Size-dependent peri-gastruloid model to investigate the regulatory factors influencing self-organization

Introduction: : Gastrulation is an early developmental process in which the body plan is determined, leading to the specification of the germ layer and orthogonal axes. Recently, gastruloids have emerged as an in vitro model of gastrulation and have proved vital to understanding inaccessible processes underlying human development. The successful formation of gastruloids from embryonic stem cell (ESC) aggregates depends on several factors, including initial cell seeding size, culture conditions, and soluble factors. While previous studies have demonstrated elongated morphogenesis along the anterior-posterior (A-P) axes with defined chemical cues in mouse embryonic stem cells, reproducibly generating these structures using human induced pluripotent stem cells (hiPSCs) remains elusive. This is due to the limited information about the regulatory factors and mechanisms that lead to tissue patterning and epithelial to mesenchymal movements involved in gastrulation. For example, failure in asymmetric breaking in gastrulation can cause congenital defects, including spina bifida and heart defects. Hence, here we seek to establish a peri-gastruloid model, which contains epiblast-like and hypoblast-like structures, mimicking peri-gastrulation development in a human-relevant system. Our research focuses on studying the chemical and homotypic cell-cell interactions during early embryo development to understand the mechanisms behind gastrulation defects.

Materials and

Methods: : To examine the role of initial aggregate size on peri-gastruloid formation, dissociated hiPSC aggregates seeded at 50 or 100 cells per microwell were stained for yes-associated protein (YAP), phosphorylated myosin light chain (pMLC), and E-cadherin at day 2 of differentiation. Developmental patterning was evaluated by immunostaining endoderm (FOXA2, GATA6), pluripotent (SOX2, OCT4), and mesoderm (Brachyury) at day 5 of differentiation. To investigate the molecular mechanisms of size-dependent patterning, aggregates were treated with small-molecule inhibitors of BMP, YAP, and myosin II. Treated aggregates were stained on day 2 for CER1 (a marker of NODAL antagonism), YAP, and PMLC, and on day 5 for FOXA2 and SOX2 to evaluate morphological and patterning outcomes. To investigate the role of cell-cell adhesion in size-dependent patterning, E-cadherin was knocked down in 2D iPSC cultures using siRNA before aggregation. Aggregates were collected on day 2 to validate knockdown efficiency by quantitative RT-PCR and confocal microscopy, targeting E-cadherin, YAP, and PMLC. In parallel, bulk RNA sequencing was performed on control and E-cadherin knockdown samples to identify transcriptional changes and signaling pathways that contribute to disrupted patterning in the peri-gastruloid model.

Results, Conclusions, and Discussions:: Our preliminary results indicate that cytoskeletal tension, chemical signaling, and mechanotransduction play critical roles in coordinating size-dependent peri-gastruloid development and morphogenesis. Notably, small aggregates exhibited augmented YAP nuclear localization and upregulated levels of BMP antagonists, which would imply a higher mechanical sensitivity and altered signaling landscape compared to large aggregates. Functional experiments confirm the participation of mechanical forces in morphogenesis coordination: inhibition of YAP activity, actomyosin contractility, or BMP signaling all interrupted spatial patterning and elongation during peri-gastruloid formation. These findings collectively demonstrate that mechanical and biochemical signals are tightly integrated in a size-dependent manner to guide early gastrulating body events. To better investigate these mechanisms, we will explore the role of mechanical forces in different-sized aggregates in regulating E-cadherin-mediated cell-cell adhesion. As E-cadherin is important for tissue integrity and mechanical signal transmission, regulation in a size-dependent manner may provide new insights into how patterning and morphogenesis are coordinated early on. In general, our work highlights aggregate size, as a determinant of elongation and patterning competence during the peri-gastruloid process. Beyond enhancing our understanding of human gastrulation-like processes, this work provides the foundation for exploring developmental pathology related to defective cell adhesion or mechanical signaling.

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