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Results for “developmental biology”

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bioRxiv2020

Specification and epigenetic resetting of the pig germline exhibit conservation with the human lineage

Investigations on the human germline and programming are challenging due to limited access to embryonic material. However, the pig as a model may provide insight on transcriptional network and epigenetic reprogramming applicable to both species. Here we show that during the pre- and early migratory stages pig primordial germ cells (PGCs) initiate large-scale epigenetic reprogramming, including DNA demethylation involving TET-mediated hydroxylation and potentially base excision repair (BER). There is also macroH2A1 depletion and increased H3K27me3, as well as X chromosome reactivation (XCR) in females. Concomitantly, there is dampening of glycolytic metabolism genes and re-expression of some pluripotency genes like those in preimplantation embryos. We identified evolutionarily young transposable elements and gene coding regions resistant to DNA demethylation in acutely hypomethylated gonadal PGCs, with potential for transgenerational epigenetic inheritance. Detailed insights into the pig germline will likely contribute significantly to advances in human germline biology, including in vitro gametogenesis.

developmental biology↗

bioRxiv2024

Substrate stiffness regulates neuronal maturation via Piezo1-mediated TTR activity

During brain development, neurons extend axons to connect to their target cells while initiating a maturation process during which neurons start expressing voltage-gated ion channels, form synapses, express synaptic transmitters and receptors, and start communicating via action potentials. Little is known about external factors regulating this process. Here, we identified environmental mechanics as an important regulator of neuronal maturation, and a molecular pathway linking tissue stiffness to this process. Using patch clamp electrophysiology, calcium imaging and immunofluorescence, we found that neurons cultured on stiffer substrates showed a delay in voltage-gated ion channel activity, spontaneous and evoked action potentials, and synapse formation. RNA sequencing and CRISPR/Cas9 knockdown revealed that the mechanosensitive ion channel Piezo1 supresses transthyretin (TTR) expression on stiffer substrates, slowing down synapse formation and consequently electrical maturation. In agreement, the stiffness of developing Xenopus laevis brain negatively correlated with local synapse densities, and stiffening of brain tissue resulted in a significant delay of synaptic activity in vivo. Our data indicate that environmental stiffness represents a fundamental regulator of neuronal maturation, which is important for the development of normal circuitry in the brain, and potentially for neurodevelopmental disorders. Furthermore, the mechanical regulation of TTR downstream of Piezo1 may be found in many other biological systems.

developmental biology↗

bioRxiv2025

Tau mediated regulation of Rho1- cytoskeletal dynamics in shaping renal tubule development in Drosophila

The present study explored the significance of the microtubule-associated protein Tau in the morphogenesis and physiological processes of Malpighian tubules (MTs) in Drosophila melanogaster. Employing genetic manipulation techniques and microscopy, we established that Tau plays a crucial role in MTs development and function. Genetic ablation and RNAi-mediated suppression of Tau led to pronounced structural abnormalities, including cystic formations, non-uniform tubule diameters, and disorganized epithelial architecture. These aberrations are accompanied by perturbations in the cytoskeletal arrangement, compromised cellular polarity, and ionic imbalances. Functional analyses revealed deficiencies in salt homeostasis and fluid secretion within Tau-deficient tubules. Attenuation of Rho1 expression mitigated the structural defects observed in Tau mutants, highlighting the importance of Tau in modulating Rho1-mediated actin-microtubule dynamics suggesting a genetic interplay between Tau and Rho1 GTPase in orchestrating tubule morphogenesis The phenotypic parallels between Tau-deficient MTs and mammalian models of polycystic kidney disease implies an evolutionarily conserved function of Tau in tubular organ biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/634261v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f4afc6org.highwire.dtl.DTLVardef@1402eb7org.highwire.dtl.DTLVardef@bb089dorg.highwire.dtl.DTLVardef@1328b2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2019

