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

Abstract

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor that regulates lipid/glucose homeostasis and adipocyte differentiation. While the role of PPARγ in adipogenesis and diabetes has been extensively studied, little is known about PPARγ function during early embryonic development. Within zebrafish, maternally-loaded pparγ transcripts are present within the first 6 h post-fertilization (hpf), and de novo transcription of zygotic pparγ commences at ~48 hpf. Since maternal pparγ transcripts are elevated during a critical window of cell fate specification, the objective of this study was to test the hypothesis that PPARγ regulates gastrulation and dorsoventral patterning during zebrafish embryogenesis. To accomplish this objective, we relied on (1) ciglitazone as a potent PPARγ agonist and (2) a splice-blocking, pparγ-specific morpholino to knockdown pparγ. We found that initiation of ciglitazone—a potent human PPARγ agonist—exposure by 4 hpf resulted in concentration-dependent effects on dorsoventral patterning in the absence of epiboly defects during gastrulation, leading to ventralized embryos by 24 hpf. Interestingly, ciglitazone-induced ventralization was reversed by co-exposure with dorsomorphin, a bone morphogenetic protein signaling inhibitor that induces strong dorsalization within zebrafish embryos. Moreover, mRNA-sequencing revealed that lipid- and cholesterol-related processes were affected by exposure to ciglitazone. However, pparγ knockdown did not block ciglitazone-induced ventralization, suggesting that PPARγ is not required for dorsoventral patterning nor involved in ciglitazone-induced toxicity within zebrafish embryos. Our findings point to a novel, PPARγ-independent mechanism of action and phenotype following ciglitazone exposure during early embryonic development.

Utilizing Zebrafish Embryos to Reveal Disruptions in Dorsoventral Patterning

“…In contrast, DMP‐treated embryos exhibited disrupted pSMAD 1/5/9 but normal mesodermal localization and somites (albeit elongated). Ciglitazone‐treated embryos did not demonstrate abnormal phenotypes or evidence of BMP signaling disruption prior to concentration‐dependent weak ventralization phenotypes at 24 hpf (Cheng et al., 2019). These data collectively suggest that the observation of DV patterning defects is not sufficient to confirm disruption of BMP signaling.…”

“…Based on prior studies from our group (Cheng, Dasgupta, Reddam, & Volz, 2019; Dasgupta et al., 2018; Dasgupta et al., 2017), we here first provide a detailed protocol for exposure and assessment of different strengths of dorsalization and ventralization phenotypes (represented as classes) that are elicited by a range of concentrations of DMP and 4′‐H, respectively, or by microinjection of a chd morpholino (Basic Protocol 1). The chemicals or morpholino can be used as positive controls for chemical testing or functional genetics, respectively.…”

Translational toxicology in zebrafish

“…approaches can be used to disprove dogma related to assumed or predicted modes of action. One recent example was the demonstration of the nonessentiality of peroxisome proliferator-activated receptor gamma (PPARg) for ciglitazone-dependent dorsoventral patterning defects in early zebrafish development [40]. Injection of an antisense oligonucleotide morpholino into single-cell stage zebrafish to transiently suppress the generation of PPARg protein revealed that defects in patterning elicited by ciglitazone exposure occurred via a PPARg>-independent mechanism [40].…”

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).…”