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bioRxiv2025

Cryopreservation of Sea Urchin (Lytechinus pictus) Embryos and Development Through Metamorphosis

BackgroundSea urchins have contributed to knowledge of fertilization, embryonic development and cell physiology for 150 years. Their evolutionary position, as basal deuterostomes, and their long background in developmental biology, motivate establishing a genetically-enabled sea urchin species. Because of its relatively short generation time of 4-6 months and ease of culture, our lab has focused on the California sea urchin Lytechinus pictus as a multigenerational model, and produced knockout and transgenic lines using this species. To ensure that diverse genetic lines can be preserved, methods must be developed to cryopreserve gametes and embryos. We have previously reported methods for cryopreservation of sperm, but robust methods to preserve embryos remained lacking. ResultsHere, we describe a relatively simple method to cryopreserve late gastrulae embryos of L. pictus. Importantly we show that, after thawing and culturing, the embryos progress through larval development, undergo metamorphosis and yield juvenile adults, indicating the method is robust. ConclusionThe cryopreservation of embryos is an important advance that will facilitate the biobanking, sharing and long-term preservation of diverse genetic lines. This method may also eventually prove useful for cryopreservation of embryos of other marine invertebrates.

developmental biology↗

bioRxiv2020

Multidimensional Chromatin Regulation of Cell Lineage Differentiation in a Metazoan Embryo

How chromatin dictates cell differentiation is an intriguing question in developmental biology. Here, a reporter gene integrated throughout the genome was used as a sensor to map the chromatin activity landscape in lineage-resolved cells during C. elegans embryogenesis. Single-cell analysis of chromatin dynamics across critical dimensions of cell differentiation was performed, including lineage, tissue, and symmetry. During lineage progression, chromatin gradually diversifies in general and exhibits switch-like changes following specific cell division, which is predictive of anterior-posterior fate asymmetry. Upon tissue differentiation, chromatin of cells from distinct lineages converge to tissue-specific states but retain "memory" of each cells lineage history, which contributes to intra-tissue heterogeneity. However, cells with a morphologically left-right symmetric organization utilize a predetermination chromatin strategy to program analogous regulatory states in early progenitor cells. Additionally, chromatin co-regulation drives the functional coordination of the genome. Collectively, this work reveals the role of multidimensional chromatin regulation in cell differentiation.

developmental biology↗

bioRxiv2015

A Branching Process to Characterize the Dynamics of Stem Cell Differentiation

The understanding of the regulatory processes that orchestrate stem cell maintenance is a cornerstone in developmental biology. Here, we present a mathematical model based on a branching process formalism that predicts average rates of proliferative and differentiative divisions in a given stem cell population. In the context of vertebrate spinal neurogenesis, the model predicts complex non-monotonic variations in the rates of pp, pd and dd modes of division as well as in cell cycle length, in agreement with experimental results. Moreover, the model shows that the differentiation probability follows a binomial distribution, allowing us to develop equations to predict the rates of each mode of division. A phenomenological simulation of the developing spinal cord informed with the average cell cycle length and division rates predicted by the mathematical model reproduces the correct dynamics of proliferation and differentiation in terms of average numbers of progenitors and differentiated cells. Overall, the present mathematical framework represents a powerful tool to unveil the changes in the rate and mode of division of a given stem cell pool by simply quantifying numbers of cells at different times.

Developmental Biology↗

bioRxiv2024

CRISPR-Cas13d as a molecular tool to achieve targeted gene expression knockdown in chick embryos

The chick embryo is a classical model system commonly used in developmental biology due to its amenability to gene perturbation experiments. Pairing this powerful model organism with cutting-edge technology can significantly expand the range of experiments that can be performed. Recently, the CRISPR-Cas13d system has been successfully adapted for use in zebrafish, medaka, killifish, and mouse embryos to achieve targeted gene expression knockdown. Despite its success in other animal models, no prior study has explored the potential of CRISPR-Cas13d in the chick. Here, we present an adaptation of the CRISPR-Cas13d system to achieve targeted gene expression knockdown in the chick embryo. As proof-of-principle, we demonstrate the knockdown of PAX7, an early neural crest marker. Application of this adapted CRISPR-Cas13d technique resulted in effective knockdown of PAX7 expression and function, comparable to knockdown achieved by translation-blocking morpholino. CRISPR-Cas13d complements preexisting knockdown tools such as CRISPR-Cas9 and morpholinos, thereby expanding the experimental potential and versatility of the chick model system.

