DEV01520 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. ClustalW alignment of 11 Dpr family members used. Species names are the same as in Fig. 1. Conserved domains are underlined. LZ, leucine zipper; PDZB, PDZ binding motif. RnDpr1, RnDpr2, FrDpr2, HsDpr2 and MmDpr2 are in silico clones. According to the Rn, dpr1 mRNA sequence has an unexpected insertion in the middle of the LZ. We expect it is not there and reflects an intron, but do not have enough information at this point to confirm this. (B) Dpr family members have low amount of overall conservation. From the ClustalW alignment, percent identity and overall similarity parentheses (taking into account conservative changes) was determined of Dpr family members. Except FRODO, which is XlDpr1b, abbreviations of species names are the same as in Fig. 1.

• Supplemental Figure 2 -

  Fig. S2. dpr1 expression at 24 and 36 hours post-fertilization. (A,B) Anterior dpr1 expression at 24 and 36 hours post-fertilization. (C) Double labeled in situ hybridization with dpr1 (blue) and dlx1(red) at 24 hours. Arrow indicated overlap of expression in the ventral telencephalon. (D) Double labeled in situ hybridization with dpr1 (blue) and hlx1 (red) (Hauptmann and Gerster, 2000). Arrow indicates overlap in the sixth transverse segment of the diencephalon. (E) Dorsal view of posterior dpr1 expression at 24 hours post-fertilization. dpr1 is expressed in the notochord. (F) dpr1 expression in the medial diencephalons at 36 hours post-fertilization. (G) dpr1 expression in the ventral neural retina and choroids fissure of the eye. In C,D, the eyes have been removed. In A-D,G, dorsal is towards the right.

• Supplemental Figure 3 -

  Fig. S3. dpr2 expression from five somites through 36 hours post-fertilization. (A) Dorsal view of posterior expression of dpr2 at five somites. Arrows indicate posterior lateral plate mesoderm. (B) Anterior dpr2 expression at 24 hours post-fertilization. Arrow indicates expression in the midcerebral vein. (C) Anterior dpr2 expression at 32 hours post-fertilization. Arrow indicates expression in the epiphysis. (D,E) Lateral anterior views of double labeled in situ hybridization with dpr2 (blue) and hlx1 (red) at 24 and 36 hours, respectively. Arrows indicate overlap of expression in the sixth transverse subdivision of the diencephalons (Hauptmann and Gerster, 2000). The eyes have been removed. (F) dpr2 expression at 20 somites. Arrow indicates expression in the ventral spinal cord. Arrowhead indicates dorsal spinal cord expression. (G) Frontal view of double labeled in situ hybridization with dpr2 (blue) and hlx1 (red) at 36 hours post-fertilization. dpr2 expression is medial to hlx1. (H,I) Dorsal views of double labeled in situ hybridization with dpr2 (blue) and flh (red). Anterior is towards the top of the page. Dpr2 is anterior to the flh expression. (J) Lateral view of posterior expression of dpr2 at 24 hours. Arrow indicates dpr2 expression in the ventral spinal cord. Arrowhead indicates dorsal spinal cord expression. (K) Dorsal view of posterior expression of dpr2 at 24 hours. Arrows indicate finbud expression. (L) Lateral view of eye at 36 hours. dpr2 is expressed in the dorsal neural retina and choroid fissure (arrow). In B-F, dorsal is towards the right. In A,J-L, anterior is towards the left.

• Supplemental Figure 4 -

  Fig. S4. Overexpression of Zebrafish Dpr2 causes the animal pigment to relocalize around the site of injection. (A) dpr1 RNA, (B) dpr2 RNA and (C) b-gal RNA injected embryos. (D) Uninjected control embryos. Arrows in B indicate aggregation of pigment. Ninety-three percent (n=40) of embryos injected with dpr2 RNA exhibited pigment relocalization compared with 0% (n=43) for dpr1 RNA, 3% (n=33) for b-gal RNA and 0% (n=50) in the uninjected control embryos.

• Supplemental Figure 5 -

  Fig. S5. Comparison of Dpr family members in Xenopus animal cap assay. RNA amounts for dpr1, dpr2, Xdpr1a, frodo/Xdpr1b and b-galactosidase were 1 ng, and for zebrafish dvl2 was 0.4 ng. All Dpr family members can synergize with zebrafish Dvl2.

• Supplemental Figure 6 -

  Fig. S6. Comparison of Dpr family members in HEK293 cells using Super(8X)TOPFlash reporter assay. (A) Zebrafish Dpr1 and Dpr2 synergize with Dvl2, while XDpr1a has no effect and FRODO/XDpr1b inhibits relative to b-galactosidase. (B) All Dpr members inhibit mouse Wnt1 activation of Super(8)TOPFlash relative to b-galactosidase. However, FRODO/XDpr1b and zebrafish Dpr1 inhibit better than do XDpr1a and zebrafish Dpr2. The data presented in Fig. 6J were taken from the experimental set depicted in B. Dpr1, Dpr2, XDpr1a, FRODO/XDpr1b or b-galactosidase (400 ng) and 20 ng of zebrafish Dvl2 (A) or 10 ng of mouse Wnt1 (B) were transfected. AU, arbitrary light units.