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Results for “developmental biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2021

The calcifying interface in a stony coral's primary polyp: An interplay between seawater and an extracellular calcifying space

Stony coral exoskeletons build the foundation to the most biologically diverse yet fragile marine ecosystems on earth, coral reefs. Understanding corals biomineralization mechanisms is therefore crucial for coral reef management and for using coral skeletons in geochemical studies. In this study, we combine in-vivo and cryo-electron microscopy with single-cell RNA-seq data to gain novel insights into the calcifying micro-environment that facilitates biomineralization in primary polyps of the stony coral Stylophora pistillata. We show an intimate involvement of seawater in this micro-environment. We further document increased tissue permeability and a highly dispersed cell packing in the tissue secreting the coral skeleton (i.e. calicoblastic). We also observe an extensive filopodial network containing carbon-rich vesicles extruding from some of the calicoblastic cells. Single-cell RNA-Seq data interrogation shows that calicoblastic cells express genes involved in filopodia and vesicle structure and function. These observations provide a new conceptual framework for resolving stony corals biomineralization processes.

developmental biology↗

bioRxiv2023

Preservation of co-expression defines the primary tissue fidelity of human neural organoids

Human neural organoid models offer an exciting opportunity for studying often inaccessible human-specific brain development; however, it remains unclear how precisely organoids recapitulate fetal/primary tissue biology. Here, we characterize field-wide replicability and biological fidelity through a meta-analysis of single-cell RNA-sequencing data for first and second trimester human primary brain (2.95 million cells, 51 datasets) and neural organoids (1.63 million cells, 130 datasets). We quantify the degree to which primary tissue cell-type marker expression and co-expression are recapitulated in organoids across 12 different protocol types. By quantifying gene-level preservation of primary tissue co-expression, we show neural organoids lie on a spectrum ranging from virtually no signal to co-expression near indistinguishable from primary tissue data, demonstrating high fidelity is within the scope of current methods. Additionally, we show neural organoids preserve the cell-type specific co-expression of developing rather than adult cells, confirming organoids are an appropriate model for primary tissue development. Overall, quantifying the preservation of primary tissue co-expression is a powerful tool for uncovering unifying axes of variation across heterogeneous neural organoid experiments.

developmental biology↗

bioRxiv2024

One probe fits all: a highly customizable modular RNA in situ hybridization platform expanding the application of SABER DNA probes

In situ hybridization (ISH) of RNA is a key method to visualize gene expression patterns in complex biological samples. The technique is indispensable for biological research related to e.g. development, disease, gene function, and the validation of novel cell types identified by single-cell sequencing methods. Especially in non-mammalian models lacking accessibility to a broad spectrum of antibodies, ISH remains a major research tool. Diverse available ISH protocols require different custom hybridization probe types, design, and/or proprietary signal detection chemistry. This makes it hard to navigate for a beginner and increases the research costs when multiple methods need to be applied. Here, we describe OneSABER - a unified open platform connecting commonly used canonical and recently developed single- and multiplex, colorimetric, and fluorescent ISH approaches. This platform uses a single type of ISH DNA probes adapted from the signal amplification by exchange reaction (SABER) method. We demonstrate applications of the proposed ISH framework in whole-mount samples of the regenerative flatworm Macrostomum lignano, advancing this animal as a powerful model for stem cell and regeneration research.

developmental biology↗

bioRxiv2022

Differential integration of activation and repression signals in a multi-enhancer system

Transcription in the early Drosophila blastoderm is coordinated by the collective action of hundreds of enhancers. Many genes are controlled by so-called "shadow enhancers," which provide resilience to environment or genetic insult, allowing the embryo to robustly generate a precise transcriptional pattern. Emerging evidence suggests that many shadow enhancer pairs do not drive identical expression patterns, however the biological significance of this remains unclear. In this study we characterize the shadow enhancer pair controlling the gene short gastrulation (sog). We removed either the intronic proximal enhancer or the upstream distal enhancer, and monitored sog transcriptional kinetics. Notably, each enhancer differs in sog spatial expression, timing of activation, and RNA Polymerase II loading rates. Additionally, modeling of individual enhancer activities demonstrates that these enhancers integrate activation and repression signals differently. While activation is due to the sum of the two enhancer activities, repression appears to depend on synergistic effects between enhancers. Finally, we examined the downstream signaling consequences resulting from the loss of either enhancer, and found changes in tissue patterning that are well explained by the differences in transcriptional kinetics measured. SUMMARY STATEMENTNon-intuitive shadow enhancer synergies are revealed by measuring transcriptional kinetics at the endogenous short gastrulation locus, giving rise to distinct patterning consequences in the dorsal ectoderm of Drosophila embryos.

