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1. Persistent and Dose-Dependent Neural and Metabolic Gene Expression Changes Induced by Transient Citalopram Exposure in Zebrafish Embryos

   https://doi.org/10.3390/ijms262311288  

   North, Ryan J; Cooper, Gwendolyn; Mears, Lucas; Bothner, Brian; Dlakić, Mensur; Merzdorf, Christa S (December 2025, International Journal of Molecular Sciences)

   Citalopram, a common selective serotonin reuptake inhibitor (SSRI), has been increasingly detected in aquatic environments due to ineffective removal and improper disposal. Although developmental exposure to SSRIs is linked to neurotoxicity, little is known about the persistence of gene expression alterations following limited exposure periods. Zebrafish embryos were exposed from 2 to 24 h post-fertilization (hpf) at concentrations of citalopram hydrobromide spanning surface water to therapeutic serum levels (0.03, 0.9, 50, and 250 μg/L), followed by removal of the citalopram and development until 48 hpf. Whole-embryo RNA sequencing at 48 hpf revealed a non-linear dose–response wherein the lowest dose resulted in the induction of the highest number of differentially expressed genes (DEGs). Gene set enrichment analyses (GSEA) and overrepresentation analyses (ORAs) showed that 0.03 μg/L citalopram caused upregulation of metabolic and developmental pathway genes, but suppressed synaptic membrane genes, whereas 0.9 μg/L resulted in strong downregulation of key neurotransmitter receptors. At 50 μg/L, genes linked to oxidative stress (glutathione metabolism and ferroptosis) were upregulated, and at 250 μg/L, stress and apoptotic processes were increased, while glutamate receptor genes were repressed. All four citalopram doses suggested synaptic and neurotransmitter alterations, implying that persistent neurodevelopmental impacts resulted from a limited early window of exposure. These data highlight that transient, low-level SSRI exposures shape long-term embryonic gene expression. 

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2. Persistent Transcriptome Alterations in Zebrafish Embryos After Discontinued Opioid Exposure

   https://doi.org/10.3390/ijms26104840  

   North, Ryan J; Cooper, Gwendolyn; Mears, Lucas; Bothner, Brian; Dlakić, Mensur; Merzdorf, Christa S (May 2025, International Journal of Molecular Sciences)

   Much attention has been paid to the public health crisis that has resulted from the opioid epidemic. Given the high number of opioid users that are of childbearing age, the impact of utero exposure is a serious concern. Unfortunately, there is little knowledge regarding the consequences of opioid exposure during early development. While neurobehavioral effects of opioid exposure are well-documented, effects of exposure on embryogenesis remain largely unexplored. To address this gap in knowledge, we investigated the effects of oxycodone and fentanyl exposure on gene expression in zebrafish (Danio rerio) embryos using whole embryo RNA sequencing. Embryos were exposed to environmentally relevant (oxycodone HCl 10.6 ng/L and fentanyl citrate 0.629 ng/L) and therapeutically relevant doses (oxycodone HCl 35.14 μg/L and fentanyl citrate 3.14 μg/L) from 2 to 24 h post-fertilization (hpf), followed by another 24 h of opioid-free development. mRNA profiling at 48 hpf revealed dose- and drug-specific gene expression changes. Lower doses of oxycodone and fentanyl both induced more differentially expressed transcripts (DETs) than higher doses, potentially indicative of opioid receptor desensitization occurring at higher concentrations. In total, 892 DETs (corresponding to 866 genes) were identified across all conditions suggesting continued differential gene expression well after cessation of opioid exposure. Gene ontology analysis revealed changes in gene expression relating to extracellular matrix (ECM) organization, cell adhesion, and visual and nervous system formation. Key pathways include those involved in axon guidance, synapse formation, and ECM biosynthesis/remodeling, all of which have potential implications on neural connectivity and sensory development. These findings demonstrate that very early developmental exposure to opioids induces persistent transcriptomic changes which may have lasting implications for vertebrate cellular functions. Overall, these data provide insights into the molecular mechanisms of opioid-induced alterations during development. 

