Ryanodine receptor-active non-dioxin-like polychlorinated biphenyls cause neurobehavioral deficits in larval zebrafish.

Although their production was banned in the United States in 1977, polychlorinated biphenyls (PCBs) continue to pose significant risks to the developing nervous system. Perinatal exposure to PCBs is associated with increased risk of neuropsychiatric disorders, perhaps due to altered patterns of dendritic arborization of central neurons. Non-dioxin-like (NDL) PCB congeners enhance dendritic arborization of developing mammalian neurons via sensitization of ryanodine receptors (RYR). Structure-activity relationships (SAR) of RYR sensitization by PCBs have been demonstrated using mammalian and rainbow trout (Oncorhynchus mykiss) tissue homogenates. The purpose of this study is to determine whether this SAR translates to developmental neurotoxicity (DNT) of PCBs in vivo, a question that has yet to be tested. To address this gap, we leveraged a zebrafish model to evaluate the developmental neurotoxicity potential of PCBs 28, 66, 84, 95, 138, and 153, congeners previously shown to have broadly different potencies towards sensitizing RYR. We first confirmed that these PCB congeners exhibited differing potency in sensitizing RYR in zebrafish muscle ranging from negligible (PCB 66) to moderate (PCB 153) to high (PCB 95) RYR activity. Next, enzymatically dechorionated embryos were statically exposed to varying concentrations (0.1-10 µM) of each PCB congener from 6 h post-fertilization to 5 days post-fertilization (dpf). Embryos were observed daily using stereomicroscopy to assess mortality and gross malformations and photomotor behavior was assessed in larval zebrafish at 3, 4, and 5 dpf. The body burden of each PCB was measured by gas chromatography. The key findings are: 1) None of these PCBs caused death or overt teratology at the concentrations tested; 2) A subset of these PCB congeners altered photomotor behavior in larval zebrafish and the SAR for PCB behavioral effects mirrored the SAR for RYR sensitization; and 3) Quantification of PCB levels in larval zebrafish ruled out the possibility that congener-specific effects on behavior were due to differential uptake of PCB congeners. Collectively, the findings from this study provide in vivo evidence in support of the hypothesis that RYR sensitization contributes to the DNT of PCBs.

CRISPR-mediated transcriptional activation with synthetic guide RNA.

The CRISPR-Cas9 system has been adapted for transcriptional activation (CRISPRa) and several second-generation CRISPRa systems (including VPR, SunTag, and SAM) have been developed to recruit different transcriptional activators to a deactivated Cas9, which is guided to a transcriptional start site via base complementarity with a target guide RNA. Multiple studies have shown the benefit of CRISPRa using plasmid or lentiviral expressed guide RNA, but the use of synthetic guide RNA has not been reported. Here we demonstrate the effective use of synthetic guide RNA for gene activation via CRISPRa. CRISPRa crRNA may be used with a canonical tracrRNA using the VPR or SunTag activation systems or with an extended tracrRNA containing an aptamer sequence for the SAM system. Transcriptional activation with synthetic crRNA:tracrRNA is comparable to activation achieved with expression vectors and combining several crRNA sequences targeting the same gene can enhance transcriptional activation. The use of synthetic crRNA is also ideal for simultaneous activation of multiple genes or use with dCas9-VPR mRNA when viral transduction is not feasible. Here, we perform a proof-of-principle arrayed screen using a CRISPRa crRNA library consisting of 153 cytokine receptor targets to identify regulators of IL-6 cytokine secretion. Together, these results demonstrate the suitability of synthetic CRISPRa guide RNA for high throughput, arrayed screening applications which allow for more complex phenotypic readouts to complement viability and drug resistance assays typically used in a pooled screening format.

Developmental Toxicity Assessment of Piperonyl Butoxide Exposure Targeting Sonic Hedgehog Signaling and Forebrain and Face Morphogenesis in the Mouse: An in Vitro and in Vivo Study.