miR-92a-3p controls cell cycle progression in zebrafish

Biological functions of micro RNAs (miRNAs) in the early stages of vertebrate development remain largely unknown. In zebrafish, miRNA miR-92a-3p is abundant in the germ cells throughout gonadal development, as well as in ovulated oocytes. Previously, we demonstrated that inhibition of miR-92a-3p in mature ovaries resulted in developmental arrest at the 1-cell stage upon fertilization of the affected oocytes. This suggested functions of miR-92a-3p in early development. In the present study, we identified wee2, an oocyte-specific protein tyrosine kinase, as a target of maternal miR-92a-3p during the early stages of zebrafish embryogenesis. Spatiotemporal co-presence of both miR-92a-3p and wee2 during early embryo development was confirmed by absolute quantification and in situ hybridization. Targeted knockdown of miR-92a-3p in embryos resulted in retarded embryonic development over the first 24 hours. Target validation assays demonstrated that miR-92a-3p interacted with the predicted wee2 3UTR binding site, which was strongly suppressed by endogenous miR-92a-3p. Our results suggest that miR-92a-3p regulates the abundance of wee2, a cyclin-dependent kinase 1 inhibitor, thus having important role in regulation of the cell cycle during cleavage stages in zebrafish.\n\nSummary statementIn zebrafish, maternal miR-92a-3p was demonstrated to suppress translation of wee2, a cyclin-dependent kinase 1 inhibitor which regulates cell cycle progression during the early stages of embryogenesis.

developmental biology↗

bioRxiv2019

Compensatory replacement of the BigH1 variant histone by canonical H1 supports normal embryonic development in Drosophila

Histone variants carry specific functions in addition to those fulfilled by their canonical counterparts. Variants of the linker Histone H1 are prevalent in vertebrates and based on the pattern of their expression, many are presumed to function during germline and the earliest zygotic stages of development. While the existence of multiple H1 variants has hampered their study in vertebrates, a single variant, BigH1, was identified in Drosophila, promising to accelerate our understanding of the biological functions of H1 and H1 variants. Here we uncovered evidence for a compensatory activity that loads maternal H1 onto BigH1-devoid chromatin. Remarkably, this H1-based chromatin state is fully functional in supporting normal embryonic development, suggesting that H1 carries the essential function of the BigH1 molecule under the same developmental context. In addition, we discovered that this compensatory replacement of BigH1 with H1 might be limited to rapidly cycling cells in early embryos.

developmental biology↗

bioRxiv2017

Morphometrics of complex cell shapes: Lobe Contribution Elliptic Fourier Analysis (LOCO-EFA)

Quantifying cell morphology is fundamental to the statistical study of cell populations, and can help us unravel mechanisms underlying cell and tissue morphogenesis. Current methods, however, require extensive human intervention, are highly sensitive to parameter choice, or produce metrics that are difficult to interpret biologically. We therefore developed a novel method, Lobe Contribution Elliptical Fourier Analysis (LOCO-EFA), which generates from digitalised cell outlines meaningful descriptors that can be directly matched to morphological features. We show that LOCO-EFA provides a tool to phenotype efficiently and objectively populations of cells by applying it to the complex shaped pavement cells of Arabidopsis thaliana wild type and speechless leaves. To further validate our method, we analysed computer-generated tissues, where cell shape can be specified in a controlled manner. LOCO-EFA quantifies deviations between the specified shape that an individual in silico cell takes up when in isolation and the resultant shape when they are allowed to interact within a confluent tissue, thereby assessing the role of cell-cell interactions on population cell shape distributions.\n\nSummary statement\n\nNovel method (LOCO-EFA) quantifies complex cell shapes, extracting meaningful biological features such as protrusion number and amplitude; here shown for plant pavement cells and validated on in silico tissues.

developmental biology↗

bioRxiv2019

Collective cell migration and residual stress accumulation: modeling consideration

