0 Q&A 184 Views Mar 20, 2023

Phagoptosis is a prevalent type of programmed cell death (PCD) in adult tissues in which phagocytes non-autonomously eliminate viable cells. Therefore, phagoptosis can only be studied in the context of the entire tissue that includes both the phagocyte executors and the targeted cells doomed to die. Here, we describe an ex vivo live imaging protocol of Drosophila testis to study the dynamics of phagoptosis of germ cell progenitors that are spontaneously removed by neighboring cyst cells. Using this approach, we followed the pattern of exogenous fluorophores with endogenously expressed fluorescent proteins and revealed the sequence of events in germ cell phagoptosis. Although optimized for Drosophila testis, this easy-to-use protocol can be adapted to a wide variety of organisms, tissues, and probes, thus providing a reliable and simple means to study phagoptosis.

0 Q&A 59 Views Mar 20, 2023

Adult stem cells not only maintain tissue homeostasis but are also critical for tissue regeneration during injury. Skeletal stem cells are multipotent stem cells that can even generate bones and cartilage upon transplantation to an ectopic site. This tissue generation process requires essential stem cell characteristics including self-renewal, engraftment, proliferation, and differentiation in the microenvironment. Our research team has successfully characterized and isolated skeletal stem cells (SSCs) from the cranial suture called suture stem cells (SuSCs), which are responsible for craniofacial bone development, homeostasis, and injury-induced repair. To assess their stemness features, we have demonstrated the use of kidney capsule transplantation for an in vivo clonal expansion study. The results show bone formation at a single-cell level, thus permitting a faithful assessment of stem cell numbers at the ectopic site. The sensitivity in assessing stem cell presence permits using kidney capsule transplantation to determine stem cell frequency by limiting dilution assay. Here, we described detailed protocols for kidney capsule transplantation and limiting dilution assay. These methods are extremely valuable both for the evaluation of skeletogenic ability and the determination of stem cell frequency.

0 Q&A 74 Views Mar 20, 2023

Successful advancement in the treatment of diabetes mellitus is not possible without well-established methodology for beta cell mass calculation. Here, we offer the protocol to assess beta cell mass during embryonic development in the mouse. The described protocol has detailed steps on how to process extremely small embryonic pancreatic tissue, cut it on the cryostat, and stain tissue slides for microscopic analysis. The method does not require usage of confocal microscopy and takes advantage of enhanced automated image analysis with proprietary as well as open-source software packages.

0 Q&A 310 Views Feb 5, 2023

Adult muscle stem cells (MuSCs) show remarkable capability in repairing injured tissues. Studying MuSCs in suitable model organisms, which show strong homology with vertebrate counterparts, helps in dissecting the mechanisms regulating their behavior. Additionally, ease of handling and access to technological tools make model organisms well suited for studying biological processes that are conserved across species. MuSCs quiescence, proliferation, and migration are regulated by various input of signals from the surrounding tissues that constitute the MuSCs niche. Observing MuSCs along with their niche in vivo through live imaging provides key information on how MuSCs behave in quiescent and activated states. Drosophila melanogaster is well known for its genetic tool arsenal and the similarity of its different biological processes with vertebrates. Hence, it is widely used to study different types of stem cells. Gained knowledge could then be extrapolated to the vertebrate/mammalian homologous systems to enhance our knowledge in stem cell fields. In this protocol, we discuss how to perform live cell imaging of Drosophila MuSCs, called adult muscle precursors (AMPs) at embryonic stages, using dual-color labelling to visualize both AMPs and the surrounding tissues. This dual-color fluorescent labelling enables the observation of in vivo behavior of two types of cells simultaneously and provides key information on their interactions. The originality of this protocol resides in its biological application to MuSCs and their niche.

