Microsurgical manipulations to isolate collectively migrating mesendoderm

Abstract

Mesendoderm mantle closure completes the gastrulation movements of the Xenopus laevis embryo and provides an unparalleled opportunity to study collective cell behaviors within a mesenchymal tissue. Free-edge sheet-like collective movements of these tissues contrast with movements of epithelial tissues in that mesendodermal cells are not constrained by tight junctions or adherens junctions, yet migrate in a coherent and persistent mode over several hours. Mesendoderm cells are the largest motile cells in the Xenopus embryo and complete a 500 µm migratory path. When mesendoderm is cultured on rigid glass substrates, these cells can exceed 100 µm in length and exhibit a highly persistent leading lamellipodia that can exceed 20 µm from tip to base. These large collectively migrating cells provide a unique imaging opportunity to visualize polarized adhesive and cytoskeletal structures with high numerical aperture objectives. Mesendodermal cells in the early embryo originate from around the entirety of the marginal zone and may also be distinguished by their source along the animal-vegetal axis. Here we use the term mesendoderm but note alternative terms for these cells can include: head mesoderm, endomesoderm, and prechordal mesoderm. This protocol summarizes microsurgical preparation of mesendoderm tissue explants and ‘windowed’ embryos. Skills needed to dissect fragments of the mesendoderm mantle are marginally greater than those needed to isolate animal cap ectoderm and can be mastered within two weeks; skills needed to isolate the mesendoderm “ring” or to prepare windowed embryos are significantly greater and may require several weeks training.

MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

Reagents

Cysteine (L-cysteine hydrochloride monohydrate; Sigma-Aldrich, C7880) (2% in 1/3X MBS)

Adjust to pH 8.0 with NaOH.

Embryo culture media (1/3X Modified Barth’s Saline (MBS))

1/3X MBS is the de facto embryo culture media used in the Keller, DeSimone, and Davidson labs where mesendoderm explant techniques described here were devised (Davidson et al., 2002; DeSimone et al., 2007; Ichikawa et al., 2020). The Winklbauer lab, also known for working with mesendoderm fragments, uses 1/10X MBS (Winklbauer, 1988) for embryo culture. Calcium concentrations in embryo culture media vary (Sive et al., 2000) and may yield qualitative differences in the abilities of tissue to remain cohesive during microsurgical manipulations.

Avoid adding phenol red if preparing for fluorescence imaging. Phenol red will introduce non-specific fluorescence that can interfere with red or near-infrared fluorophores. Explant culture media (Danilchik’s for Amy (DFA) Medium (pH 8.3) <R>) DFA explant culture media is not a random collection of odd buffers and ions but rather a solution created to match the interstitial composition of the Xenopus laevis embryo that is relatively low in chloride ions and has a slightly basic pH (Gillespie, 1983). This suitable culture media replaces conventional chloride counter ions of Na+ and K+ with gluconate ions. Endogenous interstitial buffering factors are replaced by the Good buffering agent bicine to minimize the amount of hydroxide counter ions needed to create pH 8.3. Avoid adding phenol red if preparing for fluorescence imaging. Phenol red will introduce non-specific fluorescence that can interfere with red or near-infrared fluorophores.

Fibronectin (Corning 356008; 1 mg/mL)

Aliquot and store at -20^°C.

Modified Barth’s Solution (MBS) (pH 7.4) <R>

3X stocks of MBS are pH stable and can be stored at 4^°C.

Silicone grease (high vacuum silicone grease, Dow Chemical) (Figure 1A) loaded into a 10 mL syringe

Figure 1. Tools and Schematics for Ring Explant and Windowed Embryo.

