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First published online 18 October 2006
doi: 10.1242/dev.02616

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Targeted mutation of serine 697 in the Ret tyrosine kinase causes migration defect of enteric neural crest cells

Naoya Asai^1,2, Toshifumi Fukuda³, Zaiqi Wu², Atsushi Enomoto¹, Vassilis Pachnis⁴, Masahide Takahashi^1,*, and Frank Costantini^2,*

¹ Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
² Department of Genetics and Development, Columbia University, 701 West 168th Street, New York, NY 10032, USA.
³ Laboratory of Molecular Biochemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, Tokyo 192-0392, Japan.
⁴ Division of Molecular Neurobiology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

View larger version (22K): [in a new window]   Fig. 1. Generation of RET S697A mutant mice. (A) The S697A mutation introduced in mouse RET exon 11. (B) Sequencing of amplified PCR product containing the S697A mutation site. Template DNA was prepared from clone 106 shown in (D). (C) Schematic representation of the targeting strategy. The S697A mutation was introduced in RET exon 11 and the loxP flanked neo gene in intron 11 in the targeting vector. After homologous recombination in ES cells, RETS697A-neo mutant mice were generated. The neo gene was removed to generate RETS697A mutant mice by crossing with ß-actin promoter/Cre transgenic mice. P1 and P2 indicate the forward and reverse primers for genotyping. H, HindIII; B, BamHI. (D) Southern blot analysis for RETS697A-neo and RETS697A mutations. Genomic DNA was prepared from control W9.5 ES cells (W9.5), homologous recombinant clone (clone 106), and tails of wild-type (Wt) and heterozygous RET S697A mutant mice (RETS697A/+). DNA was digested with HindIII, and analyzed with the indicated 5' external probe shown in (C). Wild-type and RETS697A-neo mutant alleles generate 9.0 kb and 4.8 kb bands, respectively. Loss of 4.8 kb band in DNA from a RETS697A/+ mutant mouse indicates successful removal of the neo gene. (E) Genotyping of mutant mice. P1 and P2 primers shown in (B) were used for genotyping by PCR. As the S697A mutant allele contains a 70 bp insertion, wild-type and mutant alleles give 302 bp and 372 bp bands, respectively. (F) RET protein expression in mutant mice. Lysates prepared from whole embryos at E11.5, newborn brain stems and stomachs were subjected to western blotting with anti-RET antibody. The 170 kDa and 150 kDa RET proteins are indicated. ß-Actin expression was investigated as a loading control. (G) Phosphorylation of Serine 697 in DRG neurons. Lysates were prepared from cultured DRG neurons isolated from wild-type and mutant mice, and subjected to western blotting with anti-RET and anti-phospho-RET(S697) antibodies (left and middle panels). The membrane was treated with alkaline phosphatase (AP) before incubation with anti-phospho-RET(S697) antibody to confirm that the detected band represents the phosphorylated protein (right panel).

View larger version (95K): [in a new window]   Fig. 2. Aganglionosis in the distal colon of RET S697A mice. Whole-mount AchE histochemistry of the distal intestine of wild-type (A-D) and homozygous S697A mutant newborns (E-H) at P3. Lower magnification views (A,E) and higher magnification views in the proximal colon (B,F), the middle colon (C,G) and the distal colon (D,H) are shown. Homozygous mutant mice exhibited marked reduction of enteric ganglia in the middle colon (G) and their absence in the distal colon (H). Arrowheads, cecum; asterisks, terminus of the colon; bars, 1 mm.

View larger version (15K): [in a new window]   Fig. 3. Numbers of ENS cells in the gut of RET S697A mutant mice. ENS cells were stained with cuprolinic blue and cell numbers were counted at each region of the small intestine (A) and the colon (B). The mean was calculated from ten views of each section of the intestine from four mice at P12. *P<0.01 compared with wild-type mice. Ho, homozygotes.

View larger version (79K): [in a new window]   Fig. 4. Impaired migration of the ENCCs in RET S697A mutant mice. Whole-mount in situ hybridization with riboprobe specific for RET on the developing guts in wild-type (A-C) and homozygous RET S697A mutant mice (D-F) at E11.5 (A,D), E12.5 (B,E) and E14.5 (C,F). (A'-C',D'-F') show high-power views of the front of the migrating RET positive ENCCs in A-F, respectively. Arrows, the front of the migrating ENCCs; arrowheads, cecum; asterisks, terminus of the hindgut. Scale bars: 500 µm.

