Biology subjects

Warmflash, A.

Publications and source records attributed to Warmflash, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Optogenetic BMP4 models symmetry breaking and primitive streak formation in hPSCs

Gastrulation begins when the epiblast epithelium breaks symmetry to form the primitive streak, but current in vitro models neither fully capture this process nor separate its mechanical and chemical inputs. Here, we introduce LightBMP, an optogenetic platform that enables spatiotemporally programmable BMP4 expression in human pluripotent stem cells. Dose and duration control reveal a sustained low-BMP regime, inaccessible to recombinant ligand, that specifies mesoderm. Spatially programmed BMP4 sources in uniform cultures mimic the radial organization of 2D gastruloids, showing that mechan-ical context and edge sensing are not required for patterning. In micropatterned colo-nies, light-defined sources break symmetry to generate a stripe-shaped, primitive-streak-like domain where cells acquire multiple mesodermal fates and undergo directed migration. Together, these results show that spatial control of a single morphogen source is sufficient to drive symmetry breaking and primitive-streak-like patterning, independent of edge constraints.

developmental biology↗

bioRxiv2026

Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

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.

genetics↗

bioRxiv2025

Comparison between the activities of canonical Wnt ligands in human pluripotent stem cell differentiation

WNT ligands are key regulators of mammalian gastrulation and anterior-posterior (A-P) patterning. The activities of different Wnt ligands that signal through the canonical pathway are often assumed to be similar due to their high structural homology. To explore potential differences, we generated human pluripotent stem cell (hPSC) lines with inducible expression of WNT3, WNT3A, WNT6, or WNT8A, enabling direct comparisons of ligand activity in multiple differentiation contexts. We find that only WNT3 and WNT3A robustly induce mesodermal markers, while WNT6 and WNT8A do not. In the context of ectodermal differentiation, WNT3, WNT3A, and WNT8 activate a similar set of targets but WNT3 and 3A do so more strongly than WNT8A. WNT6 does not induce this set of targets, however, all four ligands, including WNT6, downregulate a similar set of targets. We also observed ligand-specific differences in spatial signaling range and the ability to induce self-organized patterns of ectodermal fates including neural crest-like domains. These results highlight how signaling strength, spatial range, and diverse target genes of Wnt ligands contribute to distinct developmental outcomes in early human stem cell differentiation.

developmental biology↗

bioRxiv2025

Juxtaposition of human pluripotent stem cells with amnion-like cells is sufficient to trigger primitive streak formation

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.

developmental biology↗

bioRxiv2025

A novel self-organizing embryonic stem cell system reveals the role of WNT signaling parameters in anterior-posterior patterning of the nervous system

A Wnt activity gradient is essential for the formation of the anterior-posterior (AP) axis in all vertebrates. The relationship between the dynamics of Wnt signaling and specification of AP coordinates is difficult to study in mammalian embryos due to the inaccessibility of developing embryos and the difficulty of live imaging. Here, we developed an in vitro model of human neuroectoderm patterning, where the AP axis self-organizes along the radius of a micropatterned human pluripotent stem cell colony. We used this system to study the quantitative relationship between Wnt signaling in space and time and the resulting AP patterns. We found that rather than a smoothly varying gradient along the axis, signaling is elevated in midbrain compared to either surrounding region. The timing, rather than the amplitude or duration, of the Wnt response played the most important role in setting axial coordinates. Loss of WNT8A reduces posterior Wnt activity and anteriorizes cell fates, demonstrating that even cells exposed to high exogenous Wnt require endogenous ligand production to fully posteriorize and to position the midbrain-hindbrain boundary. These results establish a simple system for studying the patterning of the human nervous system and elucidate how cells interpret Wnt dynamics to determine their position along the AP axis.

developmental biology↗

bioRxiv2025

Dependence of cell fate potential and cadherin switching on primitive streak coordinate during differentiation of human pluripotent stem cells

