Developmental Biology

Zebrafish and medaka are valuable model vertebrates for genetic studies. The advent of CRISPR-Cas9 technology has greatly enhanced our capability to produce specific gene mutants in zebrafish and medaka. Analyzing the phenotypes of these mutants is essential for elucidating gene function, though such analyses often yield unexpected results. Consequently, providing researchers with accessible and cost-effective phenotype analysis methods is crucial. A prevalent technique for investigating calcified bone development in these species involves using transgenic fish that express fluorescent proteins labeling calcified bones; however, acquiring these fish and isolating appropriate crosses can be time-consuming. We present a comprehensive protocol for visualizing ossified bones in zebrafish and medaka larvae and juveniles using calcein and alizarin red S staining, which is both economical and efficient. This method, applicable to live specimens during the ossification of bones, avoids apparent alterations in skeletal morphology and allows for the use of different fluorescent dyes in conjunction with transgenic labeling, thus enhancing the analysis of developmental processes in calcifying bones, such as vertebrae and fin rays.

Developing a physiologically relevant in vitro model of the respiratory epithelium is critical for understanding lung development and respiratory diseases. Here, we describe a detailed protocol in which the fetal mouse proximal epithelial progenitors were differentiated into 3D airway organoids, which contain terminal-differentiated ciliated cells and basal stem cells. These differentiated airway organoids could constitute an excellent experimental model to elucidate the molecular mechanisms of airway development and epithelial cell fate determination and offer an important tool for establishing pulmonary dysplasia disease in vitro.

Osteoclasts are terminally differentiated multinucleated giant cells that mediate bone resorption and regulate skeletal homeostasis under physiological and pathological states. Excessive osteoclast activity will give rise to enhanced bone resorption, being responsible for a wide range of metabolic skeletal diseases, ranging from osteoporosis and rheumatoid arthritis to tumor-induced osteolysis. Therefore, the construction of in vitro models of osteoclast-mediated bone resorption is helpful to better understand the functional status of osteoclasts under (patho)physiological conditions. Notably, it is essential to provide an in vivo–relevant bone substrate that induces osteoclasts to generate authentic resorption lacunae and excavate bone. Here, we summarize the experimental design of a reproducible and cost-effective method, which is suitable for evaluating the regulatory mechanisms and influence of molecular agonists and antagonists as well as therapeutics on osteoclast-mediated bone-resorbing activity.

Neuroscience incorporates manipulating neuronal circuitry to enhance the understanding of intricate brain functions. An effective strategy to attain this objective entails utilizing viral vectors to induce varied gene expression by delivering transgenes into brain cells. Here, we combine the use of transgenic mice, neonatal transduction with adeno-associated viral constructs harboring inhibitory designer receptor exclusively activated by designer drug (DREADD) gene, and the DREADD agonist clozapine N-oxide (CNO). In this way, a chemogenetic approach is employed to suppress neuronal activity in the region of interest during a critical developmental window, with subsequent investigation into its effects on the neuronal circuitry in adulthood.

All living organisms require the division of a cell into daughter cells for their growth and maintenance. During cell division, both genetic and cytoplasmic contents are equally distributed between the two daughter cells. At the end of cell division, cytoplasmic contents and the plasma membrane are physically separated between the two daughter cells via a process known as cytokinesis. Hundreds of proteins and lipids involved in the cytokinetic process have been identified; however, much less is known about the mechanisms by which these molecules regulate cytokinesis, being therefore an intense area of current research. Male meiotic cytokinesis in Drosophila melanogaster testes has been shown to be an excellent model to study cytokinesis in vivo. Currently, several excellent protocols are available to study cytokinesis in Drosophila testes. However, improved methods are required to study cytokinesis under in vitro and ex vivo conditions. Here, we demonstrate a simple method to perform live imaging on individual spermatocyte cysts isolated from adult testes. We evaluate amenability of this in vitro method for treatment with pharmacological agents. We show that cytokinesis is strongly inhibited upon treatment with Dynasore, a dynamin inhibitor known to block clathrin-mediated endocytosis. In addition, we also demonstrate an ex vivo method to perform live imaging on whole mount adult testes on gas permeable membrane chambers. We believe the protocols described here are valuable tools to study cytokinetic mechanisms under various genetic and treatment conditions.