View larger version (27K): [in a new window]   Fig. 5. Impaired chemoattractant response of ENCCs in RET S697A mutant mice. (A) Gut segments were dissected out from wildtype and homozygous RET S697A mutant mice at E12.5, and cultured on collagen gels with GDNF-soaked beads (dotted circles). After 4 days of culture, immunostaining with anti-PGP9.5 antibody was carried out to visualize ENCCs. Bars, 500 µm. (B) PGP9.5-positive area was measured in the quadrant facing GDNF-soaked beads (towards) and in the opposite quadrant (away). Six explants for each experiment. *P<0.01 compared with the distal quadrant. He, heterozygotes. (C) Lamellipodia formation in migrating ENCCs. F-actin was visualized with Alexa-phalloidin staining. Arrowheads indicate lamellipodia formation. Scale bars: 20 µm. (D) Quantitative analysis of lamellipodia formation. The number of lamellipodia was counted in the migrating cell front in the proximal and distal area. The number of lamellipodia per cell surface length (1 mm) was calculated. Six explants for each experiment. *P<0.01 compared with the distal quadrant.

View larger version (22K): [in a new window]   Fig. 6. Impaired JNK signal in RET S697A mutant mice and impaired chemoattractant response of ENCCs by the JNK inhibitor. (A) DRG was dissected wild-type and homozygous RET S697A mutant mice at postnatal day 5 and cultured for 72 hours. Lysates were prepared from DRG neurons untreated or treated with GDNF (20 minutes) and were analyzed by western blotting with the indicated antibodies. In the panel of anti-pJNK, an asterisk indicates a non-specific band. Two phosphorylated JNK bands were detected (arrows). (B) Gut segments were dissected out from wild-type mice at E11.5, and cultured with GDNF-soaked beads in the absence or presence of the JNK inhibitor SP600125. After 3 days of culture, immunostaining with anti-PGP9.5 was carried out. Scale bars: 500 µm. (C) PGP9.5-positive area was measured in the towards and away quadrants. Six explants for each experiment. *P<0.01 compared with control.

View larger version (120K): [in a new window]   Fig. 7. Inhibition of migration of ENCCs in gut culture by kinase inhibitors. Embryonic guts were dissected out from wildtype mice at E11.5 and cultured in control medium (A), in medium with MEK1 inhibitor PD98059 (B), PI3K inhibitor LY294002 (C), JNK inhibitor SP600125 (D), PKA inhibitor KT5720 (E) and SRC kinase inhibitor SU6656 (F). After 3 days of culture, ENCCs were visualized by immunostaining with anti-PGP9.5 antibody. Each experiment was carried out at least twice using more than six embryonic guts. Arrows, the front of the migrating ENCCs; arrowheads, cecum; asterisks, terminus of the hindgut; bars, 200 µm. Insets show highpower views of the front of migrating ENCCs in dotted boxes of each panel. To quantify inhibitory effects, the ratios of the distance of ENCC migration to the colon length were measured and calculated. The ratios are shown in the upper right corner of each panel.

View larger version (33K): [in a new window]   Fig. 8. S697A mutation does not impair neurite outgrowth and proliferation/survival of cultured ENCCs. (A) Neurite outgrowth of cultured ENCCs with GDNF stimulation. ENCCs were dissociated from E11.5 midgut and cultured for 3 days in the absence or presence of GDNF. Neurites were visualized by immunostaining with anti-PGP9.5 antibody. (B) Quantitative analysis of cultured ENCCs. After 3 days of culture in the absence or presence of GDNF, anti-PGP9.5-positive ENCCs were counted. The mean was calculated from ten views of each of three cultures.

View larger version (47K): [in a new window]   Fig. 9. Reduced kidney size in RET S697A mutant mice. (A) The weight of the kidney as a percentage of total body weight was determined in wild-type and mutant newborn mice (P3). Means and standard errors (error bars) were calculated from 23 wild-type, 55 heterozygous and 20 homozygous mice. *P<0.01 compared with wild-type mice. (B) Hematoxylin/Eosin staining of wild-type and mutant kidneys. Kidney of mutant mice at 2 months of age shows normal histology.