A) Tools for microdissection including forceps, silicone grease dispenser, hair tools, diamond pencil, pre-cut coverslip glass bridges and silicone grease. B) Schematic of microsurgical maneuvers to make the ring explant. Sagittal and enface views of late gastrula (Stage 12 to 12.5) with a large blastocoel and broad mantle. C) Schematic of microsurgical maneuvers to make the ‘windowed’ embryo. Sagittal view of gastrula (Stage 12.5 to 13) shows narrowing mantle. The microenvironment and tissue interactions, e.g. continuity of mesendoderm with dorsal and ventral tissue, contacts between leading mesendoderm and more vegetal mesendoderm, blastocoel and lateral ectoderm are preserved in the windowed embryo. Mesendoderm mantle (stippled) with leading edge (arrowheads). (a, anterior; b, blastocoel; d, dorsal; e, ectoderm; p, posterior; v, ventral; y, yolk plug). (right) Ring explant schematic on fibronectin coated substrate (fn).

Xenopus laevis embryos at middle to late gastrula stage (stages 11 to 13) (Nieuwkoop and Faber 1967), dejellied as in Protocol: Dejellying Xenopus laevis embryos<prot4731> [Sive et al. 2007] using 2% cysteine described above

Experiments involving mid-gastrula stages may require attention to internal staging criteria described in Nieuwkoop and Faber and whether the vitelline membrane has been removed. Removal of the vitelline can cause changes in 3D morphology, such as deepening of the groove formed by bottle cells. Such changes may make embryos appear more advanced.

Equipment

Coverslip glass (24 x 40 mm, #1.5 thickness) (Figure 1A)

Custom fabricated cold plate dissecting stage (optional; see note under Method heading) The cold plate is a large-footprint aluminum and acrylic base mount for the stereoscope that passes chilled or heated water produced by a chiller-circulating water bath.

Diamond pencil (retractable; Ted Pella # 54468) (Figure 1A)

Disposable pipette

Forceps (2; Dumont #5) (Figure 1A)

Glass bottomed imaging chamber (variety of types including Cellvis or Mattek dishes, Invitrogen chambers, custom acrylic well chambers, custom nylon washer chambers (see Protocol: Chambers for Culturing and Immobilizing Xenopus Embryos and Organotypic Explants for Live Imaging<prot107649> [Chu and Davidson, 2022]) (see Protocol: Assembly of chambers for stable long-term imaging of live Xenopus tissue<prot073882> [Kim and Davidson 2013])

Hair knife (Sive et al., 2000) (Figure 1A)

Hair loop (Sive et al., 2000) (Figure 1A)

Incubator (e.g. MyTemp Mini Digital Incubator; Benchmark Scientific) (optional; see note under Method heading)

Microscope slide

Petri dishes (10 and 60 mm diameter)

Pasteur pipette, plastic, cut to remove the narrow region

Stereomicroscope (various vendors; here, Olympus SZX7 equipped with video port, Canon T3i, and video coupler MDSLR, Martin Microscope Co.) with off-axis lighting (LED dual gooseneck; here, Schott Fiber Optic Light Source and dual gooseneck)

ThermoWorks EasyLog USB Logger (TW-USB-2+; optional for each experiment but recommended to confirm consistent room temperatures; see note under Method heading)

METHOD

Xenopus laevis embryos may be raised in incubators at a range of temperatures within the normal permissive range of 14 to 26°C. Similarly, microsurgical manipulations listed below can be carried out either at room temperature or on a temperature controlled cold plate. Rates of morphogenesis and intracellular processes are very sensitive to temperature so it is critical that temperature is held constant and recorded (e.g. ThermoWorks EasyLog USB Logger).

Preparing the Embryos and the Imaging Chamber

Figure 1 shows microsurgical tools for dissection and schematics of the ring explant and the windowed embryo.

1. Prepare a dish of coverslip bridges as follows:

   1. Score and break 3x8 mm coverslip bridges with the diamond pencil using a microscope slide as a straight edge. Store the coverslip bridges in a clean Petri dish (Figure 1A).
   2. Dispense silicone grease onto the inner surface of a clean Petri dish lid and smooth into a thin layer (1 mm thick; Figure 1A).

   Broken glass and fresh coverslip bridges are extremely sharp. Handle with care. If periodically restocked, the Petri dish with the bridge fragments and silicone grease will last indefinitely and provide a ready source during microsurgery.

2. Coat the imaging chamber with fibronectin. Add 20 µg/mL of fibronectin in 1/3X MBS to the chamber and incubate overnight at 4ºC. Rinse 3 times in 1/3X MBS and fill the chamber with DFA with antibiotic-antimycotic.