developmental biology↗

bioRxiv2018

Neuropeptides required for Drosophila development under nutritional stress are regulated by the ER-Ca2+ sensor STIM

SignificanceElevation of cytosolic Ca2+ is required for the release of neuropeptides from specialised secretory neurons called neuroendocrine cells. Release of Ca2+ stored in the ER, which further triggers Ca2+ entry from the extracellular milieu is called Store-operated Ca2+ entry (SOCE). STIM, an ER protein, is a key regulator of SOCE and its contribution to neuroendocrine cell functioning is not well studied. Using Drosophila larval development under nutrient restriction as a paradigm, we identified two SOCE regulated neuropeptides, Corazonin and short Neuropeptide F. Reducing STIM alters the level of these neuropeptides in the fed as well as nutrient restricted condition. This study suggests that STIM-triggered SOCE may regulate the release of neuropeptides whose activity ultimately regulates adaptation to nutritional stress.\n\nAbstractCytosolic Ca2+ levels are tightly regulated by the sequestration of Ca2+ within the endoplasmic reticulum (ER). Loss of ER Ca2+ is sensed by STromal Interacting Molecule (STIM), whose translocation to the plasma membrane triggers Store Operated Ca2+ Entry (SOCE), and a subsequent rise in cytosolic Ca2+. Relatively little is known about SOCEs contribution to neuroendocrine cells; a neuronal sub-type that specializes in the secretion of neuropeptides (NPs) which ultimately regulate animal physiology and behavior. To investigate how SOCE regulates NPs, Drosophila development under nutrient restriction (NR) was used as the biological context. Genetic experiments identified the requirement of two SOCE-regulated NPs-corazonin (Crz) and short neuropeptide F (sNPF) - for the development of NR larvae to pupae and finally, adulthood. Overexpression of SOCE regulators was sufficient to rescue the development of NR larvae with reduced sNPF or Crz levels. To facilitate cellular investigations, a restricted set of neurons that produce sNPF and Crz, and are activated by NR, were identified. Immunohistochemistry on these neurons and mass spectrometric measurements of their projections showed that dSTIM regulates Crz and sNPF levels at steady state and in response to NR, likely by modulating their release. Genetically increasing neuronal output, in the background of reduced dSTIM, robustly rescued development of NR larvae. Because NP action fundamentally underpins how animals adapt to stimuli, regulation of NPs by STIM triggered SOCE is likely to be important. In situations where SOCE is altered, such as neurodegenerative diseases, this regulation may contribute to disease manifestation and progression.

developmental biology↗

bioRxiv2021

Omics profiling identifies MAPK/ERK pathway as a gatekeeper of nephron progenitor metabolism

Nephron endowment is defined by fetal kidney growth and critically dictates renal health in adults. Despite the advances in understanding the molecular regulation of nephron progenitors, the causes for low congenital nephron count and contribution of basic metabolism to nephron progenitor biology remain poorly understood. Here we characterized the metabolic consequences of MAPK/ERK-deficiency in nephron progenitors, whose maintenance and propagation in developing kidney critically depends on ERK activation. Our LC/MS-based metabolomics profiling identified 42 reduced metabolites, of which 26 were further supported by in vivo transcriptional characterization of MAPK/ERK-deficient nephron progenitors. This revealed a severe shortage of energy and nucleotide biosynthesis precursors, blockage in glycolysis and diminished pyruvate and proline metabolism. Utilization of in vitro kidney cultures demonstrated a dosage-specific function for glycolytic pyruvate as an energy source that controls the shape of the ureteric bud tip kwon to serve as a niche for nephron progenitor regulation. Analysis of the proline biosynthesis effects in developing kidney in vivo revealed premature loss of nephron progenitor maintenance in the absence of Pycr1/2 functions. Our results suggest that MAPK/ERK-dependent nephron progenitor metabolism functionally contributes to progenitor preservation by controlling pyruvate availability and proline metabolism in developing kidneys.

