R-loop mapping and characterization during Drosophila embryogenesis reveals developmental plasticity in R-loop signatures

R-loops are involved in transcriptional regulation, DNA and histone post-translational modifications, genome replication and genome stability. To what extent R-loop abundance and genome-wide localization is actively regulated during metazoan embryogenesis is unknown. The developmental program of Drosophila embryogenesis provides a powerful system to study address these questions due to its well-characterized developmental program, the sudden onset of zygotic transcription and available genome-wide ChIP and transcription data sets. Here, we measure the overall abundance and genome localization of R-loops in early and late-stage embryos relative to Drosophila cultured cells. We demonstrate that proper absolute R-loop levels change during embryogenesis and that resolution of R-loops is critical for embryonic development. R-loop mapping by strand-specific DRIP-seq revealed that R-loop localization is plastic across development, both in the genes which form R-loops and where they localize relative to gene bodies. Importantly, these changes are not driven by changes in the transcriptional program. Rather, negative GC skew and absolute changes in AT skew are associated with R-loop formation in Drosophila. Furthermore, we demonstrate that while some chromatin binding proteins and histone modification such as H3K27me3 are associated with R-loops throughout development, other chromatin factors associated with R-loops in a developmental specific manner. Our findings highlight the importance and developmental plasticity of R-loops during Drosophila development.

Multiview tiling light sheet microscopy for 3D high resolution live imaging

Light sheet or selective plane illumination microscopy (SPIM) is ideally suited for in toto imaging of living specimens at high temporal-spatial resolution. In SPIM, the light scattering that occurs during imaging of opaque specimens brings about limitations in terms of resolution and the imaging field of view. To ameliorate this shortcoming, the illumination beam can be engineered into a highly confined light sheet over a large field of view and multi-view imaging can be performed by applying multiple lenses combined with mechanical rotation of the sample. Here, we present a Multiview tiling SPIM (MT-SPIM) that combines the Multi-view SPIM (M-SPIM) with a confined, multi-tiled light sheet. The MT-SPIM provides high-resolution, robust and rotation-free imaging of living specimens. We applied the MT-SPIM to image nuclei and Myosin II from the cellular to subcellular spatial scale in early Drosophila embryogenesis. We show that the MT-SPIM improves the axial-resolution relative to the conventional M-SPIM by a factor of two. We further demonstrate that this axial resolution enhancement improves the automated segmentation of Myosin II distribution and of nuclear volumes and shapes.

Multiview tiling light sheet microscopy for 3D high resolution live imaging

Light sheet or selective plane illumination microscopy (SPIM) is ideally suited for in toto imaging of living specimens at high temporal-spatial resolution. In SPIM, the light scattering that occurs during imaging of opaque specimens brings about limitations in terms of resolution and the imaging field of view. To ameliorate this shortcoming, the illumination beam can be engineered into a highly confined light sheet over a large field of view and multi-view imaging can be performed by applying multiple lenses combined with mechanical rotation of the sample. Here, we present a Multiview tiling SPIM (MT-SPIM) that combines the Multi-view SPIM (M-SPIM) with a confined, multi-tiled light sheet. The MT-SPIM provides high-resolution, robust and rotation-free imaging of living specimens. We applied the MT-SPIM to image nuclei and Myosin II from the cellular to subcellular spatial scale in early Drosophila embryogenesis. We show that the MT-SPIM improves the axial-resolution relative to the conventional M-SPIM by a factor of two. We further demonstrate that this axial resolution enhancement improves the automated segmentation of Myosin II distribution and of nuclear volumes and shapes.

Mitochondrial morphology and activity regulate furrow ingression and contractile ring dynamics in Drosophila cellularization

Drp1-regulated mitochondrial fission is essential for mitochondrial distribution across the cell in cellularization during Drosophila embryogenesis. Loss of mitochondrial fission in Drp1 mutant embryos leads to defects in morphogenetic events of cell formation and contractile ring constriction in cellularization.

The dynamic transmission of positional information in stau- mutants during Drosophila embryogenesis

It has been suggested that Staufen (Stau) is key in controlling the variability of the posterior boundary of the Hb anterior domain (xHb). However, the mechanism that underlies this control is elusive. Here, we quantified the dynamic 3D expression of segmentation genes in Drosophila embryos. With improved control of measurement errors, we show that the xHb of stau– mutants reproducibly moves posteriorly by 10% of the embryo length (EL) to the wild type (WT) position in the nuclear cycle (nc) 14, and that its variability over short time windows is comparable to that of the WT. Moreover, for stau– mutants, the upstream Bicoid (Bcd) gradients show equivalent relative intensity noise to that of the WT in nc12–nc14, and the downstream Even-skipped (Eve) and cephalic furrow (CF) show the same positional errors as these factors in WT. Our results indicate that threshold-dependent activation and self-organized filtering are not mutually exclusive and could both be implemented in early Drosophila embryogenesis.

In response to Li et al.: Linker histones function in Drosophila embryogenesis

ABSTRACTIn an earlier paper (Pérez-Montero et al., 2013), we reported that the embryonic linker histone of Drosophila dBigH1 was essential for early Drosophila embryogenesis since embryos homozygous for the bigH1100 mutation showed strong defects and did not survive beyond zygotic genome activation (ZGA) at cellularization. Recent results challenge these observations since null bigH1 mutations generated by CRISPR/Cas9 methodology turn out to be homozygous viable, as reported in Li et al. (2019) and here. In this regard, Li et al. described a novel mechanism by which lack of dBigH1 is compensated by the early expression of maternal dH1. Here, we confirm this observation and show that such compensatory mechanism is not activated in bigH1100 embryos.