Readthrough-induced misincorporated amino acid ratios guide mutant-specific therapeutic approaches for two CFTR nonsense mutations.

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Premature termination codons (PTCs) represent ∼9% of CF mutations that typically cause severe expression defects of the CFTR anion channel. Despite the prevalence of PTCs as the underlying cause of genetic diseases, understanding the therapeutic susceptibilities of their molecular defects, both at the transcript and protein levels remains partially elucidated. Given that the molecular pathologies depend on the PTC positions in CF, multiple pharmacological interventions are required to suppress the accelerated nonsense-mediated mRNA decay (NMD), to correct the CFTR conformational defect caused by misincorporated amino acids, and to enhance the inefficient stop codon readthrough. The G418-induced readthrough outcome was previously investigated only in reporter models that mimic the impact of the local sequence context on PTC mutations in CFTR. To identify the misincorporated amino acids and their ratios for PTCs in the context of full-length CFTR readthrough, we developed an affinity purification (AP)-tandem mass spectrometry (AP-MS/MS) pipeline. We confirmed the incorporation of Cys, Arg, and Trp residues at the UGA stop codons of G542X, R1162X, and S1196X in CFTR. Notably, we observed that the Cys and Arg incorporation was favored over that of Trp into these CFTR PTCs, suggesting that the transcript sequence beyond the proximity of PTCs and/or other factors can impact the amino acid incorporation and full-length CFTR functional expression. Additionally, establishing the misincorporated amino acid ratios in the readthrough CFTR PTCs aided in maximizing the functional rescue efficiency of PTCs by optimizing CFTR modulator combinations. Collectively, our findings contribute to the understanding of molecular defects underlying various CFTR nonsense mutations and provide a foundation to refine mutation-dependent therapeutic strategies for various CF-causing nonsense mutations.

Visualization of real-time receptor endocytosis in dopamine neurons enabled by NTSR1-Venus knock-in mice.

Dopamine (DA) neurons are primarily concentrated in substantia nigra (SN) and ventral tegmental area (VTA). A subset of these neurons expresses the neurotensin receptor NTSR1 and its putative ligand neurotensin (Nts). NTSR1, a G protein-coupled receptor (GPCR), which classically activates Gαq/calcium signaling, is a potential route for modulating DA activity. Drug development efforts have been hampered by the receptor's complex pharmacology and a lack of understanding about its endogenous location and signaling responses. Therefore, we have generated NTSR1-Venus knock-in (KI) mice to study NTSR1 receptors in their physiological context. In primary hippocampal neurons, we show that these animals express functional receptors that respond to agonists by increasing intracellular calcium release and trafficking to endosomes. Moreover, systemic agonist administration attenuates locomotion in KIs as it does in control animals. Mapping receptor protein expression at regional and cellular levels, located NTSR1-Venus on the soma and dendrites of dopaminergic SN/VTA neurons. Direct monitoring of receptor endocytosis, as a proxy for activation, enabled profiling of NTSR1 agonists in neurons, as well as acute SN/VTA containing brain slices. Taken together, NTSR1-Venus animals express traceable receptors that will improve understanding of NTSR1 and DA activities and more broadly how GPCRs act in vivo.

Automated sample-to-answer centrifugal microfluidic system for rapid molecular diagnostics of SARS-CoV-2.

