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Aggregates of stem cells can break symmetry and self-organize into embryo-like structures with complex morphologies and gene expression patterns. Mechanisms including reaction-diffusion Turing patterns and cell sorting have been proposed to explain symmetry breaking but distinguishing between these candidate mechanisms of self-organization requires identifying which early asymmetries evolve into subsequent tissue patterns and cell fates. Here we use synthetic ‘signal-recording’ gene circuits to trace the evolution of signalling patterns in gastruloids, three-dimensional stem cell aggregates that form an anterior–posterior axis and structures resembling the mammalian primitive streak and tailbud. We find that cell sorting rearranges patchy domains of Wnt activity into a single pole that defines the gastruloid anterior–posterior axis. We also trace the emergence of Wnt domains to earlier heterogeneity in Nodal activity even before Wnt activity is detectable. Our study defines a mechanism through which aggregates of stem cells can form a patterning axis even in the absence of external spatial cues. 

ABSTRACT Many developmental processes rely on the localized activation of receptor tyrosine kinases and their canonical downstream effectors Erk and Akt, yet the specific roles played by each of these signals is still poorly understood. Gastruloids, 3D cell culture models of mammalian gastrulation and axial elongation, enable quantitative dissection of signaling patterns and cell responses in a simplified, experimentally accessible context. We find that mouse gastruloids contain posterior-to-anterior gradients of Erk and Akt phosphorylation induced by distinct receptor tyrosine kinases, with features of the Erk pattern and expression of its downstream target Snail exhibiting hallmarks of size-invariant scaling. Both Erk and Akt signaling contribute to cell proliferation, whereas Erk activation is also sufficient to induce Snail expression and precipitate profound tissue shape changes. We further uncover that Erk signaling is sufficient to convert the entire gastruloid to one of two mesodermal fates depending on position along the anteroposterior axis. In all, these data demonstrate functional roles for two core signaling gradients in mammalian development and suggest how these modules might be harnessed to engineer user-defined tissues with predictable shapes and cell fates. 

Receptor tyrosine kinases (RTKs) are major signaling hubs in metazoans, playing crucial roles in cell proliferation, migration, and differentiation. However, few tools are available to measure the activity of a specific RTK in individual living cells. Here, we present pYtags, a modular approach for monitoring the activity of a user-defined RTK by live-cell microscopy. pYtags consist of an RTK modified with a tyrosine activation motif that, when phosphorylated, recruits a fluorescently labeled tandem SH2 domain with high specificity. We show that pYtags enable the monitoring of a specific RTK on seconds-to-minutes time scales and across subcellular and multicellular length scales. Using a pYtag biosensor for epidermal growth factor receptor (EGFR), we quantitatively characterize how signaling dynamics vary with the identity and dose of activating ligand. We show that orthogonal pYtags can be used to monitor the dynamics of EGFR and ErbB2 activity in the same cell, revealing distinct phases of activation for each RTK. The specificity and modularity of pYtags open the door to robust biosensors of multiple tyrosine kinases and may enable engineering of synthetic receptors with orthogonal response programs. 

It has long been known that FGF signaling contributes to mesoderm formation, a germ layer found in triploblasts that is composed of highly migratory cells that give rise to muscles and to the skeletal structures of vertebrates. FGF signaling activates several pathways in the developing mesoderm, including transient activation of the Erk pathway, which triggers mesodermal fate specification through the induction of the gene brachyury and activates morphogenetic programs that allow mesodermal cells to position themselves in the embryo. In this review, we discuss what is known about the generation and interpretation of transient Erk signaling in mesodermal tissues across species. We focus specifically on mechanisms that translate the level and duration of Erk signaling into cell fate and cell movement instructions and discuss strategies for further interrogating the role that Erk signaling dynamics play in mesodermal gastrulation and morphogenesis. 

Many canonical signaling pathways exhibit complex time-varying responses, yet how minutes-timescale pulses of signaling interact with the dynamics of transcription and gene expression remains poorly understood. Erk-induced immediate early gene (IEG) expression is a model of this interface, exemplifying both dynamic pathway activity and a rapid, potent transcriptional response. Here, we quantitatively characterize IEG expression downstream of dynamic Erk stimuli in individual cells. We find that IEG expression responds rapidly to acute changes in Erk activity, but only in a sub-population of stimulus-responsive cells. We find that while Erk activity partially predicts IEG expression, a majority of response heterogeneity is independent of Erk and can be rapidly tuned by different mitogenic stimuli and parallel signaling pathways. We extend our findings to an in vivo context, the mouse epidermis, where we observe heterogeneous immediate-early gene accumulation in both fixed tissue and single-cell RNA sequencing data. Our results demonstrate that signaling dynamics can be faithfully transmitted to gene expression and suggest that the signaling-responsive population is an important parameter for interpreting gene expression responses. 

Markers for the endoderm and mesoderm germ layers are commonly expressed together in the early embryo, potentially reflecting cells’ ability to explore potential fates before fully committing. It remains unclear when commitment to a single-germ layer is reached and how it is impacted by external signals. Here, we address this important question in Drosophila , a convenient model system in which mesodermal and endodermal fates are associated with distinct cellular movements during gastrulation. Systematically applying endoderm-inducing extracellular signal-regulated kinase (ERK) signals to the ventral medial embryo—which normally only receives a mesoderm-inducing cue—reveals a critical time window during which mesodermal cell movements and gene expression are suppressed by proendoderm signaling. We identify the ERK target gene huckebein ( hkb ) as the main cause of the ventral furrow suppression and use computational modeling to show that Hkb repression of the mesoderm-associated gene snail is sufficient to account for a broad range of transcriptional and morphogenetic effects. Our approach, pairing precise signaling perturbations with observation of transcriptional dynamics and cell movements, provides a general framework for dissecting the complexities of combinatorial tissue patterning.