Defining the cellular complexity of the zebrafish bipotential gonad.

Zebrafish are routinely used to model reproductive development, function, and disease, yet we still lack a clear understanding of the fundamental steps that occur during early bipotential gonad development, including when endothelial cells, pericytes, and macrophage arrive at the bipotential gonad to support gonad growth and differentiation. Here, we use a combination of transgenic reporters and single-cell sequencing analyses to define the arrival of different critical cell types to the larval zebrafish gonad. We determined that blood initially reaches the gonad via a vessel formed from the swim bladder artery, which we have termed the gonadal artery. We find that vascular and lymphatic development occurs concurrently in the bipotential zebrafish gonad and our data suggest that similar to what has been observed in developing zebrafish embryos, lymphatic endothelial cells in the gonad may be derived from vascular endothelial cells. We mined preexisting sequencing datasets to determine whether ovarian pericytes had unique gene expression signatures. We identified 215 genes that were uniquely expressed in ovarian pericytes, but not expressed in larval pericytes. Similar to what has been shown in the mouse ovary, our data suggest that pdgfrb+ pericytes may support the migration of endothelial tip cells during ovarian angiogenesis. Using a macrophage-driven photoconvertible protein, we found that macrophage established a nascent resident population as early as 12 dpf and can be observed removing cellular material during gonadal differentiation. This foundational information demonstrates that the early bipotential gonad contains complex cellular interactions, which likely shape the health and function of the mature gonad.

Advancing zebrafish as a model for studying developmental neurotoxicology.

The cover photo shows the developing zebrafish nervous system at 5 days post-fertilization. Axon tracts are labeled with an anti-acetylated alpha tubulin antibody. The image, which was acquired on a Zeiss LSM 880 confocal microscope, is a maximum intensity projection of a z-stack that has been color-coded for depth. Major brain regions such as the olfactory bulb, forebrain, habenula, optic tectum, cerebellum, hindbrain, and eye are identifiable. This image is part of a study (Plavicki Lab, Brown University) focused on understanding the impact of toxicant exposures on brain development and activity with the goal of identifyingenvironmental factors that contribute to the etiology of neurodevelopmental disorders.

CNS angiogenesis and barriergenesis occur simultaneously.

The blood-brain barrier (BBB) plays a vital role in the central nervous system (CNS). A comprehensive understanding of BBB development has been hampered by difficulties in observing the differentiation of brain endothelial cells (BECs) in real-time. Here, we generated two transgenic zebrafish line, Tg(glut1b:mCherry) and Tg(plvap:EGFP), to serve as in vivo reporters of BBB development. We showed that barriergenesis (i.e. the induction of BEC differentiation) occurs immediately as endothelial tips cells migrate into the brain parenchyma. Using the Tg(glut1b:mCherry) transgenic line, we performed a genetic screen and identified a zebrafish mutant with a nonsense mutation in gpr124, a gene known to play a role in CNS angiogenesis and BBB development. We also showed that our transgenic plvap:EGFP line, a reporter of immature brain endothelium, is initially expressed in newly formed brain endothelial cells, but subsides during BBB maturation. Our results demonstrate the ability to visualize the in vivo differentiation of brain endothelial cells into the BBB phenotype and establish that CNS angiogenesis and barriergenesis occur simultaneously.

Expression of the Drosophila homeobox gene, Distal-less, supports an ancestral role in neural development.

BACKGROUND: Distal-less (Dll) encodes a homeodomain transcription factor expressed in developing appendages of organisms throughout metazoan phylogeny. Based on earlier observations in the limbless nematode Caenorhabditis elegans and the primitive chordate amphioxus, it was proposed that Dll had an ancestral function in nervous system development. Consistent with this hypothesis, Dll is necessary for the development of both peripheral and central components of the Drosophila olfactory system. Furthermore, vertebrate homologs of Dll, the Dlx genes, play critical roles in mammalian brain development. RESULTS: Using fluorescent immunohistochemistry of fixed samples and multiphoton microscopy of living Drosophila embryos, we show that Dll is expressed in the embryonic, larval and adult central nervous system and peripheral nervous system (PNS) in embryonic and larval neurons, brain and ventral nerve cord glia, as well as in PNS structures associated with chemosensation. In adult flies, Dll expression is expressed in the optic lobes, central brain regions and the antennal lobes. CONCLUSIONS: Characterization of Dll expression in the developing nervous system supports a role of Dll in neural development and function and establishes an important basis for determining the specific functional roles of Dll in Drosophila development and for comparative studies of Drosophila Dll functions with those of its vertebrate counterparts.

