Cellular Analysis of Hd mutants

2.20.1 Analysis of mesenchymal cell death

CeU death was assessed by Nile blue sulphate staining. Embryos were dissected out from their membranes in Tyrodes solution prewarmed to 37°C then sacrificed by severing the spinal chord with sharpened tungsten needles. After two quick rinses in prewarmed Tyrodes, embryos were stained in a solution of 0.005% Nile blue sulphate in Tyrodes at 37°C for 10-15 minutes. After staining, the embryos were rinsed quickly in Tyrodes at 37°C then overnight at 4°C. The following day limbs were then photographed using

Kodak T64 slide film or drawn with the aid of a dissecting microscope and attached camera or camera lucida.

2.20.2 Skeletal analysis of the limb of H d mutant embryos

The developing skeletons of mice were analysed by staining with alcian green and

clearing in either methyl salicylate or glycerol. Embryos to be cleared in methyl salicylate were removed from their extra-embryonic membranes, washed in IX phosphate-

buffered saline (PBS) and fixed overnight in a solution of 5 % trichloroacetic acid (TCA). The next day the fixative was removed and replaced with a staining solution of 0.1 % Alcian Green in acid alcohol (70% ethanol, 0.1% hydrochloric acid). The embryos were stained for 24 hours at room temperature, after which they were placed in acid alcohol overnight to remove stain differentially from non-cartilaginous structures. Prior to clearing, the embryos were dehydrated in 100 % alcohol then cleared in methyl salicylate allowing visualisation of cartilaginous elements of the developing skeletal system. Embryos were analysed and photographed with the aid of a dissection microscope and attached camera. Methyl sahcylate is a volatile and harmful substance and so a second method devised by A. Sheasby (personal communication) was carried out that uses glycerol as the clearing agent

Embryos were fixed in 5% TCA or 80% ethanol, dehydrated for an hour in 100% ethanol before being washed in acetone for 24 hours to remove any fat from the skeletons. The next day embryos were rinsed in 100% ethanol for an hour, stained in 0.1 % Alcian Green in 70% ethanol, 0.1% hydrochloric acid for three hours, then washed in running tap water for an hour. Embryos were treated with 1% aqueous potassium hydroxide (KOH) for an hour and cleared through a series of 20%, 50%, and 80% glycerol in KOH before being stored in 100% glycerol. Again, embryos were analysed and photographed with the aid of a dissection microscope and attached camera.

To allow visualisation of the skeleton of older mice, they were fixed and

processed for differential staining with alcian blue and alizarin red S which stain cartilage and bone respectively (as described above).

2.20.3 Cell culture of limb mesenchymal cells in high density micromass cultures

Limb buds were removed from embryos and washed in sterile growth medium (Appendix 1). Isolated buds from each embryo were transferred into separate dishes containing ice-cold 2% trypsin in calcium and magnesium free HBSS, and incubated on ice for 30 minutes. After trypsinisation, limbs were placed into ice-cold growth medium and stored on ice for 10 minutes before teasing away the ectoderm from the underlying limb mesenchyme with sharpened tungsten needles. After removal of the ectoderm, limb bud mesenchyme was placed into sterile microfuge tubes containing 1.0 ml of growth medium. Mesenchymal cells were then dissociated into a single cell suspension by gentle pipetting of the contents of the tube up and down a sterile glass pipette. The number of cells in each of the tubes was counted using a haemocytometer and the cell suspension subsequently adjusted to 2x10^ cells/ml by centrifugation and addition the appropriate volume of culture media (CMRL containing 10% foetal calf serum, 100 units ml"^ penicillin, lOOpg ml‘ l streptomycin, 0.25pg ml'^ fungizone and 2mM L-glutamine). Individual micromass cultures were set up in separate wells of NUNC 4-well culture dishes; lOpl of the cell suspension, containing 2x10$ cells, was pipetted as a single drop into the centre of each well. The mesenchymal cells contained in these lOpl drops were allowed to settle and adhere to the tissue culture dish by incubating for 1-1.5 hours at 37°C. The weUs of the culture dishes were then flooded with 300 pi of culture medium and incubated for six days at 37"C in 5% CO2 Cultures were fed daily with fresh or

conditioned medium.

