Dissecting the dynamics of signaling events in the BMP, WNT, and NODAL cascade during self-organized fate patterning in human gastruloids.

During gastrulation, the pluripotent epiblast self-organizes into the 3 germ layers-endoderm, mesoderm and ectoderm, which eventually form the entire embryo. Decades of research in the mouse embryo have revealed that a signaling cascade involving the Bone Morphogenic Protein (BMP), WNT, and NODAL pathways is necessary for gastrulation. In vivo, WNT and NODAL ligands are expressed near the site of gastrulation in the posterior of the embryo, and knockout of these ligands leads to a failure to gastrulate. These data have led to the prevailing view that a signaling gradient in WNT and NODAL underlies patterning during gastrulation; however, the activities of these pathways in space and time have never been directly observed. In this study, we quantify BMP, WNT, and NODAL signaling dynamics in an in vitro model of human gastrulation. Our data suggest that BMP signaling initiates waves of WNT and NODAL signaling activity that move toward the colony center at a constant rate. Using a simple mathematical model, we show that this wave-like behavior is inconsistent with a reaction-diffusion-based Turing system, indicating that there is no stable signaling gradient of WNT/NODAL. Instead, the final signaling state is homogeneous, and spatial differences arise only from boundary effects. We further show that the durations of WNT and NODAL signaling control mesoderm differentiation, while the duration of BMP signaling controls differentiation of CDX2-positive extra-embryonic cells. The identity of these extra-embryonic cells has been controversial, and we use RNA sequencing (RNA-seq) to obtain their transcriptomes and show that they closely resemble human trophoblast cells in vivo. The domain of BMP signaling is identical to the domain of differentiation of these trophoblast-like cells; however, neither WNT nor NODAL forms a spatial pattern that maps directly to the mesodermal region, suggesting that mesoderm differentiation is controlled dynamically by the combinatorial effect of multiple signals. We synthesize our data into a mathematical model that accurately recapitulates signaling dynamics and predicts cell fate patterning upon chemical and physical perturbations. Taken together, our study shows that the dynamics of signaling events in the BMP, WNT, and NODAL cascade in the absence of a stable signaling gradient control fate patterning of human gastruloids.R01 GM126122 - NIGMS NIH HHSPublished versio

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 Waddington’s landscape metaphor. However, a rational method to build these landscape models remains an open problem. Here 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

An incoherent regulatory network architecture that orchestrates B cell diversification in response to antigen signaling

B cell receptor signaling controls the expression of IRF-4, a transcription factor required for B cell differentiation. This study shows that IRF-4 regulates divergent B cell fates via a ‘kinetic-control' mechanism that determines the duration of a transient developmental state

Endothelial cells decode VEGF-mediated Ca2+ signaling patterns to produce distinct functional responses

A single extracellular stimulus can promote diverse behaviors among isogenic cells by differentially regulated signaling networks. We examined Ca2+ signaling in response to VEGF (vascular endothelial growth factor), a growth factor that can stimulate different behaviors in endothelial cells. We found that altering the amount of VEGF signaling in endothelial cells by stimulating them with different VEGF concentrations triggered distinct and mutually exclusive dynamic Ca2+ signaling responses that correlated with different cellular behaviors. These behaviors were cell proliferation involving the transcription factor NFAT (nuclear factor of activated T cells) and cell migration involving MLCK (myosin light chain kinase). Further analysis suggested that this signal decoding was robust to the noisy nature of the signal input. Using probabilistic modeling, we captured both the stochastic and deterministic aspects of Ca2+ signal decoding and accurately predicted cell responses in VEGF gradients, which we used to simulate different amounts of VEGF signaling. Ca2+ signaling patterns associated with proliferation and migration were detected during angiogenesis in developing zebrafish

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 (μCP), 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 proteins. Compared to traditional and 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, an extracellular matrix 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