A co-culture assay of embryonic zebrafish hearts to assess migration of epicardial cells in vitro.

BACKGROUND: The vertebrate heart consists of three cell layers: the innermost endothelium, the contractile myocardium and the outermost epicardium. The epicardium is vital for heart development and function, and forms from epicardial progenitor cells (EPCs), which migrate to the myocardium during early development. Disruptions in EPC migration and epicardium formation result in a number of cardiac malformations, many of which resemble congenital heart diseases in humans. Hence, it is important to understand the mechanisms that influence EPC migration and spreading in the developing heart. In vitro approaches heretofore have been limited to monolayer epicardial cell cultures, which may not fully capture the complex interactions that can occur between epicardial and myocardial cells in vivo. RESULTS: Here we describe a novel in vitro co-culture assay for assessing epicardial cell migration using embryonic zebrafish hearts. We isolated donor hearts from embryonic zebrafish carrying an epicardial-specific fluorescent reporter after epicardial cells were present on the heart. These were co-cultured with recipient hearts expressing a myocardial-specific fluorescent reporter, isolated prior to EPC migration. Using this method, we can clearly visualize the movement of epicardial cells from the donor heart onto the myocardium of the recipient heart. We demonstrate the utility of this method by showing that epicardial cell migration is significantly delayed or absent when myocardial cells lack contractility and when myocardial cells are deficient in tbx5 expression. CONCLUSIONS: We present a method to assess the migration of epicardial cells in an in vitro assay, wherein the migration of epicardial cells from a donor heart onto the myocardium of a recipient heart in co-culture is monitored and scored. The donor and recipient hearts can be independently manipulated, using either genetic tools or pharmacological agents. This allows flexibility in experimental design for determining the role that target genes/signaling pathways in specific cell types may have on epicardial cell migration.

Homeobox gene distal-less is required for neuronal differentiation and neurite outgrowth in the Drosophila olfactory system.

Vertebrate Dlx genes have been implicated in the differentiation of multiple neuronal subtypes, including cortical GABAergic interneurons, and mutations in Dlx genes have been linked to clinical conditions such as epilepsy and autism. Here we show that the single Drosophila Dlx homolog, distal-less, is required both to specify chemosensory neurons and to regulate the morphologies of their axons and dendrites. We establish that distal-less is necessary for development of the mushroom body, a brain region that processes olfactory information. These are important examples of distal-less function in an invertebrate nervous system and demonstrate that the Drosophila larval olfactory system is a powerful model in which to understand distal-less functions during neurogenesis.

Multiple modes of proepicardial cell migration require heartbeat.

