Int bcip

For double in situ localization of SmVAL13 and 7 transcripts in adult worms, parasites were initially hybridized with SmVAL13 DIG-labeled probe and SmVAL7 Fluorescein-labeled probe simultaneously. These were then incubated with anti-fluorescein antibody and the first color development step was performed using INT/BCIP (Roche) as an alkaline-phosphatase substrate for SmVAL7 transcript visualization (orange). Next, the parasites were washed several times in alkaline phosphatase buffer to remove any remaining substrate, after which the alkaline phosphatase was inactivated by fixing the parasites for 20 min in 4% formaldehyde/PBST, followed by 5× rinsing in PBST and a 30-min incubation period at 65 °C. The parasites were subsequently re-blocked for 2 h at room temperature and then incubated with anti-DIG antibody. The color was developed using BM Purple (Roche) as enzyme substrate for SmVAL13 transcript visualization (purple) and images were captured as described for germ balls. SmVAL13 DIG-labeled sense probe was used as negative control.

Embryos at different developmental stages were sampled and fixed in 4% paraformaldehyde (PFA) at 4°C overnight and then transferred in 100% MeOH. Whole-mount in situ hybridization was carried out according to a standard protocol (Thisse and Thisse, 2008 (link)). The DIG-labeled anti-sense probes were generated using a DIG RNA Labeling Kit (SP6/T7) (Roche). INT/BCIP (Roche) were used as alkaline phosphatase substrates. The primers of the probes were showed in the table of Supplementary Table S1. For embryos at or after 48 hpf, the homozygotes were separated from their siblings according to their heart edema and pectoral fin phenotype. For embryos before 48 hpf, as it was difficult to separate homozygous mutants from the heterozygous and wild-type siblings, WISH was performed for all progeny of rnf2^± parents. After the WISH, each embryo was photographed and genotyped separately. The photographs were taken under a stereomicroscope (Leica Z16 APO) with a digital camera (Leica DFC450). The number and phenotype of embryos in each group were recorded, and then the offspring produced by rnf2^± self-cross were genotyped.

Standard whole-mount in situ hybridization was performed as described previously (Thisse and Thisse, 2008 (link)). INT/BCIP (175 μg/ml; Roche) was used as alkaline phosphatase substrates. The following molecular markers were used: wnt4 and wnt11f2 (a gift from Dr. Diane Sepich, previously used in Makita et al., 1998 (link); Marlow et al., 2002 (link)).

Digoxigenin (DIG) and fluorescein (FLU)-labelled RNA probes were synthesized using T7 or T3 RNA polymerases (Promega) according to manufacturers’ instructions and supplied with DIG or FLU labelled UTP (Roche). Probes were detected with anti-DIG-AP (1:5000, Roche) or anti-FLU-AP (1:10000, Roche) antibodies and NBT/BCIP (Roche) or INT/BCIP (Roche) substrates according to standard protocols (Thisse and Thisse, 2008 (link)).

Single and two-color whole mount ISH were performed using NBT/BCIP (Roche) and INT/BCIP (Roche), as previously reported [74 (link)]. Digoxygenin- and fluorescein-labeled anti-sense RNA probes for zsyellow (ZDB-EFG-110824-1), egfp (ZDB-EFG-070117-1), nr2f1a (ZDB-GENE-980526-115), nr2f2 (ZDB-GENE-990415-252), vmhc (ZDB-GENE-991123-5), nkx2.5 (ZDB-GENE-980526-321), dlx2a (ZDB-GENE-980526-212), tbx1 (ZDB-GENE-030805-5), tcf21(ZDB-GENE-051113-88), and klf2a (ZDB-GENE-011109-1) were used. Area measurements were performed using ImageJ.

Whole-mount single and double ISH were carried out using standard methods with NBT/BCIP (Roche) and INT/BCIP (Roche) solutions [68 (link)]. Probes for the following genes were used: hoxb5b (ZDB-GENE-000823-6), dhrs3a (ZDB-GENE-040801-217), cyp26a1 (ZDB-GENE-990415-44), eng2a (ZDB-GENE-980526-167), egfp (accession number: JQ064510.1), egr2b (formerly krox20; ZDB-GENE-980526-283), and pax2a (ZDB-GENE-990415-8).