Live Imaging and Characterization of Microglia Dynamics in the Zebrafish Embryo.

Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and function. Externally developing zebrafish embryos possess optical transparency, which along with well-characterized transgenic reporter lines that fluorescently label microglia, make zebrafish an ideal vertebrate model for such studies. In this paper, we take advantage of the unique features of the zebrafish model to visualize the dynamics of microglia cells in vivo and under physiological conditions. We use confocal microscopy to record a timelapse of microglia cells in the optic tectum of the zebrafish embryo and then, extract tracking data using the IMARIS 10.0 software to obtain the cells' migration path, mean speed, and distribution in the optic tectum at different developmental stages. This protocol can be a useful tool to elucidate the physiological significance of microglia behavior in various contexts, contributing to a deeper characterization of these highly motile cells.

A cytokinetic ring-driven cell rotation achieves Hertwig's rule in early development.

Hertwig's rule states that cells divide along their longest axis, usually driven by forces acting on the mitotic spindle. Here, we show that in contrast to this rule, microtubule-based pulling forces in early Caenorhabditis elegans embryos align the spindle with the short axis of the cell. We combine theory with experiments to reveal that in order to correct this misalignment, inward forces generated by the constricting cytokinetic ring rotate the entire cell until the spindle is aligned with the cell's long axis. Experiments with slightly compressed mouse zygotes indicate that this cytokinetic ring-driven mechanism of ensuring Hertwig's rule is general for cells capable of rotating inside a confining shell, a scenario that applies to early cell divisions of many systems.

A new atlas to study embryonic cell types in Xenopus.

This paper introduces a single-cell atlas for pivotal developmental stages in Xenopus, encompassing gastrulation, neurulation, and early tailbud. Notably surpassing its predecessors, the new atlas enhances gene mapping, read counts, and gene/cell type nomenclature. Leveraging the latest Xenopus tropicalis genome version, alongside advanced alignment pipelines and machine learning for cell type assignment, this release maintains consistency with previous cell type annotations while rectifying nomenclature issues. Employing an unbiased approach for cell type assignment proves especially apt for embryonic contexts, given the considerable number of non-terminally differentiated cell types. An alternative cell type attribution here adopts a fuzzy, non-deterministic stance, capturing the transient nature of early embryo progenitor cells by presenting an ensemble of types in superposition. The value of the new resource is emphasized through numerous examples, with a focus on previously unexplored germ cell populations where we uncover novel transcription onset features. Offering interactive exploration via a user-friendly web portal and facilitating complete data downloads, this atlas serves as a comprehensive and accessible reference.

Ascidian embryonic cells with properties of neural-crest cells and neuromesodermal progenitors of vertebrates.

Neural-crest cells and neuromesodermal progenitors (NMPs) are multipotent cells that are important for development of vertebrate embryos. In embryos of ascidians, which are the closest invertebrate relatives of vertebrates, several cells located at the border between the neural plate and the epidermal region have neural-crest-like properties; hence, the last common ancestor of ascidians and vertebrates may have had ancestral cells similar to neural-crest cells. However, these ascidian neural-crest-like cells do not produce cells that are commonly of mesodermal origin. Here we showed that a cell population located in the lateral region of the neural plate has properties resembling those of vertebrate neural-crest cells and NMPs. Among them, cells with Tbx6-related expression contribute to muscle near the tip of the tail region and cells with Sox1/2/3 expression give rise to the nerve cord. These observations and cross-species transcriptome comparisons indicate that these cells have properties similar to those of NMPs. Meanwhile, transcription factor genes Dlx.b, Zic-r.b and Snai, which are reminiscent of a gene circuit in vertebrate neural-crest cells, are involved in activation of Tbx6-related.b. Thus, the last common ancestor of ascidians and vertebrates may have had cells with properties of neural-crest cells and NMPs and such ancestral cells may have produced cells commonly of ectodermal and mesodermal origins.

Mechanical stress combines with planar polarised patterning during metaphase to orient embryonic epithelial cell divisions.