developmental biology↗

bioRxiv2026

Hydrostatic pressure shapes and canalizes semicircular canal morphology to ensure vestibular function

How tissues acquire reproducible shapes to support their function is a fundamental question in developmental biology. Zebrafish semicircular canals form when epithelial pillars partition the lumen of the otic vesicle, the embryonic precursor of the inner ear, into the tubes that sense head rotation for balance. We show that hydrostatic pressure generated by the inflating otic vesicle shapes pillar geometry. Acute vesicle deflation shortens and broadens pillars, whereas pharmacologically induced inflation elongates and narrows them, effects captured by a physical model that treats the vesicle as a pressurized viscoelastic shell. Pillars initially form with variable curvature, but continued vesicle expansion drives them toward a common, straight geometry, a process accelerated by inflation and blocked by deflation. We identify Wnt signaling as crucial for sustaining this pressure by maintaining epithelial barrier integrity. Its disruption impairs pillar geometry and vestibular-dependent swimming behavior. Together, these findings identify hydrostatic pressure as a critical physical mechanism that canalizes tissue geometry to ensure robust organ formation and function.

developmental biology↗

bioRxiv2018

Quantitative study of the somitogenetic wavefront in zebrafish

A quantitative description of the molecular networks that sustain morphogenesis is one of the challenges of developmental biology. Specifically, a molecular understanding of the segmentation of the antero-posterior axis in vertebrates has yet to be achieved. This process known as somitogenesis is believed to result from the interactions between a genetic oscillator and a posterior-moving determination wavefront. Here we quantitatively study and perturb the network in zebrafish that sustains this wavefront and compare our observations to a model whereby the wavefront is due to a switch between stable states resulting from reciprocal negative feedbacks of Retinoic Acid (RA) on the activation of ERK and of ERK on RA synthesis. This model quantitatively accounts for the near linear shortening of the post-somitic mesoderm (PSM) in response to the observed exponential decrease during somitogenesis of the mRNA concentration of a morphogen (Fgf8). It also accounts for the observed dynamics of the PSM when the molecular components of the network are perturbed. The generality of our model and its robustness allows for its test in other model organisms.

developmental biology↗

bioRxiv2019

Analysis the Detailed Process of Glomerular Capillary Formation Using Immunofluorescence Perform With Ultrathick Section

BackgroundGlomerular capillary formation is one of the fundamental mysteries in renal developmental biology. However, there are still debates on this issue, and its detailed formation process has not been clarified.\n\nResultsTo resolve this problem, we performed antibody staining with ultra-thick section on embryonic and postnatal mouse kidneys to detect and analyze the development of glomerular capillaries. We found that blood vessel of the fetal kidneys expanded through proliferation and sprouting. During the comma-stage and S-shaped stages, 3-4 capillaries began to bud and migrate into the glomerular cleft, forming a capillary bed in the Bowmans capsule. Then, the capillary bed expanded into mature glomerular capillary by intussusceptive angiogenesis. The afferent and efferent arterioles were formed through pruning. The distribution of VEGFA in the nephron epithelial cells but not only in podocytes, induced multiple capillaries sprouted into the glomerular cleft. And CXCR4 played an important role in the differentiation and expansion of capillary bed into glomerular capillary.\n\nConclusionsImmunofluorescence performed with ultra-thick section allowed us to investigate the development of complex structure tissues systematically and comprehensively.

developmental biology↗

bioRxiv2026

Spatio-temporal visualisation and manipulation of endogenous Yap1 in medaka

A key requirement to address fundamental questions in developmental biology is the ability to visualise and manipulate endogenous signalling proteins in vivo. Here we generate such an enabling experimental tool for the highly conserved Hippo pathway and its transcriptional effector Yap1, which converts mechanical and chemical cues into transcriptional output during embryogenesis and regeneration. We generate and characterise a Yap1- mGreenLantern (Yap1-mGL) endogenous CRISPR/Cas9-mediated knock-in line that enables 4D analysis of Yap1 dynamics in medaka. We resolve Yap1- mGL expression and nuclear/cytoplasmic localisation in real time, revealing the onset of reporter fluorescence before gastrulation, tissue and cell-type specific Yap1-mGL localisation during somitogenesis and injury-induced Yap1 upregulation during larval spinal cord regeneration. Using the nanobody-based degron system, we degrade the Yap1 protein in vivo, recapitulating the genetic loss-of-function mutant phenotype. Combining endogenous tags with nanobody-based degrons offers a powerful approach to visualise and acutely perturb endogenous protein function in vivo.