Stress generation during collective cell migration represents one of the key factors which influence the configuration of migrating cells, viscoelasticity of multicellular systems and their inter-relation. Local generation of stress (normal and shear) is significant even in 2D (up to ~100 - 150 Pa). Compressive stress is primarily accumulated (1) within a core region of migrating cell clusters during their movement through the dense environment and (2) during the collisions of migrating cell clusters caused by uncorrelated motility. Shear stress can be significant within perturbed boundary layers around migrating clusters. Cells are more sensitive to the action of shear stress compared with compressive stress. Shear stress of a few Pa significantly influences cell state. Deeper insight into cell strategy to minimize undesirable shear stress is a priority in order to understand various biological processes such as morphogenesis, wound healing and cancer invasion. We pointed out to cause-consequence relations of these complex phenomena based on rheological modeling consideration in order to stimulate further experimental work. Cell strategy should be connected with the type and distribution of adhesion contacts such as adherens junctions and tight junctions per migrating clusters in order to (1) reinforce the cluster structure perpendicular to the direction of cell migration and (2) ensure structural elasticity of cluster in the direction of migration. These conditions lead to the stiffness inhomogeneity per single migrating clusters. Cell strategy should also be related to the state of the perturbed boundary layer around the cluster in the context of its thickness and slip effects. Statement of SignificanceCollective cell migration induces a local generation of stress (normal and shear) significant even in 2D (up to 100-150 Pa). Cells well tolerate compressive stress up to a few kPa. However, shear stress of a few Pa can induce severe damage to vimentin and keratin intermediate filament networks during 1 h, while shear stress of ~60 Pa can cause the inflammation in epithelial cells during 5.5 h. Deeper insight into cell strategy to minimize undesirable shear stress is a priority in order to understand various biological processes. Cell strategy should be connected with the type and distribution of adhesion contacts such as adherens junctions and tight junctions per migrating clusters and surrounding perturbed boundary layers.

developmental biology↗

bioRxiv2022

Mapping of the podocin proximity-dependent proteome reveals novels components of the kidney podocyte foot process

The unique architecture of glomerular podocytes is integral to kidney filtration. Interdigitating foot processes extend from the podocyte cell body, wrap around fenestrated capillaries, and form specialized junctional complexes termed slit diaphragms to create a molecular sieve. However, the full complement of proteins which maintain foot process integrity, and how this localized proteome changes with disease, remains to be elucidated. Proximity-dependent biotin identification (BioID) enables the identification of spatially localized proteomes. To this end, we developed a novel in vivo BioID knock-in mouse model. We utilized the slit diaphragm protein podocin (Nphs2) to create a podocin-BioID fusion. Podocin-BioID localizes to the slit diaphragm and biotin injection leads to podocyte-specific protein biotinylation. We isolated the biotinylated proteins and performed mass spectrometry to identify proximal interactors. Gene ontology analysis of 54 proteins specifically enriched in our podocin-BioID sample revealed cell junctions, actin binding, and cytoskeleton organization as top terms. Known foot process components were identified and we further uncovered two novel proteins: the tricellular junctional protein Ildr2 and the CDC42 and N-WASP interactor Fnbp1l. We confirmed Ildr2 and Fnbp1l are expressed by podocytes and partially colocalize with podocin. Finally, we investigated how this proteome changes with age and uncovered a significant increase in Ildr2. This was confirmed by immunofluorescence on human kidney samples and suggests altered junctional composition may preserve podocyte integrity. Together, these assays have led to new insights into podocyte biology and supports the efficacy of utilizing BioID in vivo to interrogate spatially localized proteomes in health, aging, and disease.

developmental biology↗

bioRxiv2022

Systematic characterization of Drosophila RhoGEF/GAP localizations uncovers regulators of mechanosensing and junction formation during epithelial cell division.

Cell proliferation is central to epithelial tissue development, repair and homeostasis. During cell division, small RhoGTPases control both actomyosin dynamics and cell-cell junction remodelling to faithfully segregate the duplicated genome while maintaining tissue polarity and integrity. To decipher the mechanisms of RhoGTPases spatiotemporal regulation during epithelial cell division, we generated a transgenic fluorescently tagged library for Drosophila Rho Guanine exchange factors (GEF) and GTPase activating proteins (GAP), and systematically characterized their endogenous distributions by time- lapse microscopy. Thereby, we unveiled candidate regulators of the interplay between actomyosin and junctional dynamics during epithelial cell division. Building on these findings, we uncovered that during cytokinesis, Cysts and RhoGEF4 play sequential roles in mechanosensing and de novo junction formation, respectively. We foresee that the RhoGEF/GAP library will be a key resource to understand the broad range of biological processes regulated by RhoGTPases.