3. Set up operating dish. Place sufficient DFA into three Petri dishes approximately 3–4 mm deep. One dish is for microsurgery, one dish is for rinsing fresh explants, and the last dish is for culturing or holding explants until needed.

4. Use forceps to pick up a single coverslip bridge from the dish in Step 1 and dab some silicone grease along the long edge. Place the coverslip bridge grease side down in the center of the Petri dish to be used for microsurgery. This bridge serves as a “back-stop” or aid during embryo and explant manipulation and defines a forcep-free region of the microsurgery dish.

   Forceps can scar the plastic of the Petri dish that can tear wounds on embryos and explants as they are being manipulated.

5. Select dejellied embryos from the middle to late gastrula stage (stages 11 to 13; (Nieuwkoop and Faber, 1967)) and transfer them to the operating dish with a disposable pipette.

   The goals of the experiment will dictate the precise stages needed. For instance, investigations of collective cell migration during mantle closure would require using late gastrula stages at which time the mesendoderm around the entire margin has migrated and the mantle has formed a cup with well-defined walls surrounding the floor of the blastocoel. Alternatively, if the goals of the experiment involve characterizing leading edge migration of mesendoderm, then earlier stages may suffice, as long as the mesendoderm has begun to migrate on the overlying ectoderm. The size of the yolk plug and other external criteria may not correlate with interior progressive movements of the mesendoderm mantle. With side-, or glancing-illumination from a goose-neck style lamp it is possible to observe the position of the mesendoderm mantle within intact embryos (arrowheads in Figure 2A). Under these conditions the blastocoel is dark and the edge of the mantle is detected as a lighter region. The progress of mantle closure can be more easily observed in albino embryos; see Figure 1A in (Davidson et al., 2002).

6. Remove the vitelline membrane by grabbing the membrane at the same location with two forceps (Figure 2B). The vitelline membrane is a thin flexible transparent membrane enclosing the embryo after the jelly coat has been removed. Open, or tear the membrane by slowly moving the forceps apart. This movement will create a tear in the vitelline membrane. Once the opening is large enough, release one forcep and gently shake the embryo out of the membrane with the other. Do not damage the embryo in the marginal zone or animal pole. If the vitelline membrane cannot be removed without damage, try puncturing the embryo first in the ventral pole.

   One blunt and one sharp forcep can be helpful where the blunt forcep is used to hold, or pinch the vitelline membrane, and the sharp forcep used to tear the membrane. Remove the vitelline membrane in a different region of the Petri dish than where microsurgery will be carried out since small scratches caused by forceps can in advertently wound embryos or explants.

   Depending on the microsurgeon’s speed, only remove the vitelline membrane from embryos needed within 15 min. Embryos out of their vitelline membrane for longer than 15 min will sag and may appear to be in a different stage than those remaining in the vitelline.

7. Make an incision fully encircling the pigmented region of the animal cap ectoderm. Insert the tip of a fine hair knife between the involuted mesendoderm and the overlying ectoderm and “flick” outward to make a small incision (Figure 2C). The mesendoderm should be clearly visible as large cells closely associated with ectoderm indicated by small cells. Insert the hair knife into the hole and push the tip into the embryo to emerge at another point around the margin and “flick” outward; repeat this operation through 360° of the ectoderm (Figure 2D). Use the hair loop or glass bridge fragment to brace the embryo during the flick so the embryo does not move. Rotate the embryo and continue the incision around the marginal zone. Adjust this circular incision to expose some of the mantle for intra vital imaging (e.g. with the rest of the embryo ‘intact’, see “Microsurgical Variations” section below) or most of the involuted mesoderm if a full “ring” explant is desired. See also Supplemental Movie S1 which shows a complete recording of microsurgical isolation of the mesendoderm mantle “ring”.

   “Flick cutting” is a microsurgical maneuver that slowly pushes the tip of a hair knife into a tissue followed by a rapid retraction movement that uses the side of the knife to cut through the tissue. Repeated “flick-cuts” can be used to produce and lengthen incisions.

