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bioRxiv2019

Automated high-throughput heart rate measurement in medaka and zebrafish embryos under physiological conditions

RationaleAccurate and efficient quantification of heartbeats in small fish models is an important readout to study cardiovascular biology, disease states and pharmacology at large scale. However, dependence on anesthesia, laborious sample orientation or requirement for fluorescent reporters have hampered the establishment of high-throughput heartbeat analysis. ObjectiveTo overcome these limitations, we aimed to develop a high-throughput assay with automated heart rate scoring in medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos under physiological conditions designed for genetic screens and drug discovery and validation. Methods and ResultsWe established an efficient screening assay employing automated label-free heart rate determination of randomly oriented, non-anesthetized specimen in microtiter plates. Automatically acquired bright-field data feeds into an easy-to-use HeartBeat software, a MATLAB algorithm with graphical user interface developed for automated quantification of heart rate and rhythm. Sensitivity of the assay and algorithm was demonstrated by profiling heart rates during entire embryonic development. Our analysis pipeline revealed acute temperature changes triggering rapid adaption of heart rates, which has implications for standardization of experimental layout. The approach is scalable and allows scoring of multiple embryos per well resulting in a throughput of >500 embryos per 96-well plate. In a proof of principle screen for compound testing, our assay captured concentration-dependent effects of nifedipine and terfenadine over time. ConclusionA novel workflow and HeartBeat software provide efficient means for reliable and direct quantification of heart rate and rhythm of small fish in a physiological environment. Importantly, confounding factors such as anesthetics or laborious mounting are eliminated. We provide detailed profiles of embryonic heart rate dynamics in medaka and zebrafish as reference for future assay development. Ease of sample handling, automated imaging, physiological conditions and software-assisted analysis now facilitate various large-scale applications ranging from phenotypic screening, interrogation of gene functions to cardiovascular drug development pipelines.

developmental biology↗

bioRxiv2023

A single-cell transcriptional timelapse of mouse embryonic development, from gastrula to pup

The house mouse, Mus musculus, is an exceptional model system, combining genetic tractability with close homology to human biology. Gestation in mouse development lasts just under three weeks, a period during which its genome orchestrates the astonishing transformation of a single cell zygote into a free-living pup composed of >500 million cells. Towards a global framework for exploring mammalian development, we applied single cell combinatorial indexing (sci-*) to profile the transcriptional states of 12.4 million nuclei from 83 precisely staged embryos spanning late gastrulation (embryonic day 8 or E8) to birth (postnatal day 0 or P0), with 2-hr temporal resolution during somitogenesis, 6-hr resolution through to birth, and 20-min resolution during the immediate postpartum period. From these data (E8 to P0), we annotate dozens of trajectories and hundreds of cell types and perform deeper analyses of the unfolding of the posterior embryo during somitogenesis as well as the ontogenesis of the kidney, mesenchyme, retina, and early neurons. Finally, we leverage the depth and temporal resolution of these whole embryo snapshots, together with other published data, to construct and curate a rooted tree of cell type relationships that spans mouse development from zygote to pup. Throughout this tree, we systematically nominate sets of transcription factors (TFs) and other genes as candidate drivers of the in vivo differentiation of hundreds of mammalian cell types. Remarkably, the most dramatic shifts in transcriptional state are observed in a restricted set of cell types in the hours immediately following birth, and presumably underlie the massive changes in physiology that must accompany the successful transition of a placental mammal to extrauterine life.

developmental biology↗

bioRxiv2026

Mitochondrial subpopulations in oocytes and cumulus cells exhibit distinct age-associated changes and selective plasticity in response to NMN supplementation

