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1.
Nat Methods ; 21(5): 798-803, 2024 May.
Article in English | MEDLINE | ID: mdl-38509326

ABSTRACT

Multicellular systems grow over the course of weeks from single cells to tissues or even full organisms, making live imaging challenging. To bridge spatiotemporal scales, we present an open-top dual-view and dual-illumination light-sheet microscope dedicated to live imaging of large specimens at single-cell resolution. The configuration of objectives together with a customizable multiwell mounting system combines dual view with high-throughput multiposition imaging. We use this microscope to image a wide variety of samples and highlight its capabilities to gain quantitative single-cell information in large specimens such as mature intestinal organoids and gastruloids.


Subject(s)
Organoids , Animals , Organoids/cytology , Humans , Single-Cell Analysis/methods , Microscopy/methods , Microscopy/instrumentation , Mice , Microscopy, Fluorescence/methods , Microscopy, Fluorescence/instrumentation
2.
Cell Stem Cell ; 30(6): 867-884.e11, 2023 06 01.
Article in English | MEDLINE | ID: mdl-37209681

ABSTRACT

Gastruloids are 3D structures generated from pluripotent stem cells recapitulating fundamental principles of embryonic pattern formation. Using single-cell genomic analysis, we provide a resource mapping cell states and types during gastruloid development and compare them with the in vivo embryo. We developed a high-throughput handling and imaging pipeline to spatially monitor symmetry breaking during gastruloid development and report an early spatial variability in pluripotency determining a binary response to Wnt activation. Although cells in the gastruloid-core revert to pluripotency, peripheral cells become primitive streak-like. These two populations subsequently break radial symmetry and initiate axial elongation. By performing a compound screen, perturbing thousands of gastruloids, we derive a phenotypic landscape and infer networks of genetic interactions. Finally, using a dual Wnt modulation, we improve the formation of anterior structures in the existing gastruloid model. This work provides a resource to understand how gastruloids develop and generate complex patterns in vitro.


Subject(s)
Embryo, Mammalian , Pluripotent Stem Cells , Mice , Animals , Embryo, Mammalian/metabolism , Primitive Streak/metabolism , Embryonic Development
3.
Nat Commun ; 13(1): 6133, 2022 10 17.
Article in English | MEDLINE | ID: mdl-36253406

ABSTRACT

Protein phosphorylation is a major regulatory mechanism of cellular signalling. The c-JUN proto-oncoprotein is phosphorylated at four residues within its transactivation domain (TAD) by the JNK family kinases, but the functional significance of c-JUN multisite phosphorylation has remained elusive. Here we show that c-JUN phosphorylation by JNK exhibits defined temporal kinetics, with serine63 and serine73 being phosphorylated more rapidly than threonine91 and threonine93. We identify the positioning of the phosphorylation sites relative to the kinase docking motif, and their primary sequence, as the main factors controlling phosphorylation kinetics. Functional analysis reveals three c-JUN phosphorylation states: unphosphorylated c-JUN recruits the MBD3 repressor, serine63/73 doubly-phosphorylated c-JUN binds to the TCF4 co-activator, whereas the fully phosphorylated form disfavours TCF4 binding attenuating JNK signalling. Thus, c-JUN phosphorylation encodes multiple functional states that drive a complex signalling response from a single JNK input.


Subject(s)
JNK Mitogen-Activated Protein Kinases , Proto-Oncogene Proteins c-jun , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Kinase 4/metabolism , MAP Kinase Signaling System , Phosphorylation , Proto-Oncogene Proteins c-jun/metabolism , Signal Transduction
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