Grafting Procedures 1 General

All operations were performed under a Zeiss binocular dissecting m icroscope using a S chott KL1500 fibre optic lam p for illum ination. The top layer of Sellotape was rem oved and the embryo made accessible by tearing the chorion and amnion with two pairs of fine forceps.

Tissues were cut with electrolytically sharpened tungsten needles and transferred where necessary on a flattened nickel spatula (hammered flat and the edges turned up such that a drop o f solution was m aintained with the tissue). Needles and the spatula were embedded in glass capillary tubing using A raldite resin.

W here grafts were pinned into hosts, the pins were cut from 25 jim platinum wire (Goodfellow Metals, Cambridge) and manipulated into place with fine forceps.

2.2.2 Preparation of reaggregates

In the majority of operations, leg bud mesenchyme and ectoderm were used to make reaggregates (the reasons and exceptions will be detailed later). Stage 19-21 em bryos were used for the mesenchyme donors and stage 22-24 embryos for the ectoderm donors. These stages of m esenchym e were chosen because preliminary work by Michael Richardson had suggested that they were the best for optimal morphogenesis. His study included different stage ectoderms also and, as the stage of the ectoderm did not affect the outcome, these stages were selected based on

ease o f trypsinization and capacity to hold the maximum possible am ount of mesenchyme. Reaggregates were typically grafted to the proxim al-dorsal-anterior surface of a stage 22-24 host wing. The complete procedure was as follows (Figure 2.1).

Embryos of the appropriate stage to be either mesenchyme or ectoderm donors were rem oved from the egg into a 90 mm plastic petri dish containing sterile phosphate buffered saline (PBS; 1 tablet in 100 ml distilled water; Oxoid, U nipath Ltd, Herts., England). About 8-16 mesenchyme donors were used and 8-10 ectoderm donors. Using a pair o f fine forceps and a tungsten needle the embryos were cleaned of their am nion and any residual chorion and blastodisc. H ind limb buds w ere dissected off with the tungsten needle and transferred into a 35 m m petri dish of PBS until all had been collected. The dishes were kept on ice until ready for use.

It was then necessary to separate the ectoderm al and m esenchym al com ponents o f the lim b buds. F irstly, the m esenchym e donors were cut in half along the anteroposterior axis to speed up rem oval o f their ectoderms. The PBS was rem oved and replaced with 2-3 ml of ice-cold 2% trypsin. The dish was returned to ice for 10-15 m inutes before a trypsin- stopping medium (TSM), chilled to 4°C, was added (TSM = 88% F 1 2 M e d iu m , 10% fœ ta l c a lf se ru m a n d 2% antibiotics/antim icotics, all from Gibco). After two washes in ice-cold TSM the ectoderms could readily be removed with fine forceps and discarded.

The ectoderm -free mesenchyme buds were drawn up in 100 |ll with a Gilson pipette and transferred to an Eppendorf, where 900 jll fresh TSM was added. The mesoblasts were then separated by vigorous trituration through a fine bore pipette (using a 200 )ll Gilson set to 100 |ll). To assure that the cells w ere sufficiently separated from each other, 7-8 |ll o f the suspension were placed on a haematocytometer and the number of cells counted. The suspension was inevitably very dense with cells, thus an accurate count of the total number of cells was not attempted. Instead, a count of 5 of the 25 marked squares was made to ascertain the approximate ratios of single cells to those in

sta g e 20 Stage 23 M esoderm kept Single cells . $ # Cell pellet Recom bination E ctoderm kept

Reaggregate

Schem atic diagram depicting the protocol for m aking reaggregates. The grey areas represent m esenchyme. Stage 20 leg buds are trypsinised and the m esenchym e dissociated into single cells. These cells are pelleted and placed inside the jacket trypsinised from a stage 23 leg bud. The resulting recombinant can be grafted to a host to develop.

clumps. A single cell count of <95% led to the suspension being further triturated and re-counted.

W hen a satisfactory single-cell suspension had been achieved, the cells were pelleted by m ild centrifugation (6500 rpm, 4 minutes). The pellet of cells was scraped off the bottom of the Eppendorf using a fine tungsten needle that had been bent such that the tip formed a U-shape. This was found to be the best m anner of retrieving the pellet as it caused the least dissociation. The pellet could then be sucked up in a large bore pipette and transferred to a 35 mm petri dish containing fresh TSM. To make the pellet solid enough to graft it was cut into sm all fragm ents and placed in a 5%-C02 incubator at 3 7 ± r C for 40 minutes to 1 hour to allow the mesenchyme to consolidate.

Once the mesenchyme was in the incubator, the ectoderm donors had their PBS removed, were washed twice in ice-cold 2% trypsin and transferred to ice. They were left for varying tim es depending on the batch of trypsin, ranging from 35 minutes to I hour (typically, 45 minutes). The correct trypsinisation stage could be recognised by the slight lifting of the apical ectodermal ridge from the underlying mesenchyme. Once this was seen the ectoderm donors were w ashed tw ice in ice-cold TSM and replaced on ice.

The ectoderm donors were transferred individually into the dish containing the fragments of mesenchyme. Two pairs of fine forceps were used to tease the ectodermal jackets off o f the leg buds, taking care not to rip the jackets or turn them inside out. Once the jacket had been removed, one or more pieces of the reaggregated mesenchyme were manoeuvred into the open end and positioned underneath the AER. The jackets shrink as a result o f the protease treatm ent, so the procedure had to be carried out swiftly before they were too wrinkled and small to use. It appeared, at this macroscopic level, that the ectodermal jackets w ere free from adherent m esenchym e, as they w ere translucent and the mesenchyme appeared opaque in the TSM. Any jackets that were seen to have mesenchymal cells adhering were discarded.

Once the maxim um num ber of reaggregates had been made, they were placed back into the CO2 incubator for 1 hour to

allow them to consolidate for grafting. During this tim e the presum ptive hosts were returned to the incubator to ensure they w ould be warm when grafting. It was found that the grafting procedure inevitably led to the host bleeding and grafts to a cold host significantly increased the chances of forming a substantial haematoma that prevented development.

To prepare a site for the reaggregate graft the embryo was m ade accessible as previously described. Using a sharpened tungsten needle, a cube of the host wing was removed from the dorsal surface as far proximal and anterior as possible (to avoid placing the graft near the host AER or polarising region). W ork had already shown that reaggregates developed similarly whether g ra fte d to th is site (M ich a e l R ic h a rd so n , p e rs o n a l com m unication), pinned to the anterior border o f the wing (M ichael Richardson, personal communication) or grafted to the flank mesenchyme (Ros et al., 1994).

The reaggregate was transferred in by nickel spatula and placed, open end of jacket down, into the trough. In the majority of cases this was sufficient to hold the graft in place. However, in cases where the host embryo was very deep in the egg, or where the graft was very large, the graft was pinned in place using platinum pins. This was not found to affect development of the reaggregate.

The grafts w ere allow ed to sit on the bench fo r approxim ately thirty minutes to start healing in place before returning them to the incubator to develop. They were then checked at 24 and 48 hours to ensure they were still in position and healthy.