Biology subjects

Vastenhouw, N. L.

Publications and source records attributed to Vastenhouw, N. L..

7 recordsLinked to original sources

bioRxiv2025

Multiplexed embryo profiling links cellular state to zygotic genome activation in single cells

Multicellular self-organization relies on complex interactions across multiple length scales, shaping the transcriptional activity and molecular phenotype of individual cells. Despite advances in spatial omics technologies, mapping protein state composition at high spatial resolution to capture the molecular phenotype across whole-mount structures has remained challenging. Here, we introduce high-throughput 3D iterative indirect immunofluorescence imaging (3D-4i) for the simultaneous detection of multiple proteins and protein states across the entire embryo, and a 3D-dedicated computer vision pipeline for quantifying morphological and molecular properties across subcellular, single-cell, and whole-embryo scales in hundreds of samples. Applying this pipeline to early zebrafish embryos undergoing mid-blastula transition, we determine the cell cycle phase for each cell across the embryo, allowing us to uncover the spatiotemporal dynamics by which global meta-synchronous mitotic waves transition to cell cycle desynchronization. Using statistical analysis, we find that cell cycle phase is the major source of variability in transcription within a division cycle, and combining this with analysis of key transcription factors and chromatin modifier state enables accurate prediction of transcriptional output during zygotic genome activation in individual cells. Collectively, our results demonstrate the strength of 3D-4i in quantifying multimodal effects across spatiotemporal scales and show how it can be utilized to unravel the complex contribution of causal factors that collectively drive multicellular self-organization.

systems biology↗

bioRxiv2024

Specificity in transcription factor clustering is encoded in the genome

Transcription factors (TFs) often form clusters in the nucleus. Clusters can facilitate transcription, but it remains unclear how they form. It has been suggested that clusters are seeded by the sequence-specific binding of TFs to DNA, and grow by IDR-IDR interactions that bring in more TFs. This model, however, does not explain how TFs can cluster in specific combinations. Here, we study TF clustering by quantitative imaging of Nanog, Pou5f3, and Sox19b in zebrafish embryos. Using mutant and chimaeric TFs, we show that the formation of a TF cluster requires the DBD as well as at least one of its IDRs. In contrast with the existing model, however, IDRs are not sufficient to join a pre-existing cluster. Instead, both IDR and DBD are needed. Thus, for any TF to join a cluster, motif recognition is required, which explains the specificity in cluster formation. Finally, we show that while IDRs are required to join a cluster, their amino acid sequence is interchangeable, and the DBD can confer specificity to any IDR. Taken together, our work changes the model of cluster formation and explains how specificity is achieved in the organization of transcriptional machinery in the nucleus.

cell biology↗

bioRxiv2024

Local DNA compaction creates TF-DNA clusters that enable transcription

Transcription factor (TF) clusters have been suggested to facilitate transcription. The mechanisms driving the formation of TF clusters and their impact on transcription, however, remain largely unclear. This is mostly due to the lack of a tractable system. Here, we exploit the transcriptional activation of mir430 in zebrafish embryos to simultaneously follow the dynamic formation of a large Nanog cluster, the underlying DNA, and transcription output by live imaging at high temporal and spatial resolution. We find that the formation of a Nanog cluster that can support transcription requires local DNA compaction. This brings more Nanog-binding sites into the cluster, and therefore more Nanog. Importantly, we find that Nanog stabilizes this TF-DNA cluster, which emphasizes the interdependent relationship between TFs and DNA dynamics in cluster formation. Once the Nanog-DNA cluster at the mir430 locus reaches a maximum amount of Nanog, transcription begins. This maximum is a locus-intrinsic feature, which shows that the locus self-regulates the recruitment of an optimal amount of Nanog. Our study supports a model in which endogenous TF clusters positively impact transcription and form through a combination of DNA binding and local DNA compaction.

cell biology↗

bioRxiv2022

Targeted disruption of transcription bodies causes widespread activation of transcription