BACKGROUND: Piperonyl butoxide (PBO) is a pesticide synergist used in residential, commercial, and agricultural settings. PBO was recently found to inhibit Sonic hedgehog (Shh) signaling, a key developmental regulatory pathway. Disruption of Shh signaling is linked to birth defects, including holoprosencephaly (HPE), a malformation of the forebrain and face thought to result from complex gene-environment interactions. OBJECTIVES: The impact of PBO on Shh signaling in vitro and forebrain and face development in vivo was examined. METHODS: The influence of PBO on Shh pathway transduction was assayed in mouse and human cell lines. To examine its teratogenic potential, a single dose of PBO (22-1,800mg/kg) was administered by oral gavage to C57BL/6J mice at gestational day 7.75, targeting the critical period for HPE. Gene-environment interactions were investigated using Shh+/- mice, which model human HPE-associated genetic mutations. RESULTS: PBO attenuated Shh signaling in vitro through a mechanism similar to that of the known teratogen cyclopamine. In utero PBO exposure caused characteristic HPE facial dysmorphology including dose-dependent midface hypoplasia and hypotelorism, with a lowest observable effect level of 67mg/kg. Median forebrain deficiency characteristic of HPE was observed in severely affected animals, whereas all effective doses disrupted development of Shh-dependent transient forebrain structures that generate cortical interneurons. Normally silent heterozygous Shh null mutations exacerbated PBO teratogenicity at all doses tested, including 33mg/kg. DISCUSSION: These findings demonstrate that prenatal PBO exposure can cause overt forebrain and face malformations or neurodevelopmental disruptions with subtle or no craniofacial dysmorphology in mice. By targeting Shh signaling as a sensitive mechanism of action and examining gene-environment interactions, this study defined a lowest observable effect level for PBO developmental toxicity in mice more than 30-fold lower than previously recognized. Human exposure to PBO and its potential contribution to etiologically complex birth defects should be rigorously examined. https://doi.org/10.1289/EHP5260.

Changes in thyroid hormone activity disrupt photomotor behavior of larval zebrafish.

High throughput in vitro, in silico, and computational approaches have identified numerous environmental chemicals that interfere with thyroid hormone (TH) activity, and it is posited that human exposures to such chemicals are a contributing factor to neurodevelopmental disorders. However, whether hits in screens of TH activity are predictive of developmental neurotoxicity (DNT) has yet to be systematically addressed. The zebrafish has been proposed as a second tier model for assessing the in vivo DNT potential of TH active chemicals. As an initial evaluation of the feasibility of this proposal, we determined whether an endpoint often used to assess DNT in larval zebrafish, specifically photomotor behavior, is altered by experimentally induced hyper- and hypothyroidism. Developmental hyperthyroidism was simulated by static waterborne exposure of zebrafish to varying concentrations (3-300 nM) of thyroxine (T4) or triiodothyronine (T3) beginning at 6 h post-fertilization (hpf) and continuing through 5 days post-fertilization (dpf). Teratogenic effects and lethality were observed at 4 and 5 dpf in fish exposed to T4 or T3 at concentrations >30 nM. However, as early as 3 dpf, T4 (> 3 nM) and T3 (> 10 nM) significantly increased swimming activity triggered by sudden changes from light to dark, particularly during the second dark period (Dark 2). Conversely, developmental hypothyroidism, which was induced by treatment with 6-propyl-2-thiouracil (PTU), morpholino knockdown of the TH transporter mct8, or ablation of thyroid follicles in adult females prior to spawning, generally decreased swimming activity during dark periods, although effects did vary across test days. All effects of developmental hypothyroidism on photomotor behavior occurred independent of teratogenic effects and were most robust during Dark 2. Treatment with the T4 analog, Tetrac, restored photomotor response in mct8 morphants to control levels. Collectively, these findings suggest that while the sensitivity of photomotor behavior in larval zebrafish to detect TH disruption is influenced by test parameters, this test can distinguish between TH promoting and TH blocking activity and may be useful for assessing the DNT potential of TH-active chemicals.

Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (Danio rerio).

