DEV026542 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Table S2 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Expression of celsr2 gene during gastrulation in zebrafish. celsr2 is expressed maternally (A) and is expressed ubiquitously at 50% epiboly (B) (lateral view, dorsal to the right) and at 90% epiboly dorsal view (C) and lateral view, dorsal towards the right (D).

• Supplemental Figure S2 -

  Fig. S2. celsr mutant/morphants do not either exhibit defective formation of dorsal forerunner cells or defective movement of the YSL nuclei. (A-F) Wild-type embryos (A,C,E) or MZord embryos injected with 0.4 pmoles each of celsr1a and celsr1b MOs (B,D,F) were visualised for dorsal forerunner cells with ntl probe at 70% epiboly (indicated by an arrowhead in B) or with lrdr1 probe at 90% epiboly (C,D). The embryos were injected with SYTOX green at sphere stage, and then visualised for YSL nuclei at 50% epiboly (E,F).

• Supplemental Figure S3 -

  Fig. S3. Maternal Celsr2 is required for epiboly, whereas Lyn-Celsr expression does not result in epiboly defects in wild-type embryo. (A) Wild-type embryos were injected with 300 pg RNA encoding Lyn-Celsr. (B) Embryos from crossing between MZord female with TL wild-type male were injected with 0.4 pmoles each of celsr1a and celsr1b morpholinos. The embryos were fixed at 90% epiboly for β-catenin antibody staining. An arrowhead indicates the leading edge of deep cells. Note that embryos expressing Lyn-Celsr show no epiboly phenotype (n=33).

• Supplemental Figure S4 -

  Fig. S4. Celsr-ΔC causes C&E and epiboly defects, and injection of a low dose of stbmMO, dvl2MO or Lyn-Celsr causes C&E but not epiboly defects in MZord embryos. MZord embryos (A,D) or MZord embryos injected with 0.4 pmoles stbmMO (B,E), 0.75 pmoles dvl2MO (C,F) or 20 pg Lyn-Celsr RNA (I,L). Wild-type embryos were injected with 300 pg Celsr-ΔC-HA RNA (H,K) or left uninjected (G,J). The embryos were fixed at tail-bud and subjected for in situ hybridisation with hgg1 for the prechordal plate, ntl for the notochord and dlx3 for the anterior edge of the neural plate. The results of C&E phenotypes are summerised in Table S1. Note that injection of 300 pg Celsr-ΔC-HA RNA causes C&E defects with posteriorly misplaced prechordal plate as well as epiboly defects shown by the shifted germ ring expression of ntl (indicated by an arrowhead).

• Supplemental Figure S5 -

  Fig. S5. Characterisation of Celsr-ΔN. (A-C) Wild-type embryos were injected with 150 pg Dsh-GFP together with 100 pg Celsr-ΔN-RFP RNA. Scanning for GFP (A) and RFP (B) was carried out simultaneously and merged (C). Note that Celsr-ΔN is capable of recruiting Dsh without exogenous Fz, suggesting activation of the Wnt/PCP pathway. (D,E) Celsr-ΔN causes CE defects. Wild-type embryos were injected with 100 pg Celsr-ΔN-Venus RNA, and fixed at tail-bud for staining with hgg1 for the prechordal plate, ntl for the notochord and dlx3 for the anterior edge of the neural plate (see Table S1 in the supplementary material). (F,G) Effects of Celsr-ΔN on epiboly. Injection of a high dose (300 pg) Celsr-ΔN-Venus RNA does not lead to epiboly defects (F), whereas expression of 100 pg Celsr-ΔN-Venus RNA fails to rescue the celsr mutant/morphant phenotype (G; see Table S2 in the supplementary material). An arrowhead indicates the leading edge of deep cells. (H,I) Hanging drop assays. Cells from wild-type embryos with cells from embryos expressing 300 pg Celsr-ΔN RNA, being intermingled well (H) (n=11). Expression of 50 or 100 pg Celsr-ΔN-Venus RNA fails to restore altered cell cohesive property of celsr mutant/morphant cells (I) when compared with Fig 7C. Note that there is a strong correlation of epiboly defects with altered cell cohesive properties.

• Supplemental Figure S6 -

  Fig. S6. Overexpression of Lyn-Celsr induces a planar cell polarity phenotype in the Drosophila wing. Wings of Drosophila overexpressing Lyn-Celsr (B) under the control of ptc enhancer (see Materials and methods). The region between veins 2 and 3, corresponding to the domain of ptc expression is shown. Overexpression of Lyn-Celsr only leads to a mild deflection of the hairs towards the posterior margin of the wing (B) when compared with the control fly (A). Anterior is upwards, distal is rightwards.