Biology subjects

Martinez Arias, A.

Publications and source records attributed to Martinez Arias, A..

11 recordsLinked to original sources

bioRxiv2025

N2B27 media formulations influence gastruloid development

Gastruloids are 3D aggregates of pluripotent stem cells grown in suspension culture that mimic many aspects of gastrulation and early axial elongation. The N2B27 basal medium in which mouse gastruloids are cultured can either be home-made (HM-N2B27) with materials of known origin, or commercially sourced (NDiff227), where the exact formulation is unknown. In this study we examined whether these formulations resulted in significant differences in gastruloid development. Our results reveal that while both media enable the standard gastruloid elongation, HM-N2B27 results in gastruloids that start the elongation process earlier, have higher number of cells and an increased anterior domain. RNAseq analysis showed significant differences in cell fate specification, with HM-N2B27 gastruloids exhibiting higher expression of spinal cord-related genes, while NDiff227 favours mesodermal differentiation. Furthermore, differential gene enrichment analysis suggests that changes in key signalling pathways underline the differences between HM-N2B27 and NDiff227 gastruloids. These findings highlight the importance of basal media composition for gastruloid development, underscoring the need for careful media selection during in vitro engineering of stem cell-based embryo models. Summary statementIn this work we explored the cellular and molecular sensitiveness of the mouse gastruloid model system to variations in N2B27 media formulations.

developmental biology↗

bioRxiv2025

A mosaic gastruloid model highlights the developmental stage-specific restriction of cell competition in mammalian pre-gastrulation

In early mammalian embryogenesis, the selective elimination of suboptimal cells is critical for developmental integrity. Cell competition (CC) is a cell non-autonomous quality control in which "winner" cells eliminate viable but suboptimal "loser" cells based on their relative difference in fitness. Due to its central role in fitness perception, loss of p53 results in the emergence of "supercompetitor" cells, which stand at the apex of cell competition and induce apoptosis in neighboring wild-type (WT) cells. Here, we investigate CC dynamics using mosaic 3D mouse gastruloids, an embryonic stem cell (ESC)-based in vitro model of gastrulation, composed of defined numbers of WT and p53-KO cells. In mosaic gastruloids, even low numbers of p53-KO cells robustly outcompete WT cells, and introduction of as few as two p53-KO cells is sufficient to measurably impair neighboring WT cell growth. CC in gastruloids is independent of cell proliferation rates, nutrient availability, or reactive oxygen species (ROS), and not influenced by Nodal and ERK signaling. However, we observe that Wnt and BMP signaling protect from CC, which is exclusively driven by intrinsic apoptosis, as indicated by Bcl2-mediated complete rescue of WT cells. During gastruloid development, CC is temporally restricted to a window of 48-96 hours after aggregation, mirroring embryonic days E5.5-E7.5 in the mouse. Heterochronic mosaic gastruloid experiments demonstrate that relative differences in pluripotency levels are neither necessary nor sufficient to cause supercompetition, but that CC is contingent on both competitors residing within the developmental window permissive to CC. Neither pluripotent mosaic 3D aggregates, nor 3D EpiGastruloids, which model more advanced developmental processes, display any competition, supporting the hypothesis that developmental CC is specific to the onset of gastrulation. Our findings offer insights into the mechanisms of cell fitness evaluation in mammalian embryogenesis and establish gastruloids as a powerful 3D model for investigating developmental stage-specific cell competition.

developmental biology↗

bioRxiv2025

A temporal coordination between Nodal and Wnt signalling governs the emergence of the mammalian body plan

Nodal and Wnt signalling play an important role in the emergence of the mammalian body plan, primarily by orchestrating gastrulation. While the literature suggests they cooperate to build the primitive streak, their individual contributions remain poorly understood. Using gastruloids, we found that Wnt/{beta}-catenin drives a genetic program characteristic of the late primitive streak, promoting the development of posterior body structures in a time and dose-dependent manner. Conversely, Nodal activates a distinct transcriptional module resembling the early streak. By engineering gastruloids with varying levels of Nodal signalling, we demonstrate that a decreasing temporal gradient of Nodal activity is critical for establishing the anterior body, with higher Nodal levels producing more anterior structures in a concentration-dependent manner. Our findings suggest that Nodal and Wnt act antagonistically, initiating distinct developmental modules within the primitive streak. This antagonism is likely the core mechanism driving the early body plan in mammals.

developmental biology↗

bioRxiv2024

Morphogenetic constrains in the development of gastruloids: implications for mouse gastrulation

Mammalian embryonic size is tightly controlled with checkpoints and compensatory mechanisms correcting size defects. Here, we take advantage of gastruloids, a stem cell embryoid system not subject to most size controls, to study the role of size in emergent properties of mammalian embryogenesis. We report that gastruloids exhibit robust morphology and transcriptional profiles within a size range. However, size affects the dynamics, and, outside a range of robust morphogenesis, the precision of anterior-posterior (AP) axial elongation. Gastruloid axial elongation exhibits active cellular contractility, requires planar cell polarity (PCP), adhesion and cell-cell contact remodelling. Smaller gastruloids initiate elongation earlier, correlated with an earlier Brachyury polarisation. Brachyury expression increases tissue fluidity. Axis formation is regulated by the balance of Brachyury multifoci coalescence and the timing of initiation of the elongation programme. Sizes beyond the robust range can modify relative tissue composition. Very small aggregates have increased neural fate bias, accompanied by a loss of paraxial mesoderm mediated by differences in Nodal signalling activity.