During gastrulation, the primitive streak (PS) forms and begins to differentiate into mesendodermal subtypes. This process involves an epithelial-mesenchymal transition (EMT), which is marked by cadherin switching, where E-Cadherin is downregulated, and N-Cadherin is upregulated. To understand the relationships between differentiation, EMT, and cadherin switching, we made measurements of these processes during differentiation of human pluripotent stem cells (hPSCs) to PS and subsequently to mesendoderm subtypes using established protocols, as well as variants in which signaling through key pathways including Activin, BMP, and Wnt were modulated. We found that perturbing signaling so that cells acquired identities ranging from anterior to posterior PS had little impact on the subsequent differentiation potential of cells but strongly impacted the degree of cadherin switching. The degree of E-Cadherin downregulation and N-Cadherin upregulation were uncorrelated and had different dependence on signaling. The exception to the broad potential of cells throughout the PS was the loss of definitive endoderm potential in cells with mid to posterior PS identities. Thus, cells induced to different PS coordinates had similar potential within the mesoderm but differed in cadherin switching. Consistently, E-Cadherin knockout did not alter cell fates outcomes during differentiation. Overall, cadherin switching and EMT are modulated independently of cell fate commitment in mesendodermal differentiation.

developmental biology↗

bioRxiv2024

Endogenous Nodal switches Wnt interpretation from posteriorization to germ layer differentiation in geometrically constrained human pluripotent cells

The Wnt pathway is essential for inducing the primitive streak, the precursor of the mesendoderm, as well as setting anterior-posterior coordinates. How Wnt coordinates these diverse activities remains incompletely understood. Here, we show that in Wnt-treated human pluripotent cells, endogenous Nodal signaling is a crucial switch between posteriorizing and primitive streak-including activities. While treatment with Wnt posteriorizes cells in standard culture, in micropatterned colonies, higher levels of endogenously induced Nodal signaling combine with exogenous Wnt to drive endoderm differentiation. Inhibition of Nodal signaling restores dose-dependent posteriorization by Wnt. In the absence of Nodal inhibition, micropatterned colonies undergo spontaneous, elaborate morphogenesis concomitant with endoderm differentiation even in the absence of added extracellular matrix proteins like Matrigel. Our study shows how Wnt and Nodal combinatorially coordinate germ layer differentiation with AP patterning and establishes a system to study a natural self-organizing morphogenetic event in in vitro culture.

developmental biology↗

bioRxiv2022

Combinatorial interpretation of BMP and WNT allows BMP to act as a morphogen in time but not in concentration

Secreted morphogen signals play a key role in the determination of cell fates during embryonic development. BMP signaling is essential for mammalian gastrulation, as it initiates a cascade of signals that controls the self-organized patterning of the three germ layers. Although morphogen signals are typically thought to induce cell fates in a concentration-dependent manner, development is a highly dynamic process, so it is crucial to understand how time-dependent signaling affects cellular differentiation. Here we show that varying the duration of BMP signaling in human pluripotent stem cells (hPSCs) leads to either cells remaining pluripotent, or differentiating to mesodermal or extraembryonic states, while varying the concentration does not cause efficient mesodermal differentiation at any dose. Thus, there is a morphogen effect in time but not in concentration, and an appropriately timed pulse of BMP induces hPSCs to a mesodermal fate more efficiently than sustained signaling at any concentration. Using live cell imaging of signaling and cell fate reporters together with a simple mathematical model, we show that this effect is due to a combinatorial interpretation of the applied BMP signal and induced endogenous WNT signaling. Our findings have implications for how signaling pathways control the landscape of early human development.

developmental biology↗

bioRxiv2021

Self-organized morphogenesis of a human neural tube in vitro by geometric constraints

Understanding how human embryos develop their shape is a fundamental question in physics of life with strong medical implications. However, it is challenging to study the dynamics of organ formation in humans. Animals differ from humans in key aspects, and in particular in the development of the nervous system. Conventional organoids are quantitatively unreproducible and exhibit highly variable morphology. Here we present a morphologically reproducible and scalable approach for studying human organogenesis in a dish, which is compatible with live imaging. We achieve this by precisely controlling cell fate pattern formation in 2D stem cell sheets, while allowing for self-organization of tissue shape in 3D. Upon triggering neural pattern formation, the initially flat stem cell sheet undergoes folding morphogenesis and self-organizes into a millimeter long anatomically accurate model of the neural tube, covered by epidermis. We find that neural and epidermal human tissues are necessary and sufficient for folding morphogenesis in the absence of mesoderm activity. Furthermore, we find that molecular inhibition of tissue contractility leads to defects similar to neural tube closure defects, consistent with in vivo studies. Finally, we discover that neural tube shape, including the number and location of hinge points, depends on neural tissue size. This suggests that neural tube morphology along the anterior posterior axis depends on neural plate geometry in addition to molecular gradients. Our approach provides a new path to study human organ morphogenesis in health and disease.