The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. Here, we ask how mechanics and antero-posterior (AP) patterning combine to influence the first divisions after gastrulation in the Drosophila embryonic epithelium. We analyse hundreds of cell divisions and show that stress anisotropy, notably from compressive forces, can reorient division directly in metaphase. Stress anisotropy influences the OCD by imposing metaphase cell elongation, despite mitotic rounding, and overrides interphase cell elongation. In strongly elongated cells, the mitotic spindle adapts its length to, and hence its orientation is constrained by, the cell long axis. Alongside mechanical cues, we find a tissue-wide bias of the mitotic spindle orientation towards AP-patterned planar polarised Myosin-II. This spindle bias is lost in an AP-patterning mutant. Thus, a patterning-induced mitotic spindle orientation bias overrides mechanical cues in mildly elongated cells, whereas in strongly elongated cells the spindle is constrained close to the high stress axis.

Two sequential gene expression programs bridged by cell division support long-distance collective cell migration.

The precise assembly of tissues and organs relies on spatiotemporal regulation of gene expression to coordinate the collective behavior of cells. In Drosophila embryos, the midgut musculature is formed through collective migration of caudal visceral mesoderm (CVM) cells, but how gene expression changes as cells migrate is not well understood. Here, we have focused on ten genes expressed in the CVM and the cis-regulatory sequences controlling their expression. Although some genes are continuously expressed, others are expressed only early or late during migration. Late expression relates to cell cycle progression, as driving string/Cdc25 causes earlier division of CVM cells and accelerates the transition to late gene expression. In particular, we found that the cell cycle effector transcription factor E2F1 is a required input for the late gene CG5080. Furthermore, whereas late genes are broadly expressed in all CVM cells, early gene transcripts are polarized to the anterior or posterior ends of the migrating collective. We show this polarization requires transcription factors Snail, Zfh1 and Dorsocross. Collectively, these results identify two sequential gene expression programs bridged by cell division that support long-distance directional migration of CVM cells.

A time-resolved single-cell roadmap of the logic driving anterior neural crest diversification from neural border to migration stages.

Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.

Local and global changes in cell density induce reorganisation of 3D packing in a proliferating epithelium.

Tissue morphogenesis is intimately linked to the changes in shape and organisation of individual cells. In curved epithelia, cells can intercalate along their own apicobasal axes, adopting a shape named 'scutoid' that allows energy minimization in the tissue. Although several geometric and biophysical factors have been associated with this 3D reorganisation, the dynamic changes underlying scutoid formation in 3D epithelial packing remain poorly understood. Here, we use live imaging of the sea star embryo coupled with deep learning-based segmentation to dissect the relative contributions of cell density, tissue compaction and cell proliferation on epithelial architecture. We find that tissue compaction, which naturally occurs in the embryo, is necessary for the appearance of scutoids. Physical compression experiments identify cell density as the factor promoting scutoid formation at a global level. Finally, the comparison of the developing embryo with computational models indicates that the increase in the proportion of scutoids is directly associated with cell divisions. Our results suggest that apico-basal intercalations appearing immediately after mitosis may help accommodate the new cells within the tissue. We propose that proliferation in a compact epithelium induces 3D cell rearrangements during development.

Enhancer-promoter interactions can form independently of genomic distance and be functional across TAD boundaries.

Topologically Associating Domains (TADs) have been suggested to facilitate and constrain enhancer-promoter interactions. However, the role of TAD boundaries in effectively restricting these interactions remains unclear. Here, we show that a significant proportion of enhancer-promoter interactions are established across TAD boundaries in Drosophila embryos, but that developmental genes are strikingly enriched in intra- but not inter-TAD interactions. We pursued this observation using the twist locus, a master regulator of mesoderm development, and systematically relocated one of its enhancers to various genomic locations. While this developmental gene can establish inter-TAD interactions with its enhancer, the functionality of these interactions remains limited, highlighting the existence of topological constraints. Furthermore, contrary to intra-TAD interactions, the formation of inter-TAD enhancer-promoter interactions is not solely driven by genomic distance, with distal interactions sometimes favored over proximal ones. These observations suggest that other general mechanisms must exist to establish and maintain specific enhancer-promoter interactions across large distances.