0 Q&A 456 Views Jan 20, 2023

Drosophila melanogaster is a classic model organism to study gene function as well as toxicological effects. To study gene function, the expression of a particular gene of interest is disrupted by using the widely explorable Drosophila genetic toolkit, whereas to study toxicological effects the flies are exposed to a particular toxicant through diet. These experiments often require the quantification of lethality from embryonic to adult stages, as well as the assessment of the life span in order to check the role of the gene/toxicant of interest in Drosophila. Here, we propose an experimental protocol that enables a consistent and rigorous assessment of lethality and life span of cadmium chloride (CdCl2)–exposed or genetically perturbed flies [downregulation and overexpression of the cytosolic Cu, Zn superoxide dismutase (SOD1) gene], consecutively. The protocol insists upon the requirement of one single experimental setup that is unique, distinctive, and cost-effective as it engages minimal laboratory equipment and resources. The described methods lead to the smooth observation of the embryos, their successive stagewise transition, and life span of the adult flies post eclosion. Additionally, these methods also facilitate the assessment of crawling and climbing behavioral parameters of the larvae and adults, respectively, and allow the calculation of lethal concentration (LC50) for the mentioned toxicant as well as median survival of the flies, which can be a determining factor in proceeding with further stages of experiments.

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0 Q&A 383 Views Jan 5, 2023

Utilizingresources available from the mother's body to guarantee healthy offspring growth is the fundamental reproductive strategy. Recently, we showed that a class of the largest extracellular vesicles known as exophers, which are responsible for the removal of neurotoxic components from neurons (Melentijevic et al., 2017) and damaged mitochondria from cardiomyocytes (Nicolás-Ávila et al., 2020), are released by the Caenorhabditis elegans hermaphrodite body wall muscles (BWM), to support embryonic growth (Turek et al., 2021). Employing worms expressing fluorescent reporters in BWM cells, we found that exopher formation (exophergenesis) is sex-specific and fertility-dependent. Moreover, exophergenesis is regulated by the developing embryo in utero, and exophers serve as transporters for muscle-generated yolk proteins, which can be used to nourish the next generation. Given the specific regulation of muscular exophergenesis, and the fact that muscle-generated exophers are much larger than neuronal ones and have different targeting, their identification and quantification required a modified approach from that designed for neuronal-derived exophers (Arnold et al., 2020). Here, we present a methodology for assessing and quantifying muscle-derived exophers that can be easily extended to determine their function and regulation in various biological contexts.

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0 Q&A 161 Views Jan 5, 2023

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.

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0 Q&A 699 Views Nov 20, 2022

During an animal's development, a large number of cells undergo apoptosis, a suicidal form of death. These cells are promptly phagocytosed by other cells and degraded inside phagosomes. The recognition, engulfment, and degradation of apoptotic cells is an evolutionarily conserved process occurring in all metazoans. Recently, we discovered a novel event in the nematode Caenorhabditis elegans: the double-membrane autophagosomes are recruited to the surface of phagosomes; subsequently, the outer membrane of an autophagosome fuses with the phagosomal membrane, allowing the inner vesicle to enter the phagosomal lumen and accumulate there over time. This event facilitates the degradation of the apoptotic cell inside the phagosome. During this study, we developed a real-time imaging protocol monitoring the recruitment and fusion of autophagosomes to phagosomes over two hours during embryonic development. This protocol uses a deconvolution-based microscopic imaging system with an optimized setting to minimize photodamage of the embryo during the recording period for high-resolution images. Furthermore, acid-resistant fluorescent reporters are chosen to label autophagosomes, allowing the inner vesicles of an autophagosome to remain visible after entering the acidic phagosomal lumen. The methods described here, which enable high sensitivity, quantitative measurement of each step of the dynamic incorporation in developing embryos, are novel since the incorporation of autophagosomes to phagosomes has not been reported previously. In addition to studying the degradation of apoptotic cells, this protocol can be applied to study the degradation of non-apoptotic cell cargos inside phagosomes, as well as the fusion between other types of intracellular organelles in living C. elegans embryos. Furthermore, its principle of detecting the membrane fusion event can be adapted to study the relationship between autophagosomes and phagosomes or other intracellular organelles in any biological system in which real-time imaging can be conducted.

