Fig. 7. Spatial localization of Cx43.4 transcript in whole-mount neurula and tailbud stages. Whole-mount in situ hybridization revealed expression of Cx43.4 in dorsal structures extending from head to tail (A-G). By contrast, Cx30 was localized to ectoderm including the hatching gland and the endoderm (H-I). Embryos before clearing in benzyl benzoate/benzyl alcohol were photographed on a blue background. Cleared embryos are shown on yellow background. (A-C) The signal of Cx43.4 is strongly detected in the late neurula at stage 19. (A) Cx43.4 is in the neural folds and the eye vesicles, but not in the groove between the two neural folds (see white arrow). (B) The embryo from A was cleared to demonstrate localization of Cx43.4 in the posterior, already fused neural tube, as well in the anterior open tube, the brain and eyes. (C) A side view of the cleared embryo, anterior to the left, shows that Cx43.4 is restricted to dorsal neuroectoderm: head, spinal cord and tail. (D-F) Intensified accumulation of Cx43.4 in anterior structures of the tail bud embryo at stage 25. (D) One dark band of Cx43.4 expression in the anterior fused neural tube is marked with a white arrow. (E) A side view of two embryos (anterior to the left), demonstrate head, spinal cord and tail expression of Cx43.4 (F) A side view of a cleared embryo shows expansion of Cx43.4 expression in the head and brain structures as well in a branchial arch. Clear, but relatively lower expression is seen in the spinal cord and tail. (G) A side view of stage 33 tadpoles (anterior to the left) shows strong Cx43.4 expression in head and tail structures. As indicated earlier, expression is seen also in the spinal cord. (H) Whole mount staining shows Cx30 expression in the hatching glad and the anus. This superficial staining seems similar to the Cx43.4 staining seen in D. However, the side view of the cleared embryo in I (anterior to the left) shows that hatching glad staining is ectodermal staining and is different from the neural staining as seen for Cx43.4 (compare I to F and see the nueral staining in Fig. 8B). Another significant difference between the staining patterns of the two connexins is that, unlike Cx43.4, most of Cx30 expression is in the embryonic endoderm.