Controlling organoid symmetry breaking uncovers an excitable system underlying human axial elongation

Anand, Giridhar M.; Megale, Heitor C.; Murphy, Sean H.; Weis, Theresa; Lin, Zuwan; He, Yichun; Wang, Xiao; Liu, Jia; Ramanathan, Sharad

doi:10.1016/j.cell.2022.12.043

Cited by 34 publications

(20 citation statements)

References 87 publications

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“…FGFR inhibition resulted in the most profound defects, with a ~40% reduction in major axis length and complete absence of Bra GFP enrichment in the posterior domain, consistent with the loss of Bra expression in the primitive streak of Fgfr1 -/-embryos 23 ( Figure 2B ). A similar loss in Bra GFP expression was observed in response to MEK inhibitor treatment, with a partial reduction in gastruloid length that is consistent with prior reports 11,17,18,24 ( Figure 2B ). In contrast, PI3K and Akt inhibition resulted in smaller gastruloids with a shorter major axis, yet had no impact on the posterior expression of Bra GFP ( Figure 2B ).…”

Section: Resultssupporting

confidence: 91%

“…Two canonical FGFR effector pathways, the Ras/Erk and PI3K/Akt signaling cascades, also exhibit graded activity along the anteroposterior axis of the late-gastrulation mouse embryo 12,14 . Ras/Erk signaling is a key regulator of somite formation in vertebrate species 15,16 , and has been shown in gastruloids to regulate the speed of the determination front 10,17 and the maintenance of an axial progenitor population 18 . The role of PI3K/Akt signaling during vertebrate elongation is comparatively understudied: while PI3K/Akt signaling has been implicated in the migration of mesodermal cells during chick and zebrafish gastrulation 19,20 , similar experiments have not been reported in mouse models 21 .…”

Section: Introductionmentioning

confidence: 99%

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Control of gastruloid patterning and morphogenesis by the Erk and Akt signaling pathways

Underhill

Toettcher

2023

Preprint

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Fibroblast growth factor (FGF) dependent elongation along an anterior-posterior (A-P) axis is a conserved feature of vertebrate embryogenesis. A-P axis elongation can also be reproduced in 3D cell culture models termed gastruloids, enabling dissection of this process in a controlled, minimal context. Here, we set out to determine how gastruloid posterior elongation depends on the Erk and Akt pathways, canonical downstream effectors of FGF signaling. We find that gastruloids exhibit reproducible posterior-to-anterior gradients in Erk and Akt phosphorylation that are generated independently and correlate with distinct zones of tissue movement, cell proliferation, and expression of cell motility and adhesion regulators. Pharmacological inhibition of FGFR, Erk, or Akt signaling impairs gastruloid elongation, and quantification of signaling gradients reveals how these patterns interact and scale with A-P axis length. Using global inhibitors and activators of each pathway, we find that a gradient of Ras/Erk signaling is required for the establishment of appropriately localized domains of E-cadherin, Snail, and Brachyury expression, whereas perturbing PI3K/Akt signaling alters proliferation but not patterning. Taken together, our data demonstrate that graded PI3K/Akt and Ras/Erk signaling provide spatial information to control proliferation and cell-cell adhesion during gastruloid elongation.

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Section: Resultssupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Control of gastruloid patterning and morphogenesis by the Erk and Akt signaling pathways

Underhill

Toettcher

2023

Preprint

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“…Wnt3 , Wnt3a, Fgf8 ), and their downstream targets ( Figure 6C,E ). Notably, the expression of secreted WNT inhibitors Sfrp1/2 - knockdown of which leads to a multipolar phenotype 36 - decreased with higher T mCH levels ( Figure 6E ).…”

Section: Resultsmentioning

confidence: 99%

Integrated Molecular-Phenotypic Profiling Reveals Metabolic Control of Morphological Variation in Stembryos

