Developmental Biology

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

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.

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.

Graphical abstract

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.

Graphical abstract:

Over the past years, research has made impressive breakthroughs towards the development and implementation of 3D cell models for a wide range of applications, such as drug development and testing, organogenesis, cancer biology, and personalized medicine. Opposed to 2D cell monolayer culture systems, advanced 3D cell models better represent the in vivo physiology. However, for these models to deliver scientific insights, appropriate investigation techniques are required. Despite the potential of fluorescence microscopy to visualize these models with high spatial resolution, sample preparation and imaging assays are not straightforward. Here, we provide different protocols of sample preparation for fluorescence imaging, for both matrix-embedded and matrix-free models (e.g., organoids and spheroids, respectively). Additionally, we provide detailed guidelines for imaging 3D cell models via confocal multi-photon fluorescence microscopy. We show that using these protocols, images of 3D cell culture systems can be obtained with sub-cellular resolution.

Graphical abstract:

Chromatin accessibility is a key determinant of gene expression that can be altered under different physiological and disease conditions. Skeletal muscle is made up of myofibers that are highly plastic and adaptive. Therefore, assessing the genome-wide chromatin state of myofibers under various conditions is very important to gain insight into the epigenetic state of myonuclei. The rigid nature of myofibers, as well as the low number of myonuclei that they contain, have rendered genome-wide studies with myofibers challenging. In recent years, ATAC-Seq from whole muscle and single nucleus ATAC-Seq have been performed. However, these techniques cannot distinguish between different fiber and cell types present in the muscle. In addition, due to the limited depth capacity obtained from single nucleus ATAC-Seq, an extensive comparative analysis cannot be performed. Here, we introduce a protocol where we combine the isolation of a single myofiber with OMNI ATAC-Seq. This protocol allows for genome-wide analysis of accessible chromatin regions of a selected single myofiber at a sufficient depth for comparative analysis under various physiological and disease conditions. This protocol can also allow for a specific myofiber to be selected, such as a regenerating myofiber. In the future, this protocol can help identify global changes in chromatin state under various conditions, as well as between different types of myofibers.

Graphical abstract:

Live labelling of active transcription sites is critical to our understanding of transcriptional dynamics. In the most widely used method, RNA sequence MS2 repeats are added to the transcript of interest, on which fluorescently tagged Major Coat Protein binds, and labels transcription sites and transcripts. Here we describe another strategy, using the Argonaute protein NRDE-3, repurposed as an RNA-programmable RNA binding protein. We label active transcription sites in C. elegans embryos and larvae, without editing the gene of interest. NRDE-3 is programmed by feeding nematodes with double-stranded RNA matching the target gene. This method does not require genome editing and is inexpensive and fast to apply to many different genes.

Graphical abstract:

Our ability to move and breathe requires an efficient communication between nerve and muscle that mainly takes place at the neuromuscular junctions (NMJs), a highly specialized synapse that links the axon of a motor neuron to a muscle fiber. When NMJs or axons are disrupted, the control of muscle fiber contraction is lost and muscle are paralyzed. Understanding the adaptation of the neuromuscular system to permanent or transient denervation is a challenge to understand the pathophysiology of many neuromuscular diseases. There is still a lack of in vitro models that fully recapitulate the in vivo situation, and in vivo denervation, carried out by transiently or permanently severing the nerve afferent to a muscle, remains a method of choice to evaluate reinnervation and/or the consequences of the loss of innervation. We describe here a simple surgical intervention performed at the hip zone to expose the sciatic nerve in order to obtain either permanent denervation (nerve-cut) or transient and reversible denervation (nerve-crush). These two methods provide a convenient in vivo model to study adaptation to denervation.

Graphical abstract:

The epidermis is the outermost layer of the skin. It is made up of mostly keratinocytes along with a small number of melanocytes and Langerhans cells. Melanocytes produce a pigment called melanin, which is transferred to the keratinocytes, and protects these cells from damage from UV radiation, as well as generating hair and skin colours. In this important relationship, keratinocytes exert control over melanocytes. Many questions regarding keratinocyte-melanocyte interactions have yet to be answered, and would benefit from study in model systems, to address diseases such as vitiligo and cutaneous melanoma. Most of the mouse is covered in fur and these areas lack the skin pigmenting inter-follicular epidermal (IFE) melanocytes. However, the mouse tail is pigmented analogously to human skin. Here, we present a method for isolating IFE melanocytes or keratinocytes expressing the tdTomato marker from the mouse tail, using fluorescence-activated cell sorting (FACS). The method involves firstly separating the tail skin epidermis from the dermis, and then digesting the epidermis to produce dissociated cells, which can then be sorted. These isolated cell populations can be studied using RNAseq or cultured in vitro. This protocol isolates IFE melanocytes or keratinocytes and immediately provides reasonable yields of cells, without the need to stain the cells for cell specific markers.

Craniofacial anomalies (CFA) are a diverse group of deformities, which affect the growth of the head and face. Dysregulation of cranial neural crest cell (NCC) migration, proliferation, differentiation, and/or cell fate specification have been reported to contribute to CFA. Understanding of the mechanisms through which cranial NCCs contribute for craniofacial development may lead to identifying meaningful clinical targets for the prevention and treatment of CFA. Isolation and culture of cranial NCCs in vitro facilitates screening and analyses of molecular cellular mechanisms of cranial NCCs implicated in craniofacial development. Here, we present a method for the isolation and culture of cranial NCCs harvested from the first branchial arch at early embryonic stages. Morphology of isolated cranial NCCs was similar to O9-1 cells, a cell line for neural crest stem cells. Moreover, cranial NCCs isolated from a transgenic mouse line with enhanced bone morphogenetic protein (BMP) signaling in NCCs showed an increase in their chondrogenic differentiation capacity, suggesting maintenance of their in vivo differentiation potentials observed in vitro. Taken together, our established method is useful to visualize cellular behaviors of cranial NCCs.