Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521
Performing Department
Environmental Sciences
Non Technical Summary
Early vertebrate embryogenesis is regulated by dynamic processes that have the potential to be sensitive to chemical exposure. Immediately after fertilization, the single-celled zygote undergoes cleavage to form a multicellular blastula and, during gastrulation, germ layers (ectoderm, mesoderm, and endoderm) are formed by rapid cellular proliferation; each of these germ layers have unique cell fates that give rise to different tissues and organs within the developing embryo. Chemical exposures during early development may target blastula and/or gastrula, resulting in abnormal cell migration, germ layer formation, and dorsoventral patterning. In mammals, aberrant embryogenesis during the pre-implantation period can also lead to failed uterine implantation and premature abortion of the embryo (Boué et al. 1985). In model organisms such as zebrafish andXenopus, germ layer formation is preceded by epiboly - a process dependent on microtubule dynamics and activity of cell adhesion molecules (Babb and Marrs 2004; Lepage and Bruce 2010). Epiboly is sensitive to chemicals like perfluorooctanesulfonic acid, leading to formation of embryos with structural defects (Kane et al. 1996; McCollum et al. 2011).Disruption of the normal trajectory of embryogenesis can result from targeted effects of chemicals on a network of transcriptomic, biochemical, and epigenetic pathways that regulate development. For example, epidemiological studies have shown that human exposure to chemicals like persistent organic pollutants are associated with alterations in epigenetic processes that modulate the maternal-to-zygotic transition (MZT), leading to later-life consequences such as behavioral abnormalities and cancer (Marczylo et al. 2016).Similarly, certain chemicals may target gastrulation by interfering with development-specific signaling pathways, resulting in disruption of germ layer formation, organogenesis, and dorsoventral patterning (Lange et al. 2017).Zebrafish embryos offer one of the most promising cost-effective, physiologically-relevant vertebrate models to rapidly identify chemicals disrupting early developmental landmarks such as the MZT and germ layer formation.As fish embryos are non-protected life stages and are considered alternative testing models, zebrafish embryos that are 3 days post-fertilization (dpf) or younger fall within this definition and, as such, are not protected under current animal use regulations.As a result, over the last decademy lab has utilized non-protected life stages of zebrafish (<3 dpf) to develop and deploy high-content screening assays toprioritize chemicals for hypothesis-driven, mode-of-action research as well as uncover mechanisms of developmental toxicity for pesticides, understudied high-production volume chemicals, and field-derived environmental mixtures.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
The overall objective of this project is to continue leveraging non-protected life stages of zebrafish to identify chemicals that disrupt early embryonic development.For this project, we will focus on revealing the potential for certain per- and polyfluoroalkyl substances (PFASs) to disrupt the maternal-to-zygotic transition (MZT), epiboly, germ layer formation, and/or dorsoventral patterning. PFASs have been used in a wide range of consumer products for over half a century, leading to widespread contamination of air, soil, and water following PFAS release from end-use products and persistence in the environment (https://www.epa.gov/pfas).As a result of soil contamination, PFASs have also been detected in a variety of agricultural crops (Ghisi et al. 2019).However,a better understanding of the potential effects of prenatal PFAS exposure resulting from maternal ingestion of contaminatedagricultural productsis needed since these stages may be more susceptible than later periods of development. As such, this research is well-aligned with NIFA and the UC's AES, as it is consistent with the AES's missionto "discover and disseminate research-based knowledge to ensure an abundant and nutritious food supply, protection of natural resources, healthy people and communities, and economic and ecological sustainability for the future of California, the nation, and the world."For Specific Aim 1, we will screen the toxicity of a library of 47 different PFASs within the first 24 h of development - a developmental window that includes blastula, gastrula, and segmentation.ForSpecific Aim 2, we will conduct hypothesis-driven studies to investigate the mode and mechanism(s) of action for the most potent PFAS identified within Specific Aim 1.
Project Methods
For Specific Aim 1, we will first screen a library of 47 different PFASs to assess the potential toxicity within the first 24 h of development - a developmental window that includes blastula, gastrula, and segmentation.As part of an existing collaboration with Jinyong Liu within UCR's Dept. of CEE, we have already obtained a library of47 PFASs that includes 37 linear + 10 branched PFASs.384-well glass bottom microplates will be used for all embryo bioassays and imaging protocols. Using one embryo per well, 5-hpf embryos (8/treatment) will be placed within wells containing 50 μl of vehicle or 50 μM PFAS solution and then incubated until 24 hpf at 28°C under a 14 h:10 h light:dark cycle.Each 24-hpf embryo will be imaged on our ImageXpress Micro XLS Widefield High-Content Screening System (Molecular Devices) to quantify survival and developmental abnormalities.For PFASs that induce toxicity at a limit concentration (50 μM), we will rescreen these PFASs in concentration-response format (11 concentrations/PFAS).ForSpecific Aim 2, we will investigate the mode and mechanism(s) of action for the most potent PFAS that induces reproducible, concentration-dependent effects within Specific Aim 1.To identify a sensitive window,we will initiate exposure at different stages between 0.75-24 hpf andincubated until 24 hpf as described above.Similar to our prior work (Dasgupta et al. 2017; Dasgupta et al. 2018;Vliet et al. 2018),we will conduct mRNA-sequencing to determine whether PFAS exposure disrupts the MZT, and we will determine whether PFAS exposure adversely affects epiboly, bone morphogenetic protein signaling, germ layer formation, and dorsoventral patterning.Finally, as certain PFASs are potentperoxisome proliferator-activated receptor γ (PPARγ) agonists(DeWitt 2015), we will utilize our existingsplice-blocking, PPARγ-specific morpholino to determine whether PPARγ knockdown blocks PFAS-induced toxicity during the first 24 h of development.