Thyroid hormones (THs) regulate neurodevelopment, thus TH disruption is widely posited as a mechanism of developmental neurotoxicity for diverse environmental chemicals. Zebrafish have been proposed as an alternative model for studying the role of TH in developmental neurotoxicity. To realize this goal, it is critical to characterize the normal ontogenetic expression profile of TH signaling molecules in the developing zebrafish and determine the sensitivity of these molecules to perturbations in TH levels. To address these gaps in the existing database, we characterized the transcriptional profiles of TH transporters, deiodinases (DIOs), receptors (TRs), nuclear coactivators (NCOAs), nuclear corepressors (NCORs), and retinoid X receptors (RXRs) in parallel with measurements of endogenous TH concentrations and tshß mRNA expression throughout the first five days of zebrafish development. Transcripts encoding these TH signaling components were identified and observed to be upregulated around 48-72 h post fertilization (hpf) concurrent with the onset of larval production of T4. Exposure to exogenous T4 and T3 upregulated mct8, dio3-b, trα-a, trß, and mbp-a levels, and downregulated expression of oatp1c1. Morpholino knockdown of TH transporter mct8 and treatment with 6-propyl-2-thiouracil (PTU) was used to reduce cellular uptake and production of TH, an effect that was associated with downregulation of dio3-b at 120 hpf. Collectively, these data confirm that larval zebrafish express orthologs of TH signaling molecules important in mammalian development and suggest that there may be species differences with respect to impacts of TH disruption on gene transcription.

PCB 95 promotes dendritic growth in primary rat hippocampal neurons via mTOR-dependent mechanisms.

Polychlorinated biphenyls (PCBs), and in particular non-dioxin-like (NDL) congeners, continue to pose a significant risk to the developing nervous system. PCB 95, a prevalent NDL congener in the human chemosphere, promotes dendritic growth in rodent primary neurons by activating calcium-dependent transcriptional mechanisms that normally function to link activity to dendritic growth. Activity-dependent dendritic growth is also mediated by calcium-dependent translational mechanisms involving mechanistic target of rapamycin (mTOR), suggesting that the dendrite-promoting activity of PCB 95 may also involve mTOR signaling. Here, we test this hypothesis using primary neuron-glia co-cultures derived from the hippocampi of postnatal day 0 Sprague Dawley rats. PCB 95 (1 nM) activated mTOR in hippocampal cultures as evidenced by increased phosphorylation of mTOR at ser2448. Pharmacologic inhibition of mTOR signaling using rapamycin (20 nM), FK506 (5 nM), or 4EGI-1 (1 µM), and siRNA knockdown of mTOR, or the mTOR complex binding proteins, raptor or rictor, blocked PCB 95-induced dendritic growth. These data identify mTOR activation as a novel molecular mechanism contributing to the effects of PCB 95 on dendritic arborization. In light of clinical data linking gain-of-function mutations in mTOR signaling to neurodevelopmental disorders, our findings suggest that mTOR signaling may represent a convergence point for gene by environment interactions that confer risk for adverse neurodevelopmental outcomes.

Bifenthrin causes transcriptomic alterations in mTOR and ryanodine receptor-dependent signaling and delayed hyperactivity in developing zebrafish (Danio rerio).

