Tris(1,3-dichloro-2-propyl) phosphate disrupts dorsoventral patterning in zebrafish embryos

Abstract

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.

Ciglitazone—a human PPARγ agonist—disrupts dorsoventral patterning in zebrafish

“…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.…”

“…As an embryo progresses through development, strict regulation of these various factors is required for proper dorsoventral patterning. Indeed, this process is sensitive to environmental chemicals that disrupt signaling pathways regulating dorsoventral patterning, resulting in dorsalized or ventralized embryos (Dasgupta et al, 2017).…”

Cercosporamide inhibits bone morphogenetic protein receptor type I kinase activity in zebrafish

“…However, related BMP-inhibitors such as DMH-1 and LDN-193189, sharing the same pyrazolo[1,5-a]pyrimidine core as dorsomorphin, have less offtarget effects and are potently targeting type 1 BMP receptor kinases, predominately Alk2 (Hao et al, 2010). Finally, more recent phenotype-based zebrafish embryo screens led to the discovery of other classes of small molecule inhibitors acting in the BMP-pathway (Cheng et al, 2019;Dasgupta et al, 2017;Gebruers et al, 2013;Sanvitale et al, 2013).…”

Cercosporamide inhibits bone morphogenetic protein receptor type I kinase activity in zebrafish

“…However, related BMP inhibitors such as DMH-1 and LDN-193189, sharing the same pyrazolo[1,5-a]pyrimidine core as dorsomorphin, have fewer off-target effects and are potently targeting type I BMPR kinases, predominately Alk2 ( Hao et al, 2010 ). Finally, more recent phenotype-based zebrafish embryo screens led to the discovery of other classes of small-molecule inhibitors acting in the BMP pathway ( Cheng et al, 2019 ; Dasgupta et al, 2017 ; Gebruers et al, 2013 ; Sanvitale et al, 2013 ).…”