37°C in 5% CO2 in Air

1. Thaw the required volume of HCS and RS, and inactivate the sera by heating for 30 min in a water bath at 56°C, 3.2 mL of culture medium are required for one chamber slide, and this is made up of 0.8 mL HCS, 1.6 mL RS, and 0.8 mL DMEM (1:2:1 by volume).

2. Put 0.5 mL in the first well (Note 3) and 0.9 mL in the remaining three wells. This volume is sufficient for culturing groups of 8-10 6.5-d embryos/well (Note 4) for up to 48 h (Note 5).

3. 7.5-D embryos can be cultured for up to 24 h by the static culture method.

3.1.3. Roller Bottle Culture (with Continuous Gassing) for 7.5-8.5-D Embryos

1. Embryos are cultured for 24 h in a medium of DMEM:RS (1:1 by vol). Use 1 mL of culture medium/embryo. Four to five embryos can be cultured in one 30-mL bottle. Bottles are maintained in a BTC embryo culture chamber at 37°C in 5% O2, 5% CO2, and 90% N, and are rotated on a roller/rotator at 30 rpm.

2. Embryos are transferred to fresh medium of DMEM:RS (1:3 by vol) if culturing for another 24 h is required. The gas phase is changed to 20% O2, 5% CO2, and 75% N2 (10).

3.2. Isolation of Tissue Fragments for Grafting

3.2.1. Isolation of Epiblast Fragment from 6.5-D Embryos

1. Position the embryo for easiest access to the epiblast cells required for transplantation. For example, if anterior or posterior epiblast cells are to be isolated, the embryo could be positioned with the sagittal plane in view (Note 6). However, in order to dissect cells from the lateral epiblast, the embryo is best oriented with the frontal view in sight.

2. Pin the embryo to the Petri dish using a sharp metal needle. Hold it at the site immediately adjacent to the tissue fragment required for grafting. Another tungsten needle is used to slice through the epiblast in a scissor-like action against the first needle. Another cut is then made at an angle to the first, so that the tissue fragment is released from the epiblast. The dissection is shown in Fig. 2.

Fig. 2. Schematic representation of the step taken to isolate epiblast fragments from sites adjacent to the distal cap region on the posterior side of a 6.5-d embryo. (A) Position of the pinning needle (pn) and the line of cut that will be made by the cutting needle (cn) just proximal to the required tissues (shaded area). (B) Second cut that is made to isolate the tissue fragment. Similar cutting actions are employed to isolate tissue fragments from other regions of the embryo. Curved arrows indicate the direction of slicing made by the cutting needle. Abbreviation: ps, primitive streak.

Fig. 2. Schematic representation of the step taken to isolate epiblast fragments from sites adjacent to the distal cap region on the posterior side of a 6.5-d embryo. (A) Position of the pinning needle (pn) and the line of cut that will be made by the cutting needle (cn) just proximal to the required tissues (shaded area). (B) Second cut that is made to isolate the tissue fragment. Similar cutting actions are employed to isolate tissue fragments from other regions of the embryo. Curved arrows indicate the direction of slicing made by the cutting needle. Abbreviation: ps, primitive streak.

3. The endoderm usually remains attached to the epiblast. Make several scratch marks on the bottom of the Petri dish with a metal needle. To remove the endo-derm, place the tissue fragment endoderm side down on the grid, and nudge the fragment onto the scratched surface. When the endoderm sticks to the surface, the epiblast layer can be torn away with metal needles or glass needles.

4. Cut the epiblast fragments into clumps of 5-10 cells for grafting.

3.2.2. Separation of the Germ Layers of 7.0-7.5-D Embryos

1. Cut the embryo at the junction between the embryonic and extraembryonic tissue (Fig. 3A).

2. Incubate the embryonic tissue in a trypsin-pancreatin solution (see Subheading 2., item 2) for 5-10 min or until the endoderm is loosened from the mesoderm.