developmental biology↗

bioRxiv2022

Dissecting infant leukemia developmental origins with a hemogenic gastruloid model

Current in vitro models of developmental blood formation lack spatio-temporal accuracy and weakly replicate successive waves of hematopoiesis. Herein, we describe a mouse embryonic stem cell (SC)-derived 3D hemogenic gastruloid (haemGx) that captures multi-wave blood formation, progenitor specification from hemogenic endothelium (HE), and generates hematopoietic progenitors capable of short-term engraftment of immunodeficient mice upon maturation in an in vivo niche. We took advantage of the haemGx model to interrogate the origins of infant acute myeloid leukemia (infAML). We focused on MNX1-driven leukemia, representing the commonest genetic abnormality unique to the infant group. Enforced MNX1 expression in haemGx promotes the expansion and in vitro transformation of yolk sac-like erythroid-myeloid progenitors at the HE-to-hematopoietic transition to faithfully recapitulate patient transcriptional signatures. By combining phenotypic, functional and transcriptional profiling, including at the single-cell level, we establish the haemGx as a useful new model for the study of normal and leukemic embryonic hematopoiesis.

developmental biology↗

bioRxiv2021

In vitro teratogenicity testing using a 3D, embryo-like gastruloid system

Pharmaceuticals that are intended for use in patients of childbearing potential need to be tested for teratogenicity before marketing. Several pharmaceutical companies use animal-free in vitro models which allow a more rapid selection of lead compounds and contribute to 3Rs principles ( replace, reduce and refine) by streamlining the selection of promising compounds that are submitted to further regulatory studies in animals. Currently available in vitro models typically rely on adherent monolayer cultures or disorganized 3D structures, both of which lack the spatiotemporal and morphological context of the developing embryo. A newly developed 3D gastruloid model has the potential to achieve a more reliable prediction of teratogenicity by providing a robust recapitulation of gastrulation-like events alongside morphological coordination at relatively high-throughput. In this first proof-of-concept study, we used both mouse and human gastruloids to examine a panel of seven reference compounds, with associated in vivo data and known teratogenic risk, to quantitatively assess in vitro teratogenicity. We observed several gross morphological effects, including significantly reduced elongation or decreased size of the gastruloids, upon exposure to several of the reference compounds. We also observed aberrant gene expression using fluorescent reporters, including SOX2, BRA, and SOX17, suggestive of multi-lineage differentiation defects and disrupted axial patterning. Finally, we saw that gastruloids recapitulated some of the known in vivo species-specific susceptibilities between their mouse and human counterparts. We therefore suggest that gastruloids represent a powerful tool for teratogenicity assessment by enabling relevant physiological recapitulation of early embryonic development, demonstrating their use as a novel in vitro teratogenic model system.

pharmacology and toxicology↗

bioRxiv2020

Cell state transitions and collective cell movement generate an endoderm-like region in gastruloids

Shaping the animal body plan is a complex process that involves the spatial organization and patterning of the different germ layers. Recent advances in live imaging have started to unravel the cellular choreography underlying this process in mammals, however, the sequence of events transforming an unpatterned cell ensemble into structured territories is largely unknown. Here, using gastruloids -3D aggregates of mouse embryonic stem cells-we study the formation of one of the three germ layers, the endoderm. We show that the endoderm is generated from an epiblast-like homogeneous state by a three-step mechanism: (i) a loss of E-cadherin mediated contacts in parts of the aggregate leading to the appearance of islands of E-cadherin expressing cells surrounded by cells devoid of E-cadherin, (ii) a separation of these two populations with islands of E-cadherin expressing cells flowing toward the aggregate tip, and (iii) their differentiation into an endoderm population. During the flow, the islands of E-cadherin expressing cells are surrounded by cells expressing T-Brachyury, reminiscent of the process occurring at the primitive streak. Consistent with recent in vivo observations, the endoderm formation in the gastruloids does not require an epithelial-to-mesenchymal transition, but rather a maintenance of an epithelial state for a subset of cells coupled with fragmentation of E-cadherin contacts in the vicinity, and a sorting process. Our data emphasize the role of signaling and tissue flows in the establishment of the body plan.

developmental biology↗

bioRxiv2019

Self-organised symmetry breaking in zebrafish reveals feedback from morphogenesis to pattern formation

