This work was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada and The Canada Foundation for Innovation. Thanks to Dr Paul Mains (University of Calgary, Calgary, Canada) for let-502(sb118ts) strain. Some of the strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

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The authors declare that they have no competing financial interests.

Este protocolo descreve novas métricas para caracterizar início e fases tardias de alongamento embrionário. Na seção 1, o passo crítico é a potencial presença de bactérias no teclado. Os embriões são hermeticamente fechado entre a almofada e a lamela durante a aquisição de imagem. Selagem a lâmina é necessário para evitar a dessecação dos animais durante a aquisição que dura mais do que duas horas. Para nosso conhecimento, nenhum dos cimentos usados ​​para montar almo...

Por quase 50 anos, os nematóides Caenorhabditis elegans estabeleceu-se como um modelo poderoso para estudar questões importantes no desenvolvimento, neurobiologia, evolução, interações patógeno-hospedeiro, etc. 1 A força deste modelo no estudo do desenvolvimento encontra-se em: seu ciclo de vida curta de 3 dias; a facilidade com que estes animais podem ser geneticamente alteradas; sua transparência que permite a observação de deslocamento e morfologia celular em animais vivos e seu desenvolvimento que é principalmente extra-uterina. As fases de desenvolvimento do nematóide envolvem embriogênese e quatro estágios larvais (L1 a L4), seguido por idade adulta. Durante o desenvolvimento embrionário, epidérmica morfogênese atraiu considerável atenção por sua capacidade de permitir uma melhor compreensão de como epitelial células migram como um grupo, como eles reorganizar suas junções e modificar sua morfologia individual, bem como o seu posicionamento relativo dentro de um epitélio funcional.morfogénese epidérmico é dividido em quatro fases: intercalação dorsal que consiste na reorganização das células epidérmicas dorsal, designado por hipoderme; ventral invólucro, que consiste na migração de células ventrais hipodérmicas para com a linha média ventral, assim, que encerra o embrião de uma monocamada de células epiteliais; início e alongamento final de transformação do embrião em forma de feijão em larvas vermiforme. Seguindo morfogênese, Vigia do embrião e as larvas L1 iniciar a alimentação por meio de bactérias disponíveis no seu ambiente imediato. alongamento embrionário é, por conseguinte, uma fase tardia do desenvolvimento embrionário. Consiste na extensão do embrião ao longo do seu eixo longitudinal e uma redução do seu diâmetro transversal. Isto envolve uma modificação da forma dramática das células hipodérmicas. Alongamento é dividido em um precoce e uma fase tardia. A fase inicial começa na fase vírgula e termina quando os músculos do corpo de parede começam a contrair na fase de 1,75 vezes em wdo tipo DPI (em peso) de embriões - correspondente a embriões que são 1,75 vezes em comprimento em comparação com os embriões não alongado. Morfogênese que ocorrem nessa fase são impulsionadas principalmente pela contração de feixes de filamentos de actina (FBS) localizadas no pólo apical de células hipodérmicas que impulsionam seu alongamento ao longo do eixo antero-posterior do embrião 2. A contração dos corpos estranhos é o controle por fosforilação das cadeias de miosina-luz por três quinases LET-502 / Rock, MRCK-1 e Pak-1 5. A fase final do alongamento, começa quando os músculos do corpo de parede se tornar funcional e começar a contratação. Trata-se de sinalização mecanotransdução dos músculos do corpo de parede para as células dorsal e ventral hipodérmicas e termina quando os animais chocar 3. Alongamento defeitos são geralmente caracterizadas por a percentagem de animais que morrem como embriões (letalidade embrionária; Emb) e aqueles prender o seu desenvolvimento como larvas L1 (fenótipo prisão larval; Lva) e ser significativamente menor do que em peso. A identificação do estágio da prisão de desenvolvimento requer observação microscópica de embriões mortos e presos Larvas 3-6. Recentemente, foi demonstrado que vários genes, tais como o regulador de Cdc42 / Rac efector e pix-1 e Pak-1, o controlo dos processos morfogénicas durante tanto precoce e tardia de alongamento 3,7. Nós também mostrou recentemente que os processos morfogénicas diferem ao longo do eixo ântero-posterior do embrião precoce durante o alongamento 3 7. Estes resultados motivou o desenvolvimento de novas métricas destinadas especificamente às etapas de alongamento precoce ou tardia e outras de medição, permitindo a caracterização da morfologia de embriões ao longo do seu eixo ântero-posterior durante o alongamento inicial. Estes novos métodos consistem na medição do comprimento de embriões no início e no final de alongamento precoce, bem como a largura da sua eleanúncios e caudas. 7 Dois protocolos foram também desenvolvidos para medir o comprimento das larvas recém-nascidas, sincronizados em L1 estágio 7. Os ovos dos embriões protegê-los contra o tratamento alcalino, enquanto hipoclorito larvas, e adultos bactérias presentes no meio de cultura são dissolvidos pelo tratamento. Este tratamento é, então, utilizada para purificar embriões a partir de uma população não sincronizada contendo a maioria dos adultos bem alimentados 8. restrição alimentar é usado para sincronizar as larvas recém-nascidas. Medindo a duração dessas larvas é então usada para detectar defeitos de alongamento. Esta medição é preferido em relação à medição de larvas preso em placas de cultura, porque as larvas que eclodem a partir de embriões não totalmente alongados pode recuperar a &quot;comprimento normal&quot; ao alimentar mas irá manter o seu tamanho reduzido, quando preso na ausência de comida. Aqui, apresentamos protocolos detalhados que permitem a medição da length de alongar embriões, bem como a largura da sua cabeça e cauda utilizando lapso de tempo de microscopia DIC e análise de imagem (Protocolo 1). Nós também fornecemos protocolos detalhados para medir o comprimento de larvas sincronizados usando análise de imagem (Protocolo 2) e citometria de fluxo (Protocolo 3).