developmental biology↗

bioRxiv2025

Molecular noise modulates transitions in the cell-fate differentiation landscape

Waddingtons epigenetic landscape has become one of biologys cornerstone metaphors, widely used both conceptually and computationally. Cell types are often associated with the stable stationary points or valleys of this landscape. In previous work, we showed that the molecular noise dominating sub-cellular dynamics can distort and profoundly reshape this landscape. In non-equilibrium systems, an equally profound question arises: to what extent does noise alter the transition paths between valleys in such dynamic landscapes. We tackle this question using a set of illustrative exemplars, and show that noise gives rise to paths that differ substantially from the canonical least-action paths calculated under deterministic dynamics. We dissect the dynamics of these exemplars and determine the reactive density and transition currents, which show us, respectively, where and how transitions occur for different realisations of stochastic dynamics. Our analysis unambiguously demonstrates that reaction paths for stochastic dynamics diverge non-trivially from their deterministic least-action paths or simple barrier crossing models.

developmental biology↗

bioRxiv2018

Endogenous CRISPR arrays for scalable whole organism lineage tracing

The last decade has seen a renewed appreciation of the central importance of cellular lineages to many questions in biology (especially organogenesis, stem cells and tumor biology). This has been driven in part by a renaissance in genetic clonal-labeling techniques. Recent approaches are based on accelerated mutation of DNA sequences, which can then be sequenced from individual cells to re-create a \"phylogenetic\" tree of cell lineage. However, current approaches depend on making transgenic alterations to the genome in question, which limit their application. Here, we introduce a new method which completely avoids the need for prior genetic engineering, by identifying endogenous CRISPR target arrays suitable for lineage analysis. In both mouse and zebrafish we identify the highest quality compact arrays as judged by equal base composition, 5 G sequence, minimal likelihood of residing in the functional genome, minimal off targets and ease of amplification. We validate multiple high quality endogenous CRISPR arrays, demonstrating their utility for lineage tracing. Our technique thus can produce deep and broad lineages in vivo, while removing the dependence on genetic engineering, and also avoiding the need for single-cell analysis.

developmental biology↗

bioRxiv2020

Midkine-a functions as a universal regulator of proliferation during epimorphic regeneration in adult zebrafish

Zebrafish have the ability to regenerate damaged cells and tissues by activating quiescent stem and progenitor cells or reprogramming differentiated cells into regeneration-competent precursors. Proliferation among the cells that will functionally restore injured tissues is a fundamental biological process underlying regeneration. Midkine-a is a cytokine growth factor, whose expression is strongly induced by injury in a variety of tissues across a range of vertebrate classes. Using a zebrafish Midkine-a loss of function mutant, we evaluated regeneration of caudal fin, extraocular muscle and retinal neurons to investigate the function of Midkine-a during epimorphic regeneration. In wildtype zebrafish, injury among these tissues induces robust proliferation and rapid regeneration. In Midkine-a mutants, the initial proliferation in each of these tissues is significantly diminished or absent. Regeneration of the caudal fin and extraocular muscle is delayed; regeneration of the retina is nearly completely absent. These data demonstrate that Midkine-a is universally required in the signaling pathways that convert tissue injury into the initial burst of cell proliferation. Further, these data highlight differences in the molecular mechanisms that regulate epimorphic regeneration in zebrafish.

developmental biology↗

bioRxiv2023

Extraocular muscle stem cells exhibit distinct cellular properties associated with non-muscle molecular signatures

