Regulation of cerebral cortical neurogenesis by the Pax6 transcription factor

Abstract

Understanding brain development remains a major challenge at the heart of understanding what makes us human. The neocortex, in evolutionary terms the newest part of the cerebral cortex, is the seat of higher cognitive functions. Its normal development requires the production, positioning, and appropriate interconnection of very large numbers of both excitatory and inhibitory neurons. Pax6 is one of a relatively small group of transcription factors that exert high-level control of cortical development, and whose mutation or deletion from developing embryos causes major brain defects and a wide range of neurodevelopmental disorders. Pax6 is very highly conserved between primate and non-primate species, is expressed in a gradient throughout the developing cortex and is essential for normal corticogenesis. Our understanding of Pax6's functions and the cellular processes that it regulates during mammalian cortical development has significantly advanced in the last decade, owing to the combined application of genetic and biochemical analyses. Here, we review the functional importance of Pax6 in regulating cortical progenitor proliferation, neurogenesis, and formation of cortical layers and highlight important differences between rodents and primates. We also review the pathological effects of PAX6 mutations in human neurodevelopmental disorders. We discuss some aspects of Pax6's molecular actions including its own complex transcriptional regulation, the distinct molecular functions of its splice variants and some of Pax6's known direct targets which mediate its actions during cortical development.

Keywords: BAF complex; Meis2; cell cycle; cortical lamination; differentiation; neuronal fate; neurotransmitter fate; proliferation.

FIGURE 1

Cortical germinal areas of rodents and primates. Coronal section through the developing cortex of a rodent and a primate showing progenitor types and whether they express Pax6 and/or Tbr2. CP, cortical plate; SP, subplate; IZ, intermediate zone; SVZ, subventricular zone; ISVZ, inner subventricular zone; OSVZ, outer subventricular zone; VZ, ventricular zone; aRG, apical radial glia; bRG, basal radial glia; aIPC, apical intermediate progenitor cell; bIPC, basal intermediate progenitor cell.

FIGURE 2

The gradient expression pattern of Pax6 protein in the mouse developing cortex. (A) A schematic diagram shows the Pax6 protein (red) is normally expressed in a ^highrostrolateral to ^lowcaudomedial gradient in the developing cortex during early cortical development. (B,C) immunofluorescence staining for Pax6 protein in sagittal section (B) and coronal section (C) from E12.5 mouse WT embryos (scale bars, 100 μm).

FIGURE 3

Comparison of Pax6 expression in embryonic human and mouse cortex. Images from human cortices at 8, 10, and 12 post-coital weeks (PCWs) were generated using material from the Human Developmental Biology Resource (www.hdbr.org) as part of the HuDSeN (Kerwin et al., 2010) human gene expression spatial database (http://www.hudsen.org) based at Newcastle University. PAX6 is expressed in the ventricular zone (VZ) and subventricular zone (SVZ) at 8 PCW. The SVZ divides into an outer and an inner subventricular zone (OSVZ and ISVZ), both of which continue to express PAX6. In mice, Pax6 expression is confined almost exclusively to the VZ during corticogenesis, as shown here at embryonic days (E) 13.5, 15.5, and 17.5. Additional abbreviations: CP, cortical plate; IZ, intermediate zone.

FIGURE 4

Histology of Pax6^-/- developing cortex in human and mouse. (A) Coronal section through the cortex of a fetus with a compound heterozygosity for two PAX6 mutations showing a layer of germinal cells on the surface of the cortex (arrow) and heterotopia of germinal cells within the cortex (arrowhead). Photograph taken from Schmidt-Sidor et al. (2009). (B) Coronal section through the cortex of a Pax6^-/- mutant mouse embryo showing clusters of germinal cells in the intermediate zone (arrowhead). Photograph from Caric et al. (1997).

FIGURE 5

Structure and transcriptional regulation of the PAX6 locus. Schematic map of human chromosome 11p13 shows the locations of the PAX6 and ELP4 loci, which are in an antisense orientation relative to each other. The locations of PAX6 promoters (P0, P1, Pa, and P4) are indicated by black arrows. Blue rectangles indicate PAX6 exons, while black rectangles below the line indicate ELP4 exons. The known highly conserved regulatory elements, such as E60, E100 and RB, are indicated by purple ellipses. The breakpoints of the two distal-most aniridia-associated rearrangements are indicated by “SGL” and “SIMO.” The location of the downstream regulatory region (DRR) is shown by red arrows. The known DNaseI hypersensitive sites (HS sites) within the DRR region are shown by red arrows. The schematic maps of YACs Y593 (420 kb) and Y589 (310 kb) are indicated. In transgenic mice, Y593, but not Y589, rescues the mouse Sey phenotype and homozygous Sey lethality.

FIGURE 6

Effect of loss of Pax6 on cortical progenitor cell cycle parameters. Diagram showing the cell cycle length and S-phase length of cortical progenitors at a rostral or caudal position in Wild type or Pax6^-/- embryos. At E12.5 Pax6 is normally expressed in a high rostral to low caudal gradient and loss of Pax6 results in a shortening of the cell cycle rostrally with no effect on S-phase length. At E14.5 Pax6 is expressed in a flatter rostro-caudal gradient and loss of Pax6 causes a shortening of the cell cycle both rostrally and caudally while the S-phase length remains unaffected.

FIGURE 7

Schematic representation of functional domains in Pax6 and Pax6(5a) and their “optimal” DNA binding sites. (A) Pax6 and Pax6(5a) differ in a 14 amino acid insertion in the PAI subdomain. (B) “Optimal” DNA-binding sites for Pax6 PD, PD5a and HD. P6CON and 5aCON sequences were generated by selection procedure (Epstein et al., 1994). P6CON is recognized by monomeric PD, while 5aCON contains four binding sites for PD5a that can bind up to four PD5a units. The “optimal” binding site of Pax6 HD is a dimer with typical HD binding sequences (ATTA/TAAT) separated by three nucleotides (G/T)CG (Czerny and Busslinger, 1995).

FIGURE 8

Changes in subunit composition of BAF complexes during differentiation from ES cells to neural progenitor cells. BAF complexes undergo progressive changes in subunit composition during the transition from embryonic stem cells (ESCs) to neural progenitor cells (NPCs). The BAF complex in ESCs is called esBAF; in neuronal progenitors, npBAF. As ESCs differentiate into NPCs, The esBAF complexes incorporate Brm and BAF170, while excludes BAF60B and BAF155, thereby forming the neural progenitor-specific BAF (npBAF) complexes in NPCs.