Identification of distinct vascular mural cell populations during zebrafish embryonic development.

BACKGROUND: Mural cells are an essential perivascular cell population that associate with blood vessels and contribute to vascular stabilization and tone. In the embryonic zebrafish vasculature, pdgfrb and tagln are commonly used as markers for identifying pericytes and vascular smooth muscle cells. However, the overlapping and distinct expression patterns of these markers in tandem have not been fully described. RESULTS: Here, we used the Tg(pdgfrb:Gal4FF; UAS:RFP) and Tg(tagln:NLS-EGFP) transgenic lines to identify single- and double-positive perivascular cell populations on the cranial, axial, and intersegmental vessels between 1 and 5 days postfertilization. From this comparative analysis, we discovered two novel regions of tagln-positive cell populations that have the potential to function as mural cell precursors. Specifically, we found that the hypochord-a reportedly transient structure-contributes to tagln-positive cells along the dorsal aorta. We also identified a unique mural cell progenitor population that resides along the midline between the neural tube and notochord and contributes to intersegmental vessel mural cell coverage. CONCLUSION: Together, our findings highlight the variability and versatility of tracking both pdgfrb and tagln expression in mural cells of the developing zebrafish embryo and reveal unexpected embryonic cell populations that express pdgfrb and tagln.

Cytoplasmic division cycles without the nucleus and mitotic CDK/cyclin complexes.

Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. We demonstrate in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, we present evidence that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. We postulate that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes.

Cell-cycle control: Timing is everything for the Plk1-Bub1 partnership.

Bub1 and Polo kinases are well-known multitasking regulators of mitosis. New work shows that they team up at kinetochores to determine the mitotic duration of embryonic divisions in nematodes. As is often the case, the key effector is Cdc20 activity.

maea affects head formation through ß-catenin degradation during early Xenopus laevis development.

Canonical Wnt signalling plays important roles in early embryogenesis, such as axis formation due to its activation and head formation due to its inhibition. ß-catenin protein stability is a key factor in canonical Wnt signalling. Several E3 ubiquitin ligases contribute to ß-catenin degradation through the ubiquitin/proteasome system. We characterised an E3 ubiquitin ligase gene, Xenopus laevis macrophage erythroblast attacher (maea), during early development. maea transcripts were ubiquitously detected in early embryos. The expression levels of the Wnt target genes nodal homolog 3, gene 1 (nodal3.1), and siamois homeodomain 1 (sia1), which were induced by injection with ß-catenin mRNA, were reduced by maea.S mRNA co-injection. maea.S overexpression at the anterior dorsal region enlarged head structures, whereas Maea knockdown interfered with head formation in Xenopus embryos. Maea.S decreased and ubiquitinated ß-catenin protein. ß-catenin-4KRs protein, which mutated the four lysine (K) residues known as ubiquitinated sites to arginine (R) residues, was also ubiquitinated and degraded by Maea.S. These findings suggest that Maea contributes to ß-catenin degradation by ubiquitination of unknown lysine residues in early Xenopus development.

Vasa identifies germ cells in embryos and gonads of Oryzias celebensis.

Vasa is the most studied germ cell marker that is indispensable for germ cell development in teleost fishes. Here, a vasa full-length cDNA from Oryzias celebensis was isolated. Analysis of gene expression by reversed transcription polymerase chain reaction and in situ hybridization showed the vasa transcript was maternally inherited and specifically expressed in germ cells during embryogenesis and in adult gonads. During embryogenesis, vasa mRNA was widely distributed in the embryos until the somitogenesis stage and then specifically expressed in primordial germ cells (PGCs). In the testis, vasa expression was highest in spermatogonia and gradually decreased during spermatogenesis. In ovary, vasa expression was present predominantly in immature oocytes and persisted throughout oogenesis. Constructs containing green or red fluorescence proteins and vasa 3' UTR or dnd 3' UTR, confirmed stable vasa expression in the PGCs of O. celebensis and co-expression of the two genes. In summary, the conservation of vasa expression in embryonic and adult germ cells of both sexes compared to other vertebrates suggests its function is also widely conserved.