developmental biology↗

bioRxiv2023

LINKIN-associated proteins necessary for tissue integrity during collective cell migration

Cell adhesion plays essential roles in almost every aspect of metazoan biology. LINKIN (Human: ITFG1, Caenorhabditis elegans: lnkn-1) is a conserved transmembrane protein that has been identified to be necessary for tissue integrity during migration. In C. elegans, loss of lnkn-1 results in the detachment of the lead migratory cell from the rest of the developing male gonad. Previously, three interactors of ITFG1/lnkn-1 - RUVBL1/ruvb-1, RUVBL2/ruvb-2, and alpha-tubulin - were identified by immunoprecipitation-mass spectrometry (IP-MS) analysis using human HEK293T cells and then validated in the nematode male gonad. The ITFG1-RUVBL1 interaction has since been independently validated in a breast cancer cell line model that also implicates the involvement of the pair in metastasis. Here, we showed that epitope-tagged ITFG1 localized to the cell surface of MDA-MB-231 breast cancer cells. Using IP-MS analysis, we identified a new list of potential interactors of ITFG1. Loss-of-function analysis of their C. elegans orthologs found that three of the interactors - ATP9A/tat-5, NME1/ndk-1, and ANAPC2/apc-2 - displayed migratory detachment phenotypes similar to that of lnkn-1. Taken together with the other genes whose reduction-of-function phenotype is similar to that of lnkn-1 (notably cohesion and condensin), suggests the involvement of membrane remodeling and chromosome biology in LINKIN-dependent cell adhesion and supports the hypothesis for a structural role of chromosomes in post-mitotic cells.

developmental biology↗

bioRxiv2023

Overexpression of mitochondrial fusion genes enhances resilience and extends longevity

The dynamicity of the mitochondrial network is crucial for meeting the ever-changing metabolic and energy needs of the cell. Mitochondrial fission promotes the degradation and distribution of mitochondria, while mitochondrial fusion maintains mitochondrial function through the complementation of mitochondrial components. Previously, we have reported that mitochondrial networks are tubular, interconnected and well-organized in young, healthy C. elegans, but become fragmented and disorganized with advancing age and in models of age-associated neurodegenerative disease. In this work, we examine the effects of increasing mitochondrial fission or mitochondrial fusion capacity by ubiquitously overexpressing the mitochondrial fission gene drp-1 or the mitochondrial fusion genes fzo-1 and eat-3, individually or in combination. We then measured mitochondrial function, mitochondrial network morphology, physiologic rates, stress resistance and lifespan. Surprisingly, we found that overexpression of either mitochondrial fission or fusion machinery both resulted in an increase in mitochondrial fragmentation. Similarly, both mitochondrial fission and mitochondrial fusion overexpression strains have extended lifespans and increased stress resistance, which appears to be at least partially due to the upregulation of multiple stress response pathways in these strains. Overall, our work demonstrates that increasing the expression of mitochondrial fission or fusion genes extends lifespan and improves biological resilience without promoting the maintenance of a youthful mitochondrial network morphology. This work highlights the importance of the mitochondria for both resilience and longevity.

developmental biology↗

bioRxiv2017

Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human preimplantation epiblast

Single-cell profiling techniques create opportunities to delineate cell fate progression in mammalian development. Recent studies provide transcriptome data from human preimplantation embryos, in total comprising nearly 2000 individual cells. Interpretation of these data is confounded by biological factors such as variable embryo staging and cell-type ambiguity, as well as technical challenges in the collective analysis of datasets produced with different sample preparation and sequencing protocols. Here we address these issues to assemble a complete gene expression time course spanning human preimplantation embryogenesis. We identify key transcriptional features over developmental time and elucidate lineage-specific regulatory networks. We resolve post hoc cell-type assignment in the blastocyst, and define robust transcriptional prototypes that capture epiblast and primitive endoderm lineages. Examination of human pluripotent stem cell transcriptomes in this framework identifies culture conditions that sustain a naive state pertaining to the inner cell mass. Our approach thus clarifies understanding both of lineage segregation in the early human embryo and of in vitro stem cell identity, and provides an analytical resource for comparative molecular embryology.