developmental biology↗

bioRxiv2021

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

Morphogens are signaling molecules that convey positional information and dictate cell fates during development. Little is known about how morphogen gradients are created and interpreted during mammalian embryogenesis. Here we take advantage of a human gastruloid model to visualize endogenous Nodal protein in living cells. 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 novel paradigm for tissue patterning by an activator-inhibitor pair.

developmental biology↗

bioRxiv2021

BMP-treated human embryonic stem cells transcriptionally resemble amnion cells in the monkey embryo

Human embryonic stem cells (hESCs) possess an immense potential to generate clinically relevant cell types and unveil mechanisms underlying early human development. However, using hESCs for discovery or translation requires accurately identifying differentiated cell types through comparison with their in vivo counterparts. Here, we set out to determine the identity of much debated BMP-treated hESCs by comparing their transcriptome to the recently published single cell transcriptomes of early human embryos in the study Xiang et al 2019. Our analyses reveal several discrepancies in the published human embryo dataset, including misclassification of putative amnion, intermediate and inner cell mass cells. These misclassifications primarily resulted from similarities in pseudogene expression, highlighting the need to carefully consider gene lists when making comparisons between cell types. In the absence of a relevant human dataset, we utilized the recently published single cell transcriptome of the early post implantation monkey embryo to discern the identity of BMP-treated hESCs. Our results suggest that BMP-treated hESCs are transcriptionally more similar to amnion cells than trophectoderm cells in the monkey embryo. Together with prior studies, this result indicates that hESCs possess a unique ability to form mature trophectoderm subtypes via an amnion-like transcriptional state.

developmental biology↗

bioRxiv2021

Quantifying cell transitions in C. elegans with data-fitted landscape models

Increasing interest has emerged in new mathematical approaches that simplify the study of complex differentiation processes by formalizing Waddingtons landscape metaphor. However, a rational method to build these landscape models remains an open problem. Building on pioneering work by Corson and Siggia (2012, 2017) we study vulval development in C. elegans by developing a framework based on Catastrophe Theory (CT) and approximate Bayesian computation (ABC) to build data-fitted landscape models. We first identify the candidate qualitative landscapes, and then use CT to build the simplest model consistent with the data, which we quantitatively fit using ABC. The resulting model suggests that the underlying mechanism is a quantifiable two-step decision controlled by EGF and Notch-Delta signals, where a non-vulval/vulval decision is followed by a bistable transition to the two vulval states. This new model fits a broad set of data and makes several novel predictions.

developmental biology↗

bioRxiv2021

Rapid fabrication of hydrogel micropatterns by projection stereolithography for studying self-organized developmental patterning

Self-organized patterning of mammalian embryonic stem cells on micropatterned surfaces has previously been established as an in vitro platform for early mammalian developmental studies, complimentary to in vivo studies. Traditional micropatterning methods, such as micro-contact printing (CP), involve relatively complicated fabrication procedures, which restricts widespread adoption by biologists. Here, we demonstrate a rapid method of micropatterning by printing hydrogel micro-features onto a glass-bottomed culture vessel. The micro-features are printed using a projection stereolithography bioprinter yielding hydrogel structures that geometrically restrict the attachment of cells or protein. Compared to traditional, physical photomasks, a digitally tunable virtual photomask is used in the projector to generate blue light patterns that enable rapid iteration with minimal cost and effort. We show that a protocol that makes use of this method together with LN521 coating creates a surface suitable for human embryonic stem cell (hESC) attachment and growth with minimal non-specific adhesion. We further demonstrate that self-patterning of hESCs following previously published gastrulation and ectodermal induction protocols achieves results comparable with those obtained with commercially available plates (CYTOO).

developmental biology↗

bioRxiv2019

Mesenchymal-Epithelial Transition Regulates Initiation of Pluripotency Exit before Gastrulation

The pluripotent epiblast gives rise to all tissues and organs in an adult body. Its differentiation starts at gastrulation when the epiblast generates mesoderm and endoderm germ layers through a process called epithelial-mesenchymal transition (EMT). Although gastrulation EMT coincides with loss of epiblast pluripotency, pluripotent cells in development and in vitro can adopt either mesenchymal or epithelial morphology. The relationship between epiblasts cellular morphology and its pluripotency is not well understood. In this work, using chicken epiblast and mammalian pluripotency stem cell (PSC) models, we show that PSCs undergo a mesenchymal-epithelial transition (MET) prior to EMT-associated pluripotency loss. Epiblast MET and its subsequent EMT are two distinct processes. The former, a partial MET, is associated with reversible initiation of pluripotency exit; whereas the latter, a full EMT, is associated with complete and irreversible pluripotency loss. We provide evidence that integrin-mediated cell-matrix interaction is a key player in pluripotency exit regulation. We propose that epiblast partial MET is an evolutionarily conserved process among all amniotic vertebrates and its developmental function is to mediate planar symmetry-breaking within an epithelialized epiblast, taking place after epiblast MET but before gastrulation EMT.

