105 research outputs found

    Molecular mechanisms governing primordial germ cell migration in zebrafish

    Get PDF
    In most sexually reproducing organisms primordial germ cells (pGCs) are specified early in development in places that are distinct from the region where the somatic part of the gonad develops. From their places of specification they have to migrate towards the site where they associate with somatic gonadal cells and differentiate to the gametes. The question conceming the molecular mechanisms that guide PGCs during their migration and allow them to reach their target is the focus of this work. The process was investigated in zebrafish, where the extrauterine development of the embryos their translucency allows monitoring cell migration at high resolution.Previous studies showed that zebrafish PGCs are specified in four different positions. From these positions the cells perform distinct migration steps until they arrive at their target by the end of the flTst day of development. During their migration the cells are guided by cues provided by somatic tissues.To identify the actual molecules that function as the guidance cues, a largescale antisense-oligonucleotide-based screen was carried out. In this screen, a chemokine receptor, CXCR4b and its ligand, the chemokine SDFla, were identified as proteins required for guided PGC migration. This pair of molecules had previously been shown to guide cell migration in other model organisms in a variety of developmental processes and disease. For example, SDF-l/CXCR4 signalling guides leul(Ocytes to the sites of inflarnmation or metastatic tumour ce lis to sites where they form secondary tumors.We found that in zebrafish embryos, the receptor CXCR4b is expressed in the migrating PGCs and its ligand, SDFla, is expressed in the tissues along which the PGCs migrate. Knocking down either CXCR4b or SDFla impairs PGC directed migration, which becomes evident by the inability of the cells to reach their target. Furthermore, when SDFla was expressed in ectopic sites in the embryos, PGCs arrived at these sites thus demonstrating the instructive role of this chemokine in PGC migration. Together, these results strongly suggest that SDFla provides the directional cue for PGC migration in zebrafish. These findings have since been generalized to mouse and chicken, where it was shown that CXCR4 and SDF-I play an essential role in PGC migration as weIl.Interestingly, in Drosophila a different biochemical pathway was shown to be important for providing directional cues for migrating PGCs, namely, the cholesterol/isoprenoid biosynthesis pathway. To determine whether this pathway plays a similar role in zebrafish, a 'block and rescue' pharmacogenetic approach was employed. Small chemical compounds were utilized to inhibit distinct steps in the cholesterol and isoprenoid synthesis pathway and the effect on PGC migration was examined. Using this approach, we showed that blocking HMGCoAR reductase (an enzyme that catalyses the rate limiting step in cholesterol synthesis) results in slower PGC migration. As a consequence, PGCs in treated embryos were frequently found in Abnormal locations. We then determined which components of the Cholesterol/isoprenoid biosynthetic pathway that act downstream of HMGCoAR are involved in this process. We could demonstrate that Geranylgeranyl transferase I (GGTI) activity in the isoprenoid branch of the pathway is important for optimal PGC motility

    Next-Generation Sequencing-Based Approaches for Mutation Mapping and Identification in Caenorhabditis elegans

    Get PDF
    The use of next-generation sequencing (NGS) has revolutionized the way phenotypic traits are assigned to genes. In this review, we describe NGS-based methods for mapping a mutation and identifying its molecular identity, with an emphasis on applications in Caenorhabditis elegans. In addition to an overview of the general principles and concepts, we discuss the main methods, provide practical and conceptual pointers, and guide the reader in the types of bioinformatics analyses that are required. Owing to the speed and the plummeting costs of NGS-based methods, mapping and cloning a mutation of interest has become straightforward, quick, and relatively easy. Removing this bottleneck previously associated with forward genetic screens has significantly advanced the use of genetics to probe fundamental biological processes in an unbiased manner

    C. elegans mutant identification with a one-step whole-genome-sequencing and SNP mapping strategy.

    Get PDF
    Whole-genome sequencing (WGS) is becoming a fast and cost-effective method to pinpoint molecular lesions in mutagenized genetic model systems, such as Caenorhabditis elegans. As mutagenized strains contain a significant mutational load, it is often still necessary to map mutations to a chromosomal interval to elucidate which of the WGS-identified sequence variants is the phenotype-causing one. We describe here our experience in setting up and testing a simple strategy that incorporates a rapid SNP-based mapping step into the WGS procedure. In this strategy, a mutant retrieved from a genetic screen is crossed with a polymorphic C. elegans strain, individual F2 progeny from this cross is selected for the mutant phenotype, the progeny of these F2 animals are pooled and then whole-genome-sequenced. The density of polymorphic SNP markers is decreased in the region of the phenotype-causing sequence variant and therefore enables its identification in the WGS data. As a proof of principle, we use this strategy to identify the molecular lesion in a mutant strain that produces an excess of dopaminergic neurons. We find that the molecular lesion resides in the Pax-6/Eyeless ortholog vab-3. The strategy described here will further reduce the time between mutant isolation and identification of the molecular lesion

