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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. |
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Fig. 4. LPAR6 is required for forebrain development. (A) Sequence (black letters, 5′-3′) of Xenopus lpar6 mRNA (translational start site in red) aligned with sequence for both AMO1 (pink letters 3′-5′) and AMO2 (blue letters 3′-5′). (B) In vitro translation of Xenopus lpar6 and human LPAR6 in the presence of morpholinos. Lane 1, no MO. Lane 2, control MO. Lane 3, AMO1. Lane 4, AMO2. (C-E) Stage 28, lateral view (head to left), injected with 40 ng of morpholino. (C) Normal embryo injected with control MO (100%, n=90). (D) AMO1-injected embryo with head defect (60%, n=95). (E) AMO2-injected embryo with head defect (93%, n=75). Anteroposterior axis length of control-MO-injected embryos was 4.0 mm (s.d.=0.21, n=44) and that of AMO-injected embryos 3.4 mm (s.d.=0.17, n=44). Defects in D and E are statistically significant (Fisher’s exact test, P<0.001). (F-H) Whole-mount in situ hybridisation, with antisense foxg1 probe. Stage 24, lateral view (head to left), injected with 40 ng of morpholino. (F) Normal embryo injected with control MO (100%, n=30). (G) AMO1-injected embryo with reduced foxg1 expression (60%, n=30). (H) AMO2-injected embryo with reduced foxg1 expression (87%, n=30). Defects in G and H are statistically significant (Fisher’s exact test, P<0.001). (I-K) Stage 28, lateral view (head to left), injected with 40 ng of AMO2 and 400 pg of human LPAR6 mRNA. (I) Normal embryo injected with control MO (100%, n=30). (J) AMO2-injected embryo with head defect (80%, n=35). (K) AMO2 plus hLPAR6 mRNA-injected embryo with normal head (66%, n=35). Rescue of head development in K is statistically significant (Fisher’s exact test, P<0.001). Scale bars: 400 μm. |
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Supplemental Figure 6. Inhibition of enpp2 inhibits telencephalon development. Xenopus laevis embryos were injected at the 2-cell stage with 10 ng per blastomere of enpp2 AMO and incubated until stage 25. Embryos were analysed by whole mount in situ hybridization using DIG labelled antisense RNA probes. A, B) Head of MO injected embryos stained for expression of the telencephalic marker foxg1 (arrow). Note absence of foxg1 expression in ennp2 AMO injected embryo. C, D) Head of MO injected embryos stained for expression of the ventral telencephalic marker nkx2-1 (arrow). Note strong expression of nkx2-1 in ennp2 AMO injected embryo, despite being greatly reduced in neurulae (see figure 9 of main article). E, F) Head of MO injected embryos stained for expression of the dorsal telencephalic marker emx1 (arrow). Note absence of emx1 expression in ennp2 AMO injected embryo. G, H) Head of MO injected embryos stained for expression of the eyefield marker rax. Note small spot of rax expression in the pineal gland (arrow), which is absent from ennp2 AMO injected embryo. The pineal gland is formed by the diencephalon of the newly formed brain. |
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Fig. 6. LPAR6 is required for telencephalic development. Whole-mount in situ hybridisation of tailbud embryos injected with 40 ng of morpholinos; lateral views of the head. (A,B) Telencephalon marker foxg1, with loss of expression in the AMO2-injected embryo (71%, n=35). (C,D) Dorsal telencephalon marker emx1, with loss of expression in the AMO2-injected embryo (75%, n=32). (E,F) Ventral telencephalon marker nkx2-1, with reduced expression in the AMO2-injected embryo (67%, n=33). (G,H) MHB marker en2, with normal expression in the AMO2-injected embryo (100%, n=32). All defects are statistically significant (Fisher’s exact test, P<0.001). Scale bars: 200 μm. |
