Computational protocol: The ancestral retinoic acid receptor was a low-affinity sensor triggering neuronal differentiation

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Protocol publication

[…] Sequences for in silico analyses of retinoid metabolism and signaling components were retrieved by reciprocal Basic Local Alignment Search Tool (BLAST) searches from the databases implemented at the National Center for Biotechnology Information. Species included were the cnidarian Nematostella vectensis, the mollusk Lottia gigantea, the annelid C. teleta, the amphioxus Branchiostoma floridae, the fish Danio rerio, and humans, that is, Homo sapiens. Possible orthologs from P. dumerilii were retrieved from transcriptome databases of the Arendt ( and Jékely () laboratories by reciprocal BLAST searches. Sequence alignments were initially computed using MUSCLE () and subsequently refined manually. Phylogenetic analyses were calculated with both the maximum likelihood (ML) and Bayesian inference (BI) methods. The ML trees were constructed with PhyML v3.0 using the automated substitution model selection mode (PhyML-SMS) (). Tree support for the ML analyses was assessed using an approximate likelihood-ratio test (aLRT) (). In addition, standard bootstrap support for the ML trees was calculated in 100 replicates using PhyML v2.4.5, as implemented in the MacGDE package, based on the Whelan and Goldman (WAG) substitution model and taking into account among-site rate heterogeneity with four γ-distributed categories. The BI phylogenies were calculated using MrBayes implemented in the TOPALi v2.5 package (). The BI analyses were based on the WAG substitution model taking into account the among-site rate heterogeneity with four γ-distributed categories. Posterior probability support values for the BI trees were calculated with two runs for 1,000,000 generations, with trees saved every 100 generations and with a 25% burn-in period. [...] Crystals of the PduRAR LBD–ATRA complex were obtained in 0.2 M sodium acetate, 0.1 M Hepes (pH 7.5), and 20% polyethylene glycol 3000 by the hanging drop crystallization method. Single crystals were tested, and native data were collected from one crystal cryoprotected with 30% glycerol on the ID29 beam line at the European Synchrotron Radiation Facility in Grenoble, France. Data were analyzed for protein contaminants using ContaMiner (). The crystal structure of the PduRAR LBD–ATRA complex was determined by molecular replacement using MoRDa (). MoRDa placed four molecules in the asymmetric unit, forming two canonical nuclear receptor LBD dimers. However, the produced molecular replacement model was too poor for rebuilding and refinement, and molecular replacement phases were thus used for model rebuilding with phenix_build (). The resulting model was refined using CCP4 ncsrefine (), and the obtained phases were subjected to the Buccaneer software. The mtz output file was subsequently analyzed by Parrot, using fourfold noncrystallographic symmetry. In parallel, several homology models with different ligands and helix H12 positions were built using SWISS-MODEL. These models were superimposed on the Buccaneer Protein Data Bank (PDB) file and adjusted manually to the molecule with the best electron density (model B), using only the coot_rigid_body_fit_zone to the maps of the Parrot phases. The adjusted model was then used as a template and copied into the positions of the three other molecules in the asymmetric unit (molecules A, C, and D). Each new model was manually adjusted using coot_rigid_body_fit_zone. The Refmac program was used to refine this model (all B factors were set initially to 70 Å2), using 20 steps of jelly body refinement. The Phenix software was used to obtain Translation/Libration/Screw (TLS) domains from this pdb file. These TLS domains were subsequently subjected to an analysis by the Lorestr program, and the resulting output was further refined in Refmac. The different input phases were tested by Buccaneer analyses on the final pdb, with the best results (as judged by R and Rfree improvements) having been obtained by the Lorestr phases. Parrot was run on the Buccaneer mtz, and the resulting Parrot phases and the model from the final Refmac refinement were used as bases for manual adjustments and automated refinements with Refmac and Phenix. [...] Gene expression patterns in P. dumerilii were assessed by whole-mount in situ hybridization, as previously described (). After in situ hybridization, specimens were stained with DAPI (Invitrogen by Life Technologies) and immunohistochemically labeled with an acetylated α-tubulin antibody (Sigma-Aldrich) to visualize larval morphology and nervous system architecture (). Developing P. dumerilii worms were subsequently imaged either as bright-field images with a Zeiss Axio Imager microscope and a Zeiss Axiocam MRc camera or as fluorescent images either by confocal reflection microscopy () with a Zeiss LD LCI Plan-Apochromat 25× 0.8 Oil/Glyc/Water DIC objective on a Zeiss LSM 780 NLO or by capturing the fluorescent spectra of the nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate precipitate () with an Olympus UPLSAPO 60× 1.3 silicon objective on a spinning disc confocal system Olympus IX83 (excitation, 633 nm; reflector, 785/762 nm) with an Andor iXon 888 Ultra camera. To illustrate the complete expression patterns, z sections of bright-field images were merged with the software Helicon Focus. We used Adobe Photoshop CS5.1 and Adobe Illustrator CS5 to create the final figures. [...] Developing P. dumerilii worms were treated with ATRA or 13cRA from 12 to 24 hpf, 34 to 40 hpf, 48 to 80 hpf, and 96 to 102 hpf. ATRA and 13cRA stock solutions (10 mM) were prepared in DMSO, aliquoted into amber tubes (Eppendorf), and stored at −80°C. The stock solution aliquots were defrosted and used only once. Drug treatments were performed in six-well plates in 6 ml of filtered (0.22 μm) natural seawater (NSW). NSW (3 ml) with 100 to 200 embryos was added to each well. The RA dilutions were prepared and mixed by vortexing in the remaining 3 ml of NSW before addition to the embryos. The final concentrations of the treatments were as follows: 1, 2, 4, 5, 8, and 10 μM. DMSO treatments were used as controls. After treatment, the six-well plate was covered with aluminum foil to avoid light exposure. Embryos were subsequently cultured at 18°C. At the end of the drug treatment, embryos were washed in NSW and fixed in 4% paraformaldehyde for further analysis. EdU labeling was performed before fixation and following the manufacturer’s instructions (Invitrogen). The swimming behavior of P. dumerilii prelarvae and larvae, treated with ATRA or 13cRA, was recorded, respectively, with a Nikon Digital Sight DS-Fi1 camera and a DMK 42BUC03 camera (25 frames per second; The Imaging Source) and subsequently tracked with the Fiji plug-ins TrackMate () and MTrackJ (). […]

Pipeline specifications

Software tools BLASTN, MUSCLE, PhyML, MrBayes, TOPALi, ContaMiner, CCP4, Buccaneer, Adobe Illustrator
Applications Miscellaneous, Phylogenetics, Protein structure analysis, Nucleotide sequence alignment
Organisms Platynereis dumerilii, Caenorhabditis elegans
Diseases Amino Acid Metabolism, Inborn Errors
Chemicals Tretinoin