The muscle stem cell (MuSC) population is recognized as functionally heterogeneous. Cranial muscle stem cells, which originate from head mesoderm, can have greater proliferative capacity in culture and higher regenerative potential in transplantation assays when compared to those in the limb. The existence of such functional differences in phenotypic outputs remain unresolved as a comprehensive understanding of the underlying mechanisms is lacking. We addressed this issue using a combination of clonal analysis, live imaging, and scRNA-seq, identifying critical biological features that distinguish extraocular (EOM) and limb (Tibialis anterior, TA) MuSC populations. Time-lapse studies using a MyogenintdTomatoreporter showed that the increased proliferation capacity of EOM MuSCs is accompanied by a differentiation delay in vitro. Unexpectedly, in vitro activated EOM MuSCs expressed a large array of distinct extracellular matrix (ECM) components, growth factors, and signaling molecules that are typically associated with mesenchymal non-muscle cells. These unique features are regulated by a specific set of transcription factors that constitute a coregulating module. This transcription factor network, which includes Foxc1 as one of the major players, appears to be hardwired to EOM identity as it is present in quiescent adult MuSCs, in the activated counterparts during growth and retained upon passages in vitro. These findings provide insights into how high-performing MuSCs regulate myogenic commitment by active remodeling of their local environment.

developmental biology↗

bioRxiv2023

Cadherins modulate the self-organizing potential of gastruloids

Gastruloids have recently emerged as an efficient four-dimensional model for studying some aspects of post-implantation embryonic patterning. They undergo gastrulation-like processes leading to the self-organization into highly reproducible biological objects. Here, we sought to uncover the molecular and cellular mechanism underlying this remarkable property. We report that self-organization competence is associated with a cell-specific coordination of a Cadherin switch. We find that N-Cadherin hinders gastruloids morphogenetic competence, for its inactivation leads to the formation of trunk-like structures in absence of extra-cellular matrix analogues. In contrast, E-Cadherin repression by Snai1 is critical for self-organization: Snai1 establishes a cell-specific repressive pace by triggering the repression of a pluripotency-associated transcription program and its chromatin landscape, thus allowing a proper transition from E-to N-Cadherin to occur. Altogether, this work establishes a molecular mechanism that integrates the exit from pluripotency and the pace of cell differentiation, leading to the observed self-organizing potential of gastruloids.

developmental biology↗

bioRxiv2024

Rbpms2 promotes female fate upstream of the nutrient sensing Gator2 complex component, Mios

Reproductive success relies on proper establishment and maintenance of biological sex. In many animals, including mammals, the primary gonad is initially ovary in character. We previously showed the RNA binding protein (RNAbp), Rbpms2, is required for ovary fate in zebrafish. Here, we identified Rbpms2 targets in oocytes (Rbpms2-bound oocyte RNAs; rboRNAs). We identify Rbpms2 as a translational regulator of rboRNAs, which include testis factors and ribosome biogenesis factors. Further, genetic analyses indicate that Rbpms2 promotes nucleolar amplification via the mTorc1 signaling pathway, specifically through the mTorc1-activating Gap activity towards Rags 2 (Gator2) component, Missing oocyte (Mios). Cumulatively, our findings indicate that early gonocytes are in a dual poised, bipotential state in which Rbpms2 acts as a binary fate-switch. Specifically, Rbpms2 represses testis factors and promotes oocyte factors to promote oocyte progression through an essential Gator2-mediated checkpoint, thereby integrating regulation of sexual differentiation factors and nutritional availability pathways in zebrafish oogenesis.

developmental biology↗

bioRxiv2024

Falsifying computational models of angiogenesis through quantitative comparison with in vitro models

