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bioRxiv2023

Fast Cycling Culture of the Marine Annelid Platynereis dumerilii

Platynereis dumerilii, a marine annelid, is a model animal that has gained popularity in various fields such as developmental biology, biological rhythms, nervous system organization and physiology, behaviour, reproductive biology, and epigenetic regulation. The transparency of P. dumerilii tissues at all developmental stages makes it easy to perform live microscopic imaging of all cell types. In addition, the slow-evolving genome of P. dumerilii and its phylogenetic position as a representative of the vast branch of Lophotrochozoans add to its evolutionary significance. Although P. dumerilii is amenable to transgenesis and CRISPR-Cas9 knockouts, its relatively long and indefinite life cycle, as well as its semelparous reproduction have been hindrances to its adoption as a reverse genetics model. To overcome this limitation, an adapted culturing method has been developed allowing much faster life cycling, with median reproductive age at 15 weeks instead of 6-8 months using the traditional protocol. A low worm density in boxes and a strictly controlled feeding regime are important factors for the rapid growth and health of the worms. Moreover, a genetic selection for fast-reproducing individuals has been applied to isolate a "Fast Forward" strain that can be used for egg microinjection. This culture method has several advantages, such as being much more compact, not requiring air bubbling or an artificial moonlight regime for synchronized sexual maturation, and necessitating only limited water change. A full protocol for worm care and handling is provided.

developmental biology↗

bioRxiv2022

A Chemical-mechanical Coupled Model Predicts Roles of Spatial Distribution of Morphogen in Maintaining Tissue Growth

The exact mechanism controlling cell growth remains a grand challenge in developmental biology and regenerative medicine. The Drosophila wing disc tissue serves as an ideal biological model to study growth regulation due to similar features observed in other developmental systems. The mechanism of growth regulation in the wing disc remains a subject of intense debate. Most existing models to study tissue growth focus on either chemical signals or mechanical forces only. Here we developed a multiscale chemical-mechanical coupled model to test a growth regulation mechanism depending on the spatial range of the morphogen gradient. By comparing the spatial distribution of cell division and the overall shape of tissue obtained in the coupled model with experimental data, our results show that the distribution of the Dpp morphogen can be critical in resulting tissue size and shape. A larger tissue size with a faster growth rate and more symmetric shape can be achieved if the Dpp gradient spreads in a larger domain. Together with the absorbing boundary conditions, the feedback regulation that downregulates Dpp receptors on the cell membrane allows the further spread of the morphogen away from its source region, resulting in prolonged tissue growth at a more spatially homogeneous growth rate. Summary StatementA multiscale chemical-mechanical model was developed by coupling submodels representing dynamics of a morphogen gradient at the tissue level, intracellular chemical signals, and mechanical properties at the subcellular level. By applying this model to study the Drosophila wing disc, it was found that the spatial range of the morphogen gradient affected tissue growth in terms of the growth rate and the overall shape.

developmental biology↗

bioRxiv2017

C. elegans exhibits coordinated oscillation in gene expression during development

BackgroundThe advent of in vivo automated single-cell lineaging and sequencing will dramatically increase our understanding of development. New integrative analysis techniques are needed to generate insights from single-cell developmental data.\n\nResultsWe applied novel meta-analysis techniques to the EPIC single-cell-resolution developmental gene expression dataset for C. elegans to show that a simple linear combination of the expression levels of the developmental genes is strongly correlated with the developmental age of the organism, irrespective of the cell division rate of different cell lineages. We uncovered a pattern of collective sinusoidal oscillation in gene activation, in multiple dominant frequencies and in multiple orthogonal axes of gene expression, pointing to the existence of a coordinated, multi-frequency global timing mechanism. We developed a novel method based on Fishers Discriminant Analysis (FDA) to identify linear gene expression weightings that are able to produce sinusoidal oscillations of any frequency and phase, adding to the evidence that oscillatory mechanisms likely play an important role in the timing of development. We cross-linked EPIC with gene ontology and anatomy ontology terms, employing FDA methods to identify previously unknown positive and negative genetic contributions to developmental processes and cell phenotypes.\n\nConclusionsThis meta-analysis demonstrates new evidence for direct linear and/or sinusoidal mechanisms regulating the timing of development. We uncovered a number of previously unknown positive and negative correlations between developmental genes and developmental processes or cell phenotypes. The presented novel analysis techniques are broadly applicable within developmental biology.

