Nodal is a short-range morphogen with activity that spreads through a relay mechanism in human gastruloids

Liu, Lizhong; Nemashkalo, Anastasiia; Jung, Jongwan; Chhabra, Sapna; Heemserk, I; Warmflash, Aryeh

doi:10.1101/2021.04.14.439902

Cited by 6 publications

(6 citation statements)

References 27 publications

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“…Our new direct single-molecule measurements and simulations suggest a molecular mechanism of long-range morphogen spreading in a tortuous environment, where diffusion predominantly occurs in a network of extracellular cavities. This mechanism differs from the morphogen dispersal mode recently described in a two-dimensional human embryonic stem cell culture system, in which – unlike in the three-dimensional embryo context – Nodal molecules presumably cannot be retained in extracellular cavities and are instead lost into the culturing medium 66 .…”

Section: Discussionmentioning

confidence: 64%

Single-molecule tracking of Nodal and Lefty in live zebrafish embryos supports hindered diffusion model

Kühn

Landge

Mörsdorf

et al. 2022

Preprint

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The influential hindered diffusion model postulates that the global movement of a signaling molecule through an embryo is affected by local tissue geometry and binding-mediated hindrance, but these effects have not been directly demonstratedin vivofor any signaling molecule. Nodal and Lefty are a prime example of an activator-inhibitor signaling pair whose different global diffusivities are thought to arise from differential hindrance. Here, we used single-molecule tracking of Nodal and Lefty to directly probe the tenets of the hindered diffusion model on the nanoscale. We visualized individual fluorescently-tagged Nodal and Lefty molecules in developing zebrafish embryos using reflected light-sheet microscopy. Single-particle tracking revealed molecules in three states: molecules diffusing in extracellular cavities, molecules diffusing within cell-cell interfaces, and molecules bound to cell membranes. While the diffusion coefficients of molecules were high in extracellular cavities, mobility was reduced and bound fractions were higher within cell-cell interfaces; counterintuitively, molecules nevertheless accumulated in cavities. Using agent-based simulations, we identified the geometry of the extracellular space as a key factor influencing the accumulation of molecules in cavities. For Nodal, the fraction of molecules in the bound state was larger than for Lefty, and individual Nodal molecules had binding times of tens of seconds. Together, our single-molecule measurements and simulations provide direct support for the hindered diffusion model in a developing embryo and yield unprecedented insights into the nanometer to micrometer scale transport mechanisms that together lead to macroscopic signal dispersal and gradient formation.

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Section: Discussionmentioning

confidence: 64%

Single-molecule tracking of Nodal and Lefty in live zebrafish embryos supports hindered diffusion model

Kühn

Landge

Mörsdorf

et al. 2022

Preprint

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“…4f-g). Non-phosphorylated Smad2 is evenly distributed throughout the micropattern in line with its bifunctional roles in the regulation of both self-renewal and differentiation [50][51][52] (Fig. 4d and f, Supplementary Fig.…”

Section: Single Cell Mrna and (Phospho-)protein Expression Profiles D...mentioning

confidence: 67%

“…17a). Furthermore, both phospho-Smad2 and Smad2 mRNA--i.e., the intracellular effector of NODAL pathway [50][51][52] --are more highly expressed at the edge of the micropattern (Fig. 4d and Supplementary Fig.…”

Section: Single Cell Mrna and (Phospho-)protein Expression Profiles D...mentioning

confidence: 99%

ARTseq-FISH reveals position-dependent fate decisions driven by cell cycle changes

