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bioRxiv2025

Oogenesis and germinal bed morphology of the brown anole (A. sagrei)

BackgroundThe brown anole is a model species of the genus Anolis, a squamate (encompassing lizards and snakes) group widely studied in evolutionary, behavioral, and developmental biology. Full genome annotation, the establishment of gene editing techniques, and comprehensive description of reproductive tract morphology and embryogenesis in this species, has laid the foundation for functional studies. However, analysis of brown anole oogenesis is still required and vital to optimize genome modification, mutant line establishment, and analyses of the evolution of reproductive developmental mechanisms. ResultsHere, we characterize ovary morphology and gametogenesis in the female brown anole, A. sagrei using brightfield imaging, microCT, histology staining, electron microscopy, and confocal imaging. We define 10 stages of oocyte maturation which commences inside the oogonial nest within the germinal bed and concludes with the mature follicle ready to ovulate based on follicle size, yolk-acquisition, and follicular, cellular, and basement membrane architecture. ConclusionsWe describe the complete oogenesis of the brown anole in 10 stages and report that oogenesis is highly conserved within iguanids, a suborder of lizards. With our staging framework, we lay the foundation for functional studies of oogenesis and optimized gene-editing.

developmental biology↗

bioRxiv2026

Real-Time Multi-Position and Multi-ROI Tracking with LiLiTTool for Smart Light-Sheet Microscopy in Growing Samples

Long-term live imaging of growing samples with light-sheet fluorescence microscopy provides unique insights into development, but morphogenesis often displaces features of interest outside the microscopes field of view (FOV), calling for automated methods to track these features and update the microscopes FOV in real time. Existing solutions, which typically rely on local or global intensity distributions, struggle to follow specific features robustly throughout morphogenesis, leading to truncated or incomplete datasets. Here, we present a light-sheet live tracking tool (LiLiTTool) that maintains user-defined regions of interest (ROI) within the FOV throughout extended imaging sessions. LiLiTTool uses Cotracker3, a state-of-the art deep learning-based motion predictor, augmented by sensor fusion with a trained object-detector. This enables robust compensation for drift, rotation, and deformation during morphogenesis, while meeting the timing constraints of live acquisition. We validated LiLiTool by integrating with the Viventis LS1 microscope, achieving sub-second processing and stable tracking of growing zebrafish embryos over many hours. LiLiTTool supports multi-ROI tracking in 3D, enabling simultaneous monitoring of multiple features within the same embryo and in multiple embryos during a single acquisition. LiLiTTool is modular and openly available on GitHub and as a napari plugin for post-acquisition tracking. By enabling precise, adaptive, and scalable real-time imaging, LiLiTTool advances smart microscopy approaches and provides the developmental biology community with a practical tool for capturing reliable spatio-temporal information in growing embryos or other morphogenetic systems.

developmental biology↗

bioRxiv2023

Cell Lineage-Guided Microanalytical Mass Spectrometry Reveals Increased Energy Metabolism and Reactive Oxygen Species in the Vertebrate Organizer

Molecular understanding of the vertebrate Organizer, a tissue center critical for inductive signaling during gastrulation, has so far been limited to transcripts and some proteins due to limitations in detection and sensitivity. The Spemann-Mangold Organizer (SMO) in the South African Clawed Frog (X. laevis), a popular model of development, has long been discovered to induce the patterning of the central nervous system. Molecular screens on the tissue have identified several genes, such as goosecoid, chordin, and noggin, with independent ability to establish a body axis. A comprehensive study of proteins and metabolites produced in the SMO and their functional roles has been lacking. Here, we pioneer a deep discovery proteomic and targeted metabolomic screen of the SMO in comparison to the rest of the embryo using liquid chromatography high-resolution mass spectrometry (HRMS). Quantification of [~]4,600 proteins and a panel of metabolites documented differential expression for [~]450 proteins and multiple intermediates of energy metabolism in the SMO. Upregulation of oxidative phosphorylation (OXPHOS) and redox regulatory proteins gave rise to elevated oxidative stress and an accumulation of reactive oxygen species in the Organizer. Imaging experiments corroborated these findings, discovering enrichment of hydrogen peroxide in the SMO tissue. Chemical perturbation of the redox gradient affected mesoderm involution during early tissue movements of gastrulation. HRMS expands the bioanalytical toolbox of cell and developmental biology, providing previously unavailable information on molecular classes to challenge and refine our classical understanding of the Organizer and its function during early patterning of the embryo.