A fundamental question in developmental biology is how the early embryo breaks initial symmetry to establish the spatial coordinate system later important for the organisation of the embryonic body plan. In zebrafish, this is thought to depend on the inheritance of maternal mRNAs [1-3], cortical rotation to generate a dorsal pole of beta-catenin activity [4-8] and the release of Nodal signals from the yolk syncytial layer (YSL) [9-12]. Recent work aggregating mouse embryonic stem cells has shown that symmetry breaking can occur in the absence of extra-embryonic tissue [19,20]. To test whether this is also true in zebrafish, we separated embryonic cells from the yolk and allowed them to develop as aggregates. These aggregates break symmetry autonomously to form elongated structures with an anterior-posterior pattern. Extensive cell mixing shows that any pre-existing asymmetry is lost prior to the breaking morphological symmetry, revealing that the maternal pre-pattern is not strictly required for early embryo patterning. Following early signalling events after isolation of embryonic cells reveals that a pole of Nodal activity precedes and is required for elongation. The blocking of PCP-dependent convergence and extension movements disrupts the establishment of opposing poles of BMP and Wnt/TCF activity and the patterning of anterior-posterior neural tissue. These results lead us to suggest that convergence and extension plays a causal role in the establishment of morphogen gradients and pattern formation during zebrafish gastrulation.

developmental biology↗

bioRxiv2018

Emergence of a node-like population within an in vitro derived Neural Mesodermal Progenitors (NMPs) population

The mammalian embryos Caudal Lateral Epiblast (CLE) harbours bipotent progenitors, called Neural Mesodermal Progenitors (NMPs), that contribute to the spinal cord and the paraxial mesoderm throughout axial elongation. Here we performed a single cell analysis of different in vitro NMPs populations produced either from embryonic stem cells (ESCs) or epiblast stem cells (EpiSCs) and compared them to E8.25 CLE mouse embryos. In our analysis of this region our findings challenge the notion that NMPs should coexpress Sox2 and T. We built a Support Vector Machine (SVM) based on the embryo CLE and use it as a classification model to analyse the in vitro NMP-like populations. We showed that ESCs derived NMPs are heterogeneous and contain few NMP-like cells, whereas EpiSCs derived NMPs, produce a high proportion of cells with the embryo NMP signature. Importantly, we found that the population from which the Epi-NMPs are derived in culture, contains a nodelike population, which is responsible for maintaining the expression of T in vitro. These results mimic the events in vivo and suggest a sequence of events for the NMPs emergence.

developmental biology↗

bioRxiv2018

An Epiblast Stem Cell derived multipotent progenitor population for axial extension

The Caudal Lateral Epiblast of mammalian embryos harbours bipotent progenitors that contribute to the spinal cord and the paraxial mesoderm in concert with the elongation of the body axis. These progenitors, called Neural Mesodermal Progenitors (NMPs) are identified as cells coexpressing Sox2 and T/Brachyury, a criterion used to derive NMP-like cells from Embryonic Stem Cells in vitro. However, these progenitors do not self renew, as embryonic NMPs do. Here we find that protocols that yield NMP-like cells in vitro first produce a multipotent population that, additional to NMPs, generates progenitors for the lateral plate and intermediate mesoderm. We show that Epiblast Stem Cells (EpiSCs) are an effective source for these multipotent progenitors that are further differentiated by a balance between BMP and Nodal signalling. Importantly, we show that NMP-like cells derived from EpiSCs self renew in vitro and exhibit a gene expression signature similar to that of their embryo counterparts.

developmental biology↗

bioRxiv2017

Gastruloids develop the three body axes in the absence of extraembryonic tissues and spatially localised signalling

Establishment of the three body axes is a critical step during animal development. In mammals, genetic studies have shown that a combination of precisely deployed signals from extraembryonic tissues position the anteroposterior axis (AP) within the embryo and lead to the emergence of the dorsoventral (DV) and left-right (LR) axes. We have used Gastruloids, embryonic organoids, as a model system to understand this process and find that they are able to develop AP, DV and LR axes as well as to undergo axial elongation in a manner that mirror embryos. The Gastruloids can be grown for 160 hours and form derivatives from ectoderm, mesoderm and endoderm. We focus on the AP axis and show that in the Gastruloids this axis is registered in the expression of T/Bra at one pole that corresponds to the tip of the elongation. We find that localisation of T/Bra expression depends on the combined activities of Wnt/{beta}-Catenin and Nodal/Smad2,3 signalling, and that BMP signalling is dispensable for this process. Furthermore, AP axis specification occurs in the absence of both extraembryonic tissues and of localised sources of signalling. Our experiments show that Nodal, together with Wnt/{beta}-Catenin signalling, is essential for the expression of T/Bra but that Wnt signalling has a separable activity in the elongation of the axis. The results lead us to suggest that, in the embryo, the role of the extraembryonic tissues might not be to induce the axes but to bias an intrinsic ability of the embryo to break its initial symmetry and organise its axes.\n\nOne sentence summaryCulture of aggregates of defined number of Embryonic Stem cells leads to self-organised embryo-like structures which, in the absence of localised signalling from extra embryonic tissues and under the autonomous influence of Wnt and Nodal signalling, develop the three main axes of the body.

developmental biology↗