Project description:The role of FGF is the least understood of the morphogens driving mammalian gastrulation. Here we investigated the function of FGF in a stem cell model for human gastrulation known as a 2D gastruloid. We found a ring of FGF-dependent ERK activity closely follows the emergence of primitive streak (PS)-like cells but expands further inward. We showed that this ERK activity pattern is required for PS-like differentiation and depends on localized activation of basally polarized FGF receptors (FGFR) by endogenous FGF gradients. By single cell RNA-sequencing, we identified the main receptor to be FGFR1 and the key ligands FGF2/4/17 and showed that this is similar to the human and monkey embryo but different from the mouse.
Project description:Micropatterned human pluripotent stem cells (hPSCs) treated with BMP4, known as a 2D gastruloid, are among the most widely used stem cell models for human gastrulation. Due to its simplicity and reproducibility, this system is ideal for high throughput quantitative studies of tissue patterning and has led to many insights into the mechanisms of mammalian gastrulation. However, 2D gastruloids have only been studied up to 48h. Here we extended this system to 96h.
Project description:The gastrulation process is controlled by the interplay between morphogenetic signals from BMP, WNT and NODAL pathways. Increasing evidences support an emerging role of the Hippo-YAP signaling in the cell-fate decisions that guide lineage specification in mouse and human Embryonic Stem Cells (hESCs). However, the contribution of YAP to the process of gastrulation in hESCs remains unknown. Here, we show that YAP1 regulates the specification, size and patterning of the three-germ layers. Using hESC-derived 2D-micropatterned gastruloids and directed differentiation approaches we show that YAP maintains a semi-active NODAL signaling during gastrulation essential to regulate the exit of pluripotency. In absence of YAP1, a hyperactive NODAL signaling retains SMAD2.3 in the nuclei, impeding the exit of pluripotency and the acquisition of the ectodermal gene program. Accordingly, the inhibition of NODAL signaling is sufficient to rescue the gastrulation-defective phenotype of the YAP1 KO hESCs. Our work revealed that Hippo-YAP1 signaling is an important component of the developmental network that coordinate hESC pluripotency and gastrulation.
Project description:Human pluripotent stem cell-derived kidney organoids replicate embryonic nephrogenesis in a 3D-culture system. Recent advances suggest that refining the culture environment to replicate spatiotemporal cues present during embryonic organogenesis improves patterning. Here, this paradigm was applied to FGF signalling, a key regulator of embryonic nephron progenitor maintenance, nephrogenesis and ureteric branching. Both FGF8b and FGF10 signalling is sufficient to support nephrogenesis, with each having distinct effects on nephron patterning. FGF10 enhanced the initial WT1+ mesenchymal population, leading to proximally biased nephrons, while FGF8b biased toward early distal patterning, leading to the formation of cells with connecting segment identity. The addition of both FGF8b and FGF10 together had an additive effect, leading to a balance of proximal and distal patterning. This differential patterning was retained in tissue transplanted under the murine renal capsule, with FGF8b-treated organoids displaying increased distal/connecting segments. These findings highlight plasticity during organoid nephrogenesis that can be modulated by FGF signalling and identify an approach to refine nephrogenesis toward key cell types.
Project description:Cell signaling molecules are essential drivers of gene expression patterns, which are crucial for the development of multicellular organisms. Although prior research has established the importance of the levels and distribution of these molecules for gene expression patterns during embryonic development, a complete understanding of the specific targets influenced by their activity remains elusive. This study investigates how the fibroblast growth factor (FGF), a key developmental signal, orchestrates gene expression during the organogenesis of the zebrafish lateral line. Our analysis provides a detailed catalog of genes mediated by FGF signaling and identified dose-dependent genes that consistently respond to varying levels of extracellular FGF signaling in vivo. Notably, we uncovered an unexpected group of target genes that show suppressed expression at increased levels of FGF ligand, independent of changes in extracellular signaling. Experiments utilizing mosaic mis-expression of FGF ligands demonstrate that this gene regulation occurs autonomously within individual cells. In embryos with endogenous FGF levels, these target genes are suppressed in FGF-producing cells that shows nuclear accumulation of the FGF ligand. Targeted degradation of nuclear FGF confirms its role in this suppression, while not interfering with the activity of extracellular FGF signaling in the neighboring cells. Thus, we propose a novel mechanism of gene expression patterning in which the FGF ligand is directed to the nucleus within source cells. This process autonomously regulates its signaling targets, which are induced by extracellular FGF, leading to a self-organized symmetry-breaking in FGF signaling targets. Our findings of consistent gene expression patterns across multiple tissues, together with the nuclear localization of other paracrine FGF ligands, indicate that the cell-autonomous gene regulation by nuclear-targeted ligands could be a more widespread mechanism.