developmental biology↗

bioRxiv2025

Intestinal enteroendocrine cell subtype differentiation and hormone production in zebrafish

Enteroendocrine cells (EECs) are rare sensory cells in the intestinal epithelium that coordinate digestive physiology by secreting a diverse repertoire of peptide hormones. These hormones are the main effectors of EEC function, and their characterization requires direct observation by mass spectrometry due to the specialized protein cleavage and posttranslational modifications that yield their mature forms. Based on the distinct subset of hormones they predominantly secrete, EECs can be categorized into subtypes. How each EEC subtype is specified, however, remains poorly understood. Here we describe EEC subtype differentiation and hormone production in the zebrafish. Using single-cell RNA sequencing data, we identified EEC progenitors and six EEC subtypes in zebrafish and revealed that their expression profiles are consistent across larval and adult stages. Mass spectrometry analysis of isolated zebrafish EECs identified highly processed peptides derived from 18 of 21 hormone coding genes expressed by EECs, yielding a catalog of >400 unique EEC hormone peptides. We assembled reporters for zebrafish EEC subtypes to test the lineage relationships between EEC subtypes and the EEC progenitor population, which expresses neurogenin3. Despite its essential role in mammalian EEC differentiation, we found that selective cytotoxic ablation of neurogenin3+ cells in zebrafish only reduced a subset of EEC subtypes. Finally, we discovered that selective ablation of ghrelin+ EECs reduced a different subset of EEC subtypes, together suggesting that neurogenin3+ and ghrelin+ cells serve as distinct precursors for separate EEC subtypes. We anticipate these observations and resources will facilitate future studies in the zebrafish to discern the developmental biology, physiology, and endocrinology of EEC subtypes.

developmental biology↗

bioRxiv2021

Spatially resolved whole transcriptome profiling in human and mouse tissue using Digital Spatial Profiling

Emerging spatial profiling technology has enabled high-plex molecular profiling in biological tissues, preserving the spatial and morphological context of gene expression. Here we describe expanding the chemistry for the Digital Spatial Profiling platform to quantify whole transcriptomes in human and mouse tissues using a wide range of spatial profiling strategies and sample types. We designed multiplexed in situ hybridization probe pools targeting the protein-coding genes in the human and mouse transcriptomes, hereafter referred to as the human or mouse Whole Transcriptome Atlas (WTA). We validated the human and mouse WTA using cell lines to demonstrate concordance with orthogonal gene expression profiling methods in profiled region sizes ranging from ~10-500 cells. By benchmarking against bulk RNAseq and fluorescence in situ hybridization, we demonstrate robust transcript detection possible down to ~100 transcripts per region. To assess the performance of WTA across tissue and sample types, we applied WTA to biological questions in cancer, molecular pathology, and developmental biology. We show that spatial profiling with WTA can detect expected spatial gene expression differences between tumor and tumor microenvironment, identify spatial disease-specific heterogeneity in gene expression in histological structures of the human kidney, and comprehensively map transcriptional programs in anatomical substructures of nine organs in the developing mouse embryo. Digital Spatial Profiling technology with the WTA assays provides a flexible method for spatial whole transcriptome profiling applicable to diverse tissue types and biological contexts.

genomics↗

bioRxiv2021

How Our Cells Become Our Selves: The Cellular Phylodynamic Biology of Growth and Development

