Whole embryo RNA in situ hybridization were performed as previously described [70 (link)]. In brief, E9.5 mouse embryos were fixed in 4% PFA at 4 °C overnight and then dehydrated through methanol series. DIG-labeled RNA probes were synthesized using Roche transcription kit from linearized plasmid templates by following the manufacture’s protocol. Specimens were then rehydrated, digested with proteinase K and re-fixed in 4% PFA/ 0.2% glutaraldehyde at 4 °C for 20 min. After prehybridization, hybridization with probes took place overnight in hybridization buffer at 65–70 °C. Unbonded probes were washed away. Specimens were blocked at room temperature for 1 h and then incubated with pre-absorbed alkaline phosphatase-conjugated anti-DIG Fab fragments (Roche) overnight at 4 °C. After washes, alkaline phosphatase was detected using chromogenic conversion of NBT/ BCIP (both from Roche). Probes used for detecting Fgf8 [70 (link)] was obtained or generated as cited.
Primers for cloning whole-mount in situ hybridization probe templates were En2 forward: 5′-GAAGTCGACCGCTATCACTTCACGGTGGT-3′, reverse: 5′-ACGAGAATTCACTGGCCTTTTGTTCACGGT-3′. For Wnt1, forward: 5′-TATAGTCGACGGGCATCGTGAACATAGCCT-3′, reverse: 5′-CCGTGAATTCTTGGCGCATCTCAGAGAACA-3′. Successful cloning of DNA fragments was verified by sequencing. For all the experiments, at least three embryo littermate pairs were examined.
Primers for cloning whole-mount in situ hybridization probe templates were En2 forward: 5′-GAAGTCGACCGCTATCACTTCACGGTGGT-3′, reverse: 5′-ACGAGAATTCACTGGCCTTTTGTTCACGGT-3′. For Wnt1, forward: 5′-TATAGTCGACGGGCATCGTGAACATAGCCT-3′, reverse: 5′-CCGTGAATTCTTGGCGCATCTCAGAGAACA-3′. Successful cloning of DNA fragments was verified by sequencing. For all the experiments, at least three embryo littermate pairs were examined.