Biology subjects

Buschow, R.

Publications and source records attributed to Buschow, R..

8 recordsLinked to original sources

bioRxiv2026

Nucleolus transformation and Pol1 activity pattern dual transcriptomes of neuronal subtype identity

Gene expression directs neuronal fate during development with multiple RNA polymerases in the nucleus. mRNA transcribed by Pol2 ultimately depends on the rRNA transcribed by Pol1 in the nucleolus, which generates a ribosome pool to control mRNA translation. How Pol1 activity is synchronized with the Pol2 transcriptome to direct gene expression and neuronal fate is unknown. By creating a high-resolution map of nucleoli in the developing neocortex, we discover that the nucleolus is restructured in neuronal lineages with distinct Pol1 activity. With a new method for rRNA and mRNA dual-transcriptome sequencing in single-cells, we reveal that rRNA transcription is neuronal subtype and stage specific. Rather than a general feature of neural progenitor differentiation, rRNA production peaks transiently in post-mitotic subcortical projection neurons, which have atypical nucleoli containing a single, large cavity. Pol1 inactivation redirects these neurons towards a distinct fate that expresses a hybrid of subcortical and intracortical projection neuron markers. Thus, nucleolar architecture and Pol1 activity pattern dual rRNA-mRNA transcriptome signatures of neuronal subtype identity.

neuroscience↗

bioRxiv2025

An advanced head-to-tail mouse embryo model with hypoxia-mediated neural patterning

The developing mammalian embryo is guided by the continuously changing signals that it receives from maternal tissues and its microenvironment. The dynamic cell-cell and cell-environment interactions that together shape the embryo largely remained unexplorable until the advance of stem cell-based embryo models. These revealed the self-organizing properties of cells in response to endogenous and exogenous cues. Among the latter, restricted oxygen (hypoxia) emerged as a critical microenvironmental regulator that influences cell type diversification in multicellular systems. Here we built a modular ESC-based head-to-tail model of mouse embryogenesis by developing an antero-posterior (AP) assembly strategy under hypoxia. These structures called HAP-gastruloids feature stage-appropriate anterior neural tissues that recapitulate the morphological organization and transcriptional identity of fore- and midbrain including spatial organizer regions such as the midbrain-hindbrain boundary. These anterior tissues develop in synchrony with posterior tissues such as the spinal cord, somites, and gut endoderm derivatives, ultimately yielding a unified structure. We show via genetic, environmental, and pharmacological perturbations that timed hypoxia is essential to boost anterior neural cell identities and their patterning through HIF1a and in part by modulating TGF{beta} activity. These results underline the key beneficial role of hypoxia in early development and offer a uniquely modular system to investigate antero-posterior phenotypes for basic discovery and translation.

developmental biology↗

bioRxiv2022

Discovery and characterization of LNCSOX17 as an essential regulator in human endoderm formation

Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously not annotated lncRNA that is transcribed upstream of the SOX17 gene and located within the same topologically associating domain. We termed it LNCSOX17 and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of LNCSOX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. We identified and characterized LNCSOX17 as an essential new actor in early human endoderm, thereby further expanding the list of functionally important non-coding regulators.

genomics↗

bioRxiv2022

GATA transcription factors drive initial Xist upregulation after fertilization through direct activation of a distal enhancer element

To ensure dosage compensation for X-linked genes between the sexes, one X chromosome is silenced during early embryonic development of female mammals. This process of X-chromosome inactivation (XCI) is initiated through upregulation of the RNA Xist from one X chromosome shortly after fertilization. Xist then mediates chromosome-wide gene silencing in cis and remains expressed in all cell types except the germ line and the pluripotent state, where XCI is reversed. The factors that drive Xist upregulation and thereby initiate XCI remain however unknown. We identify GATA transcription factors as potent Xist activators and demonstrate that they are essential for the activation of Xist in mice following fertilization. Through a pooled CRISPR activation screen we find that GATA1 can drive ectopic Xist expression in murine embryonic stem cells (mESCs). We demonstrate that all GATA factors can activate Xist directly via a GATA-responsive regulatory element (RE79) positioned 100 kb upstream of the Xist promoter. Additionally, GATA factors are essential for the induction of XCI in mouse preimplantation embryos, as simultaneous deletion of three members of the GATA family (GATA1/4/6) in mouse zygotes effectively prevents Xist upregulation. Thus, initiation of XCI and possibly its maintenance in distinct lineages of the preimplantation embryo is ensured by the combined activity of different GATA family members, and the absence of GATA factors in the pluripotent state likely contributes to X reactivation. We thus describe a form of regulation in which the combined action of numerous tissue-specific factors can achieve near-ubiquitous expression of a target gene.

genetics↗

bioRxiv2022

Connecting the Dots: PHF13 and cohesin promote polymer-polymer phase separation of chromatin into chromosomes

