Comparative Analysis Of Zebrafish And Planarian Model Systems For Developmental Neurotoxicity Screens Using An 87-Compound Library

There is a clear need to establish and validate new methodologies to more quickly and efficiently screen chemicals for potential toxic effects, particularly on development. The emergence of alternative animal systems for rapid toxicology screens presents valuable opportunities to evaluate how systems complement each other. In this article, we compare a chemical library of 87-compounds in two such systems, developing zebrafish and freshwater planarians, by screening for developmental neurotoxic effects. We show that the systems’ toxicological profiles are complementary to each other, with zebrafish yielding more detailed morphological endpoints and planarians more behavioral endpoints. Overall, zebrafish was more sensitive to this chemical library, yielding 86/87 hits, compared to 50/87 hits in planarians. The difference in sensitivity could not be attributed to molecular weight, Log Kow or the bioconcentration factor. Of the 87 chemicals, 28 had previously been evaluated in mammalian developmental neuro- (DNT), neuro- or developmental toxicity studies. Of the 28, 20 were hits in the planarian, and 27 were hits in zebrafish. Eighteen of the 28 had previously been identified as DNT hits in mammals and were highly associated with activity in zebrafish and planarian behavioral assays in this study. Only 1 chemical (out of 28) was a false negative in both zebrafish and planarian systems. Differences in endpoint coverage and system sensitivity illustrate the value of a dual systems approach to rapidly query a large chemical-bioactivity space and provide weight-of-evidence for prioritization of chemicals for further testing

Investigating the Roles of Lhx1a, Lhx1b, and Lhx5 in Zebrafish Spinal Cord Interneuron Specification and Function

Located in the vertebral column, the human spine is responsible for regulating body movements and receiving sensory input about pain and touch. Currently, few treatments for neurological diseases and spinal cord injuries exist, partly because we know little about how a fully functioning spinal cord is constructed. As such, studying spinal cord development, specifically neuronal specification and patterning, should be useful for developing better treatments for people with spinal cord injuries and diseases. Zebrafish are a prime model organism for studying neuronal specification because their transparent embryos develop outside the mother, allowing us to easily examine gene expression, cell movements and cell morphology during development . Furthermore, the zebrafish spinal cord has few types of interneurons compared to mammals, and each interneuron type can be recognized by its distinct morphology. I focused on V1 cells, which form in the ventral spinal cord and are functionally similar in all vertebrates. In zebrafish, V1 cells develop into CiAs, or Cicumferential Ascending interneurons, which control movement and sensory gating. Several transcription factors are expressed consistently in all vertebrate V1 cells, and my research focused on Lhx1a, Lhx1b and Lhx5. As a result of findings in mice, I predicted that knocking down these transcription factors would result in neurotransmitter deficits and potentially compromise movement ability. Additionally, a main focus of my project became assaying various experimental strategies for knocking-down Lhx1a, Lhx1b and Lhx5. These included injecting reagents into 1-4 cell stage embryos and taking advantage of a lhx1b mutant fish line. For the injections, I used morpholinos (MOs), antisense agents that either interfere with RNA transcription to protein or with RNA splicing. I also used RNA constructs which should act as either dominant activators or dominant repressors. Using in situ hybridization, I then tried to assess the impact on neurotransmitters throughout the spinal cord and specifically within CiAs. I successfully identified a PCR and restriction enzyme digest method for identifying lhx1b mutants. This was exciting as it was a completely novel method for identifying these fish. Furthermore, my results demonstrated that homozygous lhx1b mutants are viable which was previously unknown. My injection results demonstrate that lhx1b and lhx5 splice-blocking MOs are also an effective tool for knocking-down the function of these two genes. In contrast, the lhx1a MOs that I tried were not effective. My RNA injection results were inconclusive and I determined that a higher concentration was probably needed to impact Lhx1 and Lhx5 function

Analysis of MicroRNA Expression in Embryonic Developmental Toxicity Induced by MC-RR

