A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.     

Permeabilization, Staining and Culture of Living Drosophila Embryos

The organic solvent octane has been used routinely to permeabilize the hydrophobic vitelline membrane surrounding the Drosophila embryo, thereby allowing the movement of small molecules into the egg. We present evidence that hexane is a more effective permeabilizing agent than octane and compare the effects of these solvents on uniformity of permeabilization and embryonic viability. The ability of each solvent to make the embryo accessible to a range of biological stains was compared. The effect of octane versus hexane permeabilization on subsequent embryonic viability was measured at seven different stages during early embryogenesis. We found that although hexane is a superior solvent for permeabilizing the vitelline membrane, it decreases the viability of embryos exposed between 0 and 3 hr of age. Older embryos treated with either hexane or octane are usually viable. We also showed that molecules with a molecular mass of 984 Daltons or more did not diffuse into the embryo following treatment with either hexane or octane. Results presented here challenge a phase-partition model that has been proposed previously to explain the molecular basis of permeabilization of the Drosophila egg. An alternative model is described as well as an optimized protocol for permeabilizing and staining Drosophila embryos at any stage during early embryogenesis while maintaining viability for subsequent culture.     

Floral biology and ovule and seed ontogeny of Nymphaea thermarum,a water lily at the brink of extinction with potential as a model system for basal angiosperms

Background and Aims Nymphaea thermarum is a member of the Nymphaeales, of one of the most ancient lineages of flowering plants. This species was only recently described and then declared extinct in the wild, so little is known about its reproductive biology. In general, the complete ontogeny of ovules and seeds is not well documented among species of Nymphaea and has never been studied in the subgenus Brachyceras, the clade to which N. thermarum belongs.Methods Flowers and fruits were processed for brightfield, epifluorescence and confocal microscopy. Flower morphology, with emphasis on the timing of male and female functions, was correlated with key developmental stages of the ovule and the female gametophyte. Development of the seed tissues and dynamics of polysaccharide reserves in the endosperm, perisperm and embryo were examined.Key Results Pollen release in N. thermarum starts before the flower opens. Cell walls of the micropylar nucellus show layering of callose and cellulose in a manner reminiscent of transfer cell wall patterning. Endosperm development is ab initio cellular, with micropylar and chalazal domains that embark on distinct developmental trajectories. The surrounding maternal perisperm occupies the majority of seed volume and accumulates starch centrifugally. In mature seeds, a minute but fully developed embryo is surrounded by a single, persistent layer of endosperm.Conclusions Early male and female function indicate that N. thermarum is predisposed towards self-pollination, a phenomenon that is likely to have evolved multiple times within Nymphaea. While formation of distinct micropylar and chalazal developmental domains in the endosperm, along with a copious perisperm, characterize the seeds of most members of the Nymphaeales, seed ontogenies vary between and among the constituent families. Floral biology, life history traits and small genome size make N. thermarum uniquely promising as an early-diverging angiosperm model system for genetic and molecular studies.     

Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology^1,2, behavior³, and memory and learning^4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR). This technique allows investigators to study early stages of development that were previously difficult to access.     

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.     

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping^1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.     

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.     

Exploiting the Extraordinary Genetic Polymorphism of Ciona for Developmental Genetics with Whole Genome Sequencing

Studies in tunicates such as Ciona have revealed new insights into the evolutionary origins of chordate development. Ciona populations are characterized by high levels of natural genetic variation, between 1 and 5%. This variation has provided abundant material for forward genetic studies. In the current study, we make use of deep sequencing and homozygosity mapping to map spontaneous mutations in outbred populations. With this method we have mapped two spontaneous developmental mutants. In Ciona intestinalis we mapped a short-tail mutation with strong phenotypic similarity to a previously identified mutant in the related species Ciona savignyi. Our bioinformatic approach mapped the mutation to a narrow interval containing a single mutated gene, α-laminin3,4,5, which is the gene previously implicated in C. savignyi. In addition, we mapped a novel genetic mutation disrupting neural tube closure in C. savignyi to a T-type Ca²⁺ channel gene. The high efficiency and unprecedented mapping resolution of our study is a powerful advantage for developmental genetics in Ciona, and may find application in other outbred species.     

