Thirty Years' History since the Discovery of Pax6: From Central Nervous System Development to Neurodevelopmental Disorders

Abstract

Pax6 is a sequence-specific DNA binding transcription factor that positively and negatively regulates transcription and is expressed in multiple cell types in the developing and adult central nervous system (CNS). As indicated by the morphological and functional abnormalities in spontaneous Pax6 mutant rodents, Pax6 plays pivotal roles in various biological processes in the CNS. At the initial stage of CNS development, Pax6 is responsible for brain patterning along the anteroposterior and dorsoventral axes of the telencephalon. Regarding the anteroposterior axis, Pax6 is expressed inversely to Emx2 and Coup-TF1, and Pax6 mutant mice exhibit a rostral shift, resulting in an alteration of the size of certain cortical areas. Pax6 and its downstream genes play important roles in balancing the proliferation and differentiation of neural stem cells. The Pax6 gene was originally identified in mice and humans 30 years ago via genetic analyses of the eye phenotypes. The human PAX6 gene was discovered in patients who suffer from WAGR syndrome (i.e., Wilms tumor, aniridia, genital ridge defects, mental retardation). Mutations of the human PAX6 gene have also been reported to be associated with autism spectrum disorder (ASD) and intellectual disability. Rodents that lack the Pax6 gene exhibit diverse neural phenotypes, which might lead to a better understanding of human pathology and neurodevelopmental disorders. This review describes the expression and function of Pax6 during brain development, and their implications for neuropathology.

Keywords: Pax6; Sey mutant; autism spectrum disorder; brain patterning; cell proliferation; central nervous system; mouse; neural differentiation; neural stem cells; rat.

Figure 1

Schematic structure of Pax6 protein and cortical phenotypes in the Pax6 spontaneous homozygous mutant (Sey/Sey) and Pax6 splice variant (Pax6(5a)) mice. (A) Schematic structure of the Pax6 protein in the wild-type (WT), Sey/Sey, and Pax6(5a) mice. One base-pair substitution in exon 8 of the Pax6 gene causes a Gly194Stop nonsense mutation, causing a stop codon downstream at the PST site, thereby resulting in the truncated Pax6^Sey protein. The canonical PD binds via its N-terminal PAI domain to the DNA; the insertion of the 14 amino acids from exon 5a into the PAI domain leads to the mutant PD(5a) [45]. PD: paired domain, HD: homeodomain, PST: Pro-Ser-Thr rich region. (B) Schematic illustration of the head of WT and Sey/Sey mice. Pax6 shows a gradient of expression in the AP axis in WT. Sey/Sey mice cause a failure of eye and olfactory bulb formation and rostralization in the telencephalon. OB: olfactory bulb, Tel: telencephalon, M1: motor, S1: sensory, A1: auditory and V1: visual areas. (C) Schematic illustration of a WT and Sey/Sey mouse telencephalon. Sey/Sey mice cause dorsalization in the telencephalon. Cx: cortex, BG: basal ganglia. The graphical diagram has been redrawn from [9,20,46].

Figure 2

Gradient expression of key transcription factors in cortical patterning. (A) Gradient expression patterns of the major transcription factors Pax6, Emx2, Coup-TFI, Sp8, Fgf8, and Emx1 along the anteroposterior (AP) and lateromedial (LM); anterolateral (AL) and posteromedial (PM) axes. Pax6 is highly expressed along the AL axis, and weakly along the PM axis. Emx2 is expressed inversely to Pax6. (B,C) Summary of loss-of-function (B) and gain-of-function (C) by the alteration of transcription factor expression in regard to cortical patterning. Analyses of brain formation indicate that Sey/Sey mice show a reduction in the M1 and S1 areas, as well as enlargement of the A1 and V1 areas. Embryonic analyses imply that Emx2 mutants show an inverse relationship to Sey/Sey mice. The overexpression of Emx2 under the control of a Nestin promoter increases the size of the V1 area. In contrast, the overexpression of Pax6 slightly reduces the size of the S1 area. (D) The regulatory network of transcription factors and secreted molecules. The BMP and Wnt gradients increase the expression of Emx2. Fgf15 enhances the expression of Coup-TF1. Fgf8 inhibits the expression of Emx2 and Coup-TF1. TF: transcriptional factor. The graphical diagram has been redrawn from [51,54,55,56,60,61,63,66,68,69,70].

