Zhao H , Tanegashima K , Ro H , Dawid IB .

Paper published

Fig. 1. Characterization of the Lrig3 gene. (A) Regional expression of Lrig3 in stage 10 embryos. Lrig3 was expressed strongly in dorsal explants, but barely detected in other regions. The indicated genes were assayed to test quality of dissection. St10, stage 10 embryo; AC, animal cap; Veg, vegetal explant; RT-, control without reverse transcriptase. (B) Schematic drawing of the protein structure of Xenopus and zebrafish Lrig3. SP (yellow), signal peptide; LRRNT (blue box), leucine-rich repeat N-terminal domain; LRR TYP, leucine-rich repeats, typical; LRR, leucine-rich repeats; LRRCT (blue oval), leucine rich repeat C-terminal domain; IG C2 (green oval), immunoglobulin C-2 Type; TM (purple), transmembrane domain. Sequence identity between zebrafish and Xenopus Lrig3 is indicated. (C) Dendrogram of the Lrig3 family including Kekkon of Drosophila. (D,E) Subcellular distribution of Lrig3 after transfection into COS7 cells. (D) Transfected Lrig3-Flag (red) co-localized with the cis Golgi apparatus marker GM130 (green); the nucleus was stained with DAPI (blue). (E) A small proportion of Lrig3-Flag co-localized with the early endosome marker EEA1 (green). (F-M) Expression pattern of Lrig3 in Xenopus. Vegetal view at stage 10, expression in the organizer (F); stage 10 section (G). (H,I) Stage 12 (H, posterior view; I, lateral view). (J,K) Stage 15 (J, dorsal view; K, lateral view). Tailbud (stage 24, L) and tadpole (stage 32, M); expression is seen in brain, eye, somites and branchial arches. (N) Temporal expression of Lrig3 during Xenopus development. (O-R) Expression of lrig3 in zebrafish. Transcripts are present maternally (O), become localized in the forming organizer at 30% epibody (P) and subsequently in the shield (Q), and were found in brain, eye and branchial arches at 24 hours (R). (P-R) Lateral views.

Fig. 3. Knockdown of Lrig3 results in defects of the NC and its derivatives. (A-F″) In situ hybridization with the indicated markers for uninjected or two examples (′,″) of L3MO-injected embryos. One dorsal blastomere at the four-cell stage was injected with 15 ng L3MO and 100 pg lacZ mRNA, and fixed at about stage 19. The expression of hindbrain marker Krox20 (rhombomeres 3 and 5) (A-A″), NC markers Slug (B-B″), Sox9 (C-C″), Myc (D-D″) and Inca (E-E″), and pan-neural marker Sox2 (F-F″) were examined. The injected side was traced by lacZ staining. The stream of Krox20-positive cells extending from the hindbrain was absent in 97% (28 of 29 embryos) on the injected side. The expression of NC markers was reduced on the injected side in the following percentage of embryos: Slug (58%, 14 of 24), Sox9 (79%, 15 of 19), Myc (83%, 20 of 24) and Inca (65%, 17 of 26). (G) Neural induction by overexpression of Chordin (Chd) in animal caps was barely affected by L3MO. Chd (100 pg) or Chd (100 pg) plus L3MO (30 ng) were injected, caps dissected at stage 9, and assayed at equivalent stage 22. Pan neural markers Sox2 and Ncam were examined, and interference with Lrig3 splicing was verified (see also Fig. 2A); arrow indicates the predicted unspliced band (lane 4). (H-J″) NC markers, including Slug, Inca and Sox9, were examined in embryos injected with 7.5 ng L3MO in one animal dorsal blastomere at the eight-cell stage. Strong inhibition was observed for Slug (95%, 18 of 19), Inca (89%, 24 of 27) and Sox9 (73%, 19 of 26). (K-M) Cranial cartilages were reduced by knockdown of Lrig3. Ventral views of Alcian Blue stained cartilage from embryos injected with control morpholino (K) or L3MO (L,M). ba, basihyal; br, branchial; ch, ceratohyal; m, Meckel's. Percentages of embryos with reduced head cartilage are 82% (38 of 47) at 7.5 ng L3MO and 97% (31 of 32) at 15 ng L3MO. (N-P) Rescue of L3MO effect. All embryos were injected with 15 ng L3MO, and embryos in N-O″ were co-injected with 20 pg Lrig3 mRNA; in situ hybridization with Inca (N-N″) and Slug (O-O″). Percentages of strongly affected (examples in N,O), mildly affected (N′,O′) and normal (N″,O″) are shown in P.