developmental biology↗

bioRxiv2024

Mechanical control of germ cell specification in Arabidopsis anthers

A central question in developmental biology is how the germline is established. We have studied the specification of the male germ cells (GCs) within the anther1-3. Hereby, we have focused on the potential role of mechanics, an aspect of anther development which has been very poorly characterized2,4. Using a combination of live imaging and mechanical measurements, we provide evidence that GCs originate in a special micro-mechanical niche, where inner tissues exert push on outer cell layers, placing them under compression. Mechanical perturbations significantly disrupted the GC specification and patterning. Moreover, we found that the master genetic regulator SPOROCYTELESS/NOZZLE (SPL/NZZ)5,6 is central in establishing this micro-mechanical environment by softening the cell wall. The mechanical cues, in turn, stabilize the transcription of SPL/NZZ. We propose here an intrinsic growth-derived mechano-chemical feedback loop that drives germ-cell fate acquisition.

developmental biology↗

bioRxiv2020

Wnt signaling determines body axis polarity in regenerating Hydra tissue

How an animal establishes its body axis is a fundamental question in developmental biology. The freshwater cnidarian Hydra is an attractive model for studying axis formation because it is radially symmetric, with a single oral-aboral axis. It was recently proposed that the orientation of the new body axis in a regenerating Hydra is determined by the oral-aboral orientation of the actin-myosin contractile processes (myonemes) in the parent animals outer epithelial layer. However, because the myonemes are not known to possess polarity, it remained unclear how the oral-aboral polarity of the axis would be defined. As Wnt signaling is known to maintain axis polarity in Hydra and bilaterians, we hypothesized that it plays a role in axis specification in excised Hydra tissue pieces. We tested this hypothesis using pharmacological perturbations and novel grafting experiments to set Wnt-derived signals and myoneme orientation perpendicular to each other to determine which controls axis formation. Our results demonstrate that Wnt signaling is the dominant encoder of axis information, in line with its highly conserved role in anterior-posterior patterning.

developmental biology↗

bioRxiv2021

Optogenetic control of apical constriction induces synthetic morphogenesis in mammalian tissues

During embryonic development, cellular forces synchronize in space and time to generate functional tissue shapes. Apical constriction is one of these force-generating processes, and it is necessary to modulate epithelial curvature in fundamental morphogenetic events, such as neural tube folding. The emerging field of synthetic developmental biology proposes bottom-up approaches to examine the contribution of each cellular process to complex morphogenesis. However, the shortage of tools to manipulate three-dimensional (3D) shapes of mammalian tissues currently hinders the progress of the field. Here we report the development of "OptoShroom3", a new optogenetic tool that achieves fast spatiotemporal control of apical constriction in mammalian epithelia. Activation of OptoShroom3 through illumination of individual cells in an epithelial cell sheet reduced their apical surface while illumination of groups of cells caused deformation in the adjacent regions. By using OptoShroom3, we further manipulated 3D tissue shapes. Light-induced apical constriction provoked the folding of epithelial cell colonies on soft gels. Its application to murine and human neural organoids led to thickening of neuroepithelia, apical lumen reduction in optic vesicles, and flattening in neuroectodermal tissues. These results show that spatiotemporal control of apical constriction can trigger several types of 3D deformation depending on the initial tissue context.

developmental biology↗

bioRxiv2025

A method for analysing tissue motion and deformation during mammalian organogenesis

Understanding tissue morphogenesis is an important goal in developmental biology and tissue engineering. Accurately describing tissue deformation processes and how cell rearrangements contribute to these is a challenging task. Live analysis of morphogenesis in 3D is frequently used to obtain source data that allow to extract such features from developing organs. However, several limitations are encountered when applying these methodologies to mammalian embryos. The mouse embryo is the most frequently used model, but most studies use a very limited number of specimens and present only individual acquisitions due to constraints imposed by embryo culture and imaging. Here we used data from live analysis of embryonic heart development to develop methodologies that allow to 1) deduce tissue deformation and cell movements from image intensity flows, 2) stage and register data from several specimens/acquisitions to a dynamic consensus model of heart development, 3) statistically calculate and map to the model tissue growth and anisotropy and 4) generate a digital model of heart development that allows in-silico pseudo-cell fate mapping. Our methodology will greatly facilitate the understanding of morphogenetic processes underlying mammalian organogenesis.

developmental biology↗

bioRxiv2016

A gene regulatory network for apical organ neurogenesis and its spatial control in sea star embryos.