Sluijs¹,

Blay²,

Stepanov³

et al. 2022

Preprint

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Cell fate decisions are ubiquitous and play a critical role throughout development, yet how localization impacts cellular decision making remains unclear. To identify the drivers of position-dependent fate decisions at a molecular level, we developed a scalable antibody and mRNA targeting sequential fluorescence in situ hybridization (ARTseq-FISH) method capable of simultaneously profiling mRNAs, proteins and phosphoproteins in single cells at sub-micrometre spatial resolution. We studied 67 unique (phospho-)protein and mRNA targets in individual mouse embryonic stem cells (mESCs) cultured on circular micropatterns, yielding quantification of both abundance and localization of mRNAs and (phospho-)proteins during the first 48 hours of differentiation. ARTseq-FISH revealed a fate decision between continued self-renewal and differentiation that relies solely on the position of each mESC on the micropattern. Our results demonstrate that temporal changes in cell cycle orchestrate these position-dependent cell fate decisions.

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“…Access to patterned co-cultures would benefit in vitro studies to engineer cell contact-mediated signaling pathways, such as Eph/ephrin (Holland et al, 1996) or planar cell polarity (Strutt and Strutt, 2008). DNA-patterned cell colonies on BP-PA hydrogels could be used in conjunction with synthetic “sender/receiver” stem cell models (Liu et al, 2022) to interrogate the combined roles of mechanics, geometry, and diffusible signaling relays in germ layer patterning. Advanced co-culture models of tumor-stroma interactions (Shen et al, 2014) or cell competition mechanisms (Wagstaff et al, 2016) are other promising applications for this approach.…”

Section: Discussionmentioning

confidence: 99%

“…Microcontact printing or UV photopatterning approaches reliably deposit extracellular matrix (ECM) protein features that enforce geometric constraints upon tissues down to the single cell scale (∼10 μm) (Jimenez et al, 2021). Such approaches have been successfully implemented on glass, hydrogel, and elastomeric substrates (Liu et al, 2022; Smith et al, 2018; Tang et al, 2012). However, since the initial cell patterning and eventual adhesion footprints are one and the same, micro-printed ECM substrates are limited in their utility to assays in which tissues are restricted in their migration after patterning.…”

Section: Introductionmentioning

confidence: 99%

Independent control over cell patterning and adhesion on hydrogel substrates for tissue interface mechanobiology

Prahl

Porter

Liu

et al. 2022

Preprint

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Replicating organizational principles that establish fine-scale tissue structure is critical to our capacity for building functional replacement tissues. Tissue boundaries such as epithelial-mesenchymal interfaces are engines for morphogenesisin vivo. However, despite a wealth of micropatterning approaches available to control tissue size, shape, and mechanical environmentin vitro, fine-scale spatial control of cell composition within tissue constructs remains an engineering challenge. To address this, we augment DNA velcro technology for selective patterning of ssDNA-labeled cells with long-term culture on mechanically defined polyacrylamide hydrogels. We co-functionalize photoactive benzophenone-containing polyacrylamide gels (BP-PA gels) with spatially precise ssDNA features that confer temporary cell adhesion and with extracellular matrix (ECM) proteins that confer long-term adhesion. We find that co-functionalization does not compromise ssDNA patterning fidelity or cell capture, nor hydrogel mechanical properties or mechanosensitive fibroblast spreading, enabling mechanobiology studies of precise cell interfaces. We then co-pattern colonies of fibroblasts and epithelial cells to study interface formation and extracellular signal-related kinase (ERK) activity at cellular contacts. Combining DNA velcro and ECM functionalization approaches provides independent control of initial cell placement, adhesion, and mechanical environment, constituting a new tool for studying biological interfaces and for programming multicellular interactions in engineered tissues.

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Nodal is a short-range morphogen with activity that spreads through a relay mechanism in human gastruloids

Cited by 6 publications

References 27 publications

Single-molecule tracking of Nodal and Lefty in live zebrafish embryos supports hindered diffusion model

Single-molecule tracking of Nodal and Lefty in live zebrafish embryos supports hindered diffusion model

ARTseq-FISH reveals position-dependent fate decisions driven by cell cycle changes

Independent control over cell patterning and adhesion on hydrogel substrates for tissue interface mechanobiology

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