RESUMO
Systematic functional profiling of the gene set that directs embryonic development is an important challenge. To tackle this challenge, we used 4D imaging of C. elegans embryogenesis to capture the effects of 500 gene knockdowns and developed an automated approach to compare developmental phenotypes. The automated approach quantifies features-including germ layer cell numbers, tissue position, and tissue shape-to generate temporal curves whose parameterization yields numerical phenotypic signatures. In conjunction with a new similarity metric that operates across phenotypic space, these signatures enabled the generation of ranked lists of genes predicted to have similar functions, accessible in the PhenoBank web portal, for â¼25% of essential development genes. The approach identified new gene and pathway relationships in cell fate specification and morphogenesis and highlighted the utilization of specialized energy generation pathways during embryogenesis. Collectively, the effort establishes the foundation for comprehensive analysis of the gene set that builds a multicellular organism.
Assuntos
Caenorhabditis elegans , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica/métodos , Técnicas de Silenciamento de Genes , FenótipoRESUMO
The Caenorhabditis elegans embryo is an important model for analyzing mechanisms of cell fate specification and tissue morphogenesis. Sophisticated lineage-tracing approaches for analyzing embryogenesis have been developed but are labor intensive and do not naturally integrate morphogenetic readouts. To enable the rapid classification of developmental phenotypes, we developed a high-content method that employs two custom strains: a Germ Layer strain that expresses nuclear markers in the ectoderm, mesoderm and endoderm/pharynx; and a Morphogenesis strain that expresses markers labeling epidermal cell junctions and the neuronal cell surface. We describe a procedure that allows simultaneous live imaging of development in 80-100 embryos and provide a custom program that generates cropped, oriented image stacks of individual embryos to facilitate analysis. We demonstrate the utility of our method by perturbing 40 previously characterized developmental genes in variants of the two strains containing RNAi-sensitizing mutations. The resulting datasets yielded distinct, reproducible signature phenotypes for a broad spectrum of genes that are involved in cell fate specification and morphogenesis. In addition, our analysis provides new in vivo evidence for MBK-2 function in mesoderm fate specification and LET-381 function in elongation.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Endoderma/embriologia , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mesoderma/embriologia , Mesoderma/metabolismoRESUMO
We derive from statistical mechanics the Gibbs free energy of an elastic random-walk chain affected by the presence of an external electric field. Intrachain charge interactions are ignored. In addition, we find two approximations of the Helmholtz potential for this system analogous to the gaussian and Cohen-Padé approximations for an elastic strand without the presence of an electric field. Our expressions agree well with exact numerical calculations of the potential in a wide range of conditions. Our analog of the gaussian approximation exhibits distortion of the monomer density due to the presence of the electric field, and our analog of the Cohen-Padé approximation additionally includes finite chain extensibility effects. The Helmholtz potential may be used in modeling the dynamics of electrophoresis experiments.
Assuntos
Termodinâmica , Campos EletromagnéticosRESUMO
Fam20C is a secreted protein kinase mutated in Raine syndrome, a human skeletal disorder. In vertebrates, bone and enamel proteins are major Fam20C substrates. However, Fam20 kinases are conserved in invertebrates lacking bone and enamel, suggesting other ancestral functions. We show that FAMK-1, the Caenorhabditis elegans Fam20C orthologue, contributes to fertility, embryogenesis, and development. These functions are not fulfilled when FAMK-1 is retained in the early secretory pathway. During embryogenesis, FAMK-1 maintains intercellular partitions and prevents multinucleation; notably, temperature elevation or lowering cortical stiffness reduces requirement for FAMK-1 in these contexts. FAMK-1 is expressed in multiple adult tissues that undergo repeated mechanical strain, and selective expression in the spermatheca restores fertility. Informatic, biochemical, and functional analysis implicate lectins as FAMK-1 substrates. These findings suggest that FAMK-1 phosphorylation of substrates, including lectins, in the late secretory pathway is important in embryonic and tissue contexts where cells are subjected to mechanical strain.