How neural stem cells generate the correct number and type of differentiated neurons in appropriate places is an important question in developmental biology. Although nervous systems are diverse across phyla, many taxa have a larva that forms an anterior concentration of neurons, or apical organ. The number of neurons in these organs is highly variable. We show that neurogenesis in the sea star larvae begins with soxc-expressing multipotent progenitors. These give rise to restricted progenitors that express lhx2/9. Soxc- and lhx2/9-expressing cells are capable of undergoing both asymmetric divisions, which allow for progression towards a particular neural fate, and symmetric proliferative divisions. Nested concentric domains of gene expression along the anterior-posterior (AP) axis, which have been observed in a great diversity of metazoans, control neurogenesis in the sea star by promoting particular division modes and progression towards becoming a neuron. This work, therefore, explains how spatial patterning in the ectoderm controls progression of neurogenesis. Modification to the sizes of these AP territories provides a simple mechanism to explain the diversity of neuron number found among apical organs.\n\nSummary StatementThe progression of apical organ neurogenesis in the sea star is controlled by regulatory anterior-posterior patterning domains.

Developmental Biology↗

bioRxiv2017

Microfluidic Immobilization And Subcellular Imaging Of Developing Caenorhabditis elegans

C. elegans has been an essential model organism in the fields of developmental biology, neuroscience, and aging. However, these areas have been limited by our ability to visualize and track individual C. elegans worms, especially at the subcellular scale, over the course of their lifetime. Here we present a microfluidic device to culture individual C. elegans in parallel throughout post-embryonic development. The device allows for periodic mechanical immobilization of the worm, enabling 3D imaging at subcellular precision. The immobilization is sufficient to enable fluorescence recovery after photobleaching (FRAP) measurements on organelles and other substructures within the same specific cells, throughout larval development, without the use of chemical anesthetics. Using this device, we measure FRAP recovery of two nucleolar proteins in specific intestinal cells within the same worms during larval development. We show that these exhibit different fluorescence recovery as they grow, suggesting differential protein interactions during development. We anticipate that this device will help expand the possible uses of C. elegans as a model organism, enabling its use in addressing fundamental questions at the subcellular scale, including the role of phase transitions in driving spatiotemporal intracellular organization within multicellular organisms.

developmental biology↗

bioRxiv2025

Purinergic signalling and calcium dynamics: potential drivers in the onset of coordinated intestinal motility in human fetal development

Background and aimsIntestinal motility relies on inputs from multiple cells within a complex neuromuscular syncytium located in the gut wall. While it is known that motility is dependent on the development of motor patterns which govern coordinated contractile activity within the gut wall, knowledge regarding the onset of coordinated motor activity in humans is still lacking. This study assessed the emergence of coordinated motor patterns, and the molecular mechanisms underpinning developing motility, in the human fetal gut. MethodsHuman fetal gut samples (obtained via the MRC-Wellcome Trust Human Developmental Biology Resource-UK) were characterised by live imaging, spatiotemporal mapping, immunohistochemistry and RNAseq. ResultsHuman small intestinal samples displayed the presence of key cell types including enteric neurons, interstitial cells of Cajal, platelet-derived growth factor receptor alpha positive (PDGFR+) cells and smooth muscle at post conception week (PCW) 12. Between PCW12 and PCW16, functional assessment revealed a marked increase in the velocity (p= 0.0341) of propagating contractions. Subsequently, between PCW16 and PCW20 the number of contraction initiation sites reduced drastically (p=0.0053), enabling the emergence of long-distance propagating contractions. Expression analyses showed the development of coordinated motor activity was coincident with increased expression of various genes involved in calcium and purinergic signalling pathways. ConclusionsThese findings provide the first direct mechanistic evidence of the temporal development of coordinated contractile activity in the human fetal intestine, highlighting the role of calcium dynamics, purinergic signalling and interstitial cells in early stages of human motility development, potentially informing an improved understanding of the pathogenesis of gut motility disorders.

