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Morphogens are signaling molecules that convey positional information and dictate cell fates during development. Although ectopic expression in model organisms suggests that morphogen gradients form through diffusion, little is known about how morphogen gradients are created and interpreted during mammalian embryogenesis due to the combined difficulties of measuring endogenous morphogen levels and observing development in utero. Here we take advantage of a human gastruloid model to visualize endogenous Nodal protein in living cells, during specification of germ layers. We show that Nodal is extremely short range so that Nodal protein is limited to the immediate neighborhood of source cells. Nodal activity spreads through a relay mechanism in which Nodal production induces neighboring cells to transcribe Nodal. We further show that the Nodal inhibitor Lefty, while biochemically capable of long-range diffusion, also acts locally to control the timing of Nodal spread and therefore of mesoderm differentiation during patterning. Our study establishes a paradigm for tissue patterning by an activator-inhibitor pair. Studying morphogen gradient formation and reception in mammalian development is challenging. Here, the authors show with human gastruloids that Nodal activity in live cells spreads via a relay mechanism with timing that is locally controlled by Lefty, which dictates mesoderm differentiation timing.

Transitioning from pluripotency to differentiated cell fates is fundamental to both embryonic development and adult tissue homeostasis. Improving our understanding of this transition would facilitate our ability to manipulate pluripotent cells into tissues for therapeutic use. Here, we show that membrane voltage (V m ) regulates the exit from pluripotency and the onset of germ layer differentiation in the embryo, a process that affects both gastrulation and left-right patterning. By examining candidate genes of congenital heart disease and heterotaxy, we identify KCNH6 , a member of the ether-a-go-go class of potassium channels that hyperpolarizes the V m and thus limits the activation of voltage gated calcium channels, lowering intracellular calcium. In pluripotent embryonic cells, depletion of kcnh6 leads to membrane depolarization, elevation of intracellular calcium levels, and the maintenance of a pluripotent state at the expense of differentiation into ectodermal and myogenic lineages. Using high-resolution temporal transcriptome analysis, we identify the gene regulatory networks downstream of membrane depolarization and calcium signaling and discover that inhibition of the mTOR pathway transitions the pluripotent cell to a differentiated fate. By manipulating V m using a suite of tools, we establish a bioelectric pathway that regulates pluripotency in vertebrates, including human embryonic stem cells. The plasma membrane’s electrical potential is maintained by ion channels, though the impact of this potential on cell fate has not been clearly elucidated. Here they show that changes in membrane potential can affect calcium levels and mTOR in pluripotent stem cells, altering their transition from pluripotency to differentiation.

During embryonic development, diffusible signaling molecules called morphogens are thought to determine cell fates in a concentration-dependent way. Yet, in mammalian embryos, concentrations change rapidly compared to the time for making cell fate decisions. Here, we use human embryonic stem cells (hESCs) to address how changing morphogen levels influence differentiation, focusing on how BMP4 and Nodal signaling govern the cell-fate decisions associated with gastrulation. We show that BMP4 response is concentration dependent, but that expression of many Nodal targets depends on rate of concentration change. Moreover, in a self-organized stem cell model for human gastrulation, expression of these genes follows rapid changes in endogenous Nodal signaling. Our study shows a striking contrast between the specific ways ligand dynamics are interpreted by two closely related signaling pathways, highlighting both the subtlety and importance of morphogen dynamics for understanding mammalian embryogenesis and designing optimized protocols for directed stem cell differentiation. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter ).