How interphase chromatin compacts into mitotic chromosomes has eluded researchers for over a century. Here we show that PHF13, a tightly regulated H3K4me epigenetic reader, and cohesin, a mediator of chromatin architecture, cooperate to drive polymer-polymer-phase-separation (PPPS) and higher order compaction of chromatin into chromosomes. PHF13 interacts with cohesin, shows similar dynamics during mitosis, and their co-depletion dramatically impairs mitotic condensation. Mechanistically, we demonstrate that PHF13 stabilizes cohesin chromatin interactions and that itself oligomerizes, resulting in a polymer with increased chromatin avidity and the ability to bridge neighboring and distant nucleosomes. Consistently, molecular dynamic simulations modelling the ability of PHF13 and cohesin to drive chromatin phase separations recapitulated our in vivo observations and are in line with 2-step condensation models.

cell biology↗

bioRxiv2021

Hypoxia induces a transcriptional early primitive streak signature in pluripotent cells enhancing spontaneous elongation and lineage representation in gastruloids

The cellular microenvironment together with intrinsic regulators shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and appear to benefit from hypoxic culture in vitro. Yet, components of the hypoxia response and how their interplay impacts stem cell transcriptional networks and lineage choices remain poorly understood. Here we investigated the molecular effects of acute and prolonged hypoxia on distinct embryonic and extraembryonic stem cell types as well as the functional impact on differentiation potential. We find a temporal and cell type-specific transcriptional response including an early primitive streak signature in hypoxic embryonic stem (ES) cells. Using a 3D gastruloid differentiation model, we show that hypoxia-induced T expression enables symmetry breaking and axial elongation in the absence of exogenous WNT activation. Importantly, hypoxia also modulates T levels in conventional gastruloids and enhances representation of endodermal and neural markers. Mechanistically, we identify Hif1 as a central factor that mediates the transcriptional response to hypoxia in balance with epigenetic and metabolic rewiring. Our findings directly link the microenvironment to stem cell function and provide a rationale supportive of applying physiological conditions in models of embryo development

developmental biology↗

bioRxiv2021

Glucocorticoid receptor activation induces gene-specific transcriptional memory and universally reversible changes in chromatin accessibility

Glucocorticoids are stress hormones that elicit cellular responses by binding to the glucocorticoid receptor (GR), a ligand-activated transcription factor. The exposure of cells to this hormone induces wide-spread changes in the chromatin landscape and gene expression. Previous studies have suggested that some of these changes are reversible whereas others persist even when the hormone is no longer around. However, when we examined chromatin accessibility in human airway epithelial cells after hormone washout, we found that the hormone-induced changes were universally reversed after one day. Reversibility of hormone-induced changes are found for GR-occupied opening sites and also for closing sites that typically lack GR occupancy. These closing sites are enriched near repressed genes, suggesting that transcriptional repression by GR does not require nearby GR binding. Mirroring what we say in terms of chromatin accessibility, we found that transcriptional responses to hormone are universally reversable. Moreover, priming of cells by a previous exposure to hormone, in general, did not alter the transcriptional response to a subsequent encounter of the same cue. Interestingly, despite the short-lived nature of hormone-induced changes in the chromatin landscape, we identified a single gene, ZBTB16, that displays transcriptional memory manifesting itself as a more robust transcriptional response upon repeated hormone stimulation. Single-cell analysis revealed that the more robust response is driven by a higher probability of primed cells to activate ZBTB16 and by a subset of cells that express the gene at levels that are higher than the induction levels observed for naive cells. Although our study shows that hormone-induced changes are typically reversable, exposure to hormone can induce gene-specific changes in the response to subsequent exposures which may play a role in habituation to stressors and changes in glucocorticoid sensitivity.

genomics↗

bioRxiv2020

Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites

Post-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes inaccessible to deep analysis in vivo. Mouse embryonic stem cells (mESCs) can form aggregates reflecting the post-occipital embryo (gastruloids), but lacking proper morphogenesis. Here we show that embedding of aggregates derived from mESCs in an extracellular matrix compound results in Trunk-Like-Structures (TLS) with a high level of organization comprising a neural tube and somites. Comparative single-cell RNA-seq analysis demonstrates that TLS execute gene-regulatory programs in an embryo-like order, and generate primordial germ cell like cells (PGCLCs). TLS lacking Tbx6 form ectopic neural tubes, mirroring the embryonic mutant phenotype. ESC-derived trunk-like structures thus constitute a novel powerful in vitro platform for investigating lineage decisions and morphogenetic processes shaping the post-implantation embryo. One sentence summaryA platform for generating trunk-like-structures with precursors of spinal cord, bone and muscle from stem cells in a dish

developmental biology↗