Villaronga Luque,

Savill,

López-Anguita

et al. 2023

Preprint

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SUMMARYMammalian stem-cell-based models of embryo development (stembryos) hold great promise in basic and applied research. However, considerable phenotypic variation despite identical culture conditions limits their potential. The biological processes underlying this seemingly stochastic variation are poorly understood. Here, we investigate the roots of this phenotypic variation by intersecting transcriptomic states and morphological history of individual stembryos across stages modeling post-implantation and early organogenesis. Through machine learning and integration of time-resolved single-cell RNA-sequencing with imaging-based quantitative phenotypic profiling, we identify early features predictive of the phenotypic end-state. Leveraging this predictive power revealed that early imbalance of oxidative phosphorylation and glycolysis results in aberrant morphology and a neural lineage bias that can be corrected by metabolic interventions. Collectively, our work establishes divergent metabolic states as drivers of phenotypic variation, and offers a broadly applicable framework to chart and predict phenotypic variation in organoid systems. The strategy can be leveraged to identify and control underlying biological processes, ultimately increasing the reproducibility of in vitro systems.HighlightsTime-resolved single-cell RNA-sequencing and imaging-based quantitative charting of hundreds of individual stembryos generates molecular and phenotypic fingerprintsMachine learning and integration of molecular and phenotypic fingerprints identifies features and biological processes predictive of phenotypic end-stateEarly imbalance of oxidative phosphorylation and glycolysis results in aberrant morphology and cellular compositionMetabolic interventions tune stembryo end-state and can correct derailment of differentiation outcomes

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“…These genes represent the defining characteristics of NMPs and the neuronally committed posterior progenitors that are derived from NMPs. 12,32,33 As a first step, we aimed to identify the temporal point during human ES cell differentiation when the activation of Wnt/β-catenin promotes NMP markers without yet inducing somitogenesis and mesendoderm genes. We analyzed RNA-seq data where the Wnt/β-catenin pathway was activated via CHIR99021 treatment and by employing human ES cells harbouring stably integrated β-catenin ΔN90 variant that is responsive to doxycycline.…”

Section: Resultsmentioning

confidence: 99%

Capture of Human Neuromesodermal and Posterior Neural Tube Axial Stem Cells

Kelle,

Ugur,

Rusha

et al. 2024

Preprint

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The spinal cord, nerves, and skeletal muscles arise from neuromesodermal progenitors (NMPs). We have developed a growth-factor screening strategy, utilizing ES and iPS cells, facilitating the indefinite self-renewal of two types of human axial stem cells (AxSCs), closely resembling mouse NMPs (NM-AxSCs) and posterior neural tube progenitors (N-AxSCs). Under specific regimens—Wnt/CHIR99021, FGF2, and TGF-β inhibitor SB431542 (CFS) and excluding FGF2 (CS), respectively—these AxSCs self-renew and sustain telomeres. Single cell transcriptomics and proteomics have revealed expression of posterior growth-zone and dorsoventral neural tube markers in NM-AxSCs, and correspondingly, differentiation to a wide spectrum of neural tube neurons and myocytes. N-AxSCs rapidly matured into dorsal sensory subsets and neural crest. Crucially, neither AxSC type produces teratomas, and analogous mouse NM-AxSCs integrated successfully into the neural tube and somites. Capturing of AxSCs from patient and GMP ES / iPS cells without transgenesis unveils ontogeny and promises modeling and therapy in neuropathies.

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Controlling organoid symmetry breaking uncovers an excitable system underlying human axial elongation

Cited by 34 publications

References 87 publications

Control of gastruloid patterning and morphogenesis by the Erk and Akt signaling pathways

Control of gastruloid patterning and morphogenesis by the Erk and Akt signaling pathways

Integrated Molecular-Phenotypic Profiling Reveals Metabolic Control of Morphological Variation in Stembryos

Capture of Human Neuromesodermal and Posterior Neural Tube Axial Stem Cells

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