Transcriptional regulation of cell fate determination in the ventral forebrain orchestrated by homeobox transcription factors
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2024
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Melbourne Authors
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Paediatrics (RCH)
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PhD thesis
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This item is embargoed and will be available on 2027-05-29.
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© 2024 Fung Leung
Abstract
Throughout embryonic development, neural stem cells (NSCs) in the forebrain can acquire neuronal, oligodendroglial, or astrocytic cell fates. During early gestation, NSCs predominantly differentiate into neurons, whereas glial cell fates are adopted later. In the developing mouse ventral forebrain, the neural-glial switch occurs at around E13.5-E15.5 and is regulated by a number of transcription factors (TFs). Amongst these TFs is the distalless homeobox 2 (Dlx2) TF, which is an important regulator of the neuronal-glial cell fate switch in the ganglionic eminence (GE), a ventral forebrain structure. DLX2 promotes GABAergic interneuron differentiation and migration, while repressing differentiation of oligodendroglial precursor cells (OPCs). DLX2 promotes interneuron differentiation partly through promoting expression of Gad1/Gad2, genes important for GABA synthesis. DLX2 also regulates interneuron tangential migration where the absence of Dlx1/Dlx2 results in a phenotypic loss of migration across the pallial-subpallial boundary. On the contrary, the mechanism by which DLX2 regulates glial fate is less well understood. Hence, investigating the DLX2 regulatory network is crucial for gaining further insights into the cell fate determination process in the ventral forebrain.
In this thesis, a multiomic approach was adopted to investigate the DLX2 gene regulatory network during embryonic neurogenesis at E12.5-E14.5. DLX2 direct gene targets were identified in E13.5 mouse GE using DLX2 CUT&RUN combined with H3K27ac ChIPseq to identify active promoters and enhancers. Gene expression and chromatin accessibility changes in the GE at this embryonic age was also assessed to examine the regulatory effect of DLX2 on its gene targets. Multiomic snRNAseq and snATACseq were performed using the same cell input, harvested from E12.5-E14.5 embryonic wildtype and Dlx1/Dlx2 double knockout mutants (Dlx1/Dlx2-/-) mouse 6 GEs to examine changes to cell fates and gene regulatory networks. Cell fate changes and gene expression dysregulation in embryonic mouse forebrain in the absence of Dlx1/Dlx2 were also assessed using single cell whole genome spatial transcriptomics. This enabled the exploration of the temporal-spatial context of the DLX2 gene regulatory network in the developing GE.
It was found that DLX2 directly represses the Notch signalling pathway and a range of glial differentiation promoting TFs (Sox9, Olig2, Nfia) in the developing GE. Single-cell analysis revealed that in the Dlx1/Dlx2-/- GE there was an increased in number of OPCs and ventral intermediate progenitors while the number of GABAergic interneurons was reduced. A subpopulation of GABAergic interneurons expressing Sst and Npy, however, was not significantly altered. Through gene regulatory network analysis, it was found that NKX2-1 likely acts independently of DLX1/DLX2 in promoting differentiation of GABAergic interneurons, especially the subpopulation that expresses Sst and Npy. The increased in numbers of OPCs and ventral intermediate progenitors in Dlx1/Dlx2-/- GE correlated with the increased Notch signalling and activities of glial promoting TFs (SOX9, NFIA, RFX4, SOX10). Whole genome based single-cell spatial transcriptomics further characterized that the dysregulated Notch signalling in the Dlx1/Dlx2-/- GE was specific to the subventricular zone. These converging data demonstrate that DLX1/DLX2 controls the neural-glial switch in part by targeting Notch signalling genes in addition to key neural fate determining TFs.
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Keywords
Neurodevelopment, Gene regulatory networks, Forebrain development, Notch signalling pathway