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First published online 17 November 2004
doi: 10.1242/dev.01524

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Molecular dissection of Pax6 function: the specific roles of the paired domain and homeodomain in brain development

¹ GSF-National Research Center for Environment and Health, Institute for Stem Cell Research, Neuherberg, Germany
² Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany
³ MD Anderson Cancer Center, Houston, TX, USA
⁴ GSF-National Research Center for Environment and Health, Institute of Developmental Genetics, Neuherberg, Germany
⁵ GSF-National Research Center for Environment and Health, Institute of Human Genetics, Neuherberg, Germany

View larger version (15K): [in a new window]   Fig. 1. Schematic drawings of the Pax6 mRNA in wild type (WT) and mice harboring mutations in the distinct DNA-binding domains of Pax6. (A) The mRNA of Pax6 consists of two DNA binding-domains, the paired domain (PD) and the homeodomain (HD). The PD is subdivided in two independent DNA-binding domains, the N-terminal PAI and the C-terminal RED domain. The PAI domain is able to bind to P6CON (Pax6 consensus site) and 5aCON (Pax6(5a) consensus site), whereas the RED domain binds exclusively to 5aCON. By means of alternative splicing, 14 amino acids are inserted into the N-terminal PAI domain (see yellow box) and thereby abolish its DNA binding. The transactivation domain (TA), important for gene activation, is located at the C-terminal end of Pax6. (B) The Pax6Aey18 mutant is characterized by a large deletion in the PD (exon 5a and exon 6), whereas the HD and TA are still present. (C) The Pax6(5a)–/– lacks exon 5a in the PD due to gene targeting (Singh et al., 2002), while the rest of the PD, HD and TA are unaffected. (D) Pax64Neu mutant mice bear a point mutation in the HD that abolishes DNA binding of the HD, while PD and TA are intact (Favor et al., 2001). (E) The Pax6Sey mutant mice are characterized by a truncated form of Pax6, lacking the HD and TA. These mutants have a comparable phenotype to the full gene deletion (Pax6–/–) and are therefore referred to as functional null alleles. PD, paired domain; HD, homeodomain; TA, PST-rich transactivator domain.

View larger version (56K): [in a new window]   Fig. 5. Retroviral overexpression of Pax6(5a) reveals a distinct influence on cell proliferation without effects on cell fate. (A) Corresponding micrographs of a clone of cells (indicated by arrow), isolated from the embryonic day (E) 14 cortex, infected with the BAG control retrovirus (upper row), Pax6-containing retrovirus (middle row) and Pax6(5a)-containing virus (lower row), cultured for 7 days in vitro and immunostained against ß-galactosidase (ß-gal, green), NeuN (red). Scale bar: 50 µm. (B) Schematic drawings of the retroviral constructs used for control, Pax6 (canonical form of PD) and Pax6(5a) overexpression together with the marker gene lacZ. (C) Histogram depicting the clone type of either control (BAG), Pax6- or Pax6(5a)-infected cells isolated from E14 WT (bars to the left) or Pax6Sey–/– mutant cortex (bars to the right) and cultured for 7 days. Note that the percentage of pure neuronal clones (blue bars) increases significantly (compared to WT control, t-test, see Materials and methods; error bars=s.e.m.) in the cells transduced with virus containing Pax6 with the canonical form of the PD, at the expense of the mixed (yellow bars) and pure non-neuronal clones (red bars). In contrast, Pax6(5a) exerted no effect on the clone type, even in the absence of functional Pax6 in Pax6Sey–/– cortical cells. (D) Histogram depicting the mean size of clones (=the number of ß-galactosidase-positive cells per clone, i.e. the number of cells generated by a single infected precursor) in the cultures described in C. Note that the clone size was reduced after transduction of cortical cells with Pax6 and Pax6(5a). Pax6(5a) was sufficient to reduce cell proliferation, suggesting that this effect is mediated by the 5aCON site. Numbers of clones analyzed: WT Ctrl: 395, WT Pax6: 243, WT Pax6(5a): 268, Pax6Sey–/– Ctrl: 353, Pax6Sey–/– Pax6: 229, Pax6Sey–/– Pax6(5a): 219.

