Cytokinetic abscission is part of the midblastula transition in early zebrafish embryogenesis

Animal cytokinesis ends with the formation of a thin intercellular membrane bridge that connects the two newly formed sibling cells, which is ultimately resolved by abscission. While mitosis is completed within 15 min, the intercellular bridge can persist for hours, maintaining a physical connection between sibling cells and allowing exchange of cytosolic components. Although cell–cell communication is fundamental for development, the role of intercellular bridges during embryogenesis has not been fully elucidated. In this work, we characterized the spatiotemporal characteristics of the intercellular bridge during early zebrafish development. We found that abscission is delayed during the rapid division cycles that occur in the early embryo, giving rise to the formation of interconnected cell clusters. Abscission was accelerated when the embryo entered the midblastula transition (MBT) phase. Components of the ESCRT machinery, which drives abscission, were enriched at intercellular bridges post-MBT and, interfering with ESCRT function, extended abscission beyond MBT. Hallmark features of MBT, including transcription onset and cell shape modulations, were more similar in interconnected sibling cells compared to other neighboring cells. Collectively, our findings suggest that delayed abscission in the early embryo allows clusters of cells to coordinate their behavior during embryonic development.

Structures of the germline-specific Deadhead and thioredoxin T proteins from Drosophila melanogaster reveal unique features among thioredoxins

Thioredoxins (Trxs) are ubiquitous enzymes that regulate the redox state in cells. In Drosophila, there are two germline-specific Trxs, Deadhead (Dhd) and thioredoxin T (TrxT), that belong to the lethal(3)malignant brain tumor signature genes and to the `survival network' of genes that mediate the cellular response to DNA damage. Dhd is a maternal protein required for early embryogenesis that promotes protamine–histone exchange in fertilized eggs and midblastula transition. TrxT is testis-specific and associates with the lampbrush loops of the Y chromosome. Here, the first structures of Dhd and TrxT are presented, unveiling new features of these two thioredoxins. Dhd has positively charged patches on its surface, in contrast to the negatively charged surfaces commonly found in most Trxs. This distinctive charge distribution helps to define initial encounter complexes with DNA/RNA that will lead to final specific interactions with cofactors to promote chromatin remodeling. TrxT contains a C-terminal extension, which is mostly unstructured and highly flexible, that wraps the conserved core through a closed conformation. It is believed that these new structures can guide future work aimed at understanding embryo development and redox homeostasis in Drosophila. Moreover, due to their restricted presence in Schizophora (a section of the true flies), these structures can help in the design of small-molecular binders to modulate native redox homeostasis, thereby providing new applications for the control of plagues that cause human diseases and/or bring about economic losses by damaging crop production.

High-resolution single-cell 3D-models of chromatin ensembles during Drosophila embryogenesis

Single-cell chromatin studies provide insights into how chromatin structure relates to functions of individual cells. However, balancing high-resolution and genome wide-coverage remains challenging. We describe a computational method for the reconstruction of large 3D-ensembles of single-cell (sc) chromatin conformations from population Hi-C that we apply to study embryogenesis in Drosophila. With minimal assumptions of physical properties and without adjustable parameters, our method generates large ensembles of chromatin conformations via deep-sampling. Our method identifies specific interactions, which constitute 5–6% of Hi-C frequencies, but surprisingly are sufficient to drive chromatin folding, giving rise to the observed Hi-C patterns. Modeled sc-chromatins quantify chromatin heterogeneity, revealing significant changes during embryogenesis. Furthermore, >50% of modeled sc-chromatin maintain topologically associating domains (TADs) in early embryos, when no population TADs are perceptible. Domain boundaries become fixated during development, with strong preference at binding-sites of insulator-complexes upon the midblastula transition. Overall, high-resolution 3D-ensembles of sc-chromatin conformations enable further in-depth interpretation of population Hi-C, improving understanding of the structure-function relationship of genome organization.

