DEV023978 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Right nasal pit extirpation removes SP8 expression. SP8 is restricted to the nasal placode and following extirpation there is complete ablation of expression on the treated side (A,B). (A,B) Right-hand side. (A′,B′) Frontal views. (A′′′,B′′′) Untreated, contralateral left side. (A′′,B′′) Sections of the same embryo as in the adjacent panels. (B-B′′′) Regrowth of epithelium has occurred but SP8 is not reinduced. Scale bars: 500 μm for whole-mount in situs; 200 μm for the sections.

• Supplemental Figure S2 -

  Fig. S2. Nasal pit extirpation at stage 20 causes mild defects of the nasal capsule. (A) Lateral view, mandible removed showing deficient nasal bone (arrow). (A′) Dorsal view showing the loss of the nasal bone and partial loss of the nasal conchae. The premaxilla including nasal processes and the overall length of the upper beak are unaffected. nb, nasal bone; nc, nasal conchae; npp, nasal process of premaxillary bone; pmx, premaxilla. Scale bar: 5 mm.

• Supplemental Figure S3 -

  Fig. S3. Gene expression in the grafted nasal pit and recipient embryo. (A,A′) SP8 expression in the host embryo is strong in the nasal pit and the limb ectoderm. Graft has not been pinned but Neutral Red-stained epithelium is visible (arrowhead). No SP8 transcripts are detected in the graft. (B,B′) SP8 is expressed in the olfactory epithelium of a stage 24 embryo but not in the graft (arrowhead). The staple marks the position of the graft. (C-D′) PAX7 is very strongly expressed in the lateral nasal mesenchyme (arrow) but not surrounding the pinned graft (arrowhead, C,D′). np, nasal pit. Scale bars: 1 mm for the low-power views; 500 μm for the high-power views.

• Supplemental Figure S4 -

  Fig. S4. Ectopic pieces of bone and cartilage are induced by stage 20 and stage 26 nasal pits grafted to stage 20 hosts. (A) A proximal graft induced an ectopic process of the quadratojugal, which fused with the squamosal (box). Insets show schematic of grafting protocol. (A′) Graft was dissected from skull and coverslipped for photography. Squamosal process is fused to the proximal quadratojugal. Staple used to retain the graft is present in this specimen (arrowhead). The normal secondary cartilage present on the quadratojugal is visible (arrow). (B) Isolated ectopic bone and cartilage was induced in another graft. (C) A more distal placement of the nasal pit induced ectopic structures near the maxillary bone (box). (C′) An ectopic process projects from the jugal bone. (D) Ectopic bone and cartilage in the soft tissue over the eye (box). (D′) Two pieces of bone and one piece of cartilage fragment have formed. (E) An ectopic process on the proximal end of the quadratojugal. The normal secondary cartilage at the tip of the quadratojugal is indicated by the arrow. g, graft; j, jugal; mxb, maxillary bone; p, palatine; q, quadrate; qj, quadratojugal; rap, retroarticular process; s, squamosal; so, scleral ossicles. Scale bars: in A, 5 mm for A,C,D; 1 mm in A′,B,C′,D′,E.

• Supplemental Figure S5 -

  Fig. S5. Nasal pit grafts to the lateral plate mesoderm of the flank. Sixteen hours after grafting a stage 20 nasal pit to the flank of a stage 15 host, it was hybridized with two probes, one for the epithelium and one for the mesenchyme. (A,B) HOXB1 expression in the lateral plate mesoderm is present surrounding the graft (arrows), while the graft did not maintain SP8 expression. (B′) Section through the graft showing the invagination into the flank mesenchyme. (C,D) Another marker of nasal epithelium, DLX5, was not expressed in the graft and there is no change in the expression of HOXB9 in the host mesenchyme. The faint red staining of the graft is retained Neutral Red. Scale bars: 500 μm in A,B; 250 μm in B′; 1 mm in C,D.