Over the last few decades, the pyrethroid insecticide bifenthrin has been increasingly employed for pest control in urban and agricultural areas, putting humans and wildlife at increased risk of exposure. Exposures to nanomolar (nM) concentrations of bifenthrin have recently been reported to alter calcium oscillations in rodent neurons. Neuronal calcium oscillations are influenced by ryanodine receptor (RyR) activity, which modulates calcium-dependent signaling cascades, including the mechanistic target of rapamycin (mTOR) signaling pathway. RyR activity and mTOR signaling play critical roles in regulating neurodevelopmental processes. However, whether environmentally relevant levels of bifenthrin alter RyR or mTOR signaling pathways to influence neurodevelopment has not been addressed. Therefore, our main objectives in this study were to examine the transcriptomic responses of genes involved in RyR and mTOR signaling pathways in zebrafish (Danio rerio) exposed to low (ng/L) concentrations of bifenthrin, and to assess the potential functional consequences by measuring locomotor responses to external stimuli. Wildtype zebrafish were exposed for 1, 3 and 5 days to 1, 10 and 50 ng/L bifenthrin, followed by a 14 d recovery period. Bifenthrin elicited significant concentration-dependent transcriptional responses in the majority of genes examined in both signaling cascades, and at all time points examined during the acute exposure period (1, 3, and 5 days post fertilization; dpf), and at the post recovery assessment time point (19 dpf). Changes in locomotor behavior were not evident during the acute exposure period, but were observed at 19 dpf, with main effects (increased locomotor behavior) detected in fish exposed developmentally to bifenthrin at 1 or 10 ng/L, but not 50 ng/L. These findings illustrate significant influences of developmental exposures to low (ng/L) concentrations of bifenthrin on neurodevelopmental processes in zebrafish.

Opportunities and challenges for using the zebrafish to study neuronal connectivity as an endpoint of developmental neurotoxicity.

Chemical exposures have been implicated as environmental risk factors that interact with genetic susceptibilities to influence individual risk for complex neurodevelopmental disorders, including autism spectrum disorder, schizophrenia, attention deficit hyperactivity disorder and intellectual disabilities. Altered patterns of neuronal connectivity represent a convergent mechanism of pathogenesis for these and other neurodevelopmental disorders, and growing evidence suggests that chemicals can interfere with specific signaling pathways that regulate the development of neuronal connections. There is, therefore, a growing interest in developing screening platforms to identify chemicals that alter neuronal connectivity. Cell-cell, cell-matrix interactions and systemic influences are known to be important in defining neuronal connectivity in the developing brain, thus, a systems-based model offers significant advantages over cell-based models for screening chemicals for effects on neuronal connectivity. The embryonic zebrafish represents a vertebrate model amenable to higher throughput chemical screening that has proven useful in characterizing conserved mechanisms of neurodevelopment. Moreover, the zebrafish is readily amenable to gene editing to integrate genetic susceptibilities. Although use of the zebrafish model in toxicity testing has increased in recent years, the diverse tools available for imaging structural differences in the developing zebrafish brain have not been widely applied to studies of the influence of gene by environment interactions on neuronal connectivity in the developing zebrafish brain. Here, we discuss tools available for imaging of neuronal connectivity in the developing zebrafish, review what has been published in this regard, and suggest a path forward for applying this information to developmental neurotoxicity testing.

Simultaneous quantification of T4, T3, rT3, 3,5-T2 and 3,3'-T2 in larval zebrafish (Danio rerio) as a model to study exposure to polychlorinated biphenyls.

Environmental toxicants that interfere with thyroid hormone (TH) signaling can impact growth and development in animals and humans. Zebrafish represent a model to study chemically induced TH disruption, prompting the need for sensitive detection of THs. Simultaneous quantification of 3,3',5-triiodo-l-thyronine (T3), thyroxine (T4), 3,3',5'-triiodo-l-thyronine (rT3), 3,5-diiodo-l-thyronine (3,5-T2) and 3,3'-diiodo-l-thyronine (3,3'-T2) in zebrafish larvae was achieved by ultra-performance liquid chromatography-tandem mass spectrometry in positive ion mode. Solid-phase extraction with SampliQ cartridges and derivatization with 3 m hydrochloric acid in n-butanol reduced matrix effects. Derivatized compounds were separated on an Acquity UPLC BEH C18 column with mobile phases consisting of 0.1% acetic acid in deionized water and 0.1% acetic acid in methanol. The limits of detection ranged from 0.5 to 0.6 pg injected on column. The method was validated by evaluating recovery (77.1-117.2%), accuracy (87.3-123.9%) and precision (0.5-12.4%) using diluted homogenized zebrafish embryos spiked with all target compounds. This method was then applied to zebrafish larvae collected after 114 h of exposure to polychlorinated biphenyls (PCBs), including PCB 28, PCB 66 and PCB 95, or the technical mixture Aroclor 1254. Exposure to PCB 28 and PCB 95 increased the T4:T3 ratio and decreased the T3:rT3 ratio, demonstrating that this method can effectively detect PCB-induced alterations in THs.