BACKGROUND: The outermost layer of the vertebrate heart, the epicardium, forms from a cluster of progenitor cells termed the proepicardium (PE). PE cells migrate onto the myocardium to give rise to the epicardium. Impaired epicardial development has been associated with defects in valve development, cardiomyocyte proliferation and alignment, cardiac conduction system maturation and adult heart regeneration. Zebrafish are an excellent model for studying cardiac development and regeneration; however, little is known about how the zebrafish epicardium forms. RESULTS: We report that PE migration occurs through multiple mechanisms and that the zebrafish epicardium is composed of a heterogeneous population of cells. Heterogeneity is first observed within the PE and persists through epicardium formation. Using in vivo imaging, histology and confocal microscopy, we show that PE cells migrate through a cellular bridge that forms between the pericardial mesothelium and the heart. We also observed the formation of PE aggregates on the pericardial surface, which were released into the pericardial cavity. It was previously reported that heartbeat-induced pericardiac fluid advections are necessary for PE cluster formation and subsequent epicardium development. We manipulated heartbeat genetically and pharmacologically and found that PE clusters clearly form in the absence of heartbeat. However, when heartbeat was inhibited the PE failed to migrate to the myocardium and the epicardium did not form. We isolated and cultured hearts with only a few epicardial progenitor cells and found a complete epicardial layer formed. However, pharmacologically inhibiting contraction in culture prevented epicardium formation. Furthermore, we isolated control and silent heart (sih) morpholino (MO) injected hearts prior to epicardium formation (60 hpf) and co-cultured these hearts with "donor" hearts that had an epicardium forming (108 hpf). Epicardial cells from donor hearts migrated on to control but not sih MO injected hearts. CONCLUSIONS: Epicardial cells stem from a heterogeneous population of progenitors, suggesting that the progenitors in the PE have distinct identities. PE cells attach to the heart via a cellular bridge and free-floating cell clusters. Pericardiac fluid advections are not necessary for the development of the PE cluster, however heartbeat is required for epicardium formation. Epicardium formation can occur in culture without normal hydrodynamic and hemodynamic forces, but not without contraction.

Metabolomic changes following GenX and PFBS exposure in developing zebrafish.

Short chain per- and polyfluoroalkyl substances (PFAS), including hexafluoropropylene oxide dimer acid (GenX) and perfluorobutane sulfonate (PFBS), are replacement chemicals for environmentally persistent, long-chain PFAS. Although GenX and PFBS have been detected in surface and ground water worldwide, few studies provide information on the metabolic alterations or risks associated with their exposures. In this study, larval zebrafish were used to investigate the toxicity of early-life exposure to GenX or PFBS. Zebrafish were chronically exposed from 4 h post-fertilization (hpf) to 6 days post-fertilization (dpf) to 150 µM GenX or 95.0 µM PFBS. Ultra-high-performance liquid chromatography paired with high-resolution mass spectrometry was used to quantify uptake of GenX and PFBS into zebrafish larvae and perform targeted and untargeted metabolomics. Our results indicate that PFBS was 20.4 % more readily absorbed into the zebrafish larvae compared to GenX. Additionally, PFBS exposure significantly altered 13 targeted metabolites and 21 metabolic pathways, while GenX exposure significantly altered 1 targeted metabolite and 17 metabolic pathways. Exposure to GenX, and to an even greater extent PFBS, resulted in a number of altered metabolic pathways in the amino acid metabolism, with other significant alterations in the carbohydrate, lipid, cofactors and vitamins, nucleotide, and xenobiotics metabolisms. Our results indicate that GenX and PFBS impact the zebrafish metabolome, with implications of global metabolic dysregulation, particularly in metabolic pathways relating to growth and development.

Loss of developmentally derived Irf8+ macrophages promotes hyperinnervation and arrhythmia in the adult zebrafish heart.

Recent developments in cardiac macrophage biology have broadened our understanding of the critical functions of macrophages in the heart. As a result, there is further interest in understanding the independent contributions of distinct subsets of macrophage to cardiac development and function. Here, we demonstrate that genetic loss of interferon regulatory factor 8 (Irf8)-positive embryonic-derived macrophages significantly disrupts cardiac conduction, chamber function, and innervation in adult zebrafish. At 4 months post-fertilization (mpf), homozygous irf8st96/st96 mutants have significantly shortened atrial action potential duration and significant differential expression of genes involved in cardiac contraction. Functional in vivo assessments via electro- and echocardiograms at 12 mpf reveal that irf8 mutants are arrhythmogenic and exhibit diastolic dysfunction and ventricular stiffening. To identify the molecular drivers of the functional disturbances in irf8 null zebrafish, we perform single cell RNA sequencing and immunohistochemistry, which reveal increased leukocyte infiltration, epicardial activation, mesenchymal gene expression, and fibrosis. Irf8 null hearts are also hyperinnervated and have aberrant axonal patterning, a phenotype not previously assessed in the context of cardiac macrophage loss. Gene ontology analysis supports a novel role for activated epicardial-derived cells (EPDCs) in promoting neurogenesis and neuronal remodeling in vivo. Together, these data uncover significant cardiac abnormalities following embryonic macrophage loss and expand our knowledge of critical macrophage functions in heart physiology and governing homeostatic heart health.