As cynobacterial blooms frequently occur in fresh waters throughout the world, microcystins (MCs) have caused serious damage to both wildlife and human health. MCs are known to have developmental toxicity, however, the possible molecular mechanism is largely unknown. This is the first toxicological study to integrate post-transcriptomic, proteomic and bioinformatics analysis to explore molecular mechanisms for developmental toxicity of MCs in zebrafish. After being microinjected directly into embryos, MC-RR dose-dependently decreased survival rates and increased malformation rates of embryos, causing various embryo abnormalities including loss of vascular integrity and hemorrhage. Expressions of 31 microRNAs (miRNAs) and 78 proteins were significantly affected at 72 hours post-fertilisation (hpf). Expressions of miR-430 and miR-125 families were also significantly changed. The altered expressions of miR-31 and miR-126 were likely responsible for the loss of vascular integrity. MC-RR significantly reduced the expressions of a number of proteins involved in energy metabolism, cell division, protein synthesis, cytoskeleton maintenance, response to stress and DNA replication. Bioinformatics analysis shows that several aberrantly expressed miRNAs and proteins (involved in various molecular pathways) were predicted to be potential MC-responsive miRNA-target pairs, and that their aberrant expressions should be the possible molecular mechanisms for the various developmental defects caused by MC-RR

The homology of odontodes in gnathostomes: insights from Dlx gene expression in the dogfish, Scyliorhinus canicula

<p>Abstract</p> <p>Background</p> <p>Teeth and tooth-like structures, together named odontodes, are repeated organs thought to share a common evolutionary origin. These structures can be found in gnathostomes at different locations along the body: oral teeth in the jaws, teeth and denticles in the oral-pharyngeal cavity, and dermal denticles on elasmobranch skin. We, and other colleagues, had previously shown that teeth in any location were serially homologous because: i) pharyngeal and oral teeth develop through a common developmental module; and ii) the expression patterns of the <it>Dlx </it>genes during odontogenesis were highly divergent between species but almost identical between oral and pharyngeal dentitions within the same species. Here we examine <it>Dlx </it>gene expression in oral teeth and dermal denticles in order to test the hypothesis of serial homology between these odontodes.</p> <p>Results</p> <p>We present a detailed comparison of the first developing teeth and dermal denticles (caudal primary scales) of the dogfish (<it>Scyliorhinus canicula</it>) and show that both odontodes develop through identical stages that correspond to the common stages of oral and pharyngeal odontogenesis. We identified six <it>Dlx </it>paralogs in the dogfish and found that three showed strong transcription in teeth and dermal denticles (<it>Dlx3</it>, <it>Dlx4 </it>and <it>Dlx5</it>) whereas a weak expression was detected for <it>Dlx1 </it>in dermal denticles and teeth, and for <it>Dlx2 </it>in dermal denticles. Very few differences in <it>Dlx </it>expression patterns could be detected between tooth and dermal denticle development, except for the absence of <it>Dlx2 </it>expression in teeth.</p> <p>Conclusions</p> <p>Taken together, our histological and expression data strongly suggest that teeth and dermal denticles develop from the same developmental module and under the control of the same set of <it>Dlx </it>genes. Teeth and dermal denticles should therefore be considered as serial homologs developing through the initiation of a common gene regulatory network (GRN) at several body locations. This mechanism of heterotopy supports the 'inside and out' model that has been recently proposed for odontode evolution.</p

Dopaminergic Neuronal Loss and Dopamine-Dependent Locomotor Defects in Fbxo7-Deficient Zebrafish