Testing for SARS-CoV-2 is one of the most important assets in COVID-19 management and mitigation. At the onset of the pandemic, SARS-CoV-2 testing was uniquely performed in central laboratories using RT-qPCR. RT-qPCR relies on trained personnel operating complex instrumentation, while time-to-result can be lengthy (e.g., 24 to 72 h). Now, two years into the pandemic, with the surge in cases driven by the highly transmissible Omicron variant, COVID-19 testing capabilities have been stretched to their limit worldwide. Rapid antigen tests are playing an increasingly important role in quelling outbreaks by expanding testing capacity outside the realm of clinical laboratories. These tests can be deployed in settings where repeat and rapid testing is essential, but they often come at the expense of limited accuracy and sensitivity. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) provides a number of advantages to SARS-CoV-2 testing in standard laboratories and at the point-of-need. In contrast to RT-qPCR, RT-LAMP is performed at a constant temperature, which circumvents the need for thermal cycling and translates into a shorter analysis time (e.g., <1 h). In addition, RT-LAMP is compatible with colorimetric detection, facilitating visualization and read-out. However, even with these benefits, RT-LAMP is not yet clinically deployed at its full capacity. Lack of automation and integration of sample preparation, such as RNA extraction, limits the sensitivity and specificity of the method. Furthermore, the need for cold storage of reagents complicates its use at the point of need. The developments presented in this work address these limitations: We describe a fully automated SARS-CoV-2 detection method using RT-LAMP, which also includes up-front lysis and extraction of viral RNA, performed on a centrifugal platform with active pneumatic pumping, a disposable, all-polymer-based microfluidic cartridge and lyophilized reagents. We demonstrate that the limit of detection of the RT-LAMP assay itself is 0.2 copies per µL using N and E genes as target sequences. When combined with integrated RNA extraction, the assay sensitivity is 0.5 copies per µL, which is highly competitive to RT-qPCR. We tested the automated assay using 12 clinical swab specimens from patients and were able to distinguish positive and negative samples for SARS-CoV-2 within 60 min, thereby obtaining 100% agreement with RT-qPCR results.

Multifunctional magnetic nanoparticle cloud assemblies for in situ capture of bacteria and isolation of microbial DNA.

We investigate the formation of suspended magnetic nanoparticle (MNP) assemblies (M-clouds) and their use for in situ bacterial capture and DNA extraction. M-clouds are obtained as a result of magnetic field density variations when magnetizing an array of micropillars coated with a soft ferromagnetic NiP layer. Numerical simulations suggest that the gradient in the magnetic field created by the pillars is four orders of magnitude higher than the gradient generated by the external magnets. The pillars therefore serve as the sole magnetic capture sites for MNPs which accumulate on opposite sides of each pillar facing the magnets. Composed of loosely aggregated MNPs, the M-cloud can serve as a porous capture matrix for target analyte flowing through the array. The concept is demonstrated by using a multifunctional M-cloud comprising immunomagnetic NPs (iMNPs) for capture of Escherichia coli O157:H7 from river water along with silica-coated NPs for subsequent isolation and purification of microbial DNA released upon bacterial lysis. Confocal microscopy imaging of fluorescently labeled iMNPs and E. coli O157:H7 reveals that bacteria are trapped in the M-cloud region between micropillars. Quantitative assessment of in situ bacterial capture, lysis and DNA isolation using real-time polymerase chain reaction shows linear correlation between DNA output and input bacteria concentration, making it possible to confirm E. coli 0157:H7 at 103 cells per mL. The M-cloud method further provides one order of magnitude higher DNA output concentrations than incubation of the sample with iMNPs in a tube for an equivalent period of time (e.g., 10 min). Results from assays performed in the presence of Listeria monocytogenes (at 106 cells per mL each) suggest that non-target organisms do not affect on-chip E. coli capture, DNA extraction efficiency and quality of the eluted sample.

Mitochondria of teleost radial glia: A novel target of neuroendocrine disruption by environmental chemicals?

