Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

The following protocol uses chicken embryos at less than ten days of incubation. All experiments were performed in accordance with King's College London, UK and the UK Home Office animal care guidelines.

1. Preparation Required Prior to Chick Embryo Operation

1. Gather the following surgical instruments required for the operations, for harvesting and for fixing the chick embryos: Two pairs of No. 5 watchmakers forceps, one pair of strong forceps, e.g., type AA, curved scissors with blades 2.5 cm long, blunt ended curved forceps with ends approximately 0.5 cm long, and also two pairs of No. 4 watchmakers forceps to use while making foil barriers (1.6.3).
2. Make a micro knife from a steel pin (0.3 mm across) sharpened using oil on a sharpening stone. Using a binocular microscope, sharpen the tip of the pin to a flat blade. Place the pin in a needle holder and protect its sharp end from being knocked and dented.
   NOTE: Sheathing the knife with a 1,000 µl pipette tip is a simple option for this.
3. Prepare 70% ethanol in a plastic wash bottle. Use a squirt of this on a paper tissue to wipe the surgical instruments and the micro knife before and after use to sterilize and clean them.
4. Buy 5 cm wide clear sticky tape and a suitable dispenser. Find or manufacture a rest suitable to hold a chicken egg steady on its side.
5. Make and store solutions for soaking beads.
   1. Prepare a 0.35 mg/ml solution of Fibroblast Growth Factor 4 (FGF4) protein from 1 mg/ml frozen stock by diluting it with water and store at -20 °C in 30 µl aliquots.
   2. Prepare solutions (either 0.05 or 0.1 mg/ml) of all-trans-retinoic acid (RA) diluted with dimethyl sulfoxide (DMSO) in dark microcentrifuge tubes from a 5 mg/ml stock. Store the stock and 100 µl aliquots at -20 °C.
   3. Prepare solutions (either 2.5 or 5 mg/ml) of BMS 493 (an inverse agonist of RARs) diluted with DMSO in dark microcentrifuge tubes from a 10 mg/ml stock. Store the stock and 100 µl aliquots at -20 °C.
6. Make barriers from aluminum foil.
   NOTE: The foil used to make the barriers is standard catering foil (measured by micrometer as 0.013 mm thick). Any domestic aluminum foil would likely be suitable.
   1. Cut a 1 cm square piece of aluminum foil and sterilize it by wiping it with 70% ethanol on a tissue. Place it onto a 6 cm sterile plastic Petri dish. Discard the lid and make a lid for the dish from foil.
      NOTE: This will prevent the cut barriers from later sticking to the plastic lid of the dish due to static.
   2. Place the Petri dish on top of a stage graticule (1 cm long split into 100 sections) under a binocular microscope with magnification set to 1.6X. Using a No.15 blade small scalpel, cut strips of the foil to an exact width, e.g., 0.7 mm or 1.3 mm.
   3. Separate the foil strips using two pairs of No. 4 watchmakers forceps. Then make a right-angled bend in the center of the foil barrier such that it resembles a hinge of the specific width.
      NOTE: This shape of barrier is taken from ^{9, 10}. A hinged barrier stays in place better than a straight, flat barrier. No. 4 forceps are relatively blunt and are used because they are less likely to make dents or scores in the foil barriers.
   4. Line up the barriers in the center of the Petri dish with one side flat against the dish and the other pointing up out of the dish, moving the remaining uncut foil to one side. Prepare 20 or more barriers before operation day. Place the foil lid over the dish and store the barriers in a place where they will not be disturbed.
      NOTE: Even a slight knock can tip the barriers over or mean that they stick with static to the sides of the dish.
7. Incubate fertilized chicken eggs on their sides on cardboard egg trays at 38 °C for the appropriate length of time before operation day such that the embryos will be at the desired ⁸ stage.