Our lives begin with 1 cell, then 2, then 4, then the trillion cell adult, comprised of cell lineages, tissues, organs. How does this occur? Examination in numbers of cells, N, Cellular Phylodynamics, revealed two previously unappreciated processes: UNI-GROWTH, the slowing of growth that occurs as we become larger, caused by fewer cells dividing, captured by the Universal Mitotic Fraction and Universal Growth Equations, with accuracy confirmed for 13 species, including nematodes, mollusks, and vertebrates; and ALLO-GROWTH, the creation of body parts from Founder Cells, captured by the Cellular Allometric Growth Equation, which describes mitotic expansion by Cell-Heritable change in the Cell Cycle Time. These equations can generate cell lineage approximations, bringing the power of coalescent theory to developmental biology.

developmental biology↗

bioRxiv2026

An optimized workflow for spatial transcriptomics across early development in Xenopus

How gene expression patterns change spatially as the embryo transitions from simple to complex structures remains a major developmental biology question. Recently developed imaging-based spatial transcriptomics (ST) enable mapping expression of multiple gene at a single-cell resolution. Although Xenopus is a key model in embryology there is no established ST pipeline, and commercially available techniques face many challenges (sample preparation, probe design, cell segmentation). Furthermore, the highly diverse cell shapes and sizes across developmental stages and between different tissues represent major hurdles to accurately defining cells. Here, we describe an optimized workflow for ST in blastula-to-tailbud-stage frog embryos using Merscope, commercial MERFISH (Multiplexed Error-Robust Fluorescence In Situ Hybridization) originally designed for standard mammalian tissues. With stringent quality control and tailored computational pipelines, we optimize this technology for robust, semi-quantitative profiling of spatial transcriptomic landscapes in non-mammalian embryos. Reliable tissue preservation and cell-segmentation enable high-resolution mapping of gene expression during the development of a complex multi-tissue organization. This versatile strategy applies broadly to various dynamic systems, from embryos of various model organisms to complex and heterogeneous organs in mammals. Summary statementThis Single-cell Spatial Transcriptomics pipeline and reference atlas in Xenopus - a model organism in embryology - overcome technical challenges and resolve dynamic changes in patterning during development.

developmental biology↗

bioRxiv2024

Sex-biased gene expression precedes sexual dimorphism in the agonadal annelid Platynereis dumerilii

Gametogenesis is the process by which germ cells differentiate into mature sperm and oocytes, cells essential for sexual reproduction. The sex-specific molecular programs that drive spermatogenesis and oogenesis can also serve as sex identification markers. Platynereis dumerilii is a research organism that has been studied in many areas of developmental biology. However investigations often disregard sex, as P. dumerilii juveniles lack sexual dimorphism. The molecular mechanisms of gametogenesis in the segmented worm P. dumerilii are also largely unknown. In this study, we used RNA sequencing to investigate the transcriptomic profiles of gametogenesis in P. dumerilii juveniles. Our analysis revealed that sex-biased gene expression becomes increasingly pronounced during the advanced developmental stages, particularly during the meiotic phases of gametogenesis. We identified conserved genes associated with spermatogenesis, such as dmrt1, and a novel gene psmt, that is associated with oogenesis. Additionally, putative long non-coding RNAs were upregulated in both male and female gametogenic programs. This study provides a foundational resource for germ cell research in P. dumerilii, markers for sex identification, and offers comparative data to enhance our understanding of the evolution of gametogenesis mechanisms across species. Summary statementThis study provides insights into the mechanisms of gametogenesis in Platynereis dumerilii through comparative transcriptomics, unveiling sex-biased genes, including conserved and novel genes, governing this largely unexplored process.

developmental biology↗

bioRxiv2026

Whole-Embryo 3D Quantification Reveals Conserved Topological Design and Scaling of Germ Layers in Xenopus