The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if and how they affect gene expression. Here, we disrupted the formation of two prominent endogenous transcription bodies that mark the onset of zygotic transcription in zebrafish embryos and analysed the effect on gene expression using enriched SLAM-Seq and live-cell imaging. We find that the disruption of transcription bodies results in downregulation of hundreds of genes, providing experimental support for a model in which transcription bodies increase the efficiency of transcription. We also find that a significant number of genes are upregulated, counter to the suggested stimulatory effect of transcription bodies. These upregulated genes have accessible chromatin and are poised to be transcribed in the presence of the two transcription bodies, but they do not go into elongation. Live-cell imaging shows that the disruption of the two large transcription bodies enables these poised genes to be transcribed in ectopic transcription bodies, suggesting that the large transcription bodies sequester a pause release factor. Supporting this hypothesis, we find that CDK9, the kinase that releases paused polymerase II, is highly enriched in the two large transcription bodies. Importantly, overexpression of CDK9 in wild type embryos results in the formation of ectopic transcription bodies and thus phenocopies the removal of the two large transcription bodies. Taken together, our results show that transcription bodies regulate transcription genome-wide: the accumulation of transcriptional machinery creates a favourable environment for transcription locally, while depriving genes elsewhere in the nucleus from the same machinery.

cell biology↗

bioRxiv2022

Actin filaments accumulate in the nucleus during interphase and remain in the vicinity of condensing chromosomes during prophase to metaphase in zebrafish early embryos

In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies have shown that actin filaments that form in the nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated the dynamics of nuclear actin in zebrafish (Danio rerio) embryos. In early zebrafish embryos up to around the high stage, UtrCH-sfGFP increasingly accumulated in nuclei during the interphase and reached a peak during the prophase. After nuclear envelope breakdown (NEBD), patches of UtrCH-sfGFP remained in the vicinity of condensing chromosomes during the prometaphase to metaphase. When zygotic transcription was inhibited by injecting -amanitin, the nuclear accumulation of UtrCH-sfGFP was still observed at the sphere and dome stages, suggesting that zygotic transcription may induce a decrease in nuclear F-actin. The accumulation of F-actin in nuclei may contribute to proper mitotic progression of large cells with rapid cell cycles in zebrafish early embryos, by assisting in NEBD, chromosome congression, and/or spindle assembly. Summary statementFilamentous actin accumulates in the nucleus of zebrafish early embryos and forms patches associating with condensing chromosomes during prophase.

cell biology↗

bioRxiv2019

Chromatin accessibility established by Pou5f3, Sox19b and Nanog primes genes for activity during zebrafish genome activation

In many organisms, early embryonic development is driven by maternally provided factors until the controlled onset of transcription during zygotic genome activation. The regulation of chromatin accessibility and its relationship to gene activity during this transition remains poorly understood. Here, we generated chromatin accessibility maps from genome activation until the onset of lineage specification. During this period, chromatin accessibility increases at regulatory elements. This increase is independent of RNA polymerase II-mediated transcription, with the exception of the hyper-transcribed miR-430 locus. Instead, accessibility often precedes the transcription of associated genes. Loss of the maternal transcription factors Pou5f3, Sox19b, and Nanog, which are known to be required for zebrafish genome activation, results in decreased accessibility at regulatory elements. Importantly, the accessibility of regulatory regions, especially when established by Pou5f3, Sox19b and Nanog, is predictive for future transcription. Our results show that the maternally provided transcription factors Pou5f3, Sox19b, and Nanog open up chromatin and prime genes for activity during zygotic genome activation in zebrafish.

developmental biology↗

bioRxiv2017

MS Western, a method of multiplexed absolute protein quantification is a practical alternative to western blotting

Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the sub-femtomole level in whole cell or tissue lysates without metabolic or chemical labelling and without using specific antibodies. MS Western relies upon GeLC-MS/MS and quantifies proteins by in-gel co-digestion with an isotopically labelled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance.

biochemistry↗