发育生物学

Vertebrate embryogenesis is a highly dynamic process involving coordinated cell and tissue movements that generate the final embryonic body plan. Many of these movements are difficult to image at high resolution because they occur deep within the embryo along the midline, causing light scattering and requiring longer working distances. Here, we present an explant-based method to image transverse cross sections of living zebrafish embryos. This method allows for the capture of all cell movements at high-resolution throughout the embryonic trunk, including hard-to-image deep tissues. This technique offers an alternative to expensive or computationally difficult microscopy methods.

Key features

• Generates intact zebrafish explants with minimal tissue disturbance.

• Allows for live imaging of deep tissues normally obscured by common confocal microscopy techniques.

• Immobilizes tissues for extended periods required for time-lapse imaging.

• Utilizes readily available reagents and tools, which can minimize the time and cost of the procedure.

Graphical overview

Advances in imaging technology offer new opportunities in developmental biology. To observe the development of internal structures, microtome cross-sectioning followed by H&E staining on glass slides is a common procedure; however, this technique can be destructive, and artifacts can be introduced during the process. In this protocol, we describe a less invasive procedure with which we can stain insect samples and obtain reconstructed three-dimensional images using micro-computed tomography, or micro-CT (µCT). Specifically, we utilize the fungus-farming ambrosia beetle species Euwallacea validus to observe the morphology of mycangia, a critical internal organ with which beetles transport fungal symbionts. Not only this protocol is ideal to observe mycangia, our staining/scanning procedure can also be applied to observe other delicate tissues and small organs in arthropods.

Graphical abstract

Currently, there are several in vitro protocols that focus on directing human induced pluripotent stem cell (hiPSC) differentiation into either the cardiac or pulmonary lineage. However, these systemsprotocols are unable to recapitulate the critical exchange of signals and cells between the heart and lungs during early development. To address this gap, here we describe a protocol to co-differentiate cardiac and pulmonary progenitors within a single hiPSC culture by temporal specific modulation of Wnt and Nodal signaling. Subsequently, human cardio-pulmonary micro-tissues (μTs) can be generated by culturing the co-induced cardiac and pulmonary progenitors in 3D suspension culture. Anticipated results include expedited alveolarization in the presence of cardiac cells, and segregation of the cardiac and pulmonary μTs in the absence of exogenous Wnt signaling. This protocol can be used to model cardiac and pulmonary co-development, with potential applications in drug testing, and as a platform for expediting the maturation of pulmonary cells for lung tissue engineering.

Aging and neuronal deterioration constitute important risk factors for the development of neuronal-related diseases, such as different dementia. The nematode Caenorhabditis elegans has emerged as a popular model system for studying neurodegeneration diseases, due to its complete neuronal connectivity map. DiI is a red fluorescent dye that can fill the worm amphid neurons and enables the visualization of their neurodegeneration over time. This protocol provides an efficient, fast, and safe method to stain worm amphid neurons to highlight the chemosensory structures of live nematodes.

In this study, we present a detailed protocol for live imaging and quantitative analysis of floral meristem development in Aquilegia coerulea, a member of the buttercup family (Ranunculaceae). Using confocal microscopy and the image analysis software MorphoGraphX, we were able to examine the cellular growth dynamics during floral organ primordia initiation, and the transition from floral meristem proliferation to termination. This protocol provides a powerful tool to study the development of the meristem and floral organ primordia, and should be easily adaptable to many plant lineages, including other emerging model systems. It will allow researchers to explore questions outside the scope of common model systems.

In the Japanese rhinoceros beetle Trypoxylus dichotomus, various candidate genes required for a specific phenotype of interest are listed by next-generation sequencing analysis. Their functions were investigated using RNA interference (RNAi) method, the only gene function analysis tool for T. dichotomus developed so far. The summarized procedure for the RNAi method used for T. dichotomus is to synthesize double-stranded RNA (dsRNA), and inject it in larvae or pupae of T. dichotomus. Although some dedicated materials or equipment are generally required to inject dsRNA in insects, the advantage of the protocol described here is that it is possible to inject dsRNA in T. dichotomus with one syringe.

