The role of Nuclear Factor One transcription factors in cerebellar development

Abstract

The cerebellum is a unique and often overlooked component of the central nervous system, with a lobular and multi-layered structure. It consists of a dense interwoven network of interacting neurons and glia with only one major output; through Purkinje neuron afferent projections. Upwards of 80% of the cells in the cerebellum are cerebellar granule neurons (CGNs). CGNs arise from highly proliferative, MATH1+ progenitors (CGNPs) in the embryonic brain, found in a germinal region called the rhombic lip (RL). RL progenitors respond to mitogenic signals and cues initiated by transcription factors and migrate along the cerebellar anlage, forming the external granule layer (EGL). Mitogens such as Sonic Hedgehog (SHH), encourage these progenitors to proliferate in the EGL, before they differentiate and migrate inwards forming the internal granule layer (IGL). This migration results in eventual disintegration of the EGL early in postnatal development. One family of transcription factors identified to play a role in self-renewal and differentiation of stem and progenitor populations is the Nuclear Factor One (NFI) family.NFIA, NFIB and NFIX are expressed throughout the central nervous system (CNS) and are essential for normal development. NFI-mutant mice are embryonic or postnatal lethal, with a number of developmental defects. In the cerebellum, loss of NFIX has been shown to cause delays in the development of both neurons and glia. As the cerebellum consists of almost a dozen different cell types, the first step of this thesis was to examine the cell-type specific expression of NFIX. Immunohistochemistry and co-immunofluorescence analysis revealed that NFIX is strongly expressed in proliferative progenitors in the RL, embryonic and postnatal EGL, as well as in post-mitotic CGNs in the IGL. NFIX was also expressed in Bergmann glia, and GFAP expressing astrocytes in the IGL and white matter. NFIX was expressed in stellate and basket interneurons, as well as in subpopulations of unipolar brush cells, microglia and oligodendrocytes. Interestingly, NFIX was not expressed in Purkinje neurons, despite strong NFIX expression in the ventricular and nuclear transitory zone of the embryonic cerebellum. Lastly, analysis revealed the presence of four NFIX transcript variants in isolated CGNPs. Together these findings suggest NFIX may play an extensive role in cerebellar development, especially in the maturation of CGNs.Nfix-/- mice display delays in cerebellar granule neuron progenitor (CGNP) differentiation, yet the mechanisms behind this delay are yet to be determined. Using co-immunofluorescence staining with granule neuron and proliferative markers, we determined that a delay EGL differentiation is present at P15, with a higher number of proliferative and mitotically active CGNPs. Furthermore, in vitro analysis revealed increased proliferation of Nfix-/- neurospheres, compared to wild-type controls. As CGNPs are the cell of origin of some medulloblastomas (MB), and loss of NFIA expression has led to poorer outcomes in mouse models of MB, we decided to look at NFIX expression in human MB cell lines. NFIX expression was markedly reduced, suggesting that NFIX is crucial for CGNP differentiation. Lastly, to identify mechanistic determinants of this developmental delay, we performed both ChIP-seq for NFIX and RNA-seq on Nfix-/- and control CGNPs at P7. Combining these data with a DNase I hypersensitive site dataset (accessible chromatin) and an RNA-seq dataset from Math1-/- CGNPs, we revealed 578 directly regulated gene targets of NFIX in CGNPs, of which 90 were co-ordinately regulated by Math1. One of these downstream targets was the Reelin-pathway scaffold protein Itsn1. We showed that like Nfix, loss of Itsn1 results in a delay in CGNP differentiation, potentially exacerbated Nfix-mediated manipulation of Itsn1 binding partner Dab1. Lastly, we also found that ITSN1 expression was downregulated in human MB cell lines. Collectively, this showed that NFI-mediation of downstream targets genes is a crucial in the regulation of CGNP differentiation,Previous analysis has shown that NFI expression overlaps in progenitor populations in the developing CNS, suggesting a redundant, compensatory or synergistic mechanism may direct progenitor cell differentiation. Indeed, using co-immunofluorescence staining we found that NFIA and X expression overlaps in both CGNPs, postnatal CGNs and Bergmann glia throughout postnatal development. As co-expression suggests the potential for an overlapping role in development, we performed RNA-seq in Nfiafl/fl; Math1-cre+ CGNPs, and compared this with our Nfix-/- RNA-seq dataset from the previous chapter, to reveal co-ordinately mis-regulated gene targets of NFIA and NFIX. Then, combining these data with a DNase I hypersensitive site dataset, and NFIX, NFIA and NFIB ChIP-seq datasets in P7 CGNPs, we reveal 304 directly regulated gene targets of NFIA and NFIX, of which 283 show co-ordinate regulation, Additionally, 282 of these contain an NFIB-associated ChIP-peak. Further examination of these targets using Gene Ontology reveal co-ordinate regulation of genes involved in nervous system development and cell differentiation, as well as a suite of other transcription factors.Collectively, this thesis presents an in-depth characterisation of both the expression and multifaceted roles of NFI transcription factors in the postnatal cerebellum.