Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a high-production volume organophosphate flame retardant widely used within the United States. Within zebrafish, initiation of TDCIPP exposure at 0.75 h post-fertilization (hpf) results in genome-wide alterations in methylation during cleavage (2 hpf) as well as epiboly delay or arrest (at higher concentrations) during late-blastula and early-gastrula (4-6 hpf). To determine whether these TDCIPP-induced effects were associated with impacts on the transcriptome, embryos were exposed to vehicle (0.1% DMSO) or 2 μM TDCIPP from 0.75 hpf to 6 hpf, and total RNA was extracted from triplicate embryo pools per treatment and hybridized onto duplicate Affymetrix Zebrafish Gene 1.0 ST Arrays per RNA sample. Based on transcriptome-wide profiling, TDCIPP resulted in a significant impact on biological pathways involved in dorsoventral patterning and bone morphogenetic protein (BMP) signaling. Consistent with pathway-level responses, TDCIPP exposure also resulted in strongly dorsalized embryos by 24 hpf – a phenotype that mimicked the effects of dorsomorphin, a potent and selective BMP inhibitor. Moreover, the majority of dorsalized embryos were preceded by epiboly arrest at 6 hpf. Our microarray data also revealed that the expression of sizzled (szl) – a gene encoding a secreted Frizzled-related protein that limits BMP signaling – was significantly decreased by nearly 4-fold at 6 hpf. Therefore, we used a splice-blocking morpholino to test the hypothesis that knockdown of szl phenocopies TDCIPP-induced delays in epiboly progression. Interestingly, contrary to our hypothesis, injection of szl MOs did not affect epiboly progression but, similar to chordin (chd) morphants, resulted in mildly ventralized embryos by 24 hpf. Overall, our findings suggest that TDCIPP-induced epiboly delay may be independent of szl expression and function, and that TDCIPP-induced dorsalization may – similar to dorsomorphin – be due to interference with BMP signaling during early zebrafish.
Overall design: Zebrafish embryos were exposed to 0.1% DMSO or 2 µM TDCIPP from 0.75 hpf. Embryos (25 per replicate) were collected at either 2 or 6 hpf, transferred from beakers to 2-mL cryovials, snap-frozen in liquid nitrogen, and stored at -80ºC. These experiments resulted in three independent replicate samples for each time point and treatment group, resulting a total of 12 samples for RNA extractions. Total RNA was extracted from pooled embryos using a SV Total RNA Isolation System (Promega). After elution of RNA in 100 μL nuclease-free water, total RNA concentrations, 260/280 ratios, and 260/230 ratios were quantified using a NanoDrop ND-2000 spectrophotometer (ThermoFisher Scientific) and then stored at -80°C; total RNA quality was also confirmed using an Agilent 2100 Bioanalyzer system. Total RNA samples were amplified and biotinylated using GeneChip WT PLUS Reagent Kit (Affymetrix). Briefly, 100 ng of total RNA per sample was reverse-transcribed into ds-cDNA using NNN random primers containing a T7 RNA polymerase promoter sequence; cDNA quality was then confirmed using an Agilent 2100 Bioanalyzer system. T7 RNA polymerase was then added to cDNA samples to amplify RNA, and then RNA was reverse-transcribed to ss-DNA and degraded using RNase H. ss-DNA molecules were then fragmented and terminally labelled with biotin. Amplified and labeled samples were hybridized onto duplicate Zebrafish Gene 1.0 ST Arrays (Affymetrix) for 16 h at 45°C using a GeneChip Hybridization Oven 640 and a GeneChip Hybridization, Wash, and Stain Kit (Affymetrix); these arrays were constructed by Affymetrix based on the danRer6/Zv9 genome build and contained 1,255,682 probes representing 59,302 unique genes. Hybridized arrays were washed and stained using GeneChip Fluidics Stations 450 (Affymetrix). Arrays (24 total) were then scanned using a GeneChip Scanner 3000 7G system and computer workstation equipped with GeneChip Command Console 4.0 software (Affymetrix).
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