8. Gently remove the animal cap from the remainder of the mantle (Figure 2E). Use the hair knife to gently lift the animal cap from the mesendoderm. Removing the cap will expose the leading edge of the mantle and the open cavity of the blastocoel. Some damage to mesendoderm or mesoderm can be tolerated as long as the leading edge is not wounded.

Figure 2. Steps of Microdissection.

A) Select embryos. B) Remove vitelline membrane. C) Begin incision through ectoderm. D) Complete incision around 360° of the embryo, the full extent of the ectoderm from dorsal to ventral. Note the slight anterior-ward offset of the incision (y, yolk plug) due to the ventral-ward position of the blastocoel. Remove the ectoderm (e) by peeling from underlying mesoderm (not shown, see also Supplemental Movie S1 which shows a complete recording of microsurgical isolation of the mesendoderm mantle “ring’“). E) Removal of the ectoderm reveals mesendoderm mantle (leading edge, arrowheads) and blastocoelic space (b). F) Remove the mantle through a 360° incision that cuts across the mesendoderm into the blastocoelic space. G) Separate the ‘ring’ of the mesendoderm mantle from the remaining embryo. Full microsurgical manipulation can be followed in the Supplemental Movie S1.

Microsurgical Variations

At this point the protocol branches based on specific goals as follows: 1. Single fragments of the leading edge can be isolated for the study of collective migration or multiple fragments can be arranged for confrontation, e.g. where two opposing leading edges collide needed to test contact inhibition of locomotion (Ichikawa et al., 2020). (Step 9). 2. The entire mantle can be isolated from the embryo for studies of closure in the absence of the blastocoel (Step 10). 3. The entire mantle and remainder of the embryo, with blastocoel intact, can be positioned for intra vital imaging (Step 11). This configuration preserves contact between more vegetal mesendoderm and ectoderm and may retain the contents of blastocoel. 4. The mesendoderm can be isolated together with the marginal zone explant (Step 12).

In all of these cases, mesendoderm and accompanying tissues are cultured on a fibronectin-coated substrate. Protein localization and dynamics in lamellipodia, medio-basal cortex, cell-substrate and cell-cell adhesions are accessible to live cell confocal imaging by a high numerical aperture objective (e.g. 63x or 40x oil-immersion objective) with short working distances (< 150 µm). Simple cell boundaries or labeled nuclei can be imaged with 20x air or long working distance water immersion objectives. Time-lapses collected from a simple stereoscope can be used to record and quantify rates of tissue migration.

1. Isolate fragments of mesendoderm. For this preparation, select embryos when the mantle has progressed within 30 to 60° of the animal pole.

   1. Use a hair knife to cut segments from the mantle from the leading edge to the vegetal margin of the blastocoel floor. Separate the mantle segment from the embryo by rolling the embryo and “flick-cutting” the embryo from the mantle segment.
   2. Immediately transfer the explant using a plastic Pasteur pipette that has been trimmed to remove the narrow region. Move the explant to a dish of fresh DFA and then transfer to the imaging chamber. Do not “expel” the explant from the pipette, instead let the explant fall into the dish or chamber under gravity (if needed rotate the pipette to dislodge a stuck explant).
   3. Use a glass coverslip bridge with a dab of silicone grease on both ends to gently compress the mantle segment so that the face of the explant that had previously faced the blastocoel roof is brought into contact with the fibronectin substrate. Position the bridge side-ways over the explant and then pivot the fragment and iteratively compress each end of the bridge over the dab. Too much compression can cause lysis and cell death. Too little compression will fail to bring the mesendoderm into contact with the fibronectin substrate. Adequate compression typically produces a maximum of 10 to 20% strain as measured by the lengthening or widening of the compressed explant with minimal cell rupture.
      The ectoderm-contacting surface can be identified throughout manipulations as the flatter surface of the mesendoderm. Leading edge migration rates of fragments are nearly 1/2 to 1/3 the rate of the intact ring, however, increased rates can be achieved by positioning fragments so they converge (e.g. “in-the-round” configuration in Fig. 10, (Davidson et al., 2002)).
      Proceed to Step 13.
2. Isolate the entire rim of mesendoderm mantle, a.k.a. the ‘ring’ explant. For this preparation, allow the mantle to progress within 25 to 45° of the animal pole. With proper lighting from the side and a dark background, mantle progression may be observed as a lighter region surround a dark shadow, e.g. the blastocoel, under the animal cap. However, mantle progress can also be tracked by sampling embryos at regular time points and removing their animal cap ectoderm.