BackgroundMitochondrial dysfunction is a leading contributor to the decline in oocyte quality associated with maternal aging. Prior investigations of mitochondrial function in the ovarian follicle have largely treated the mitochondrial pool as a homogeneous population, reporting aggregate values that may obscure biologically meaningful differences between distinct mitochondrial subpopulations. The present study addresses this limitation by characterizing mitochondrial subpopulation dynamics in oocytes and cumulus granulosa cells at single-organelle resolution using fluorescence-activated mitochondria sorting (FAMS). ResultsAnalysis of the aggregate mitochondrial population in mouse oocytes revealed no significant age-associated differences in mitochondrial DNA copy number or membrane potential, a result that would previously have been interpreted as evidence of minimal age-related mitochondrial change. Subpopulation analysis revealed this conclusion to be incomplete: aged oocytes showed significantly elevated mitochondrial DNA copy number specifically within the high membrane potential and small mitochondrial subpopulations, with no significant differences in the low membrane potential or large subpopulations. NMN supplementation normalized mitochondrial DNA copy number in the high membrane potential and small subpopulations toward young levels while producing an opposing effect in large mitochondria, demonstrating subpopulation-specific rather than uniform rejuvenation. In cumulus cells, significant age-associated changes were detectable at the aggregate level, including a reduction in mitochondrial DNA copy number and an elevation in membrane potential, and subpopulation analysis further resolved these findings. The age-associated reduction in cumulus cell mitochondrial DNA copy number was driven predominantly by the high membrane potential subpopulation. NMN supplementation exerted opposing effects on small and large cumulus cell mitochondrial subpopulations, increasing mitochondrial DNA copy number above both young and aged levels in small mitochondria while further reducing it below aged levels in large mitochondria. ConclusionsViewing the mitochondrial pool as a heterogeneous mixture of functionally distinct subpopulations rather than a uniform population reveals age-associated alterations in oocytes and cumulus cells that are undetectable by aggregate analysis. NMN supplementation exerts subpopulation-specific effects in both cell types, identifying specific mitochondrial subtypes as more precise targets for future mechanistic investigation of age-associated infertility than the mitochondrial pool considered in aggregate.

developmental biology↗

bioRxiv2022

Bat pluripotent stem cells reveal unique entanglement between host and viruses

Bats have evolved features unique amongst mammals, including flight, laryngeal echolocation, and certain species have been shown to have a unique immune response that may enable them to tolerate viruses such as SARS-CoVs, MERS-CoVs, Nipah, and Marburg viruses. Robust cellular models have yet to be developed for bats, hindering our ability to further understand their special biology and handling of viral pathogens. To establish bats as new model study species, we generated induced pluripotent stem cells (iPSCs) from a wild greater horseshoe bat (Rhinolophus ferrumequinum) using a modified Yamanaka protocol. Rhinolophids are amongst the longest living bat species and are asymptomatic carriers of coronaviruses, including one of the viruses most closely related to SARS-CoV-2. Bat induced pluripotent stem (BiPS) cells were stable in culture, readily differentiated into all three germ layers, and formed complex embryoid bodies, including organoids. The BiPS cells were found to have a core pluripotency gene expression program similar to that of other species, but it also resembled that of cells attacked by viruses. The BiPS cells produced a rich set of diverse endogenized viral sequences and in particular retroviruses. We further validated our protocol by developing iPS cells from an evolutionary distant bat species Myotis myotis (greater mouse-eared bat) non-lethally sampled in the wild, which exhibited similar attributes to the greater horseshoe bat iPS cells, suggesting that this unique pluripotent state evolved in the ancestral bat lineage. Although previous studies have suggested that bats have developed powerful strategies to tame their inflammatory response, our results argue that they have also evolved mechanisms to accommodate a substantial load of endogenous viral sequences and suggest that the natural history of bats and viruses is more profoundly intertwined than previously thought. Further study of bat iPS cells and their differentiated progeny should advance our understanding of the role bats play as virus hosts, provide a novel method of disease surveillance, and enable the functional studies required to ascertain the molecular basis of bats unique traits.

developmental biology↗

bioRxiv2023

On the independent irritability of goldfish eggs and embryos -- a living communication on the rhythmic yolk contractions in goldfish

Rhythms play an important role in the precise spatiotemporal regulation of biological processes during development and patterning of embryos. We here investigate the rhythmic contractions of the yolk during early development of the goldfish Carassius auratus. We quantify these contractions and record robust and persistent rhythmic yolk movements that are not seen in closely-related species (common carp Cyprinus carpio and zebrafish Danio rerio). We report that yolk contractions are an intrinsic emergent property of the egg, i.e. goldfish eggs are independently irritable / excitable. These contractions do not require sperm entry / fertilization nor cell division, and they notably emerge at a precise time -- suggesting that goldfish eggs are able to measure elapsed time from what we infer to be egg activation. We further show that these rhythmic contractions persist even in yolk in isolation. As the yolk itself is known to confer critical cues for early dorsoventral (DV) patterning of teleost embryos, we hypothesize that its contractions in goldfish may influence the patterning process of this species. Indeed, we find that embryos of the naturally more ventralized twin-tail goldfish strain Oranda display altered yolk contraction dynamics (i.e. faster contractions). We also present that the period of yolk contractions is independent of ChdS, a key gene involved in DV pattening and linked to the twin-tail phenotype, but is instead a trait that is maternal in origin. We aim to uncover whether the yolk contractions happening during early development of domesticated goldfish are the licensing process which permit the emergence of novel patterning phenotypes naturally-observed in this species (e.g. twin-tail and dorsal-finless strains) and which instead have not been found among closely-related species (e.g. common carp) whose yolks do not contract. This manuscript is here published as a living communication (as described in Gnaiger (2021)). The authors intend to share findings when they are available, encourage feedback and discussion, and invite knowledge exchange and collaboration.

