Deciphering the Regulatory Logic of Cortical Development

Informations projet

EpiCortex

N° de convention de subvention: 101044469

DOI

10.3030/101044469

Date de signature de la CE 27 Juin 2022

Date de début 1 Janvier 2023

Date de fin 31 Decembre 2027

Financé au titre de

• European Research Council (ERC)

Coût total

€ 1 999 643,00

Contribution de l’UE

€ 1 999 643,00

Coordonné par

HELMHOLTZ ZENTRUM MUENCHEN DEUTSCHES FORSCHUNGSZENTRUM FUER GESUNDHEIT UND UMWELT GMBH
Germany

Periodic Reporting for period 1 - EpiCortex (Deciphering the Regulatory Logic of Cortical Development)

Période du rapport: 2023-01-01 au 2025-06-30

Résumé du contexte et des objectifs généraux du projet

The mammalian cortex, with its complexity and central role in higher cognitive functions, presents one of the most intricate challenges in developmental biology. Abnormalities in cortical development are implicated in a range of neuropsychiatric disorders, yet our understanding of the molecular mechanisms driving neuronal subtype specification remains limited. The EpiCortex project was conceived to tackle this gap by integrating advanced single-cell multiomics techniques—including scRNA-seq, scATAC-seq, and Hi-C—with functional assays like in vivo MPRA and CRISPR perturbations.

At its core, the project seeks to decode the molecular logic underlying temporal cell fate decisions in the developing cortex. By comprehensively mapping the regulatory landscape—spanning gene expression, chromatin topology, and epigenetic modifications—the project aims to illuminate how distinct neuronal identities emerge during development. This includes identifying key cis-regulatory elements, understanding enhancer–promoter interactions, and delineating the dynamics of DNA methylation and chromatin accessibility.

The expected impact of this research is multifold. Scientifically, it will provide unprecedented insights into the fundamental processes governing neuronal differentiation and the interplay of multiple regulatory layers. Methodologically, the development of novel tools such as the cell-type–specific in vivo MPRA and 3DRAM-seq positions the project at the cutting edge of functional genomics, with potential applications across diverse tissues and model organisms. Furthermore, the findings have significant translational potential; a deeper understanding of cortical development may inform therapeutic strategies for neuropsychiatric diseases and aid in the design of regenerative approaches.

In summary, EpiCortex sets the stage for a paradigm shift in how we understand and manipulate the regulatory networks that control cell fate decisions in the brain, with implications that extend to both basic science and clinical applications.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

Our project was built upon a comprehensive multiomic strategy to decode the molecular logic underlying neuronal subtype specification in the mammalian cortex. Key technical and scientific activities performed include:

- Multiomic Profiling:
We generated high-resolution single-cell RNA and chromatin accessibility datasets from tens of thousands of cells, enabling us to map the regulatory landscape of cortical development. This robust dataset has allowed us to dissect progenitor heterogeneity and chart differentiation trajectories.

- Computational Advances:
A novel computational framework was developed to identify lineage trajectories and temporally regulated genes and enhancers. This tool has been instrumental in unraveling the dynamic regulatory networks that govern neural differentiation.

- Development of 3DRAM-seq:
We introduced 3DRAM-seq—a method that simultaneously profiles chromatin accessibility, DNA methylation, and 3D genome organization. Applied to human cortical organoids as a conceptual follow-up to our original model, this technique has provided unprecedented insights into enhancer–promoter interactions and epigenetic dynamics during neuronal specification.

- In Vivo Functional Assays:
We successfully adapted the massively parallel reporter assay (MPRA) for in vivo, cell-type–specific measurement of enhancer activity. This approach was further refined for use in human cortical organoids, enabling the profiling of thousands of human enhancers and leading to the discovery of novel principles of transcription factor binding and synergy.

- Functional Perturbation Studies:
Utilizing CRISPR-based methods, we performed functional perturbations to assess the importance of cis-regulatory elements. These studies revealed insights into the robustness of gene regulatory networks, including compensatory mechanisms such as "shadow" enhancers.

- Collaborative Extensions:
Complementary investigations employing similar experimental and computational approaches were conducted to study direct neuronal reprogramming. These efforts uncovered novel mechanisms of epigenome remodeling and identified essential regulatory factors critical for cellular reprogramming.

At the end of the project, the outcomes include a robust multiomic dataset, novel methodological tools (3DRAM-seq and in vivo MPRA), and significant insights into the regulatory dynamics of neuronal specification. These achievements not only advance our fundamental understanding of cortical development but also provide a valuable foundation for future research in regenerative medicine and neuropsychiatric disorders.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

Our work has produced transformative results that push the boundaries of current methodologies and scientific understanding in the field of developmental neurobiology:

- Technological Breakthroughs:
The introduction of 3DRAM-seq represents a significant advance in multiomic profiling by integrating chromatin accessibility, DNA methylation, and 3D genome organization into a single assay. Similarly, the in vivo MPRA assay, with its unique cell-type specificity, offers a new paradigm for high-throughput functional assessment of enhancers directly in their native tissue context. These tools extend beyond conventional approaches and open new avenues for systematic studies of gene regulation.

- Scientific Impacts:
Our integrated multiomic approach has provided novel insights into enhancer–promoter interactions and the epigenetic mechanisms driving neuronal specification. The computational models and perturbation studies have advanced our understanding of how regulatory networks function, particularly in the context of redundant enhancer activity and compensatory mechanisms. Moreover, our collaborative extension into neuronal reprogramming has identified key regulatory factors, further broadening the impact of our research.

At the end of the project, our results have not only advanced the state-of-the-art in developmental neurobiology and epigenetics but also laid the groundwork for future innovations in understanding and manipulating gene regulatory networks. These achievements are expected to drive further research, foster international collaborations, and ultimately contribute to new therapeutic strategies for neurodevelopmental and neuropsychiatric disorders.