Developmental Biology

Repetitive increases of intracellular calcium ions (Ca²⁺ oscillations) control cellular functions in various biological events, including meiotic resumption after fertilization. Sperm-derived substances enter the cytoplasm of mature oocytes by sperm fusion, causing Ca²⁺ oscillations. Sperm-independent Ca²⁺ oscillations are also induced in immature oocytes isolated from the ovaries of neonatal to adult mice. The presence of Ca²⁺ oscillations may contribute to subsequent oocyte quality; however, its physiological role and molecular mechanism are unclear. Here, we describe a method of collecting immature oocytes from the ovaries of juvenile (12, 15, and 21 days after birth) and adult mice and monitoring their Ca²⁺ oscillations. Since mouse oocytes are larger than other types of cells, they are a useful model for studying spatiotemporal patterns and the mechanism of Ca²⁺ oscillations in various types of cells. This method can be applied to other rodents due to similarities in oocyte size and developmental processes. Furthermore, the use of various fluorescent probes enables visualization of organelle rearrangement. The mechanism of interaction between oocytes and somatic cells differs between juvenile and adult mice. Therefore, two distinct methods are employed for oocyte collection.

Nowadays, the use of 3D cultures (organoids) is considered a valuable experimental tool to model physiological and pathological conditions of organs and tissues. Organoids, retaining cellular heterogeneity with the presence of stem, progenitor, and differentiated cells, allow the faithful in vitro reproduction of structures resembling the original tissue. In this context, the growth of endometrial organoids allows the generation of 3D cultures characterized by a hollow lumen, secretory activity, and apicobasal polarity and displaying phenotypical modification in response to hormone stimulation. However, a limitation in currently used models is the absence of stromal cells in their structure; as a result, they miss epithelial–stromal interactions, which are crucial in endometrial physiology. We developed a novel 3D model to generate endometrial organoids grown in floating Matrigel^TM droplets in the presence of standard culture medium. From a structural point of view, these novel floating 3D cultures develop as gland-like structures constituted by epithelial cells organized around a central lumen and retain the expression of endometrial and decidual genes, like previously published organoids, although with a phenotype resembling hormonally differentiated structures. Importantly, floating organoids retain stromal cells which grow in close contact with the epithelial cells, localized within the internal or external portion of the organoid structure. In summary, we present a simple and rapid model for generating 3D endometrial organoids that preserve epithelial–stromal cell interactions, promoting the formation of differentiated organoids and enabling the study of reciprocal modulation between epithelium and stroma.

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.

Centrosomes are vital eukaryotic organelles involved in regulating cell adhesion, polarity, mobility, and microtubule (MT) spindle assembly during mitosis. Composed of two centrioles surrounded by the pericentriolar material (PCM), centrosomes serve as the primary microtubule-organizing centers (MTOCs) in proliferating cells. The PCM is crucial for MT nucleation and centriole biogenesis. Centrosome numbers are tightly regulated, typically duplicating once per cell cycle, during the S phase. Deregulation of centrosome components can lead to severe diseases. While traditionally viewed as stable structures, centrosomes can be inactivated or disappear in differentiating cells, such as epithelial cells, muscle cells, neurons, and oocytes. Despite advances in understanding centrosome biogenesis and function, the mechanisms maintaining mature centrosomes or centrioles, as well as the pathways regulating their inactivation or elimination, remain less explored. Studying centrosome maintenance is challenging as it requires the uncoupling of centrosome biogenesis from maintenance. Tools for acute spatial-temporal manipulation are often unavailable, and manipulating multiple components in vivo is complex and time-consuming. This study presents a protocol that decouples centrosome biogenesis from maintenance, allowing the study of critical factors and pathways involved in the maintenance of the integrity of these important cellular structures.