2. Preparation of Chick Embryos and Beads on Operation Day

1. 'Window' incubated chicken eggs.
   NOTE: The following method for opening eggs was passed on by Dennis Summerbell (not published).
   1. With the egg lying on its side, pierce the blunt end (air sac end) of the egg with type AA strong forceps making sure that the inner shell membrane has been broken. To do this grasp the forceps firmly together and use a controlled stabbing motion while holding the egg with the free hand.
   2. Take the egg, still lying on its side, in both hands (one either side) turn it through 180° along the horizontal axis and replace it in the egg tray such that the side that was uppermost is now next to the tray.
      NOTE: This loosens the embryo from the shell membranes and will mean that the embryo is less likely to be cut when the shell above the embryo is removed.
   3. Take a piece of clear tape approximately 5 cm square and stick this closely over the uppermost surface of the egg to stop the eggshell from crumbling onto the embryo when the egg is opened.
   4. Use the curved scissors to gently break through the shell and its membranes in the center of the uppermost part of the egg, make a very small hole with one blade of the scissors so that air can enter the egg over the embryo (which lies just under the uppermost part of the shell).
      NOTE: By just breaking the shell membranes and not breaking any of the membranes covering the embryo, air entering the egg will separate the embryo from the shell and the embryo will come to rest on top of the yolk about 0.5-1 cm from the shell.
   5. Use the curved scissors to enlarge the hole in the shell to a circle with a diameter of approximately 1 cm. Remove the piece of excised shell and cover the hole in the egg with a piece of clear tape approximately 5 cm square. To easily remove this tape, press the tape to stick it only at the lip of the hole, leaving the remaining tape free. The free tape can then be grasped to unseal the egg.
2. Stage embryos
   1. Following incubation at 38 °C (see step 1.7) and windowing (step 2.1), use a binocular microscope to stage the chick embryos according to ⁸. Do this just before the eggs are sealed following windowing. Write the stage of each embryo on the eggshell. Return the eggs to the incubator.
      NOTE: Embryos at stages between 8 and 15 are suitable for barrier and bead experiments. Embryos are more likely to survive if they have not been left out of the incubator for too long prior to operation.
3. Soaking beads prior to operation.
   1. FGF4
      1. Take the number of affinity chromatography gel beads (150-300 µm) that stick to the end of a 1,000 µl pipette tip and wash them in 1 ml phosphate buffered saline (PBS) in a microcentrifuge tube to remove the azide they are stored in. Centrifuge for 5 sec, discard the PBS and repeat the process twice.
      2. Soak the gel beads in a 30 µl drop of 0.35 mg/ml FGF4 protein on a sterile Petri dish on ice for 30 min. Pick beads approximately 150 µm in diameter with No. 5 microforceps for insertion into the embryo.
         NOTE: Handle ion exchange resin beads used in the following steps with No. 5 microforceps and grip them very gently because the beads are brittle and can shatter if squeezed too tightly. This warning applies to 2.3.2-2.3.3 and all resin bead operations.
   2. RA
      1. Defrost an aliquot of 0.05 or 0.1 mg/ml all-trans-RA diluted with DMSO and place a 100 µl drop on a sterile Petri dish (9 cm diameter), which has previously been covered in foil to protect the RA from light. Take a 10 µl pipette tip and pick up some chloride form ion exchange resin beads on its end and soak these in the RA drop protected from light at room temperature for 20 min.
      2. Rinse the beads in three successive drops of 100 µl Dulbecco's Modified Eagle Medium (DMEM) placed on the same Petri dish at least 1 cm apart.
         NOTE: The beads take up the phenol red dye from the DMEM. This method is based on ⁵.
      3. Pick beads approximately 100 µm in diameter (beads range in size from 75-180 µm when wet) for insertion into the embryo, using a microscope stage graticule slide under the Petri dish and No.5 microforceps to hold the beads. When picking up the bead from the 100 µl drop of DMEM, first place the bead down onto the Petri dish and remove any remaining DMEM from it either by pushing the bead out of the drop or by placing the closed forceps next to the bead (capillary action draws the liquid away from the bead).
   3. BMS 493
      1. As for the RA (2.3.2), soak ion exchange resin beads in a 100 µl drop of 2.5 or 5.0 mg/ml BMS 493 in DMSO at room temperature for 20 min then rinse in 100 µl drops of DMEM. Pick beads approximately 100 µm in diameter for insertion into the embryo.
   4. DMSO control
      1. As for the RA (2.3.2), soak ion exchange resin beads in a 100 µl drop of DMSO at room temperature for 20 min then rinse in 100 µl drops of DMEM. Pick beads approximately 100 µm in diameter for insertion into the embryo.