Figure 4

The function of Pax6 and downstream genes. (A) Subcellular gene localization in radial glial (RG) cells. Genes localized in the cell nucleus, cytoplasm, and apical and basal endfeet. The graphical diagram has been redrawn from [135]. (B) Downstream genes regulated by Pax6 and their roles. Pax6 regulates Fabp7, Ninein, and Lewis X, which are involved in stem cell self-renewal. Pax6 induces Ngn2, Tbr2, and Dmrta1, which induce neural differentiation. (C) The Pax6–Ninein network regulates interkinetic nuclear migration (INM) during cell-cycle progression in neuroepithelial cells. The microtubule cytoskeleton plays an important role in INM, in which the nuclei of neuroepithelial cells move apically during G2 phase and basally during G1 phase. Ninein, downstream of Pax6, anchors microtubules during elevator movements to control the dynamics of INM. The graphical diagram has been redrawn from [99]. (D) Pax6–Notch interaction for neurogenic programs in the developing mouse cortex. Pax6-dependent neural differentiation by Notch signaling inhibition generates deeper layer (DL) neurons in the early neurogenic phase. Pax6-dependent self-renewal of the RG cells which give rise to upper layer (UL) neurons occurs in the middle/late neurogenic phase when Notch signaling is absent. In this way, the Pax6–Notch pathway coordinates the balance between self-renewal and neural differentiation. The graphical diagram has been redrawn from [127].

Figure 5

Linage and gene expression of NSCs in the adult hippocampal dentate gyrus. Pax6 is expressed in quiescent and active NSCs and intermediate progenitor cells (IPCs). The graphical diagram has been redrawn from [20,88,91,136,137,138,139,140,141,142,143,144].

Figure 3

Neural stem cell (NSC) differentiation in the embryonic cortex. (A) Neuroepithelial cells undergo symmetrical cell division to increase the population of neural stem cells (NSCs, proliferation phase). When brain development progresses, neuroepithelial cells elongate their processes stretching from the apical surface of the ventricular zone (VZ) to the basal tip (basal endfoot) at the pia surface; these cells are now called radial glial (RG) cells. They undergo asymmetrical cell division and induce intermediate progenitor cells (IPCs), as well as neurons. Intermediate progenitors migrate to the subventricular zone and differentiate into neurons. Subsequently, these neurons migrate towards the basal side. After producing neurons, RG cells produce glia, i.e., astrocytes and oligodendrocytes. The graphical diagram has been redrawn from [80]. (B) Expression of Pax6, Tbr2, Tuj1 in the developing cortex at E11.5, E14.5, and E17.5: Pax6 (red), Tbr2 (blue), and Tuj1 (green). These proteins are expressed in the VZ, subventricular zone (SVZ), intermediate zone (IZ), and cortical plate (CP), respectively. Scale bar: 100 μm. (C) The transition of the relative volume of brain subdivisions in the developing cortex. The Pax6-positive VZ area gradually narrowed during development.

Figure 6

Expression patterns of Pax6 in the mouse brain and the regional volume decrease in the Pax6 heterozygous mutant (rSey²/+) rat brain. (A) Expression patterns of Pax6 in the adult mouse brain have been redrawn from [25,88,160,161]. The nomenclature and subdivided brain regions are based on previous literature [162]. (B,C) The sex differences in regional volume decrease in the brain of the rSey²/+ rat compared to the WT using a deformation-based morphometry analysis of MRI data [160]. Pink and blue represent the clusters of regional volume decreases in the brain of female (B) and male (C) rSey²/+ rats compared to WT rats, respectively. The pink region is larger than the blue region. The graphical diagram has been redrawn from [160]. Abbreviations; AMG: amygdala, DG: dentate gyrus, HPC: hippocampus, ICx: isocortex, PrC: precommissural nucleus, SVZ: subventricular zone.