    Comparative genetic, proteomic and phosphoproteomic analysis of C. <i>elegans </i>embryos with a focus on <i>ham</i>-1/STOX and <i>pig</i>-1/MELK in dopaminergic neuron development

    Get PDF
    Asymmetric cell divisions are required for cellular diversity and defects can lead to altered daughter cell fates and numbers. In a genetic screen for C. elegans mutants with defects in dopaminergic head neuron specification or differentiation, we isolated a new allele of the transcription factor HAM-1 [HSN (Hermaphrodite-Specific Neurons) Abnormal Migration]. Loss of both HAM-1 and its target, the kinase PIG-1 [PAR-1(I)-like Gene], leads to abnormal dopaminergic head neuron numbers. We identified discrete genetic relationships between ham-1, pig-1 and apoptosis pathway genes in dopaminergic head neurons. We used an unbiased, quantitative mass spectrometry-based proteomics approach to characterise direct and indirect protein targets and pathways that mediate the effects of PIG-1 kinase loss in C. elegans embryos. Proteins showing changes in either abundance, or phosphorylation levels, between wild-type and pig-1 mutant embryos are predominantly connected with processes including cell cycle, asymmetric cell division, apoptosis and actomyosin-regulation. Several of these proteins play important roles in C. elegans development. Our data provide an in-depth characterisation of the C. elegans wild-type embryo proteome and phosphoproteome and can be explored via the Encyclopedia of Proteome Dynamics (EPD) - an open access, searchable online database

    A novel piggybac transposon inducible expression system identifies a role for akt signalling in primordial germ cell migration

    Get PDF
    In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC) lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development

    The Mechanism for Primordial Germ-Cell Migration Is Conserved between Japanese Eel and Zebrafish

    Get PDF
    Primordial germ cells (PGCs) are segregated and specified from somatic cells during early development. These cells arise elsewhere and have to migrate across the embryo to reach developing gonadal precursors. Several molecules associated with PGC migration (i.e. dead-end, nanos1, and cxcr4) are highly conserved across phylum boundaries. However, since cell migration is a complicated process that is regulated spatially and temporally by multiple adaptors and signal effectors, the process is unlikely to be explained by these known genes only. Indeed, it has been shown that there are variations in PGC migration pattern during development among teleost species. However, it is still unclear whether the actual mechanism of PGC migration is conserved among species. In this study, we studied the migration of PGCs in Japanese eel (Anguilla japonica) embryos and tested the migration mechanism between Japanese eel and zebrafish (Danio rerio) for conservation, by transplanting eel PGCs into zebrafish embryos. The experiments showed that eel PGCs can migrate toward the gonadal region of zebrafish embryos along with endogenous PGCs, even though the migration patterns, behaviors, and settlements of PGCs are somewhat different between these species. Our results demonstrate that the migration mechanism of PGCs during embryonic development is highly conserved between these two distantly related species (belonging to different teleost orders)

    Identification of factors required for meristem function in Arabidopsis using a novel next generation sequencing fast forward genetics approach

    Get PDF
    <p>Abstract</p> <p>Background</p> <p>Phenotype-driven forward genetic experiments are powerful approaches for linking phenotypes to genomic elements but they still involve a laborious positional cloning process. Although sequencing of complete genomes now becomes available, discriminating causal mutations from the enormous amounts of background variation remains a major challenge.</p> <p>Method</p> <p>To improve this, we developed a universal two-step approach, named 'fast forward genetics', which combines traditional bulk segregant techniques with targeted genomic enrichment and next-generation sequencing technology</p> <p>Results</p> <p>As a proof of principle we successfully applied this approach to two Arabidopsis mutants and identified a novel factor required for stem cell activity.</p> <p>Conclusion</p> <p>We demonstrated that the 'fast forward genetics' procedure efficiently identifies a small number of testable candidate mutations. As the approach is independent of genome size, it can be applied to any model system of interest. Furthermore, we show that experiments can be multiplexed and easily scaled for the identification of multiple individual mutants in a single sequencing run.</p

    Lensfree Fluorescent On-Chip Imaging of Transgenic Caenorhabditis elegans Over an Ultra-Wide Field-of-View