developmental biology↗

bioRxiv2021

Regeneration of the larval sea star nervous system by wounding induced respecification to the sox2 lineage

The ability to restore lost body parts following traumatic injury is a fascinating area of biology that challenges current understanding of the ontogeny of differentiation. The origin of new cells needed to regenerate lost tissue, and whether they are pluripotent stem cells, tissue-specific stem cells or have de- or trans- differentiated, remains one of the most important open questions in regeneration. Additionally, it is not clearly known whether developmental gene regulatory networks (GRNs) are reused to direct specification in these cells or whether regeneration specific networks are deployed. Echinoderms, including sea stars, have extensive ability for regeneration and have therefore been the subject of many thorough studies on the ultrastructural and molecular properties of cells needed for regeneration. However, the technologies for obtaining transgenic echinoderms are limited and tracking cells involved in regeneration, and thus identifying the cellular sources and potencies has proven challenging. In this study we develop new transgenic tools for cell tracking in the regenerating bipinnaria larva of the sea star Patira minaita. We show that the larval serotonergic nervous system can regenerate following decapitation. Using a BAC-transgenesis approach with photoconvertible fluorescent proteins, we show that expression of the pan ectodermal marker, sox2, is induced in previously sox2 minus cells at the wound site, even when cell division is inhibited. sox2+ cells give rise to new sox4+ neural precursors that then proceed along an embryonic neurogenesis pathway to reform the anterior nervous systems. sox2+ cells contribute to only neural and ectoderm lineages, indicating that these progenitors maintain their normal, embryonic lineage restriction. This indicates that sea star larval regeneration uses a combination of existing lineage restricted stem cells, as well as respecification of cells into neural lineages, and at least partial reuse of developmental GRNs to regenerate their nervous system.

developmental biology↗

bioRxiv2024

A single-cell transcriptomics atlas for the parasitic nematode Heligmosomoides bakeri: Extrapolating model organism information to non-model systems

Single-cell atlases aim to collect the gene expression information for every cell type in an organism but can be challenging to perform in non-model organisms. To try to circumvent the problem of having no verified cell type markers in the parasitic nematode Heligmosomoides bakeri to use for an atlas, we attempted to use orthologs of verified markers from the closely related model organism Caenorhabditis elegans. This resulted in a useful comparison between the two worms for each of the cell types recovered in preliminary H. bakeri single-cell RNA-sequencing. For H. bakeri males and females, robustly recovered cell types include the gametes, embryos, and male intestine, while hypodermis, neurons, muscles, and pharyngeal cells were under-represented cell types. The two worms appear to have a similar hypodermis, cuticle, eggshell, and spermatogenesis process. On the other hand, putative cell identities and cell cycle scores suggest the intestine and muscle cells in H. bakeri may still be cycling and dividing, unlike in C. elegans. Additionally, embryogenesis and early development appear to be quite different between the two worms, with only eight out of 94 confirmed paternal contributions to the embryo in C. elegans (with an ortholog) predicted to also be paternal contributions in H. bakeri. Overall, this new dataset allowed me to move beyond the presence or absence of orthologs to include their tissue specificity and expression level similarities and differences when comparing these two worms to better identify biological processes and traits in a parasitic nematode that are modelled well by C. elegans.

developmental biology↗

bioRxiv2024

Mechanochemical Patterning Localizes the Organizer of a Luminal Epithelium

The spontaneous emergence of tissue patterns is often attributed to biochemical reaction-diffusion systems. In Hydra tissue regeneration, the formation of a Wnt signaling center serves as a well-known example of such a process. However, despite extensive research, a strictly biochemical mechanism for self-organization in Hydra remains elusive. In this study, we investigated mechanical stimuli and identified a positive feedback loop between Wnt signaling and tissue stretching. We developed a mathematical model of mechanochemical pattern formation in a closed elastic shell, representing regenerating Hydra epithelial spheroids. Our model explains how mechanical forces drive axis formation and predicts the organizers location under various perturbations, providing a more comprehensive understanding of the spatial dynamics involved. Validation by partially confining regenerating tissues showed that the organizer indeed forms in regions with the greatest stretching potential. This work highlights a novel mechanochemical mechanism for luminal epithelium patterning, suggesting that mechanical forces, in addition to biochemical signals, play a crucial role in tissue regeneration and axis specification. Our findings offer broader implications for the role of mechanical forces in tissue organization in various biological systems, opening new avenues for investigating mechanochemical feedback in development and regeneration.

