Digoxigenin 11 utp

Synthesis of capped RNA in vitro was accomplished with SP6 or T7 RNA polymerase (New England Biolabs, Ipswich, MA, USA), and m⁷G(5′)ppp(5′)G RNA cap analog (New England Biolabs) as described previously [9 (link)]. Synthetic RNA incorporating fluorescent or DIG labels were made using ribonucleotides mixed with ChromaTide^® Alexa Fluor^® 488-5-UTP- (Thermo Fisher Scientific) or Digoxigenin-11-UTP- (Sigma-Aldrich, Burlington, MA, USA) during in vitro transcription as per manufacturers’ protocol. The free labels were removed by Sephadex G-50 quick spin columns (Sigma-Aldrich).
In vitro translation was carried out using the Flexi^® Rabbit reticulocyte lysate and the nonradioactive FluoroTect™ Green_Lys in vitro translation labeling as per the manufacturer’s protocol (Promega, Madison, WI, USA). In brief, approximately 100–200 ng RNA transcripts were used in a 15 μL-reaction with indicated recombinant RuV proteins. The results were resolved on 10 or 12% SDS-polyacrylamide gels and visualized using a Typhoon 9400 Imager (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Data analysis was accomplished with ImageQuant software (GE Healthcare Life Sciences).

Embryos were fixed in 4% paraformaldehyde in PBS with 0.1% Tween-20 (PFA) at 4°C overnight and stored in 100% methanol at −20°C until required. All whole-mount in situ hybridization (WISH) was carried out according to standard protocols (Lauter et al. 2011 ). Antisense probes for hnrnpul1, hnrnpul1l, scxa, hand2, tbx5, foxd3, and sox10 were produced by in vitro transcription using T7 polymerase (Roche), in the presence of digoxigenin-11-UTP (Sigma), from PCR fragments amplified from embryonic zebrafish cDNA (Supplementary Table 4). Antisense probes for gli3 and col1a1a were a gift from Peng Huang and produced from plasmid clones. Images were taken on a Zeiss Stemi SV 11 microscope with a Zeiss Axiocam HRc camera. Area and length measurements were completed in ImageJ using the line and measure tools or Zen Blue (Zeiss) using the line tool. Confocal images were collected on a Zeiss LSM700 confocal microscope and assembled in ImageJ and Adobe Photoshop.

Following the general formatting of gene symbols, chicken genes will be abbreviated with upper‐case letters (e.g., TH1), while those in Xenopus and zebrafish will be abbreviated with lower‐case letters (e.g., th1). In order to refer to the homologous genes throughout the three species or throughout vertebrates, we will use upper‐case (e.g., TH1) in this article.
Chicken and Xenopus TH and TPH genes were cloned into pCRII Vector (Invitrogen/Thermo Fisher Scientific Inc.) or StrataClone (Agilent Technologies, Santa Clara, CA), after PCR amplification of the transcripts using specific primers (Table 2). Zebrafish th2 and tph1a had already been used in previous publications (Bellipanni, Rink, & Bally‐Cuif, 2002; Yamamoto et al., 2010, 2011). Antisense and sense RNA probes were synthesized by in vitro transcription using T3, T7, or Sp6 RNA polymerase (Promega, Madison, WI) and labeled with fluorescein‐12‐UTP or digoxigenin‐11‐UTP (Sigma‐Aldrich Co. LLC./Roche). Probes were purified using Nucleospin RNA clean‐UP kit (Macherey‐Nagel, Hoerdt, France) and analyzed by gel electrophoresis to confirm the size.