developmental biology↗

bioRxiv2021

Mathematical modeling of Erk activity waves in regenerating zebrafish scales

Erk signaling regulates cellular decisions in many biological contexts. Recently, we have reported a series of Erk activity traveling waves that coordinate regeneration of osteoblast tissue in zebrafish scales. These waves originate from a central source region, propagate as expanding rings, and impart cell growth, thus controlling tissue morphogenesis. Here, we present a minimal reaction-diffusion model for Erk activity waves. The model considers three components: Erk, a diffusible Erk-activator, and an Erk-inhibitor. Erk stimulates both its activator and inhibitor, forming a positive and negative feedback loop, respectively. Our model shows that this system can be excitable and propagate Erk activity waves. Waves originate from a pulsatile source which is modeled by adding a localized basal production of the activator that switches the source region from an excitable to an oscillatory state. As Erk activity periodically rises in the source, it can trigger an excitable wave which travels across the entire tissue. Analysis of the model finds that positive feedback controls the properties of the traveling wavefront and that negative feedback controls the duration of Erk activity peak and the period of Erk activity waves. The geometrical properties of the waves facilitate constraints on the effective diffusivity of the activator, indicating that waves are an efficient mechanism to transfer growth factor signaling rapidly across a large tissue. Significance statementSignaling waves represent a possible mechanism of spatiotemporal organization of multicellular tissues. We have recently shown that waves of activity of the kinase Erk control osteoblast regeneration in adult zebrafish scales. Here, we present a detailed characterization of a mathematical model of these signaling waves. We show that a source region poised in an oscillatory state can broadcast traveling waves of Erk activity in the surrounding excitable tissue. The dynamics of the source control the number and frequency of waves. Geometrical arguments support the notion that excitable Erk waves are an effective mechanism to transport growth factor signaling across a large regenerating tissue.

developmental biology↗

bioRxiv2024

A double ovulation protocol for Xenopus laevis produces doubled fertilisation yield and moderately transiently elevated corticosterone levels without loss of egg quality

The African claw-toed frog, Xenopus laevis, is a well-established laboratory model for the biology of vertebrate fertilisation and development. For ovulation, X. laevis females are usually injected with chorionic gonadotropin, whereupon they lay typically hundreds to thousands of eggs in a day. Animals are re-used after being rested for a minimum of three months. The literature suggests that adult females can lay much larger numbers of eggs in a short period. Here, we compared the standard "single ovulation" protocol with a "double ovulation" protocol, in which females were ovulated, then re-ovulated after seven days and then rested for three months before re-use. We quantified egg number, fertilisation rate (development to cleavage stage), and corticosterone secretion rate (a measure of stress response) over seven 3-month cycles. We found no differences in egg number-per-ovulation or fertilisation, and no long-term changes in any measures over the trial period. Corticosterone secretion was elevated by ovulation, similarly for the single ovulation as for the first ovulation in the double-ovulation protocol, but more highly for the second ovulation (to a level comparable to that seen following shipment). However, both groups exhibited the same baseline secretion rates by the time of the subsequent cycle. Double ovulation is thus transiently more stressful/demanding than single ovulation but within levels routinely experienced by laboratory X. laevis. Noting that "stress hormone" corticosterone/cortisol secretion is linked to physiological processes, such as ovulation, that are not necessarily harmful to the individual, we suggest that the benefits of a doubling in egg yield-per-cycle per animal without loss of egg quality or signs of acute or long-term harm may outweigh the relatively modest and transient corticosterone elevation we observed. The double ovulation protocol therefore represents a potential new standard practice for promoting the "3Rs" (animal use reduction, refinement and replacement) mission for Xenopus research.

developmental biology↗

bioRxiv2020

Proteasome-mediated regulation of Cdhr1a by Siah1 modulates photoreceptor development and survival in zebrafish.