developmental biology↗

bioRxiv2024

Spatiotemporal distribution of the glycoprotein pherophorin II reveals stochastic geometry of the growing ECM of Volvox carteri

The evolution of multicellularity involved the transformation of a simple cell wall of unicellular ancestors into a complex, multifunctional extracellular matrix (ECM). A suitable model organism to study the formation and expansion of an ECM during ontogenesis is the multicellular green alga Volvox carteri, which, along with the related volvocine algae, produces a complex, self-organized ECM composed of multiple substructures. These self-assembled ECMs primarily consist of hydroxyproline-rich glycoproteins, a major component of which is pherophorins. To investigate the geometry of the growing ECM, we fused the yfp gene with the gene for pherophorin II (PhII) in V. carteri. Confocal microscopy reveals PhII:YFP localization at key structures within the ECM, including the boundaries of compartments surrounding each somatic cell and the outer surface of the organism. Image analysis during the life cycle allows the stochastic geometry of those growing compartments to be quantified. We find that their areas and aspect ratios exhibit robust gamma distributions and exhibit a transition from a tight polygonal to a looser acircular packing geometry with stable eccentricity over time, evoking parallels and distinctions with the behavior of hydrated foams. These results provide a quantitative benchmark for addressing a general, open question in biology: How do cells produce structures external to themselves in a robust and accurate manner?

developmental biology↗

bioRxiv2021

A single cell transcriptional roadmap for human pacemaker cell differentiation

Each heartbeat is triggered by the sinoatrial node, the natural pacemaker of the heart. Animal models have revealed that pacemaker cells share a common progenitor with the (pro)epicardium, and that the pacemaker cardiomyocytes further diversify into "transitional", "tail" and "head" subtypes. However, the underlying molecular mechanisms are poorly understood. Here, we studied the differentiation of human induced pluripotent stem cells into pacemaker cardiomyocytes. Single cell RNA sequencing identified the presence of myocardial populations resembling subtypes present in the formed sinoatrial node, and in addition revealed a side population of (pro)epicardial cells. Time-course trajectory analysis uncovered a role for WNT signaling in determining myocardial versus proepicardial cell fate. We experimentally demonstrate that presence of WNT signaling prior to the branching point of a common progenitor enhances proepicardial cell differentiation at the expense of myocardial pacemaker cells. Furthermore, we uncover a role for TGF{beta} and WNT signaling in differentiation towards transitional and head pacemaker subtypes, respectively. Our findings provide new biological insights into human pacemaker differentiation, open avenues for complex disease modeling and inform regenerative approaches.

developmental biology↗

bioRxiv2023

New tools reveal PCP-dependent polarized mechanics in the cortex and cytoplasm of single cells during convergent extension

Understanding biomechanics of biological systems is crucial for unraveling complex processes like tissue morphogenesis. However, current methods for studying cellular mechanics in vivo are limited by the need for specialized equipment and often provide limited spatiotemporal resolution. Here we introduce two new techniques, Tension by Transverse Fluctuation (TFlux) and in vivo microrheology, that overcome these limitations. They both offer time-resolved, subcellular biomechanical analysis using only fluorescent reporters and widely available microscopes. Employing these two techniques, we have revealed a planar cell polarity (PCP)-dependent mechanical gradient both in the cell cortex and the cytoplasm of individual cells engaged in convergent extension. Importantly, the non-invasive nature of these methods holds great promise for its application for uncovering subcellular mechanical variations across a wide array of biological contexts. SummaryNon-invasive imaging-based techniques providing time-resolved biomechanical analysis at subcellular scales in developing vertebrate embryos.

developmental biology↗