Single whole-mount in situ hybridisation was performed as described [31 (link)]. Embryos were photographed with a LEICA Z6 APO stereoscope coupled to a LEICA DFC490 camera.
Digoxigenin-labelled antisense RNA probes were synthesised from DNA templates of deltaC [32 (link)], her1 [33 (link)], her7 [34 (link)], mespb [35 (link)], pcdh8 [36 (link)], myod1 [37 (link)], cb1045 [38 (link)], spaw [39 (link)], pitx2 [40 (link)], myl7 [41 (link)], cxcl12b [25 (link)], etv2 [42 (link)], foxj1b [19 (link)], foxc1b [43 (link)], dmrt2a [4 (link)], cyp1a, which was cloned in pGEM-T Easy (Promega) using the pF-cyp1a (5’-ATGGCTCTGACTATTCTTCCAATATTGGG-3′) and pR-cyp1a (5’-CTAGAACCCAGGCTGTGGTGTGACCCGA-3′) and pxdc1b, which was cloned in pGEM-T Easy (Promega) using the pF-pxdc1b (5’-ATGGCATCGGCGATTTTTGAGGGCA-3′) and pR-pxdc1b (5’-AAGTCAGTTTCAAAAGGAACCAGA-3′).
For HA immunohistochemistry, the embryos were fixed after heat-shock in 4% paraformaldehyde overnight, incubated with rat anti-HA antibody (1:200, 3F10, Roche #11867423001) followed by anti-rat Alexa Fluor 594 (1:500, Thermo Fisher #A11007). F-actin and nuclei were detected with Alexa Fluor 488-Phalloidin (1:400, Thermo Fisher #A12379) and DAPI (10 μL.mL− 1), respectively. Embryos were photographed with a Zeiss LSM 510 Meta.
Digoxigenin-labelled antisense RNA probes were synthesised from DNA templates of deltaC [32 (link)], her1 [33 (link)], her7 [34 (link)], mespb [35 (link)], pcdh8 [36 (link)], myod1 [37 (link)], cb1045 [38 (link)], spaw [39 (link)], pitx2 [40 (link)], myl7 [41 (link)], cxcl12b [25 (link)], etv2 [42 (link)], foxj1b [19 (link)], foxc1b [43 (link)], dmrt2a [4 (link)], cyp1a, which was cloned in pGEM-T Easy (Promega) using the pF-cyp1a (5’-ATGGCTCTGACTATTCTTCCAATATTGGG-3′) and pR-cyp1a (5’-CTAGAACCCAGGCTGTGGTGTGACCCGA-3′) and pxdc1b, which was cloned in pGEM-T Easy (Promega) using the pF-pxdc1b (5’-ATGGCATCGGCGATTTTTGAGGGCA-3′) and pR-pxdc1b (5’-AAGTCAGTTTCAAAAGGAACCAGA-3′).
For HA immunohistochemistry, the embryos were fixed after heat-shock in 4% paraformaldehyde overnight, incubated with rat anti-HA antibody (1:200, 3F10, Roche #11867423001) followed by anti-rat Alexa Fluor 594 (1:500, Thermo Fisher #A11007). F-actin and nuclei were detected with Alexa Fluor 488-Phalloidin (1:400, Thermo Fisher #A12379) and DAPI (10 μL.mL− 1), respectively. Embryos were photographed with a Zeiss LSM 510 Meta.
Free full text: Click here