Congenital retinal dystrophies are a major cause of unpreventable and incurable blindness worldwide. Mutations in CDHR1, a retina specific cadherin, are associated with cone-rod dystrophy. The ubiquitin proteasome system (UPS) is responsible for mediating orderly and precise targeting of protein degradation to maintain biological homeostasis and coordinate proper development, including retinal development. Recently, our lab uncovered that the seven in absentia (Siah) family of E3 ubiquitin ligases play a role in optic fissure fusion, and identified Cdhr1a as a potential target of Siah. Using two-color whole mount in situ hybridization and immunohistochemistry, we detected siah1 and cdhr1a co-expression as well as protein co-localization in the retinal outer nuclear layer (ONL), and more precisely in the connecting cilium of rods and cones between 3-5 days post fertilization (dpf). We confirmed that Siah1 targets Cdhr1a for proteasomal degradation by co-transfection and co-immunoprecipitation in cell culture. To analyze the functional importance of this interaction, we created two transgenic zebrafish lines that express siah1 or an inactive siah1 (siah1{Delta}RING) under the control of the heat shock promoter to modulate Siah activity during photoreceptor development. Overexpression of siah1, but not siah1{Delta}RING, resulted in a decrease in the number of rods and cones at 72 hours post fertilization (hpf). The number of retinal ganglion cells, amacrine and bipolar was not affected by Siah1 overexpression, and there was no significant reduction of proliferating cells in the Siah1 overexpressing retina. We did however detect increased cell death, confirmed by an increase in the number of TUNEL+ cells in the ONL, which was proteasome-dependent, as MG132 treatment rescued the cell death phenotype. Lastly, reduction in rods and cones resulting from increased Siah1 expression was rescued by injection of cdhr1 mRNA, and to an even greater extent by injection of a Siah1-insensitive cdhr1a variant mRNA. Taken together, our work provides the first evidence that Cdhr1a plays a role during early photoreceptor development and that Cdhr1a is regulated by Siah1 via the UPS. This work provides new avenues for investigation into the roles of CDHR1, and now also Siah1, in the predisposition and pathogenesis of inherited cone-rod dystrophy.

developmental biology↗

bioRxiv2023

A mechanohydraulic model supports a role for plasmodesmata in cotton fiber elongation

Plant cell growth depends on turgor pressure, the cell hydrodynamic pressure, which drives expansion of the extracellular matrix (the cell wall). Turgor pressure regulation depends on several physical, chemical and biological factors, including: vacuolar invertases, which modulate osmotic pressure of the cell, aquaporins, which determine the permeability of the plasma membrane to water, cell wall remodeling factors, which determine cell wall extensibility (inverse of effective viscosity), and plasmodesmata, which are membrane-lined channels that allow free movement of water and solutes between cytoplasms of neighbouring cells, like gap junctions in animals. Plasmodesmata permeability varies during plant development and experimental studies have correlated changes in the permeability of plasmodesmal channels to turgor pressure variations. Here we study the role of plasmodesmal permeability in cotton fiber growth, a type of cell that increases in length by at least 3 orders of magnitude in a few weeks. We incorporated plasmodesma-dependent movement of water and solutes into a classical model of plant cell expansion. We performed a sensitivity analysis to changes in values of model parameters and found that plasmodesmal permeability is among the most important factors for building up turgor pressure and expanding cotton fibers. Moreover, we found that non-monotonic behaviors of turgor pressure that have been reported previously in cotton fibers cannot be recovered without accounting for dynamic changes of the parameters used in the model. Altogether, our results suggest an important role for plasmodesmal permeability in the regulation of turgor pressure. Significance StatementThe cotton fiber is among the plant cells with the highest growth rates. In cultivars, a single fiber cell generally reaches a few centimeters in length. How such size is achieved is still poorly understood. In order to tackle this question, we built a comprehensive mathematical model of fiber elongation, considering cell mechanics and water entry into the cell. Model predictions agree with experimental observations, provided that we take into account active opening and closure of plasmodesmata, the nano-channels that connect the fiber with neighboring cells. Because cotton fiber length is a key factor for yarn quality, our work may help understanding the mechanisms behind an important agronomic trait.

developmental biology↗

bioRxiv2018

Interplay scenarios between CatSper and CaV channels in sea urchin sperm flagellum signaling cascades triggered by egg peptides: a mathematical modelling approach

Intracellular calcium ([Ca2+]i) is a basic, versatile and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of cytosolic [Ca2+]i spikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelop and temporal organisation of the [Ca2+]i-spike trains. The signalling network comprises an initial membrane hyperpolarisation/repolarisation produced by an upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules leading to pHi, voltage and [Ca2+]i fluctuations. The upstream module outputs were fitted to kinetic data on cGMP activity and early membrane potential changes measured in bulk cell populations. Two candidate modules featuring voltage-dependent Ca2+-channels link these outputs to the downstream dynamics and can independently explain the typical decaying envelop and the progressive spacing of the spikes. In the first module, [Ca2+]i-spike trains require the concerted action of a classical CaV-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pHi-dependent, [Ca2+]i-inactivated CatSper dynamics alone. The model predicts that these two modules interfere with each other and produce unreasonable dynamics when present at similar proportions, which suggests that one may predominate over the other in vivo. To assess these alternatives, several quantitative predictions were derived from each module and confronted to experimental observations. We show that the [Ca2+]I dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the CatSper module but not by one including the pH-independent CaV and BK module. We conclude in favour of the module containing CatSper.

