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Multiple effects of artemin on sympathetic neurone generation, survival and growth

¹ Department of Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, Summerhall Square, Edinburgh EH9 1QH, UK
² Department of Molecular Oncology, Genentech, 1 DNA Way, South San Francisco, California CA 94010, USA

View larger version (12K): [in a new window]   Fig. 1. Graph of the number of sympathetic neurones present in E14 dissociated SCG cultures after 48 hours incubation with a range of concentrations of artemin expressed as a percentage of the number of neurones counted 6 hours after plating. The means and standard errors (minus survival in control cultures) of data obtained from three petri dishes for each condition are shown. Similar results were obtained in two separate sets of cultures.

View larger version (32K): [in a new window]   Fig. 2. Stacking bar charts of representative cumulative cohort experiments set up from the SCG and SG of E12 to E16 embryos (SCG at E12, E14 and E16, and SG at E13 and E15). The cells were grown in either defined medium alone (control cultures) or medium supplemented with 10 ng/ml artemin. An initial cohort comprising all the neurones in a 12x12 mm grid in the centre of each dish was identified 6 hours after plating. The number of surviving neurones in this cohort was monitored at 6 hourly intervals and is expressed as a percentage of the initial cohort at 6 hours (black bars). New neurones that were generated in the grid between 6 and 12 hours, 12 and 18 hours, 18 and 24 hours, 24 and 30 hours, 30 and 36 hours are expressed as a percentage of the initial cohort, and their survival was likewise subsequently monitored at 6 hourly intervals after their appearance (patterned and white bars).

View larger version (20K): [in a new window]   Fig. 3. (A) Graph of the number of new neurones generated between 6 and 36 hours after plating in dissociated cultures of sympathetic ganglia of E12 to E16 embryos in defined medium alone (control) or medium supplemented with 10 ng/ml artemin. Bar charts of the number of BrdU-labelled neurones in dissociated E12 SCG (B) and E13 (C) stellate ganglion cultures grown either in defined medium alone (control) or medium supplemented with 10 ng/ml artemin. In E12 cultures, BrdU was added 2 hours after plating and the cultures were fixed and stained 16 hours after plating. In E13 cultures, BrdU was added 24 hours after plating and the cultures were fixed and stained 12 hours later. The means and standard errors are shown (n=3-6 for each condition).

View larger version (21K): [in a new window]   Fig. 4. Generation of GFR3 knockout mice by homologous recombination. (A) Gene targeting vector. A Neo cassette replaced a fragment containing exon one, deleting the start codon and the signal sequence of GFR3. H, HindIII; E, EcoRI; X, XhoI. (B) Southern blot screening of targeted ES clones. Genomic DNA was digested with HindIII. The probe, upstream of the short arm (shown in A), detected the 11 kb wild type and 7 kb mutant fragments. (C) PCR genotyping of tail DNA prepared from offspring of intercrossing heterozygous mice. Tail DNA was amplified with a primer set specific for the Neo gene, which detects the mutant allele, and a primer set that hybridises within the sequence that is deleted in the mutant and therefore detects the wild-type allele.

View larger version (31K): [in a new window]   Fig. 5. Bar charts of (A) the total number of ßIII-tubulin-positive and (B) the percent of ßIII-tubulin-positive cells that are PCNA positive in E14 wild-type and GRF3-/- embryos. The means and standard errors are shown (data derived from six wild-type and five GRF3-/- embryos).

View larger version (22K): [in a new window]   Fig. 6. Graphs of the percent survival of P0 to P60 SCG neurones grown in either defined medium alone (control) or in medium supplemented with 10 ng/ml artemin, 10 ng/ml NGF or artemin plus NGF. The number of neurones surviving 24, 48, 72 and 92 hours after plating is expressed as a percentage of the number of neurones counted 3 to 6 hours after plating. The means and standard errors are shown (n=6-9 for each condition).

View larger version (18K): [in a new window]   Fig. 7. Graph summarising the developmental changes in the percent survival of SCG neurones grown in either defined medium alone (control) or in medium supplemented with 10 ng/ml artemin, 10 ng/ml NGF or artemin plus NGF in cultures established from P0 to P60 mice. The means and standard errors are shown (n=6-9 for each condition).

View larger version (47K): [in a new window]   Fig. 8 Bar charts of the numbers of surviving neurones in cultures of E14 and P60 SCG grown for 48 and 72 hours, respectively, in either defined medium alone (control) or in medium supplemented with 100 ng/ml anti-GFR3 antibody, 10 ng/ml NGF, 10 ng/ml artemin, anti-GFR3 plus NGF or anti-GFR3 plus artemin. The means and standard errors are shown (n=6-9 for each condition).

View larger version (33K): [in a new window]   Fig. 9. Bar charts of the total length of the neurite arbours growing from SCG neurones in cultures established from E14 to P60 animals after 48 hours incubation with NGF alone or NGF plus artemin (both at 10 ng/ml). The means and standard errors are shown (n=4-9 for each condition).