Notch signaling regulates neural stem cell quiescence entry and exit in Drosophila.

Stem cells enter and exit quiescence as part of normal developmental programs and to maintain tissue homeostasis in adulthood. Although it is clear that stem cell intrinsic and extrinsic cues, local and systemic, regulate quiescence, it remains unclear whether intrinsic and extrinsic cues coordinate to control quiescence and how cue coordination is achieved. Here, we report that Notch signaling coordinates neuroblast intrinsic temporal programs with extrinsic nutrient cues to regulate quiescence in Drosophila. When Notch activity is reduced, quiescence is delayed or altogether bypassed, with some neuroblasts dividing continuously during the embryonic-to-larval transition. During embryogenesis before quiescence, neuroblasts express Notch and the Notch ligand Delta. After division, Delta is partitioned to adjacent GMC daughters where it transactivates Notch in neuroblasts. Over time, in response to intrinsic temporal cues and increasing numbers of Delta-expressing daughters, neuroblast Notch activity increases, leading to cell cycle exit and consequently, attenuation of Notch pathway activity. Quiescent neuroblasts have low to no active Notch, which is required for exit from quiescence in response to nutrient cues. Thus, Notch signaling coordinates proliferation versus quiescence decisions.

The effect of P2X7R- mediated Ca2+ and MAPK signaling in OPG-induced duck embryo osteoclasts differentiation and adhesive structure damage.

Various factors cause animal bone malnutrition disease during intensive culture. Osteoclasts play an important role in regulating bone metabolism disease. Osteoprotegerin (OPG) modulates osteoclast function; however, the mechanism underlying this effect is unknown. Therefore, the present study aimed to explore whether OPG affects duck embryo osteoclast function via purinergic receptor P2X7. OPG significantly inhibited duck embryo osteoclast differentiation and bone resorption, and suppressed F-actin formation. In addition, OPG remarkably impaired duck embryo osteoclasts' adhesive structure. After OPG treatment, the expression of P2X7R significantly reduced, the ATP level and Ca2+-ATPase activity decreased rapidly, and concomitantly suppressed calcium and MAPK signaling. A438079 (a selective P2X7R inhibitor) significantly inhibited duck embryo osteoclast differentiation and bone resorption, and the phosphorylation of Ca2+ regulated proteins (CAM, CAMKII, CAMKIV) and MAPKs (ERK, JNK, and P38) were markedly suppressed. Pretreatment of duck embryo osteoclasts with BzATP, a P2X7R agonist, activated Ca2+ and MAPK signaling. BzATP alleviated OPG-induced duck embryo osteoclast differentiation and adhesive structure damage, and recovered the distribution of adhesion-related proteins in mature duck embryo osteoclasts. Thus, P2RX7-mediated Ca2+ and MAPK signaling has a key function in OPG-induced duck embryo osteoclast differentiation and adhesive structure damage. P2X7R might be an ideal target to treat bone diseases through regulating bone cell activation.

A novel deep learning-based 3D cell segmentation framework for future image-based disease detection.