developmental biology↗

bioRxiv2024

Comprehensive characterization of mitochondrial bioenergetics at different larval stages reveals novel insights about the developmental metabolism of Caenorhabditis elegans

Mitochondrial bioenergetic processes are fundamental to development, stress responses, and health. Caenorhabditis elegans is widely used to study developmental biology, mitochondrial disease, and mitochondrial toxicity. Oxidative phosphorylation generally increases during development in many species, and genetic and environmental factors may alter this normal trajectory. Altered mitochondrial function during development can lead to both drastic, short-term responses including arrested development and death, and subtle consequences that may persist throughout life and into subsequent generations. Understanding normal and altered developmental mitochondrial biology in C. elegans is currently constrained by incomplete and conflicting reports on how mitochondrial bioenergetic parameters change during development in this species. We used a Seahorse XFe24 Extracellular Flux (XF) Analyzer to carry out a comprehensive analysis of mitochondrial and non-mitochondrial oxygen consumption rates (OCR) throughout larval development in C. elegans. We optimized and describe conditions for analysis of basal OCR, basal mitochondrial OCR, ATP-linked OCR, spare and maximal respiratory capacity, proton leak, and non-mitochondrial OCR. A key consideration is normalization, and we present and discuss results as normalized per individual worm, protein content, worm volume, mitochondrial DNA (mtDNA) count, nuclear DNA (ncDNA) count, and mtDNA:ncDNA ratio. Which normalization process is best depends on the question being asked, and differences in normalization explain some of the discrepancies in previously reported developmental changes in OCR in C. elegans. Broadly, when normalized to worm number, our results agree with previous reports in showing dramatic increases in OCR throughout development. However, when normalized to total protein, worm volume, or ncDNA or mtDNA count, after a significant 2-3-fold increase from L1 to L2 stages, we found small or no changes in most OCR parameters from the L2 to the L4 stage, other than a marginal increase at L3 in spare and maximal respiratory capacity. Overall, our results indicate an earlier cellular shift to oxidative metabolism than suggested in most previous literature.

developmental biology↗

bioRxiv2015

Measuring forces and stresses in situ in living tissues

Development, homeostasis and regeneration of tissues result from the interaction of genetics and mechanics. Kinematics and rheology are two main classes of measurements respectively providing deformations and mechanical properties of a material. They are now applied to living tissues and have contributed to the better understanding of their mechanics. Due to the complexity of living tissues, however, a third class of mechanical measurements, that of in situ forces and stresses, appears to be increasingly important to elaborate realistic models of tissue mechanics. We review here several emerging techniques of this class, their fields of applications, their advantages and limitations, and their validations. We argue that they will strongly impact on our understanding of developmental biology in the near future.

Developmental Biology↗

bioRxiv2024

A feather star is born: embryonic development and nervous system organization in the crinoid Antedon mediterranea

BackgroundCrinoids belong to the phylum Echinodermata, marine invertebrates with a highly derived pentaradial body plan. As the only living members of the Pelmatozoa, the sister group to other extant echinoderms, crinoids are in a key phylogenetic position to reconstruct the evolutionary history of this phylum. However, the development of crinoids has been scarcely investigated, limiting their potential for comparative studies. Many crinoids are difficult to collect in the wild and embryo manipulation is challenging. Conversely, the Mediterranean feather star Antedon mediterranea can be found in shallow waters and has been used for experimental studies, most notably to investigate regeneration. ResultsThe aim here was to establish A. mediterranea as an experimental system for developmental biology. To accomplish this, we set up a method for culturing embryos in vitro from zygote to hatching larva stage that allowed us to define a developmental timeline and a standardized staging system for this species. We then optimized protocols to characterize the development of the main structures of the feather star body plan, using a combination of microscopy techniques and whole mount immunohistochemistry and in situ hybridization chain reaction. Focusing on the nervous system, we show that the larval apical organ includes a combination of serotonergic, GABAergic and glutamatergic neurons that form under the influence of a conserved anterior molecular signature. The larval neural plexus is instead composed of glutamatergic neurons and develops during the formation of the ciliary bands. Larval neurons disappear at metamorphosis, and the ectoneural and entoneural components of the adult nervous system develop early in post-metamorphic stages. Furthermore, the oral ectoderm that contains the ectoneural system acquires an "anterior" signature expressing Six3/6 and Lhx2/9 orthologs. ConclusionsOur results deepen our knowledge on crinoid development and provide new techniques to investigate feather star embryogenesis, promoting the use of A. mediterranea in developmental and evolutionary biology. This in turn will pave the way for the inclusion of crinoids in comparative studies to understand the origin of the echinoderm body plan and clarify many unanswered questions on deuterostome evolution.

