Whole-Mount In Situ Hybridization
For mouse analysis, CD-1 mouse embryos were collected after timed mating between embryonic day (E) 7.5 and E8.0 following standard procedures. Midday on the day of plug detection was defined as 0.5 dpc. In addition, embryos were staged by somite counting. Sense and antisense probes for Ccdc151 were made from a 709 bp pCRII-TOPO construct (RefSeq accession number NM_001163787.1, nt 1031–1740, transcript variant 1) made by TOPO TA cloning (Invitrogen) after amplification from complementary DNA. Probes were synthesized with digoxigenin NTPs (Roche) after template linearization with HindIII (sense) or NotI (antisense) before RNA synthesis with T7 or SP6 RNA polymerases, respectively. For whole-mount in situ hybridization, staged embryos were fixed overnight at 4°C in 4% paraformaldehyde in 1× PBS. Whole-mount in situ hybridization (WISH) was then performed according to standard procedures with minor modifications.27 Stained samples were transferred into 80% glycerol, and images were captured using a Scion CFW-1310C color digital camera mounted on an Axioskop 2 plus microscope (Zeiss) and Image-Pro Express.
For zebrafish analysis, the full-length ccdc151 cDNA (GenBank ID BC124606.1, Open Biosystems) was cloned using standard methods into pCS2 for sense and antisense probe transcription, with RNA probes transcribed from linearized plasmid templates using DIG-labeled nucleotides and used in a standard WISH protocol. Southpaw expression was investigated as previously described.46