Thesis, FRANZEN Rachelle

Abstract

The cerebral cortex is the outer layer of the brain, responsible of higher cognitive functions. It is populated by two main types of neurons: i – the excitatory projection neurons which, during development, migrate radially through the cortex; and ii – the inhibitory interneurons (cINs) that originate ventrally at the medial and the caudal ganglionic eminence (MGE and CGE, respectively) and preoptic area and migrate tangentially to the developing cortex. cINs migrate via nucleokinesis: a saltatory movement characterized by a nuclear jump. During this process, they respond to extracellular diffusible cues released by surrounding cells, which allows intercellular communication that will influence their trajectory and promote their survival. Cortical interneurons also secrete signals that will influence neighboring cells, such as intermediate progenitors (IPs), by promoting their proliferation (REF). The identity of these cues has not yet been elucitated, and whether they are conserved between mice and human remains unknown. The aim of my thesis is to characterize a novel human ventral forebrain organoid model to study cINs migration, and to set a protocol to study the diffusible cues that modulate their migration. Here, I characterized them by immunolabaling to identify ventral progenitors (NKX2.1, SOX2), immature interneurons (TUJ1), MGE mature cINs (SST28, GAD67), and oligodendrocyte precursor cells (OPCs) (SOX10). In addition I performed RT-qPCRs at different time points of the protocol to track the maturation of cINs. Results revealed that gene expression is stable between cell lines, including some genes of interest such as NKX2.1, DLX5, DCX and GAD67, further confirming the ventral forebrain identity. The second part of my thesis focuses on developing a protocol to collect the secretome of migrating cINs derived from human ventral organoids. The secretome refers to the complete set of proteins actively secreted by a cell into the extracellular space. To achieve this goal, GFP+ cINs were isolated from other progenitor populations by FACS after infection of the organoids with a DLX5/6-GFP lentivirus. Once sorted and put in culture, cells undergo two starvation periods before medium collection. The collected samples were sent to the mass spectrometry platform at the University of Liège for analysis. This platform will provide a list of proteins specifically secreted by interneurons. Identified candidates will subsequently be screen bibliographically and analyse by bioinformatic approaches to assess their potential roles in the crosstalk occurring between IPs and cINs. The protocol is now established, and we expect to identify proteins associated with cell proliferation or migration.