Fig. 9. Knockdown of all three PG1 genes affects the development of neural crest and its derivatives. Non-injected controls (NIC; A,E,G,I) and embryos injected on the left-hand side with Hoxd1 MO insensitive mRNA (B,J), all three Hox PG1 morpholinos (A1/B1/D1MO; C,F,H,K) or a combination of both (D,L) were analyzed for neural crest gene expression, or stained with alcian blue to show the craniofacial structures. The embryos shown in A-F are early tailbud stage embryos shown from the dorsal side with anterior to the top. With the PG1 MOs, neural crest cells fail to migrate away from the hindbrain, as shown by Xslug (C: 86%, n=29) and Xsnail (F: 100%, n=14) expression. This is rescued (22% of embryos with restricted Xslug expression, n=9) by Hoxd1 overexpression (D) and simple overexpression leads to an expanded Xslug domain (B: 100%, n=10). Slightly later tailbud embryos analyzed for dll4 are shown from the left-hand side, with anterior to the left, and in the triple PG1 knockdown dll4 expression in the branchial arches is restricted to close to the hindbrain (50%) or completely lost (G,H: 44%, n=16). Alcian blue was used to stain the cartilage of stage 49, embryos (I-L) shown from the ventral side to show the craniofacial structures (injected side on the right:^*). The complete gill area is missing in the triple morpholino injected embryos (K: 82%, n=11) and this is rescued (26% of embryos with missing gill, n=19) when Hoxd1 mRNA is co-injected (L). C, ceratohyal; G, gill cartilage; M, Meckel's cartilage. Dotted line indicates midline.