developmental biology↗

bioRxiv2024

Transcriptomic comparison of in vitro models of the human placenta

Studying the human placenta through in vitro cell culture methods is necessary due to limited access and amenability of human placental tissue to certain experimental methods as well as distinct anatomical and physiological differences between animal and human placentas. Selecting an in vitro culture model of the human placenta is challenging due to representation of different trophoblast cell types with distinct biological roles and limited comparative studies that define key characteristics of these models. Therefore, the aim of this research was to create a comprehensive transcriptomic comparison of common in vitro models of the human placenta compared to bulk placental tissue from the CANDLE and GAPPS cohorts (N=1083). We performed differential gene expression analysis on publicly available RNA sequencing data from 6 common in vitro models of the human placenta (HTR-8/SVneo, BeWo, JEG-3, JAR, Primary Trophoblasts, and Villous Explants) and compared to CANDLE and GAPPS bulk placental tissue or cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast cell types derived from bulk placental tissue. All in vitro placental models had a substantial number of differentially expressed genes (DEGs, FDR<0.01) compared to the CANDLE and GAPPS placentas (Average DEGs=10,873), and the individual trophoblast cell types (Average DEGs=5,346), indicating that there are vast differences in gene expression compared to bulk and cell-type specific human placental tissue. Hierarchical clustering identified 53 gene clusters with distinct expression profiles across placental models, with 22 clusters enriched for specific KEGG pathways, 7 clusters enriched for high-expression placental genes, and 7 clusters enriched for absorption, distribution, metabolism, and excretion genes. In vitro placental models were classified by fetal sex based on expression of Y-chromosome genes that identified HTR-8/SVneo cells as being of female origin, while JEG-3, JAR, and BeWo cells are of male origin. Overall, none of the models were a close approximation of the transcriptome of bulk human placental tissue, highlighting the challenges with model selection. To enable researchers to select appropriate models, we have compiled data on differential gene expression, clustering, and fetal sex into an accessible web application: "Comparative Transcriptomic Placental Model Atlas (CTPMA)" which can be utilized by researchers to make informed decisions about their selection of in vitro placental models.

developmental biology↗

bioRxiv2024

From womb to crib: How fetal activity patterns in utero reveal postnatal sleep behavior

ImportanceThe human circadian system, a critical biological mechanism governing the sleep-wake cycle, begins to oscillate before birth. Distinct fetal behavioral states have been described using short-duration ultrasound recordings, but the alignment of fetal activity with day and night cycles (i.e., diurnal rhythms) remains poorly understood due to the lack of long-term monitoring. Accordingly, a substantial knowledge gap exists concerning the evolution of fetal diurnal rhythms into infant sleep-wake cycles. ObjectiveTo investigate the development of fetal diurnal rhythms and their evolution into postnatal infant sleep, as well as the role of maternal factors and melatonin in shaping early circadian entrainment. MethodsIn this cohort study, conducted in 2022 and 2023, we collected over 20 000 hours of wearable acceleration and temperature data from 32 fetuses and their mothers during continuous 5-day monitoring in the third trimester of pregnancy. The cohort was subsequently followed longitudinally from the prenatal period through 6 months postpartum, with infant sleep outcomes assessed at three key time points (first postnatal weeks, 3 months, and 6 months) using sleep diaries and questionnaires. The first postnatal assessment additionally included breast milk and infant stool sample collection. ResultsMulti-level modelling revealed early diurnal rhythms in fetuses that align with maternal activity and circadian rhythms. Random Forest analyses identified fetal day/night sleep duration ratio - a simplified proxy for fetal circadian entrainment - as the strongest predictor of postnatal infant sleep, with fetuses preferring nighttime sleep maintaining this preference postnatally. Maternal sleep regularity (i.e., consistent sleep patterns characterized by low day-to-day variability in sleep timing) during pregnancy also predicted infant sleep, associating with a stronger preference for nighttime sleep in infants. Conclusions and relevanceThese findings highlight the potential influence of intrauterine and maternal factors on the evolution of circadian entrainment. Our study contributes to a broader understanding of the earliest emergence of circadian rhythms, supporting future research on their long-term health impacts. Maternal sleep regularity stands out as the earliest modifiable target for interventions, offering an actionable pathway to promote infant circadian entrainment, with potential long-term benefits for family dynamics and well-being.

developmental biology↗

bioRxiv2025

High-Yield Induced Ovulation in Adult Fat-Tailed Dunnarts by PMSG Treatment Combined with Estrus Synchronization