In teleost fish, radial glial cells (RGCs) are progenitor cells for neurons and the major cell type synthesizing neuroestrogens. We hypothesized that chemical exposure impairs mitochondrial bioenergetics of RGCs, which then may lead to downstream consequences for neuroestrogen production. Here we provide proof of concept that mitochondria of RGCs can be perturbed by fungicides. We isolated RGCs from a mixed sex population of goldfish (Carassius auratus) and measured metabolic capacity of primary cells to a model mitotoxin fluazinam, a broad-spectrum fungicide that inhibits mitochondria electron transport chain (or ETC) Complex I. Using immunocytochemistry and real-time PCR, we demonstrate that the goldfish primary cell cultures are highly enriched for glia after multiple passages. Cytotoxicity assays revealed that glia treated with >25 µM fluazinam for 24 and 48-h showed reduced viability. As such, metabolic assays were conducted with non-cytotoxic concentrations (0.25-12.5 µM). Fluazinam did not affect oxygen consumption rates of RGCs at 24 h, but after 48 h, oligomycin induced ATP-linked respiration was decreased by both 6.25 and 12.5 µM fluazinam. Moreover, concentrations as low as 0.25 µM disrupted the mitochondrial membrane potential of RGCs, reflecting strong uncoupling effects of the fungicide on mitochondria. Here we provide proof of concept that mitochondrial bioenergetics of teleostean RGCs can be responsive to agrochemicals. Additional studies are required to address low-dose exposures in vivo and to determine if metabolic disruption impairs neuroendocrine functions of RGCs. We propose this mechanism constitutes a novel aspect of neuroendocrine disruption, significant because dysregulation of neuron-glia communication is expected to contribute to neuroendocrine disruption.

Allosteric folding correction of F508del and rare CFTR mutants by elexacaftor-tezacaftor-ivacaftor (Trikafta) combination.

Based on its clinical benefits, Trikafta - the combination of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor), and the gating potentiator VX-770 (ivacaftor) - was FDA approved for treatment of patients with cystic fibrosis (CF) carrying deletion of phenylalanine at position 508 (F508del) of the CF transmembrane conductance regulator (CFTR) on at least 1 allele. Neither the mechanism of action of VX-445 nor the susceptibility of rare CF folding mutants to Trikafta are known. Here, we show that, in human bronchial epithelial cells, VX-445 synergistically restores F508del-CFTR processing in combination with type I or II correctors that target the nucleotide binding domain 1 (NBD1) membrane spanning domains (MSDs) interface and NBD2, respectively, consistent with a type III corrector mechanism. This inference was supported by the VX-445 binding to and unfolding suppression of the isolated F508del-NBD1 of CFTR. The VX-661 plus VX-445 treatment restored F508del-CFTR chloride channel function in the presence of VX-770 to approximately 62% of WT CFTR in homozygous nasal epithelia. Substantial rescue of rare misprocessing mutations (S13F, R31C, G85E, E92K, V520F, M1101K, and N1303K), confined to MSD1, MSD2, NBD1, and NBD2 of CFTR, was also observed in airway epithelia, suggesting an allosteric correction mechanism and the possible application of Trikafta for patients with rare misfolding mutants of CFTR.

Secretoneurin is a secretogranin-2 derived hormonal peptide in vertebrate neuroendocrine systems.

Secretogranin-2 (SCG2) is a large precursor protein that is processed into several potentially bioactive peptides, with the 30-43 amino acid central domain called secretoneurin (SN) being clearly evolutionary conserved in vertebrates. Secretoneurin exerts a diverse array of biological functions including regulating nervous, endocrine, and immune systems in part due to its wide tissue distribution. Expressed in some neuroendocrine neurons and pituitary cells, SN is a stimulator of the synthesis and release of luteinizing hormone from both goldfish pituitary cells and the mouse LßT2 cell line. Neuroendocrine, paracrine and autocrine signaling pathways for the stimulation of luteinizing hormone release indicate hormone-like activities to regulate reproduction. Mutation of the scg2a and scg2b genes using TALENs in zebrafish reduces sexual behavior, ovulation, oviposition, and fertility. A single injection of the SNa peptide enhanced reproductive outcomes in scg2a/scg2b double mutant zebrafish. Evidence in goldfish suggests a new role for SN to stimulate food intake by actions on other feeding-related neuropeptides. Expression and regulation of the Scg2a precursor mRNA in goldfish gut also supports a role in feeding. In rodent models, SN has trophic-like properties promoting both neuroprotection and neuronal plasticity and has chemoattractant properties that regulate neuroinflammation. Data obtained from several cellular models suggest that SN binds to and activates a G-protein coupled receptor (GPCR), but a bona fide SN receptor protein needs to be identified. Other signaling pathways for SN have been reported which provides alternatives to the GPCR hypothesis. These include AMP-activated protein kinase (AMPK), extracellular signal-regulated kinases (ERK), mitogen-activated protein kinase (MAPK)and calcium/calmodulin-dependent protein kinase II in cardiomyocytes, phosphatidylinositol 3-kinase (PI3K) and Akt/Protein Kinase B (AKT, and MAPK in endothelial cells and Janus kinase 2/signal transducer and activator of transcription protein (JAK2-STAT) signaling in neurons. Some studies in cardiac cells provide evidence for cellular internalization of SN by an unknown mechanism. Many of the biological functions of SN remain to be fully characterized, which could lead to new and exciting applications.

Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics.

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBC subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.

Mutation-specific dual potentiators maximize rescue of CFTR gating mutants.

BACKGROUND: The potentiator ivacaftor (VX-770) has been approved for therapy of 38 cystic fibrosis (CF) mutations (∼10% of the patient population) associated with a gating defect of the CF transmembrane conductance regulator (CFTR). Despite the success of VX-770 treatment of patients carrying at least one allele of the most common gating mutation G551D-CFTR, some lung function decline and P. aeruginosa colonization persist. This study aims at identifying potentiator combinations that can considerably enhance the limited channel activity of a panel of CFTR gating mutants over monotherapy. METHODS: The functional response of 13 CFTR mutants to single potentiators or systematic potentiator combinations was determined in the human bronchial epithelial cell line CFBE41o- and a subset of them was confirmed in primary human nasal epithelia (HNE). RESULTS: In six out of thirteen CFTR missense mutants the fractional plasma membrane (PM) activity, a surrogate measure of CFTR channel gating, reached only ∼10-50% of WT channel activity upon VX-770 treatment, indicating incomplete gating correction. Combinatorial potentiator profiling and cluster analysis of mutant responses to 24 diverse investigational potentiators identified several compound pairs that improved the gating activity of R352Q-, S549R-, S549N-, G551D-, and G1244E-CFTR to ∼70-120% of the WT. Similarly, the potentiator combinations were able to confer WT-like function to G551D-CFTR in patient-derived human nasal epithelia. CONCLUSION: This study suggests that half of CF patients with missense mutations approved for VX-770 administration, could benefit from the development of dual potentiator therapy.

Biased Signaling of the Mu Opioid Receptor Revealed in Native Neurons.

G protein-coupled receptors are key signaling molecules and major targets for pharmaceuticals. The concept of ligand-dependent biased signaling raises the possibility of developing drugs with improved efficacy and safety profiles, yet translating this concept to native tissues remains a major challenge. Whether drug activity profiling in recombinant cell-based assays, traditionally used for drug discovery, has any relevance to physiology is unknown. Here we focused on the mu opioid receptor, the unrivalled target for pain treatment and also the key driver for the current opioid crisis. We selected a set of clinical and novel mu agonists, and profiled their activities in transfected cell assays using advanced biosensors and in native neurons from knock-in mice expressing traceable receptors endogenously. Our data identify Gi-biased agonists, including buprenorphine, and further show highly correlated drug activities in the two otherwise very distinct experimental systems, supporting in vivo translatability of biased signaling for mu opioid drugs.

Secretoneurin A Directly Regulates the Proteome of Goldfish Radial Glial Cells In Vitro.