   1. Remove the animal ectoderm of the blastocoel roof so that the edge of the incision is even with the floor of the blastocoel. Insert a fine hair loop into the mesendoderm with the tip emerging from the blastocoel floor (Figure 2F). Use a gentle sawing motion, removing the mesendoderm by using the hair tool to cut as it is pulled out of the tissue. This maneuver will cut the rim of the mesendoderm free from the vegetal half of the embryo.
   2. Rotate the embryo through the full 360° to dissect the entire mesendoderm mantle from the embryo (Figure 2G). The ring explant is exceptionally delicate and easily torn. Immediately transfer the explant to a dish of fresh DFA and then transfer to the culture or imaging chamber.
   3. Use a glass coverslip bridge to gently compress the explant so that the face that had bound the blastocoel roof (typically smooth) is brought into contact with the fibronectin substrate. Application of too much compression will cause fluid in the center of the explant to ‘blow-out’ and break the ring of the mesendoderm mantle. Proceed to Step 13.
3. Isolate an intra vital or ‘windowed’ embryo preparation of mesendoderm mantle with the remainder of embryo intact. For this preparation the mantle should have progressed within 20 to 30° of the animal pole. Progress can be tracked as described in Step 10.

   1. For best results remove only a small patch of ectoderm to expose 100 to 300 µm of the mesendoderm. The mantle and embryo are extremely delicate and may be transferred immediately to the imaging chamber without an intervening rinse in fresh DFA.
   2. Position the embryo with the mesendoderm mantle facing the fibronectin substrate. Take a broad coverslip bridge with large dollop of silicone grease on either end and gently compress the embryo. The mantle should adhere to the fibronectin with the cell face that had been contacting the blastocoel roof brought to contact the fibronectin substrate. Too much compression will cause the blastocoelic fluid to ‘blow-out’ a portion of the mesendoderm mantle.
      Proceed to Step 13.
4. Isolate mesendoderm together with the marginal zone explant.

   1. This manipulation is better described as a variation of the marginal zone explant (see Protocol: Microsurgical Methods to Isolate and Culture the Early Gastrula Dorsal Marginal Zone<prot097360> [Davidson 2022]). In this variation, bottle cells and a few rows of sub-blastoporal endoderm are not removed from the marginal zone explant. Deep cells under the retained endoderm are still considered mesendoderm and adhere strongly to a fibronectin coated substrate. Within the embryo, the smaller size and anterior fate of these cells differ from the larger, ventral-fated mesendoderm at the leading edge cells of the mantle. The mesendoderm isolated with the marginal zone expresses genes common to head mesoderm and anterior endoderm (Davidson et al., 2004).
   2. Mesendoderm cells at the vegetal edge of marginal zone explants, like preparations of the mesendoderm mantle, are larger than mesoderm cells and migrate in a collective manner similar to the leading edge mesendoderm from the mantle. Leading edge mesendoderm cells in marginal zone explants extend broad lamellipodia and migrate collectively away from the marginal zone, exhibiting qualitatively similar morphologies, polarity, and collective migratory behaviors to leading edge mesendoderm cells isolated from the mantle preparations.
      Proceed to Step 13.

