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bioRxiv2020

Planarian CREB-binding protein (CBP) gene family regulates stem cell maintenance and differentiation

The regulation of stem cells plasticity and differentiation is still an open question in developmental biology. CBP (CREB-binding protein)/p300 is a conserved gene family which functions as a transcriptional co-activator and shows an important role in a wide range of cellular processes, such as cell death, DNA damage response and tumorigenesis. Moreover, CBPs have an acetyl transferase activity that is relevant as histone and non-histone acetylation results in changes in chromatin architecture and protein activity that affects gene expression. Many studies have shown the conserved functions of CBP/p300 on stem cell proliferation and differentiation. The planarian Schmidtea mediterranea is an excellent model to study in vivo the molecular mechanism underlying stem cell differentiation during regeneration. We have identified five different Smed-cbp genes in S. mediterranea that show different expression patterns. Functional analyses indicate that Smed-cbp-2 seems to be essential for stem cell maintenance and cell survival. On the other hand, the silencing of Smed-cbp-3 results in the growth of apparently normal blastemas; however, these remain largely depigmented and undifferentiated. Smed-cbp-3 silencing affects the differentiation of several cell lineages including neural, epidermal, digestive and excretory cell types. Finally, we have analyzed the predicted interactomes of CBP-2 and CBP-3 as an initial step to better understand their function on planarian stem cell biology.

developmental biology↗

bioRxiv2021

Inferring and perturbing cell fate regulomes in human cerebral organoids

Self-organizing cerebral organoids grown from pluripotent stem cells combined with single-cell genomic technologies provide opportunities to explore gene regulatory networks (GRNs) underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin profiling data over a dense time course covering multiple phases of organoid development including neuroepithelial formation, patterning, brain regionalization, and neurogenesis. We identify temporally dynamic and brain region-specific regulatory regions, and cell interaction analysis reveals emergent patterning centers associated with regionalization. We develop Pando, a flexible linear model-based framework that incorporates multi-omic data and transcription binding site predictions to infer a global GRN describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoid. We find that certain factors regulate the abundance of cell fates, whereas other factors impact neuronal cell states after differentiation. We show that the zinc finger protein GLI3 is required for cortical fate establishment in humans, recapitulating previous work performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify a regulome central to the dorsoventral telencephalic fate decision. This regulome suggests that Notch effectors HES4/5 are direct GLI3 targets, which together coordinate cortex and ganglionic eminence diversification. Altogether, we provide a framework for how multi-brain region model systems and single-cell technologies can be leveraged to reconstruct human brain developmental biology.

developmental biology↗

bioRxiv2024

Post-embryonic tail development through molting of the freshwater shrimp Neocaridina denticulata

BackgroundUnderstanding postembryonic morphogenesis through molting in arthropods has recently become a focus of developmental biology. The hierarchical mechanisms of epithelial sheet folds play a significant role in this process. Drosophila is a well-studied model for holometabolous insects, with extensive research on imaginal disc growth. While developmental processes in other arthropods have been described, live imaging of morphological changes is challenging due to the macroscopic movements and hard cuticles. Neocaridina denticulata, a crustacean, presents unique tail morphogenesis through molting, which makes it the potential model. This study investigated the development of the tail in Neocaridina denticulata through histological analysis and in vivo live imaging using fluorescent probes. This study also performed long-read sequencing of the whole genome for future genetic tools. ResultsThe tail of Neocaridina was found to undergo two major changes with the first ecdysis. Firstly, the branches of the uropods are cleared, and secondly, the telson undergoes convergent elongation. Cross-sectional analysis revealed that uropod and telson branching occurs immediately after hatching in the form of cuticle branching. The surface structure of the developmental tail suggested that telson elongation is achieved by the extension of anisotropic furrows in the cuticle during ecdysis. Anisotropy of cuticle furrows was associated with the epithelial cell shape, and the anisotropy of cell shape was found to occur during development from post-hatching. We also established an in vivo live imaging system with UV-LED resin and detected the changes of tail development over time. in vivo live imaging analysis revealed that telson contraction occurs gradually prior to ecdysis. Furthermore, we have also provided a draft genome of Neocaridina. ConclusionNeocaridina denticulata is a valuable model for studying morphogenesis in arthropods through molting. The tail undergoes complex changes involving cuticle branching, anisotropic furrows, and cellular dynamics. in vivo live imaging system provides insights into the developmental process, and the draft genome enhances the potential for genetic tools in future studies. This research contributes to the understanding of arthropod morphogenesis and provides a foundation for further developmental and cytological investigations in Neocaridina.

