C dsd115 microscope

Embryos were staged by morphological criteria as described (Kimmel et al., 1995 (link)). Whole-mount in situ hybridizations were carried out as described (Thisse et al., 1993 (link)) and antisense probes generated for the following probes: cyp26a1 (Shelton et al., 2006 (link)), krox20 (Oxtoby and Jowett, 1993 (link)) and crestin, snai1b, dlx2a and mitfa (Stewart et al., 2006 (link)). The twist1a cDNA was generated by one-step RT-PCR and cloned into pGEM-T Easy plasmid (Promega) using primers: Forward 5′-GCAATCTGAGCTTTTCCAGAGG-3′, Reverse 5′-ATCCTTATTTTCGCCCTTG-3′. Anti-sense twist1a probe was generated using T7 polymerase after linearization with Spe1. The hoxb1a probe was generated with SP6 polymerase after linearization with EcoRV. Embryos were imaged using a Nikon C-DSD115 microscope with an Olympus DP72 camera. Identical settings were used to obtain in situ images within data sets. Brightness and contrast for final images were adjusted equally across data sets using Photoshop CS4.

Confocal images were acquired using an Olympus Fluoview FV1200 confocal microscope and Olympus FV10-ASW v4.1 software. Olympus UPlanSApo 60X/1.20W and Olympus UPlanSApo 10X/0.45 objectives were used in this study. For all confocal imaging, embryos were embedded on cover slips in 1% low melt agarose. For analysis of filopodia dynamics, z-stacks of the leading edge of NC streams 1–2 (cranial, n = 6) or of trunk NC cells between somites 6–8 (trunk, n = 6) in 26 hpf Tg(sox10:rfpmb) and Tg(sox10:rfpmb); fscn1a MZ embryos were acquired every 4 minutes for one hour using the 60X water objective (S1–S2 Movies). To monitor NC migration and individual cell behaviors, z-stacks were acquired every 25 minutes for 18 hours using the 10X objective (S3–S4 Movies). Widefield fluorescent images were acquired on an Olympus SZX16 microscope configured with an Olympus DP72 camera. Brightfield images were taken using a Nikon C-DSD115 microscope configured with an Olympus DP72 camera. Prism 6, ImageJ 1.46r, Adobe Photoshop CS5 and CS6, and Adobe Illustrator CS6 were used to generate figures.

Confocal images were acquired using an Olympus Fluoview FV1000XY, FV10i or FV1200 confocal microscopes and Olympus FV10-ASW v4.1 software. All imaging was performed using Olympus UPlanSApo 60X water and Olympus UPlanSApo 10X objectives. Embryos were embedded in 1% low melt agarose on cover slips for all confocal imaging. For analysis of filopodia dynamics, z-stacks of the leading edge of NC stream 3 in 26 hpf Tg(sox10:rfpmb) embryos injected with tp53MO or tp53MO plus fscn1aMO (n = 5 of each) were acquired every 2 minutes for 1 hour using the 60X water objective. For analysis of NC stream depth, z-stacks were acquired of NC stream 3 in 26 hpf Tg(sox10:rfpmb; sox10:h2a-gfp) embryos injected with tp53MO or tp53MO plus fscn1aMO. Cranial NC migration was imaged in 22, 25, 28 and 36 hpf Tg(sox10:GFP) embryos using a Zeiss Axiovert 200 inverted microscope configured with an Olympus DP72 camera. Widefield fluorescent images were acquired on an Olympus SZX16 microscope configured with an Olympus DP72 camera. Brightfield images were taken using a Nikon C-DSD115 microscope configured with an Olympus DP72 camera. Prism 6, ImageJ 1.46r, Adobe Photoshop CC 2014–2015, and Adobe Illustrator CC 2014–2015 were used to generate figures.