Sox9b is required for epicardium formation and plays a role in TCDD-induced heart malformation in zebrafish.

Activation of the transcription factor aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents the formation of the epicardium and leads to severe heart malformations in developing zebrafish (Danio rerio). The downstream genes that cause heart malformation are not known. Because TCDD causes craniofacial malformations in zebrafish by downregulating the sox9b gene, we hypothesized that cardiotoxicity might also result from sox9b downregulation. We found that sox9b is expressed in the developing zebrafish heart ventricle and that TCDD exposure markedly reduces this expression. Furthermore, we found that manipulation of sox9b expression could phenocopy many but not all of the effects of TCDD at the heart. Loss of sox9b prevented the formation of epicardium progenitors comprising the proepicardium on the pericardial wall, and prevented the formation and migration of the epicardial layer around the heart. Zebrafish lacking sox9b showed pericardial edema, an elongated heart, and reduced blood circulation. Fish lacking sox9b failed to form valve cushions and leaflets. Sox9b is one of two mammalian Sox9 homologs, sox9b and sox9a. Knock down of sox9a expression did not cause cardiac malformations, or defects in epicardium development. We conclude that the decrease in sox9b expression in the heart caused by TCDD plays a role in many of the observed signs of cardiotoxicity. We find that while sox9b is expressed in myocardial cells, it is not normally expressed in the affected epicardial cells or progenitors. We therefore speculate that sox9b is involved in signals between the cardiomyocytes and the nascent epicardial cells.

Defining the cellular complexity of the zebrafish bipotential gonad.

Zebrafish are routinely used to model reproductive development, function, and disease, yet we still lack a clear understanding of the fundamental steps that occur during early bipotential gonad development, including when endothelial cells, pericytes, and macrophage cells arrive at the bipotential gonad to support gonad growth and differentiation. Here, we use a combination of transgenic reporters and single-cell sequencing analyses to define the arrival of different critical cell types to the larval zebrafish gonad. We determined that blood initially reaches the gonad via a vessel formed from the swim bladder artery, which we have termed the gonadal artery. We find that vascular and lymphatic development occurs concurrently in the bipotential zebrafish gonad and our data suggest that similar to what has been observed in developing zebrafish embryos, lymphatic endothelial cells in the gonad may be derived from vascular endothelial cells. We mined preexisting sequencing data sets to determine whether ovarian pericytes had unique gene expression signatures. We identified 215 genes that were uniquely expressed in ovarian pericytes that were not expressed in larval pericytes. Similar to what has been shown in the mouse ovary, our data suggest that pdgfrb+ pericytes may support the migration of endothelial tip cells during ovarian angiogenesis. Using a macrophage-driven photoconvertible protein, we found that macrophage established a nascent resident population as early as 12 dpf and can be observed removing cellular material during gonadal differentiation. This foundational information demonstrates that the early bipotential gonad contains complex cellular interactions, which likely shape the health and function of the mature, differentiated gonad.

Exposure to the aryl hydrocarbon receptor agonist dioxin disrupts formation of the muscle, nerves, and vasculature in the developing jaw.