developmental biology↗

bioRxiv2025

Enhanced Multiplexed Single-Cell RNA-Sequencing for Accurate Detection of Treatment Effects Without Batch Correction in the Avian Embryonic Model

Single-cell RNA sequencing has revolutionized our ability to explore cellular heterogeneity and is a powerful tool to study the impact of environmental perturbations on multiple cell states. However, environmental perturbations can be subtle, and the associated biological effects could be masked by experimental noise and bioinformatic processing, especially when the samples are generated separately. Multiplexing strategies have been developed to label each sample and process them together to reduce experimental noise, but existing multiplexing methods often fall short for non-human and non-mouse models in established single-cell RNA sequencing protocols like the BD Rhapsody technology. To address this gap, we combined Lipid Modified Oligonucleotide (LMO) cell tagging with the BD Rhapsody platform to achieve efficient and scalable multiplexing of early embryonic chick cells under different environmental conditions. This species-agnostic LMO approach overcomes limitations of antibody-based multiplexing methods that are often restricted to human and mouse systems, and the only multiplexing option available with the BD Rhapsody system. In our study, we successfully compartmentalized and multiplexed chick embryonic cells under different treatment conditions, analyzing up to 40,000 viable cells per experiment. This strategy minimized experimental noise, eliminating the need for bioinformatics-based batch correction. As a result, we were able to achieve high-quality transcriptomic profiling with minimal loss of critical biological information and identified subtle biological differences that were masked when using data integration pipelines. Our workflow provides an adaptable, robust solution for LMO-tagged single-cell analyses of complex non-human models with the BD Rhapsody technology and opens new avenues for developmental biology research by accurately capturing treatment-induced effects in embryonic tissues.

developmental biology↗

bioRxiv2026

Capturing self-renewing multipotent neural crest stem cells from human pluripotent stem cells

During embryonic development, neural crest cells (NCCs) represent a multipotent population characterized by an inherently transient nature, rapidly differentiating into various lineages. This instability has presented a fundamental challenge, as it is exceedingly difficult to maintain these cells in a stable, multipotent state in vitro. In this study, we report a robust culture system dependent on Wnt and FGF signaling that enables the long-term (>6 months) expansion of human iPSC-derived neural crest stem cells (NCSCs). These NCSCs retain their self-renewal and differentiation capacity, validated at the single-cell clonal level. ATAC-seq analysis indicated that posterior NCSCs maintain a more permissive chromatin structure at neuronal gene loci. Furthermore, ChIP-seq analysis revealed that the key transcription factor SOX10 binds to the regulatory regions of genes involved in both maintenance and differentiation. This system provides a stable source of human NCSCs, offering a valuable platform for developmental biology, disease modeling, and regenerative medicine.

developmental biology↗

bioRxiv2024

inTRACKtive - A Web-Based Tool for Interactive Cell Tracking Visualization

We introduce inTRACKtive, an innovative web-based tool for interactive visualization and sharing of large 3D cell tracking datasets, eliminating the need for software installations or data downloads. Built with modern web technologies, inTRACKtive enables researchers to explore cell-tracking results from terabyte-scale microscopy data, conduct virtual fate-mapping experiments, and share these results via simple hyperlinks. The platform powers the Virtual Embryo Zoo, an online resource showcasing cell-tracking datasets from state-of-the-art light-sheet embryonic microscopy of six model organisms. inTRACKtives open-source code allows users to visualize their own data or host customized viewer instances. By providing easy access to complex tracking datasets, inTRACKtive offers a versatile, interactive, collaborative tool for developmental biology.

developmental biology↗

bioRxiv2024

Spatio-temporal Reconstruction of Gene Expression Patterns in Developing Mice

Understanding gene regulation in organism development is crucial in biology. Techniques like whole-mount in situ hybridization can reveal spatial gene expression in organs and tissues. However, capturing time-lapse images of gene expression dynamics in embryos developing in utero, such as mice, remains technically challenging beyond the early stages. To address this, we present a method to integrate static snapshots of gene expression patterns across limb developmental stages, creating a continuous 2D reconstruction of gene expression patterns over time. This method interpolates small tissue regions over time to create smooth temporal trajectories of gene expression. We successfully applied it to a number of key genes in limb development, including Sox9, Hand2, and Bmp2. This approach enables a detailed spatio-temporal mapping of gene expression, providing insights into developmental mechanisms. By estimating Gene Expression Patterns at previously unobserved time points, it facilitates the comparison of these patterns across samples. The reconstructed trajectories offer high-quality data that will be useful to guide computational modeling and machine learning, advancing the study of developmental biology in systems where real-time imaging is technically difficult or impossible.