Radial glial cells (RGCs) are the main macroglia in the teleost brain and have established roles in neurogenesis and neurosteroidogenesis. They are the only brain cell type expressing aromatase B (cyp19a1b), the enzyme that synthesizes estrogens from androgen precursors. There are few studies on the regulation of RGC functions, but our previous investigations demonstrated that dopamine stimulates cyp19a1b expression in goldfish RGCs, while secretoneurin A (SNa) inhibits the expression of this enzyme. Here, we determine the range of proteins and cellular processes responsive to SNa treatments in these steroidogenic cells. The focus here is on SNa, because this peptide is derived from selective processing of secretogranin II in magnocellular cells embedded within the RGC-rich preoptic nucleus. Primary cultures of RGCs were treated (24 h) with 10, 100, or 1,000 nM SNa. By using isobaric tagging for relative and absolute quantitation and a Hybrid Quadrupole Obritrap Mass Spectrometry system, a total of 1,363 unique proteins were identified in RGCs, and 609 proteins were significantly regulated by SNa at one or more concentrations. Proteins that showed differential expression with all three concentrations of SNa included H1 histone, glutamyl-prolyl-tRNA synthetase, Rho GDP dissociation inhibitor γ, vimentin A2, and small nuclear ribonucleoprotein-associated protein. At 10, 100, and 1,000 nM SNa, there were 5, 195, and 489 proteins that were downregulated, respectively, whereas the number of upregulated proteins were 72, 44, and 51, respectively. Subnetwork enrichment analysis of differentially regulated proteins revealed that processes such as actin organization, cytoskeleton organization and biogenesis, apoptosis, mRNA processing, RNA splicing, translation, cell growth, and proliferation are regulated by SNa based on the proteomic response. Moreover, we observed that, at the low concentration of SNa, there was an increase in the abundance of proteins involved in cell growth, proliferation, and migration, whereas higher concentration of SNa appeared to downregulate proteins involved in these processes, indicating a dose-dependent proteome response. At the highest concentration of SNa, proteins linked to the etiology of diseases of the central nervous system (brain injuries, Alzheimer disease, Parkinson's disease, cerebral infraction, brain ischemia) were also differentially regulated. These data implicate SNa in the control of cell proliferation and neurogenesis.

Secretoneurin-A inhibits aromatase B (cyp19a1b) expression in female goldfish (Carassius auratus) radial glial cells.

In the teleost brain, radial glial cells (RGCs) are the main macroglia and are stem-like progenitors that express key steroidogenic enzymes, including the estrogen-synthesizing enzyme, aromatase B (cyp19a1b). As a result, RGCs are integral to neurogenesis and neurosteroidogenesis, however little is known about the regulatory factors and signaling mechanisms that control these functions. A potential new role of the secretogranin II-derived neuropeptide secretoneurin A (SNa) in the control of goldfish (Carassius auratus) RGC function is the subject of this study. Immunohistochemistry revealed a close neuroanatomical relationship between RGCs and soma of SNa-immunoreactive magnocellular and parvocellular neurons in the preoptic nucleus of female goldfish. Five hours following intracerebroventricular injection of 0.2ng/g SNa cyp19a1b mRNA levels were decreased by 86% (P<0.05) in the hypothalamus and by 88% (P<0.05) in the telencephalon. In vitro, 24 h incubation with 500nM SNa decreased cyp19a1b mRNA by 51% (P<0.05) in cultured RGCs. These data provide evidence that SNa can regulate aromatase expression in goldfish RGCs. By regulating neuroestrogen production in RGCs SNa may therefore be implicated in the control of major estrogen-dependent functions of the preoptic region such as reproductive behavior and osmoregulation.

Secretoneurin A regulates neurogenic and inflammatory transcriptional networks in goldfish (Carassius auratus) radial glia.

Radial glial cells (RGCs) are the most abundant macroglia in the teleost brain and have established roles in neurogenesis and neurosteroidogenesis; however, their transcriptome remains uncharacterized, which limits functional understanding of this important cell type. Using cultured goldfish RGCs, RNA sequencing and de novo transcriptome assembly were performed, generating the first reference transcriptome for fish RGCs with 17,620 unique genes identified. These data revealed that RGCs express a diverse repertoire of receptors and signaling molecules, suggesting that RGCs may respond to and synthesize an array of hormones, peptides, cytokines, and growth factors. Building upon neuroanatomical data and studies investigating direct neuronal regulation of RGC physiology, differential gene expression analysis was conducted to identify transcriptional networks that are responsive to the conserved secretogranin II-derived neuropeptide secretoneurin A (SNa). Pathway analysis of the transcriptome indicated that cellular processes related to the central nervous system (e.g., neurogenesis, synaptic plasticity, glial cell development) and immune functions (e.g., immune system activation, leukocyte function, macrophage response) were preferentially modulated by SNa. These data reveal an array of new functions that are proposed to be critical to neuronal-glial interactions through the mediator SNa.