Recessive mutations in the F-box only protein 7 gene (FBXO7) cause PARK15, a Mendelian form of early-onset, levodopa-responsive parkinsonism with severe loss of nigrostriatal dopaminergic neurons. However, the function of the protein encoded by FBXO7, and the pathogenesis of PARK15 remain unknown. No animal models of this disease exist. Here, we report the generation of a vertebrate model of PARK15 in zebrafish. We first show that the zebrafish Fbxo7 homolog protein (zFbxo7) is expressed abundantly in the normal zebrafish brain. Next, we used two zFbxo7-specific morpholinos (targeting protein translation and mRNA splicing, respectively), to knock down the zFbxo7 expression. The injection of either of these zFbxo7-specific morpholinos in the fish embryos induced a marked decrease in the zFbxo7 protein expression, and a range of developmental defects. Furthermore, whole-mount in situ mRNA hybridization showed abnormal patterning and significant decrease in the number of diencephalic tyrosine hydroxylase-expressing neurons, corresponding to the human nigrostriatal or ventral tegmental dopaminergic neurons. Of note, the number of the dopamine transporter-expressing neurons was much more severely depleted, suggesting dopaminergic dysfunctions earlier and larger than those due to neuronal loss. Last, the zFbxo7 morphants displayed severe locomotor disturbances (bradykinesia), which were dramatically improved by the dopaminergic agonist apomorphine. The severity of these morphological and behavioral abnormalities correlated with the severity of zFbxo7 protein deficiency. Moreover, the effects of the co-injection of zFbxo7- and p53-specific morpholinos were similar to those obtained with zFbxo7-specific morpholinos alone, supporting further the contention that the observed phenotypes were specifically due to the knock down of zFbxo7. In conclusion, this novel vertebrate model reproduces pathologic and behavioral hallmarks of human parkinsonism (dopaminergic neuronal loss and dopamine-dependent bradykinesia), representing therefore a valid tool for investigating the mechanisms of selective dopaminergic neuronal death, and screening for modifier genes and therapeutic compounds

Lessons from morpholino-based screening in zebrafish

Morpholino oligonucleotides (MOs) are an effective, gene-specific antisense knockdown technology used in many model systems. Here we describe the application of MOs in zebrafish (Danio rerio) for in vivo functional characterization of gene activity. We summarize our screening experience beginning with gene target selection. We then discuss screening parameter considerations and data and database management. Finally, we emphasize the importance of off-target effect management and thorough downstream phenotypic validation. We discuss current morpholino limitations, including reduced stability when stored in aqueous solution. Advances in MO technology now provide a measure of spatiotemporal control over MO activity, presenting the opportunity for incorporating more finely tuned analyses into MO-based screening. Therefore, with careful management, MOs remain a valuable tool for discovery screening as well as individual gene knockdown analysis

Organ-targeted high-throughput in vivo biologics screen identifies materials for RNA delivery

Therapies based on biologics involving delivery of proteins, DNA, and RNA are currently among the most promising approaches. However, although large combinatorial libraries of biologics and delivery vehicles can be readily synthesized, there are currently no means to rapidly characterize them in vivo using animal models. Here, we demonstrate high-throughput in vivo screening of biologics and delivery vehicles by automated delivery into target tissues of small vertebrates with developed organs. Individual zebrafish larvae are automatically oriented and immobilized within hydrogel droplets in an array format using a microfluidic system, and delivery vehicles are automatically microinjected to target organs with high repeatability and precision. We screened a library of lipid-like delivery vehicles for their ability to facilitate the expression of protein-encoding RNAs in the central nervous system. We discovered delivery vehicles that are effective in both larval zebrafish and rats. Our results showed that the in vivo zebrafish model can be significantly more predictive of both false positives and false negatives in mammals than in vitro mammalian cell culture assays. Our screening results also suggest certain structure–activity relationships, which can potentially be applied to design novel delivery vehicles.National Institutes of Health (U.S.) (Transformative Research Award R01 NS073127)National Institutes of Health (U.S.) (Director's Innovator Award DP2 OD002989)David & Lucile Packard Foundation (Award in Science and Engineering)Sanofi Aventis (Firm)Foxconn International Holdings Ltd.Hertz Foundation (Fellowship)University Grants Committee (Hong Kong, China) (Early Career Award 125012)National Natural Science Foundation (China) (81201164)ITC (ITS/376/13)Chinese University of Hong Kong (Grant 9610215)Chinese University of Hong Kong (Grant 7200269

Organ-targeted high-throughput in vivo biologics screen identifies materials for RNA delivery