3. Transfer the embryonic fragment to three changes of PB1 + 10% FCS to stop the enzyme digestion.

3. Separate the germ layers as shown in Fig. 3B-D.

4. Cut the isolated germ layers into clumps of 5-10 cells using glass needles.

3.2.3. Isolation of Mesoderm and Premigratory Neural Crest Cells of 8.5-D Embryos

1. Remove the yolk sac and the amnion using fine watchmaker forceps.

2. Bisect the embryo using tungsten needles along its midline.

ectoderm endoderm

Line of cut of the ectoderm

Line of cut of the ectoderm

mesoderm + endoderm

Fig. 3. Schematic representation of steps of germ layer separation of 7.0- to 7.5-d embryos. (A) Tissue organization of the 7.5-d embryo. The dashed line marks the position of the first cut at the junction between the embryonic and extraembryonic parts of the egg cylinder. (B) Embryonic portion of the embryo that reveals the anatomical relationship of the three germ layers. A metal needle is pushed into the amniotic cavity and pins the egg cylinder in the upright position. The second metal needle is then brought inside the amniotic cavity, and the embryo is cut along the anterior side (indicated by the dashed line) by slicing the second needle through all germ layers. (C) Embryo opened out flat and ready for enzyme digestion. (D) Ectoderm layer that has been loosened by enzymatic digestion. The ectoderm can be lifted up and torn away from the mesoderm using needles. It is not possible to separate the germ layers at the site of the primitive streak. The ectoderm is therefore cut along the dashed line where no further separation from the underlying primitive streak could be made. The mesoderm layer can be separated from the endoderm in the same manner, followed again by cutting them close to the primitive streak. The remaining tissue is the primitive streak (PS).

mesoderm + endoderm

Fig. 3. Schematic representation of steps of germ layer separation of 7.0- to 7.5-d embryos. (A) Tissue organization of the 7.5-d embryo. The dashed line marks the position of the first cut at the junction between the embryonic and extraembryonic parts of the egg cylinder. (B) Embryonic portion of the embryo that reveals the anatomical relationship of the three germ layers. A metal needle is pushed into the amniotic cavity and pins the egg cylinder in the upright position. The second metal needle is then brought inside the amniotic cavity, and the embryo is cut along the anterior side (indicated by the dashed line) by slicing the second needle through all germ layers. (C) Embryo opened out flat and ready for enzyme digestion. (D) Ectoderm layer that has been loosened by enzymatic digestion. The ectoderm can be lifted up and torn away from the mesoderm using needles. It is not possible to separate the germ layers at the site of the primitive streak. The ectoderm is therefore cut along the dashed line where no further separation from the underlying primitive streak could be made. The mesoderm layer can be separated from the endoderm in the same manner, followed again by cutting them close to the primitive streak. The remaining tissue is the primitive streak (PS).

3. Make transverse cuts (using metal needles) along the neuromeric junctions to isolate wedge-shaped fragments containing the tissue to be transplanted by (7,8). For example, to isolate somitomere IV and the middle hindbrain neural crest cells, transverse cuts should be made at the preotic sulcus and the otic sulcus.

4. Incubate the embryonic fragments in the trypsin-pancreatin solution for 20 min at 37°C. When the mesoderm and ectoderm layers are loosened, separate the tissue layers using glass needles.

5. Dissect the isolated mesoderm or neuroectoderm into small clumps of to 10 cells (see Note 7).

3.3. Labeling and Grafting of Cell in 6.5-D Embryos

3.3.1. Making Pipets

3.3.1.1. Angled Pipets

1. Make holding, grafting, and injection pipets as described in Subheadings 3.3.1.2. and 3.3.1.3.

2. Heat the pipet, over a flame, 1-2 cm from the tip of the injection pipet. It is heated until it is bent to an angle of approx 100°.

3. Turn the pipet 180°, and heat the pipet at a position 1-2 cm further proximal of the first bend. Again the pipet is bent at an angle of approx 100° (Fig. 1E).

3.3.1.2. Making Holding Pipets

The holding pipets are made using Leitz thick-wall glass capillaries. The internal diameter of holding pipets should be 10-50 |m depending on the size of the embryo used for the experiment.

1. Hold the middle portion of the capillary over a small flame until the glass begins to melt.

2. Take the capillary away from the flame, and pull from both ends to produce a fine segment of capillary.

3. Break the fine capillary with a diamond pencil.

4. Polish the holding pipets on a microforge. A small glass bead is melted onto the platinum wire of the microforge. Heat the glass bead up by increasing the electric current until the platinum wire glows red hot. Bring the tip of the pipet as close as possible to the glowing glass melts under the heat of the glass bead. Retract the pipet from the glass bead when a polished tip is produced (Fig. 1A, see Note 8).

3.3.1.3. Making Injection and Grafting Pipets

Injection pipets with an inner diameter of about 1-2 |m are made from thin-wall glass capillaries. The internal diameter of grafting pipets is larger and varies according to the size of the clumps of cells to be grafted.

1. Pull glass capillaries on a vertical pipet puller to produce pipets with a fine tip, a long shaft, and a short shoulder.

injection pipette glass b heated glass b heated platinum wire platinum wire

glass bead retracts as it cools -►

glass bead retracts as it cools -►

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