Deciphering the gene-regulatory mechanisms shaping inhibitory neuronal diversity

• Applicant: Dr. Christian Mayer
• Subject Area: Developmental Neurobiology ; Developmental Biology ; Molecular and Cellular Neurology and Neuropathology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 549328218

The mammalian telencephalon contains a tremendous diversity of GABAergic projection neuron and interneuron types that originate in a germinal zone of the embryonic basal ganglia, the ganglionic eminence. It is unclear how this diversity arises from a pool of undifferentiated progenitor cells. Many risk genes for neurodevelopmental disorders encode proteins that regulate important aspects of GABAergic neuron development. A deeper understanding of GABAergic neuron development is essential for comprehending the impacts of genetic mutations in neurodevelopmental disorders. During development, each cell makes a series of fate decisions. They involve complex gene regulatory networks consisting of transcription factors, co-regulators, enhancers and promoters. The result of these interactions is that cells switch between cell states, each defined by a specific gene expression profile. This proposal aims to elucidate the gene regulatory mechanisms that mediate developmental fate decisions leading to cardinal classes of GABAergic precursors through three aims: Aim 1 is to construct a representation that enables the prediction of transcription factors and co-regulators crucial for cell fate decisions. To achieve this, we will bring together existing scRNA-seq and scATAC-seq data from the ganglionic eminence, and use state-of-the-art data analysis tools to reconstruct developmental trajectories, discern fate bifurcation points, and decipher the gene regulatory networks underlying bifurcation points. Aim 2 involves an affinity purification mass spectrometry experiment to identify proteins associated with selected cis-regulatory elements. We have carefully chosen six short genomic sequences from different enhancer regions to design biotinylated synthetic oligos. These oligos will be used to perform a pull-down of associated proteins from nuclear lysates of different precursor states. This will allow us to investigate the composition of transcriptional complexes formed at genomic enhancers. The findings will illuminate the mechanisms through which enhancers integrate inputs from cell-type-specific factors to guide lineage-specific gene expression. Lastly, Aim 3 seeks to study the functional role of candidate transcription factors and cofactors during cell fate decisions. We will employ our CRISPR perturbation sequencing method from Dvoretskova et al. to perturb transcription factors and co-regulators in a sparse population of GABAergic precursors in mouse embryos. Subsequent scRNA-seq combined with barcode lineage tracing will elucidate the impact of perturbations on developmental trajectories and fate choices. In conclusion, this proposal aims to unravel the gene regulatory networks underlying the diversification of GABAergic neurons in the mammalian telencephalon. The research will advance our understanding of how genomic information is translated into diverse cell fates during neural development, shedding light on neurodevelopmental disorders.

• DFG Programme: Research Grants