The fat-tailed dunnart, Sminthopsis crassicaudata, is an emerging laboratory-based marsupial model for research on comparative biology, reproduction and conservation. In females, the reproductive cycle lasts 31 days and approximately 10 oocytes are ovulated per cycle. Assisted reproductive technologies (ART) play a crucial role in marsupial conservation, but developing protocols to harvest large numbers of oocytes remains a key challenge. Specifically, producing sufficient mature metaphase II (MII) oocytes in dunnarts continues to be difficult. Ovarian follicle stimulation is common practice to achieve superovulation in many species and typically requires treatment of prepubertal female animals to avoid the impacts of endogenous hormone cycling. Alternatively, adult females can be stimulated during the intermediate or follicular phase. In this study, we aimed to develop a high-yield induced-ovulation protocol to collect higher number of MII oocytes from adult, cycling, female dunnarts. We first synchronized the female dunnart reproductive cycles using luteinizing hormone-releasing hormone (LHRH). The reproductive cycles were monitored by examining cytology of vaginal lavage samples. After administering four LHRH injections given at three-day intervals, 88.9% (n=36) of the adult female dunnarts responded to the treatment, with their estrous cycles synchronized at the diestrous stage. We then induced ovarian follicle development through two PMSG injections over 6 days, followed by hCG administration to trigger ovulation. By combining estrous cycle synchronization with PMSG stimulation, we achieved 77.8% (n=36) ovulation success and obtained an average of 20.1{+/-}9.1 (n=28) MII oocytes per adult dunnart. These data demonstrated that estrous cycle synchronization followed by the PMSG-hCG treatment yields consistent, highly efficient induced-ovulation in adult dunnarts. This approach of combining estrus synchronization and follicle stimulation to produce sufficient MII oocytes for ART purpose could be applied to other valuable marsupial species to support conservation efforts. Summary SentenceSuperovulation and robust production of mature MII oocytes can be induced by 10-day estrus synchronization using LHRH followed by 6-day PMSG stimulation of follicle growth in adult fat-tailed dunnarts, a marsupial species. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/671172v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@14c6685org.highwire.dtl.DTLVardef@decf75org.highwire.dtl.DTLVardef@64330org.highwire.dtl.DTLVardef@12538a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2019

Analysis of Finnish blue mussel (Mytilus edulis L.) shell: Biomineral ultrastructure, organic-rich interfacial matrix and mechanical behavior

Studying various marine biomineralized ultrastructures reveals the appearance of common architectural designs and building blocks in materials with fascinating mechanical properties that match perfectly to their biological tasks. Advanced mechanical properties of biological materials are attributed to evolutionary optimized molecular architectures and structural hierarchy. One example which has not yet been structurally investigated in great detail is the shell of Mytilus edulis L. (Finnish blue mussel) found in the archipelago of SW-Finland. Through a combination of various state-of-the-art techniques such as high-resolution electron microscopy imaging, Fourier-transformed infrared spectroscopy, powder X-ray diffraction, synchrotron wide-angle X-ray diffraction, nanoindentation and protein analysis, both the inorganic mineralized components as well as the organic-rich matrix were extensively characterized. We found very similar ultra-architecture across the shell of M. edulis L. as compared to the widely studied and closely related M. edulis. However, we also found interesting differences, for instance in the thickness and degree of orientation of the mineralized layers indicating dissimilar properties and related alterations in the biomineralization processes. Our results show that the shell of M. edulis L. has a gradient of mechanical properties, with the increase in the stiffness and the hardness from anterior to the posterior region of the shell. The shell is made from distinct and recognizable mineralized layers each varying in thickness and microstructural features. At posterior regions of the shell, moving from dorsal to ventral side, these layers are an oblique prismatic layer, a prismatic layer and a nacreous layer, in which the oblique prismatic layer is found to be the main and thickest mineralized layer of the shell. Probing the calcified rods in the oblique prismatic layer using high resolution SEM imaging revealed opening of channels with a diameters of 40-50 nm and lengths up to a micrometer extending through the rods. The chitin and protein have been found to be the main component of the organic-rich interfacial matrix as expected. Protein analysis showed two abundant proteins with sizes around 100 kD and 45 kD which likely not only regulates biomineralization and adhesion of the crystals but also governing the intrinsic-extrinsic toughening in the shell. Overall, this detailed analysis provides new structural insights into biomineralization of marine shells in general.

developmental biology↗

bioRxiv2024

Age-associated growth control modifies leaf proximodistal symmetry and enables leaf shape diversification