developmental biology↗

bioRxiv2018

Quantitative comparison of the anterior-posterior patterning system in the embryos of five Drosophila species

Complex spatiotemporal gene expression patterns direct the development of the fertilized egg into an adult animal. Comparisons across species show that, in spite of changes in the underlying regulatory DNA sequence, developmental programs can be maintained across millions of years of evolution. Reciprocally, changes in gene expression can be used to generate morphological novelty. Distinguishing between changes in regulatory DNA that lead to changes in gene expression and those that do not is therefore a central goal of evolutionary developmental biology. Quantitative, spatially-resolved measurements of developmental gene expression patterns play a crucial role in this goal, enabling the detection of subtle phenotypic differences between species and the development of computations models that link the sequence of regulatory DNA to expression patterns. Here we report the generation of two atlases of cellular resolution gene expression measurements for the primary anterior-posterior patterning genes in Drosophila simulans and Drosophila virilis. By combining these data sets with existing atlases for three other Drosophila species, we detect subtle differences in the gene expression patterns and dynamics driving the highly conserved axis patterning system and delineate inter-species differences in the embryonic morphology. These data sets will be a resource for future modeling studies of the evolution of developmental gene regulatory networks.

systems biology↗

bioRxiv2018

Noise-resistant developmental reproducibility in vertebrate somite formation

The reproducibility of embryonic development is remarkable, although molecular processes are intrinsically stochastic at the single-cell level. How the multi-cellular system resists the inevitable noise to acquire developmental reproducibility constitutes a fundamental question in developmental biology. Toward this end, we focused on vertebrate somitogenesis as a representative system, because somites are repeatedly reproduced within a single embryo whereas such reproducibility is lost in segmentation clock gene-deficient embryos. However, the effect of noise on developmental reproducibility has not been fully investigated, because of the technical difficulty in manipulating the noise intensity in experiments. In this study, we developed a computational model of ERK-mediated somitogenesis, in which bistable ERK activity is regulated by an FGF gradient, cell-cell communication, and the segmentation clock, subject to the intrinsic noise. The model simulation generated our previous in vivo observation that the ERK activity was distributed in a step-like gradient in the presomitic mesoderm, and its boundary was posteriorly shifted by the clock in a stepwise manner, leading to regular somite formation. Here we showed that this somite regularity was robustly maintained against the noise. Removing the clock from the model predicted that the stepwise shift of the ERK activity occurs at irregular timing with irregular distance owing to the noise, resulting in somite size variation. Through theoretical analysis, we presented a mechanism by which the clock reduces the inherent somite irregularity observed in clock-deficient embryos. Therefore, this study indicates a novel role of the segmentation clock in noise-resistant developmental reproducibility.

developmental biology↗

bioRxiv2023

Automated staging of zebrafish embryos with KimmelNet

The zebrafish (Danio rerio), is an important biomedical model organism used in many disciplines. The phenomenon of developmental delay in zebrafish embryos has been widely reported as part of a mutant or treatment-induced phenotype. However, the detection and quantification of these delays is often achieved through manual observation with reference to staging guides, which is both time-consuming and subjective. We recently reported a machine learning-based classifier, capable of quantifying the developmental delay between two populations of zebrafish embryos. Here, we build on that work by introducing a deep learning-based model (KimmelNet) that has been trained to predict the age (hours post fertilisation) of populations of zebrafish embryos. We show that when KimmelNet is tested on 2D brightfield images of zebrafish embryos, the predictions generated agree closely with those expected from established approaches to staging. Random sampling of the test data demonstrate that KimmelNet can be used to detect developmental delay between two populations with high confidence based on as few as 100 images of each population. Finally, we show that KimmelNet generalises to previously unseen data, with limited transfer learning improving this performance significantly. With the ability to analyse tens of thousands of standard brightfield microscopy images on a timescale of minutes, we envisage that KimmelNet will be a valuable resource for the developmental biology community. Furthermore, the approach we have used could easily be adapted to generate models for other organisms.