During angiogenesis, endothelial cells expand the vasculature by migrating from existing blood vessels, proliferating and collectively organizing into new capillaries. In vitro and in vivo experimentation is instrumental for identifying the molecular players and cell behaviour that regulate angiogenesis. Alongside experimental work, computational and mathematical models of endothelial cell network formation have helped to analyse if the current molecular and cellular understanding of endothelial cell behaviour is sufficient to explain the formation of endothelial cell networks. As input, the models take (a subset of) the current knowledge or hypotheses of single cell behaviour and capture it into a dynamical, mathematical description. As output, they predict the multicellular behaviour following from the actions of many individual cells, i.e., formation of a vascular-like network. Paradoxically, computational modelling based on different assumptions, i.e., completely different, sometimes non-intersecting sets of observed single cell behaviour, can reproduce the same angiogenesis-like multicellular behaviour, making it practically impossible to decide which, if any, of these models is correct. Here we present dynamical analyses of time-lapses of in vitro endothelial cell network formation experiments and compare these with dynamic analyses of three mathematical models: (1) the cell elongation model; (2) the contact-inhibited chemotaxis model; and (3) the mechanical cell-cell communication model. We extract a variety of dynamical characteristics of endothelial cell network formation using a custom time-lapse video analysis pipeline in ImageJ. We compare the dynamical network characteristics of the in vitro experiments to those of the cellular networks produced by the computational models. We test the response of the in silico dynamical cell network characteristics to changes in cell density and make related changes in the in vitro experiments. Of the three computational models that we have considered, the cell elongation model best captures the remodelling phase of in vitro endothelial cell network formation. Furthermore, in the in vitro model, the final size and number of lacunae in the network are independent of the initial cell density. This observation is also reproduced in the cell elongation model, but not in the other two models that we have considered. Altogether, we present an approach to model validation based on comparisons of time-resolved data and variations of model conditions. Author SummaryUnderstanding how blood vessels grow and organise into well-structured networks is crucial for many clinical applications, from wound healing to cancer treatment. The growth of blood vessels, known as angiogenesis, involves endothelial cells migrating, proliferating, and forming new vascular structures. Angiogenesis is studied through biological experiments, but mathematical models are also used to test whether our understanding of cell behaviour is sufficient to explain cell network formation. Models assuming chemotaxis, elongated cell shape, contact inhibition or mechanical cell-cell communication often produce similar results despite the large differences in the underlying assumptions, making it difficult to determine which model is most accurate. In this study, we analysed time-lapse videos of endothelial cell network formation and compared these with three computational models: (1) the cell elongation model, (2) the contact-inhibited chemotaxis model, and (3) the mechanical cell-cell communication model. By examining the dynamics of network formation and testing how they change with cell density, we found that the cell elongation model best captures key aspects of the real-life cell network remodelling process. This approach highlights the importance of time-resolved data in evaluating computational models and provides a framework for refining our understanding of angiogenesis.

developmental biology↗

bioRxiv2025

OpenEMMU: a versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

5-Ethynyl-2-deoxyuridine (EdU) has transformed DNA replication and cell cycle analyses through fast and efficient click chemistry detection. However, commercial EdU kits are expensive, contain proprietary reagents, are suboptimal for antibody multiplexing, and are challenging to use with larger biological specimens, highlighting the need for innovation. Here, we report Open-source EdU Multiplexing Methodology for Understanding DNA replication dynamics (OpenEMMU), an optimized, affordable, and user-friendly click chemistry resource using off-the-shelf reagents. OpenEMMU improves the efficiency, brightness, and multiplexing capabilities of EdU thymidine analog staining with both non-conjugated and conjugated antibodies across various cell types. We validated its effectiveness for fluorescent imaging of nascent DNA in developing embryos and organs, including the embryonic heart and forelimbs, and in 3D hiPSC-derived cardiac organoids. OpenEMMU also enabled 3D imaging of DNA synthesis in zebrafish larvae at fine resolution. This opens new avenues for insights into organismal development and growth, cell proliferation, and DNA replication, with precision and flexibility that surpass traditional methods. TeaserAn open-source and cost-effective resource for DNA replication profiling and cell cycle analysis using click chemistry.

developmental biology↗

bioRxiv2025

Modeling Montbeillards height data of a human male

Growth is a dynamic activity of simultaneous biological processes happening at multiple time-scales varying from orders of fractions of a second to several years. Rather than modeling growth with differential equations, this multiple time-scale dynamics is modeled using a simpler algebraic approach that involves continued fraction of the linear time scale. This algebraic approach offers models that are infinitely differentiable like an exponential function but also robust and superposable like linear equations. Thus, unique insights into growth dynamics can be obtained without much need of a calculus background. Growth of bacterial colonies, yeast cultures, Drosophila population, mean individual attributes of Helianthus and rats have already been modeled using this approach. In this work, we extend the modeling procedure to individual human growth using Montbeillards height measurements of his son starting from birth upto almost 18 years of age. Good fits are obtained on the data and growth rates are estimated directly from the model. Thus, this methodology provides generic, flexible, simpler and more interpretable growth models.

developmental biology↗

bioRxiv2026

PPI-Rescue: restoration of eye development via functional reconstitution of a transcription complex.

