Developmental Biology

X-ray computed microtomography (µCT) is a powerful tool to reveal the 3D structure of tissues and organs. Compared with the traditional sectioning, staining, and microscopy image acquisition, it allows a better understanding of the morphology and a precise morphometric analysis. Here, we describe a method for 3D visualization and morphometric analysis by µCT scanning of the embryonic heart of iodine-stained E15.5 mouse embryos.

Visualization of cell structure with fluorescent dye for characterizing cell size, shape, and arrangement is a common method to study tissue morphology and morphogenesis. In order to observe shoot apical meristem (SAM) in Arabidopsis thaliana by laser scanning confocal microscopy, we modified the pseudo-Schiff propidium iodide staining method by adding a series solution treatment to stain the deep cells. The advantage of this method is mainly reflected by the direct observation of the clearly bounded cell arrangement and the typical three-layer cells in SAM without the traditional tissue slicing.

A basic function of the nervous system is to confer the ability to detect external stimuli and generate appropriate behavioral and physiological responses. These can be modulated when parallel streams of information are provided to the nervous system and neural activity is appropriately altered. The nematode Caenorhabditis elegans utilizes a simple and well characterized neural circuit to mediate avoidance or attraction responses to stimuli, such as the volatile odorant octanol or diacetyl (DA), respectively. Aging and neurodegeneration constitute two important factors altering the ability to detect external signals and, therefore, changing behavior. Here, we present a modified protocol to assess avoidance or attraction responses to diverse stimuli in healthy individuals and Caenorhabditis elegans models associated with neurodegenerative diseases.

Accidental wounding of the peripheral nervous system leads to acute neural dysfunction. Normally, chronic deficits are overcome because peripheral nerves naturally regenerate. However, various genetic and metabolic defects can impair their natural regenerative capacity, which may be due to neuron-extrinsic mechanisms. Therefore, characterizing the behavior of multiple cells during nerve injury and repair in vivo is a pressing need in regenerative medicine. Here, we detail a method for precise wounding of sensory axons in zebrafish, followed by high-resolution in toto long-term quantitative videomicroscopy of neurons, Schwann cells, and macrophages. This protocol can be easily adapted to study the effects of targeted genetic or metabolic disruptions in zebrafish and other suitable organisms, as well as for screening pharmacological agents with therapeutic potential.

Graphical overview

Skeletal muscle is the most abundant tissue in the human body and has a tremendous capability to regenerate in response to muscle injuries and diseases. Induction of acute muscle injury is a common method to study muscle regeneration in vivo. Cardiotoxin (CTX) belongs to the family of snake venom toxins and is one of the most common reagents to induce muscle injury. Intramuscular injection of CTX causes overwhelming muscle contraction and lysis of myofibers. The induced acute muscle injury triggers muscle regeneration, allowing in-depth studies on muscle regeneration. This protocol describes a detailed procedure of intramuscular injection of CTX to induce acute muscle injury that could be also applied in other mammalian models.

The developing cerebral cortex of mammals is generated from nascent pyramidal neurons, which radially migrate from their birthplace in the ventral part of the neural tube to the cortical surface. Subtle aberrations in this process may cause significant changes in cortical structure and lead to developmental neurological disorders. During pyramidal neuron migration, we recently showed that the migrating neuron, which bypasses its last preceding neuron, is critical for its proper positioning and contributes to cerebral cortex thickness. Studying this process requires an imaging system with single-cell resolution and a prolonged observation window. Therefore, we built a system to maintain an organotypic brain slice on the stage of a Leica SP5 confocal microscope, which facilitated high-resolution imaging over a 12-hour time-lapse observation period of cellular events during neuron migration. Here, we share our protocol along with guidelines for overcoming difficulties during the setup. This protocol facilitates the observation of, but is not limited to, neurodevelopmental and pathological processes occurring during neuron migration.

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.

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.

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.

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.