developmental biology↗

bioRxiv2024

A community-oriented, data-driven resource to improve protocol design for cardiac modelling from human pluripotent stem cells

Protocol design and benchmarking is central to optimising model development using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs). By applying data mining to decades of research and hundreds of peer reviewed studies, we evaluate how protocol variables associate with common properties of cardiac functional and physiological maturation. This resource is publicly accessible through CMPortal, a community-oriented website that provides data-driven tools for researchers to navigate leverage decades of knowledge for benchmarking protocol designs and outcomes for their dedicated applications in developmental biology, disease modelling, and drug screening.

developmental biology↗

bioRxiv2020

GATA4 regulates epithelial morphogenesis in the developing mouse stomach to promote establishment of a glandular columnar epithelium

The transcription factor GATA4 is broadly expressed in nascent foregut endoderm. As development progresses, GATA4 is lost in the domain giving rise to the stratified squamous epithelium of the esophagus and forestomach (FS), while it is maintained in the domain giving rise to the simple columnar epithelium of the hindstomach (HS). Differential GATA4 expression within these domains coincides with the onset of distinct tissue morphogenetic events, suggesting a role for GATA4 in diversifying foregut endoderm into discrete esophageal/FS and HS tissues. By eliminating GATA4 in the developing HS or maintaining GATA4 in the developing FS, we identified GATA4 as an essential, principal regulator of simple columnar epithelium morphogenesis within the developing HS. GATA4- deficient HS epithelium adopted FS-like fate, and conversely, GATA4- expressing FS epithelium adopted HS-like fate. Underlying structural changes in these epithelia were broad changes in gene expression networks attributable to GATA4 directly activating or repressing expression of HS or FS defining transcripts. Our data implicate GATA4 as having a primary role in suppressing an esophageal/FS transcription factor network during HS development to promote a columnar epithelium. Moreover, GATA4-dependent phenotypes in developmental mutants reflected changes associated with Barretts esophagus, suggesting that developmental biology can provide insight into human disease mechanisms.

developmental biology↗

bioRxiv2024

Quantitative Resolving Cell Fate in the Early Embryogenesis of Caenorhabditis elegans

The nematode Caenorhabditis elegans exhibits an invariant cell lineage during its development where the gene-molecular network that regulates the development is crucial for the biological process. While there are many molecular cell atlases describing the phenomena and key molecules involved in cell transformation, the underlying mechanisms from a systems biology perspective have received less attention. Based on an endogenous molecular-cellular theory that relates the molecular mechanisms to biological phenotypes, we constructed a model of core endogenous network to describe the early stages of embryonic development of the nematode. Different cell types and intermediate cell states during development from zygotes to founder cells correspond to the steady states of the network as a nonlinear stochastic dynamical system. Connections between steady states form a topological landscape that encompasses known developmental lineage trajectories. By regulating the expression of agents in the network, we quantitatively simulated the effects of the Wnt and Notch signaling pathway on cell fate transitions and predicted the possible trajectories of transdifferentiation of the AB cell across the lineage. The success of the current study may help advance our understanding of the fundamental principles of developmental biology and cell fate determination, offering an effective tool for quantitative analysis of cellular processes.

developmental biology↗

bioRxiv2018

In vitro characterization of the human segmentation clock

The vertebral column is characterized by the periodic arrangement of vertebrae along the anterior-posterior (AP) axis. This segmental or metameric organization is established early in embryogenesis when pairs of embryonic segments called somites are rhythmically produced by the presomitic mesoderm (PSM). The tempo of somite formation is controlled by a molecular oscillator known as the segmentation clock 1,2. While this oscillator has been well characterized in model organisms 1,2, whether a similar oscillator exists in humans remains unknown. We have previously shown that human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells can differentiate in vitro into PSM upon activation of the Wnt signaling pathway combined with BMP inhibition3. Here, we show that these human PSM cells exhibit Notch and YAP-dependent oscillations4 of the cyclic gene HES7 with a 5-hour period. Single cell RNA-sequencing comparison of the differentiating iPS cells with mouse PSM reveals that human PSM cells follow a similar differentiation path and exhibit a remarkably coordinated differentiation sequence. We also demonstrate that FGF signaling controls the phase and period of the oscillator. This contrasts with classical segmentation models such as the \"Clock and Wavefront\" 1,2,5, where FGF merely implements a signaling threshold specifying where oscillations stop. Overall, our work identifying the human segmentation clock represents an important breakthrough for human developmental biology.