Structures of the germline-specific Deadhead and Thioredoxin T proteins from Drosophila melanogaster reveal unique features among Thioredoxins

ABSTRACTThioredoxins (Trxs) are ubiquitous enzymes that regulate the redox state in cells. In Drosophila, there are two germline-specific Trxs, Deadhead (Dhd) and TrxT. Both proteins belong to the L(3)mbt malignant brain tumor signature and to the MMS survival network of genes that mediate the cellular response to DNA damage. Dhd is a maternal protein required for early embryogenesis that promotes protamine-histone exchange in fertilized eggs and midblastula transition. TrxT is testis-specific and associates with the lampbrush loops of the Y chromosome.Here we present the first structures of Dhd and TrxT that unveil new features of these Thioredoxins. Dhd is highly positively charged, unusual in canonical Trxs. This positively charged surface can facilitate its approximation to DNA and to protamine oligomers, to promote chromatin remodeling. On the other hand, TrxT contains a C-terminal extension, mostly unstructured and highly flexible, which wraps the conserved core through a closed conformation. This extension partially covers the catalytic site and modulates the redox activity of the protein.The information provided by these structures can guide future work aimed at understanding how redox inputs modulate the initial steps of embryo development in Drosophila and may help in the design of molecular inhibitors through a structure-based approach.HighlightsWe have determined the first structures of the germline-specific Trxs Dhd and TrxT.Dhd has a highly positively charged surface that facilitates its approximation to DNA and protamine oligomers, to promote chromatin remodeling.TrxT contains a C-terminal extension, highly unusual in canonical Trxs, mostly unstructured and highly flexible.The TrxT C-terminal extension partially covers the catalytic site and modulates the redox activity of the protein.The differences observed in Thioredoxins can help in fine-tuning specific molecules to be active against selected insect species.

Cytokinetic abscission is part of the mid-blastula transition switch in early zebrafish embryogenesis

ABSTRACTAnimal cytokinesis ends with the formation of a thin intercellular membrane bridge connecting the two newly formed sibling cells that is ultimately resolved by abscission. While mitosis is completed within 15 minutes, the intercellular bridge can persist for hours, maintaining a physical connection between sibling cells and allowing exchange of cytosolic components. Although cell-cell communication is fundamental for development, the potential role of intercellular bridges during embryogenesis have not been fully elucidated. Here, we found that in early zebrafish (Danio rerio) embryogenesis, abscission is delayed and cells do not resolve their intercellular bridges until midblastula transition (MBT), giving rise to the formation of small inter-connected cell clusters. Interestingly, abscission commences during the MBT switch, which is manifested by cell cycle elongation, loss of synchronized divisions and genome activation. Moreover, depletion of Chmp4bb which is an essential ESCRT-III component for scission, delayed abscission beyond the MBT switch. Hallmark features of MBT, including transcription onset and cell shape changes, were similar in sibling cells connected by intercellular bridges, proposing a role for intercellular bridges in maintaining cell-cell communication in the embryo. Taken together, our data suggest that abscission is part of the cellular changes that occur during MBT and that cells coordinate their behavior during this critical embryonic phase through persisted intercellular bridges.Significance StatementIn this work we show that the last step of cytokinesis, termed abscission, is inhibited in early zebrafish embryos. As a result, sibling cells remain connected to one another for several cycles and mutually time their developmental progress including transcription onset. Abscission commences at the 10th cell cycle, when embryos enter the midblastula transition (MBT) switch in which embryonic cells become individualized and exhibit the characteristics of mature cells. Our data suggest that abscission is part of the MBT switch and that embryonic sibling cells mutually time their developmental progress by maintaining physical connections between them in the early embryo.

Quantitative capillary zone electrophoresis-mass spectrometry reveals the N-glycome developmental plan during vertebrate embryogenesis

The Xenopus laevis N-glycome undergoes massive reprogramming after the midblastula transition and the onset of neuronal development.