Traditional tissue dissociation methods for bulk- and single-cell sequencing use various protease and/or collagenase combinations at temperatures ranging from 28 to 37 °C, which cause transcriptional cell stress that may alter data interpretation. Such artifacts can be reduced by dissociating cells in cold-active proteases, but few studies have shown that this improves cell-type specific transcription, particularly in tissues hypersensitive to mechanical integrity and extracellular matrix (ECM) interactions. To address this, we have dissociated zebrafish tendons and ligaments in subtilisin A at 4 °C and compared the results with 37 °C collagenase dissociation using bulk RNA sequencing. We find that high-temperature collagenase dissociation causes general cell stress in tendon fibroblasts (tenocytes) as reported in previous studies with other cell types, but also that high temperature specifically downregulates hallmark genes involved in tenocyte specification and ECM production in vivo. Our results suggest that cold-protease dissociation reduces transcriptional artifacts and increases the robustness of RNA-sequencing datasets such that they better reflect native in vivo tissue microenvironments.

One of the major factors contributing to aging and age-related diseases is the well-understood decline in the function of adult stem cells. Quantifying the degree of aging in adult stem cells is essential for advancing anti-aging mechanisms and developing anti-aging agents. However, no systematic approach to this exists. In this study, we developed a method to quantitatively assess the degree of aging in adult intestinal stem cells using a Drosophila midgut model and two aging markers. First, aging was induced in Drosophila with the desired genotype, and the anti-aging agent was administered 7 days before dissection. Then, the levels of two intestinal stem cell aging markers found in Drosophila (PH3 and γ-tubulin) were measured using immunohistochemistry. Finally, fluorescence microscopy was employed to count the number of aging markers and take images, which were analyzed using image analysis software. Using this approach, we quantitatively analyzed the effects of anti-aging agents on the aging of adult intestinal stem cells. This methodology is expected to significantly expedite the development of anti-aging agents and substantially reduce the research costs associated with aging-related studies.

Our goal is to understand how hematopoietic stem cell precursors emerge from vessels and to visualize their settling in developmental and more definitive niches that will persist in the adult. For this, we use as a biological model the zebrafish, which offers invaluable advantages owing to its transparency and small size, allowing high-resolution imaging and investigations of the entire animal. In vertebrate species, precursors of hematopoietic stem cells emerge from arterial vessels, mainly from the ventral side of the dorsal aorta. From there, they can either reside in the underlying vascular niche and/or pass through the vein to enter the blood circulation and conquer the caudal hematopoietic tissue, a functional equivalent to the fetal liver in mammals. Here, we provide experimental details of a protocol we have recently optimized to identify, based on mRNA in situ hybridization, precursors of hematopoietic stem cells while still embedded in the aortic wall (at the embryonic stage) as well as when they reside in specific niches a few days after emergence (at the early larval stage). Our experimental approach uses RNAscope technology, which allows combining high-sensitivity mRNA detection with high-resolution fluorescence confocal imaging to achieve spatial transcriptomics. Importantly, the small size of the probes allows better penetration inside tissues, which is a significant improvement in comparison to long mRNA probes; this is an invaluable advantage for reaching deeply embedded niches such as the ones of the pronephros region in the larva and, in addition, provides an increased signal-to-noise ratio.

The fate mapping technique is essential for understanding how cells differentiate and organize into complex structures. Various methods are used in fate mapping, including dye injections, genetic labeling (e.g., Cre-lox recombination systems), and molecular markers to label cells and track their progeny. One such method, the FlashTag system, was originally developed to label neural progenitors. This technique involves injecting carboxyfluorescein diacetate succinimidyl ester (CFSE) into the lateral ventricles of mouse embryos, relying on the direct uptake of dye by cells. The injection of CFSE into the lateral ventricle allows for the pulse labeling of mitotic (M-phase) neural progenitors in the ventricular zone and their progeny throughout the brain. This approach enables us to trace the future locations and differentiation paths of neural progenitors. In our previous study, we adapted this method to selectively label central nervous system–associated macrophages (CAMs) in the lateral ventricle by using a lower concentration of CFSE compared to the original protocol. Microglia, the brain's immune cells, which play pivotal roles in both physiological and pathological contexts, begin colonizing the brain around embryonic day (E) 9.5 in mice, with their population expanding as development progresses. The modified FlashTag technique allowed us to trace the fate of intraventricular CAMs, revealing that certain populations of microglia are derived from these cells. The optimized approach offers deeper insights into the developmental trajectories of microglia. This protocol outlines the modified FlashTag method for labeling intraventricular CAMs, detailing the CFSE injection procedure, evaluation of CFSE dilution, and preparation of tissue for immunohistochemistry.