developmental biology↗

bioRxiv2025

Learning a mechanical growth model of flower morphogenesis

Morphogenesis, the process by which organs take their shape, is a fundamental problem in biology and requires a complex combination of genetic, biochemical, and mechanical signals. The data that we collect for this process are usually multi-scale and multi-modal, typically image timeseries of tissue morphology and transcription data. As the data becomes larger and more diverse, the harder it becomes to gain meaningful understanding in terms of theoretical models. Here, and tackling this problem in the developing flower organ, we learn a mechanical model of growth starting from combined morphology and transcription data across the tissue by firstly learning and connecting independent representations for the two spaces and secondly refining the representation using the mechanical state of the tissue to learn a mechanical growth model. This approach can link multiple scales, in this case proposing the physical action of genes, and can act as a vehicle to accommodate diverse data and hypotheses in developmental biology of plants and beyond.

developmental biology↗

bioRxiv2024

TiFM2.0 - Versatile mechanical measurement and actuation in live embryos

During development, spatial-temporally patterned tissue-level stresses and mechanical properties create diverse tissue shapes. To understand the mechanics of small-scale embryonic tissues, precisely controlled sensors and actuators are needed. Previously, we reported a control-based approach named tissue force microscopy (TiFM1.0), which combines dynamic positioning and imaging of an inserted cantilever probe to directly measure and impose forces in early avian embryos. Here we present an upgraded system (TiFM2.0) that utilises interferometer positioning to minimise probe holder footprint, enhancing accessibility and imaging signal. This new design enables a double-probe configuration for bidirectional stretching, compression and stress propagation experiments. As proof-of-concept, we showcase a variety of examples of TiFM2.0 applications in chicken and zebrafish embryos, including the characterization of mechanical heterogeneities important for the morphogenesis of the chicken posterior body axis. We also present simplified designs and protocols for the replication of TiFM systems with minimal custom engineering for developmental biology labs.

developmental biology↗

bioRxiv2020

A practical staging atlas to study embryonic development of Octopus vulgaris under controlled laboratory conditions

BackgroundOctopus vulgaris has been an iconic cephalopod species for neurobiology research as well as for cephalopod aquaculture. It is one of the most intelligent and well-studied invertebrates, possessing both long- and short-term memory and the striking ability to perform complex cognitive tasks. Nevertheless, how the common octopus developed these uncommon features remains enigmatic. O. vulgaris females spawn thousands of small eggs and remain with their clutch during their entire development, cleaning, venting and protecting the eggs. In fact, eggs incubated without females usually do not develop normally, mainly due to biological contamination (fungi, bacteria, etc.). This high level of parental care might have hampered laboratory research on the embryonic development of this intriguing cephalopod. ResultsHere, we present a completely parameter-controlled artificial seawater standalone egg incubation system that replaces maternal care and allows successful embryonic development of a small-egged octopus species until hatching in a laboratory environment. We also provide a practical and detailed 1 staging atlas based on bright-field and light sheet fluorescence microscopy imaging for precise monitoring of embryonic development. The atlas has a comparative section to benchmark stages to the different scales published by Naef (1928), Arnold (1965) and Boletzky (2016). Finally, we provide methods to monitor health and wellbeing of embryos during organogenesis. ConclusionBesides introducing the study of O. vulgaris embryonic development to a wider community, this work can be a high-quality reference for comparative evolutionary developmental biology.

developmental biology↗

bioRxiv2026

Measuring developmental information encoded by a dynamicallandscape

During embryogenesis, as cells proliferate and assemble into tissues, they undergo a sequence of transitions between distinct molecular states eventually giving rise to a cellular population consisting of an appropriate distribution of specific functional cell types. Recent progress on the dynamics underlying decision-making in developmental landscape makes it feasible to start analysing the amount of information involved in constructing such systems. To explore this we introduce the notion of potency of a developmental landscape and attempt to calculate it for two development systems of current interest, in-vitro differentiation of epiblast-like cells into neural and mesodermal progenitors and the worm vulva patterning system. Our approach integrates concepts from developmental biology, information theory and dynamical systems to estimate both the number and identity of signalling regimes that give rise to distinguishable temporal response patterns.

developmental biology↗