This protocol describes how to differentiate and image human embryonic stem cells on micropatterned colonies to create radially organized domains of the germ layers mimicking embryonic gastrulation in vitro . Fate allocation in the gastrulating embryo is spatially organized as cells differentiate into specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. Although embryoid bodies and organoids can exhibit some spatial organization of differentiated cells, methods that generate embryoid bodies or organoids do not yield consistent and fully reproducible results. Here, we describe a micropatterning approach in which human embryonic stem cells are confined to disk-shaped, submillimeter colonies. After 42 h of BMP4 stimulation, cells form self-organized differentiation patterns in concentric radial domains, which express specific markers associated with the embryonic germ layers, reminiscent of gastrulating embryos. Our protocol takes 3 d; it uses commercial microfabricated slides (from CYTOO), human laminin-521 (LN-521) as extracellular matrix coating, and either conditioned or chemically defined medium (mTeSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The protocol is appropriate for personnel with basic stem cell culture training. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation.

Stem cells maintain a dynamic dialog with their niche, integrating biochemical and biophysical cues to modulate cellular behavior. Yet, the transcriptional networks that regulate cellular biophysical properties remain poorly defined. Here, we leverage human pluripotent stem cells (hPSCs) and two morphogenesis models – gastruloids and pancreatic differentiation – to establish ETV transcription factors as critical regulators of biophysical parameters and lineage commitment. Genetic ablation of ETV1 or ETV1/ETV4/ETV5 in hPSCs enhances cell-cell and cell-ECM adhesion, leading to aberrant multilineage differentiation including disrupted germ-layer organization, ectoderm loss, and extraembryonic cell overgrowth in gastruloids. Furthermore, ETV1 loss abolishes pancreatic progenitor formation. Single-cell RNA sequencing and follow-up assays reveal dysregulated mechanotransduction via the PI3K/AKT signaling. Our findings highlight the importance of transcriptional control over cell biophysical properties and suggest that manipulating these properties may improve in vitro cell and tissue engineering strategies. Stem cell interactions involve biochemical and biophysical cues, but the transcriptional control of biophysical traits is unclear. This study identifies ETVs as regulators of adhesion in hPSCs and pancreatic progenitors, shaping lineage decisions.

Studies in the mouse have established that communication between the trophectoderm and the epiblast is crucial for initiating gastrulation. In the primate embryo, the amnion rather than the trophectoderm is directly juxtaposed to the epiblast and may play this role. To model the interactions between the amnion and epiblast, we differentiated human pluripotent stem cells (hPSCs) to amnion-like cells (AMLCs) and juxtaposed them in a controlled manner with undifferentiated hPSCs, which represent the epiblast. We found that juxtaposition between these cell types is sufficient to initiate a range of cell behaviors associated with gastrulation including organized differentiation to primitive streak and downstream mesendodermal cell fates and directed cell migration out of the primitive streak region. Performing knockout experiments specifically in either the epiblast or amnion compartment revealed intricate crosstalk that is required to properly initiate gastrulation. In particularly, using knockouts of NODAL we show that Nodal signaling in both the amnion and epiblast is required for gastrulation patterning. Finally, we show that inductive ability is a transient property acquired during amnion differentiation, and that cells that differentiate from this inductive state acquire an extraembryonic mesenchyme identity. This study establishes a system to study epiblast-amnion communication and shows that this communication is sufficient to initiate gastrulation in the epiblast.

Understanding human specific mechanisms of cell fate control is essential for advancing developmental biology and regenerative medicine. Here, we identify the ILF2/3 complex as a critical regulator of primate cell fate transitions. Using genetically and epigenetically engineered gastruloids and adult stem cells, we show that ILF2/3 is required for gastrulation in primates but not mice, and for differentiation of adult progenitor cells. Mechanistically, ILF2/3 directly binds Alu elements in chromatin-associated RNAs and shields them from ADAR1-mediated adenosine-to-inosine (A-to-I) editing. Acute ILF2/3 degradation increases A-to-I editing at Alu elements, but not murine retrotransposons, leading to aberrant splicing and nonsense-mediated decay of transcripts encoding key chromatin regulators in primate cells. This in turn destabilizes the epigenetic landscape and blocks lineage commitment across all three germ layers. Re-expression of correctly spliced chromatin regulators rescues differentiation defects in ILF2/3-deficient cells, functionally linking Alu editing control to chromatin regulation and cell fate. These findings define an evolutionary mechanism that restrains retrotransposon-associated RNA editing to preserve proteome integrity and enable primate-specific developmental programs.

