Developmental Biology

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Authors: Hazel L. Sive, Robert M. Grainger and Richard M. Harland

This protocol was adapted from “Microdissection,” Chapter 10, in Early Development of Xenopus laevis by Hazel L. Sive, Robert M. Grainger, and Richard M. Harland. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000.

INTRODUCTION

The basic Keller explant is a rectangle of dorsal mesendoderm and ectoderm from an early-gastrula-stage Xenopus laevis embryo. It is ~60° to 90° wide, extending from the bottle cells to the animal pole. This protocol describes how to dissect, assemble, and cultivate Keller explants. The purpose of Keller explants was initially to allow observation of gastrulation movements, particularly convergent extension, in culture. This is difficult to do when explants curl up, but in Keller sandwiches, the explants are cultured flat, either as a single sheet (open-face explant) or more frequently as two sheets sandwiched together with their inner surfaces apposed (closed sandwich). Explants are cultured beneath a coverslip fragment or a glass bridge resting on silicone vacuum grease until the desired stage, usually during or after neurulation. Instead of involuting beneath the ectoderm, mesoderm elongates in a plane with adjacent ectoderm. Explants are made at the onset of gastrulation before significant vertical juxtaposition of ectoderm and mesoderm has occurred.

RELATED INFORMATION

For further information on the generation and use of Keller explants, please see Keller and Danilchik (1998). A general introduction to explant and transplant assays in Xenopus can be found in Microdissection: Explant and Transplant Assays in Xenopus laevis.

MATERIALS

Reagents

  1. Sater’s modified blastocoel buffer (SMBB)
  2. Xenopus laevis embryos, early gastrula (stage 10 to 10+)

Equipment

  1. Coverslips
  2. Dissection tools, including eyebrow knife, hair loop, and forceps (see Embryo Dissection and Micromanipulation Tools)
  3. Petri dishes
  4. Syringe (5 ml) to dispense vacuum grease
  5. Vacuum grease

METHOD

  1. Dejelly the embryos and remove the vitelline envelope as described in Dejellying Xenopus laevis Embryos and Removing the Vitelline Membrane from Xenopus laevis Embryos.
    • To avoid damaging the dorsal side or animal cap, remove the vitelline membrane from the ventral side of the embryo.
  2. Place the devitellinated embryo animal pole down, with the dorsal side (bottle cells) toward the hand in which the eyebrow knife is held (the “cutting” hand).
  3. Using a hair loop or forceps to hold the embryo in place, poke the tip of the eyebrow hair downward at one end of the line of bottle cells. Make a radial cut toward the animal pole, as shown in Figure 1A (slit 1).

Figure 1

Figure 1. Preparation of a Keller explant, for use in sandwich or open-face explant assays. (A) Initial cuts, 1-3; (B) peel back of mesendoderm and secondary cut 4; (C) trimming explant.

  • This cut is easily achieved by cutting against the base of the Petri dish, or by flicking the knife upward.

4.Make a similar cut at the opposite end of the bottle cell line (Fig. 1A, slit 2). Turn the embryo over, that is, animal pole facing up.

If the cuts have not reached the animal pole, complete the cuts using one of the following approaches:

  • i. Insert the tip of the eyebrow knife into one of the cuts, under the region that needs to be cut. Then press the hair loop against the tip of the eyebrow hair. This will complete the cut by pinching the tissue between the two hairs.
  • ii. Alternatively, hold the region adjacent to the cutting edge with the hair loop, and gently flick upward with the eyebrow hair. This trick requires some practice, but, once mastered, it is a clean and fast way to cut through thin tissues.

5.Using one of the cutting methods described in Step 4, make a horizontal cut at the animal end of the dissection to complete a rectangle with the previous two incisions (Fig. 1A, slit 3).

6.Hold the embryo down with a hair loop. Using an eyebrow knife, peel back the dorsal tissues to the dorsal blastopore lip (Fig. 1B). Use the edge of the eyebrow hair to deepen the cleft formed by the involuting head mesoderm to allow peeling back to the lip.

  • In very early gastrulae, the cleft has yet to form, thus it must be cut with the knife. This removes some of the head mesoderm from the noninvoluted chordamesoderm.

7.Flip the embryo over (vegetal pole up), and, with the edge of the eyebrow hair, cut downward along or just below the bottle cells, all the way through to the blastocoel. This is the bottom edge of the rectangle (Fig. 1B, slit 4).

  • Do not cut off the bottle cells.

8.Remove any pieces of involuting head mesoderm that are left on the inner side of the outer mesoderm cells (Fig. 1C). Place the rectangle inner surface down, and trim off any material that protrudes beyond the bottle cells. Then, turn the explant over, and use the tip of the eyebrow knife to gently pick off any loose head mesoderm cells.

9.Trim the explant into a regular rectangle of the desired dimensions.

For an open-face explant, proceed to Step 10; for a closed sandwich, proceed to Step 11.

10.For an open-face explant:

  • i. Transfer the explant to a new dish of Sater’s modified blastocoel buffer (SMBB), and lay the tissue flat, with the side of interest uppermost.
  • ii. Using a 5-ml syringe, put a dab of vacuum grease at each end of a sterile rectangle of coverslip (~5 × 10 mm) or glass bridge.
    • The grease serves to hold the coverslip in place on top of the explant and should be used sparingly.
  • iii. Gently place the coverslip on top of the explant. Exert slight pressure to flatten the explant, but do not damage the cells. Avoid contaminating the explant with vacuum grease.

11.To make a closed sandwich:

  • i. Using two rectangular explants of the same size (from Step 9), immediately press the inner sides together, making sure that the bottle cells are aligned.
    • If explants are left in medium for more than a few minutes, they will not adhere well.
  • ii. Trim the edges of the sandwich so that the explants are exactly the same size. Set them aside in a dissecting dish.
    • The dissecting dish should be kept relatively free from debris, and thus, it is advisable to prepare sandwiches one at a time.
  • iii. Transfer the sandwiches to a clean dish, and gently press each one under a coverslip (as described in Step 10.ii). Do not squash the sandwich!
    • Make sure that the layers of the sandwich are well aligned and that the explant is flat. If explants are out of register, the layers will not elongate efficiently.

12.Culture the explants in SMBB at 15°C-23°C, depending on the desired rate of development.

  • To monitor explant development, include three to four control embryos with intact vitelline membranes in the same dish as the explants. If the incubation time exceeds 24 hours, replace the medium every day.

TROUBLESHOOTING

  1. Problem: The mesoderm of late-teen-stage explants begins to dissociate. [Step 12]
    • Solution: Reduce the salt concentration of the medium as follows:
      • Replace half of the volume with H2O containing antibiotics (e.g., gentamycin at 0.05 mg/ml).
      • Alternatively, replace the entire volume with 0.5X MBS containing antibiotics

REFERENCES

  1. Keller R., Danilchik M. (1988) Regional expression, pattern and timing of convergence and extension during gastrulation of Xenopus laevis. Development 103:193–209.

DOI

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