Explant cultures
Neural plate explants from Hamburger-Hamilton (HH) stage 10 chick embryos (Hamburger and Hamilton, 1951) were isolated essentially as described (Yamada et al., 1993). After dissection, explants were cultured in collagen matrix (Vitrogen) or fibronectin (Boehringer Mannheim)-treated 10 mm tissue culture wells, with F12 medium containing penicillin/streptomycin, glutamine (Speciality Media), 2 mM glucose and Mito+ Serum Extender (Collaborative Biomedical Products). Neural tube tissue from stage 10 chick embryos was dissected, treated with dispase and then cultured on fibronectin (Boehringer Mannheim)-treated chamber slides or 10 mm tissue culture wells.
To quantify the emigration of neural crest cells from explants cultured in collagen gels, 16 pairs of explants (with and without C3) were analyzed under a dissecting microscope. To determine the number of slug+ and DAPI+ cells, each explant was sectioned serially at 10 mm, stained and the number of cells in each section was then counted. A total of 3 pairs of explants (with and without C3) were analyzed. The concentration of C3 used in these neural plate explant assays was ~50 mg/ml. For the analysis of neural crest delamination, C3 (150-200 mg/ml) was added to the medium at the beginning of the culture period. For the analysis of neural crest migration, C3 (~100 mg/ml) was added to the culture media 6 hours after plating. To measure the rate of cell migration, the distances between the center of each explant and cells that had migrated farthest away from the explant were recorded at each time point (~40 cells/explant). A total of 3 pairs of explants (with and without C3) were measured.
Differential display screen
A differential display screen was performed essentially as described (Liang et al., 1993; Liang and Pardee, 1992) using a-[33P]dATP (Amersham) and a combination of nineteen 5¢ primers and three 3¢ primers. The sequences of 5¢ primers used are:
1. 5¢-CTGATCCATG-3¢,
2. 5¢-CTTGATTGCC-3¢,
3. 5¢-CTGCTCTCAA-3¢,
4. 5¢-CTAGACTAGC-3¢,
5. 5¢-GATCGATGGA-3¢,
6. 5¢-GTCATGCACA-3¢,
7. 5¢ AGCTTCGATC-3¢,
8. 5¢ ACTGGATTCG-3¢,
9. 5¢-TGGATCCAAG-3¢,
10. 5¢-TCACGTAAGC-3¢,
11. 5¢-TACAACGAGG-3¢,
12. 5¢-GACCGCTTGT-3¢,
13. 5¢-GGAGGGTGTT-3¢,
14. 5¢-GAACGGACTC-3¢,
15. 5¢-AGCGCCATTG-3¢,
16. 5¢-CTTCACCCGA-3¢,
17. 5¢-GGGATATCGG-3¢,
18. 5¢-TTCCCGGGTT-3¢,
19. 5¢-AATGGCGCAG-3¢.
The sequence of 3¢ primers used are: T12MA, T12MG, T12MC, (M=A or G or C).
This screen generated 121 PCR fragments of 200-700 bp in length that were detected at higher levels in cultured [d] or [i]+ Dsl1 explants when compared to [i] explants cultured alone. These 121 fragments were used as probes to screen Southern blots of PCR-amplified [i], [d] and [i]+ Dsl1 cDNAs using the original primer combinations by which these fragments were derived. After this second round of screening, the number of positive bands was reduced to 30. These DNA fragments were then used as probes to isolate cDNA clones from a stage 17 chick neural tube cDNA library (Basler et al., 1993). cDNAs corresponding to 25 of the original 30 PCR-derived fragments were isolated and used to generate probes for determining the expression patterns of the corresponding genes by in situ hybridization.
In situ hybridization
HH stage 10 and 20 chick embryos were processed for whole-mount in situ hybridization performed as described (Thery et al., 1995). HH stage 24 chick embryos were fixed, embedded and sectioned at 10 mm first before processing for in situ hybridization. Generation of anti-rhoB antibody To generate an antibody against chick rhoB, the 3¢ coding region of rhoB cDNA (amino acids 114-196) was subcloned into pQE42 expression vector (Qiagen). The resulting fusion protein consists of dehydrofolate reductase (DHFR) and the C-terminal of rhoB. Purified protein was injected (100 mg/mouse) every 3 weeks into Balb/C mice. After six injections, spleen cells were fused with NS1 myeloma cells for generation of monoclonal antibodies. One clone directed against rhoB, mAb 4H7, was obtained.