How embryos with markedly different absolute sizes and cell numbers establish comparable tissue organization during development remains a fundamental question in developmental biology. To address this question, we compared two closely related Xenopus species that differ substantially in embryonic size, Xenopus laevis and Xenopus tropicalis. We generated a whole-embryo quantitative 3D atlas of cell allocation, spatial organization, and mitotic dynamics at key time points between gastrulation to tailbud stages. Using tissue clearing, and 3D imaging we tracked single-nucleus coordinates across developmental milestones to resolve how body plans adapt to organismal scale. We show that embryonic scaling is not achieved through simple proportional changes in cell number. Instead, the smaller X. tropicalis embryo is characterized by a distinct high-density tissue organization associated with a persistently higher mitotic index (~1.4-fold higher than in X. laevis at both gastrula and tailbud stages). Across development, this is accompanied by a near-doubling of cell number in X. tropicalis without a proportional increase in embryo volume. We quantify tissue organization and find species-specific cellular architectures during gastrulation that largely converge by the tailbud stage. At this stage, homologous tissues display broadly similar structural profiles despite persistent differences in embryo size, cell number, and density. Together, our findings reveal that closely related vertebrate embryos can follow distinct cellular organization trajectories while converging toward comparable tissue architecture, providing a quantitative framework for understanding robust body plan formation across divergent physical scales.

Developmental Biology↗

bioRxiv2020

DNA methylation during development and regeneration of the annelid Platynereis dumerilii

BackgroundMethylation of cytosines in DNA (5mC methylation) is a major epigenetic modification that modulates gene expression and is important for embryonic development and cell reprogramming in vertebrates. In mammals, 5mC methylation in promoter regions is linked to transcriptional repression. Transcription regulation by 5mC methylation notably involves the Nucleosome Remodeling and Deacetylase complex (NuRD complex) which bridges DNA methylation and histone modifications. Less is known about roles and mechanisms of 5mC methylation in non-vertebrate animals. In this paper, we study 5mC methylation in the marine annelid worm Platynereis dumerilii, an emerging evolutionary and developmental biology model capable of regenerating the posterior part of its body upon amputation. The regenerated region includes both differentiated structures and a growth zone consisting of stem cells required for the continuous growth of the worm. ResultsUsing in silico and experimental approaches, we show that P. dumerilii displays a high level of DNA methylation comparable to that of mammalian somatic cells. 5mC methylation in P. dumerilii is dynamic along the life cycle of the animal and markedly decreases at the transition between larval to post-larval stages. We identify a full repertoire of mainly singlecopy genes encoding the machinery associated to 5mC methylation or members of the NuRD complex in P. dumerilii and show, through phylogenetic analyses, that this repertoire is close to the one inferred for the last common ancestor of bilaterians. These genes are dynamically expressed during P. dumerilii development, growth and regeneration. Treatment with the DNA hypomethylating agent Decitabine, impairs P. dumerilii larval development and regeneration, and has long-term effects on post-regenerative growth by affecting the functionality of stem cells of the growth zone. ConclusionsOur data indicate high-level of 5mC methylation in the annelid P. dumerilii, highlighting that this feature is not specific to vertebrates in the bilaterian clade. Analysis of DNA methylation levels and machinery gene expression during development and regeneration, as well as the use of a chemical inhibitor of DNA methylation, suggest an involvement of 5mC methylation in P. dumerilii development, regeneration and stem cell-based post-regenerative growth. We also present data indicating that P. dumerilii constitutes a promising model to study biological roles and mechanisms of DNA methylation in non-vertebrate bilaterians and to provide new knowledge about evolution of the functions of this key epigenetic modification in bilaterian animals.

developmental biology↗

bioRxiv2025

Automated Image-Based Profiling of Pluripotent Stem Cell Colonies

Quantitative image analysis is essential for advancing stem cell biology, developmental studies, and drug discovery, yet most workflows still rely on manual or semi-quantitative scoring that is slow, subjective, and poorly scalable. A major challenge is converting complex colony morphologies into reproducible, high-dimensional datasets. To address this gap, we developed ColonyQuant, an open-source platform that integrates automated colony segmentation, alkaline phosphatase (AP) intensity quantification, morphometric profiling, and statistical classification into a single workflow. ColonyQuant computes per-colony functional readouts alongside comprehensive shape descriptors, capturing both staining intensity and structural features in a unified framework. Applied to embryonic stem cells (ESCs) treated with a selective KDM4 histone-demethylase inhibitor, ColonyQuant revealed dose-dependent reductions in colony area and integrated AP signal, together with systematic remodeling of morphometric metrics. Multivariate analyses robustly stratified treatment groups and identified intensity and solidity as principal features capturing dose-dependent colony responses. By transforming subjective scoring into objective, scalable, and biologically interpretable phenotyping, ColonyQuant provides a reproducible platform for stem cell research and high-content screening.