developmental biology↗

bioRxiv2025

Evidence for chronological diversification of spinal neuron subtypes by a shared sequence of transcription factors

The mechanisms underlying the generation of the immense diversity of neuronal cell types remains a fundamental question of developmental biology. In the spinal cord, different "cardinal classes" of neurons that share a common molecular identity are produced from spatially segregated progenitor domains. Within many such classes, a stereotyped sequence of divergent neuronal types of related function is generated over time, raising the question of the molecular mechanisms that control this process. Here, we show that the successive expression of mouse transcription factors Onecut2, Pou2f2 and Pou3f1 within the cardinal classes giving rise to motor and sensory circuits, correlates with the emergence of sequentially generated subpopulations of neurons within those domains. We demonstrate that the genetic loss of Pou2f2 results in impaired development of two early-born motor neuron columns and re-specification of anterolateral system projection neurons as a later-born subset. Similarly, we show that Pou3f1 expression is required for the normal development of later-born subsets of motor neurons and anterolateral system projection neurons. Together, our observations provide functional evidence that horologic diversification of output neurons of spinal motor and sensory circuits are driven by a conserved sequential order of expression of transcription factors.

developmental biology↗

bioRxiv2026

EpiCure (Epithelial Curation): a versatile and handy tool for curation of epithelial segmentation

Investigating single-cell dynamics and morphology in tissues and embryos requires highly accurate quantitative analysis of microscopy images. Despite significant advances in the field of bioimage analysis, even the most sophisticated segmentation and tracking algorithms inevitably produce errors (e.g. : over segmentation, missing objects, miss-connected objects). Although error rate may be small, their propagation throughout a time-lapse sequence has catastrophic effects on the accuracy of tracking and extraction of single cell parameters. Extracting single cell temporal information in the context of tissue/embryo requires thus expert curation to identify and correct segmentation errors. In the movies commonly used in developmental biology and stem cell research, both the number of imaged cells and the duration of recording are large, making this manual correction task extremely time-consuming. This has now become a major bottleneck in the fields of development, stem cell biology and bioimage analysis. We present here EpiCure (Epithelial Curation), a versatile tool designed to streamline and accelerate manual curation of segmentation and tracking in 2D movies of large epithelial tissues. EpiCure uses temporal information and morphometric parameters to automatically identify segmentation and tracking errors and provides user-friendly tools to correct them. It focuses on ergonomics and offers several visualization options to help navigating in movies of tissue covering a large number of cells, speeding up the detection of errors and their curation. EpiCure is highly interoperable and supports input from a wide range of segmentation tools. It also includes multiple export filters, enabling seamless integration with downstream analysis pipelines. In this paper, using movies from several animal models, we highlight the importance of curating cell segmentation and tracking for accurate downstream analysis, and demonstrate how EpiCure helps the curation process for extracting accurate single cell dynamics and cellular events detection, making it faster and amenable on large dataset.

developmental biology↗

bioRxiv2018

Electroporation of short hairpin RNAs for rapid and efficient gene knockdown in the starlet sea anemone, Nematostella vectensis

A mechanistic understanding of evolutionary developmental biology requires the development of novel techniques for the manipulation of gene function in phylogenetically diverse organismal systems. Recently, gene-specific knockdown by microinjection of short hairpin RNA (shRNA) has been applied in the sea anemone Nematostella vectensis, a cnidarian model organism. Due to the unusual architecture of the cnidarian microRNA processing pathway, the shRNA approach is unusually effective for sequence-specific knockdown of a gene of interest. However, the time- and labor-intensive process of microinjection limits access to this technique and its application in large scale experiments. To address this issue, here we present an electroporation protocol for shRNA delivery into Nematostella eggs. This method leverages the speed and simplicity of electroporation, enabling users to manipulate gene expression in hundreds of Nematostella eggs or embryos within minutes. We provide a detailed description of the experimental procedure, including reagents, electroporation conditions, preparation of Nematostella vectensis eggs, and follow-up care of experimental animals. Finally, we demonstrate the knockdown of several endogenous and exogenous genes with known phenotypes and discuss the potential applications of this method.

developmental biology↗