Developmental Biology

Maternal mRNAs and proteins are produced during oogenesis by more than 60% of zebrafish genes. They are indispensable for fertilization and early embryogenesis. Generation and analysis of the maternal mutant is the most direct way to characterize the maternal function of the specific gene. However, due to the lethality of zygotic mutants, the maternal function of most genes in zebrafish remains elusive. Several methods have been developed to circumvent this obstacle, including mRNA rescue, germ-line replacement, oocyte microinjection in situ, mosaic mutation, and bacterial artificial chromosome (BAC)-mediated conditional rescue. Here, we provide an alternative approach to generate zebrafish maternal mutants rapidly and efficiently by introducing four tandem sgRNA expression cassettes into Tg(zpc:zcas9) embryos. This method is more technically feasible and cost- and time-effective than other established methods.

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.

The mammary gland undergoes functional, developmental, and structural changes that are essential for lactation and reproductive processes. An overview of such unique tissue can offer clearer insights into mammary development and tumorigenesis. Compared to traditional methods, mouse mammary gland whole mount is a pivotal technique that provides three-dimensional structural perspectives on gland morphology and developmental stages, offering an inexpensive and accessible approach. This protocol outlines the tissue isolation of the mouse mammary gland and provides detailed instructions for whole-mount staining and analysis. Mammary gland tissues are carefully dissected from euthanized mice and stained with Carmine Alum to highlight the ductal structures, enabling detailed visualization of the branching patterns and morphological changes. Light microscopy is used to capture a panoramic image of the stained mammary gland, enabling the quantitative analysis of terminal end buds (TEBs) and bifurcated TEBs to further investigate mammary gland remodeling. This method can provide invaluable insights, particularly in the study of mammary gland morphogenesis and tumorigenesis, underscoring its significance in both basic research and clinical applications.

Enzyme-catalyzed proximity labeling is a potent technique for the discernment of subtle molecular interactions and subcellular localization, furnishing contextual insights into the protein of interest within cells. Although ascorbate peroxidase2 (APEX2) has proven effective in this approach when overexpressed, its compatibility with endogenous proteins remains untested. We improved this technique for studying native protein–protein interactions in live Drosophila ovary tissue. Through CRISPR/Cas9 genome editing, APEX2 was fused with the endogenous dysfusion gene. By pre-treating the tissue with Triton X-100 to enhance biotin-phenol penetration, we successfully identified multiple proteins interacting with the native Dysfusion proteins that reside on the inner nuclear membrane. Our protocol offers a comprehensive workflow for delineating the interactome networks of ovarian components in Drosophila, aiding future studies on endogenous protein–protein interactions in various tissues of other animals.

In this protocol, we focused on analyzing internal branches of Drosophila class IV neurons. These neurons are characterized by their highly branched axons and dendrites and intricately tile the larval body. As Drosophila larvae progress through developmental stages, the dendritic arbors of Class IV neurons undergo notable transformations. As Drosophila larvae develop, their Class IV dendritic arbors grow. In the initial 24 h after egg laying (AEL), the dendrites are smaller than segments. During the subsequent 24 h of the first instar larval stage, dendritic arbors outpace segment growth, achieving tiling. After 48 h, arbors and segments grow concurrently. Epidermal cells near Class IV dendrites expand in proportion to segment growth. This observation suggested that Class IV cells might grow via branch dilation—uniformly elongating branches, akin to Class I cells [1,2]. To understand whether the class IV complex arbor structure is formed by dilation or simply from growing tips, we developed this protocol to introduce a systematic approach for quantitatively assessing the growth dynamics of internal branches.

Calcium signalling in the endocardium is critical for heart valve development. Calcium ion pulses in the endocardium are generated in response to mechanical forces due to blood flow and can be visualised in the beating zebrafish heart using a genetically encoded calcium indicator such as GCaMP7a. Analysing these pulses is challenging because of the rapid movement of the heart during heartbeat. This protocol outlines an imaging analysis method used to phase-match the cardiac cycle in single z-slice movies of the beating heart, allowing easy measurement of the calcium signal.

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

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