Biological shape diversity is often manifested in modulation of organ symmetry and modification of the patterned elaboration of repeated shape elements.1-5 Whether and how these two aspects of shape determination are coordinately regulated is unclear.5-7 Plant leaves provide an attractive system to investigate this problem, because they usually show asymmetries along their proximodistal axis, along which they can also produce repeated marginal outgrowths such as serrations or leaflets.1 One case of leaf shape diversity is heteroblasty, where the leaf form in a single genotype is modified with progressive plant age.8-11 In Arabidopsis thaliana, a plant with simple leaves, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9) controls heteroblasty by activating CyclinD3 expression, thereby sustaining proliferative growth and retarding differentiation in adult leaves.12 However, the precise significance of SPL9 action for leaf symmetry and marginal patterning is unknown. By combining genetics, quantitative shape analyses, and time-lapse imaging, we show that, in A. thaliana, proximodistal symmetry of the leaf blade decreases in response to an age-dependent SPL9 expression gradient, and that SPL9 action coordinately regulates the distribution and shape of marginal serrations and overall leaf form. Using comparative analyses, we demonstrate that heteroblastic growth reprogramming in Cardamine hirsuta, a complex-leafed relative of A. thaliana, also involves prolonging the duration of cell proliferation and delaying differentiation. We further provide evidence that SPL9 enables species-specific action of homeobox genes that promote leaf complexity. In conclusion, we identify an age-dependent layer of organ proximodistal growth regulation that modulates leaf symmetry and has enabled leaf shape diversification. In BriefAge-dependent regulation of proliferative growth and differentiation along the proximodistal axis of leaves modulates organ symmetry and marginal patterning in heteroblasty, and enabled the action of homeobox genes in complex leaf shape evolution. HighlightsO_LIAn age-dependent growth gradient underpins regulation of A. thaliana leaf symmetry C_LIO_LISPL9-mediated heteroblastic growth control potentiates leaf histogenic capacity C_LIO_LIA common growth framework underlies heteroblasty in complex and simple leaves C_LIO_LISPL9 enables the action of homeobox genes that promote leaf complexity C_LI

developmental biology↗

bioRxiv2022

Model of neural induction in the ascidian embryo

How cell specification can be controlled in a reproducible manner is a fundamental question in development biology. In ascidians, a group of marine invertebrate chordates, geometry plays a key role in achieving this control. Here, we use mathematical modeling to demonstrate that geometry dictates the neural-epidermal cell fate choice in the 32-cell stage ascidian embryo by a two-step process involving first the modulation of ERK signaling and second, the expression of the neural marker gene, Otx. The model describes signal transduction by the ERK pathway that is stimulated by FGF and repressed by ephrin, and ERK-mediated control of Otx gene expression, which involves both an activator and an inhibitor of transcription. Considering the measured area of cell surface contacts with FGF- or ephrin-expressing cells as inputs, the solutions of the model reproduce the experimental observations about ERK activation and Otx expression in the different cells under normal and perturbed conditions. Sensitivity analyses and computations of Hill coefficients allow to quantify the robustness of the specification mechanism controlled by cell surface area and to identify the respective role played by each signaling input. Simulations also predict in which conditions the dual control of gene expression by an activator and an inhibitor that are both under the control of ERK can induce a robust ON/OFF control of neural fate induction. Author summaryThe development of a single cell zygote into a multicellular embryo occurs thanks to the combination of cell division and cell specification. The latter process corresponds to the progressive acquisition by the embryonic cells of their final physiological and functional characteristics, which rely on well-defined signaling-controlled genetic programs. The origin of the great robustness of cell specification remains poorly understood. Here, we address this question in the framework of the embryonic neural fate induction in ascidians, which are marine invertebrates. At the 32-cells stage, four cells identified by their precise location in the embryo adopt neural fate. On the basis of experimental observations, we develop a mathematical model that predicts that the choice between the neural or epidermal fate is controlled by the cell surface areas of the cells in contact with two antagonistic signals, FGF and ephrin. Our findings provide a computational confirmation of the major role played by the geometry of the embryo in controlling cell lineage acquisition during ascidian development.

developmental biology↗

bioRxiv2024

Computing hematopoiesis plasticity in response to genetic mutations and environmental stimulations

