Biology subjects

Estaras, C.

Publications and source records attributed to Estaras, C..

5 recordsLinked to original sources

bioRxiv2025

YAP levels regulate anteroposterior elongation of hESC-derived gastruloids

Human embryonic stem cells (hESCs) can self-organize into anteroposterior patterned three-dimensional structures, characterized by polarized expression of CDX2 and GATA6, known as human 3D gastruloids (h3D-gastruloids). This patterning emerges through hESC-intrinsic mechanisms that remain poorly understood. Here, we demonstrate that the formation and elongation of h3D-gastruloids is modulated by the activity levels of the Hippo pathway effector YAP. Using complementary chemical and genetic perturbations, we show that elevated nuclear YAP activity inhibits gastruloid elongation, whereas YAP inhibition enhances axial elongation relative to controls. Single-cell RNA sequencing (scRNA-seq) analyses reveal that YAP activation disrupts the establishment of distinct anterior GATA6 and posterior CDX2 poles. Conversely, YAP inhibition promotes clearer segregation of these domains, facilitating symmetry breaking and elongation. Finally, we developed a high-throughput platform for h3D-gastruloid generation, enabling the production of over 500 gastruloids per well. Together, our findings uncover a role for YAP in orchestrating the three-dimensional organization of human gastruloids.

developmental biology↗

bioRxiv2025

YAP1 and QSER1 are Key Modulators of Embryonic Signaling Pathways in the Mammalian Epiblast

YAP1 signaling is essential for development but its specific roles in early embryogenesis remain poorly understood. To shed light on this, we analyzed YAP1s role in regulating the pluripotency of the mammalian epiblast, using scRNAseq approaches. Conditional deletion of Yap1 in the mouse epiblast (Sox2-Cre) altered the expression of signaling genes, including Nodal, Wnt3, and Fgf8. Accordingly, Yap1 loss led to enhanced differentiation of the epiblast toward primitive streak lineages, as evidenced by the upregulation of T/Brachyury and Eomes genes. Furthermore, a proximity labeling assay in human pluripotent stem cells, followed by biochemical assays and molecular modeling predictions, revealed that YAP1 cooperates with QSER1 protein to regulate lineage genes. Our analysis shows that YAP1:TEAD4 enhancers recruit QSER1 to prevent RNA Polymerase II recruitment. Accordingly, QSER1 depletion, similar to YAP1, increases NODAL gene expression and leads to hyperactive NODAL signaling in human 2D-gastruloids. Overall, our findings define a role of YAP1 in the epiblast in vivo and uncovered an interplay with QSER1 controlling the activity of developmental signaling pathways in pluripotent cells.

developmental biology↗

bioRxiv2024

A Retinoic Acid:YAP1 signaling axis controls atrial lineage commitment

Vitamin A/Retinoic Acid (Vit A/RA) signaling is essential for heart development. In cardiac progenitor cells (CPCs), RA signaling induces the expression of atrial lineage genes while repressing ventricular genes, thereby promoting the acquisition of an atrial cardiomyocyte cell fate. To achieve this, RA coordinates a complex regulatory network of downstream effectors that is not fully identified. To address this gap, we applied a functional genomics approach (i.e scRNAseq and snATACseq) to untreated and RA-treated human embryonic stem cells (hESCs)-derived CPCs. Unbiased analysis revealed that the Hippo effectors YAP1 and TEAD4 are integrated with the atrial transcription factor enhancer network, and that YAP1 is necessary for activation of RA-enhancers in CPCs. Furthermore, in vivo analysis of control and conditionally YAP1 KO mouse embryos (Sox2-cre) revealed that the expression of atrial lineage genes, such as NR2F2, is compromised by YAP1 deletion in the CPCs of the second heart field. Accordingly, we found that YAP1 is required for the formation of an atrial chamber but is dispensable for the formation of a ventricle, in hESC-derived patterned cardiac organoids. Overall, our findings revealed that YAP1 is a non-canonical effector of RA signaling essential for the acquisition of atrial lineages during cardiogenesis.

developmental biology↗

bioRxiv2024

Single-cell RNA sequencing of mutant whole mouse embryos: from the epiblast to the end of gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, we designed a robust pipeline to perform single-cell and nuclei analysis on mouse embryos from E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to WT perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, we present a methodology designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with FAST genotyping protocol (3 hours) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. We also include guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula. SUMMARYWe establish a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

developmental biology↗

bioRxiv2021

YAP1 Regulates the Self-organized Fate Patterning of hESCs-Derived Gastruloids

During gastrulation, the coordinated activity of BMP, WNT and NODAL signaling pathways guide the differentiation of the pluripotent epiblast into the three germinal layers. Recent studies underline the role of the Hippo-effector YAP1 regulating WNT and NODAL signaling pathways and repressing mesoendodermal differentiation in human embryonic stem cells (hESCs). However, the contribution of YAP1 to the cell-fate patterning decisions that transform the epiblast in a three-germ layer gastrula remains unknown. We address this question by analyzing micropatterned 2D-gastruloids derived from hESCs, in the presence and absence of YAP1. Our findings show that YAP1 is necessary for gastrulation. YAP1 KO-gastruloids display reduced ectoderm layer and enlarged mesoderm and endoderm layers, compared to WT. Furthermore, YAP1 regulates the self-organized patterning of the hESCs, as the discrete position of the three germ layers is altered in the YAP1 KO-gastruloids. Our epigenome (single-nuclei ATACseq) and transcriptome (RNA-seq) analysis revealed that YAP1 directly represses the chromatin accessibility and transcription of key genes in the NODAL pathway, including the NODAL and FOXH1 genes. In WT gastruloids, a gradient of NODAL: SMAD2.3 signaling from the periphery to the center of the colony regulates the exit of pluripotency toward endoderm, mesoderm and ectoderm, respectively. Hence, in the absence of YAP1, a hyperactive NODAL signaling retains SMAD2.3 in the nuclei impeding the self-organized differentiation of hESCs. Accordingly, the partial inhibition of NODAL signaling is sufficient to rescue the differentiation and pattern -defective phenotypes of the YAP1 KO gastruloids. Our work revealed that YAP1 is a master regulator of NODAL signaling, essential to instruct germ layer fate patterning in human gastruloids.

developmental biology↗