2.20.4 Preparation of conditioned medium and treatment of cultures with growth factors

Conditioned medium was prepared from cultures of 12.5 dpc putative +/+, Hd/+, and Hd/Hd mesenchymal cells. Cultures were set up as normal. However after 24 hours of incubation, the medium was removed and mixed with fresh medium a to concentration of 50-75%, this was then used to feed other cultures. Micromass cultures of 12.5 dpc limb bud mesenchyme were set up as described, and fed daily with medium containing

10 ng/ml of TG Fpl (obtained from P. Martin), FGF2 or FGF4 (obtained from R and D Systems).

2.20.5 Fixing and staining of micromass cultures

Culture medium was removed, each well rinsed with 500|il of PBS, and then 500|il of fresh, filtered 4% paraformaldehyde in PBS (PFA) was added. Cultures were fixed overnight at 4“C, after which the PFA was removed and cultures were washed in filtered PBS. In some instances, cultures were photographed at high magnification using an inverted phase contrast microscope prior to staining for cartilage.

Cultures were stained by replacing the PBS with a staining solution of 1% alcian blue in O.IN hydrochloric acid for 4 hours at room temperature. The staining solution was removed and stained cultures were washed three times in fresh, filtered PBS, before being dehydrated through a series of 50%, 70%, 90%, and 100% alcohol (5 minutes each). Cultures were photographed under a dissecting microscope and then stored under

100% glycerol at 4 T .

2.20.6 Estimation of the amount of cartilage produced in culture

To estimate the amount of cartilage produced by each of the cultures, the number of cartilage nodules in each culture as shown by alcian blue staining was counted. The number of nodules in cultures originating from the same embryo were added together and then divided by the number of cultures to give an average value for that embryo.

2.20.7 Scanning Electron Microscopy

9.5-14.5 dpc Hd/Hd, Hd/+ , and +/+ embryos were washed in IX PBS after dissection from the uterus and removal of extra-embryonic membranes. Embryos were then rinsed in filtered Tyrodes solution and fixed and stored at 4°C in modified Tyrodes (Tyrodes containing 1% glutaraldehyde). After post-fixation in 1% osmium tetroxide in O.IM phosphate buffer for 1 hour at 4°C, the specimens were dehydrated in graded alcohols and rinsed thoroughly in amyl acetate. Specimens were then subjected to critical point

coating, the distal tips of several limb buds were removed in order to see the

mesenchyme, by tapping the distal end of the limb bud with a tungsten needle. These specimens were then sputter coated with gold particles as normal. All specimens were analysed using a Hitachi S-530 scanning electron microscope and photographed onto medium format, Ilford FP4 black and white film.

2.20.8 Araldite Histology

Embryos or isolated limbs were fixed in half strength Kamovsky's (Appendix I) overnight at 4°C. Prior to embedding in araldite resin, specimens were washed for 20 minutes each in 50% and 70% alcohol, then stained for 45 minutes in 0.5% alcian green in acid alcohol, washed for 30 minutes in 90% alcohol, then washed four times in 100% alcohol each for 15 minutes. Once fully dehydrated, specimens were cleared in propylene oxide (3 X 10 minute washes) then soaked for 45 minutes in 50:50 propylene oxide: araldite resin. Specimens were then rotated overnight in araldite resin at room temperature before being embedded in fresh resin and placed at 60°C overnight to harden the resin. Limbs were sectioned at 1pm using an ultracut microtome, approximately 5 out of every 10 sections were taken until the limbs were fully sectioned. Photographs were taken using a Zeiss Axiovert 405M microscope with an inbuilt camera and Ilford FP4 film.

Chapter 3

An introduction to the mouse mutant hypodactyly {Hd)i a