Coordinated d-cyclin/Foxd1 activation drives mitogenic activity of the Sonic Hedgehog signaling pathway.

Sonic Hedgehog (Shh) signaling plays key regulatory roles in embryonic development and postnatal homeostasis and repair. Modulation of the Shh pathway is known to cause malformations and malignancies associated with dysregulated tissue growth. However, our understanding of the molecular mechanisms by which Shh regulates cellular proliferation is incomplete. Here, using mouse embryonic fibroblasts, we demonstrate that the Forkhead box gene Foxd1 is transcriptionally regulated by canonical Shh signaling and required for downstream proliferative activity. We show that Foxd1 deletion abrogates the proliferative response to SHH ligand while FOXD1 overexpression alone is sufficient to induce cellular proliferation. The proliferative response to both SHH ligand and FOXD1 overexpression was blocked by pharmacologic inhibition of cyclin-dependent kinase signaling. Time-course experiments revealed that Shh pathway activation of Foxd1 is followed by downregulation of Cdkn1c, which encodes a cyclin-dependent kinase inhibitor. Consistent with a direct transcriptional regulation mechanism, we found that FOXD1 reduces reporter activity of a Fox enhancer sequence in the second intron of Cdkn1c. Supporting the applicability of these findings to specific biological contexts, we show that Shh regulation of Foxd1 and Cdkn1c is recapitulated in cranial neural crest cells and provide evidence that this mechanism is operational during upper lip morphogenesis. These results reveal a novel Shh-Foxd1-Cdkn1c regulatory circuit that drives the mitogenic action of Shh signaling and may have broad implications in development and disease.

Transcriptomic profiling of mTOR and ryanodine receptor signaling molecules in developing zebrafish in the absence and presence of PCB 95.

The mechanistic target of rapamycin (mTOR) and ryanodine receptor (RyR) signaling pathways regulate fundamental processes of neurodevelopment, and genetic mutations within these pathways have been linked to neurodevelopmental disorders. While previous studies have established that these signaling molecules are expressed in developing zebrafish, a detailed characterization of the ontogenetic profile of these signaling molecules is lacking. Thus, we evaluated the spatiotemporal expression of key transcripts in mTOR and RyR signaling pathways in wildtype zebrafish at 24, 72 and 120 hours post fertilization (hpf). We further determined whether transcriptional profiles of a subset of genes in both pathways were altered by exposure to PCB 95 (2,2',3,5',6-pentachlorobiphenyl), a pervasive environmental contaminant known to cause developmental neurotoxicity in mammalian systems via RyR-dependent mechanisms. Quantitative PCR revealed that transcription generally increased across development. Genes in the signaling pathway upstream of the mTORC1 complex, and the RyR-paralogs, ryr2a and ryr3, were robustly upregulated, and in situ hybridization of ryr3 coincided with a transcriptional shift from muscle to neuronal tissue after 24 hpf. Static waterborne exposure to PCB 95 beginning at 6 hpf significantly altered transcription of genes in both pathways. These changes were concentration- and time-dependent, and included downregulation of rptor, a member of the mTORC1 complex, at both 72 and 120 hpf, and increased transcript levels of the RyR paralog ryr2b and downstream target of RyR signaling, Wingless-type 2ba (wnt2ba) at 72 hpf. The detailed transcriptomic profiling of key genes within these two signaling pathways provides a baseline for identifying other environmental factors that modify normal spatiotemporal expression patterns of mTOR and RyR signaling pathways in the developing zebrafish, as illustrated here for PCB 95.

Gli2 gene-environment interactions contribute to the etiological complexity of holoprosencephaly: evidence from a mouse model.

