Periodic Reporting for period 4 - CERDEV (Transcriptional controls over cerebellar neuron differentiation and circuit assembly)
Reporting period: 2022-08-01 to 2023-01-31
The CERDEV project tackles the complex molecular principles underlying the development of cerebellar neurons and their subtype-specific identities, which are essential for proper brain function but remain poorly understood. The cerebellum, a highly conserved brain structure across mammals, plays a vital role in modulating motor outputs, making understanding these principles crucial for society and potentially informing future research on cerebellar disorders. The project's overall objectives are to investigate the transcriptional programs governing the generation, diversity, and developmental circuit assembly of cerebellar neurons, with a particular focus on Purkinje cells (PCs), granule cells (GCs), and molecular layer interneurons (MLIs).To achieve these objectives, we used cutting-edge techniques such as FlashTag, single-cell RNA sequencing and ATAC-seq, and spatial transcriptomics to create a pioneering 4D atlas of the mouse cerebellum during development. This atlas provides a comprehensive insight into the cerebellum's development by placing cells within their spatial and temporal context, offering a more complete understanding of cerebellar neuron diversity. Furthermore, we developed in silico methods to study the postnatal differentiation and circuit assembly of neurons, providing valuable insights into cerebellar disorders and potentially informing future studies on neuron development in other brain areas. Overall, the CERDEV project contributes significantly to our understanding of the intricate relationships between neuron birth, molecular diversity, and spatial distribution, paving the way for advances in neuroscience and the study of neurological conditions.
In the first part of the project, we successfully used the FlashTag method to label cerebellar progenitors and newborn neurons. By combining various techniques, we created a groundbreaking 4D-atlas of the mouse cerebellum, covering embryonic and postnatal development. This atlas offers a comprehensive understanding of cerebellum development by placing cells within their spatial and temporal context. Our findings also identified Neurod2 as a key transcription factor responsible for the birthdate of GABAergic cerebellar neurons. In the second part of the project, we investigated the complex regional diversity of Purkinje cells (PCs) during development. We found that the identity and distribution of PCs are determined by their birthdate, and cell cycle kinetics of progenitor cells may contribute to fate restriction. Our analysis revealed preferential clustering of PCs based on their spatial distribution in the cerebellar cortex and spatially-defined enrichment of molecular markers. Finally, we faced initial delays to developed the last part, leading to the development of the FlashTag 2.0 approach to track and genetically manipulate newborn neurons using lipid nanoparticles. By examining datasets from the mouse telencephalic brain, we generated a comprehensive neuronal atlas of the developing mouse somatosensory cortex and constructed a ligand-receptor atlas between cell types, which revealed interactions that may guide the stereotypical lamination and connectivity of neuronal cell types.
The newly generated 4D atlas and our Ligand/receptor model will reveal previously unknown programs supporting neuronal development and neuronal assembly. Beyond their fundamental importance in understanding how cerebellar circuits are established and genetically controlled, we are certain that these findings will pave the road for future studies aiming at understanding how miss-wirings occur in mouse models of cerebellar disorders.