3. Barrier and Bead Operations

Figure 1. Schematics of Chick Embryos of Various Stages Showing Where Barriers and or Beads Should be Placed. (A) Stage 13 chick embryo schematic showing barrier position (green line) between somites and LPM at the presumptive wing level (somites 15-20). (B) The same schematic as in (A), indicating where RA bead (red circle) should be placed prior to barrier insertion. (C) Schematic showing barrier position (green line) at the presumptive leg level (somites 26-32) in a stage 15 embryo. (D) The same schematic as in (C), indicating where an RA bead (red circle) should be placed. (E) Schematic indicating where BMS 493 beads (purple circles) should be placed in the LPM of a stage 15 embryo. (F) Schematic diagram of a stage 9 chick embryo indicating barrier position (green line) between the presomitic mesoderm and LPM at the presumptive wing level. (G) Schematic diagram of a stage 9 embryo showing the positions of a barrier (green line) and RA bead (red circle). (H) Schematic diagram of a stage 10 chick embryo showing barrier position (green line) at the presumptive leg level. (I) Schematic diagram showing BMS 493 bead position (purple circle) in stage 10 leg level LPM. This figure has been modified from ⁷. Please click here to view a larger version of this figure.

1. Barrier inserted at stage 13 to prevent wing bud initiation.
   1. Take a stage 13 embryo from the incubator and place the egg on an egg rest under a binocular microscope set to 3.2X magnification or higher if preferred. Remove the upper layer of clear tape.
      1. Using a steel micro knife, make an incision through the vitelline membrane and lateral plate mesoderm (LPM) adjacent to somites 15 to 20 (Figure 1A) on the right side of the embryo (somites have not all formed at this stage). Cut adjacent to the last four somites formed and continue the cut two somite lengths caudally.
      2. Pick up the barrier (0.7-1 mm wide) with No. 5 microforceps at the end protruding from the Petri dish and twist the hand to insert the free end of the barrier into the incision. The hinge shaped barrier lies flat on the vitelline membrane above the LPM with half of it protruding downwards through the incision distal to the somites. As soon as possible seal up the egg with clear tape and return it to the incubator.
         NOTE: The micro knife and forceps become sticky on contact with the vitelline membrane. It is important to wipe them clean with 70% ethanol before attempting a subsequent operation. This applies to all subsequent operation descriptions and to bead implantations.
      3. If a bead is to be inserted in conjunction with a barrier; firstly, take the bead in No. 5 microforceps and insert it into the cut face of the LPM on the distal side of the incision and then insert the barrier into the incision proximal to the bead (Figure 1B). Seal the egg and return it promptly to the incubator.
2. Barrier inserted at stage 15 to prevent leg bud initiation.
   NOTE: An example of the result of this operation on Tbox 4 transcription factor (Tbx4) expression is shown in Figure 2D and 2E.
   1. Take a stage 15 embryo from the incubator and place the egg on an egg rest under a binocular microscope set to 3.2X magnification or higher if preferred. Remove the upper layer of clear tape.
      1. Using a steel micro knife make an incision through the vitelline membrane and LPM adjacent to somites 26 to 32 (Figure 1C) on the right side of the embryo (somites have not all formed at this stage). Cut adjacent to the last two somites formed and continue the cut five somite lengths caudally.
      2. Pick up the barrier (1.2-1.3 mm wide) with No. 5 microforceps at one end and twist the barrier to insert the free end into the incision. As soon as possible seal up the egg with clear tape and return it to the incubator.
         NOTE: The hinge shaped barrier lies flat on the vitelline membrane above the LPM with half of it protruding downwards through the incision distal to the somites.
      3. If a bead is to be inserted in conjunction with a barrier, firstly, take the bead in No. 5 microforceps and insert it into the cut face of the LPM on the distal side of the incision and then insert the barrier into the incision proximal to the bead (Figure 1D). Seal the egg and return it promptly to the incubator.
      4. If a bead or beads are to be inserted without a barrier, e.g., BMS 493 beads to the leg region at stage 15, do not make the long incision (3.2.2.1). Make a small incision through the vitelline membrane and into the LPM at the position the bead is to be placed. Take the bead in No. 5 microforceps and insert it into the hole in the LPM. Push it in a little further with the closed ends of the forceps (Figure 1E).
3. Barrier inserted at stage 9 to prevent wing bud induction and initiation.
   1. Take a stage 8 or 9 embryo from the incubator and place the egg on an egg rest under a binocular microscope set to 3.2X magnification or higher if preferred.
      1. Using a steel micro knife make an incision through the vitelline membrane and LPM lateral and rostral to the primitive streak, at the caudal end of the embryo in line with the somites (Figure 1F) about 5 somite lengths long.
      2. Pick up the barrier (0.7-1 mm wide) with No. 5 microforceps at the end protruding from the Petri dish and twist the hand to insert the free end of the barrier into the incision. As soon as possible seal up the egg with clear tape and return it to the incubator.
         NOTE: The hinge shaped barrier lies flat on the vitelline membrane above the LPM with half of it protruding downwards through the incision.
      3. If a bead is to be inserted in conjunction with a barrier; firstly, take the bead in No. 5 microforceps and insert it into the cut face of the LPM on the distal side of the incision and then insert the barrier into the incision proximal to the bead (Figure 1G). Seal the egg and return it promptly to the incubator.
4. Barrier inserted at stage 10 to prevent leg bud induction and initiation.
   NOTE: An example of the result of this operation on Tbx4 expression is shown in Figure 2G.
   1. Take a stage 10 embryo from the incubator and place the egg on an egg rest under a binocular microscope set to 3.2X magnification or higher if preferred.
      1. Using a steel micro knife make an incision through the vitelline membrane and LPM lateral to the primitive streak, at the caudal end of the embryo in line with the somites (Figure 1H) approximately 6 somite lengths long.
      2. Pick up the barrier (1.2 mm wide) with No. 5 microforceps at the end protruding from the Petri dish and twist the hand to insert the free end of the barrier into the incision. As soon as possible seal up the egg with clear tape and return it to the incubator.
         NOTE: The hinge shaped barrier lies flat on the vitelline membrane above the LPM with half of it protruding downwards through the incision.
      3. If a bead is to be inserted without a barrier, e.g., BMS 493 bead to the leg region at stage 10, make a small incision through the vitelline membrane and into the LPM at the position the bead is to be placed. Take the bead in No. 5 microforceps and insert it into the hole in the LPM. Push it in a little further with the closed ends of the forceps (Figure 1I).
5. 24 hr or more after operating check the position of the barrier compared with the emerging contralateral limb bud. Discard any embryos in which the barrier is clearly not opposite the left limb bud. In this case the barrier has been wrongly positioned.

