Materials and Methods 1 Cell tracing experiments

3.2.1.1 Quail-chick chimeras

Q uail-chick chim eras have been much used in developm ental biology due to the fact that the more dense packing of chromatin in quail cell nuclei make them easily distinguishable from chick cells in cell mixtures (Le Douarin, 1973).

The reaggregates were made as normal (Chapter Two), except for the following (Figure 3.1). Quail and chick embryos were harvested and a nick was made in the anterior margin of the lim bs used for mesenchyme donors, to act as a marker. The limbs were de-ectodermed as usual with 2% trypsin. A piece of m esenchym e approxim ating to the polarising region was then excised. The chick limb mesenchyme (without polarising region) was then mixed with the quail polarising region. This effectively substituted the chick polarising region for a quail one. The chimeric mixture was triturated together and reaggregates made as normal. Reaggregates were harvested at different time points and the positions o f the quail cells visualised with H arris's haematoxylin (section 2.3.2.3).

Chick Quail

F ig u re 3.1

Schematic diagram showing experimental protocol for quail-chick chim aeras. E m pty limb outline represents chick limb, grey limb re p re sen ts quail lim b. The delin e ate d sq u a re s rep re s e n t the polarising regions. The polarising regions are excised from both chick and quail, and that from the quail is mixed with the rest o f the lim b fro m c h ic k . T h e resulting c h im a e r a is tritu ra te d and reaggregated as normal.

3.2.1.2 DU

The lipophilic dye, D il (M olecular Probes, UK), was used as an o th er way of visualising polarising region cells in the reaggregate. This dye is incorporated into cell membranes, does not spread to neighbouring cells, but is passed on to progeny.

The normal reaggregate procedure was followed, except, once again, the polarising region was excised. In this experiment, these cells were triturated out in a pre-spun (to remove crystals) solution o f 0.002 mg ml-^ D il in TSM (from 3 mg ml-^ stock in dimethylformamide) and left to stand for 30 minutes. The cells w ere centrifuged and w ashed in TSM tw ice to rem ove all rem aining crystals of Dil. The cells were reaggregated with the rest of the limb cells and the whole mix triturated, spun and the pellet incubated for 1 hour to consolidate.

R eaggregates were made and grafted as normal. They w ere harvested at 24 and 48 hours and exam ined under e p iflu o re sc e n c e m icro sco p y to v isu a lise la b elle d cells. Photographs were taken on 400X Tungsten colour slide film. 3.2.2 Wholemount staining for cell death

To visualise cell death, embryos were dissected out into a dish of Tyrode's salt solution (137 mM NaCl; 2.68 mM KCl; 1.36 mM C a C h ; 0.49 mM M gCli 6H2O; 0.36 mM NaH2P04 H2O; 1% glucose; 12 mM NaHCOs) and placed in 0.005% Nile Blue A in Tyrode's for 20 minutes at 37 “C. Next they were washed 3 times in fresh Tyrode's at room tem perature, before storing at 4°C. They were photographed on Kodak 64T colour slide film.

3.2.3 Wholemount staining for nerve supplv

Embryos with their reaggregates were harvested at varying times after grafting and cleaned o f their extraem bryonic membranes and viscera. The silver staining itself was kindly performed by Steven Harsum, a postgraduate student with Jonathon Clarke and Paul M artin (Anatomy and Developmental Biology, UCL). The procedure for this was as follows.

Embryos were fixed in B odian’s fixative (75% absolute alcohol, 15% distilled water, 5% formalin and 5% glacial acetic acid) for 12 hours to 3 days and placed in 70% alcohol for 2 days or m ore (at least four changes in this time). All further steps involved agitating the embryos on a shaking platform. They were washed for 1 hour in distilled water and incubated at 37°C for 36 hours in 1% protargol (a protein-silver conjugate) with 0.4 g of copper wire per 20 mis of water. This step binds both silver and copper onto the nerves to stain them. The next series o f washes were at 4°C. The stain was reduced and fixed on the nerves with 1% hydroquinone plus 7.5% anhydrous sodium sulphate in water (made the previous night). There follow ed four 30 m inute washes in water before transferring them to 1% gold chloride (acidified with 3 drops acetic acid per 100 mis water) for 1 hour. The gold chloride is drained and re-used. Again, they were washed four times for 15 minutes each.

T he rest o f the procedure was perform ed at room temperature, again with agitation. The embryos were bleached in freshly prepared 5% potassium ferricyanide in w ater for 1 hour and washed a further four times, 15 minutes each, in water. They were soaked for 30 minutes in freshly prepared 5% sodium thiosulphate with 2% potassium hydroxide in water, followed by a further four 15 minutes washes in water. The specimens were dehydrated through sequential alcohols, cleared in m ethyl salicylate for viewing and photographed on Kodak 64T tungsten colour slide film.

