Fluorescent Whole-Mount In Situ Hybridization

For fluorescent whole mount in situ hybridization (FISH), we followed the protocol outlined in Cole and Arnone, 2009 (link); Perillo et al., 2016 (link). Signal was developed with fluorophore-conjugated tyramide (Perkin Elmer, Cat. #:NEL752001KT; RRID:AB_2572409). Labeled probes were transcribed from linearized DNA using digoxigenin-11-UTP (Sigma Aldrich, Cat. #:11277073910), or labeled with DNP (Mirus Bio, Cat. #:MIR3825) following kit instructions. Probes were synthesized using primers in Supplementary file 3. 20 to 40 samples were imaged with a Zeiss 800 confocal microscope from the Brown University Leduc Bioimaging Core Facility (RRID:SCR_017861).