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doi: 10.1242/10.1242/dev.00217

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The guanine nucleotide exchange factor C3G is necessary for the formation of focal adhesions and vascular maturation

¹ Development and Neurobiology, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia
² Max-Planck-Institute of Biophysical Chemistry, Goettingen, Germany

View larger version (19K): [in a new window]   Fig. 1. The C3G locus and the mutated allele C3Ggt. (A) The 124 kb C3G locus comprises 24 exons. The start of translation is encoded by exon 1. The gene trap insertion occurred in intron 1. Intronic probe 1 is used for Southern analysis of genomic DNA (probe 1). The major product of the native locus is a 1095 aa protein with a carboxy-terminal CDC25-like catalytic domain (black box) and 4 internal Crk-SH3 domain-binding sites (black stripes) and one p130cas-SH3 binding site (grey box). Probe 2 used for northern analysis and in situ hybridisation of paraffin sections of embryos is indicated (probe 2). The gene trap disrupts the coding region after the first 19 codons (black arrowhead). The mutated allele, C3Ggt, codes for a fusion between the first 19 aa of C3G, ß-galactosidase (ß-gal) and neomycin phosphotransferase (neoR). (B) Southern analysis of genomic DNA isolated from embryos of heterozygous intercrosses hybridised with probe 1 as indicated in A. Bands of 12 kb and 16 kb for the wild-type and the C3Ggt mutant allele, respectively. Homozygous mutant, mt; heterozygous, ht; wild type, wt.

View larger version (83K): [in a new window]   Fig. 2. The mRNA and protein products of the wild-type and the mutant C3G alleles. (A) Northern analysis of total RNA isolated from E10.5 embryos of C3Ggt/+ heterozygous intercrosses. 10 µg of total RNA were loaded per lane. The genotype of the embryos is indicated above, as wild type (wt), heterozygous mutant (ht) and homozygous mutant (mt) for the gene trap mutation in the C3G locus. (Upper panel) Hybridisation with the C3G probe 2 as indicated in Fig. 1A. (Middle panel) hybridisation with a lacZ-specific probe. (Lower panel) ethidium bromide staining of the 18S rRNA. Note the absence of detectable C3G mRNA in homozygous mutant embryos by this method. The lacZ probe detected a fusion mRNA of the expected size of 4.7 kb (169 bases 5' UTR and coding sequence 5' of the gene trap insertion of the C3G locus and 4.42 kb mRNA product of pGT1.8geo plus polyadenylation tail). (B) RT-PCR of total RNA isolated from macroscopically normal E10.5 embryos of heterozygous intercrosses. Oligo-dTTP was used to generate cDNA. Lanes 1, 2, 3, 4 and 5: cDNA of wild-type embryos used diluted 1:10 (1), 1:50 (2), 1:100 (3), 1:500 (4), 1:1000 (5). Lanes 6, 7, 8, 9 and 10: cDNA of homozygous C3Ggt/gt embryo was used diluted 1:10 (6), 1:50 (7), 1:100 (8), 1:500 (9) 1:1000 (10). Lane 11: control without cDNA template. M=100 bp ladder, intense band=500 bp. Note that even the 1:1000 diluted wild-type RNA yielded a prominent RT-PCR product, whereas the amount of product yielded from 1:10 and 1:50 dilution of the homozygous RNA was small. (C) PCR of undiluted homozygous mutant (mt) and wild-type cDNA (wt) as in B, but using primers spanning exons 1-21 representing all coding exons of the C3G locus. Note the presence of a small amount of product from homozygous template indicating the generation of normal protein coding mRNA from the homozygous mutant allele. Lanes 1 and 3 are controls. M=1 kb ladder; intense band=5 kb. (D) Western analysis of cell lysates of primary embryonic fibroblasts isolated from E10.5 homozygous, heterozygous and wild-type littermates. 40 µg of lysate were loaded per lane. Genotypes are wild type (wt), homozygous mutant (mt) and heterozygous (ht). C3G protein bands were detected with an anti-C3G antibody in the expected position (compare to Posern et al., 2000). Note the two prominent C3G bands in wild-type and heterozygous cell lysates. The two weak bands visible in homozygous lysate are likely to be residual C3G protein. They comprise no more than 5% of the normal level of C3G protein as determined by densitometry.

