Neuronal maturation is an intricate and tightly-controlled process in neurodevelopment, during which neuronal cells acquire their mature molecular, morphological and electrophysiological properties to function as the central components of the nervous system. However, the cell-intrinsic mechanisms and transcriptional programs that govern it remain poorly understood. To address this gap in knowledge, the objective of this thesis was to identify the transcription factors (TFs) that control neuronal maturation and elucidate the pathways through which they exert their function.

The present work takes advantage of the recent discovery that Neurogenin 2 (NGN2) overexpression in induced pluripotent stem cells results in their rapid conversion into excitatory induced neurons (iNeurons) that are morphologically complex and electrophysiologically active after 2 weeks in culture. In addition, the inducible control of NGN2 expression from genomic safe harbour sites has provided a deterministic model system, enabling the generation of pure neuronal cultures within days, with minimal variability. Culturing iNeurons together with primary rat glia results in more mature networks with early synchronous burst activity, as measured on multi-electrode arrays (MEAs).

Candidate master-regulators of iNeuron maturation were identified using TF motif prediction tools and a dataset combining ATAC-seq and RNA-seq analysis of a granular time-course experiment during NGN2 reprogramming, in the presence and absence of glia. Predicted TFs were found to also be expressed during human cortical development and mutations in them are enriched in neurodevelopmental disorders, suggesting potential functional roles in vivo.

To simultaneously investigate the function of 25 potential master-regulator TFs, I conducted a pooled CRISPR knock-out (KO) screen with single-cell transcriptome readout (CROP-seq screen) in iNeurons cultured with glia. Differential expression analysis implicated TFs in cellular processes hypothesized to promote neuronal maturation, including autophagy, oxidative stress response, cellular respiration and lipid metabolism. To assess the effects of transcription factor KO on the maturity of iNeurons, single-cell RNA-sequencing reads were analysed with a composite metric, namely neuronal maturity index, which suggested incomplete transcriptome transition towards mature states for a number of gene KOs.

I next generated KO lines for the transcription factors CUX2, ZNF384, FOSL2, PATZ1 and ONECUT2 for further in-depth investigation. MEA analysis demonstrated notable impairments in iNeuron electrophysiology, while neurite and synaptic connectivity profiling revealed stunted neurite outgrowth and synapse maturation, respectively. Lastly, the investigation of cell metabolism revealed that PATZ1-KO iNeurons displayed decreased cellular respiration, ROS levels and autophagic activity, which may be directly linked to the maturation deficits.

In conclusion, the present work highlights the importance of transcriptional regulation in the process of neuronal maturation. It identified and confirmed the functional role of a number of transcription factors, enabling not only a mechanistic understanding of neuronal maturation but also providing a basis for further research in neurodevelopmental conditions.