Obtaining embryos
Xenopus embryos were obtained by in vitro fertilization (Smith and Slack, 1983). Briefly, adult Xenopus females were induced to ovulate by an injection of 150 units of Folligon (pregnant mares' serum), up to a week before collection, followed by an injection of 500 units of human chorionic gonadotrophin 12-16 hours before eggs were required. Eggs were collected from 1 x MMR solution into which the induced females were placed. The eggs were fertilised by rubbing macerated testis, dissected from a sacrificed male, over them. Males were sacrificed by injection of 3-aminobenzoic acid ethyl ester (MS-222) and subsequent destruction of the circulatory system. Testes were dissected and kept in 60% L-15 media (Sigma) at 4°C for up to a week. After
fertilisation, the embryos were flooded with 10% normal amphibian medium (NAM; Slack, 1984). After 30 min the embryos were dejellied using 2% cysteine hydrochloride (pH 7.9-8.1) and staged according to Nieuwkoop and Faber (1967).
Micro-injection and culture of Xenopus embryos
After dejellying, embryos were transferred to 1% agarose coated dishes containing 4% Ficoll in 75% NAM. Embryos were injected using a Narishige IM 300 compressed nitrogen gas microinjector using needles made from glass capillaries and with the help of a micromanipulator (Micro Instruments). Embryos were injected at the one-cell to two-cell stage with RNA/DNA dissolved in water, as described by Smith (1993). Typical injection volumes were 5-10 nl, which was estimated by injecting a drop of water into oil and measuring the diameter using a graticule. By treating the drop as a sphere it is possible to calculate the volume of the drop (A sphere of diameter 267 pm has a volume of 10 nl).
Animal cap dissection and recombinations
For animal cap assays, animal pole explants (known as animal caps) were dissected from embryos at stage 8.5. The vitelline membrane was removed using number 5 watchmaker forceps and a square piece of tissue was dissected from the animal-most quarter of the embryo using the forceps as scissors. Explants were cultured in 75% NAM in 1% agarose coated dishes.
Chapter 2. M aterials & M ethods
When dexamethasone (DEX) or cycloheximide (CHX) was included in the culture medium, explants were cultured in 75% NAM containing 0.1% bovine serum albumin (BSA). DEX (Sigma) was dissolved in ethanol at 2 mM and then diluted to a final concentration of 1 pM in 75% NAM + 0.1% BSA. CHX was applied at a concentration of 10 ItM.
Recombination experiments were carried out as described by Jones et alj^ Animal caps were cut as described above and two individual caps were then juxtaposed - a "source" cap from an embryo injected with a test mRNA and an Fluorescein-injected "responding" cap - and allowed to heal for at least 5-10 min. The recombinants were cultured in 75% NAM until sibling embryos reached stage 10.5, at which point they were fixed in MEMFA and processed by in situ hybridisation for expression of Xbra RNA (In situ hybridisation).
Incorporation of [^®S]-Met
To assess the effectiveness of CHX treatment, the inhibition of protein synthesis was assayed by the incorporation of [^^S]-Met. Groups of 10 control animal caps were cultured in 75% NAM containing 0.1% BSA and 0,37 MBq/ml [^^S]-Met, either with or without CHX. These caps were collected and frozen at stage 10.5 in the same manner as sample caps. To assay the incorporation of radiolabel, the caps were disrupted in 200 p,l extraction buffer by vigorous vortexing. 250 pi BSA (1 mg/ml) and 1 ml 10% ice-cold TCA were added, the mixture vortexed again and incubated on ice for 20 min.
The samples were then passed through a Whatman GF/C glass-flbre filter. The filter was washed five times with 2 ml 10% TCA and then three times with 3 ml ethanol. The filter was allowed to dry at room temperature for 20 min and was then placed in 1 ml of scintillation fluid and counted in a scintillation counter. The values for the two samples were then used to calculate a percentage inhibition of translation. Only experiments where inhibition was over 90% were used in RNase protection analyses.
Extraction buffer
20 mM Tris'HCl (pH 7.4) 100 mM NaCl
1 mM EDTA 0.5% SDS
Chapter 2. M aterials & M ethods
In situ hybridisation
Whole-mount in situ hybridisation was performed as described by Harland (1991), except that BM purple was used as substrate and RNase treatment was omitted. Probe labeling
Probes were labelled with digoxygenin (DIG)-ll-UTP (Roche) in a standard in vitro transcription reaction using the appropriate RNA polymerase (see Vectors and
constructs). Reactions contained 2.5 pg/ml linearised DNA template, 400 mM DTT, 1 x DIG-labelling mix (Roche), 40 U RNasin, 50 U specific RNA polymerase and buffer. The reaction was incubated at 37°C for 2 hours, then treated with 20 U RNase-free DNase I at 37°C for 15 min to digest the template. The resulting labelled RNA was then purified over CHROMASPIN-100 columns to remove unincorporated nucleotides. Probes were used at 500 ng/ml in hybridisation buffer.
Hybridisation
Embryos were processed in 5 ml glass vials. They were fixed at the appropriate stage in MBMFA for 1 hour at room temperature. Before fixation a hole was poked in the blastocoel to prevent non-specific trapping of probe or antibody. After fixation the embryos were washed twice in 100% ethanol or methanol and then stored at -20°C.
Embryos were first rehydrated by incubation for 2-5 min in solutions of decreasing percentage alcohol until they were completely rehydrated (75% EtOH, 50% EtOH, 25% EtOH). They were then washed three times in PBST (1 x PBS + 0.1% Tween-20) for 5 min each and incubated for 10 min in 5 fig/ml proteinase K in PBST. Embryos were then rinsed twice in 0.1 M triethanolamine (pH 7-8) to which acetic anhydride is also added twice (2.5 p.l/ml triethanolamine) and incubated for 5 min each time. This
treatment blocks charged groups within the tissue and decreases background (Hayashi et al., 1978). The embryos were then washed twice in PBST for 5 min, refixed in MEMFA for 20 min at room temperature and washed five times in PBST for 5 min. The PBST was then replaced by 500 pi hybridisation buffer and embryos were then incubated at 60°C for 2 hours to prehybridise. After 2 hours the DIG-labelled probe was added to hybridisation buffer at a concentration of 500 ng/ml and incubated at 60°C overnight.
Chapter 2. M aterials & M ethods