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.

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.

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.

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.

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.

Influence of Incorporation of Different dn-Electron Metal Cations into Biologically Active System on Its Biological and Physicochemical Properties.

Three new compounds, namely [HL]2+[CuCl4]2-, [HL]2+[ZnCl4]2-, and [HL]2+[CdCl4]2- (where L: imipramine) were synthesized and their physicochemical and biological properties were thoroughly investigated. All three compounds form isostructural, crystalline systems, which have been studied using Single-Crystal X-ray diffraction analysis (SC-XRD) and Fourier-transform infrared spectroscopy (FTIR). The thermal stability was investigated using thermogravimetric analysis (TGA) and melting points for all compounds have been determined. Magnetic measurements were performed in order to study the magnetic properties of the compounds. The above mentioned techniques allowed us to comprehensively examine the physicochemical properties of the newly obtained compounds. The biological activity was investigated using the number of Zebrafish tests, as it is one of the most common models for studying the impact of newly synthesized compounds on the central nervous system (CNS), since this model is very similar to the human CNS.

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.

Correct regionalization of a tissue primordium is essential for coordinated morphogenesis.

During organ development, tubular organs often form from flat epithelial primordia. In the placodes of the forming tubes of the salivary glands in the Drosophila embryo, we previously identified spatially defined cell behaviors of cell wedging, tilting, and cell intercalation that are key to the initial stages of tube formation. Here, we address what the requirements are that ensure the continuous formation of a narrow symmetrical tube from an initially asymmetrical primordium whilst overall tissue geometry is constantly changing. We are using live-imaging and quantitative methods to compare wild-type placodes and mutants that either show disrupted cell behaviors or an initial symmetrical placode organization, with both resulting in severe impairment of the invagination. We find that early transcriptional patterning of key morphogenetic transcription factors drives the selective activation of downstream morphogenetic modules, such as GPCR signaling that activates apical-medial actomyosin activity to drive cell wedging at the future asymmetrically placed invagination point. Over time, transcription of key factors expands across the rest of the placode and cells switch their behavior from predominantly intercalating to predominantly apically constricting as their position approaches the invagination pit. Misplacement or enlargement of the initial invagination pit leads to early problems in cell behaviors that eventually result in a defective organ shape. Our work illustrates that the dynamic patterning of the expression of transcription factors and downstream morphogenetic effectors ensures positionally fixed areas of cell behavior with regards to the invagination point. This patterning in combination with the asymmetric geometrical setup ensures functional organ formation.

Unique pattern of histogenesis of the parakeratinized epithelium on lingual prominence in the domestic goose embryos (Anser anser f. domestica).

A triangular lingual prominence (LP) is a characteristic part of the tongue in Anseriformes containing adipose tissue. The parakeratinized epithelium (PEp) covers the LP. Studies aimed to describe the histogenesis of PEp during the process of the intensive formation of the LP in domestic goose during embryonic period and to determine the structural readiness to perform a protective function. The study were conducted by using LM, SEM and TEM technique. The results revealed that on day 16th the undifferentiated epithelium of LP transformed into the typical avian multilayered epithelium. Contrary to pattern of histogenesis of parakeratinized epithelium on the lingual body, on the medial and lateral areas of the elongating and bulging LP were formed epithelial furrows. Which around 20th day, on lateral areas of LP deepened up to half of epithelium, whereas on the medial area began to fade. The ultrastructure of cells lying in furrows indicated progressive apoptosis-like degeneration. On the 25th day, shallow furrows were only present on lateral areas, where bulging of LP was continued. Whereas the epithelium on medial area started cornification by the accumulation of cytokeratin fibers. Lack of the periderm during the development of the PEp of the LP indicated its endodermal origin.

A live-imaging protocol to track cell movement in the Xenopus embryo.

Tracking individual cell movement during development is challenging, particularly in tissues subjected to major remodeling. Currently, most live imaging techniques in Xenopus are limited to tissue explants and/or to superficial cells. We describe here a protocol to track immature multiciliated cells (MCCs) moving within the inner epidermal layer of a whole embryo. In addition, we present a data processing protocol to uncouple the movements of individual cells from the coplanar drifts of the tissue in which they are embedded. For complete details on the use and execution of this protocol, please refer to Chuyen et al. (2021).

Male-biased protein expression in primordial germ cells, identified through a comparative study of UAS vectors in Drosophila.

In Drosophila, three types of UAS vectors (UASt, UASp, and UASz) are currently available for use with the Gal4-UAS system. They have been used successfully in somatic cells and germline cells from ovaries. However, it remains unclear whether they are functional in the germline cells of embryos, larvae, and adult testes. In this study, we found that all three types of UAS vectors were functional in the germline cells of embryos and larvae and that the UASt and UASz vectors were active in the germline of the distal tip region in adult testes. Moreover, we observed that protein expression from the UAS vectors was male-biased in germline cells of late embryos, whereas their respective mRNA expression levels were not. Furthermore, O-propargyl-puromycin (OPP) staining revealed that protein synthesis was male-biased in these germline cells. In addition, GO terms related to translation and ribosomal maturation were significantly enriched in the male germline. These observations show that translational activity is higher in male than in female germline cells. Therefore, we propose that male-biased protein synthesis may be responsible for the sex differences observed in the early germline.