Assuntos
Caenorhabditis elegans/enzimologia , Caseína Quinase I/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Caseína Quinase I/genética , Células HEK293 , HumanosRESUMO
C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80-100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.
Assuntos
Caenorhabditis elegans/embriologia , Visualização de Dados , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Ensaios de Triagem em Larga Escala/métodos , Interpretação de Imagem Assistida por Computador/métodos , Morfogênese , Animais , Caenorhabditis elegans/fisiologiaRESUMO
To ensure timely cytokinesis, the equatorial actomyosin contractile ring constricts at a relatively constant rate despite its progressively decreasing size. Thus, the per-unit-length constriction rate increases as ring perimeter decreases. To understand this acceleration, we monitored cortical surface and ring component dynamics during the first cytokinesis of the Caenorhabditis elegans embryo. We found that, per unit length, the amount of ring components (myosin, anillin) and the constriction rate increase with parallel exponential kinetics. Quantitative analysis of cortical flow indicated that the cortex within the ring is compressed along the axis perpendicular to the ring, and the per-unit-length rate of cortical compression increases during constriction in proportion to ring myosin. We propose that positive feedback between ring myosin and compression-driven flow of cortex into the ring drives an exponential increase in the per-unit-length amount of ring myosin to maintain a high ring constriction rate and support this proposal with an analytical mathematical model.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas Contráteis/metabolismo , Citocinese/fisiologia , Retroalimentação Fisiológica/fisiologia , Mecanotransdução Celular/fisiologia , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Fenômenos Biomecânicos , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans/genética , Proteínas Contráteis/genética , Embrião não Mamífero , Expressão Gênica , Cinética , Miosinas/genética , Pressão , ReologiaRESUMO
The one-cell Caenorhabditis elegans embryo offers many advantages for mechanistic analysis of cell division processes. Conservation of key genes and pathways involved in cell division makes findings in C. elegans broadly relevant. A key technical advantage of this system is the ability to penetrantly deplete essential gene products by RNA interference (RNAi) and replace them with wild-type or mutant versions expressed at endogenous levels from single copy RNAi-resistant transgene insertions. This ability to precisely perturb essential genes is complemented by the inherently highly reproducible nature of the zygotic division that facilitates development of quantitative imaging assays. Here, we detail approaches to generate targeted single copy transgene insertions that are RNAi-resistant, to engineer variants of individual genes employing transgene insertions as well as at the endogenous locus, and to in situ tag genes with fluorophores/purification tags. We also describe imaging assays and common image analysis tools employed to quantitatively monitor phenotypic effects of specific perturbations on meiotic and mitotic chromosome segregation, centrosome assembly/function, and cortical dynamics/cytokinesis.
Assuntos
Caenorhabditis elegans/embriologia , Divisão Celular , Técnicas Citológicas/métodos , Embrião não Mamífero/citologia , Alelos , Animais , Sistemas CRISPR-Cas/genética , Caenorhabditis elegans/genética , Engenharia Genética , Microtúbulos/metabolismo , Mutação/genética , Interferência de RNA , Reprodutibilidade dos Testes , TransgenesRESUMO
An analytic expression is proposed for the primitive-path length of entangled polymer chains. The expression is derived from statistical mechanics of a chain that is a random walk with randomly scattered entanglements. The only parameters are the number of Kuhn steps in the chain and a dimensionless parameter beta that contains information about the entanglement density and Kuhn step size. The expression is found to compare very favorably with numerical results recently found from examining topological constraints in microscopic simulations. The comparison also predicts well the plateau modulus of polyethylene, suggesting that the slip-link model is a viable intermediate in the search for true ab initio rheology predictions. Since the expression is analytic, it can be used to make predictions where the simulations cannot reach, and hence is applicable for coarse graining.