Human exposure to environmental pollutants can disrupt embryonic development and impact juvenile and adult health outcomes by adversely affecting cell and organ function. Notwithstanding, environmental contamination continues to increase due to industrial development, insufficient regulations, and the mobilization of pollutants as a result of extreme weather events. Dioxins are a class of structurally related persistent organic pollutants that are highly toxic, carcinogenic, and teratogenic. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent dioxin compound and has been shown to induce toxic effects in developing organisms by activating the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor targeted by multiple persistent organic pollutants. Contaminant-induced AHR activation results in malformations of the craniofacial cartilages and neurocranium; however, the mechanisms mediating these phenotypes are not well understood. In this study, we utilized the optically transparent zebrafish model to elucidate novel cellular targets and potential transcriptional targets underlying TCDD-induced craniofacial malformations. To this end, we exposed zebrafish embryos at 4 h post fertilization to TCDD and employed a mixed-methods approach utilizing immunohistochemistry staining, transgenic reporter lines, fixed and in vivo confocal imaging, and timelapse microscopy to determine the targets mediating TCDD-induced craniofacial phenotypes. Our data indicate that embryonic TCDD exposure reduced jaw and pharyngeal arch Sox10+ chondrocytes and Tcf21+ pharyngeal mesoderm progenitors. Exposure to TCDD correspondingly led to a reduction in collagen type II deposition in Sox10+ domains. Embryonic TCDD exposure impaired development of tissues derived from or guided by Tcf21+ progenitors, namely: nerves, muscle, and vasculature. Specifically, TCDD exposure disrupted development of the hyoid and mandibular arch muscles, decreased neural innervation of the jaw, resulted in compression of cranial nerves V and VII, and led to jaw vasculature malformations. Collectively, these findings reveal novel structural targets and potential transcriptional targets of TCDD-induced toxicity, showcasing how contaminant exposures lead to congenital craniofacial malformations.

Environmentally relevant uptake, elimination, and metabolic changes following early embryonic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish.

Dioxin and dioxin-like compounds are ubiquitous environmental contaminants that induce toxicity by binding to the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. The zebrafish model has been used to define the developmental toxicity observed following exposure to exogenous AHR ligands such as the potent agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD). While the model has successfully identified cellular targets of TCDD and molecular mechanisms mediating TCDD-induced phenotypes, fundamental information such as the body burden produced by standard exposure models is still unknown. We performed targeted gas chromatography (GC) high-resolution mass spectrometry (HRMS) in tandem with non-targeted liquid chromatography (LC) HRMS to quantify TCDD uptake, model the elimination dynamics of TCDD, and determine how TCDD exposure affects the zebrafish metabolome. We found that 50 ppt, 10 ppb, and 1 ppb waterborne exposures to TCDD during early embryogenesis produced environmentally relevant body burdens: 38 ± 4.34, 26.6 ± 1.2, and 8.53 ± 0.341 pg/embryo, respectively, at 24 hours post fertilization. TCDD exposure was associated with the dysregulation of metabolic pathways that are associated with the AHR signaling pathway as well as pathways shown to be affected in mammals following TCDD exposure. In addition, we discovered that TCDD exposure affected several metabolic pathways that are critical for brain development and function including glutamate metabolism, chondroitin sulfate biosynthesis, and tyrosine metabolism. Together, these data demonstrate that existing exposure methods produce environmentally relevant body burdens of TCDD in zebrafish and provide insight into the biochemical pathways impacted by toxicant-induced AHR activation.

Exposure to the persistent organic pollutant 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) disrupts development of the zebrafish inner ear.