Gene regulation of adult skeletogenesis in starfish and modifications during gene network co-option.

The larval skeleton of the echinoderm is believed to have been acquired through co-option of a pre-existing gene regulatory network (GRN); that is, the mechanism for adult skeleton formation in the echinoderm was deployed in early embryogenesis during echinoderm diversification. To explore the evolutionary changes that occurred during co-option, we examined the mechanism for adult skeletogenesis using the starfish Patiria pectinifera. Expression patterns of skeletogenesis-related genes (vegf, vegfr, ets1/2, erg, alx1, ca1, and clect) suggest that adult skeletogenic cells develop from the posterior coelom after the start of feeding. Treatment with inhibitors and gene knockout using transcription activator-like effector nucleases (TALENs) suggest that the feeding-nutrient sensing pathway activates Vegf signaling via target of rapamycin (TOR) activity, leading to the activation of skeletogenic regulatory genes in starfish. In the larval skeletogenesis of sea urchins, the homeobox gene pmar1 activates skeletogenic regulatory genes, but in starfish, localized expression of the pmar1-related genes phbA and phbB was not detected during the adult skeleton formation stage. Based on these data, we provide a model for the adult skeletogenic GRN in the echinoderm and propose that the upstream regulatory system changed from the feeding-TOR-Vegf pathway to a homeobox gene-system during co-option of the skeletogenic GRN.

Membrane architecture and adherens junctions contribute to strong Notch pathway activation.

The Notch pathway mediates cell-to-cell communication in a variety of tissues, developmental stages and organisms. Pathway activation relies on the interaction between transmembrane ligands and receptors on adjacent cells. As such, pathway activity could be influenced by the size, composition or dynamics of contacts between membranes. The initiation of Notch signalling in the Drosophila embryo occurs during cellularization, when lateral cell membranes and adherens junctions are first being deposited, allowing us to investigate the importance of membrane architecture and specific junctional domains for signalling. By measuring Notch-dependent transcription in live embryos, we established that it initiates while lateral membranes are growing and that signalling onset correlates with a specific phase in their formation. However, the length of the lateral membranes per se was not limiting. Rather, the adherens junctions, which assemble concurrently with membrane deposition, contributed to the high levels of signalling required for transcription, as indicated by the consequences of α-Catenin depletion. Together, these results demonstrate that the establishment of lateral membrane contacts can be limiting for Notch trans-activation and suggest that adherens junctions play an important role in modulating Notch activity.

The biological regulation of sea urchin larval skeletogenesis - From genes to biomineralized tissue.

Biomineralization is the process in which soft organic tissues use minerals to produce shells, skeletons and teeth for various functions such as protection and physical support. The ability of the cells to control the time and place of crystal nucleation as well as crystal orientation and stiffness is far beyond the state-of-the art of human technologies. Thus, understanding the biological control of biomineralization will promote our understanding of embryo development as well as provide novel approaches for material engineering. Sea urchin larval skeletogenesis offers an excellent platform for functional analyses of both the molecular control system and mineral uptake and deposition. Here we describe the current understanding of the genetic, molecular and cellular processes that underlie sea urchin larval skeletogenesis. We portray the regulatory genes that define the specification of the skeletogenic cells and drive the various morphogenetic processes that occur in the skeletogenic lineage, including: epithelial to mesenchymal transition, cell migration, spicule cavity formation and mineral deposition into the spicule cavity. We describe recent characterizations of the size, motion and mineral concentration of the calcium-bearing vesicles in the skeletogenic cells. We review the distinct specification states within the skeletogenic lineage that drive localized skeletal growth at the tips of the spicules. Finally, we discuss the surprising similarity between the regulatory network and cellular processes that drive sea urchin skeletogenesis and those that control vertebrate vascularization. Overall, we illustrate the novel insights on the biological regulation and evolution of biomineralization, gained from studies of the sea urchin larval skeletogenesis.

Cell-cell contact landscapes in Xenopus gastrula tissues.

Molecular and structural facets of cell-cell adhesion have been extensively studied in monolayered epithelia. Here, we perform a comprehensive analysis of cell-cell contacts in a series of multilayered tissues in the Xenopus gastrula model. We show that intercellular contact distances range from 10 to 1,000 nm. The contact width frequencies define tissue-specific contact spectra, and knockdown of adhesion factors modifies these spectra. This allows us to reconstruct the emergence of contact types from complex interactions of the factors. We find that the membrane proteoglycan Syndecan-4 plays a dominant role in all contacts, including narrow C-cadherin-mediated junctions. Glypican-4, hyaluronic acid, paraxial protocadherin, and fibronectin also control contact widths, and unexpectedly, C-cadherin functions in wide contacts. Using lanthanum staining, we identified three morphologically distinct forms of glycocalyx in contacts of the Xenopus gastrula, which are linked to the adhesion factors examined and mediate cell-cell attachment. Our study delineates a systematic approach to examine the varied contributions of adhesion factors individually or in combinations to nondiscrete and seemingly amorphous intercellular contacts.