DEV027284 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (A-C) Whole-mount in situ hybridization showing the expression of Rspo3. (A) Expression in the atrial aspect of the developing heart (outlined) (E9.5). (B) Expression in the truncus arteriosus of a dissected heart of E12.5. (C) Expression in the umbilical cord (arrowhead) E12.5. (D-F) In situ hybridization on transverse cryosections of E9.5 placenta showing expression of indicated genes in endothelial cells (VEGFR2 positive) of the chorionic plate and blood vessels of the labyrinth (arrow and arrowhead, respectively). Abbreviations: h, heart; ta, trunkus arteriosus.

• Supplemental Figure S2 -

  Fig. S2. (A-I) Rspo3 expression in sites of vasculogenesis and angiogenesis. Rspo3, VEGF, Msr and Scl expression by in situ hybridization in stage 23 and stage 27 Xenopus tropicalis, anterior leftwards. Rspo3 and msr expression are detected in vasculogenic precurors such as the vbi and the dlp. (F,G) Vegfa is expressed in all somites (future intersomitic veins) and is co-expressed with Rspo3 in the lateral plate, the pronephric sinus and the branchial arch. Abbreviations: ba, branchial arches; dnt, dorsal neural tube; dlp, dorsal lateral plate; pn, pronephros; sm, somites; tb, tailbud mesoderm; vbi, ventral blood island; vvn, vitelline vein network.

• Supplemental Figure S3 -

  Fig. S3. (A,B) Construction of Rspo3 mutant mice. (A) Targeting strategy to delete exon 1, encoding the signal sequence. The position of the Southern hybridization probes is shown by horizontal bars (5′ probe and 3′ probe), the position of PCR primers for genotyping (PCR wild type and PCR knockout) is marked by arrowheads. Crossed lines show regions of homologous recombination; hatched lines indicate equivalent genomic loci. Homology regions of ∼5 and 7 kb containing (respectively) promoter, exon 1, and introns 1 and 2 were used for recombination. A neomycin cassette flanked by a 3′ loxP site was inserted downstream of exon 1 and a 5′ loxP site was inserted into the promoter region. The fragment flanked by two loxP sites was then removed by cre-recombinase. (B) (a-d) Validation of Rspo3 targeting. Validation of homologous recombination in ES cells by Southern hybridization of genomic DNA. Restriction of genomic DNA with HpaI (H) resulted in the following diagnostic fragments: wild type Rspo3 allele, 11.3 kbp; targeted (floxed) allele, 5.6 kbp and 7.9 kbp; knockout allele, 3.8 kbp. (a) Detection of homologous recombination with both 5′ external and 3′ external probes. (b) Detection of Cre deletion with the 3′ external probe. (c) genotyping by PCR, the 333 and 198 bp bands represent amplicons obtained with wild-type primers and co -rimers, respectively. (d) RT-PCR analysis of Rspo3 expression in E9.5 embryos. Abbreviations: H, HpaI.

• Supplemental Figure S4 -

  Fig. S4. qPCR analysis of wild-type and mutant samples. (A) Allantois E8.5; (B) chorionic plate E9.5. Relative expression of Msr and VEGFA to β-actin is shown, each bar represents an average for three samples. Note the decreased expression of Vegfa and Msr in allantois and chorionic plate in Rspo3-null mutants