Supplementary MaterialsSupplemental Information 1: Supplemental Numbers S1CS5. 24 hpf. Oddly enough, ciglitazone-induced ventralization was reversed by co-exposure with dorsomorphin, a bone tissue morphogenetic proteins signaling inhibitor that induces solid dorsalization within zebrafish embryos. Furthermore, mRNA-sequencing exposed that lipid- and cholesterol-related procedures were suffering from contact with ciglitazone. However, ppar knockdown did not block ciglitazone-induced ventralization, suggesting that PPAR is not required for dorsoventral patterning nor involved in ciglitazone-induced toxicity within zebrafish embryos. Our findings point to a novel, PPAR-independent mechanism of action and phenotype following ciglitazone exposure during early embryonic development. (NP_005027.2; NP_006229.1; NP_056953.2), (NP_035274.2; NP_035275.1; NP_035276.2), (NP_037328.1; NP_037273.2; NP_001138838.1), and (NP_001154805.1 (a); NP_001096037.1 (b); XP_699900.6 (a); NP_571543.1 (b); NP_571542.1) were obtained from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Sequences were aligned using the Multiple Sequence Alignment Tool within Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/), and the aligned file was used to generate a cladogram within Clustal Omega. Pairwise sequence alignments were also performed to obtain percent amino acid similarity using EMBOSS Matcher (https://www.ebi.ac.uk/Tools/psa/emboss_matcher/). The following default options were used for all pairwise alignments: Matrix = BLOSUM62; Gap Open = 1; Gap Extend = 4; and Alternatives = 1. Embryo exposures and phenotyping Embryos were sorted and exposed to either vehicle (0.2% DMSO) or ciglitazone (9.375, 12.5, 15, or 20 M) from 4 to 24 hpf in glass petri dishes (20 embryos per replicate; three replicates per treatment). Ciglitazone concentrations were selected based on the maximum tolerated concentration (based on survival as an endpoint) in zebrafish embryos following a 4C24 hpf exposure. At 24 hpf, embryos were imaged under transmitted light at 2 magnification using a Leica MZ10 F stereomicroscope equipped with a DMC2900 camera and assessed for survival and dorsoventral patterning abnormalities (ventralization, dorsalization, or delayed development). Following previously described protocols (Dasgupta et al., 2017), ventralized embryos were defined as embryos with a swollen yolk sac extension; dorsalized embryos were defined as embryos with a tail deformity; and delayed embryos were defined as embryos that phenocopied embryos at a developmental stage prior to 24 hpf. TC-DAPK6 Morpholino injections Morpholino antisense oligos were synthesized and obtained from Gene Tools, Inc. (Philomath, OR, USA). A fluorescein-tagged splice-blocking MO was designed to target the first exon-intron boundary (E1I1) of zebrafish ppar-specific pre-mRNA (NCBI Gene ID: 557037), leading to insertion of intron 1 within ppar mRNA (ppar-MO sequence: 5-TCAGCTCCTCTCTGACACTTACCAG-3). We did not rely on a ppar-specific translational MO due to the lack of a commercially available PPAR-specific antibody that cross reacts with zebrafish PPAR and, as such, inability to confirm knockdown of PPAR protein. Gene Tools standard fluorescein-tagged unfavorable control MO (nc-MO)a MO that targets a human TC-DAPK6 -globin intron mutationwas used in order to account for potential non-target MO toxicity, and a zebrafish-specific, fluorescein-tagged chordin MO (chd-MO sequence: 5-ATCCACAGCAGCCCCTCCATCATCC-3) was used as a positive control for disruption of dorsoventral patterning (ventralization) at 24 hpf. Water injections were performed in order to account for potential toxicity associated with injection-related stress. MO stock solutions (1 mM) were prepared by resuspending lyophilized MOs in molecular biology-grade (MBG) water, and stocks were stored at Rabbit polyclonal to ABCA5 room temperature in the dark. Working solutions of nc-MOs and ppar-MOs were diluted to 0.5 mM in MBG water and working solutions of chd-MOs were diluted to 0.125 mM in MBG water. Fertilized (1- to 8-cell stage Newly, or before 1.25 hpf) zebrafish embryos were microinjected with MOs (~three nL per embryo) utilizing a motorized Eppendorf Injectman NI2 and FemtoJet 4x much like previously described protocols (McGee et al., 2013; Dasgupta et al., TC-DAPK6 2017). At 3 hpf, MO delivery in embryos was verified utilizing a Leica MZ10 F stereomicroscope built with a DMC2900 camcorder along with a GFP filtration system cube; nonfluorescent.