Molecular diversification of inhibitory neurons during development

Project Information

GIDE

Grant agreement ID: 803984

DOI

10.3030/803984

Project closed

EC signature date 9 October 2018

Start date 1 January 2019

End date 31 December 2024

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 493 382,00

EU contribution

€ 1 493 382,00

Coordinated by

MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Germany

Periodic Reporting for period 4 - GIDE (Molecular diversification of inhibitory neurons during development)

Reporting period: 2023-07-01 to 2024-12-31

Summary of the context and overall objectives of the project

The human brain contains a vast diversity of neurons that must assemble into functional circuits to regulate behavior, perception, and cognition. A crucial component of this network is inhibitory neurons, which balance excitatory activity and shape neural computations. Disruptions in inhibitory neuron development have been linked to neurodevelopmental disorders such as autism and schizophrenia, but the fundamental mechanisms that generate different inhibitory neuron types remain poorly understood.

This lack of understanding presents a major gap in neuroscience. To build a fully functional brain, inhibitory neurons must acquire precise identities and integrate into circuits in a highly coordinated manner. If the developmental programs guiding this process are altered, the resulting miswiring of brain networks can lead to cognitive and behavioral dysfunction. Identifying the mechanisms that control inhibitory neuron diversification is therefore not only essential for understanding brain function but also for uncovering the origins of neurological and psychiatric disorders.

The objective of this project was to determine how inhibitory neurons acquire their distinct identities during brain development. To achieve this, we aimed to reconstruct the lineage relationships between individual inhibitory neurons by using genetic barcoding approaches. By tagging progenitor cells with unique molecular barcodes, we could track how their descendant neurons diversified into distinct subtypes. This approach provided a means to directly test whether inhibitory neuron diversity is determined by lineage relationships established during early development or emerges progressively as neurons mature.

Beyond lineage tracing, the project also aimed to define the gene regulatory programs that control inhibitory neuron differentiation. A key goal was to identify which transcription factors and regulatory elements govern fate decisions, and how these genetic programs interact with neuronal activity to refine inhibitory neuron identity. Understanding these processes is crucial because it provides a molecular framework for how inhibitory neurons are generated and functionally integrated into neural circuits.

By addressing these objectives, this project aimed to bridge a major gap in developmental neuroscience, providing fundamental insights into how inhibitory neurons form and how alterations in these processes contribute to brain disorders.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

This project successfully addressed its main objectives, advancing our understanding of how inhibitory neurons acquire their distinct identities during brain development. By combining molecular lineage tracing, single-cell genomics, and functional perturbation approaches, we investigated the genetic and regulatory mechanisms that shape inhibitory neuron diversity.

A key achievement was the development of genetic barcodes to reconstruct the lineage relationships of inhibitory neurons. By introducing unique molecular barcodes into progenitor cells, we were able to track how their descendant neurons diversified into distinct subtypes. This approach provided new insights into the extent to which inhibitory neuron identity is influenced by lineage relationships versus regulatory programs that act as neurons mature (Bandler et al., Nature, 2022; Bandler et al., Current Opinion in Neurobiology, 2023).

To define the molecular mechanisms underlying inhibitory neuron differentiation, we applied single-cell transcriptomic and functional perturbation approaches to identify key transcription factors and gene regulatory elements that govern fate decisions. By integrating genetic perturbations with single-cell sequencing, we mapped transcriptional programs that control inhibitory neuron fate and identified enhancers that activate lineage-specific gene expression. These findings refine our understanding of how combinatorial transcription factor interactions drive inhibitory neuron diversity, with a particular focus on differential transcription factor binding to determine lineage-specific gene expression (Dvoretskova et al., Nature Neuroscience, 2024).

We also examined the role of neuronal activity in inhibitory neuron development by selectively altering membrane potential. Our results indicate that, in contrast to excitatory neurons, inhibitory neuron maturation is less dependent on membrane potential changes during early differentiation (Bright et al., BioRxiv, 2025).

The findings of this project contribute to both basic neuroscience and the study of neurodevelopmental disorders by providing insights into how inhibitory neuron diversity emerges. Since inhibitory neuron dysfunction is implicated in conditions such as autism and schizophrenia, understanding the mechanisms underlying their development may help inform future research on these disorders. The results have been published in peer-reviewed journals, presented at international conferences, and shared with other research groups. The methodologies and genetic tools developed here are now being applied in diverse biological systems, including studies on brain tumors and non-mammalian models. Through these efforts, this project has established a foundation for further studies on inhibitory neuron development and its broader implications in neuroscience.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

This project advanced the study of inhibitory neuron development by resolving key open questions about how these neurons acquire distinct identities. Previous models were based on population-level analyses, leaving uncertainty about how individual progenitors contribute to inhibitory neuron diversity. By implementing genetic barcoding and single-cell transcriptomic approaches, this work provided a high-resolution view of lineage relationships and the molecular programs shaping inhibitory neuron fate.

A major step forward was the ability to track inhibitory neurons from progenitor stages to their differentiated states. Genetic barcodes enabled the reconstruction of clonal family trees, revealing lineage influences on fate decisions. Combined with functional perturbations and enhancer analysis, this work identified key transcriptional programs and regulatory elements controlling inhibitory neuron differentiation, offering a more mechanistic understanding than previously available.

This project also examined membrane potential in mitotic progenitors, revealing differences between inhibitory and excitatory neuron lineages. While excitatory neuron development is influenced by membrane potential changes, inhibitory neuron progenitors displayed distinct electrical properties early in development, suggesting differences in how these neuronal classes acquire their identities.

The methodologies developed — combining lineage tracing, single-cell sequencing, and gene perturbations — have set a foundation for future studies and are now being adapted to research on neurodevelopmental disorders and comparative models of brain evolution. This project has provided both new biological insights and widely applicable experimental tools that will support future investigations in neuroscience and beyond.

Clonal divergence of ventrally derived inhibitory neurons