Cytokinesis occurs at the final step of cell division and leads to the separation of daughter cells. It requires assembly and constriction of the actomyosin contractile ring. The phases of assembly and constriction of the contractile ring show an increase in tension in the actomyosin complex. The measurement of tension in the contractile ring is of interest to probe the mechanics of contractile ring formation. Drosophila cellularization is a powerful genetic model system to study the mechanisms regulating actomyosin contractility during contractile ring constriction. Cellularization occurs in the interphase of syncytial cycle 14, where the plasma membrane extends around individual nuclei and forms complete cells with the help of a contractile ring at the bottom. The contractile ring forms at the furrow tip during the extension around individual nuclei and its assembly requires the coordinated action of cytoskeletal and plasma membrane-associated proteins. Laser ablation of the contractile ring enables the measurement of the contractility of the actomyosin network during cytokinesis. This protocol outlines the method used for estimating the contractility at the actomyosin ring during cellularization by laser ablation, in both control and mutant embryos for a Rho guanosine triphosphatase activating protein (RhoGAP) containing protein called GRAF (GTPase regulator associated with focal adhesion kinase-1). Physical cutting of the contractile ring by a two-photon laser at 800 nm leads to the displacement of the actomyosin ring edges, at a rate dependent upon the tension. This can be carried out at distinct steps of the contractile ring assembly during furrow extension in cellularization. Quantification of the extent of displacement and recoil velocity of movement of the edges at different stages of cellularization provides a quantitative measure of contractility in the system. This protocol describes the experimental procedure containing the preparation of live embryos, optimization of laser power, acquisition settings, and post-acquisition analysis of actomyosin contractility during Drosophila cellularization.

Mitochondria are relatively small, fragmented, and abundant in the large embryos of Drosophila, Xenopus and zebrafish. It is essential to study their distribution and dynamics in these embryos to understand the mechanistic role of mitochondrial function in early morphogenesis events. Photoactivation of mitochondrially tagged GFP (mito-PA-GFP) is an attractive method to highlight a specific population of mitochondria in living embryos and track their distribution during development. Drosophila embryos contain large numbers of maternally inherited mitochondria, which distribute differently at specific stages of early embryogenesis. They are enriched basally in the syncytial division cycles and move apically during cellularization. Here, we outline a method for highlighting a population of mitochondria in discrete locations using mito-PA-GFP in the Drosophila blastoderm embryo, to follow their distribution across syncytial division cycles and cellularization. Photoactivation uses fluorophores, such as PA-GFP, that can change their fluorescence state upon exposure to ultraviolet light. This enables marking a precise population of fluorescently tagged molecules of organelles at selected regions, to visualize and systematically follow their dynamics and movements. Photoactivation followed by live imaging provides an effective way to pulse label a population of mitochondria and follow them through the dynamic morphogenetic events during Drosophila embryogenesis.

RNA sequencing allows for the quantification of the transcriptome of embryos to investigate transcriptional responses to various perturbations (e.g., mutations, infections, drug treatments). Previous protocols either lack the option to genotype individual samples, or are laborious and therefore difficult to do at a large scale. We have developed a protocol to extract total nucleic acid from individual zebrafish embryos. Individual embryos are lysed in 96-well plates and nucleic acid is extracted using SPRI beads. The total nucleic acid can be genotyped and then DNase I treated to produce RNA samples for sequencing. This protocol allows for processing large numbers of individual samples, with the ability to genotype each sample, which makes it possible to undertake transcriptomic analysis on mutants at timepoints before the phenotype is visible.

Graphic abstract:

Extraction of total nucleic acid from individual zebrafish embryos for genotyping and RNA-seq.

Lymphatic vessels are abundant in the skin where they regulate interstitial fluid uptake and immune surveillance. Defects in dermal lymphatic vessels, such as fewer vessels and abnormal lymphatic vessel coverage with mural cells, are frequently associated with lymphedema and other lymphatic disorders. Whole-mount immunohistochemistry allows the visualization of dermal lymphatic vessels and identifies morphogenetic defects. Most dermal lymphatic vessels start growing during embryogenesis from lymph sacs that are located close to the axilla towards the dorsal and ventral midlines. Here, we present an approach that we have developed to permeabilize, immunolabel, clear, and visualize the lymphatic vessels. These simple and inexpensive techniques reproducibly generate images of dermal lymphatic vessels with great clarity.