Key features

• In vitro method to study male meiotic cytokinesis in dissected spermatocyte cysts.

• In vitro method allows acute treatment with various pharmacological agents to study cytokinesis.

• Ex vivo method to image male meiosis cytokinesis in intact adult testes.

• Requires 15–60 min to set up and could be imaged up to 6–12 h.

Graphical overview

In vitro and ex vivo live imaging of male meiotic cytokinesis in adult Drosophila testes

The African killifish Nothobranchius furzeri is an attractive research organism for regeneration- and aging-related studies due to its remarkably short generation time and rapid aging. Dynamic changes in cell proliferation are an essential biological process involved in development, regeneration, and aging. Quantifying the dynamics of cell proliferation in these contexts facilitates the elucidation of the attendant underlying mechanisms. Whole-mount and cryosectioning sample preparation are the preferred approaches to investigate the distribution of cellular structures, cell–cell communication, and spatial gene expression within tissues. Using African killifish caudal fin regeneration as an example, we describe an efficient and detailed protocol to investigate cell proliferation dynamics in both space and time during caudal fin regeneration. The quantification of cell proliferation was achieved through high-resolution immunofluorescence of the proliferation marker Phospho-Histone H3 (H3P). We focused on the characterization of epithelial and mesenchymal proliferation in three-dimensional space at two regeneration time points. Our protocol provides a reliable tool for comparing cell proliferation under different biological contexts.

Key features

• Elaborates in detail the method used by Wang et al. (2020) to quantify whole-organ mitotic events during tail fin regeneration in vertebrates.

• Enables proliferation analysis of millimeter-sized homeostatic and regenerating tissues.

• Three-day alternative method to whole mount using cryosections.

• Allows automatic quantification using ImageJ macros and R scripts.

Graphical overview

The hypothalamus is an evolutionarily ancient part of the vertebrate ventral forebrain that integrates the dialogue between environment, peripheral body, and brain to centrally govern an array of physiologies and behaviours. Characterizing the mechanisms that control hypothalamic development illuminates both hypothalamic organization and function. Critical to the ability to unravel such mechanisms is the skill to isolate hypothalamic tissue, enabling both its acute analysis and its analysis after explant and culture. Tissue explants, in which cells develop in a manner analogous to their in vivo counterparts, are a highly effective tool to investigate the extrinsic signals and tissue-intrinsic self-organising features that drive hypothalamic development. The hypothalamus, however, is induced and patterned at neural tube stages of development, when the tissue is difficult to isolate, and its resident cells complex to define. No single molecular marker distinguishes early hypothalamic progenitor subsets from other cell types in the neural tube, and so their accurate dissection requires the simultaneous analysis of multiple proteins or mRNAs, techniques that were previously limited by antibody availability or were arduous to perform. Here, we overcome these challenges. We describe methodologies to precisely isolate early hypothalamic tissue from the embryonic chick at three distinct patterning stages and to culture hypothalamic explants in three-dimensional gels. We then describe optimised protocols for the analysis of embryos, isolated embryonic tissue, or cultured hypothalamic explants by multiplex hybridisation chain reaction. These methods can be applied to other vertebrates, including mouse, and to other tissue types.

Key features

• Detailed protocols for enzymatic isolation of embryonic chick hypothalamus at three patterning stages; methods can be extended to other vertebrates and tissues.

• Brief methodologies for three-dimensional culture of hypothalamic tissue explants.

• Optimised protocols for multiplex hybridisation chain reaction for analysis of embryos, isolated embryonic tissues, or explants.

