Infinity5 camera

Fragments of chordin and Hox genes were isolated as previously described^{24 (link)} using gene-specific oligonucleotides and a T7 adaptor. Riboprobes were synthesized using a T7 MEGAscript kit (ThermoFisher, AM1334) and stored at a concentration of 50 ng µl^–1 in hybridization buffer at –20 °C. Whole-mount in situ hybridization in embryonic, larval and juvenile stages were conducted as described elsewhere^{24 (link),26 (link)}. Antibody staining in larval stages of O. fusiformis, Magelona spp. and C. teleta was carried out as previously described^{23 (link),115 (link)} using the following antibodies: mouse anti-acetyl-α-tubulin antibody, clone 6-11B-1, 1:800 dilution (Sigma-Aldrich, MABT868, RRID: AB_2819178) and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 647, 1:800 dilution (Thermo Fisher Scientific, A-21235, RRID: AB_2535804). Differential interface contrast images of the colorimetric in situ were obtained using a Leica 560 DMRA2 upright microscope equipped with an Infinity5 camera (Lumenera). Fluorescently stained samples were scanned using a Nikon CSU-W1 spinning disk confocal microscope.

Differential interface contrast (DIC) images were taken with a Leica CTRMIC compound scope couple with an Infinity5 camera (Lumenera). Confocal laser scanning microscopy (CLSM) images were taken with a Leica SP5. CLSM Z-stack projections were built with ImageJ2 [81 (link)]. To reduce some of the background noise at early stages, the DAPI and phalloidin channels were multiplied and square rooted. This product was subtracted from the original images, as previously described [17 (link)]. Autofluorescent dust and precipitates near the animals were digitally removed using Photoshop CC version 14.0 (Adobe Systems, Inc.). DIC images were digitally stacked with Helicon Focus 7 (HeliconSoft). Brightness and contrast were edited with Adobe Photoshop and figures built with Adobe Illustrator CC (Adobe Systems, Inc.).