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3. Persistent Metabolic Changes Are Induced by 24 h Low-Dose Lead (Pb) Exposure in Zebrafish Embryos

   https://doi.org/10.3390/ijms26031050  

   Cooper, Gwendolyn; North, Ryan; Hunt-Smith, Tyler; Larson, James; Rennie, Madison; Bailey, Marguerite L; Scarlata, Suzanne; Merzdorf, Christa S; Bothner, Brian (February 2025, International Journal of Molecular Sciences)

   Lead (Pb) is a heavy metal associated with a range of toxic effects. Relatively few studies attempt to understand the impact of lead on development from a mechanistic perspective. Danio rerio (zebrafish) embryos are a model organism for studying the developmental consequences of exposure to chemical agents. This study examined the metabolome of developing zebrafish embryos exposed to 5 ppb, 15 ppb, 150 ppb, and 1500 ppb Pb concentrations during the first 24 h post fertilization, followed by 24 h of unexposed development and harvest at 48 h. Untargeted metabolomics and multivariate analysis revealed that various Pb exposures differentially affected the embryonic metabolome. Pathway analyses showed the dysregulation of biopterin, purine, alanine, and aspartate metabolism. Inductively coupled plasma mass spectrometry demonstrated Pb accumulation in embryos. Additionally, decreases in oxidation–reduction ratios were observed in 5–150 ppb groups but not in the 1500 ppb exposure group. This finding, along with several metabolite abundances, suggests a hormetic effect of Pb concentrations on the developing zebrafish metabolome. Together, these data reveal persistent global changes in the embryonic metabolome, pin-point biomarkers for Pb exposure, unveil dose-dependent relationships, and reflect Pb-induced changes in cellular energy. This work highlights aberrant processes and persistent changes underlying low-dose heavy metal exposure during early development. 

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4. Developmental exposure to domoic acid targets reticulospinal neurons and leads to aberrant myelination in the spinal cord

   https://doi.org/10.1038/s41598-023-28166-2  

   Panlilio, Jennifer M.; Hammar, Katherine M.; Aluru, Neelakanteswar; Hahn, Mark E. (December 2023, Scientific Reports)

   Abstract Harmful algal blooms (HABs) produce neurotoxins that affect human health. Developmental exposure of zebrafish embryos to the HAB toxin domoic acid (DomA) causes myelin defects, loss of reticulospinal neurons, and behavioral deficits. However, it is unclear whether DomA primarily targets myelin sheaths, leading to the loss of reticulospinal neurons, or reticulospinal neurons, causing myelin defects. Here, we show that while exposure to DomA at 2 dpf did not reduce the number of oligodendrocyte precursors prior to myelination, it led to fewer myelinating oligodendrocytes that produced shorter myelin sheaths and aberrantly wrapped neuron cell bodies. DomA-exposed larvae lacked Mauthner neurons prior to the onset of myelination, suggesting that axonal loss is not secondary to myelin defects. The loss of the axonal targets may have led oligodendrocytes to inappropriately myelinate neuronal cell bodies. Consistent with this, GANT61, a GLI1/2 inhibitor that reduces oligodendrocyte number, caused a reduction in aberrantly myelinated neuron cell bodies in DomA-exposed fish. Together, these results suggest that DomA initially alters reticulospinal neurons and the loss of axons causes aberrant myelination of nearby cell bodies. The identification of initial targets and perturbed cellular processes provides a mechanistic understanding of how DomA alters neurodevelopment, leading to structural and behavioral phenotypes. 

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5. Diapause and Anoxia-Induced Quiescence Are Unique States in Embryos of the Annual Killifish, Austrofundulus limnaeus

   https://doi.org/10.3390/biom15040515  

   Clouser, Patrick R; Riggs, Claire L; Romney, Amie_L T; Podrabsky, Jason E (April 2025, Biomolecules)

   Diapause is a state of developmental and metabolic dormancy that precedes exposure to environmental stresses. Yet, diapausing embryos are typically stress-tolerant. Evidence suggests that diapausing embryos “prepare” for stress as part of a gene expression program as they enter dormancy. Here, we investigate if diapause II embryos of the annual killifish Austrofundulus limnaeus, which can survive for hundreds of days of anoxia, can mount a transcriptomic response to anoxic insult. Bulk RNAseq was used to characterize the transcriptomes of diapause II embryos exposed to normoxia, 4 h and 24 h anoxia, and 2 h and 24 h normoxic recovery from anoxia. Differential expression and gene ontology analyses were used to probe for pathways that may mitigate survival. Transcriptional factor analysis was used to predict potential mediators of this response. Diapausing embryos exhibited a robust transcriptomic response to anoxia and recovery that returns to near baseline conditions after 24 h. Anoxia induced an upregulation of genes involved in the integrated stress response, lipid metabolism, p38mapk kinase signaling, and apoptosis. Developmental and mitochondrial genes decreased. We conclude that diapause II embryos mount a robust transcriptomic stress response when faced with anoxic insult. This response is consistent with mediating expected challenges to cellular homeostasis in anoxia. 

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