View larger version (124K): [in a new window]   Fig. 3. Expression pattern of the C3G gene. (A-D) ß-galactosidase reporter activity in whole heterozygous (A) and wild-type E11.5 embryo (B), and in a 16 µm paraffin sagittal section of an E11.5 heterozygote embryo (C) and in a small cephalic blood vessel at E12.5 (D). (E-L) Radioactive in situ hybridisation using an antisense C3G riboprobe (E,F, probe 2, Fig. 1A) and a sense control probe (G,H) on paraffin sections of E12.5 wild-type embryos and the antisense probe on sections of an E11.5 wild type (I,J) or homozygous mutant embryo (K,L). Ubiquitous expression of the C3G locus is shown by the reporter activity and by in situ hybridisation. Note specifically presence of reporter activity and endogenous gene activity in all cells including blood vessel endothelial cells and blood vessel surrounding cells in both small (D,J) and large blood vessels (I). Also note the absence of silver grains in the sections of homozygous embryos (K,L). Arrows point to blood vessel surrounding cells, arrowheads to endothelial cells (D,I,J). Bar represents 70 µm in A,B,C,E,F,G,H and 10 µm in I,J,K,L.

View larger version (82K): [in a new window]   Fig. 4. C3Ggt/gt mutant phenotype. Homozygous mutant embryos at E11.5 (A-C), E13.5 (D,E), E14.5 (F, dead), and (G-I) Haematoxylin and Eosinstained paraffin sections at E11.5. Note the formation of haemorrhages in the majority of cases (E10.5-E11.5), which initiated near the hindbrain neuroepithelium (arrows in A,B,G). These haemorrhages enlarged (H) and finally the neuroepithelium ruptured (arrow in I). In other cases, embryos exhibited massive subcutaneous oedema and haemorrhagic oedema at E13.5 (arrows in D,E). Bar represents 77 µm in A-C, 150 µm in D,E, 190 µm in F, 106 µm in G, 66 µm in H and 80 µm in I.

View larger version (96K): [in a new window]   Fig. 5. Examination of blood vessel formation and maturation in the head area. (A,C,E,G,I,K) Sections of homozygous C3G mutant embryos at E11.0 and (B,D,F, H,J,L) sections of wild-type littermate controls. A-D,K,L are paraffin sections, E-J are frozen sections. SMA immunostaining for vascular pericytes and smooth muscle cells using horseradish peroxidase histochemistry (A-D) or fluorescent labelling (G,H) and nuclear counterstaining with Methyl Green (A-D) or bis-benzimide (G-J). PECAM 1 immunostaining for vascular endothelial cells (E,F,I,J). Nidogen immunostaining for one of the proteins produced by endothelial cells (K,L). G,I and H,J are neighbouring sections of the basilar artery. Note the reduction of SMA staining in mutant embryos (A,C,G) and the lack of cohesion between the few SMA-positive cells (A). Lack of SMA-staining cells is more complete in small blood vessels (A,C) than in larger blood vessels (G). Arrowheads point to blood vessels and capillaries in C and D. However, even larger blood vessels have fewer SMA-staining cells (G compared with H). In contrast, vascular endothelial cells are unaffected by the C3G mutation. All embryos were alive at the time of tissue preparation. Bar represents 28 µm in A,B, 56 µm in C,D,G,H,I,J and 110 µm in E,F,K,L.