Holoprosencephaly (HPE) is a common and severe human developmental abnormality marked by malformations of the forebrain and face. Although several genetic mutations have been linked to HPE, phenotypic outcomes range dramatically, and most cases cannot be attributed to a specific cause. Gene-environment interaction has been invoked as a premise to explain the etiological complexity of HPE, but identification of interacting factors has been extremely limited. Here, we demonstrate that mutations in Gli2, which encodes a Hedgehog pathway transcription factor, can cause or predispose to HPE depending upon gene dosage. On the C57BL/6J background, homozygous GLI2 loss of function results in the characteristic brain and facial features seen in severe human HPE, including midfacial hypoplasia, hypotelorism and medial forebrain deficiency with loss of ventral neurospecification. Although normally indistinguishable from wild-type littermates, we demonstrate that mice with single-allele Gli2 mutations exhibit increased penetrance and severity of HPE in response to low-dose teratogen exposure. This genetic predisposition is associated with a Gli2 dosage-dependent attenuation of Hedgehog ligand responsiveness at the cellular level. In addition to revealing a causative role for GLI2 in HPE genesis, these studies demonstrate a mechanism by which normally silent genetic and environmental factors can interact to produce severe outcomes. Taken together, these findings provide a framework for the understanding of the extreme phenotypic variability observed in humans carrying GLI2 mutations and a paradigm for reducing the incidence of this morbid birth defect.

A Simple and Reliable Method for Early Pregnancy Detection in Inbred Mice.

The study of normal and abnormal development typically requires precise embryonic staging. In mice, this task is accomplished through timed matings and the detection of a copulation plug. However, the presence of a plug is not a definitive indicator of true pregnancy, particularly in inbred mice, in which false-pregnancy rates have been reported to be 50% or higher, depending on the strain. This high rate poses considerable financial and animal use burdens because manipulation of the putative dam is often required before pregnancy can be confirmed by palpation or visual inspection. To address this problem, we examined weight gain in a population of 275 wildtype C57BL/6J mice (age, 12 wk or older) between the time of plug detection and during early embryogenesis (gestational days 7 to 10). In this population, assessing pregnancy according to the presence of a plug alone yielded a 37.1% false-positive rate. Pregnant mice gained an average of 3.49 g, whereas non-pregnant mice gained only 1.15 g. Beginning at gestational day 7.75, implementing an optimal weight-gain discrimination threshold of 1.75 g reduced the false-positive rate to 10.5%, without excluding any pregnant mice. These results were consistent with those from younger (age, 8 wk) wildtype C57BL/6J and FVB/NTac female mice, suggesting broad applicability of this method across age and strain. Our findings provide a simple and effective method for reducing animal use and study costs.

Definition of critical periods for Hedgehog pathway antagonist-induced holoprosencephaly, cleft lip, and cleft palate.

The Hedgehog (Hh) signaling pathway mediates multiple spatiotemporally-specific aspects of brain and face development. Genetic and chemical disruptions of the pathway are known to result in an array of structural malformations, including holoprosencephaly (HPE), clefts of the lip with or without cleft palate (CL/P), and clefts of the secondary palate only (CPO). Here, we examined patterns of dysmorphology caused by acute, stage-specific Hh signaling inhibition. Timed-pregnant wildtype C57BL/6J mice were administered a single dose of the potent pathway antagonist vismodegib at discrete time points between gestational day (GD) 7.0 and 10.0, an interval approximately corresponding to the 15th to 24th days of human gestation. The resultant pattern of facial and brain dysmorphology was dependent upon stage of exposure. Insult between GD7.0 and GD8.25 resulted in HPE, with peak incidence following exposure at GD7.5. Unilateral clefts of the lip extending into the primary palate were also observed, with peak incidence following exposure at GD8.875. Insult between GD9.0 and GD10.0 resulted in CPO and forelimb abnormalities. We have previously demonstrated that Hh antagonist-induced cleft lip results from deficiency of the medial nasal process and show here that CPO is associated with reduced growth of the maxillary-derived palatal shelves. By defining the critical periods for the induction of HPE, CL/P, and CPO with fine temporal resolution, these results provide a mechanism by which Hh pathway disruption can result in "non-syndromic" orofacial clefting, or HPE with or without co-occurring clefts. This study also establishes a novel and tractable mouse model of human craniofacial malformations using a single dose of a commercially available and pathway-specific drug.

