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First published online January 11, 2008
doi: 10.1242/10.1242/dev.013763

Development 135, 589-598 (2008)
Published by The Company of Biologists 2008
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Disruption of PDGFR{alpha}-initiated PI3K activation and migration of somite derivatives leads to spina bifida

Elizabeth A. Pickett, Gregory S. Olsen and Michelle D. Tallquist*

Department of Molecular Biology, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.


Figure 1
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  		Fig. 1. Induction of spina bifida by imatinib-mesylate. (A-F) Representative Safranin-O stained transverse sections through the lumbar and lumbar-sacral regions of chick embryos (HH stage 36). (A,D) Vehicle treated, and (B,E) 25 µM and (C,F) 50 µM imatinib-mesylate treated. The lamina (indicated by an arrow in A,D) failed to form across the dorsal midline in imatinib-treated embryos (indicated by arrowheads in B,C,E,F), resulting in spina bifida. Images show the most advanced arch formation found in serial sections of each treatment group. Scale bars: 200 µm. nt, neural tube; vb, vertebral body. Values listed below each column indicate the number of open vertebrae in all serial sections through the lower thoracic, lumbar and sacral region in each embryo. The number of embryos examined is indicated in parentheses. *Two out of four embryos treated with 50 µM imatinib-mesylate also exhibited failure of ventral closure.

 

Figure 2
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  		Fig. 2. Spina bifida in PDGFR{alpha} mutant embryos. Skeletal preparations of E18.5 embryos display defects in lumbar vertebrae formation. (A) Control, (B) PDGFR{alpha}CKO (C) PDGFR{alpha}PI3K/PI3K and (D) PDGFR{alpha}TKO. Headings at the top of each column indicate the bone isolated. L, lumbar; T, thoracic. The lumbar vertebral arches form normally in A and B but fail to progress in C and D. Vertebral arch development of the thoracic vertebrae and limbs was normal in all embryos. Arrows indicate the extent that Alcian Blue cartilage can be detected. Scale bars: 1 mm. Asterisks indicate location of bones lost during dissection. sp, spinous process; l, lamina; vb, vertebral body; r, rib. Data representative of skeletal preparations performed on E18.5 and P1 embryos: wild type (n=10); PDGFR{alpha}CKO (n=4); PDGFR{alpha}PI3K/PI3K (n=10); and PDGFR{alpha}TKO (n=9).

 

Figure 3
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  		Fig. 3. Timing of vertebral arch defect. (A,B,E,F,I,J) Safranin-O stained transverse sections through lumbar vertebrae at the indicated ages. (A,E,I) are control and (B,F,J) are PDGFR{alpha}PI3K/PI3K embryos. Safranin-O sections are representative of the furthest progression of the vertebral arch in the lumbar region at each time-point. By E15.5 the development of the vertebral arch in the mutant lags behind that of the control littermate, and failure of the vertebral arch to fully form was observed at E16.5. Arrowhead indicates condensing cartilage that fails to stain with Safranin-O. Black arrows denote furthest progression of vertebral arch. (C,D,G,H) H&E (in black and white) of (C,G) control and (D,H) PDGFR{alpha}PI3K/PI3K embryos at the indicated ages. (K,L) Hematoxylin and Eosin staining of E18.5 (K) control and (L) PDGFR{alpha}TKO. Asterisks indicate the lamina of the vertebral arch. White arrows indicate region of mesenchymal cells dorsal to the neural tube and their absence in the mutant embryos. Scale bars: 100 µm, except C,D,G,H (20 µm).

 

Figure 4
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  		Fig. 4. Loss of PDGFR{alpha}-PI3kinase signaling results in defective migration. (A,A') PDGFR{alpha}GFP/+, (B,B') PDGFR{alpha}PI3K/GFP and (C,C') PDGFR{alpha}TKO/GFP. PDGFR{alpha}-expressing cells (as detected by PDGFR{alpha}GFP) are present in the perichondrium and in the mesenchyme surrounding the vertebral arch. (A,A') Heterozygote control section is at the level of the perichondrium and therefore displays more PDGFR{alpha}-positive chondrocytes. The PDGFR{alpha}-positive mesenchyme adjacent to the vertebral arch extends just beyond the tip of the arch in the heterozygote control, but has failed to advance in the (B,C) mutants. Sections are representative of furthest advancement of the vertebral arch and adjacent mesenchyme population in two embryos of each genotype. (A'-C') DAPI fluorescence of the sections in A-C. Adjacent mesenchyme is outlined. Asterisks denote the tip of the vertebral arch. Arrow indicates mesenchyme. va, vertebral arch; nt, neural tube. Scale bar: 100 µm. (D) In vitro migration assay. Heterozygous cells (grey bars) migrated in a dose-dependent manner in response to PDGF-AA and PDGF-BB ligands, whereas mutant cells (striped bars) were unable to migrate in response to PDGF-AA and showed a decreased response to PDGF-BB. Both genotypes were able to migrate in response to 10% serum. This experiment is representative of three independent experiments.

 

Figure 5
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  		Fig. 5. Disruption of PI3K and PAK pathway activation in PDGFR{alpha}PI3K/PI3K cells. (A-C) Western blot analysis of whole cell lysates from primary mesenchymal cells. (A) PDGFR{alpha} is expressed and ERK1/2 is phosphorylated in PDGFAA and serum-stimulated control and PDGFR{alpha}PI3K/PI3K cells. (B) Akt and S6K1 are not phosphorylated in PDGFAA-stimulated PDGFR{alpha}PI3K/PI3K cells. (C) PAK1 is not phosphorylated in PDGFAA stimulated PDGFR{alpha}PI3K/PI3K cells. Loading controls (immediately beneath the sample) were either actin (for the PDGFR{alpha}) or stripped blots that were reblotted for the unphosphorylated form of the protein. Stimulation conditions: st, starved; AA, 10 ng/ml PDGF-AA; se, 10% serum. Blots are representative of three independent experiments.

 

Figure 6
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  		Fig. 6. Effects of PDGFR{alpha}PI3K/PI3K mutation on Rac1 activation and paxillin localization. (A) Western blot for GTP-bound Rac1 and total Rac1 in control and PDGFR{alpha}PI3K/PI3K cells. Upper blot demonstrates the time course of Rac1 activation in control cells. Lower blot illustrates the failure of PDGFR{alpha}PI3K/PI3K cells to stimulate Rac1 activation when stimulated with PDGFAA. Cells were stimulated for 10 minutes. Results are representative of three independent experiments. (B) Immunocytochemistry for paxillin localization in control and PDGFR{alpha} mutant cells. Genotypes are indicated on the left. Cells were plated for two hours, starved for 24 hours, and then stimulated for 30 minutes with the indicated stimulation. Images are representative of five independent experiments and represent the majority of cells within the culture for each stimulation. Scale bar: 50 µm.

 




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