View larger version (109K): [in a new window]   Fig. 2. Neurogenesis and cell proliferation in the cerebral cortex of mice harboring mutations in the distinct DNA-binding domains of Pax6. (A-E) Micrographs show coronal sections of the lateral cerebral cortex immunostained for NeuN (red) (neuronal marker) and PH3 (green) (marker for cells in M-phase) at embryonic day (E)14 in the respective mouse mutants as indicated in the panels. Note that the band of NeuN-positive cells is reduced in the PD mutant Pax6Aey18–/– (B) compared to WT cortex (A), indicating a reduced neurogenesis that appears comparable in extent to the phenotype in the functional null Pax6Sey–/– (E). In contrast, no such changes could be observed for the cortex of Pax6(5a)–/– (C) or the HD-mutant Pax64Neu–/– (D). In the mutants with an impaired neurogenesis (the PD mutant Pax6Aey18–/– and Pax6Sey–/–), we observed an increase in precursors (green PH3-positive cells in B,E), while no changes in comparison to WT (A) were seen in the cortex of Pax6(5a)–/– (C) and Pax64Neu–/– (D). Note that the PH3-positive cells were mostly increased in the subventricular zone (SVZ, arrowhead in A; increase in B and E), but not in the VZ (arrow in A). Thus, the PD of Pax6 is necessary and sufficient to mediate the effects of Pax6 on neurogenesis and cell proliferation in the cerebral cortex, while targets of the HD seem to play no role in these aspects. The dashed white line (A-E) indicates the ventricular surface of the cortex. MZ, marginal zone; CP, cortical plate; SVZ, subventricular zone; VZ, ventricular zone; CTX, cortex; GE, ganglionic eminence. Scale bar: 100 µm.

View larger version (85K): [in a new window]   Fig. 3. Dorso-ventral patterning in the telencephalon of mice harboring mutations in the distinct DNA-binding domains of Pax6. Micrographs of coronal sections of the lateral telencephalon at embryonic day (E) 14. (A-D) In WT CTX, but not GE, precursors are Ngn2-immunopositive (red), while precursors in the GE, but not the CTX, are Mash1-immunoreactive (green in A-D). Panels from littermate mutant mice are depicted in the right column (A'-D'). Note that Ngn2-immunoreactivity is not detectable in the CTX of mutant mice with a large deletion in the PD (see Fig. 1B), the Pax6Aey18–/– mice (A') and the functional null allele Pax6Sey–/– (E'), while it is unaffected in Pax6(5a)–/– (B') and Pax64Neu–/– (C'). Conversely, Mash1-immunoreactivity spreads ectopically into the cortex of Pax6Aey18–/– (A') and Pax6Sey–/– (D') mice, but is not changed in Pax6(5a)–/– (B') or Pax64Neu–/– (C') telencephalon. Thus, the PD of Pax6 seems to be necessary and sufficient to exert patterning of the telencephalon. The dashed white line (A,B,B',C,C') indicates the ventricular surface. CTX, cortex; GE, ganglionic eminence. Scale bar: 100 µm.

View larger version (82K): [in a new window]   Fig. 4. SFRP2 expression in the pallio-subpallial boundary between the dorsal (CTX) and ventral (GE) telencephalon of mice harboring mutations in the distinct DNA-binding domains of Pax6. Micrographs depict in situ-hybridization for SFRP2 mRNA in coronal sections of the telencephalon of WT and mutant littermates as indicated in the panel at embryonic day (E) 14. Note that SFRP2 expression is highest at the boundary between CTX and GE in WT mice (A,B,C,D; arrowhead in A), and Pax6(5a)–/– (B'), while its expression is lost at this position in the Pax6 mutation with a large deletion in the PD Pax6Aey18–/– (A'), the mutation with the defect HD Pax64Neu–/– (C') and the functional null allele Pax6Sey–/– (D'). Thus, both, DNA binding of the PD and the HD of Pax6 are required for SFRP2 expression at the pallial-subpallial boundary. CTX, cortex; GE, ganglionic eminence. Scale bar: 100 µm.

View larger version (28K): [in a new window]   Fig. 6. Summary of the specific roles of the Pax6 DNA-binding domains in telencephalic development. Analysis of the Pax6Aey18–/– mutant showed that the PD of Pax6 (A,B) is important for the regulation of neurogenesis, proliferation and regionalization in the developing telencephalon and that it contributes to the formation of the pallio-subpallial boundary. The lack of a phenotype in neurogenesis and proliferation in the forebrain of Pax64Neu–/– mice showed that the HD has no role in these processes (A), but is involved in some aspects of the pallio-subpallial boundary formation (SFRP2 expression). The Pax6(5a) isoform (B) is generated by alternative splicing leading to the insertion of a 14 AA insert in the N-terminal PAI subdomain of the PD, abolishing DNA binding and thereby favoring DNA binding via the RED domain. Retroviral overexpression revealed the anti-proliferative role of the Pax6(5a) isoform in cortical cells.

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