Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Summary

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Abstract

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

Introduction

Xenopus laevis embryos have been utilized extensively to identify the mechanisms by which embryonic cells acquire their specific fates because their eggs are large enough to permit microsurgical approaches. Additionally, they develop externally without the need for nutritional supplementation of the culture medium because each cell contains a rich intracellular supply of yolk platelets that provide an intrinsic energy store. An important asset for studying the mechanisms by which cell fate is determined is the comprehensive set of fate maps of the cleavage stage blastomeres (from 2- through 32-cell stages) ^{1, 2, 3, 4, 5, 6}. These maps were constructed by microinjecting a detectable molecule into a single, identifiable blastomere and monitoring later in development which tissues are populated by the labeled progeny. Consistent fate maps are possible because the cardinal axes of the embryos can be reliably identified in many embryos. First, in all wild type embryos the animal hemisphere is pigmented, whereas the vegetal hemisphere is not. Second, the entry of the sperm at fertilization causes a contraction of the animal hemisphere pigmentation towards the future ventral side; in many embryos a pigmentation difference therefore can be used to discriminate between dorsal and ventral sides. Third, the first cleavage furrow approximates the mid-sagittal plane in most embryos, and thus can be used to identify the right and left sides of the embryo. The fate maps also rely on the fact that naturally fertilized eggs frequently cleave in regular patterns that make each blastomere identifiable across a large population of embryos. While there is variability within and between clutches of eggs regarding pigmentation and cleavage patterns, using selection procedures described herein allows cells with prescribed fates to be identified with about 90% accuracy.

Cell fates can be determined during embryogenesis by several mechanisms. Intrinsic factors, such as differentially inherited cytoplasmic mRNAs or proteins, contribute to several aspects of early patterning. For example, specific maternal mRNAs determine which cells will contribute to the germ line, become the endoderm, or contribute to the dorsal body axis (reviewed in ⁷). Extrinsic factors provided locally by neighboring cells or more distantly from an embryonic signaling center, are responsible for inducing specific tissue types and patterning nearly every organ system. Examples of signaling centers include the organizer/node in the gastrula that induces the neural ectoderm and patterns the mesoderm, and the zone of polarizing activity that patterns the anterior-posterior axis of the limb bud. Although fate maps identify the precursors of the different organs and reveal the developmental path taken by their descendants in the normal embryo, they cannot distinguish between intrinsic and extrinsic influences on those cells. They also do not reveal the full developmental potential of a cell, whose descendants differentiate in the complex signaling environment of the embryo. Two experimental approaches can test whether a cell's fate is determined by intrinsic factors or is subsequently influenced by external factors: 1) transplantation of the cell to a novel location in the embryo; or 2) removal of the cell from the embryo followed by culture in the absence of exogenous signals.

Both experimental approaches have been feasible in Xenopus because the cells are large enough to be manually separated. For example, numerous studies have deleted single cells from embryos (to change cell-cell interactions) or transplanted cells to novel locations in host embryos to test for fate changes (reviewed in ^{7, 8, 9}). In addition, the second approach of explanting small numbers of cells from different regions of the embryo into culture to elucidate inductive tissue interactions in the absence of exogenous factors is possible because the cells of the Xenopus embryo are filled with an intracellular nutrient store, the yolk platelets. Therefore, they can be cultured for a few days in a defined salt medium without nutritional or growth factor supplementation of the culture medium. We have used this approach to show that dorsal animal blastomeres have an autonomous ability to produce neural and dorsal mesodermal tissues due to maternally inherited mRNAs ^{10, 11}, and others showed that mesoderm specification of 32-cell blastomeres relies on both intrinsic and extrinsic information ¹². An advantage of culturing cells as explants is that the medium can also be supplemented with defined signaling factors to determine which cell-to-cell communication pathway might influence the fate of the explanted cell ^{12, 13, 14}. In addition, one can inject the blastomere prior to culture with mRNA to over-express a gene, or with anti-sense oligonucleotides to prevent the translation of endogenous mRNAs. These, gain- and loss-of-function analyses can identify which molecules are required for an autonomously expressed fate. To analyze the fate of the explant, identification of specific cell types can be performed by standard gene expression (e.g., in situ hybridization, RT-PCR) and immunocytochemical assays. This protocol provides a simple, yet powerful, way to distinguish between intrinsic and extrinsic mechanisms that regulate how an embryonic cell develops into specific tissues.

Protocol

1. Preparation of Instruments, Culture Media and Dishes

1. Sharpen four forceps using Alumina abrasive film. Do this under a dissecting microscope to monitor the tip size. One pair of forceps serves as a back-up in case a tip is damaged during the procedure. Autoclave all four forceps and store in a sterile container.
2. Make 500 ml each of 0.1X and 1.0X culture medium (either Marc's Modified Ringers [MMR] or Modified Barth's Saline [MBS]; recipes in ¹⁵) and filter sterilize. We routinely use MBS (1X = 88 mM NaCl; 1 mM KCl, 0.7 mM CaCl₂, 1 mM MgSO₄, 5 mM HEPES [pH 7.8], 2.5 mM NaHCO₃). Store at 14-18 °C for months.
3. Make a 2% agarose solution in culture medium (2 g electrophoresis grade agarose in 100 ml 1X culture medium in a screw cap glass bottle). Autoclave to dissolve agarose. This can be stored at 4 °C for months, and microwaved to liquefy the agarose when it is next needed.
4. While the agarose is liquid, pour about 0.5 ml onto the bottom of each well of a sterile 24-well culture plate. This will prevent the explants from sticking to the plastic.
5. While the agarose is liquid, pour about 2-3 ml onto the bottom of two to three 60 mm Petri dishes, and swirl gently to make sure the bottom is covered. These will serve as dissection dishes and the agarose prevents embryos from sticking to the plastic once their membranes are removed.
6. When the agarose has cooled and hardened, flame the tip of a 6" Pasteur pipette until it melts into a ball, and lightly touch it on the surface of the agarose in each well of the culture plate. This will create a shallow depression into which the explant will be placed.
7. Fill each well of the culture plate with 1X sterile culture medium.
8. When the agarose has cooled and hardened, fill the dissection dish with diluted culture medium (0.1X MMR or 0.1X MBS) to facilitate blastomere separation.

