发育生物学

Region-specific RNA sequencing is a powerful approach for investigating tissue differentiation and dynamic changes in gene expression during embryonic development. The chicken embryo has long served as an important model system in developmental biology. However, the limited availability of tissue-specific reporter lines makes region-specific RNA-seq approaches particularly valuable in this organism. Here, we applied photo-isolation chemistry-based RNA sequencing (PIC-RNA-seq) to the somatic lateral plate mesoderm (sLPM) before the emergence of limb bud progenitor cells (LPCs) and to early LPCs in chicken embryos. These analyses revealed the upregulation of multiple genes, including Hox genes, in LPCs, suggesting the initiation of their regional patterning program. This workflow enables visualization of dynamic changes in the gene expression profile of LPCs and should also be applicable to other tissues in avian embryos.

Limb development requires the coordination of multiple cell types, including the limb bud mesoderm and surface ectoderm, by the apical ectodermal ridge (AER), a specialized signaling center secreting numerous morphogens. Characterizing these cell–cell interactions is crucial for understanding limb morphogenesis, but they are challenging to study in vivo. Furthermore, existing in vitro models do not capture the multilineage complexity of the limb. We recently developed a robust 7-day differentiation protocol using mouse embryonic stem cells (mESCs) to generate heterogeneous cultures containing cells with characteristics of the limb bud mesoderm, surface ectoderm, and AER. Dissociating and reaggregating these cultures in low attachment 96-well plates forms budoids, organoids that display certain limb bud–like features. Budoids undergo chondrogenesis-mediated symmetry breaking and elongation within 5 days of culture. Altogether, our protocols have enabled the study of cell–cell interactions in limb development and provide an easily scalable model adaptable for various applications, including drug testing and congenital disorder modeling.

Embryonic blood formation encompasses the independent generation of different cell types in distinct cellular and anatomical environments, reflecting highly coordinated specific hierarchies of interacting tissues. Despite widespread use of embryonic stem cells (ESC) and induced pluripotent stem cell (iPSC)-based models to attempt to capture blood development in vitro and generate hematopoietic stem cells (HSC), a system that fully captures the spatial and temporal complexity of embryonic hematopoiesis is still lacking. In recent years, gastruloid models have emerged as powerful representations of early development, demonstrating self-organizing behaviors such as symmetry breaking, elongation, multi-axis formation, somitogenesis, and early organogenesis, with striking parallels to embryonic processes. Here, we present a protocol to generate hemogenic gastruloids (haemGx) from mouse ESC (mESC) that closely recapitulates the multi-stage, multi-niche process of blood formation and generates developmentally accurate hematopoietic progenitors. The haemGx model has been proven valuable in understanding embryonic hematopoiesis, as well as an in vitro model of forms of infant leukemia with an embryonic, in utero origin.

Neural tube closure is a critical process that transforms the neural plate, an open epithelial tissue, into the closed tube that serves as the structural basis of the central nervous system. Defects in this process are among the most common and severe developmental diseases in the human population, with failures in cranial closure accounting for approximately one-third of total defects. However, the cell and tissue mechanisms that drive cranial closure remain opaque relative to the better studied process of spinal closure, in large part due to the unique challenges in characterizing cranial tissues. Here, we present protocols for quantifying cell dynamics and tissue-level remodeling events that enable highly spatiotemporally resolved investigations of the causes of cranial closure defects in mouse embryos. These include brightfield morphometric approaches, fluorescent staining and confocal imaging, and quantitative pipelines to analyze these image-based datasets. At the conclusion of these approaches, users will be able to quantify several parameters of overall tissue shape in the cranial neural tissues and produce rich quantitative datasets about cell-level parameters, particularly apical cell area. These can be used to identify correlative and causative differences between mutants and control embryos. Given their flexibility, many of these approaches can be generalized to other tissue morphogenetic contexts.

N⁶-methyladenosine (m⁶A) is the most abundant internal modification in mRNA and is regulated primarily by the balance between the METTL3 methylase complex and two demethylases, FTO (fat mass and obesity-associated protein) and ALKBH5 (α-ketoglutarate-dependent dioxygenase alkB homolog). Reflecting this prevalence, m⁶A participates in virtually every step of RNA metabolism, influencing a wide range of physiological and pathological processes. The first step in studying m⁶A is genome-wide mapping, typically performed by m⁶A-seq, which sequences RNA fragments immunoprecipitated with an m⁶A-specific antibody. This is followed by identification of RRACH motifs (R = A or G; H = A, C, or U) within these sequences, with m⁶A being located at the third nucleotide. The second step involves mutating the putative m⁶A sites to establish a causal link between the modification and downstream biological effects. Since the mapping step has been covered in several detailed protocols, this article focuses on the second step—mutagenesis of RRACH motifs and subsequent functional analysis of the mutations by ectopic expression. The 3′ untranslated region (UTR) of the mouse Runx2 gene is used as an example. The mutant and wild-type sequences are inserted into a luciferase reporter vector and transfected into 293FT cells to evaluate how loss of m⁶A affects luciferase protein levels. The same reporter plasmids are also used in an RNA stability assay with a transcription inhibitor. Although site-specific demethylation of endogenous mRNA would be preferable, it remains technically challenging despite many attempts. Thus, ectopic expression of the mutated target gene remains a widely used and practical alternative.

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.

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.

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.