Project description:The neuroectoderm is patterned along a rostral-caudal axis in response to localized factors in the embryo, but exactly how these factors act as positional information for this patterning is not yet fully understood. Here, using the self-organizing properties of mouse embryonic stem cell (ESC), we report that ESC-derived neuroectoderm self-generates a Six3+ rostral and a Irx3+ caudal bipolarized patterning. In this instance, localized Fgf signaling performs dual roles, as it regulates Six3+ rostral polarization at an earlier stage and promotes Wnt signaling at a later stage. The Wnt signaling components are differentially expressed in the polarized tissues, leading to genome-wide Irx3+ caudal-polarization signals. Surprisingly, differentially expressed Wnt agonists and antagonists have essential roles in orchestrating the formation of a balanced rostral-caudal neuroectoderm pattern. Together, our findings provide key processes for dynamic self-patterning and evidence that a temporally and locally regulated interaction between Fgf and Wnt signaling controls self-patterning in ESC-derived neuroectoderm.
Project description:The role of FGF is the least understood of the morphogens driving mammalian gastrulation. Here we investigated the function of FGF in a stem cell model for human gastrulation known as a 2D gastruloid. We found a ring of FGF-dependent ERK activity that closely follows the emergence of primitive streak (PS)-like cells but expands further inward. We showed that this ERK activity pattern is required for PS-like differentiation and that loss of PS-like cells upon FGF receptor inhibition can be rescued by directly activating ERK. We further demonstrated that the ERK-ring depends on localized activation of basally localized FGF receptors (FGFR) by endogenous FGF gradients. We confirm and extend previous studies in analyzing expression of FGF pathway components, showing the main receptor to be FGFR1 and the key ligands FGF2/4/17, similar to the human and monkey embryo but different from the mouse. In situ hybridization and scRNA-seq revealed that FGF4 and FGF17 expression colocalize with the PS marker TBXT but only FGF17 is maintained in nascent mesoderm and endoderm. FGF4 and FGF17 reduction both reduced ERK activity and differentiation to PS-like cells and their derivatives, indicating overlapping function. Thus, we have identified a previously unknown role for FGF-dependent ERK signaling in 2D gastruloids and possibly the human embryo, driven by a mechanism where FGF4 and FGF17 signal through basally localized FGFR1 to induce PS-like cells.
Project description:Ectodermal patterning is required for the establishment of multiple components of the vertebrate body plan. Previous studies have demonstrated that precise combinations of extracellular signals determine the induction of neural or neural crest progenitors. Yet, we still have a limited understanding of how the response to inductive signals is optimized to generate the proper transcriptional output in target cells. Here we show that post-transcriptional attenuation of signaling gradients is essential for the formation of the neural crest. We found that neural crest cells display enhanced expression of Dicer, which promotes the maturation of a set of cell-type-specific miRNAs. These miRNAs target multiple components of the FGF signaling pathway, a central player in the process of neural induction in avian embryos. Loss of neural crest miRNAs prevented the attenuation of this neuralizing signal, leading to the expansion of the neural plate at the expense of neural crest cells. Thus, the post-transcriptional attenuation of FGF signaling is pre-requisite for neural crest specification. These findings demonstrate how post-transcriptional repression may reshape signaling gradients to set the boundaries between distinct spatial domains of progenitor cells.