developmental biology↗

bioRxiv2023

Tracking Early Mammalian Organogenesis - Prediction and Validation of Differentiation Trajectories at Whole Organism Scale

Early organogenesis represents a key step in animal development, where pluripotent cells divide and diversify to initiate formation of all major organs. Here, we used scRNA-Seq to profile over 300,000 single cell transcriptomes sampled in 6 hour intervals from mouse embryos between E8.5 and E9.5. Combining this dataset with our previous E6.5 to E8.5 atlas resulted in a densely sampled time course of over 400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction at full organismal scale identified complex waves of blood and endothelial development, including a new molecular programme for somite-derived endothelium. To assess developmental fates across the primitive streak, we dissected the E7.5 primitive streak into four adjacent regions, performed scRNA- Seq and predicted cell fates computationally. We next defined early developmental state/fate relationships experimentally by a combination of orthotopic grafting, microscopic analysis of graft contribution as well as scRNA-Seq to transcriptionally determine cell fates of the grafted primitive streak regions after 24h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with the fates predicted computationally, thus demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in both developmental biology as well as guide the in vitro production of cells for organ regeneration and repair.

developmental biology↗

bioRxiv2023

Microinjection, gene knockdown, and CRISPR-mediated gene knock-in in the hard coral, Astrangia poculata

Cnidarians have become valuable models for understanding many aspects of developmental biology including the evolution of body plan diversity, novel cell type specification, and regeneration. Most of our understanding of gene function during early development in cnidarians comes from a small number of experimental systems including the sea anemone, Nematostella vectensis. Few molecular tools have been developed for use in hard corals, limiting our understanding of this diverse and ecologically important clade. Here, we report the development of a suite of tools for manipulating and analyzing gene expression during early development in the northern star coral, Astrangia poculata. We present methods for gene knockdown using short hairpin RNAs, gene overexpression using exogenous mRNAs, and endogenous gene tagging using CRISPR-mediated gene knock-in. Combined with our ability to control spawning in the laboratory, these tools make A. poculata a tractable experimental system for investigative studies of coral development. Further application of these tools will enable functional analyses of embryonic patterning and morphogenesis across Anthozoa and open new frontiers in coral biology research. Summary StatementThis study reports the development of the first transgenic knock-in coral, providing the opportunity to track the behavior of various cell types during early coral development.

developmental biology↗

bioRxiv2021

Development of larvae of the Australian blowfly, Calliphora augur (Diptera: Calliphoridae), at constant temperatures

Calliphora augur (Diptera: Calliphoridae) is a common carrion-breeding blowfly of forensic, medical and agricultural importance in eastern Australia. Despite this, detailed information on the developmental biology of C. augur is lacking. Here, we present the first comprehensive study on the development of all three larval instars of C. augur, fed on sheeps liver, at constant temperatures of 15, 20, 25, 30 and 35{degrees}C. We provide thermal summation models describing instar duration, as well as 95% prediction intervals for larval length at each constant temperature, enabling the age of larvae of C. augur to be estimated from their developmental stage and their average length. These data provide a basis for the application of this species to forensic investigations.

developmental biology↗

bioRxiv2025

TGFβ-dependent upregulation of OCIAD2 is essential for epithelial-to-mesenchymal transition during mesendoderm differentiation

