DEV02250 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1A
• Supplemental Figure 1B -

  Fig. S1. Comparison of Cvl2 proteins from different species. (A) ClustalW alignment of Cvl2 from Danio rerio (d.r.), Homo sapiens (h.s.), Mus musculus (m.m.) and Drosophila melanogaster (d.m.). Residues conserved compared with Danio rerio are boxed, conserved cysteines are shaded grey, the proteolytic clevage site is indicated as a black bar. (B) Alignment of CR domains 1-5 of zebrafish Cvl2 and CR1-4 of Chordin, conserved cysteines are shaded gray.

• Supplemental Figure 2 -

  Fig. S2. Spatial expression of crossveinless2 at later developmental stages. Developmental stage is indicated in the lower left corner. (A,C-M,O-S) In situ hybridization with a cvl2 antisense probe, (B) double in situ staining with cvl2 in red and floating head (flh) in blue, and (N) in situ hybridization with a collagen X probe. (A,E) Lateral views, (B) frontal optical cross-section showing co-expression of cvl2 and flh in the epiphysis. (C,D) Dorsal view of (C) wild type and (D) casanova mutant. Expression in the pharyngeal endoderm is absent in casanova mutants, (F) optical cross-section of the tail at the level of the yolk extension showing expression in medially migrating neural crest cells, (G,I,K,L) dorsal views, focal planes of K,L are indicated in J. (K) Expression in putative medioventral cartilage condensations (see also J). (L) Expression in more dorsolateral areas that separate the arches (compare also with J). (M-P) Ventral views, expression domains of cvl2 and the bmp target gene collagen X overlap. (O) Higher magnification of the area of the hypophyseal fenestre showing that cvl2 is not expressed in the stacked mature chondrocytes of the trabeculae. (P) Higher magnification of the hyoid arch as indicated in M. cvl2 expression is restricted to tissue surrounding the cartilage. cl, cleithrum; dm, dorsal diencephalic midline; em, eye muscles; en, endoderm; enp, endodermal pouches; ep, epiphysis; fc, finbud cartilage; fe, facial ectoderm; g, gut; hy, hyoid arch; le, lens; nc, neural crest; pe, pharyngeal endoderm; pll, posterior lateral line ganglion; op, operculum; ov, otic vesicle; t, trabeculae; vm, ventral mesoderm; vsn, visceral sensory neurons

• Supplemental Figure 3 -

  Fig. S3. Loss of Cvl2 causes dorsalization and rescues the ventral tail fin duplication of ogon/sizzled (ogo) mutants. (A,C,E,G,I,J) Control embryos, (B,D,F,H,K) cvl2 MO injected. (A-H) Immunostainings or whole-mount in situ hybridization with antibodies or probes indicated in lower left corners. (A-F) Eighty percent epiboly stage, dorsal towards the right. (A,B) Vegetal views of embryos also shown in Fig. 2C,D; (C-F) Lateral views; insets in E,F show lateral views of same embryos. (G,H) Ten-somite stage, dorsal views. In the injected embryo, the anterior somites are laterally expanded. (I-K) Live embryos at 36 hpf, lateral views; insets show magnified ventral views on tail. There is duplication of the ventral tail fin in the ogon mutant (J), which is rescued close to wild-type condition (I) after injection of cvl2 MO (K). The small eyes phenotype of the ogon mutant results from brain necrosis, which is most probably not due to the loss of sizzled, but to a thus far unidentified other gene deleted in the used m60 deficiency allele. Accordingly, it is not rescued upon injection of cvl2 MO.