Biology subjects

Holtackers, R.

Publications and source records attributed to Holtackers, R..

3 recordsLinked to original sources

bioRxiv2023

Multiscale chromatin dynamics and high entropy in plant iPSC cultures

Plant protoplasts constitute the starting material to induce pluripotent cell masses in vitro competent for tissue regeneration. Dedifferentiation is associated with large-scale chromatin reorganisation and massive transcriptome reprogramming, characterized by stochastic gene expression. How this cellular variability reflects on chromatin organisation in individual cells and what are the factors influencing chromatin transitions during culturing is largely unknown. High-throughput imaging and a custom, supervised image analysis protocol extracting over 100 chromatin features unravelled a rapid, multiscale dynamics of chromatin patterns which trajectory strongly depends on nutrients availability. Decreased abundance in H1 (linker histones) is hallmark of chromatin transitions. We measured a high heterogeneity of chromatin patterns indicating an intrinsic entropy as hallmark of the initial cultures. We further measured an entropy decline over time, and an antagonistic influence by external and intrinsic factors, such as phytohormones and epigenetic modifiers, respectively. Collectively, our study benchmarks an approach to understand the variability and evolution of chromatin patterns underlying plant cell reprogramming in vitro.

molecular biology↗

bioRxiv2023

Morphodynamics of human early brain organoid development

Brain organoids enable the mechanistic study of human brain development, and provide opportunities to explore self-organization in unconstrained developmental systems. Here, we establish long-term, live light sheet microscopy on unguided brain organoids generated from fluorescently labeled human induced pluripotent stem cells, which enables tracking of tissue morphology, cell behaviors, and subcellular features over weeks of organoid development. We provide a novel dual-channel, multi-mosaic and multi-protein labeling strategy combined with a computational demultiplexing approach to enable simultaneous quantification of distinct subcellular features during organoid development. We track Actin, Tubulin, plasma membrane, nucleus, and nuclear envelope dynamics, and quantify cell morphometric and alignment changes during tissue state transitions including neuroepithelial induction, maturation, lumenization, and brain regionalization. Based on imaging and single-cell transcriptome modalities, we find that lumenal expansion and cell morphotype composition within the developing neuroepithelium are associated with modulation of gene expression programs involving extracellular matrix (ECM) pathway regulators and mechanosensing. We show that an extrinsically provided matrix enhances lumen expansion as well as telencephalon formation, and unguided organoids grown in the absence of an extrinsic matrix have altered morphologies with increased neural crest and caudalized tissue identity. Matrixinduced regional guidance and lumen morphogenesis are linked to the WNT and Hippo (YAP1) signaling pathways, including spatially restricted induction of the Wnt Ligand Secretion Mediator (WLS) that marks the earliest emergence of nontelencephalic brain regions. Altogether, our work provides a new inroad into studying human brain morphodynamics, and supports a view that matrix-linked mechanosensing dynamics play a central role during brain regionalization.

developmental biology↗

bioRxiv2021

Maturation of human intestinal epithelium from pluripotency in vitro

Methods to generate human intestinal tissue from pluripotent stem cells (PSCs) open new inroads into modeling intestine development and disease. However, current protocols require organoid transplantation into an immunocompromised mouse to achieve matured and differentiated epithelial cell states. Inspired by developmental reconstructions from primary tissues, we establish a regimen of inductive cues that enable stem cell maturation and epithelial differentiation entirely in vitro. We show that the niche factor Neuregulin1 (NRG1) promotes morphological change from proliferative epithelial cysts to matured epithelial tissue in three-dimensional cultures. Single-cell transcriptome analyses reveal differentiated epithelial cell populations, including diverse secretory and absorptive lineages. Comparison to multi-organ developmental and adult intestinal cell atlases confirm the specificity and maturation state of cell populations. Altogether, this work opens a new direction to use in vitro matured epithelium from human PSCs to study human intestinal epithelium development, disease, and evolution in controlled culture environments.

cell biology↗