Dehydroabietic acid cytotoxicity in goldfish radial glial cells in vitro.

Dehydroabietic acid (DHAA) is a resin acid present in aquatic environments shown to induce cellular and molecular damage in aquatic animals. In this study, the cytotoxicity of DHAA on primary cultured goldfish radial glial cells (RGCs), an important component of the central nervous system, was evaluated. Here, it is reported that a concentration of 20mg/L DHAA affected cellular morphology and expression of genes involved in RGC steroidogenesis and metabolism. Higher concentration exposures of DHAA (40mg/L) lead to RGC death based on a lactate dehydrogenase leakage assay. Together, these data have implications in understanding the effects of DHAA on an integral central nervous system cell type important for neurogenesis, steroidogenesis and structural support. Due to the continuous presence of DHAA into water systems, results from this study provide indications as to the potential impacts of DHAA and demonstrate the importance of this class of chemicals on aquatic organisms.

Proteomic profiling reveals dopaminergic regulation of progenitor cell functions of goldfish radial glial cells in vitro.

UNLABELLED: Radial glial cells (RGCs) are stem-like cells found in the developing and adult central nervous system. They function as both a scaffold to guide neuron migration and as progenitor cells that support neurogenesis. Our previous study revealed a close anatomical relationship between dopamine neurons and RGCs in the telencephalon of female goldfish. In this study, label-free proteomics was used to identify the proteins in a primary RGC culture and to determine the proteome response to the selective dopamine D1 receptor agonist SKF 38393 (10µM), in order to better understand dopaminergic regulation of RGCs. A total of 689 unique proteins were identified in the RGCs and these were classified into biological and pathological pathways. Proteins such as nucleolin (6.9-fold) and ependymin related protein 1 (4.9-fold) were increased in abundance while proteins triosephosphate isomerase (10-fold) and phosphoglycerate dehydrogenase (5-fold) were decreased in abundance. Pathway analysis revealed that proteins that consistently changed in abundance across biological replicates were related to small molecules such as ATP, lipids and steroids, hormones, glucose, cyclic AMP and Ca(2+). Sub-network enrichment analysis suggested that estrogen receptor signaling, among other transcription factors, is regulated by D1 receptor activation. This suggests that these signaling pathways are correlated to dopaminergic regulation of radial glial cell functions. Most proteins down-regulated by SKF 38393 were involved in cell cycle/proliferation, growth, death, and survival, which suggests that dopamine inhibits the progenitor-related processes of radial glial cells. Examples of differently expressed proteins including triosephosphate isomerase, nucleolin, phosphoglycerate dehydrogenase and capping protein (actin filament) muscle Z-line beta were validated by qPCR and western blot, which were consistent with MS/MS data in the direction of change. This is the first study to characterize the RGC proteome on a large scale in a vertebrate species. These data provide novel insight into glial protein networks that are associated with neuroendocrine function and neurogenesis in the teleost brain. BIOLOGICAL SIGNIFICANCE: While the role of radial glial cells in organizing brain structure and neurogenesis has been well studied, protein profiling experiments in this unique cell type has not been conducted. This study is the first to profile the proteome of goldfish radial glial cells in culture and to study the regulation of progenitor functions of radial glial cells by the neurotransmitter dopamine. This study provides the foundation for molecular network analysis in fish radial glial cells, and identifies cellular processes and signaling pathways in these cells with roles in neurogenesis and neuroendocrine function. Lastly, this study begins to characterize signatures and biomarkers for specific neuroendocrine and neurogenesis disruptors.