View larger version (71K): [in a new window]   Fig. 6. PDGF response and paxillin and vinculin distribution in C3G mutant MEFs. FITC-phalloidin staining for filamentous actin of homozygous C3G mutant MEFs (A,B) or wild-type MEFs (C), plated onto gelatine-coated coverslips in 24-well plates in DME plus 10% FBS, serum-starved for 16 hours and treated for 10 minutes with 10 ng/ml PDGF-BB. Note the formation of rings of filamentous actin. Actin rings were significantly more numerous (B) and larger (A) in C3G mutant MEFs as compared to wild-type MEFs (C). (D) Graphic representation of the PDGF-BB response of C3G mutant (gt/gt) vs. wild-type MEFs. For a<b, a<c and b<c P<0.001. Paxillin (E,F) and vinculin (G,H) immunostaining of C3G mutant and wild-type MEFs. Mutant MEFs plated onto gelatine-coated cover slips in DMEM plus 10% FBS and serum-starved for 16 hours formed considerably fewer paxillin-positive focal adhesions, which were also reduced in size (E compared with F). This phenomenon can also be seen in cultures without serum in figure 8. Likewise, vinculin-positive cell adhesions are reduced in number and size (G compared with H). Bar represents 27 µm in A,B,C,E,F and 11 µm in G,H.

View larger version (69K): [in a new window]   Fig. 7. C3G mutant MEFs showed impaired cell adhesion and spreading and enhanced cell migration. (A,B) Cells were plated in serum-free medium onto substrates as indicated. (A) One hour after plating, non-adherent cells were removed by washing. Attached cells were stained and counted by photometric absorption. C3G mutant MEFs showed a 66% reduction in adhesion to laminin (*, P<0.03) and a 23% reduction in adhesion to gelatine (**, P<0.001). Adhesion to fibronectin appeared normal. (B) Forty-eight hours after plating, few C3G mutant cells remained attached to laminin and gelatine and no cell spreading had occurred. Lack of spreading on fibronectin was also evident. Bar represents 185 µm in all panels. (C) Monolayer wounding assay in serum-containing medium on tissue culture plastic. Lower panel immediately after wounding, upper panel 7 hours later. The mutant cells covered 20% more surface area than the controls in 7 hours Bar represents 400 µm in all panels (0.59 mm2±0.03 vs 0.49 mm2±0.02, *, P<0.02, along 4.4 mm wound edge). (D) Graphic representation of the cell migration assays.

View larger version (55K): [in a new window]   Fig. 8. Integrin ß1 and paxillin distribution in C3G mutant MEFs cultured without serum. Integrin ß1 (A,B,G,H) and paxillin (C,D,I,J) immunostaining of MEFs plated on laminin (A-F) or fibronectin (G-L). (E,F,K,L) Merged images with nuclear counterstain bis-benzimide. Note the punctate staining of integrin ß1 in wild-type MEFs on both substrates (B,H), which was lost in MEFs lacking normal C3G expression (A,G). In wild-type MEFs integrin ß1 focal points were also paxillin-positive. Arrowheads in B,D,F and H,J,L point to prominent examples. In contrast, C3G mutant MEFs on laminin and fibronectin exhibited an aggregation of integrin ß1 near the nucleus (A,G). Paxillin was poorly distributed in C3G mutant MEFs on laminin (C,D) and more normally distributed in mutant cells on fibronectin (I,J). A few foci of integrin ß1 and paxillin co-localisation were observed in C3G mutant MEFs (A,C,E, arrowhead). However, paxillin- and integrin ß1-positive foci were by far more numerous in the controls on both laminin (F) and on fibronectin (L). Bar represents 14 µm in all panels.

View larger version (60K): [in a new window]   Fig. 9. Integrin ß3 and ß1 distribution in C3G mutant MEFs cultured with serum. (A-D) Integrin ß3 distribution was unaffected by the C3G mutation in the absence (A,B) or the presence (C,D) of serum. Integrin ß3 foci were more numerous and smaller than integrin ß1-positive foci (D,F). (E-K) We investigated integrin ß1 and paxillin distribution in MEFs plated in the presence of serum, because the morphology of C3G mutant MEFs was highly abnormal without serum on all substrates (see Fig. 8). Although C3G mutant MEFs appeared healthy when plated onto gelatine-coated cover slips in the presence of serum, integrin ß1 foci were similarly reduced in number (compare E and F) as in culturs without serum (compare Fig. 8A with B and 8G with H). Only very few integrin ß1-positive foci coincided with paxillin staining (J, yellow) as compared to controls (K). Arrowheads point to examples. Bar represents 14.5 µm in all panels.