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Fig. 5. XtSulf1/2 double knockdown embryos show impaired early migration of the cranial neural crest. Xenopus tropicalis embryos were injected at two and four-cell stage into the animal hemisphere. Sibling embryos were cultured until NF stage 15 (top panels), stage 22 (middle panels) and NF stage 30 (bottom panels) and fixed for in situ hybridisation. This figure shows control uninjected embryos (E, J, O, T, Y, D′); embryos injected with 30 ng of control morpholino oligo (CMO) (A, C, F, K, P, U, Z, E′); embryos injected with 10 ng of antisense morpholino oligo (AMO) targeted against XtSulf1 (G, L, Q, V, A′, F′); embryos injected with 20 ng of AMO targeted against XtSulf2 (H, M, R, W, B′, G′); and embryos co-injected with 10 ng of AMO targeted against XtSulf1and 20 ng of AMO targeted against XtSulf1(B, D, I, N, S, X, C′, H′). Top panels (A–D) are dorsal views, the others are lateral views with anterior to the left. At stage 15, the expression of Slug marks the prospective neural crest at the boundary of the neural plate and the non-neural ectoderm (A, B). MyoD marks the dorsal, paraxial mesoderm (C, D). The expression of these genes is not effected when embryos are injected into the animal hemisphere with AMOs targeting XtSulf1 and XtSulf2 (B, D). At stage 22, NeuroD is expressed in the profundal, trigeminal and antero-dorsal lateral line placodes and lens (E,F). In single XtSulf1 knockdown, NeuroD expression in these placodes is reduced and disorganised (G), while in double Sulf knockdown, NeuroD expression is further reduced and restricted to a very small dorsal region (L). Sox8 and Twist are expressed in the mandibular (around the eye), hyoid and branchial arches (J, K, O, P; three arrow heads) at NF stage 22. In single XtSulf1 and XtSulf2 knockdown embryos the expression of these markers in the mandibular arch appears normal, but the expression in the hyoid and branchial arches does not extend as far ventrally as controls (L, M, R, Q; arrowhead and line). The anterior and posterior branchial arches are no longer visible as two distinct entities. In double Sulf1/2 knockdowns, Sox8 and Twist expression in the mandibular arch is normal, but expression in the hyoid and branchial arches is further restricted dorsally and the anterior and posterior branchial arches appear as one fused domain (N and S, arrowhead and line). At NF stage 30, NeuroD is expressed normally in the neural placodes and pineal gland (T,U). In XtSulf1 single knockout, expression in the antero-dorsal lateral line and trigeminal placodes migrate abnormally, all other expression is absent (V). In XtSulf2 knockouts, NeuroD expression is absent from the posterior lateral line placode, and expression in the trigeminal and antero-dorsal lateral line placode appear closer together (W). In double Sulf knockouts, NeuroD is expressed abnormally in the antero-dorsal lateral line and trigeminal placodes (X). Sox8 and Twist are expressed in the mandibular, hyoid and branchial arches (Y, Z, D′, E′). In single knockouts, the expression in the mandibular arch appears normal, but the expression in the hyoid and branchial arches is restricted dorsally. Furthermore, the anterior and posterior branchial arches appear fused together (A′, B′, F′, G′). In double Sulf knockouts, Sox8 and Twist expression domains are restricted dorsally in the hyoid and branchial arches, and the expression is fused in the anterior and posterior branchial arches (C′, H′). The effects of XtSulf 1/2 knockdown on CNC migration as assayed by the expression of the markers shown in this figure are summarised in Table 1. |
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Fig. 6. Targeting XtSulf1/2 knockdown to neural tissue disrupts cranial neural crest migration and does not affect mesoderm differentiation. X. tropicalis embryos were injected into the two dorsal blastomeres in the animal hemisphere at the 8-cell stage with 30 ng of control morpholino (CMO) or with 10 ng of S1 AMO together with 20 ng of S2 AMO (S1/S2 AMO). At stage 16, CMO injected (A, B) and S1/S2 AMO injected (C, D) embryos show normal expression of slug (CMO = 78% normal, n = 23; S1/S2 AMO = 59% normal, n = 17) and MyoD (CMO = 90% normal, n = 20, S1/S2 AMO = 65% normal; n = 23). At stage 22, twist expression is disrupted in knockdown embryos (F, 66% disrupted twist expression, n = 15) and does not extend as far ventrally as seen in those injected with control morpholino (E). The expression of myoD and α-cardiac actin in CMO injected embryos at stage 22 are shown in G and I. There is normal expression of myoD (66%, n = 18) and α-cardiac actin (64%, n = 14) in S1/S2 knockdown embryos (H and J). (K) The expression of twist in embryos injected with control morpholino at stage 30. (L) Twist expression is disrupted in knockdown embryos (69%, n = 16). The expression of myoD and α-cardiac actin in CMO injected embryos at stage 30 are shown in M and O. There is normal somite expression of myoD (83%, n = 12) and α-cardiac actin (80%, n = 10) in stage 30 S1/S2 knockdown embryos (N and P). |