developmental biology↗

bioRxiv2017

A defined platform of human peri-gastrulation-like biological fate patterning reveals coordination between Reaction-Diffusion and Positional-Information

How position dependent cell fate acquisition occurs during embryogenesis has been a central question in developmental biology. To study this process, we developed a defined, high-throughput assay using BMP4 to induce peri-gastrulation-like fate patterning in geometrically constrained human pluripotent stem cell colonies. We observed that, upon BMP4 treatment, phosphorylated SMAD1 (pSMAD1) activity in the colonies organized into a radial gradient - an observation mechanistically compliant with a BMP4-NOGGIN Reaction-Diffusion (RD) model. Consequent fate acquisition occurred as a function of both the pSMAD1 signaling strength, and induction time - consistent with the Positional-Information (PI) paradigm. Our findings implicate coordination between RD and PI underlying the peri-gastrulation-like fate patterning. This model not only predicts experimental results of perturbing key parameters like colony size, and BMP4 dose, but also identifies experimental conditions that rescue patterning in colonies of sizes that have been reported to be patterning-reticent, and recapitulate RD-like periodic patterns in large colonies.

developmental biology↗

bioRxiv2024

Zebrafish arterial valve development occurs through direct differentiation of second heart field progenitors

AimsBicuspid Aortic Valve (BAV) is the most common congenital heart defect, affecting at least 2% of the population. The embryonic origins of BAV remain poorly understood, limiting the identification of assays for validating patient variants and ultimately causative genes for BAV. In both human and mouse, the left and right leaflets of the arterial valves arise from the outflow tract cushions, with interstitial cells originating from neural crest cells and endocardial-to-mesenchymal transition (EndoMT). In contrast, an EndoMT-independent mechanism of direct differentiation by cardiac progenitors from the second heart field (SHF) is responsible for the formation of the anterior and posterior leaflets. Defects in either of these developmental mechanisms can result in BAV. Although zebrafish have been suggested as a model for human variant testing, their naturally bicuspid arterial valve has not been considered suitable for understanding human arterial valve development. Here, we have set out to investigate to what extent the processes involved in arterial valve development are conserved in zebrafish and ultimately, whether functional testing of BAV variants could be carried out in zebrafish. Methods and ResultsUsing a combination of live imaging, immunohistochemistry and Cre-mediated lineage tracing, we show that the zebrafish arterial valve primordia develop directly from undifferentiated SHF progenitors with no contribution from EndoMT or neural crest, in keeping with the human and mouse anterior and posterior leaflets. Moreover, once formed, these primordia share common subsequent developmental events with all three mammalian arterial valve leaflets. ConclusionsOur work highlights a conserved ancestral mechanism of arterial leaflet formation from the SHF and identifies that development of the zebrafish arterial valve is distinct from that of the atrioventricular valve. Crucially, this confirms the utility of zebrafish for understanding the development of specific BAV subtypes and arterial valve dysplasia, offering potential for high-throughput variant testing. Translational PerspectiveLarge genomic studies of patients with Bicuspid Aortic Valve (BAV) have identified numerous variants predicted to be causative, yet due to a lack of suitable, in vivo functional assays, advancement of genetic testing, discussion of risk to family members and accurate prognosis is not yet widely possible. Here, we show that zebrafish demonstrate a high level of conservation in arterial valve development with the intercalated leaflets in human, establishing zebrafish as a suitable in vivo model that can begin to overcome the disconnect between clinical genetics and developmental biology.

developmental biology↗

bioRxiv2021

Quantitative comparison of in vitro and in vivo embryogenesis at a single cell resolution

