发育生物学

Congenital renal disorders, such as the Potter sequence, result from renal dysgenesis. To explore a prenatal therapeutic approach for fetuses with kidney insufficiency, we established an in utero transplantation protocol using donor fetal kidneys. Although numerous rodent studies have reported cellular injections into fetal recipients, no protocol to date has described whole-organ transplantation during gestation. Here, we present a step-by-step method for grafting donor fetal kidneys (embryonic day 14.0–16.5) into allogeneic rat fetuses at embryonic day 18.0–18.5, resulting in term neonates that retain the grafts postnatally. A 15–16 G needle preloaded with the donor kidney is inserted transuterinely, depositing the organ into the subcutaneous space of the fetus. Four days later, the term pups are delivered naturally and evaluated for graft development. This protocol enables organ-level transplantation and longitudinal assessment of graft maturation within the unique fetal environment, which differs markedly from adult settings in terms of growth factor availability and immune reactivity. To our knowledge, this is the first protocol to successfully achieve whole-organ transplantation directly into fetuses in utero. Therefore, the model provides a valuable platform for studying developmental organogenesis, fetal immunology, and regenerative strategies that leverage embryonic cues.

Underwater noise is a growing source of anthropogenic pollution in aquatic environments. However, few studies have evaluated the impact of underwater noise on aquatic invertebrates. More importantly, studies involving early developmental stages have been poorly addressed. Significant limitations are due to the lack of standardized protocols for working in the laboratory. Particularly, the design of uniform procedures in the laboratory is important when working with species that inhabit short-term changing habitats, such as estuaries, which makes it difficult to carry out repeated experiments in the natural habitat. Besides, controlling for environmental variables is also important when assessing the effect of a stressor on the physiological parameters of individuals. This experimental protocol addresses that gap by offering an adaptable laboratory-based method to evaluate sublethal physiological responses to sound exposure under highly controlled conditions. Here, we present a reproducible and accessible laboratory protocol to expose crabs to recorded boat noise and evaluate physiological responses using oxidative stress biomarkers. The method is designed for ovigerous females, as we evaluated the effects on embryos and early life stages (i.e., larvae), but it can be readily adapted to different life stages of aquatic invertebrates. A key strength of this protocol is its simplicity and flexibility: animals are exposed to noise using submerged transducers under well-controlled laboratory conditions, ensuring consistency and repeatability. Following exposure, tissues or whole-body samples can be processed for a suite of oxidative stress biomarkers—glutathione-S-transferase (GST), catalase (CAT), lipid peroxidation (LPO), and protein oxidation. These biomarkers are highly responsive, cost-effective indicators that provide a sensitive and early readout of sublethal stress. Together, the exposure and analysis steps described in this protocol offer a powerful and scalable approach for investigating the physiological impacts of underwater noise in crustaceans and other aquatic invertebrates.

Primary cilia are evolutionarily conserved organelles that play critical roles in brain development. In the developing cortex, neural progenitors extend their primary cilia into the ventricular surface, where the cilia act as key signaling hubs. However, visualizing these cilia in a systematic and intact manner has been challenging. The commonly used cryostat sectioning only provides a limited snapshot of cilia on individual sections, and this process often disrupts the ciliary morphology. By contrast, the previously established whole-mount technique has been shown to preserve ciliary architecture in the adult mouse cortex. Here, we adapt and optimize the whole-mount approach for embryonic and neonatal brain, allowing robust visualization of ciliary morphology at the ventricular surface during development. This protocol describes step-by-step procedures for whole-mounting and immunostaining delicate embryonic and neonatal mouse cortices, enabling direct visualization of cilia in neural progenitors in the developing brain.