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with more than 150 of economic value. It is a major pest of greenhouse crops, especially in Solanaceae and Cucurbitaceae (e.g., tomatoes, eggplants, peppers, cucumbers, zucchini) and greenhouse ornamentals (e.g., roses, chrysanthemum, carnations), annual field crops (such as maize, cotton, soybean, and sugar beet), and in perennial cultures (alfalfa, strawberries, grapes, citruses, and plums)^1,2. In addition to the extreme polyphagy that makes it an important agricultural pest, T. urticae has a tendency to develop resistance to a wide array of insecticides and acaricides that are used for its control^3-7.T. urticae is an excellent experimental organism, as it has a rapid life cycle (7 days at 27 °C) and can be easily maintained at high density in the laboratory. Methods to assay gene expression (including in situ hybridization and antibody staining) and to inactivate expression of spider mite endogenous genes using RNA interference have been developed^8-10. Recently, the whole genome sequence of T. urticae has been reported, creating an opportunity to develop this pest herbivore as a model organism with equivalent genomic resources that already exist in some of its host plants (Arabidopsis thaliana and the tomato Solanum lycopersicum)¹¹. Together, these model organisms could provide insights into molecular bases of plant-pest interactions.Here, an efficient method for quick and easy collection of a large number of adult female mites, their application on an experimental plant host, and the assessment of the plant damage due to spider mite feeding are described. The presented protocol enables fast and efficient collection of hundreds of individuals at any developmental stage (eggs, larvae, nymphs, adult males, and females) that can be used for subsequent experimental application.     

Polyembryony in non-apomictic citrus genotypes

Background and Aims

Adventitious embryony from nucellar cells is the mechanism leading to apomixis in Citrus sp. However, singular cases of polyembryony have been reported in non-apomictic genotypes as a consequence of 2x × 4x hybridizations and in vitro culture of isolated nucelli. The origin of the plants arising from the aforementioned processes remains unclear.

Methods

The genetic structure (ploidy and allelic constitution with microsatellite markers) of plants obtained from polyembryonic seeds arising from 2x × 4x sexual hybridizations and those regenerated from nucellus culture in vitro was systematically analysed in different non-apomictic citrus genotypes. Histological studies were also conducted to try to identify the initiation process underlying polyembryony.

Key Results

All plants obtained from the same undeveloped seed in 2x × 4x hybridizations resulted from cleavage of the original zygotic embryo. Also, the plants obtained from in vitro nucellus culture were recovered by somatic embryogenesis from cells that shared the same genotype as the zygotic embryos of the same seed.

Conclusions

It appears that in non-apomictic citrus genotypes, proembryos or embryogenic cells are formed by cleavage of the zygotic embryos and that the development of these adventitious embryos, normally hampered, can take place in vivo or in vitro as a result of two different mechanisms that prevent the dominance of the initial zygotic embryo.     

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster. In contrast to the syncytial cleavages of Drosophila, the early cleavages of Parhyale are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.     

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries and testes^3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.     

Trends in flower symmetry evolution revealed through phylogenetic and developmental genetic advances

A striking aspect of flowering plant (angiosperm) diversity is variation in flower symmetry. From an ancestral form of radial symmetry (polysymmetry, actinomorphy), multiple evolutionary transitions have contributed to instances of non-radial forms, including bilateral symmetry (monosymmetry, zygomorphy) and asymmetry. Advances in flowering plant molecular phylogenetic research and studies of character evolution as well as detailed flower developmental genetic studies in a few model species (e.g. Antirrhinum majus, snapdragon) have provided a foundation for deep insights into flower symmetry evolution. From phylogenetic studies, we have a better understanding of where during flowering plant diversification transitions from radial to bilateral flower symmetry (and back to radial symmetry) have occurred. From developmental studies, we know that a genetic programme largely dependent on the functional action of the CYCLOIDEA gene is necessary for differentiation along the snapdragon dorsoventral flower axis. Bringing these two lines of inquiry together has provided surprising insights into both the parallel recruitment of a CYC-dependent developmental programme during independent transitions to bilateral flower symmetry, and the modifications to this programme in transitions back to radial flower symmetry, during flowering plant evolution.     

Detrimental effects of an autosomal selfish genetic element on sperm competitiveness in house mice

Female multiple mating (polyandry) is widespread across many animal taxa and indirect genetic benefits are a major evolutionary force favouring polyandry. An incentive for polyandry arises when multiple mating leads to sperm competition that disadvantages sperm from genetically inferior mates. A reduction in genetic quality is associated with costly selfish genetic elements (SGEs), and studies in invertebrates have shown that males bearing sex ratio distorting SGEs are worse sperm competitors than wild-type males. We used a vertebrate model species to test whether females can avoid an autosomal SGE, the t haplotype, through polyandry. The t haplotype in house mice exhibits strong drive in t heterozygous males by affecting spermatogenesis and is associated with homozygous in utero lethality. We used controlled matings to test the effect of the t haplotype on sperm competitiveness. Regardless of mating order, t heterozygous males sired only 11% of zygotes when competing against wild-type males, suggesting a very strong effect of the t haplotype on sperm quality. We provide, to our knowledge, the first substantial evidence that polyandry ameliorates the harmful effects of an autosomal SGE arising through genetic incompatibility. We discuss potential mechanisms in our study species and the broader implications for the benefits of polyandry.     

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.