3.2.4 Scanning electron microscopv

Embryos for scanning electron microscopy were harvested and fixed in M odified Tyrode's fixative (941 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl, 0.49 mM M gC l2, 1% sucrose, 12 mM NaHCO], 3.62 mM NaH2?04 H2O, 1% gluteraldehyde, in water). They were washed through several changes of distilled w ater lasting at least 2 hours (up to overnight) and several washes of absolute alcohol lasting at least 2 hours (up to overnight) before placing them in amylacetate for approximately 20 minutes.

The specimens were dried on a Baltzer critical point dryer, mounted on metal studs and sputter coated with gold in a Polaron E5000. Specimens were examined on an Hitachi S-530 scanning electron microscope and photographed on Ilford FP4 Plus film. 3.3 R e s u lts

3.3.1 Early appearance and histology 3.3.1.1 General observations

Reaggregates were examined 24 and 48 hours after grafting. As early as 24 hours it was usually easy to identify whether grafts would develop or not. Grafts that would not go on to develop generally failed for one of two reasons: firstly, the graft would appear white and stunted, a result of its failure to vascularise; secondly, the grafts would be swollen by massive haematomas that could often encom pass the whole reaggregate. As stated in Chapter 2, the latter was more common in grafts to a cold host. The reasons for the form er are unclear, but may be due to interference in the host-graft interface, perhaps by excess ectoderm from the jacket interposing between the mesenchyme.

Grafts that did take grew little in the first 24 hours. The beginnings o f vascularisation were visible, although no m ajor vessels were yet apparent. By 48 hours, growth was rapid. A characteristic axe-head shape was often observed and a vascular marginal sinus was visible. These were more pronounced by 72 hours (Figure 3.2).

T hroughout these first 48 hours, an AER was alm ost alw ays apparent (Figure 3.3). It was som etim es uneven, appearing ruffled or corrugated, although this did not appear to have an effect on how well these grafts grew or patterned.

3.3.1.2 Early histology

The observations in this section are a combination of results from reaggregates made specifically for histology and those specimens used for in-situ hybridisation to tissue sections (Chapter Five).

Figure 3.2

72 hour reaggregate graft in ovo. Open arrow indicates AER; arrow indicates vascular marginal sinus. Proximal reaggregate is

Each observation was based on specimens from at least three separate experiments, and was seen in virtually all of those that were judged to be developing well (i.e. showed polarised growth and an AER, and not massive areas of mesenchymal necrosis or haem atom as). In general, about 80% o f reaggregate grafts survived, with 70% of these showing good development.

At 24 hours after grafting, reaggregates tended to have a rounded shape (Figure 3.4A). The m esenchym e appeared generally hom ogeneous at this point, with some blood vessels visible, but no vascular sinus. The AERs were generally in good condition, appearing still pseudo-stratified where visible (Figure 3.4B).

By 48 hours after grafting, outgrowth was now apparent in the proximodistal polarisation of the graft (Figure 3.4C). AERs were almost invariably healthy looking, pseudo-stratified and a marginal sinus had generally been established (Figure 3.4C, D). In m ost grafts by this stage, proximal mesenchym e was now distinct from distal as pre-cartilage condensations appear (Figure 3.4C). Note the ventral bias of the AER (Figure 3.4C, D), characteristic of normal limbs.

By 72 hours, well defined cartilage was present, often show ing a branched p attern (Figure 3.4E). AERs had disappeared and a good vascular network had been established. At all these stages, the vasculature developing does so at a distance o f roughly 75-100 pm from the ectoderm, leaving an avascular m argin also characteristic o f norm al lim b developm ent (for example. Figure 3.4C, D; Feinberg et al., 1983).

3.3.2 A description of tissues present

3.3.2.1 The development of the skeleton

Skeletal development in normal and reaggregated hind limb buds was exam ined using Alcian green staining. A lthough p re­ cartilage condensations are visible in section earlier than Alcian green staining can be detected, the latter technique allows an easy com parison of the tim ing o f appearance of differentiating cartilage in normal and reaggregated hindlimb mesenchyme.

Figure 3.3

Scanning electron micrographs of reaggregates, 24 hours after grafting. Arrows indicate AERs; scale bars indicate 100 pm. In A, proxim al reaggregate is down and the anteroposterior axis

Figure 3.4

Histological preparations of reaggregate grafts.

A - 24 hour reaggregate, h, host; r, reaggregate; arrow indicates AER. Proximal reaggregate is left. Scale bar = 1 0 0 |lm

B - 24 hour reaggregate. Short arrow indicates pseudo­ stratified AER; long arrow indicates basement membrane. Scale bar = 20 juim

C - 48 hour reaggregate. Short arrow indicates AER; long arrow indicates vascular marginal sinus. Proximal reaggregate if right, dorsal is up. Scale bar = 200 |im

D - 48 hour reaggregate. Arrows indicate AER, note the ventral bias. Dorsal is up, anteroposterior is left/right. Scale bar = 100 jiim

E - 72 hour reaggregate. Arrowhead indicates cartilage bifurcation point. The thick black lines running through the

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