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, 52 (18), 10820-10828

Tris(1,3-dichloro-2-propyl) Phosphate Exposure During the Early-Blastula Stage Alters the Normal Trajectory of Zebrafish Embryogenesis

Tris(1,3-dichloro-2-propyl) Phosphate Exposure During the Early-Blastula Stage Alters the Normal Trajectory of Zebrafish Embryogenesis

Subham Dasgupta et al. Environ Sci Technol.

Abstract

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is an organophosphate flame retardant used around the world. Within zebrafish, we previously showed that initiation of TDCIPP exposure during cleavage (0.75 h post-fertilization, hpf) results in epiboly disruption at 6 hpf, leading to dorsalized embryos by 24 hpf, a phenotype that mimics the effects of dorsomorphin (DMP), a bone morphogenetic protein (BMP) antagonist that dorsalizes embryos in the absence of epiboly defects. The objective of this study was to (1) investigate the role of BMP signaling in TDCIPP-induced toxicity during early embryogenesis, (2) identify other pathways and processes targeted by TDCIPP, and (3) characterize the downstream impacts of early developmental defects. Using zebrafish as a model, we first identified a sensitive window for TDCIPP-induced effects following exposure initiation at 0.75 hpf. We then investigated the effects of TDCIPP on the transcriptome during the first 24 h of development using mRNA sequencing and amplicon sequencing. Finally, we relied on whole-mount immunohistochemistry, dye-based labeling, and morphological assessments to study abnormalities later in embryonic development. Overall, our data suggest that the initiation of TDCIPP exposure during early blastula alters the normal trajectory of early embryogenesis by inducing gastrulation defects and aberrant germ-layer formation, leading to abnormal tissue and organ development within the embryo.

Figures

Figure 1.
Figure 1.
Sensitive window of exposure and effects of 4’H pre-treatment on TDCIPP-induced epiboly defects at 6 hpf. 2–3 hpf represents the most sensitive window for TDCIPP-induced effects on epiboly at 6 hpf (A). 4’H pre-treatment partially blocks TDCIPP-induced epiboly defects at 6 hpf (B). 100% of embryos were normal across all DMSO control replicates. Bars with dissimilar letters are significantly different (p<0.05).
Figure 2.
Figure 2.
Effect of TDCIPP on activation of the zygotic genome. Based on normalized read counts, TDCIPP exposures induce a slight (albeit non significant) ~1-hr delay in activation of zygotic genome at 3 hpf.
Figure 3.
Figure 3.
Impact of TDCIPP on BMP signaling. While exposure to 0.625 μM DMP decreased BMP specific transcripts between 4 and 12 hpf, these transcripts are not affected by exposure to 3.12 μM TDCIPP (A). The only exception was sizzled (szl), where mRNA levels were strongly decreased in both treatments (B). Immunostaining with anti phospho SMAD 1/5/9 also showed minimal effects of TDCIPP on BMP gradients (C); DMSO-and TDCIPP-treated embryos showed a normal BMP gradient from ventral to dorsal side at 8 hpf, whereas DMP treated embryos showed a disrupted gradient. Asterisk denotes significant difference relative to time matched vehicle controls (padj<0.05).
Figure 4.
Figure 4.
TDCIPP-induced effects on mesoderm development and differentiation. mRNA levels of mesoderm regulating genes (tbx6, tbx16, tbx6l and msgn1) were decreased in 3.12 μM TDCIPP treatments between 10 and 12 hpf (beginning of somitogenesis), whereas DMP did not affect these transcripts (A). Immunostaining with anti-VegT (tbx16) antibody at 12 hpf revealed normal patterning of paraxial mesodermal tissues and localization towards the tail bud at 12 hpf in DMSO-and DMP-treated embryos, but disrupted localization in 3.12 μM TDCIPP-treated embryos (B). Yellow arrow points to tail bud and white arrow points to notochord. Higher magnification of the embryonic structure revealed that, while TDCIPP-treated embryos at 12 hpf lacked a well-developed notochord and somites were absent, DMP-treated embryos possessed somites in the tail that are broader and more radially extended than somites within vehicle controls (red arrow) (C). Asterisk denotes significant difference relative to time matched vehicle controls (padj<0.05).
Figure 5.
Figure 5.
TDCIPP-induced effects on hemoglobin levels as well as cardiac and ocular development. TDCIPP-treatments induce a concentration-dependent decrease in hemoglobin levels at 72 hpf when treatments are initiated at 0.75 hpf (N=20 embryos per treatment). Butafenacil was used as a positive control for chemically induced anemia (A). TDCIPP exposure from 0.75–24 hpf results in a concentration dependent increase in pericardial area at 72 hpf (B) and concentration dependent decrease in ocular area (C) and ocular pigmentation (D) at 48 hpf. N=15 embryos per treatment in each case. Insets show occurrence of tube hearts (B) and lack of pigmentation (D) in representative embryos treated with 3.12 mM TDCIPP. In all cases, asterisk denotes significant difference relative to vehicle controls (p<0.05), and black line denotes the treatment mean.

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