DEV015073 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Protein sequence comparisons. Alignment of Lrig3 from human (NP_700356), mouse (NP_796126), chicken (XP_416055), Xenopus (EU126151) and zebrafish (EU126152). Identical amino acids are shaded in black and similar amino acids are shaded in grey. Percentage identity of the extracellular domain, transmembrane domain and intracellular domain from Xenopus Lrig3 is compared to other species. ID, identical.

• Supplemental Figure S2 -

  Fig. S2. Upregulation of Lrig3 by Chordin and tBMPRI and by activin treatment. Animal caps were dissected from stage 9 embryos and treated with 250 pM (lane 3) and 500 pM (lane 4) activin, respectively, until the sibling embryos developed to stage 11.5. The induction of Xbra was examined as a positive control. Lrig3 was moderately upregulated in the activin-treated animal caps; AC, untreated animal caps. Injection with 100 pg Chordin (Chd) or 1 ng truncated BMP receptor I (tBMPRI) strongly induced the expression of Lrig3 (lanes 5-10). The animal caps were cultured until the sibling embryo reached stage 25. H4 was used for a loading control. RT-, control without reverse transcriptase.

• Supplemental Figure S3 -

  Fig. S3. Depletion of Lrig3 by L3MO inhibited NC formation. Embryos were injected with 15 ng L3MO into one dorsal blastomere at the four-cell stage. The expression of the retinal marker Rx2A (A-A′′) and the NC markers Ap2a (B-B′′), Traf4 (C-C′′) and Twist (D-D′′) was examined at the late neurula stage; the injection side was visualized by lacZ staining. Rx2a was modestly decreased on the injected side (48%, 13 of 27 embryos). Traf4 (80%, 16 of 20), Twist (83%, 25 of 30), and Ap2a (80%, 16 of 20) were strongly inhibited.

• Supplemental Figure S4 -

  Fig. S4. Mesoderm formation was not substantially affected by L3MO. The mesoderm markers Xbra, Chordin, Goosecoid (Gsc), and Cerberus were examined by whole-mount in situ hybridization at stage 11 in embryos injected dorsally at the four-cell stage with 30 ng L3MO.

• Supplemental Figure S5 -

  Fig. S5. Injection of L3MO inhibited trigerminal nerve formation. One dorsal blastomere of 4-cell stage embryos was injected with either control MO (A,C) or L3MO (B,D), and allowed to develop to stage 35. Two branches of the trigeminal nerve, the ophthalmic and mandibular, were visualized by whole-mount in situ hybridization with Synuclein γ. The expression of Synuclein γ was reduced on the Lrig3-depleted side (77%, 20 of 26 embryos), while embryos injected with control MO showed normal expression (92%, 12 of 13).

• Supplemental Figure S6 -

  Fig. S6. Lrig3 moderately enhanced canonical Wnt signaling as measured by Topflash luciferase assay. Wnt3a (100 ng/ml) and LiCl (20 mM) were used to stimulate Wnt signaling. LRP6 enhanced, while Axin decreased, the Topflash activity induced by LiCl. Lrig3 affected LiCl-induced activity only slightly, but increased Topflash activity induced by Wnt3a in a dose-dependant manner. RLU, relative luciferase unit.

• Supplemental Figure S7 -

  Fig. S7. Interaction between Xfgfr1 and Lrig3 were examined by co-immunopriciptation (IP). HEK293T cells were co-transfected with Xfgfr1 and Lrig3-Flag, lysed and immunopriciptated with anti-Flag antibody (M2), anti-XFGFR1 antibody or mouse IgG. Xfgfr1 was detected after IP with anti-Flag (left gel), while Lrig3-Flag was detected after IP with anti-Xfgfr1 (right gel). WCL, whole cell lysate.