5. Imaging mesendoderm. Due to the rapid closure of 360° mantle preparations from Steps 10 and 11 should be imaged as soon as possible. Leading edge mesendoderm cells within these geometrically confined preparations can migrate at 300 µm/h and will resume migration in a highly persistent manner with large stable lamellipodia immediately after they are brought in contact with fibronectin. Once dissected, either as a tissue ring or in a windowed embryo, the mesendoderm mantle will collapse unless provided a fibronectin coated substrate. Collective migration will continue until cells at the leading edge contact cells migrating from the other side. In a classic display of contact inhibition of locomotion, leading edge cells that contact opposing cells will retract their lamellipodia within seconds, cease directed migration, and extend short-lived protrusions in random directions. Collective migration of mesendoderm cells in tissues prepared from Steps 9 and 12 can be imaged within 15 – 30 minutes after they have attached to the fibronectin substrate. Leading edge mesendoderm cells in these preparations will continue to exhibit persistent directed migration at 100 µm/h for 5 to 10 hours after isolation.

DISCUSSION

Mesendoderm explants may be dissected together with marginal zone tissues, as an isolated fragment, as an isolated ring, or nearly intact in a windowed embryo (Davidson et al., 2002) and can be used as a source of mesenchymal cells to study collective migration distinct from migration of epithelia. Mesendoderm cells in explants exhibit collective migration that is thought to occur in vivo on the inner surface of the blastocoel roof (Nakatsuji and Johnson, 1982; Winklbauer and Nagel, 1991; Winklbauer et al., 1992; Winklbauer and Keller, 1996) (Ichikawa et al., 2020). High resolution imaging of migratory cells in the mantle can reveal both adhesive and cytoskeletal dynamics due to the remarkably large size of mesendoderm cells, often longer than 100 µm during later stages of migration and mantle closure. Portions of the mesendoderm can be excised and used to study migration of cell clusters in gradients of extracellular matrix or in gradients of chemo-attractant (Nagel et al., 2004). Remarkably, mesendoderm masses can also be separated into single cells using calcium- and magnesium-free culture media to investigate single cell migration and contact inhibition (Ichikawa et al., 2020).

Mesendoderm cells are large and easily distinguished from smaller lateral plate or dorsal mesodermal derivatives. Furthermore, mesendoderm can be identified by expression of Sox17 (Hudson et al., 1997). It is possible to inadvertently include dorsal mesodermal progenitors with the mantle but this contamination can be readily detected in explants cultured to equivalent stage 23 by immunofluorescence with tor70 (notochord) or 12/101 (somitic mesoderm) at later stages. Thus, mesendoderm explants offer the opportunity to study mass mesenchymal migration, collective movements, and single cell movements from an easily isolated source.

From their origins around the marginal zone, mesendoderm cells migrate on the inner fibronectin-coated surface of the blastocoel as the leading edge of the mesendoderm ‘mantle’ converges toward the animal pole (Ewald et al., 2004). As the mesendoderm mantle converges, cells at the leading edge form a perimeter shaped like a shrinking circle or ellipse. Mantle closure completes the major tissue internal movements of gastrulation by positioning mesodermal cells into the ventral-most regions of the gastrula and early neurula. After mantle closure, mesendoderm seals off the connection between the blastocoel and the animal cap ectoderm.

There is ongoing debate concerning the nature of cues driving directional migration of mesendoderm (Davidson et al., 2002; Nagel et al., 2004). It has been suggested that mechanical cues drive directional migration through ‘cohesio-taxis’ as polarized protrusions from the leading free-edge of mesendoderm cells can be initiated by tension directed in the opposite direction at the rear of the cell (Weber et al., 2012; Sonavane et al., 2017). C-cadherin (CDH3) regulates both cell-cell adhesion and non-junctional signaling to alter persistence, velocity, and contact inhibition of locomotion (Ichikawa et al., 2020). The native substrate of the mesendoderm, i.e. the extracellular matrix coat of the blastocoel animal cap ectoderm includes fibrillar fibronectin assembled by deep ectoderm cells. It has been proposed that mesendoderm may follow directional cues embedded in this extracellular matrix using a form of haptotaxis (Winklbauer and Nagel, 1991). Alternatively, mesendoderm may also follow chemotactic cues secreted by the animal cap ectoderm (Nagel et al., 2004; Smith et al., 2009; Nagel and Winklbauer, 2018). Mesendoderm explants offer the unique opportunity to test these and other models and elucidate principles that integrate molecular pathways, biomechanics, and mechanobiology.