The activity of transcription factors (TFs) can be modulated through protein-protein interactions (PPIs) with cofactors which can serve to enhance, repress or alter the specificity of TF function. Current genetic and molecular approaches such as knockout and overexpression studies are likely to affect all possible activities of a TF, presenting a dilemma when attempting to parse out how TFs function within a cell. Hence, there is a need to devise a technique that will allow for mechanistic insight to be gained into the function(s) of discrete transcription complexes. Herein we report ''PPI-Rescue'', an approach that leverages bespoke mutations in TFs and the selective reconstitution of specific complexes to study the molecular properties and biological functions of discrete transcription complexes in vitro and in vivo. We demonstrate PPI-Rescue using the conserved Drosophila transcription factor Sine oculis (So) and cofactor Eyes Absent (Eya). To validate this approach, we engineered a missense mutation, So{Delta}Eya (C110S), that selectively abrogates PPI-Eya to show that PPI-Eya is necessary for the transcriptional activity of So in cultured cells and to promote eye formation in Drosophila. Using heterologous leucine zipper domains, we specifically reconstituted the So{Delta}Eya-Eya PPI and show that this complex is transcriptionally active in cells and can rescue eye development. PPI-Rescue was also used to restore transcriptional activity to So{Delta}PPI (V111E), an allele that mimics the human SIX1(V17E) allele associated with Branchio-Oto-Renal syndrome. PPI-Rescue thus represents an innovative approach to understanding the complex molecular logic of transcription complexes in both development and disease.

developmental biology↗

bioRxiv2020

TGF-beta/Activin ligand Myoglianin couples muscle growth to the initiation of metamorphosis

Although the mechanisms that control growth are now well understood, the mechanism by which animals assess their body size remains one of the great puzzles in biology. The final larval instar of holometabolous insects, after which growth stops and metamorphosis begins, is specified by a threshold size. We investigated the mechanism of threshold size assessment in the tobacco hornworm, Manduca sexta. The threshold size was found to change depending on the amount of exposure to poor nutrient conditions whereas hypoxia treatment consistently led to a lower threshold size. Under these various conditions, the mass of the muscles plus integuments was correlated with the threshold size. Furthermore, the expression of myoglianin (myo) increased at the threshold size in both M. sexta and Tribolium castaneum. Knockdown of myo in T. castaneum led to larvae that underwent supernumerary larval molts and stayed in the larval stage permanently even after passing the threshold size. We propose that increasing levels of Myo produced by the growing tissues allow larvae to assess their body size and trigger metamorphosis at the threshold size.

developmental biology↗

bioRxiv2022

Cell- and non-cell-autonomous ARF3 coordinates meristem proliferation and organ patterning in Arabidopsis

In cell-cell communication, non-cell-autonomous transcription factors play vital roles in controlling plant stem cell fate. We previously reported that AUXIN RESPONSE FACTOR 3 (ARF3), a member of the ARF family with critical roles in floral meristem maintenance and determinacy, has a distinct accumulation pattern that differs from the expression domain of its encoding gene in the shoot apical meristem (SAM). However, the biological meaning of this difference is obscure. Here, we demonstrate that ARF3 expression is mainly activated at the periphery of the SAM by auxin, where ARF3 cell-autonomously regulates the expression of meristem-organ boundary-specific genes, such as CUP-SHAPED COTYLEDON1-3 (CUC1-3), BLADE ON PETIOLE1-2 (BOP1-2) and TARGETS UNDER ETTIN CONTROL3 (TEC3) to determine organ patterning. We also show that ARF3 is translocated into the organizing center, where it represses cytokinin activity and WUSCHEL expression to regulate meristem activity non-cell-autonomously. Therefore, ARF3 acts as a molecular link that mediates the interaction of auxin and cytokinin signaling in the SAM while coordinating the balance between meristem maintenance and organogenesis. Our findings reveal an ARF3-mediated coordination mechanism through cell-cell communication in dynamic SAM maintenance.

developmental biology↗