Therapies based on biologics involving delivery of proteins, DNA, and RNA are currently among the most promising approaches. However, although large combinatorial libraries of biologics and delivery vehicles can be readily synthesized, there are currently no means to rapidly characterize them in vivo using animal models. Here, we demonstrate high-throughput in vivo screening of biologics and delivery vehicles by automated delivery into target tissues of small vertebrates with developed organs. Individual zebrafish larvae are automatically oriented and immobilized within hydrogel droplets in an array format using a microfluidic system, and delivery vehicles are automatically microinjected to target organs with high repeatability and precision. We screened a library of lipid-like delivery vehicles for their ability to facilitate the expression of protein-encoding RNAs in the central nervous system. We discovered delivery vehicles that are effective in both larval zebrafish and rats. Our results showed that the in vivo zebrafish model can be significantly more predictive of both false positives and false negatives in mammals than in vitro mammalian cell culture assays. Our screening results also suggest certain structure–activity relationships, which can potentially be applied to design novel delivery vehicles.National Institutes of Health (U.S.) (Transformative Research Award R01 NS073127)National Institutes of Health (U.S.) (Director's Innovator Award DP2 OD002989)David & Lucile Packard Foundation (Award in Science and Engineering)Sanofi Aventis (Firm)Foxconn International Holdings Ltd.Hertz Foundation (Fellowship)University Grants Committee (Hong Kong, China) (Early Career Award 125012)National Natural Science Foundation (China) (81201164)ITC (ITS/376/13)Chinese University of Hong Kong (Grant 9610215)Chinese University of Hong Kong (Grant 7200269

The gene regulatory basis of genetic compensation during neural crest induction.

The neural crest (NC) is a vertebrate-specific cell type that contributes to a wide range of different tissues across all three germ layers. The gene regulatory network (GRN) responsible for the formation of neural crest is conserved across vertebrates. Central to the induction of the NC GRN are AP-2 and SoxE transcription factors. NC induction robustness is ensured through the ability of some of these transcription factors to compensate loss of function of gene family members. However the gene regulatory events underlying compensation are poorly understood. We have used gene knockout and RNA sequencing strategies to dissect NC induction and compensation in zebrafish. We genetically ablate the NC using double mutants of tfap2a;tfap2c or remove specific subsets of the NC with sox10 and mitfa knockouts and characterise genome-wide gene expression levels across multiple time points. We find that compensation through a single wild-type allele of tfap2c is capable of maintaining early NC induction and differentiation in the absence of tfap2a function, but many target genes have abnormal expression levels and therefore show sensitivity to the reduced tfap2 dosage. This separation of morphological and molecular phenotypes identifies a core set of genes required for early NC development. We also identify the 15 somites stage as the peak of the molecular phenotype which strongly diminishes at 24 hpf even as the morphological phenotype becomes more apparent. Using gene knockouts, we associate previously uncharacterised genes with pigment cell development and establish a role for maternal Hippo signalling in melanocyte differentiation. This work extends and refines the NC GRN while also uncovering the transcriptional basis of genetic compensation via paralogues

Neurodegeneration and Epilepsy in a Zebrafish Model of CLN3 Disease (Batten Disease)

The neuronal ceroid lipofuscinoses are a group of lysosomal storage disorders that comprise the most common, genetically heterogeneous, fatal neurodegenerative disorders of children. They are characterised by childhood onset, visual failure, epileptic seizures, psychomotor retardation and dementia. CLN3 disease, also known as Batten disease, is caused by autosomal recessive mutations in the CLN3 gene, 80–85% of which are a ~1 kb deletion. Currently no treatments exist, and after much suffering, the disease inevitably results in premature death. The aim of this study was to generate a zebrafish model of CLN3 disease using antisense morpholino injection, and characterise the pathological and functional consequences of Cln3 deficiency, thereby providing a tool for future drug discovery. The model was shown to faithfully recapitulate the pathological signs of CLN3 disease, including reduced survival, neuronal loss, retinopathy, axonopathy, loss of motor function, lysosomal storage of subunit c of mitochondrial ATP synthase, and epileptic seizures, albeit with an earlier onset and faster progression than the human disease. Our study provides proof of principle that the advantages of the zebrafish over other model systems can be utilised to further our understanding of the pathogenesis of CLN3 disease and accelerate drug discovery