Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice

Serup, Palle; Gustavsen, Carsten R.; Klein, Tino; Potter, Leah A.; Lin, Robert; Mullapudi, Nandita; Wandzioch, Ewa; Hines, Angela; Davis, Ashley; Bruun, Christine; Engberg, Nina; Petersen, Dorthe R.; Peterslund, Janny M.L.; MacDonald, Raymond J.; Grapin-Botton, Anne; Magnuson, Mark A.; Zaret, Kenneth S.

doi:10.1242/dmm.009696

Cited by 21 publications

(27 citation statements)

References 44 publications

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“…The observation that EBs can form multiple contact loci or an entire cap contact locus when confined in micropools raised the question whether the potential to express Bry is limited to specific areas on the outer layer of the EB, to the entire shell, or is uniform throughout the EB volume. Growing EBs in hanging drops, we first confirmed that a canonical Wnt activation reporter (SuTOP-CFP, [21]) showed co-localization with Bry, in agreement with observations that Bry is activated via the canonical Wnt pathway ( Fig. 2A) [9,22].…”

Section: Brachyury Competent Shell Is Formed Prior To Onsetsupporting

confidence: 87%

Prediction and control of symmetry breaking in embryoid bodies by environment and signal integration

Sagy

Slovin

Allalouf

et al. 2018

Preprint

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During early embryogenesis, mechanical signals, localized biochemical signals and neighboring cell layers interaction coordinate around anteroposterior axis determination and symmetry breaking. Deciphering their relative roles, which are hard to tease apart in vivo, will enhance our understanding of how these processes are driven. In recent years, in vitro 3D models of early mammalian development, such as embryoid bodies (EBs) and gastruloids, were successful in mimicking various aspects of the early embryo, providing high throughput accessible systems for studying the basic rules shaping cell fate and morphology during embryogenesis. Using Brachyury (Bry), a primitive streak and mesendoderm marker in EBs, we study how contact, biochemical and neighboring cell cues affect the positioning of a primitive streak-like locus, determining the AP axis. We show that a Bry-competent layer must be formed in the EB before Bry expression initiates, and that Bry onset locus selection depends on contact points of the EB with its surrounding. We can maneuver Bry onset to occur at a specific locus, a few loci, or in an isotropic peripheral pattern. By spatially separating contact and biochemical signal sources, we show these two modalities can be integrated by the EB to generate a single Bry locus. Finally, we show Foxa2+ cells are predictive of the future location of Bry onset, demonstrating an earlier symmetry-breaking event. By delineating the temporal signaling pathway dependencies of Bry and Foxa2, we were able to selectively abolish either, or spatially decouple the two cell types during EB differentiation. These findings demonstrate multiple inputs integration during an early developmental process, and may prove valuable in directing in vitro differentiation.

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Section: Brachyury Competent Shell Is Formed Prior To Onsetsupporting

confidence: 87%

Prediction and control of symmetry breaking in embryoid bodies by environment and signal integration

Sagy

Slovin

Allalouf

et al. 2018

Preprint

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“…) (Srinivas et al, 2001), Gt(ROSA)26Sor tm2Mgn (ROSA26 mCherry ) , ROSA26 228.3TF [unpublished, contains a ʻ−228' deletion of the ROSA26 promoter similar to that described by Serup et al (2012)] and Setd5…”

Section: Lslyfpmentioning

confidence: 96%

Setd5 is essential for mammalian development and the co-transcriptional regulation of histone acetylation

et al. 2016

Self Cite

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SET domain-containing proteins play a vital role in regulating gene expression during development through modifications in chromatin structure. Here we show that SET domain-containing 5 (Setd5) is divergently transcribed with Gt(ROSA26)Sor, is necessary for mammalian development, and interacts with the PAF1 co-transcriptional complex and other proteins. Setd5-deficient mouse embryos exhibit severe defects in neural tube formation, somitogenesis and cardiac development, have aberrant vasculogenesis in embryos, yolk sacs and placentas, and die between embryonic day 10.5 and 11.5. Setd5-deficient embryonic stem cells have impaired cellular proliferation, increased apoptosis, defective cell cycle progression, a diminished ability to differentiate into cardiomyocytes and greatly perturbed gene expression. SETD5 co-immunoprecipitates with multiple components of the PAF1 and histone deacetylase-containing NCoR complexes and is not solely required for major histone lysine methylation marks. In the absence of Setd5, histone acetylation is increased at transcription start sites and near downstream regions. These findings suggest that SETD5 functions in a manner similar to yeast Set3p and Drosophila UpSET, and that it is essential for regulating histone acetylation during gene transcription.

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“…Please click here to view a larger version of this figure. 19,22,27,30,[36][37][38][39][40] have been characterized for the formation of aggregates and, although subtle differences between cell lines are expected and have been observed, all the above lines show similar dynamics in terms of elongation and morphology. In terms of gene expression, the expression pattern is specific to the gene expressed, but within each cell line, the expression pattern is generally consistent over a number of passages.…”

Section: Aggregate Imaging and Quantitative Image Analysismentioning

confidence: 99%

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour <em>In Vitro</em>

Baillie-Johnson

Brink

Balayo

et al. 2015

JoVE

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We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/β-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

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Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice

Cited by 21 publications

References 44 publications

Prediction and control of symmetry breaking in embryoid bodies by environment and signal integration

Prediction and control of symmetry breaking in embryoid bodies by environment and signal integration

Setd5 is essential for mammalian development and the co-transcriptional regulation of histone acetylation

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour <em>In Vitro</em>

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