    Get PDF
    We demonstrate lensfree on-chip fluorescent imaging of transgenic Caenorhabditis elegans (C. elegans) over an ultra-wide field-of-view (FOV) of e.g., >2–8 cm2 with a spatial resolution of ∼10µm. This is the first time that a lensfree on-chip platform has successfully imaged fluorescent C. elegans samples. In our wide-field lensfree imaging platform, the transgenic samples are excited using a prism interface from the side, where the pump light is rejected through total internal reflection occurring at the bottom facet of the substrate. The emitted fluorescent signal from C. elegans samples is then recorded on a large area opto-electronic sensor-array over an FOV of e.g., >2–8 cm2, without the use of any lenses, thin-film interference filters or mechanical scanners. Because fluorescent emission rapidly diverges, such lensfree fluorescent images recorded on a chip look blurred due to broad point-spread-function of our platform. To combat this resolution challenge, we use a compressive sampling algorithm to uniquely decode the recorded lensfree fluorescent patterns into higher resolution images, demonstrating ∼10 µm resolution. We tested the efficacy of this compressive decoding approach with different types of opto-electronic sensors to achieve a similar resolution level, independent of the imaging chip. We further demonstrate that this wide FOV lensfree fluorescent imaging platform can also perform sequential bright-field imaging of the same samples using partially-coherent lensfree digital in-line holography that is coupled from the top facet of the same prism used in fluorescent excitation. This unique combination permits ultra-wide field dual-mode imaging of C. elegans on a chip which could especially provide a useful tool for high-throughput screening applications in biomedical research

    An Evolutionarily Conserved Arginine Is Essential for Tre1 G Protein-Coupled Receptor Function During Germ Cell Migration in Drosophila melanogaster

    Get PDF
    BACKGROUND: G protein-coupled receptors (GPCRs) play central roles in mediating cellular responses to environmental signals leading to changes in cell physiology and behaviors, including cell migration. Numerous clinical pathologies including metastasis, an invasive form of cell migration, have been linked to abnormal GPCR signaling. While the structures of some GPCRs have been defined, the in vivo roles of conserved amino acid residues and their relationships to receptor function are not fully understood. Trapped in endoderm 1 (Tre1) is an orphan receptor of the rhodopsin class that is necessary for primordial germ cell migration in Drosophila melanogaster embryos. In this study, we employ molecular genetic approaches to identify residues in Tre1 that are critical to its functions in germ cell migration. METHODOLOGY/PRINCIPAL FINDINGS: First, we show that the previously reported scattershot mutation is an allele of tre1. The scattershot allele results in an in-frame deletion of 8 amino acids at the junction of the third transmembrane domain and the second intracellular loop of Tre1 that dramatically impairs the function of this GPCR in germ cell migration. To further refine the molecular basis for this phenotype, we assayed the effects of single amino acid substitutions in transgenic animals and determined that the arginine within the evolutionarily conserved E/N/DRY motif is critical for receptor function in mediating germ cell migration within an intact developing embryo. CONCLUSIONS/SIGNIFICANCE: These structure-function studies of GPCR signaling in native contexts will inform future studies into the basic biology of this large and clinically important family of receptors

    Hypoxia Impairs Primordial Germ Cell Migration in Zebrafish (Danio rerio) Embryos

    Get PDF
    Background: As a global environmental concern, hypoxia is known to be associated with many biological and physiological impairments in aquatic ecosystems. Previous studies have mainly focused on the effect of hypoxia in adult animals. However, the effect of hypoxia and the underlying mechanism of how hypoxia affects embryonic development of aquatic animals remain unclear. Methodology/Principal Findings: In the current study, the effect of hypoxia on primordial germ cell (PGC) migration in zebrafish embryos was investigated. Hypoxic embryos showed PGC migration defect as indicated by the presence of mis-migrated ectopic PGCs. Insulin-like growth factor (IGF) signaling is required for embryonic germ line development. Using real-time PCR, we found that the mRNA expression levels of insulin-like growth factor binding protein (IGFBP-1), an inhibitor of IGF bioactivity, were significantly increased in hypoxic embryos. Morpholino knockdown of IGFBP-1 rescued the PGC migration defect phenotype in hypoxic embryos, suggesting the role of IGFBP-1 in inducing PGC mis-migration. Conclusions/Significance: This study provides novel evidence that hypoxia disrupts PGC migration during embryonic development in fish. IGF signaling is shown to be one of the possible mechanisms for the causal link between hypoxia and PGC migration. We propose that hypoxia causes PGC migration defect by inhibiting IGF signaling through the induction of IGFBP-1
    corecore