Emergence of the subapical domain is associated with the midblastula transition

Epithelial domains and cell polarity are determined by polarity proteins which are associated with the cell cortex in a spatially restricted pattern. Early Drosophila embryos are characterized by a stereotypic dynamic and de novo formation of cortical domains. For example, the subapical domain emerges at the transition from syncytial to cellular development during the first few minutes of interphase 14. The dynamics in cortical patterning is revealed by the subapical markers Canoe/Afadin and ELMO-Sponge, which widely distributed in interphase 13 but subapically restricted in interphase 14. The factors and mechanism determining the timing for the emergence of the subapical domain have been unknown. In this study, we show, that the restricted localization of subapical markers depends on the onset of zygotic gene expression. In contrast to cell cycle remodeling, the emergence of the subapical domain does not depend on the nucleo-cytoplasmic ratio. Thus, we define cortical dynamics and specifically the emergence of the subapical domain as a feature of the midblastula transition.Author summaryMidblastula transition is a paradigm of a developmental transition. Multiple processes such as cell cycle, cell mobility, onset of zygotic gene expression, degradation of maternal RNA and chromatin structure are coordinated to lead to defined changes in visible morphology. The midblastula transition in Drosophila embryos is associated with a change from fast nuclear cycles to a cell cycle mode with gap phase and slow replication, a strong increase in zygotic transcription and cellularization. The timing of the processes associated with the midblastula transition are controlled by the onset of zygotic gene expression or the nucleocytoplasmic ratio. Here we define the patterning of cortical domains, i. e. the emergence of a subapical domain as a novel feature of the midblastula transition whose appearance is controlled by the onset of zygotic transcription but not the nucleocytoplasmic ratio. Our findings will help to gain further understanding of the coordination of complex developmental processes during the midblastula transition.

Altering nuclear import in early Xenopus laevis embryos affects later development

ABSTRACTMore than just a container for DNA, the nucleus carries out a wide variety of critical and highly regulated cellular functions. One of these functions is nuclear import, and in this study we investigate how altering nuclear import impacts developmental progression and organismal size. During early Xenopus laevis embryogenesis, the timing of a key developmental event, the midblastula transition (MBT), is sensitive to nuclear import factor levels. How might altering nuclear import and MBT timing in the early embryo affect downstream development of the organism? We microinjected X.laevis two-cell embryos to increase levels of importin α or NTF2, resulting in differential amounts of nuclear import factors in the two halves of the embryo. Compared to controls, these embryos exhibited delayed gastrulation, curved neural plates, and bent tadpoles with different sized eyes. Furthermore, embryos microinjected with NTF2 developed into smaller froglets compared to control microinjected embryos. We propose that altering nuclear import and size affects MBT timing, cell size, and cell number, subsequently disrupting later development. Thus, altering nuclear import early in development can affect function and size at the organismal level.

Developmentally regulated H2Av buffering via dynamic sequestration to lipid droplets in Drosophila embryos

Regulating nuclear histone balance is essential for survival, yet in early Drosophila melanogaster embryos many regulatory strategies employed in somatic cells are unavailable. Previous work had suggested that lipid droplets (LDs) buffer nuclear accumulation of the histone variant H2Av. Here, we elucidate the buffering mechanism and demonstrate that it is developmentally controlled. Using live imaging, we find that H2Av continuously exchanges between LDs. Our data suggest that the major driving force for H2Av accumulation in nuclei is H2Av abundance in the cytoplasm and that LD binding slows nuclear import kinetically, by limiting this cytoplasmic pool. Nuclear H2Av accumulation is indeed inversely regulated by overall buffering capacity. Histone exchange between LDs abruptly ceases during the midblastula transition, presumably to allow canonical regulatory mechanisms to take over. These findings provide a mechanistic basis for the emerging role of LDs as regulators of protein homeostasis and demonstrate that LDs can control developmental progression.