In Vitro Model of Vertebrate Trunk Development

Academic Background

The development of the vertebrate trunk is a highly coordinated process involving the generation and organization of multiple cell types. At the core of this process are progenitor cells located in the posterior of the embryo, which differentiate into tissues such as the neural tube, somites, and notochord through complex signaling networks. The notochord, a defining feature of chordates, not only provides mechanical support during embryonic development but also regulates the patterning of surrounding tissues through the secretion of signaling molecules. However, existing in vitro models, such as those using pluripotent stem cells (PSCs), while capable of mimicking certain aspects of trunk development, often lack the notochord and its dependent tissues, such as the floor plate of the neural tube. This limitation has hindered the application of these models in studying the mechanisms underlying vertebrate trunk development.

To address this gap, a research team from The Francis Crick Institute conducted single-cell transcriptomic analysis to map the molecular landscape of trunk development in chick embryos. Based on this map, they developed a novel in vitro model that successfully induced the differentiation of human pluripotent stem cells into three-dimensional trunk structures containing a notochord. This study not only sheds light on the mechanisms of notochord formation in vertebrates but also provides a new tool for future research on tissue patterning in physiologically relevant environments.

Research Team and Publication Information

The study was conducted by Tiago Rito, Ashley R. G. Libby, Madeleine Demuth, Marie-Charlotte Domart, Jake Cornwall-Scoones, and James Briscoe from The Francis Crick Institute in London, UK. The research paper, titled “Timely TGFβ signalling inhibition induces notochord,” was published online in Nature on November 1, 2024.

Research Process and Experimental Design

1. Single-Cell Transcriptomic Analysis

The research team first performed single-cell transcriptomic analysis on the caudal regions of chick embryos at stages corresponding to 4, 7, 10, and 13 somites (Hamburger-Hamilton stages 8–11). Using Louvain clustering and marker gene expression analysis, they defined multiple cell types, including the primitive streak, early mesoderm, presomitic mesoderm, lateral plate mesoderm, and notochord cells. Notochord cells were identified by the expression of specific marker genes such as TBXT, NOTO, SHH, and CHRD.

2. Construction of the In Vitro Model

Based on the transcriptomic data from chick embryos, the research team developed an in vitro model using human embryonic stem cells (hESCs) to mimic trunk development. They first activated Wnt and FGF signaling pathways in monolayer cultures while inhibiting BMP and Nodal signaling, successfully inducing SOX2+TBXT+ neuromesodermal progenitors (NMPs). Subsequently, the researchers cultured hESCs on micropatterned laminin substrates and observed that the cells formed an ordered pattern of gene expression under geometric constraints, with SOX2-high neural progenitor cells in the center and TBXT-high mesodermal cells at the periphery.

3. Regulation of Signaling Pathways

To further dissect the role of signaling pathways in cell fate determination, the research team analyzed the dynamics of YAP, ERK1/2, and Wnt signaling during the development of neuruloids. They found that YAP inactivation promoted Wnt signaling activation and TBXT expression, while sustained MAPK pathway activity maintained TBXT expression at the periphery. Additionally, the timing of BMP and Nodal signaling inhibition was found to be critical for the formation of notochord-like cells. Delayed inhibition of BMP and Nodal signaling significantly increased the proportion of TBXT+FOXA2+ notochord-like cells.

4. Generation of 3D Trunk Organoids

Based on these findings, the research team applied the signaling conditions to three-dimensional cultures, successfully generating trunk organoids (notoroids) containing a notochord. These organoids elongated over time, with TBXT+ notochord-like structures forming in the interior and SOX2+ neuroepithelial cells in the outer layer. Single-cell RNA sequencing further validated the presence of cell types such as notochord cells, paraxial mesoderm, and neural progenitors in the organoids.

Key Findings

1. Single-Cell Transcriptomic Analysis: The research team successfully mapped the molecular landscape of trunk development in chick embryos, defining multiple progenitor populations and their spatial distribution, particularly the transcriptional signatures of notochord progenitors.

2. Construction of the In Vitro Model: By modulating Wnt, FGF, BMP, and Nodal signaling, the researchers induced the differentiation of human pluripotent stem cells into three-dimensional trunk structures containing a notochord.

3. Regulation of Signaling Pathways: The study revealed that YAP inactivation and sustained MAPK pathway activity are key factors in TBXT expression and notochord formation. Additionally, the timing of BMP and Nodal signaling inhibition significantly influenced the proportion of notochord-like cells.

4. Generation of 3D Trunk Organoids: The research team successfully generated trunk organoids containing a notochord, which exhibited tissue structures and gene expression patterns similar to those observed in vivo.

Conclusions and Significance

Through single-cell transcriptomic analysis and the construction of an in vitro model, this study elucidated the molecular mechanisms underlying notochord formation in vertebrates and developed a novel model capable of mimicking key processes in human trunk development. This research not only provides new insights into vertebrate trunk development but also offers an important tool for future studies on tissue patterning and disease modeling in physiologically relevant environments.

Research Highlights

1. Molecular Mechanisms of Notochord Formation: The study revealed the synergistic role of YAP, Wnt, and FGF signaling in notochord formation, particularly the promotion of TBXT expression by YAP inactivation.

2. Development of a Novel In Vitro Model: The research successfully constructed three-dimensional trunk organoids containing a notochord, filling a gap in existing in vitro models regarding the notochord and its dependent tissues.

3. Timing of Signaling Regulation: The study demonstrated that the timing of BMP and Nodal signaling inhibition is critical for notochord formation, providing important guidance for optimizing in vitro differentiation conditions.

Additional Valuable Information

The research team also detailed the cell types and tissue structures of the three-dimensional trunk organoids through single-cell RNA sequencing and imaging analysis, further validating the physiological relevance of the model. Additionally, the study found that notochord-like cells could regulate the patterning of surrounding neural tissues through the secretion of SHH signaling molecules, consistent with in vivo developmental processes.

In summary, this study provides new tools and insights for research on vertebrate trunk development, with significant scientific and practical value.