Cell segmentation plays a crucial role in understanding, diagnosing, and treating diseases. Despite the recent success of deep learning-based cell segmentation methods, it remains challenging to accurately segment densely packed cells in 3D cell membrane images. Existing approaches also require fine-tuning multiple manually selected hyperparameters on the new datasets. We develop a deep learning-based 3D cell segmentation pipeline, 3DCellSeg, to address these challenges. Compared to the existing methods, our approach carries the following novelties: (1) a robust two-stage pipeline, requiring only one hyperparameter; (2) a light-weight deep convolutional neural network (3DCellSegNet) to efficiently output voxel-wise masks; (3) a custom loss function (3DCellSeg Loss) to tackle the clumped cell problem; and (4) an efficient touching area-based clustering algorithm (TASCAN) to separate 3D cells from the foreground masks. Cell segmentation experiments conducted on four different cell datasets show that 3DCellSeg outperforms the baseline models on the ATAS (plant), HMS (animal), and LRP (plant) datasets with an overall accuracy of 95.6%, 76.4%, and 74.7%, respectively, while achieving an accuracy comparable to the baselines on the Ovules (plant) dataset with an overall accuracy of 82.2%. Ablation studies show that the individual improvements in accuracy is attributable to 3DCellSegNet, 3DCellSeg Loss, and TASCAN, with the 3DCellSeg demonstrating robustness across different datasets and cell shapes. Our results suggest that 3DCellSeg can serve a powerful biomedical and clinical tool, such as histo-pathological image analysis, for cancer diagnosis and grading.

Live 3D imaging and mapping of shear stresses within tissues using incompressible elastic beads.

To investigate the role of mechanical constraints in morphogenesis and development, we have developed a pipeline of techniques based on incompressible elastic sensors. These techniques combine the advantages of incompressible liquid droplets, which have been used as precise in situ shear stress sensors, and of elastic compressible beads, which are easier to tune and to use. Droplets of a polydimethylsiloxane mix, made fluorescent through specific covalent binding to a rhodamin dye, are produced by a microfluidics device. The elastomer rigidity after polymerization is adjusted to the tissue rigidity. Its mechanical properties are carefully calibrated in situ, for a sensor embedded in a cell aggregate submitted to uniaxial compression. The local shear stress tensor is retrieved from the sensor shape, accurately reconstructed through an active contour method. In vitro, within cell aggregates, and in vivo, in the prechordal plate of the zebrafish embryo during gastrulation, our pipeline of techniques demonstrates its efficiency to directly measure the three dimensional shear stress repartition within a tissue.

Yolk platelets impede nuclear expansion in Xenopus embryos.

During metazoan early embryogenesis, the intracellular properties of proteins and organelles change dynamically through rapid cleavage. In particular, a change in the nucleus size is known to contribute to embryonic development-dependent cell cycle and gene expression regulation. Here, we compared the nuclear sizes of various blastomeres from developing Xenopus embryos and analyzed the mechanisms that control the nuclear expansion dynamics by manipulating the amount of intracellular components in a cell-free system. Nuclear expansion was slower in blastomeres from vegetal hemispheres during a longer interphase than in those from animal hemispheres. Furthermore, upon recapitulating interphase events by manipulating the concentration of yolk platelets, which are originally rich in the vegetal blastomeres, in cell-free cytoplasmic extracts, nuclear expansion and DNA replication became slower than that in normal yolk-free conditions. Under these conditions, the supplemented yolk platelets accumulated around the nucleus in a microtubule-dependent manner and impeded the organization of the endoplasmic reticulum network. Overall, we propose that yolk platelets around the nucleus reduce membrane supply from the endoplasmic reticulum to the nucleus, resulting in slower nuclear expansion and cell cycle progression in the yolk-rich vegetal blastomeres.

The primitive streak and cellular principles of building an amniote body through gastrulation.

The primitive streak, a transient embryonic structure, marks bilateral symmetry in mammalian and avian embryos and helps confer anterior-posterior and dorsal-ventral spatial information to early differentiating cells during gastrulation. Its recapitulation in vitro may facilitate derivation of tissues and organs with in vivo­like complexity. Proper understanding of the primitive streak and what it entails in human development is key to achieving such research objectives. Here we provide an overview of the primitive streak and conclude that this structure is neither conserved nor necessary for gastrulation or early lineage diversification. We offer a model in which the primitive streak is viewed as part of a morphologically diverse yet molecularly conserved process of spatial coordinate acquisition. We predict that recapitulation of the primitive streak is dispensable for development in vitro.