Dioxins are a class of highly toxic and persistent environmental pollutants that have been shown through epidemiological and laboratory-based studies to act as developmental teratogens. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent dioxin congener, has a high affinity for the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. TCDD-induced AHR activation during development impairs nervous system, cardiac, and craniofacial development. Despite the robust phenotypes previously reported, the characterization of developmental malformations and our understanding of the molecular targets mediating TCDD-induced developmental toxicity remains limited. In zebrafish, TCDD-induced craniofacial malformations are produced, in part, by the downregulation of SRY-box transcription factor 9b ( sox9b ), a member of the SoxE gene family. sox9b , along with fellow SoxE gene family members sox9a and sox10 , have important functions in the development of the otic placode, the otic vesicle, and, ultimately, the inner ear. Given that sox9b in a known target of TCDD and that transcriptional interactions exist among SoxE genes, we asked whether TCDD exposure impaired the development of the zebrafish auditory system, specifically the otic vesicle, which gives rise to the sensory components of the inner ear. Using immunohistochemistry, in vivo confocal imaging, and time-lapse microscopy, we assessed the impact of TCDD exposure on zebrafish otic vesicle development. We found exposure resulted in structural deficits, including incomplete pillar fusion and altered pillar topography, leading to defective semicircular canal development. The observed structural deficits were accompanied by reduced collagen type II expression in the ear. Together, our findings reveal the otic vesicle as a novel target of TCDD-induced toxicity, suggest that the function of multiple SoxE genes may be affected by TCDD exposure, and provide insight into how environmental contaminants contribute to congenital malformations. Highlights: The zebrafish ear is necessary to detect changes in motion, sound, and gravity.Embryos exposed to TCDD lack structural components of the developing ear.TCDD exposure impairs formation of the fusion plate and alters pillar topography.The semicircular canals of the ear are required to detect changes in movement.Following TCDD exposure embryos fail to establish semicircular canals.

Exposure to the persistent organic pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) disrupts development of the zebrafish inner ear.

Dioxins are a class of highly toxic and persistent environmental pollutants that have been shown through epidemiological and laboratory-based studies to act as developmental teratogens. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent dioxin congener, has a high affinity for the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. TCDD-induced AHR activation during development impairs nervous system, cardiac, and craniofacial development. Despite the robust phenotypes previously reported, the characterization of developmental malformations and our understanding of the molecular targets mediating TCDD-induced developmental toxicity remains limited. In zebrafish, TCDD-induced craniofacial malformations are produced, in part, by the downregulation of SRY-box transcription factor 9b (sox9b), a member of the SoxE gene family. sox9b, along with fellow SoxE gene family members sox9a and sox10, have important functions in the development of the otic placode, the otic vesicle, and, ultimately, the inner ear. Given that sox9b is a known target of TCDD and that transcriptional interactions exist among SoxE genes, we asked whether TCDD exposure impaired the development of the zebrafish auditory system, specifically the otic vesicle, which gives rise to the sensory components of the inner ear. Using immunohistochemistry, in vivo confocal imaging, and time-lapse microscopy, we assessed the impact of TCDD exposure on zebrafish otic vesicle development. We found exposure resulted in structural deficits, including incomplete pillar fusion and altered pillar topography, leading to defective semicircular canal development. The observed structural deficits were accompanied by reduced collagen type II expression in the ear. Together, our findings reveal the otic vesicle as a novel target of TCDD-induced toxicity, suggest that the function of multiple SoxE genes may be affected by TCDD exposure, and provide insight into how environmental contaminants contribute to congenital malformations.