2. Selection of Embryos

1. Obtain fertilized eggs and remove the jelly coats according to standard protocols ¹⁵. Transfer them to 0.5X culture medium in a 100 mm Petri dish.
2. When embryos reach the 2-cell stage, sort those in which the first cleavage furrow bisects the lightly pigmented area in the animal hemisphere (Figure 1) into a separate dish. These will be the donors for the explants. Keep the remaining 2-cell embryos in a separate dish next to the donor embryos throughout the procedure to serve as sibling controls to stage the explants.

3. Preparation of Explants

1. Place 5-10 embryos in a dissection dish, but only work on one embryo at a time.
2. Position the first embryo so the transparent vitelline membrane can be seen; it is separated by a clear space (perivitelline space) above the surface of the animal pole of the embryo. Using a sharpened forceps in one's subdominant hand (left hand if one is right handed), grasp the vitelline membrane above the perivitelline space. Using a sharpened forceps in the other hand, grasp the membrane close to the first forceps tip, and gently pull in opposite directions to peel the membrane away. Make the initial grasp at a distance from the cell that is to be dissected, so that the target cell is not damaged during removal of the membrane. One can tell that the membrane has been removed because the embryo will flatten.
3. Grab one neighboring cell with the forceps in the subdominant hand and use this cell as a "handle" so the cell to be dissected is not directly touched. With the forceps in the other hand, gently pull the remaining neighboring cells away from the desired blastomere. If midline cells, which share the same fate, are targeted, they can be removed together as a pair. Finally, dissect away the "handle" cell.
4. Pick up the blastomere (or blastomere pair), with a sterile, glass Pasteur pipette, avoiding air bubbles and excessive suction. The pipette can be fire-polished to remove sharp edges that can damage the cell, but we do not routinely do this. Place the tip of the Pasteur pipette under the surface of the culture medium in a well of the explant culture dish, and gently expel the blastomere. It should slide into the shallow depression by gravity. The same blastomere from more than one embryo can be combined into a single explant.
5. Repeat this procedure with the remaining embryos in the dissection dish, one-by-one. After about 10 embryos, the dissection dish will be filled with cellular debris. When this occurs, change to a fresh dissection dish.
6. After all dissections are done, check whether the explants are in the shallow depressions. If not, they can be gently pushed into the depression with a hair loop that has been sterilized in 70% ethanol and air dried.
7. About an hour after the last dissection, remove debris surrounding the healed explants with a sterile, glass Pasteur pipette.
8. Culture the plate of explants at 14-20 °C next to the Petri dish containing the sibling, control embryos. Sibling embryos will indicate the stage of development of the blastomere explants.

4. Harvesting Explants for Analysis

1. Harvest the explants when the siblings reach the desired developmental stage. These explants survive quite well even if some of the cells disintegrate. Therefore, if there is a cloudy mass in the culture well, explore it with a hair loop or forceps to determine if a healthy explant is buried within.
2. Pick up explants in a small volume of culture solution with a glass Pasteur pipette, and gently expel them into a fixative or lysis buffer appropriate for the assay to be conducted.

Results

The ability of this assay to accurately assess the developmental potential of the cell relies upon dissecting out the correct blastomere based on the fate maps ^{1, 2, 3, 4, 5, 6}. Therefore, it is critical to choose embryos with the correct pigmentation pattern at the 2-cell stage, as illustrated in Figure 1, that subsequently conform to regular cleavage patterns, as illustrated in Figure 2. If one observes the sorted embryos as they reach the required cleavage stage and s...

Discussion

The most critical steps for successful culturing of individual blastomeres are: 1) correctly identifying the blastomere of interest; 2) maintaining a sterile, healthy culture; 3) dissecting cells at the correct part of the cell cycle; and 4) developing the necessary manual dexterity to prevent damage during dissection and transfer to the culture well.

To manipulate specific blastomeres, it is essential to be able to identify the cardinal axes. In Xenopus embryos, the dorsal sid...

Materials

Name	Company	Catalog Number	Comments
Alumina abrasive film	Thomas Scientific	#6775E-38	Course (12 μm), for major repairs of forceps tips
Alumina abrasive film	Thomas Scientific	#6775E-46	Medium (3 μm), for fine sharpening of forceps tips
Alumina abrasive film	Thomas Scientific	#6775E-54	Fine (0.3 μm), for polishing of forceps tips
Forceps: Dumont, Dumoxel Biologie #5	Fine Science Tools	#11252-30	These have the fine tips that do not need sharpening when first purchased. Corrosive resistant so they can be autoclaved.
Gentamicin solution	Sigma	G1397	Add to medium on same day as use