Microarray validation. In situ hybridization and RT-PCR validation of differential gene expression in plxnA2 and sema6A morphants at 18 somites. A–E: Top, middle, and bottom panels show uninjected control, plxnA2 MO injected, and sema6A MO injected embryos, respectively. Left and right panels show lateral and dorsal views, respectively, for each probe under each condition. Colored bars show corresponding heat map expression changes for each gene. UIC, uninjected control. A,B: Additional right panels show transverse 20 μm sections through 18 somite stage brain and eye vesicles processed for in situ hybridization with rasl11b and shtn-1 probes. Lines in whole-mounts indicate corresponding section locations. A′–E′: RT-PCR gels and quantification of gene expression changes for each gene as a ratio to ef1α control levels (rasl11b control 0.1137 ± 0.04, N = 3, rasl11b sema6A MO 0.34 ± 0.02, N = 3, **P < 0.01; rasl11b plxnA2 MO 0.32 ± 0.01, N = 3, **P < 0.01; shtn-1 control 0.08 ± 0.01, N = 3, shtn-1 sema6A MO 0.28 ± 0.01, N = 3, **P < 0.01; shtn-1 plxnA2 MO 0.24 ± 0.03, N = 3, **P < 0.01; dcdc2b control 0.06 ± 0.004, N = 3, dcdc2b sema6A MO 0.13 ± 0.02 N = 3, *P < 0.05; dcdc2b plxnA2 MO 0.24 ± 0.02, N = 3, ***P < 0.001; mmp2 control 0.004 ± 0.001, N = 5, mmp2 sema6A MO 0.21 ± 0.07, N = 5, *P < 0.05; mmp2 plxnA2 MO 0.27 ± 0.06, N = 5, **P < 0.01; rbl2 control 0.01 ± 0.002, N = 4, rbl2 sema6A MO 0.17 ± 0.02, N = 4, ***P < 0.001; rbl2 plxnA2 MO 0.15 ± 0.03, N = 4, **P < 0.01; one-way ANOVA, multiple comparisons test. Error bars indicate SEM). B, brain. E, eye.
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