Cell plasticity (CP), describing a dynamic cell state, plays a crucial role in maintaining homeostasis during organ morphogenesis, regeneration and damage-to-repair biological process. Single-cell-omics datasets provide unprecedented resource to empowers analysis on CP. Hematopoiesis offers fertile opportunities to develop quantitative methods for understanding CP with rich supports from experimental ground-truths. In this study we generated high-quality lineage-negative (Lin-) single-cell RNA-sequencing datasets under various conditions and introduced a working pipeline named Snapdragon to interrogate naive and disturbed plasticity of hematopoietic stem and progenitor cells (HSPCs) with mutational or environmental challenges. Utilizing embedding methods UMAP or FA, a continuum of hematopoietic development is visually observed in wildtype where the pipeline confirms a very low Proportion of hybrid-cells (Phc, with bias range: 0.4-0.6) on a transition trajectory. Upon Tet2 mutation, a driver of leukemia, or treatment of DSS, an inducer of colitis, Phc is increased and plasticity of HSPCs was enhanced. Quantitative analysis indicates that Tet2 mutation enhances HSC self-renewal capability while DSS treatment results in an enhanced myeloid-skewing trajectory, suggesting their similar but different consequences. We prioritized several transcription factors (i.e the EGR family) and signaling pathways (i.e. receptors IL1R1 and ADRB, inflammation and sympathy-sensing respectively) which are responsible for Phc alterations. CellOracle-based simulation suggests that knocking-out EGR regulons or pathways of IL1R1 and ADRB partially reverses Phc promoted by Tet2 mutation and inflammation. In conclusion, the study provides high-quality datasets with single-cell transcriptomic matrices for diversified hematopoietic simulations and a computational pipeline Snapdragon for quantifying disturbed Phc and CP. (247 words) HighlightsO_LITo guide CP analysis, we introduce a quantizable parameter Phc and a pipeline Snapdragon, which discriminate naive and disturbed hematopoiesis; C_LIO_LIThe Snapdragon pipeline analysis on Tet2+/-Lin- cells demonstrates many novel insights, including enhanced HSC plasticity and increased PHC; similar trends are observed in inflammatory Lin- cells; C_LIO_LIRegulon analysis suggests that transcriptional factor EGR1 is significantly activated to elevated the HSC plasticity and change hematopoietic trajectory; C_LIO_LIStress-response-related signaling pathways mediated by receptors IL1R1 or ADRB were obviously activated in the challenged hematopoiesis; C_LIO_LICellOracle-based simulation suggests that knocking-out EGR regulons or pathways of IL1R1 and ADRB partially reverses Phc promoted by Tet2 mutation and inflammation. C_LI

developmental biology↗

bioRxiv2025

F-53B damages cell viability leading to impaired root development in Arabidopsis

The emerging contaminant chlorinated polyfluoroalkyl ether sulfonate (Cl-PFAES/F-53B) has been detected in many plant species, however, the impact of F-53B on the development in plants and the underpinning mechanisms remain largely unelucidated. In this study, we demonstrate by molecular biology, cytogenetics, and fluorescence microscopy mythologies that F-53B impairs cell viability leading to disrupted root development in Arabidopsis (Arabidopsis thaliana). We show that F-53B suppresses root length and the inhibitory effect is pronunanced at increasing concentrations. F-53B disrupts cell wall and microtubule-mediated cell plate formation and configuration in meristem cells, revealing its toxicity to cell division and cytoskeleton organization. F-53B induces cell death predominantly at the meristematic zone, however, gene exression and genetic studies indicate that F-53B does not trigger DNA double-strand breaks, implying a divergent effect on DNA stability across species. Moreover, we show that F-53B damages nuclei stability and viability, and induces Autophagy-Related 8 protein foci formation implying that it triggers autophagy-mediated cell responses. Taken together, this study provides cellular and molecular insights into the toxicity of F-53B to plant cells, which highlights its damage to agriculture and ecology security that deserve imperative environmental management actions. Environmental ImplicationF-53B has been detected in many plant species, which raises increasing concerns on its toxicity to plants and thus agriculture and ecology safety. However, the toxicity of F-53B to development in plants and the underpinning mechanisms remain largely unelucidated. Here, we demonstrate that F-53B damages nuclei stability and viability leading to cell death and impaired root development in the model plant species Arabidopsis thaliana. Our study provides cytogenetic and molecular insights into the F-53B toxicity to plant cells, which highlights imperative environmental management actions and can be referenced for developing F-53B control and management policies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/653422v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@159b211org.highwire.dtl.DTLVardef@1b99a95org.highwire.dtl.DTLVardef@53b964org.highwire.dtl.DTLVardef@b089be_HPS_FORMAT_FIGEXP M_FIG C_FIG One-sentence summaryF-53B triggers DNA double-strand break-free cell unviability leading to impaired meristematic cell division and root development in plants. HIGHLIGHTSO_LIF-53B impairs root development in Arabidopsis. C_LIO_LIF-53B prohibits mitotic cell division at root tips. C_LIO_LIF-53B damages cell viability predominantly at the meristematic zone in seedlings. C_LIO_LIF-53B does not induce DNA double-strand breaks in Arabidopsis seedlings. C_LIO_LIF-53B damages nuclei viability and stability in seedlings. C_LI

developmental biology↗