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novel metrics to characterize embryonic elongation of the nematode caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Cabeça-, Cauda e Rácio de cabeça / cauda de largura são métricas robusto. Os protocolos aqui descritos têm sido utilizados com sucesso para caracterizar a função dos reguladores e dos efectores de Rho GTPases pix-1, Pak-1 e deixou-502 durante o alongamento precoce ...

Watch this Scientific Journal Video about Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans at JoVE.com

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Novas métricas para Caracterizar Embryonic Alongamento do nemátodo<em&gt; Caenorhabditis elegans</em

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

novel-metrics-to-characterize-embryonic-elongation-nematode

Research

JoVE Journal

Neuroscience

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

6.0K visualizações.  Université du Québec à Montréal. Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo. 

Vídeo: Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Medição Caenorhabditis elegans Expectativa de Vida em meios sólidos

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Biologia

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as self-fertilizing hermaphrodites, which produce large broods of progeny-when males are present, they can produce even larger broods of cross progeny. Errors in meiosis, fertilization, and embryogenesis can be rapidly assessed as phenotypes of sterility, reduced fertility, or embryonic lethality. This article describes a method to determine embryonic viability and brood size in C. elegans. We demonstrate how to set up this assay by picking a worm onto an individual Modified Youngren's, Only Bacto-peptone (MYOB) plate, establish the appropriate timeframe to count viable progeny and non-viable embryos, and explain how to accurately count live worm specimens. This technique can be used to determine viability in self-fertilizing hermaphrodites as well as cross-fertilization by mating pairs. These relatively simple experiments are easily adoptable for new researchers, such as undergraduate students and first-year graduate students.

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Here, we present a general method to determine the embryonic viability and total number of embryos produced (brood) using the model organism C. elegans.

Mensuração da Viabilidade Embrionária e do Tamanho da Ninhada em Caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as ...

Genética

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Microscopia de folha de luz isotrópica e análise de linhagem de células automatizadas para catalogar Caenorhabditis elegans Embryogenesis com resolução subcelular

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Biologia do Desenvolvimento

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Medindo a orientação e a motilidade do esperma dentro do intervalo reprodutivo hermafrodita dos elegans de Caenorhabditis

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

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&#8211;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.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

Um método de imagem semi-alta-fonte e um kit de ferramentas de visualização de dados para analisar o desenvolvimento embrionário de C. elegans

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

Microscopia 4D: Desvendando caenorhabditis elegans Desenvolvimento Embrionário usando Microscopia Nomarski

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Imagem C. elegans Os embriões utilizando um microscópio de epifluorescência e Software de Código Aberto

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Rastreamento e quantificação processos de desenvolvimento em<em&gt; C. elegans</em&gt; Como usar as ferramentas de código aberto

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Abordagens quantitativas para o estudo de estruturas celulares e morfologia organelle em Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Lifespan Analysis: Measuring C. elegans Longevity

Source: Cornwell, A. B., et al. <a href="https://www.jove.com/video/57819/the-replica-set-method-high-throughput-approach-to-quantitatively">The Replica Set Method: A High-throughput Approach to Quantitatively Measure&#160;Caenorhabditis elegans&#160;Lifespan</a>.&#160;J. Vis. Exp.&#160; (2018).
This video describes a traditional, plate-based method of measuring the lifespan of C. elegans. The example protocol measures the lifespan of worms treated with RNAi.

Novas métricas para Caracterizar Embryonic Alongamento do nemátodo

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