Dopamine D1 receptor activation regulates the expression of the estrogen synthesis gene aromatase B in radial glial cells.

Radial glial cells (RGCs) are abundant stem-like non-neuronal progenitors that are important for adult neurogenesis and brain repair, yet little is known about their regulation by neurotransmitters. Here we provide evidence for neuronal-glial interactions via a novel role for dopamine to stimulate RGC function. Goldfish were chosen as the model organism due to the abundance of RGCs and regenerative abilities of the adult central nervous system. A close anatomical relationship was observed between tyrosine hydroxylase-positive catecholaminergic cell bodies and axons and dopamine-D1 receptor expressing RGCs along the ventricular surface of telencephalon, a site of active neurogenesis. A primary cell culture model was established and immunofluorescence analysis indicates that in vitro RGCs from female goldfish retain their major characteristics in vivo, including expression of glial fibrillary acidic protein and brain lipid binding protein. The estrogen synthesis enzyme aromatase B is exclusively found in RGCs, but this is lost as cells differentiate to neurons and other glial types in adult teleost brain. Pharmacological experiments using the cultured RGCs established that specific activation of dopamine D1 receptors up-regulates aromatase B mRNA through a cyclic adenosine monophosphate-dependent molecular mechanism. These data indicate that dopamine enhances the steroidogenic function of this neuronal progenitor cell.

Comparative analysis of goitrogenic effects of phenylthiourea and methimazole in zebrafish embryos.

Craniofacial malformations, reduced locomotion and induction of genes encoding for enzymes involved in thyroid hormone synthesis were assessed using methimazole and N-phenylthiourea in zebrafish embryos. Gene expression, the most sensitive endpoint (EC50_MMI=372-765µM, EC50_PTU=7.6-8.6µM), was analysed in wild-type and in a transgenic strain, tg(tg:mCherry), expressing mCherry fluorescence protein under the control of the thyroglobulin gene. Reduction of locomotion and craniofacial malformations were observed at one or two orders of magnitude above concentrations affecting gene expression, respectively. Both effects could be linked to the malformations caused by reduced thyroxin levels. Our results show that due to the presence of the autoregulatory loop of the hypothalamus-pituitary-thyroid axis, various molecular initiating events of thyroid disruption are amenable for the zebrafish embryo. We propose the tg(tg:mCherry) bioassay as a sensitive tool in medium scale screening of goitrogens, given the minimal effort for sample preparation and analysis of gene expression.

Peripheral chemoreceptors in fish: A brief history and a look ahead.

The story of control of cardiorespiratory reflexes by peripheral chemoreceptors includes a chapter on evolution in large part because of the work of Prof. William K. Milsom. Bill has reminded us to think comparatively about O2 and CO2/H(+) sensing. We present a brief review of the fish gill and O2 chemoreceptors, as well as recent results from our laboratory, that were discussed at a symposium in honour of Prof. Milsom's extensive career. In a series of papers from the Milsom laboratory from 1986 to 1995, it was demonstrated that the fish gill is a major site of chemosensory discharge during hypoxia, and that this response is sensitive to multiple neurochemicals involved in chemosensing. These and other more recent studies by Bill et al. are now fundamental and have helped to shape the field as it is today. At the cellular level, we have shown that chemosensitive neuroepithelial cells (NECs) of the gills may possess unique adaptations compared to their mammalian homologues. In addition, we used injection of the styryl dye, FM1-43, to identify gill NECs in zebrafish and demonstrate increased vesicular activity in NECs in vitro during acute stimulation. In vivo, we have identified 5-HT2, 5-HT3, dopaminergic and nicotinic receptor activity involved in the hyperventilatory response in developing zebrafish. With this model we have also traced the fate of mitotic cells in the gills, and demonstrated the regeneration of resected gill filaments and replacement of O2-sensitive NECs.