developmental biology↗

bioRxiv2019

A novel self-organizing embryonic stem cell system reveals signaling logic underlying the patterning of human ectoderm

During development, the ectoderm is patterned by a combination of BMP and WNT signaling. Research in model organisms has provided substantial insight, however, there are currently no systems to study this patterning in humans. Further, the complexity of neural plate border specification has made it difficult to transition from discovering the genes involved to deeper mechanistic understanding. Here, we develop an in vitro model of human ectodermal patterning, in which hESCs self-organize to form robust and quantitatively reproducible patterns corresponding to the dorsal-ventral axis of the embryo. Using this platform, we show that the duration of endogenous WNT signaling is a crucial control parameter, and that cells sense relative levels of BMP and WNT signaling in making fate decisions. These insights allowed us to develop an improved protocol for placodal differentiation. Thus, our platform is a powerful tool for studying human ectoderm patterning and for improving directed differentiation protocols.

developmental biology↗

bioRxiv2018

The timing of signaling events in the BMP, WNT, and Nodal cascade determines self-organized fate patterning in human gastruloids

During gastrulation, the pluripotent epiblast is patterned into the three germ layers, which form the embryo proper. This patterning requires a signaling cascade involving the BMP, WNT and NODAL pathways; however, how these pathways regulate one another in space and time to generate cell-fate patterns remains unknown. Using a human gastruloid model, we show that BMP signaling initiates a wave of WNT signaling, which, in turn, initiates a wave of NODAL signaling. While WNT propagation depends on continuous BMP activity, NODAL propagates independently of upstream signals. We further show that the duration of BMP signaling determines the position of mesodermal differentiation while WNT and NODAL synergize to achieve maximal differentiation. The waves of both WNT and NODAL signaling activity extend farther into the colony than mesodermal differentiation. Combining dynamic measurements of signaling activity with mathematical modeling revealed that the formation of signaling waves is inconsistent with WNT and NODAL forming a stable spatial pattern in signaling activities, and the final signaling state is spatially homogeneous. Thus, dynamic events in the BMP, WNT, and NODAL signaling cascade, in the absence of a signaling gradient, have the potential to mediate epiblast patterning.

developmental biology↗

bioRxiv2018

WNT ligands stimulate transient signaling in human pluripotent cells and synergize with TGF-β ligands to stimulate sustained signaling during differentiation

WNT/{beta}-catenin signaling is crucial to all stages of life. It controls early morphogenetic events in embryos, maintains stem-cell niches in adults, and is disregulated in many types of cancer. Despite its ubiquity, little is known about the dynamics of signal transduction or whether it varies across contexts. Here we probe the dynamics of signaling by monitoring nuclear accumulation of {beta}-catenin, the primary transducer of canonical WNT signals, using quantitative live-cell imaging. We show that {beta}-catenin signaling responds adaptively to constant WNT signaling in pluripotent stem cells, and that these dynamics become sustained upon differentiation. Varying dynamics were also observed in the response to WNT in commonly used mammalian cell-lines. Signal attenuation in pluripotent cells is controlled by both intra- and extra-cellular negative regulation of WNT signaling. TGF{beta}-superfamily ligands Activin and BMP, which coordinate with WNT signaling to pattern the gastrula, increase the {beta}-catenin response in a manner independent of their ability to induce new WNT-ligand production. Our results reveal how variables external to the pathway, including differentiation status and crosstalk with other pathways, dramatically alter WNT/{beta}-catenin dynamics.

developmental biology↗

bioRxiv2018

S-MiRAGE: A quantitative, secreted RNA-based reporter of gene expression and cell persistence

Non-destructive measurements of cell persistence and gene expression are crucial for longitudinal research studies and for prognostic assessment of cell therapies. Here we describe S-MiRAGE, a platform that utilizes small secreted RNA molecules as sensitive and quantitatively accurate reporters of cellular processes. We demonstrate the utility of S-MiRAGE by monitoring the differentiation status of human embryonic stem cells in vitro, and tumor growth in a mouse model in vivo.

bioengineering↗