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, 5, e4156

Tris(1,3-dichloro-2-propyl) Phosphate Disrupts Dorsoventral Patterning in Zebrafish Embryos


Tris(1,3-dichloro-2-propyl) Phosphate Disrupts Dorsoventral Patterning in Zebrafish Embryos

Subham Dasgupta et al. PeerJ.


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 processes involved in dorsoventral patterning and bone morphogenetic protein (BMP) signaling. Consistent with these 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 not be driven by decreased szl expression, and that TDCIPP-induced dorsalization may-similar to dorsomorphin-be due to interference with BMP signaling during early zebrafish development.

Keywords: Dorsoventral patterning; Embryo; Flame retardant; Public health; TDCIPP; Zebrafish.

Conflict of interest statement

The authors declare there are no competing interests.


Figure 1
Figure 1. TDCIPP disrupts biological processes that regulate BMP signaling and dorsoventral patterning.
(A) Volcano plot showing transcriptome-wide responses following exposure to 2 µM TDCIPP from 0.75 to 6 hpf. x axis = log2 (fold-change); y axis = −log10 (p-value). Blue and red dots denote genes with a FDR p < 0.1 and p < 0.05, respectively. (B) Annotated transcripts with statistically significant fold changes (FDR p < 0.05) following exposure to 2 µM TDCIPP from 0.75 to 6 hpf. Data are presented as mean fold-change ± standard deviation relative to vehicle controls. N, three independent replicate samples per treatment group and two duplicate arrays per replicate sample.
Figure 2
Figure 2. TDCIPP-exposed embryos phenocopy embryos exposed to dorsomorphin from 0.75 to 24 hpf.
(A) Representative 24-hpf images of normal embryos, delayed embryos, Class I–V levels of dorsalization, and split yolk embryos following exposure to TDCIPP or DMP. (B) Distribution of dorsalization classes following exposure to TDCIPP from 0.75 to 24 hpf. (C) Distribution of dorsalization classes following exposure to DMP from 0.75 to 24 hpf. Depending on the TDCIPP concentration (1.56 or 3.12 µM), the severity of dorsalization varied from mild (Class I) to strong (Class V).
Figure 3
Figure 3. TDCIPP-induced dorsalization at 24 hpf is preceded by epiboly arrest at 6 hpf.
(A) Representative images of three epiboly phenotypes (normal, delayed, and arrested) at 6 hpf. Untreated embryos are normally at the germ ring or shield stage by 6 hpf. Epiboly-delayed embryos only progress to the dome and 30% epiboly stage by 6 hpf; epiboly-arrested embryos stall at the high stage by 6 hpf. (B) TDCIPP exposure results in a concentration-dependent increase in epiboly-arrested embryos, whereas DMP-treated embryos show no epiboly defects at 6 hpf. (C) Compared to <10% for normal and delayed embryos, 76% of embryos with epiboly arrest are dorsalized at 24 hpf.
Figure 4
Figure 4. szl and chd morphants do not phenocopy TDCIPP-exposed embryos at 6 and 24 hpf.
(A) Knockdown of szl or chd results in no effect on epiboly initiation or progression at 6 hpf. Grey bars represent normal embryos. (B) Similar to chd morphants (95% ventralized), 84% of szl morphants are ventralized at 24 hpf. Grey bars represent normal embryos whereas black bars represent ventralized embryos. (C) Representative images of normal vs. ventralized embryos at 24 hpf. Ventralized embryos are characterized by a decrease in dorsal structures (head, eye, etc.) and expansion of ventral structures.

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