Graphical overview

Neovascular diseases of the retina, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), are proliferative retinopathies involving the growth of new blood vessels on the retina, which in turn causes impairment and potential loss of vision. A drawback of conventional angiogenesis assays is that they are not representative of the angiogenic processes in the retina. In the retina, the new blood vessels grow (from pre-existing blood vessels) and migrate into a non-perfused region of the eye including the inner limiting membrane of the retina and the vitreous, both of which contribute to vision loss. The Matrigel Duplex Assay (MDA) measures the migration of angiogenic capillaries from a primary Matrigel layer to a secondary Matrigel layer, which resembles the pathological angiogenesis in AMD and DR. The methodology of MDA is comprised of two steps. In the first step, the human retinal microvascular endothelial cells (HRMECs) are mixed with phenol red–containing Matrigel (in a 1:1 ratio) and seeded in the center of an 8-well chamber slide. After 24 h, a second layer of phenol red–free Matrigel is overlaid over the first layer. Over the course of the next 24 h, the HRMECs invade from the primary Matrigel layer to the secondary layer. Subsequently, the angiogenic sprouts are visualized by brightfield phase contrast microscopy and quantified by ImageJ software. The present manuscript measures the angiogenesis-inhibitory activity of the Src kinase inhibitor PP2 in primary HRMECs using the MDA. The MDA may be used for multiple applications like screening anti-angiogenic drugs, measuring the pro-angiogenic activity of growth factors, and elucidating signaling pathways underlying retinal angiogenesis in normal and disease states.

Graphical overview

The interaction of RNA with specific RNA-binding proteins (RBP) leads to the establishment of complex regulatory networks through which gene expression is controlled. Careful consideration should be given to the exact environment where a given RNA/RBP interplay occurs, as the functional responses might depend on the type of organism as well as the specific cellular or subcellular contexts. This requisite becomes particularly crucial for the study of long non-coding RNAs (lncRNA), as a consequence of their peculiar tissue-specificity and timely regulated expression. The functional characterization of lncRNAs has traditionally relied on the use of established cell lines that, although useful, are unable to fully recapitulate the complexity of a tissue or organ. Here, we detail an optimized protocol, with comments and tips, to identify the RNA interactome of given RBPs by performing cross-linking immunoprecipitation (CLIP) from mouse embryonal hearts. We tested the efficiency of this protocol on the murine pCharme, a muscle-specific lncRNA interacting with Matrin3 (MATR3) and forming RNA-enriched condensates of biological significance in the nucleus.

Key features

• The protocol refines previous methods of cardiac extracts preparation to use for CLIP assays.

• The protocol allows the quantitative RNA-seq analysis of transcripts interacting with selected proteins.

• Depending on the embryonal stage, a high number of hearts can be required as starting material.

• The steps are adaptable to other tissues and biochemical assays.

Graphical overview

Identification of RNA/protein interactions from developing hearts

Congenital heart disease (CHD) is often associated with myogenic defects. During heart development, cardiomyocyte growth requires essential cues from extrinsic factors such as insulin-like growth factor 2 (IGF-2). To determine whether and how growth factors account for embryonic cardiomyocyte proliferation, isolation followed by culturing of embryonic cardiomyocytes can be utilized as a useful tool for heart developmental studies. Current protocols for isolating cardiomyocytes from the heart do not include a cardiomyocyte-specific reporter to distinguish cardiomyocytes from other cell types. To optimize visualization of cardiomyocyte proliferation, our protocol utilizes a Tnnt2-promoter-driven H2B-GFP knock-in mouse model (TNNT2^H2B-GFP/+) for in vitro visualization of nuclear-tagged cardiomyocyte-specific fluorescence. A cardiomyocyte-specific genetic reporter paired with an effective proliferation assay improves the reproducibility of mechanistic studies by increasing the accuracy of cell identification, proliferated cell counting, and cardiomyocyte tracking.

Key features

• This protocol refines previous methods of cardiomyocyte isolation to specifically target embryonic cardiomyocytes.

• UsesH2B-GFP/+cardiomyocyte reporters as identified by Yan et al. (2016).

• Traces cell proliferation with Phospho-Histone 3 (p-H3) assay.

• Has applications in assessing the role of growth factors in cardiomyocyte proliferation.

Graphical overview