Deciphering mechanisms that govern lineage commitment of human pluripotent stem cells (hPSCs) is essential for optimizing differentiation strategies. Here, we investigated the role of the mitochondrial protein OCIAD2 in hPSC differentiation. We show that OCIAD2 is transiently upregulated during mesendoderm differentiation, in a TGF{beta}/Activin A-dependent manner. OCIAD2 depletion impairs mesendoderm induction and specification, resulting in an incomplete epithelial-to-mesenchymal transition (EMT). Through transcriptome analysis, immunoblotting and localization studies on OCIAD2-depleted (KO) or overexpressing (OV) human embryonic stem cells (hESCs), we identified OCIAD2 as a positive regulator of TGF{beta} signalling. Additionally, KO hESCs exhibited downregulated fatty acid oxidation (FAO) genes, indicating dysregulated lipid metabolism. Pharmacological restoration of FAO improved mesendoderm differentiation capacity in KO, suggesting a key role for OCIAD2 in coordinating metabolism and TGF{beta} signalling. We propose that OCIAD2 regulates EMT by integrating metabolic cues with TGF{beta} pathway activation. Our findings provide insight into how mitochondrial proteins regulate lineage commitment and EMT, with broader implications for both developmental biology and tumor progression. HighlightsO_LIOCIAD2 expression peaks during mesendoderm specification of hESCs C_LIO_LIOCIAD2 depletion transcriptionally represses TGF{beta} signalling and impedes EMT C_LIO_LILoss of OCIAD2 leads to hyperfused mitochondria and downregulation of FAO genes C_LIO_LIPharmacologic supplementation with acetate enhances mesendoderm specification C_LI

developmental biology↗

bioRxiv2017

A simple bioreactor-based method to generate kidney organoids from pluripotent stem cells

Kidney organoids generated from human pluripotent stem cells have the potential to revolutionize how kidney development and injury are studied. Current protocols are technically complex and suffer from poor reproducibility and high reagent costs restricting scalability. To overcome these issues, we have established a simple, inexpensive and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day (d) 8 and show optimal tissue morphology at d14. A comparison with fetal human kidney suggests that d14 organoid renal structures most closely resemble capillary loop stage nephrons. We show that deletion of HNF1B, a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant human renal development.

developmental biology↗

bioRxiv2024

Spatial patterning of the epigenome during vertebrate gastrulation

A central question in developmental biology is how embryonic cells acquire and store positional information during pattern formation. In vertebrates, this process begins with the localized activation of signaling systems that mediate axial specification. How these spatial cues are recorded within the regulatory landscape of cells has remained unclear. Here, we report that the chromatin landscape of embryonic cells is spatially patterned during gastrulation. Using spatially resolved genomic analysis in avian embryos, we observed that the epigenome becomes organized in gradients of accessibility and activity along the embryonic axes. These chromatin gradients are established within the loci of developmental genes at the onset of gastrulation and can be used to infer the position of cells in the embryo. Our results show that axial specification involves the spatial organization of the epigenome, linking patterns of chromatin activity to the emergence of the embryonic body plan.

developmental biology↗

bioRxiv2023

BirthSeq, a new method to isolate and analyze dated cells from any tissue in vertebrates

Embryonic development is a complex and dynamic process that unfolds over time and involves the production of increasing numbers of cells, as well as the diversification of different cell types. The impact of developmental time on the formation of the central nervous system is well-documented, with evidence showing that time plays a critical role in establishing the identity of neuronal subtypes. However, the study of how time translates into genetic instructions driving cell fate is limited by the scarcity of suitable experimental tools. We introduce BirthSeq, a new method for isolating and analyzing cells based on their birth date. This innovative technique allows for in vivo labeling of cells, isolation via FACS, and analysis using high-throughput techniques. We demonstrate the effectiveness of BirthSeq for single-cell RNA sequencing and novel spatially resolved transcriptomic approaches in brain development across three vertebrate species (mouse, chick, and gecko). Overall, BirthSeq provides a versatile tool for studying any tissue in any vertebrate organism, helping to fill the necessity in developmental biology research by targeting cells and their temporal cues. SUMMARY STATEMENTBirthSeq allows the isolation and investigation of alive cells according to their birthdate, in any kind of tissue and vertebrate species.

developmental biology↗