During embryonic development, diffusible signaling molecules called morphogens are thought to determine cell fates in a concentration-dependent manner, and protocols for directed stem cell differentiation are based on this picture. However, in the mammalian embryo, morphogen concentrations change rapidly compared to the time for making cell fate decisions. It is unknown how changing ligand levels are interpreted, and whether the precise timecourse of ligand exposure plays a role in cell fate decisions. Nodal and BMP4 are morphogens crucial for gastrulation in vertebrates. Each pathway has distinct receptor complexes that phosphorylate specific signal transducers, known as receptor-Smads, which then complex with the shared cofactor Smad4 to activate target genes. Here we show in human embryonic stem cells (hESCs) that the response to BMP4 signaling indeed is determined by the ligand concentration, but that unexpectedly, the expression of many mesodermal targets of Activin/Nodal depends on rate of concentration increase. In addition, we use live imaging of hESCs with GFP integrated at the endogenous SMAD4 locus to show that a stem cell model for the human embryo generates a wave of Nodal signaling. Cells experience rapidly increasing Nodal specifically in the region of mesendoderm differentiation. We also demonstrate that pulsatile stimulation with Activin induces repeated strong signaling and enhances mesoderm differentiation. Our results break with the paradigm of concentration-dependent differentiation and demonstrate an important role for morphogen dynamics in the cell fate decisions associated with mammalian gastrulation. They suggest a highly dynamic picture of embryonic patterning where some cell fates depend on rapid concentration increase rather than absolute levels, and point to ligand dynamics as a new dimension to optimize protocols for directed stem cell differentiation.

Primate genomes harbor over a million Alu retrotransposons, yet how cells limit their impact on transcriptome stability during lineage specification remains unclear. Here, we show that the chromatin-associated ILF2/3 complex suppresses Alu hyper-editing, a function required for gastrulation and adult stem-cell differentiation in primates but not mice. Mechanistically, ILF2/3 directly binds Alu elements in nascent RNAs and shields them from ADAR1-mediated adenosine-to-inosine (A-to-I) editing. Acute ILF2/3 degradation increases A-to-I editing at Alu elements, but not murine retrotransposons, leading to aberrant splicing and nonsense-mediated decay of transcripts encoding key chromatin regulators in primate cells. This in turn destabilizes the epigenetic landscape and blocks lineage commitment across all three germ layers. Re-expression of correctly spliced chromatin regulators rescues differentiation defects in ILF2/3-deficient cells, functionally linking Alu editing control to chromatin regulation and cell fate. These findings define an evolutionary mechanism that restrains retrotransposon-associated RNA editing to preserve proteome integrity and enable primate-specific developmental programs.Primate genomes harbor over a million Alu retrotransposons, yet how cells limit their impact on transcriptome stability during lineage specification remains unclear. Here, we show that the chromatin-associated ILF2/3 complex suppresses Alu hyper-editing, a function required for gastrulation and adult stem-cell differentiation in primates but not mice. Mechanistically, ILF2/3 directly binds Alu elements in nascent RNAs and shields them from ADAR1-mediated adenosine-to-inosine (A-to-I) editing. Acute ILF2/3 degradation increases A-to-I editing at Alu elements, but not murine retrotransposons, leading to aberrant splicing and nonsense-mediated decay of transcripts encoding key chromatin regulators in primate cells. This in turn destabilizes the epigenetic landscape and blocks lineage commitment across all three germ layers. Re-expression of correctly spliced chromatin regulators rescues differentiation defects in ILF2/3-deficient cells, functionally linking Alu editing control to chromatin regulation and cell fate. These findings define an evolutionary mechanism that restrains retrotransposon-associated RNA editing to preserve proteome integrity and enable primate-specific developmental programs.