Immunohistochemistry
Chick embryos were dissected and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C for 2 hours, washed with PBS and equilibrated with 30% sucrose in 0.1 M PB, then mounted in OCT embedding medium. Neural plate explants in collagen gels were fixed for 20 minutes at 4°C, washed and embedded as described above. All tissues were sectioned on a cryostat at 10 mm. Neural tube explants grown on chamber slides were fixed for 20 minutes and washed with PBS. For staining with mAb 4H7 (anti-rhoB), sections were treated with 6 M guanidine HCl in 0.1 M PB for 10 minutes at 20°C, washed with PBS, blocked with 10% goat serum and 0.1% Triton X-100 in PBS.
mAb 4H7 was used at 1:40 dilution, mAb HNK-1 was used at 1:10 dilution, mAb 1E6 (anti-slug) was used at 1:100 dilution in 1% heat-inactivated goat serum and 0.1% Triton X-100 in PBS. Rhodamine-conjugated phalloidin (Molecular Probes) was used at 1:100 dilution. FITC- and Cy3-conjugated goat anti-mouse secondary antibodies were used as directed (Jackson Immuno Research Laboratory).
Western blot analysis
Plasmids containing human rhoA (L63), rac1 (V12) and cdc42 cDNAs in pGEX-2T vectors were provided by Dr Alan Hall. These pGEX fusion plasmids were transformed into BL21 bacterial cells while the pQE fusion plasmid (rhoB) was transformed into M15 bacterial cells. Bacterial lysate containing the chick rhoB (L114-L196)/DHFR, human rhoA/GST, rac1/GST and cdc42/GST fusion proteins were separated on 12% SDS-PAGE gels. Proteins were transferred to nitrocellulose filters using Tris/glycine/SDS transfer buffer. After blocking, mAb 4H7 was used at 1:6000 dilution and incubated with membranes at 4°C overnight. Alkaline phosphatase-conjugated antimouse IgG (Boehringer Mannheim) was used at a 1:400 dilution. The chromogenic reaction was performed with xphosphate/ 5-bromo-4-cholro-3-indolyl-phosphate (BCIP) and 4-nitro blue tetrazolium chloride (NBT) (Boehringer Mannheim).
RT-PCR assay
Four explants per collagen gel were collected in 1 ml Trizol reagent (Gibco BRL) and RNA was purified according to the manufacturer’s protocol. RNA from each sample was divided equally for RT reactions and for controls lacking RT. 1-4 ml of the 30 ml total volume of the RT reaction was used for each PCR reaction. Amplification was performed at 17-18 cycles. PCR products were transferred to Hybond- N+ membranes (Amersham) and hybridized with corresponding probes. Quantitation of each product was performed with a phosphoimager ‘Storm 860’ (Molecular Dynamics). The sequence for each primer pair is:
rhoB 5¢: 5¢-ATGGCCGCCATCCGCAAGAAGCTG-3¢.
rhoB 3¢: 5¢-CTATAGGACCTTGCAGCAATTGATGC-3¢.
rhoA 5¢: 5¢-GCAGCTATGGCAGCCATTCG-3¢.
rhoA 3¢: 5¢-GACTTTTTCTTGCCACGCCG-3¢.
cadherin6B 5¢: 5¢-GGCTCTTATTGCCATTCTTCTC-3¢.
cadherin6B 3¢: 5¢-TTAAGAGTCTTTGTCACTGTCCAT- 3¢.
cadherin7 5¢: 5¢-GAGCCCTGATAGCAATATTGGCT-3¢.
cadherin7 3¢: 5¢-CTATGAGTATAAGCAGTCTGGACC-3¢.
slug 5¢: 5¢-ACCCCATTACTGTGTGGACTACAA-3¢.
slug 3¢: 5¢-TTGGATCTGTCTGCGAAAGCCCTG-3¢.
s17 5¢: 5¢-AGAAGGCGGCGCGGGTGATCATCG-3¢.
s17 3¢: 5¢-GTTTATTGTAAAAGCAACATAACG-3¢.
Purification of C3 exotoxin
A plasmid encoding C3 toxin was provided by Dr Larry Feig. C3 protein was purified as described (Dillon and Feig, 1995). Centriprep 10 (Amicon) columns were used to change the buffer to F12 medium and to concentrate the protein. The amount of protein was quantified with a Bio- Rad protein assay using BSA as the standard. Assay of rho inhibition by C3-mediated ADPribosylation To analyze the C3-mediated inhibition of rho activity [d] explants and [i] explants cultured with BMP (Dsl1) were treated with ~50 mg/ml C3. Samples were collected in 0.2% Triton X-100, 50 mM Tris pH 7.4, 5 mM MgCl2, 50 mM NaCl, and 1 mM PMSF at 4.5 hours and 9 hours after plating. For pNT explants, C3 was added at 150-200 mg/ml, samples were collected at 3 hours and 6 hours after plating. For aNT explants, ~100 mg/ml C3 was added 6 hours after planting. Neural tube explants were removed with a tungsten needle at 3 hours and 6 hours after the addition of C3, and migrating neural crest cells were collected. ADP-ribosylation assays were performed as described (Ridley and Hall, 1992). Quantitation was performed with a phosphoimager ‘Storm 860’ (Molecular Dynamics).
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