Evaluation of Neural Regulation and Microglial Responses to Brain Injury in Larval Zebrafish Exposed to Perfluorooctane Sulfonate.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are biopersistent, global pollutants. Although some in vitro and epidemiological studies have explored the neurotoxic potential of perfluorooctane sulfonate (PFOS), a prevalent PFAS congener, it is unknown how developmental PFOS exposure affects neuronal signaling, microglia development, and microglial-neuron communication. OBJECTIVES: We sought to determine the extent to which PFOS exposure disrupts brain health, neuronal activity, and microglia-neuron communication during development. In addition, although PFOS impairs humoral immunity, its impact on innate immune cells, including resident microglia, is unclear. As such, we investigated whether microglia are cellular targets of PFOS, and, if so, whether disrupted microglial development or function could contribute to or is influenced by PFOS-induced neural dysfunction. METHODS: Zebrafish were chronically exposed to either a control solution [0.1% dimethyl sulfoxide (DMSO)], 7µM PFOS, 14µM PFOS, 28µM PFOS, or 64µM perfluorooctanoic acid (PFOA). We used in vivo imaging and gene expression analysis to assess microglial populations in the developing brain and to determine shifts in the microglia state. We functionally challenged microglia state using a brain injury model and, to assess the neuronal signaling environment, performed functional neuroimaging experiments using the photoconvertible calcium indicator calcium-modulated photoactivatable ratiometric integrator (CaMPARI). These studies were paired with optogenetic manipulations of neurons and microglia, an untargeted metabolome-wide association study (MWAS), and behavioral assays. RESULTS: Developmental PFOS exposure resulted in a shift away from the homeostatic microglia state, as determined by functional and morphological differences in exposed larvae, as well as up-regulation of the microglia activation gene p2ry12. PFOS-induced effects on microglia state exacerbated microglia responses to brain injury in the absence of increased cell death or inflammation. PFOS exposure also heightened neural activity, and optogenetic silencing of neurons or microglia independently was sufficient to normalize microglial responses to injury. An untargeted MWAS of larval brains revealed PFOS-exposed larvae had neurochemical signatures of excitatory-inhibitory imbalance. Behaviorally, PFOS-exposed larvae also exhibited anxiety-like thigmotaxis. To test whether the neuronal and microglial phenotypes were specific to PFOS, we exposed embryos to PFOA, a known immunotoxic PFAS. PFOA did not alter thigmotaxis, neuronal activity, or microglial responses, further supporting a role for neuronal activity as a critical modifier of microglial function following PFOS exposure. DISCUSSION: Together, this study provides, to our knowledge, the first detailed account of the effects of PFOS exposure on neural cell types in the developing brain in vivo and adds neuronal hyperactivity as an important end point to assess when studying the impact of toxicant exposures on microglia function. https://doi.org/10.1289/EHP12861.

Assessing CaMPARI as new approach methodology for evaluating neurotoxicity.

Developmental exposure to environmental toxicants has been linked to the onset of neurological disorders and diseases. Despite substantial advances in the field of neurotoxicology, there remain significant knowledge gaps in our understanding of cellular targets and molecular mechanisms that mediate the neurotoxicological endpoints associated with exposure to both legacy contaminants and emerging contaminants of concern. Zebrafish are a powerful neurotoxicological model given their high degree sequence conservation with humans and the similarities they share with mammals in micro- and macro-level brain structures. Many zebrafish studies have effectively utilized behavioral assays to predict the neurotoxic potential of different compounds, but behavioral phenotypes are rarely able to predict the brain structures, cell types, or mechanisms affected by chemical exposures. Calcium-modulated photoactivatable ratiometric integrator (CaMPARI), a recently developed genetically-encoded calcium indicator, undergoes a permanent green to red switch in the presence of elevated intracellular Ca2+ concentrations and 405-nm light, which allows for a "snapshot" of brain activity in freely-swimming larvae. To determine whether behavioral results are predictive of patterns of neuronal activity, we assessed the effects of three common neurotoxicants, ethanol, 2,2',3,5',6-pentachlorobiphenyl (PCB 95), and monoethylhexyl phthalate (MEHP), on both brain activity and behavior by combining the behavioral light/dark assay with CaMPARI imaging. We demonstrate that brain activity profiles and behavioral phenotypes are not always concordant and, therefore, behavior alone is not sufficient to understand how toxicant exposure affects neural development and network dynamics. We conclude that pairing behavioral assays with functional neuroimaging tools such as CaMPARI provides a more comprehensive understanding of the neurotoxic endpoints of compounds while still offering a relatively high throughput approach to toxicity testing.

Proper modulation of AHR signaling is necessary for establishing neural connectivity and oligodendrocyte precursor cell development in the embryonic zebrafish brain.