Stem cell engineering is a powerful platform to address numerous challenges in regenerative medicine and developmental biology. Typically, engineered populations are derived by exposing pluripotent stem cells to a series of signaling cues meant to recapitulate developmental milestones, such as the induction of the primitive streak. Morphologic, genetic, transcriptomic, and functional differences between fully differentiated in vivo and engineered populations have long been recognized. However, the correspondence between engineered and native embryonic progenitors has been relatively less well characterized, largely due to their transient and highly heterogenous nature, and therefore many studies have relied on expression of a few canonical markers to ensure that their cells are on the correct path. To address this challenge, we first generated an in vivo gastrulation mouse single cell RNA sequencing (scRNA-seq) reference data set and used it to train a collection of computational tools for comparing cell types, states, regulators, and trajectories across studies. Then we used these tools to evaluate a commonly used protocol designed to promote mesoderm derivation, as well as four previously published biomimetic protocols. Finally, we diversified our toolkits to reach a broader scientific community by implementing our primary analysis tool in Python and as an interactive web application.

developmental biology↗

bioRxiv2026

The XenCart Protocol: A Method for Alcian Blue Labeling and Quantitative Analysis of Craniofacial Cartilage in Xenopus

Craniofacial birth defects, such as cleft lip and palate, are among the most common congenital anomalies and often arise from disruptions in early facial patterning. Many of these defects are linked to environmental teratogens, yet such exposures cannot be directly tested in humans, making animal models essential for evaluating developmental risks. Xenopus laevis offers a powerful solution: its tadpoles develop externally, share deeply conserved craniofacial patterning mechanisms with humans, and provide an accessible platform for uncovering how environmental exposures reshape facial structures during development. Here, we present the XenCart Protocol, a reproducible workflow for Alcian Blue staining and quantitative morphometric analysis of Xenopus craniofacial cartilage. This method provides clear visualization of individual cartilage elements and can be readily applied to investigate genetic or environmental perturbations. The Xenopus craniofacial skeleton contains distinct cartilaginous structures that perform key biomechanical functions and share strong homology with regions of the human craniofacial skeleton. These similarities allow direct comparison of developmental outcomes across vertebrates. As part of a CURE-based undergraduate course, the XenCart Protocol was used to measure jaw cartilage dimensions in tadpoles exposed to an emerging teratogen, e-liquids used in vaping. E-liquid exposure caused consistent reductions across major craniofacial cartilages, including shorter Meckels cartilage, narrowed infrarostral width, decreased basihyobranchial and ceratohyal dimensions, and reduced suprarostral angles, reflecting an overall shift toward a smaller, more compact craniofacial morphology. These patterns suggest potential disruption of neural crest cell migration or signaling pathways for craniofacial cartilage development, mechanisms that, if similarly affected in humans, could contribute to midfacial narrowing, jaw underdevelopment, or increased vulnerability to conditions such as orofacial clefts. The ability to detect robust, structure-specific differences highlights the sensitivity of the protocol and its strong alignment with student-led research. These findings also pinpoint the precise regions of the jaw most affected by e-liquid exposure, providing a foundation for uncovering the developmental mechanisms driving these craniofacial changes. In summary, the XenCart Protocol provides a standardized, scalable method for quantifying craniofacial cartilage development and offers a powerful platform for both mechanistic research and undergraduate training in developmental biology and toxicology.

developmental biology↗

bioRxiv2022

Sequential addition of neuronal stem cell temporal cohorts generates a feed-forward circuit in the Drosophila larval nerve cord

Understanding how circuits self-assemble starting from neuronal stem cells is a fundamental question in developmental biology. Here, we addressed how neurons from different lineages wire with each other to form a specific circuit motif. To do so, we combined developmental genetics--Twin spot MARCM, Multi-color Flip Out, permanent labeling--with circuit analysis--calcium imaging, connectomics, and network science analyses. We find many lineages are organized into temporal cohorts, which are sets of lineage-related neurons born within a tight time window, and that temporal cohort boundaries have sharp transitions in patterns of input connectivity. We identify a feed-forward circuit motif that encodes the onset of vibration stimuli. This feed-forward circuit motif is assembled by preferential connectivity between temporal cohorts from different neuronal stem cell lineages. Further, connectivity does not follow the often-cited early-to-early, late-to-late model. Instead, the feed-forward motif is formed by sequential addition of temporal cohorts, with circuit output neurons born before circuit input neurons. Further, we generate multiple new tools for the fly community. Ultimately, our data suggest that sequential addition of neurons (with outputs neurons being oldest and input neurons being youngest) could be a fundamental strategy for assembling feed-forward circuits.