0 Q&A 813 Views Oct 20, 2022

When understanding the neuronal function of a specific neural circuit, single-cell level photoablation of a targeted cell is one of the useful experimental approaches. This protocol describes a method to photoablate specific motor neurons via the mini singlet oxygen generator (miniSOG2), a light–oxygen–voltage (LOV)-based optogenetic tool used for ablating targeted cells in arbitrary areas. MiniSOG2 could induce the cell death pathway by generating reactive oxygen species (ROS) upon blue light illumination. Photoablation of a specific cell using the miniSOG2 was performed to show that, in Ciona intestinalis type A (Ciona robusta), a single pair of motor neurons, MN2/A10.64, is necessary to drive their tail muscle contraction. The membrane targeted miniSOG2 combined with neuron-specific promoter (pSP-Neurog::miniSOG2-CAAX) was electroplated into the Ciona egg and transiently expressed at specific neurons of the embryo. MN2 labeled with pSP-Neurog:mCherry-CAAX was irradiated using a 440-nm laser from the lateral side for 10 min to ablate its neural function. The behavior of the embryo before and after the irradiation was recorded with a high-speed camera.

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0 Q&A 704 Views Oct 20, 2022

Infertility has become a major public health problem, with a male factor involved in about half the cases. Mice are the most widely used animal model in reproductive biology research laboratories, but changes in sperm parameters in mice can be subtle and, in the absence of official guidelines, it is important that analyses are carried out in a strict and reproductive manner. This protocol successively details the different steps required to obtain spermatozoa under good conditions, the measurement of sperm motility using a Computer Assisted Sperm Analysis System (CASA) device, the calculation of sperm concentration in the epididymides using a sperm counting cell, and the examination of sperm morphology. The combination of these assays provides an overview of the basic sperm parameters in mice. This is both a diagnostic and a decision-making tool for researchers to orient their scientific strategy according to the observed abnormalities.

0 Q&A 1001 Views Sep 20, 2022

The activity of numerous autophagy-related proteins depends on their phosphorylation status, which places importance on understanding the responsible kinases and phosphatases. Great progress has been made in identifying kinases regulating autophagy, but much less is known about the phosphatases counteracting their function. Genetic screens and modern proteomic approaches provide powerful tools to identify candidate phosphatases, but further experiments are required to assign direct roles for candidates. We have devised a novel protocol to test the role of purified phosphatases in dephosphorylating specific targets in situ. This approach has the potential to visualize context-specific differences in target dephosphorylation that are not easily detected by lysate-based approaches such as Western blots.

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0 Q&A 1121 Views Sep 5, 2022

Skeletal muscle stem cells differentiated from human-induced pluripotent stem cells (hiPSCs) serve as a uniquely promising model system for investigating human myogenesis and disease pathogenesis, and for the development of gene editing and regenerative stem cell therapies. Here, we present an effective and reproducible transgene-free protocol for derivation of human skeletal muscle stem cells, iMyoblasts, from hiPSCs. Our two-step protocol consists of 1) small molecule-based differentiation of hiPSCs into myocytes, and 2) stimulation of differentiated myocytes with growth factor-rich medium to activate the proliferation of undifferentiated reserve cells, for expansion and cell line establishment. iMyoblasts are PAX3+/MyoD1+ myogenic stem cells with dual potential to undergo muscle differentiation and to self-renew as a regenerative cell population for muscle regeneration both ex vivo and in vivo. The simplicity and robustness of iMyoblast generation and expansion have enabled their application to model the molecular pathogenesis of Facioscapulohumeral Muscular Dystrophy and Limb-Girdle Muscular Dystrophies, to both ex vivo and in vivo muscle xenografts, and to respond efficiently to gene editing, enabling the co-development of gene correction and stem cell regenerative therapeutic technologies for the treatment of muscular dystrophies and muscle injury.

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0 Q&A 1627 Views Aug 20, 2022

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.