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Fig. 5. LPAR6 is required for neural development. Whole-mount in situ hybridisation analysis of MO injected neurulae. (A-J) Dorsal views of neurulae injected with 20 ng of morpholino into a single blastomere at the two-cell stage. Head at the top and injected side (asterisk) on the right. (A,B) Neural plate marker sox2, with increased width on the AMO2-injected side (70%, n=40). (C,D) Epidermal marker k81a1, with decreased expression on the AMO2-injected side (75%, n=40). (E,F) Neural crest marker snai2, with reduced expression on the AMO2-injected side (69%, n=35). (G,H) Skeletal muscle marker myod1, with no defect (100%, n=38). (I,J) Posterior neural plate marker cdx4, with increased width on the AMO2-injected side (80%, n=40). (K-X) Anterodorsal views of neurulae injected with 40 ng of morpholino. (K,L) Telencephalon marker foxg1, with reduced expression in the AMO2-injected embryo (79%, n=38). (M,N) Hindbrain marker egr2, with normal expression in the AMO2-injected embryo (100%, n=35). (O,P) MHB marker en2, with normal expression in the AMO2-injected embryo (100%, n=35). Expression usually moved anteriorly (74%, n=35). (Q,R) Eyefield marker rax, with reduced expression in the AMO2-injected embryo (70%, n=40). (S,T) Eyefield marker pax6, with reduced expression in the AMO2-injected embryo (60%, n=30). (U,V) Anterior neural plate marker fgf8, with reduced expression in AMO2-injected embryos at the ANR (black arrow) and MHB (white arrow) (100%, n=23). (W,X) Whole-mount immunostaining for dpERK, with reduced ERK activity in AMO2-injected embryos at the ANR (black arrow), MHB (white arrow) and branchial arches (white arrowhead) (92%, n=36). All defects are statistically significant (Fisher’s exact test, P<0.001). Scale bars: 200 μm. |
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Fig. 9. ENPP2 is required for forebrain development. (A) Sequence of Xenopus enpp2a (black lettering, 5′-3′) and enpp2b (green lettering, 5-3′), translational start site in red, aligned with sequence for AMO (blue letters, 3′-5′). (B) Western blot analysis of Xenopus embryos injected with 1 μg of enpp2a.myc mRNA and 40 ng of morpholino. (C,D) Stage 28, lateral views (head to right) injected with 20 ng of either control MO or enpp2-AMO. Anteroposterior axis length of control-morpholino-injected embryos was 4.0 mm (s.d.=0.13, n=45) and that of AMO-injected embryos 2.9 mm (s.d.=0.22, n=45). (E-P) Whole-mount in situ hybridisation analysis of neurulae injected with 20 ng of either control (CMO) or enpp2-AMO. All embryos are viewed from anterodorsal perspective. (E,F) Telencephalon marker foxg1, with reduced expression in AMO-injected embryo (57%, n=82) (G,H) Ventral telencephalon marker nkx2-1, with reduced expression in AMO-injected embryo (59%, n=70). (I,J) Dorsal telencephalon marker emx1, with reduced expression in AMO-injected embryo (72%, n=53). (K,L) Eyefield marker rax, with normal expression in AMO-injected embryo (100%, n=77). (M,N) MHB marker en2, with reduced expression in AMO-injected embryo (16%, n=73 - 6% in controls, n=50). (O,P) Anterior neural plate marker fgf8, with reduced expression in AMO-injected embryo in both ANR (black arrow) and MHB (white arrow) (71%, n=21). (Q,R) Whole-mount immunolocalisation for dpERK. Note reduced ERK activity in AMO-injected embryos in the ANR (black arrow), the MHB (white arrow) and the branchial arches (white arrowhead) (96%, n=26). All defects are statistically significant (Fisher’s exact test, P<0.001) except for en2 (M,N). Scale bars: 600 μm in C,D; 200 μm in E-P. |