developmental biology↗

bioRxiv2023

Mesoscopic Oblique Plane Microscopy via Light-sheet Mirroring

Understanding the intricate interplay and inter-connectivity of biological processes across an entire organism is important in various fields of biology, including cardiovascular research, neuroscience, and developmental biology. Here, we present a mesoscopic oblique plane microscope (OPM) that enables whole organism imaging with high speed and subcellular resolution. A microprism underneath the sample enhances the axial resolution and optical sectioning through total internal reflection of the light-sheet. Through rapid refocusing of the light-sheet, the imaging depth is extended up to threefold while keeping the axial resolution constant. Using low magnification objectives with a large field of view, we realize mesoscopic imaging over a volume of 3.7x1.5x1 mm3 with [~]2.3 microns lateral and [~]9.2 microns axial resolution. Applying the mesoscopic OPM, we demonstrate in vivo and in toto whole organism imaging of the zebrafish vasculature and its endothelial nuclei, and blood flow dynamics at 12 Hz acquisition rate, resulting in a quantitative map of blood flow across the entire organism.

biophysics↗

bioRxiv2025

Fluorescent protein-based ticker tapes for multiplexed recordings of transcriptional histories in single cells in culture and in vivo

Recording and imaging of promoter activities in real time is critical for deciphering cellular states and dynamic signaling crosstalk, but technologies capable of simultaneously capturing multiple transient events in living cells are lacking. Here, we custom-design fluorescent protein-based ticker tapes (FPTT) for multiplexed and longitudinal recording of physiological activities in single cells. FPTT integrates multi-spectral monomeric fluorescent proteins with self-assembling protein fibers, enabling massively parallel analysis of signaling dynamics under varying cellular conditions. Using FPTT, we were able to log dose-dependent and reversible transcriptional histories of endogenous cFos signaling in primary hippocampus neurons at a 3-hour temporal resolution via biological timestamps and an extended recording time of over 8 days. Furthermore, we expanded the imaging toolset by engineering genetically encoded FPTT variants for human NF{kappa}B, JAK/STAT3, mTOR, NFAT and cAMP signaling, allowing for the quantification of potential crosstalk between cFos and NF{kappa}B pathways in neurons, multiplexed recording of STAT3- and cAMP-specific promoters during drug-induced liver injury in mice, as well as simultaneous analysis of up to four major signaling pathways involved in experimental T-cell activation. This platform advances single-cell analysis by providing a versatile tool to investigate transcriptional histories and signaling interplay across diverse biological contexts, with broad applications in developmental biology and disease modeling.

synthetic biology↗

bioRxiv2018

A meta-analysis of Planarian: an animal model useful to understand the effect of magnetic field

The planarians research since the first description until nowadays has a broad spectrum of topics. The present paper deals on a meta-analysis which identified as a problem the lack of consolidate information and data analysis about the use of planarian as animal model, and specifically the use of magnetic field in regeneration experiments. To conduct the meta-analysis 1255 papers published since 1955 to 2017 were analysed, identifying the Stem cell biology and regeneration as the most published topic, with 276 papers, followed by molecular and cellular analyses (238), genetics (175), inter and transdisciplinary research (107), ecotoxicological evaluations (102), animal model (67), ecological and biological studies (56), magnetic field (57), developmental biology (32) and RNA regulation (31).Other statistics and metrics indicators were taken in to account like total of papers and distribution per year, distribution of paper by journals and selection of main journals according to the number of published papers, most cited papers, authors and countries and distribution of papers by countries. Finally, were analysed those papers with Planarians research using magnetic field, all of them published during the last three years. It was an evidence that this topic is becoming a trending with rising interest, being the most reported species to study the magnetic field effects Dugesia spp. and Girardia sp.\n\nSummary statementThis work presents a meta-analysis which allows consolidating information and a better understanding of trends in Planarian researches, emphasizing in the use of magnetic field.

