Developmental Biology

The female reproductive tract is comprised of different regions, each with distinctive physiological characteristics. One of them is the fallopian tubes, which are vital for human reproductive health and success. The ability to model their function and physiology is of utmost importance. So far, in vitro models have been based on a few immortalized or cancer cell lines derived from fallopian tube cells that lacked differentiated, specialized cell types and did not allow for the study of cancer initiation due to their implicit biases. Organoids, in contrast, overcome these limitations and provide an advanced, three-dimensional system for the study of healthy fallopian tube physiology and pathology. Fallopian tube organoids are comprised of epithelial progenitors that can be enriched using chemical or hormonal treatment into the different cell types that are found in the in vivo tissue, namely detyrosinated-tubulin-positive ciliated cells or paired-box protein 8 (PAX8)-positive secretory cells. This protocol provides a step-by-step guide for the establishment and maintenance of a long-term culture of organoids from healthy human fallopian tube tissue. The organoid model described here closely mimics the in vivo physiology and anatomy of human fallopian tube epithelium and provides a comprehensive basis for future studies on its underlying molecular characteristics and possible pathology.

Communication between motor neurons and muscles is established by specialized synaptic connections known as neuromuscular junctions (NMJs). Altered morphology or numbers of NMJs in the developing muscles can indicate a disease phenotype. The distribution and count of NMJs have been studied in the context of several developmental disorders in different model organisms, including zebrafish. While most of these studies involved manual counting of NMJs, a few of them employed image analysis software for automated quantification. However, these studies were primarily restricted to the trunk musculature of zebrafish. These trunk muscles have a simple and reiterated anatomy, but the cranial musculoskeletal system is much more complex. Here, we describe a stepwise protocol for the visualization and quantification of NMJs in the ventral cranial muscles of zebrafish larvae. We have used a combination of existing ImageJ plugins to develop this methodology, aiming for reproducibility and precision. The protocol allows us to analyze a specific set of cranial muscles by choosing an area of interest. Using background subtraction, pixel intensity thresholding, and watershed algorithm, the images are segmented. The binary images are then used for NMJ quantification using the Analyze Particles tool. This protocol is cost-effective because, unlike other licensed image analyzers, ImageJ is open-source and available free of cost.

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.

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

Immotile cilia of crown cells at the node of mouse embryos are required for sensing leftward fluid flow that gives rise to the breaking of left-right (L-R) symmetry. The flow-sensing mechanism has long remained elusive, mainly because of difficulties inherent in manipulating and precisely analyzing the cilium. Recent progress in optical microscopy and biophysical analysis has allowed us to study the mechanical signals involving primary cilia. In this study, we used high-resolution imaging with mechanical modeling to assess the membrane tension in a single cilium. Optical tweezers, a technique used to trap sub-micron-sized particles with a highly focused laser beam, allowed us to manipulate individual cilia. Super-resolution microscopy allowed us to discern the precise localization of ciliary proteins. Using this protocol, we provide a method for applying these techniques to cilia in mouse embryonic nodes. This method is widely applicable to the determination of mechanical signals in other cilia.

Embryonic development is a complex process integrating cell fate decisions and morphogenesis in a spatiotemporally controlled manner. Previous studies with model organisms laid the foundation of our knowledge on post-implantation development; however, studying mammalian embryos at this stage is a difficult and laborious process. Early attempts to recapitulate mammalian development in vitro begun with embryoid bodies (EBs), in which aggregates of mouse embryonic stem cells (mESCs) were shown to differentiate into spatially arranged germ layers. A more revised version of EBs, gastruloids, improved the germ layer differentiation efficiency and demonstrated cell fate patterning on multiple axes. However, gastruloids lack anterior neural progenitors that give rise to brain tissues in the embryo. Here, we report a novel culture protocol to coax mESCs into post-implantation epiblast-like (EPI) aggregates in high throughput on bioengineered microwell arrays. We show that upon inhibition of the Wnt signaling pathway, EPI aggregates establish an extended axial patterning, leading to co-derivation of anterior neural progenitors and posterior tissues. Our approach is amenable to large-scale studies aimed at identifying novel regulators of gastrulation and anterior neural development that is currently out of reach with existing embryoid models. This work should contribute to the advancement of the nascent field of synthetic embryology, opening up exciting perspectives for various applications of pluripotent stem cells in disease modeling and tissue engineering.

Key features

• A new gastruloid culture system to model post-implantation mouse embryonic development in vitro

• High-throughput formation of epiblast-like aggregates on hydrogel microwells

• Builds upon conventional gastruloid cultures and provides insight into the role of Wnt signaling for the formation of anterior neural tissues

Graphical overview

The trachea tube is the exclusive route to allow gas exchange between the external environment and the lungs. Recent studies have shown the critical role of mesenchymal cells in tracheal tubulogenesis. Improved methods for studying the dynamics of the tracheal mesenchyme development are needed to investigate the cellular and molecular mechanisms during tracheal tubulogenesis. Here, we describe a detailed protocol for a systematic analysis of tracheal tube development to enable observing tracheal smooth muscle (SM) and cartilage ring formation. We describe immunostaining, confocal and stereomicroscopy imaging, and quantitative methods to study the process of tracheal SM and cartilage ring development, including SM cell alignment, polarization, and changes in cell shape as well as mesenchymal condensation. The technologies and approaches described here not only improve analysis of the patterning of the developing trachea but also help uncover the mechanisms underlying airway disease. This protocol also provides a useful technique to analyze cell organization, polarity, and nuclear shape in other organ systems.

Cellular protrusions are fundamental structures for a wide variety of cellular behaviors, such as cell migration, cell–cell interaction, and signal reception. Visualization of cellular protrusions in living cells can be achieved by labeling of cytoskeletal actin with genetically encoded fluorescent probes. Here, we describe a detailed experimental procedure to visualize cellular protrusions in medaka embryos, which consists of the following steps: preparation of Actin-Chromobody-GFP and α-bungarotoxin mRNAs for actin labeling and immobilization of the embryo, respectively; microinjection of the mRNAs into embryos in a mosaic fashion to sparsely label individual cells; removal of the hard chorion, which hampers observation; and visualization of cellular protrusions in the embryo with a confocal microscope. Overall, our protocol provides a simple method to reveal cellular protrusions in vivo by confocal microscopy.