Genetic fate specification of inhibitory neurons

Ho, May Cheng-Ching (2024): Genetic fate specification of inhibitory neurons. Dissertation, LMU München: Fakultät für Biologie

Abstract

Understanding how an enormous diversity of neuronal cell types is generated has been a major objective of neurobiology. This task is particularly challenging in the case of inhibitory neurons because they migrate long distances during development. It is believed that a combi-nation of intrinsic factors and external signals influence progenitor cells to differentiate into distinct types of inhibitory cells, such as interneurons or long-range projection neurons. To tackle this issue, one approach involves examining the clonal relationships between inhibitory cell types in the brain. In this thesis, I established a single-cell RNA sequencing compatible, lineage-tracing method, TrackerSeq, that enables both the identity of a neuron and its developmental history to be retrieved simultaneously to analyze developmental relationships of inhibitory cell types in the mouse brain. TrackerSeq achieves this by tagging progenitors with inheritable DNA barcodes followed by transcriptome sequencing at a later time point to ana-lyze developmental relationships of inhibitory cell types in the mouse brain. Using TrackerSeq, I found different inhibitory cell types occupying different regions in the brain shared inherited the same lineage barcodes, suggesting that mitotic progenitors can give rise to different cell types. Subsequently, I explored whether specific transcription factors expressed in inhibitory neurons, such as Meis2 and Lhx6, play a crucial role in determining the fate of inhibitory cell types. Single-cell sequencing compatible perturbation methods, like tCROP-seq, have emerged as an effective way to interrogate the impact of these factors on the outcome of neuronal fates. In a typical tCROP-seq protocol, sgRNAs are delivered to cycling progenitors via in utero electropo-ration to introduce fameshift mutations in genes of interest, followed by sequencing of neurons at a later timepoint. By analyzing the tCROP-seq data obtained from perturbing Meis2, I observed that interneu-ron genes were upregulated in projection neuron cell types, leading to an increased proportion of interneurons. Interestingly, perturbing Lhx6 had the opposite effect. These findings suggest that when Meis2 is perturbed, progenitor cells originally destined to become projection neurons may instead differentiate into interneurons. To confirm this possibility, I employed TrackerSeq barcodes to tag non-perturbed and Meis2-perturbed cells. The analysis revealed that Meis2-perturbed mitotic cells shared more clones with interneurons than projection neurons, providing further evidence that Meis2 perturbation promotes the preferential differentiation of progenitor cells into interneurons. My findings reveal that specification of inhibitory subtypes already takes place at the pro-genitor stage and require the expression of select transcription factors. Gaining a better under-standing of how genetic programs such as lineage and transcription factor expression influence subtype specification can improve our modeling of neurodevelopmental disorders.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: neurodevelopment, single-cell sequencing, genomics
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 7. Februar 2024
• 1. Bericht­erstatter:in: Klein, Rüdiger