cell biology↗

bioRxiv2025

RNAi Gene knockdown of R-opsin and F-actin in the marineannelid Streblospio benedicti by delivering dsRNA

BackgroundRNA interference (RNAi) is a genetic tool to disrupt the expression of selected genes by delivering dsRNA into a specific tissue of an organism, resulting in a gene knockdown. We apply RNAi methods to the marine polychaete Streblospio benedicti, which is a model system for studying evolutionary and developmental biology, which currently has no established methods for gene expression manipulation. ResultsHere we describe a RNAi gene knockdown methodology using two different approaches depending on developmental stage. We feed bacteria expressing dsRNA to early swimming larvae and microinject in vitro transcribed dsRNA in juveniles. We used two genes for testing: R-opsin and F-actin. For both developmental stages, gene knockdown was validated using qPCR (quantitative Real-Time PCR). For larvae, we also visualized reduction in RNA expression using Hybridization Chain Reaction (HCR) in situ hybridization. ConclusionsWe show that both larval feeding and juvenile microinjections are sufficient delivery mechanisms for dsRNA to result in a RNAi gene knockdown. The efficiency can vary depending on the gene and timing, but this is typical for RNAi based approaches.

developmental biology↗

bioRxiv2019

Molecular and genetic regulation of pig pancreatic islet cell development

Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases like diabetes mellitus. Similarities of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet {beta}-, - and {delta}-cells followed by transcriptome analysis (RNA-Seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet {beta}-cell functional maturation, including evidence of fetal -cell GLP-1 production and signaling to {beta}-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases. Summary StatementThis study reveals transcriptional, signaling and cellular programs governing pig pancreatic islet development, including striking similarities to human islet ontogeny, providing a novel resource for advancing human islet replacement strategies.

developmental biology↗

bioRxiv2019

Establishment of morphological atlas of Caenorhabditis elegans embryo with cellular resolution using deep-learning-based 4D segmentation

Cell lineage consists of cell division timing, cell migration and cell fate, which are highly reproducible during the development of some nematode species, including C. elegans. Due to the lack of high spatiotemporal resolution of imaging technique and reliable shape-reconstruction algorithm, cell morphology have not been systematically characterized in depth over development for any metazoan. This significantly inhibits the study of space-related problems in developmental biology, including cell segregation, cell-cell contact and cell shape change over development. Here we develop an automated pipeline, CShaper, to help address these issues. By quantifying morphological parameters of densely packed cells in developing C. elegans emrbyo through segmentation of fluorescene-labelled membrance, we generate a time-lapse framework of cellular shape and migration for C. elegans embryos from 4-to 350-cell stage, including a full migration trajectory, morphological dynamics of 226 cells and 877 reproducible cell-cell contacts. In combination with automated cell tracing, cell-fate associated cell shape change becomes within reach. Our work provides a quantitative resource for C. elegans early development, which is expected to facilitate the research such as signaling transduction and cell biology of division.

developmental biology↗

bioRxiv2025

ATAC-seq of Low-Input and Cryopreserved Primordial Germ Cells Reveals Functional Enhancers

Cell fate specification during development is orchestrated by dynamic changes in gene expression, driven by the accessibility of cis-regulatory elements. To profile such functional regulatory elements at the genome-wide level, bulk ATAC-seq is a powerful method. Yet, its application has still been technically limited in rare cell types. Here, we optimized and titrated the ATAC-seq protocol for cultured chicken primordial germ cells (PGCs) and obtained high-quality chromatin accessibility profiles from as few as 200 cells, even after cryopreservation. Integrative analysis of our ATAC-seq with published RNA-seq and tissue ATAC-seq datasets identified over 10,000 PGC-specific accessible chromatin regions (ACRs), many absent from somatic tissues. Reporter assays demonstrated activity of selected enhancers in cultured PGCs, while transcriptome integration revealed preferential association of PGC-specific ACRs with genes highly expressed at embryonic day 2.5. In vivo transplantation confirmed that these enhancers are active in an early-stage-specific manner, but become silenced upon gonadal settlement. In conclusion, this streamlined method provides a practical and cost-effective approach to the epigenomic analysis of scarce cell populations, facilitating future investigations in developmental biology, germline research, and beyond.

developmental biology↗