DEV02123 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Surgical procedure followed for EZ-I ablation. (1) Two bilateral excisions were performed in ovo on the superficial ectoderm, A, on each side of the neural tube of 5-somite embryos from the prosencephalic to the mesencephalic level. The ectoderm was then moved laterally to gain free access to more profound parts of the embryo. (2) A very fine curved tungsten microknife was then passed under the neural tube, B, and the notochord, resulting in their separation from the dorsal foregut endoderm. A transversal incision was then performed on the neural plate at the level of the posterior mesencephalon. The neural epithelium was reclined rostrally in order to gain access to the endoderm. (3) The ventral endoderm was separated from the ventral ectoderm by passing between the two tissues a very fine curved tungsten microknife. The rostral-most attachment between the ventral endoderm and the anterior neural fold was then cut. At this point, the ventral endoderm was free of any attachment and we could easily remove the EZ-I by performing a transversal incision at a distance of about 150 µm from its rostral end about 100 µm in front of the anterior intestinal portal vein. For grafting experiments, a cut was performed in the midline of the dissected EZ-I before reimplanting. AIP, anterior intestinal port; NT, neural tube.

• Supplemental Figure S2 -

  Fig. S2. Supernumerary EZ-I graft close to the ocular region induces ectopic Gli1 expression. An EZ-1 fragment from a 4-somite quail embryo was ectopically grafted into the presumptive latero-ocular territory of a stage-matched chick embryo. Immunoperoxidase detection of Gli1 was performed 24 hours after transplantation (∼HH18). (A) Ectopic expression of Gli1 (dark staining delineated by arrows) can be seen on this lateral view of an operated embryo. (B) Absence of Gli1 expression in an unoperated embryo. 1^stpa, first pharyngeal arch; ey, eye.

• Supplemental Figure S3 -

  Fig. S3. Effects of cyclopamine on craniofacial development. (A) Dorsal schematic view of a 5-somite chick embryo showing the implantation of a cyclopamine-loaded agarose bead in contact with EZ-I. (B) Frontal view of the chondrocranium of a cyclopamine-treated HH34 embryo (day 8). (B′) Drawing highlighting the elements of the nasal capsule: red, ectethmoid (ect); green, infraorbital septum (is). Meckel and ectethmoid cartilages are present, but the mesethmoid is absent. (C-D′) Right and left lateral views with corresponding drawing (D′; same colour code as B′) of a cyclopamine-treated embryo more clearly showing the normal development of first PA derivatives. I and II, endoderm zones I and II; ect, ectethmoid; LE, left eye; Mc, Meckel’s cartilage; r1-r8, rhombomeres 1 to 8.