Serotonin regulates calcium homeostasis in lactation by epigenetic activation of hedgehog signaling.

Calcium homeostasis during lactation is critical for maternal and neonatal health. We previously showed that nonneuronal/peripheral serotonin [5-hydroxytryptamine (5-HT)] causes the lactating mammary gland to synthesize and secrete PTHrP in an acute fashion. Here, using a mouse model, we found that genetic inactivation of tryptophan hydroxylase 1 (Tph1), which catalyzes the rate-limiting step in peripheral 5-HT synthesis, reduced circulating and mammary PTHrP expression, osteoclast activity, and maternal circulating calcium concentrations during the transition from pregnancy to lactation. Tph1 inactivation also reduced sonic hedgehog signaling in the mammary gland during lactation. Each of these deficiencies was rescued by daily injections of 5-hydroxy-L-tryptophan (an immediate precursor of 5-HT) to Tph1-deficient dams. We used immortalized mouse embryonic fibroblasts to demonstrate that 5-HT induces PTHrP through a sonic hedgehog-dependent signal transduction mechanism. We also found that 5-HT altered DNA methylation of the Shh gene locus, leading to transcriptional initiation at an alternate start site and formation of a variant transcript in mouse embryonic fibroblasts in vitro and in mammary tissue in vivo. These results support a new paradigm of 5-HT-mediated Shh regulation involving DNA methylation remodeling and promoter switching. In addition to having immediate implications for lactation biology, identification and characterization of a novel functional regulatory relationship between nonneuronal 5-HT, hedgehog signaling, and PTHrP offers new avenues for the study of these important factors in development and disease.

The influences of parental diet and vitamin E intake on the embryonic zebrafish transcriptome.

The composition of the typical commercial diet fed to zebrafish can dramatically vary. By utilizing defined diets we sought to answer two questions: 1) How does the embryonic zebrafish transcriptome change when the parental adults are fed a commercial lab diet compared with a sufficient, defined diet (E+)? 2) Does a vitamin E-deficient parental diet (E-) further change the embryonic transcriptome? We conducted a global gene expression study using embryos from zebrafish fed a commercial (Lab), an E+ or an E- diet. To capture differentially expressed transcripts prior to onset of overt malformations observed in E- embryos at 48h post-fertilization (hpf), embryos were collected from each group at 36hpf. Lab embryos differentially expressed (p<0.01) 946 transcripts compared with the E+ embryos, and 2656 transcripts compared with the E- embryos. The differences in protein, fat and micronutrient intakes in zebrafish fed the Lab compared with the E+ diet demonstrate that despite overt morphologic consistency, significant differences in gene expression occurred. Moreover, functional analysis of the significant transcripts in the E- embryos suggested perturbed energy metabolism, leading to overt malformations and mortality. Thus, these findings demonstrate that parental zebrafish diet has a direct impact on the embryonic transcriptome.

Effect of nitric oxide on anterior segment physiology in monkeys.

PURPOSE: To determine the effect of the nitric oxide donor, sodium nitroprusside (SNP), and the nitric oxide synthase (NOS) inhibitor, L-nitro-arginine-methylester (L-NAME), on IOP, mean arterial pressure (MAP), pupil diameter (PD), refraction (Rfx), aqueous humor formation (AHF), and outflow facility (OF) in monkeys. METHODS: Monkeys were treated with single or multiple topical treatments of 500 µg SNP or L-NAME to one eye. IOP was determined by Goldmann applanation tonometry, PD with vernier calipers in room light, Rfx by Hartinger coincidence refractometry, AHF by fluorophotometry, and MAP with a blood pressure monitor. OF was determined by two-level constant pressure perfusion following anterior chamber exchange. RESULTS: Following four topical treatments with 500 µg SNP, 30 minutes apart, IOP was significantly decreased from 2 to 6 hours compared with the contralateral control with the maximum IOP reduction of 20% at 3 hours (P < 0.001). PD, Rfx, and AHF were unchanged. Effects on MAP were variable. OF after SNP exchange was significantly increased by 77% (P < 0.05) at 10(-3) M. Topical L-NAME had no effect on IOP, PD, Rfx, or MAP. CONCLUSIONS: Enhancement of nitric oxide concentration at targeted tissues in the anterior segment may be a useful approach for IOP reduction for glaucoma therapy. Additional studies are warranted before conclusions can be made regarding the effect of NOS inhibition on ocular physiology in nonhuman primates.

