Molecular dissection of Pax6 DNA-binding domains and their roles in mouse cerebral cortex development

Generation of the brain depends on proper regulation of progenitor
proliferation and differentiation during development. Many such
factors known to affect proliferation and differentiation are
transcription factors. In particular, the transcription factor Pax6
has received much attention because of its potency to control
various aspects of brain development. During development of the
telencephalon Pax6 regulates patterning, cell proliferation and
neurogenesis, but how Pax6 mediates and coordinates these diverse
functions at the molecular level is not well understood. It has
previously been demonstrated that the homeodomain of Pax6 plays a
role in establishing the pallial-subpallial boundary. However it is
not involved in other processes during telencephalic development as
shown by the analysis of Pax64NEU mice, which are characterized by
a point-mutation in the DNA-binding helix of the homeodomain. In
order to gain more insights into the molecular network underlying
the mild homeodomain function in the developing telencephalon,
transcriptome analysis with Pax64NEU mice was performed. Almost no
transcriptional changes were detected, suggesting that
transcriptional regulation by the homeodomain of Pax6 has no major
impact on forebrain development. Additionally, these results
implied that the majority of effects exerted through Pax6 during
telencephalic development are mediated by the bipartite
paired-domain (PD). Therefore the main focus of this thesis was to
examine the specific roles of the Pax6 paired-domain and its
individual DNA-binding subdomains (PAI and RED) during forebrain
development. The role of these DNA-binding domains was examined
using mice with point-mutations in the PAI (Pax6Leca4, N50K) and
RED (Pax6Leca2 R128C) subdomains and showed that the mutations in
these subdomains exert opposing roles regulating proliferation in
the developing cortex. While the mutated PAI domain resulted in
reduced proliferation of both apical and basal progenitors, the
mutated RED domain provoked increased proliferation. However, the
PAI domain largely mediates the neurogenic function of Pax6.
Additionally, genome-wide transcriptome analysis was able to
unravel the key signatures mediated by the distinct domains. In
summary, Pax6 exerts its key roles during forebrain development by
use of distinct subdomains to regulate proliferation and
differentiation. Thus Pax6 is able to coordinate and fine tune
patterning, neurogenesis and proliferation in a simultaneous manner
in different radial glial subpopulations. The transcriptional
regulation through Pax6 may not only be restricted to protein
coding genes, but may also include control of microRNA (miRNA)
expression. Such small RNA molecules have recently been implicated
in proliferation and differentiation during development, however
expression and the role of single microRNAs is still poorly
understood. Towards this end, miRNA expression profiling was
performed using an embryonic stem cell differentiation system at
different stages of neuronal differentiation in order to identify
new miRNAs involved in radial glia specification and
differentiation. This analysis revealed a number of microRNAs
induced during differentiation from neural progenitors to neurons.
Most strikingly only four miRNA candidates were found with
exclusively high expression in progenitor cells. These data suggest
that also Pax6 may play a role in transcriptional regulation beyond
mRNAs.