In mammals, the semen is ejaculated into the female reproductive tract, and the sperm travel to the oviduct to fertilize the egg. A comprehensive understanding of the pre- and post-ejaculatory intrauterine environment is one of the key points for overcoming infertility; however, the dynamics of the intrauterine environment and its physiological role in the uterus, namely in the internal fertilization process, remain unclear. Conventional methods for collecting uterine fluids from the uterus post-ejaculation of mice show challenges regarding the ambiguous ejaculation timing. Here, we established a method for a mating environment with exact ejaculation timing. We also created a simple method for collecting pre- and post-ejaculatory uterine fluid without using forceps. Our methods achieved time-dependent biochemical and histological analyses of uterine fluids to provide fundamental information regarding protein composition and uterine structure changes during pre- and post-ejaculation. This protocol is suitable for analyzing temporal changes in reproductive phenomena, thereby contributing to elucidating the physiological role of the uterus in the process of intrauterine fertilization.

Zebrafish are a powerful model for investigating vascular and lymphatic biology due to their genetic tractability and optical transparency. While translating ribosome affinity purification (TRAP) has been widely applied in other systems, its application in zebrafish has remained limited. Here, we present an optimized TRAP protocol for isolating ribosome-associated mRNAs from endothelial cells in vivo, without the need for cell dissociation or sorting. Using a novel transgenic zebrafish line, which expresses HA-tagged Rpl10a under the mrc1a promoter, we enriched actively translating endothelial transcripts. Differential expression analysis revealed robust upregulation of vascular and lymphatic genes including flt4, kdrl, and lyve1b. This approach captures the endothelial cell translatome with high specificity and offers a robust platform for investigating the molecular mechanisms of endothelial biology under genetic, environmental, or toxicological perturbations.

Adipose cells vary functionally, with white adipocytes storing energy and brown/beige adipocytes generating heat. Mouse and human subcutaneous white adipose tissue (WAT)-derived stromal vascular fraction (SVF) provides mesenchymal stem cells (MSCs) that can be differentiated into thermogenic adipocytes using pharmacological cocktails. After six days of browning induction, these cells exhibited significant upregulation of thermogenic markers (UCP1, Cidea, Dio2, PRDM16) along with adipogenic genes (PPARγ, aP2), showing enhanced thermogenic potential. This in vitro system offers a practical platform to study adipogenesis and thermogenic regulation.

Peripheral nerve injuries (PNIs) often result in incomplete functional recovery due to insufficient or misdirected axonal regeneration. Balanced regeneration of myelinated A-fibers and unmyelinated C-fibers is essential for functional recovery, making it crucial to understand their differential regeneration patterns to improve PNI treatment outcomes. However, immunochemical staining does not clearly differentiate between A- and C-fiber axons in whole-mount nerve preparations. To overcome this limitation, we developed a modified protocol by optimizing the immunostaining to restrict the antibody access to myelinated axons. This enables visualization of A-fibers by myelin sheath labeling, while allowing selective staining of unmyelinated C-fiber axons. As a result, A- and C-fibers can be reliably distinguished, facilitating accurate analysis of their regeneration in both normal and post-injury conditions. Combined with confocal microscopy, this approach supports efficient screening of whole-mount nerve preparations to evaluate fiber density, spatial distribution, axonal sprouting, and morphological characteristics. The refined technique provides a robust tool for advancing PNI research and may contribute to the development of more effective therapeutic strategies for nerve repair.

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.

Telomere shortening is a hallmark of human aging, and telomerase regulation plays a critical role in cellular proliferation and replicative senescence. In human cells, telomere length imposes a limit on proliferative potential, a phenomenon known as the Hayflick limit. However, species-specific differences in telomere dynamics and telomerase regulation between humans and mice present challenges to using mice as accurate models for human telomere-related research. To address this limitation, we engineered a humanized telomerase gene (hmTert) in mice by replacing the non-coding sequences within the mouse Tert locus (mTert) with corresponding regulatory sequences from the human TERT gene. Breeding of these genetically modified mice resulted in progressive telomere shortening over successive generations, ultimately reaching human-like lengths (below 10 kb). This protocol outlines the development of this humanized telomere mouse model, referred to as HuT mice, offering a robust platform for studying human telomere biology and aging-related diseases.

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.