Chronic vitamin E deficiency promotes vitamin C deficiency in zebrafish leading to degenerative myopathy and impaired swimming behavior.

We hypothesized that zebrafish (Danio rerio) undergoing long-term vitamin E deficiency with marginal vitamin C status would develop myopathy resulting in impaired swimming. Zebrafish were fed for 1 y a defined diet without (E-) and with (E+) vitamin E (500 mg α-tocopherol/kg diet). For the last 150 days, dietary ascorbic acid concentrations were decreased from 3500 to 50 mg/kg diet and the fish sampled periodically to assess ascorbic acid concentrations. The ascorbic acid depletion curves were faster in the E- compared with E+ fish (P < 0.0001); the estimated half-life of depletion in the E- fish was 34 days, while in it was 55 days in the E+ fish. To assess swimming behavior, zebrafish were monitored individually following a "startle-response" stimulus, using computer and video technology. Muscle histopathology was assessed using hematoxylin and eosin staining on paramedian sections of fixed zebrafish. At study end, E- fish contained 300-fold less α-tocopherol (p < 0.0001), half the ascorbic acid (p = 0.0001) and 3-fold more malondialdehyde (p = 0.0005) than did E+ fish. During the first minute following a tap stimulus (p < 0.05), E+ fish swam twice as far as did E- fish. In the E- fish, the sluggish behavior was associated with a multifocal, polyphasic, degenerative myopathy of the skeletal muscle. The myopathy severity ranged from scattered acute necrosis to widespread fibrosis and was accompanied by increased anti-hydroxynonenal staining. Thus, vitamin E deficiency in zebrafish causes increased oxidative stress and a secondary depletion of ascorbic acid, resulting in severe damage to muscle tissue and impaired muscle function.

Novel liquid chromatography-mass spectrometry method shows that vitamin E deficiency depletes arachidonic and docosahexaenoic acids in zebrafish (Danio rerio) embryos.

To test the hypothesis that embryogenesis depends upon α-tocopherol (E) to protect embryo polyunsaturated fatty acids (PUFAs) from lipid peroxidation, new methodologies were applied to measure α-tocopherol and fatty acids in extracts from saponified zebrafish embryos. A solid phase extraction method was developed to separate the analyte classes, using a mixed mode cartridge (reverse phase, π-π bonding, strong anion exchange), then α-tocopherol and cholesterol were measured using standard techniques, while the fatty acids were quantitated using a novel, reverse phase liquid chromatography-mass spectrometry (LC-MS) approach. We also determined if α-tocopherol status alters embryonic lipid peroxidation products by analyzing 24 different oxidized products of arachidonic or docosahexaenoic (DHA) acids in embryos using LC with hybrid quadrupole-time of flight MS. Adult zebrafish were fed E- or E+ diets for 4 months, and then were spawned to obtain E- and E+ embryos. Between 24 and 72 hours post-fertilization (hpf), arachidonic acid decreased 3-times faster in E- (21 pg/h) compared with E+ embryos (7 pg/h, P<0.0001), while both α-tocopherol and DHA concentrations decreased only in E- embryos. At 36 hpf, E- embryos contained double the 5-hydroxy-eicosatetraenoic acids and 7-hydroxy-DHA concentrations, while other hydroxy-lipids remained unchanged. Vitamin E deficiency during embryogenesis depleted DHA and arachidonic acid, and increased hydroxy-fatty acids derived from these PUFA, suggesting that α-tocopherol is necessary to protect these critical fatty acids.