2,3,7,8-tetrachlorodibenzo-[p]-dioxin (TCDD) is a persistent global pollutant that exhibits a high affinity for the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. Epidemiological studies have associated AHR agonist exposure with multiple human neuropathologies. Consistent with the human data, research studies using laboratory models have linked pollutant-induced AHR activation to disruptions in learning and memory as well as motor impairments. Our understanding of endogenous AHR functions in brain development is limited and, correspondingly, scientists are still determining which cell types and brain regions are sensitive to AHR modulation. To identify novel phenotypes resulting from pollutant-induced AHR activation and ahr2 loss of function, we utilized the optically transparent zebrafish model. Early embryonic TCDD exposure impaired embryonic brain morphogenesis, resulted in ventriculomegaly, and disrupted neural connectivity in the optic tectum, habenula, cerebellum, and olfactory bulb. Altered neural network formation was accompanied by reduced expression of synaptic vesicle 2. Loss of ahr2 function also impaired nascent network development, but did not affect gross brain or ventricular morphology. To determine whether neural AHR activation was sufficient to disrupt connectivity, we used the Gal4/UAS system to express a constitutively active AHR specifically in differentiated neurons and observed disruptions only in the cerebellum; thus, suggesting that the phenotypes resulting from global AHR activation likely involve multiple cell types. Consistent with this hypothesis, we found that TCDD exposure reduced the number of oligodendrocyte precursor cells and their derivatives. Together, our findings indicate that proper modulation of AHR signaling is necessary for the growth and maturation of the embryonic zebrafish brain.

2,3,7,8-Tetrachlorodibenzo-p-dioxin exposure disrupts development of the visceral and ocular vasculature.

The aryl hydrocarbon receptor (AHR) has endogenous functions in mammalian vascular development and is necessary for mediating the toxic effects of a number of environmental contaminants. Studies in mice have demonstrated that AHR is necessary for the formation of the renal, retinal, and hepatic vasculature. In fish, exposure to the prototypic AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces expression of the AHR biomarker cyp1a throughout the developing vasculature and produces vascular malformations in the head and heart. However, it is not known whether the vascular structures that are sensitive to loss of AHR function are also disrupted by aberrant AHR activation. Here, we report that TCDD-exposure in zebrafish disrupts development of 1) the subintestinal venous plexus (SIVP), which vascularizes the developing liver, kidney, gut, and pancreas, and 2) the superficial annular vessel (SAV), an essential component of the retinal vasculature. Furthermore, we determined that TCDD exposure increased the expression of bmp4, a key molecular mediator of SIVP morphogenesis. We hypothesize that the observed SIVP phenotypes contribute to one of the hallmarks of TCDD exposure in fish - the failure of the yolk sac to absorb. Together, our data describe novel TCDD-induced vascular phenotypes and provide molecular insight into critical factors producing the observed vascular malformations.

sox9b is required in cardiomyocytes for cardiac morphogenesis and function.

The high mobility group transcription factor SOX9 is expressed in stem cells, progenitor cells, and differentiated cell-types in developing and mature organs. Exposure to a variety of toxicants including dioxin, di(2-ethylhexyl) phthalate, 6:2 chlorinated polyfluorinated ether sulfonate, and chlorpyrifos results in the downregulation of tetrapod Sox9 and/or zebrafish sox9b. Disruption of Sox9/sox9b function through environmental exposures or genetic mutations produce a wide range of phenotypes and adversely affect organ development and health. We generated a dominant-negative sox9b (dnsox9b) to inhibit sox9b target gene expression and used the Gal4/UAS system to drive dnsox9b specifically in cardiomyocytes. Cardiomyocyte-specific inhibition of sox9b function resulted in a decrease in ventricular cardiomyocytes, an increase in atrial cardiomyocytes, hypoplastic endothelial cushions, and impaired epicardial development, ultimately culminating in heart failure. Cardiomyocyte-specific dnsox9b expression significantly reduced end diastolic volume, which corresponded with a decrease in stroke volume, ejection fraction, and cardiac output. Further analysis of isolated cardiac tissue by RT-qPCR revealed cardiomyocyte-specific inhibition of sox9b function significantly decreased the expression of the critical cardiac development genes nkx2.5, nkx2.7, and myl7, as well as c-fos, an immediate early gene necessary for cardiomyocyte progenitor differentiation. Together our studies indicate sox9b transcriptional regulation is necessary for cardiomyocyte development and function.