4. Harvesting Embryos, Fixation and Preparation for Wholemount In Situ Hybridization (WISH)

1. Harvesting embryos post operation.
   1. Harvest embryos at stage 19-23 such that limb bud morphology is clearly visible in both the operated and contralateral control sides.
      1. Using curved scissors cut a larger hole in the eggshell. Cut around the embryo while holding the large blood vessel to the left of the embryo with microforceps. Use this grip of the vessel to remove the embryo and place it in a Petri dish of phosphate buffered saline (PBS) pH 7.3.
      2. Under a binocular microscope, dissect away extra embryonic vessels and membranes using a combination of curved scissors and no. 5 microforceps. Remove the head using the scissors.
2. Fixation of embryos post harvesting.
   1. Using curved blunt forceps, lift the embryo and place it in 5 ml 4% paraformaldehyde (PFA) in PBS at room temperature for two hr rocking and then at 4 °C overnight in a 20 ml glass bottle with a screw cap.
      Caution: PFA is a serious hazard to health. It is a harmful, corrosive irritant and the powder must be prevented from coming into contact with skin, eyes and lungs. Use personal protective clothing and a fume hood while preparing the solution. Once it is a 4% solution, gloves, eye protection and a lab coat must be worn.
      NOTE: It is important to get the embryos into fix quickly to best preserve mRNA integrity.
3. Removal of barriers prior to wholemount in situ hybridization.
   NOTE: Barriers are removed because they are sharp and can damage embryos as they go through the in situ hybridization protocol, especially if several embryos are stained at once.
   1. Following fixation, place the embryo in a dish of PBS under a binocular microscope.
   2. Using two pairs of No. 5 microforceps, place the left pair closely either side of the barrier in the operated side of the embryo. Grasp the barrier with the right pair and gently pull the barrier from the embryo using the left pair to keep the embryo down and to prevent any tissue from adhering to the barrier as it is removed.
4. Carry out WISH essentially as described in ¹¹.