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First published online 3 January 2007
doi: 10.1242/dev.02764

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Controlled overexpression of Pax6 in vivo negatively autoregulates the Pax6 locus, causing cell-autonomous defects of late cortical progenitor proliferation with little effect on cortical arealization

¹ Genes and Development Group, Centres for Integrative Physiology and Neuroscience Research, Hugh Robson Building, George Square, University of Edinburgh, Edinburgh EH8 9XD, UK.
² Division of Reproductive and Developmental Sciences, Genes and Development Group, Centres for Integrative Physiology and Reproductive Biology, Hugh Robson Building, George Square, University of Edinburgh, Edinburgh EH8 9XD, UK.
³ MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, UK.

View larger version (57K): [in this window] [in a new window]   Fig. 1. Pax6 protein levels are increased in PAX77 mice. (A) Western-blots of E12.5 wild-type, PAX77+ and PAX77+/+ telencephalic-protein extracts with anti-Pax6 and anti-ß-actin antibodies. (B) Quantitation of the two isoforms, 48 kDa Pax6(5a) and 46 kDa Pax6, in E12.5 wild-type PAX77+ and PAX77+/+ telencephalons. For each sample, the intensity of the Pax6 and Pax6(5a) bands was divided by the intensity of the ß-actin band to account for loading differences and values were calculated relative to the mean value for wild-type Pax6, which was assigned a value of 1 (mean±s.e.m. are shown; n=3 in all cases). Asterisks indicate statistically significant differences from wild type (Student's t-test, P<0.05). There was no significant difference between the levels of Pax6 and Pax6(5a) in PAX77+/+ and PAX77+ embryonic brains. (C-E) Immunohistochemistry on sagittal sections through the cortex showing Pax6 expression in (C) wild-type (WT), (D) PAX77+ and (E) PAX77+/+ E12.5 embryos. The rostro-lateralhigh to caudo-mediallow gradient of Pax6 expression is conserved in the cortex of PAX77 mice. Scale bar: 200 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 2. Telencephalic expression of tau-GFP from Y1123 is regulated by differences in the levels of Pax6 expression. Expression is shown in coronal sections from (A) Pax6+/+, (B) PAX77 and (C) Pax6-/- E14.5 embryos. Scale bar: 200 µm. (D-H) Expression of tau-GFP quantified with flow cytometry. (D-G) Examples of frequency histograms of cell number against GFP fluorescence for samples of cells from the brains of (D) wild-type embryos and (E-G) embryos containing Y1123 on (E) a wild-type background, (F) a Pax6-/- background and (G) a PAX77 background. (H) Histogram showing the mean fluorescence of cells in gate B (see D-G) from four to six embryos with each Y1123-containing genotype. All differences are significant (P<0.001; Student's t-test).

View larger version (89K): [in this window] [in a new window]   Fig. 3. Pax6 overexpression affects late cortical progenitor proliferation. (A-C) Coronal sections at (A) rostral, (B) central and (C) caudal levels of the cortex (left hemisphere shown) of an E12.5 wild-type embryo labeled with anti-BrdU (brown). Cell counts were made in 100 µm-wide sampling boxes (red boxes). (D,E) Examples of anti-BrdU labelling (brown) of coronal sections of the cortex of (D) a wild-type and (E) a PAX77 embryo at E12.5. (F) The proportion of AP cells in S-phase along the cortex of PAX77 embryos at E12.5 is not different from that of wild type. (G,H) Example of anti-BrdU labeling (brown) of coronal sections of the cortex of (G) a wild-type and (H) a PAX77 embryo at E15.5. (I) The proportion of AP cells in S-phase in the rostral and central cortex of PAX77 embryos at E15.5 is significantly decreased compared with wild type. (J,K) Example of anti-phosphorylated histone H3 labeling (brown) of coronal sections of the cortex of (J) a wild-type and (K) a PAX77 embryo at E15.5. (L) The density of AP cells in M-phase in the rostral and central cortex of PAX77 embryos at E15.5 is significantly decreased compared with wild type. All sections shown are counterstained with cresyl violet. MZ, marginal zone; VZ, ventricular zone; SVZ, subventricular zone. Scale bars: 50 µm in A-E, 70 µm in G,H,J,K.

View larger version (95K): [in this window] [in a new window]   Fig. 4. PAX77 cells are under-represented in the cortical proliferative layers of PAX77+;Tg-wild-type;Tg+ chimeric embryos at E16.5. (A,B,F,G) Coronal sections through the cortex of a PAX77+;Tg-wild-type;Tg+ chimera at E16.5. (A,B) Tg+ cells (marked with brown dots) and Tg- cells were counted in the proliferative layers (VZ and SVZ), intermediate zone (IZ) and cortical plate (CP). (C-E) Ratios of observed/expected contributions of Tg- (i.e. PAX77) cells in (C) VZ and SVZ, (D) IZ and (E) CP. (C) PAX77;Tg- cells are significantly under-represented in the proliferative layers along the chimeric cortex and (D) in the IZ of the rostral chimeric cortex. (F,G) Example of Tg+ (arrow) and Tg- (arrowhead) apical progenitors in M-phase labelled with anti-phosphorylated histone H3 (grey). Scale bars: 50 µm in A; 10 µm in B,F,G.

View larger version (60K): [in this window] [in a new window]   Fig. 5. Pax6 overexpression affects the formation of superficial cortical layers. (A,B) Coronal sections through the cortex of (A) a wild-type and (B) a PAX77 mouse at P7, stained with cresyl violet. Borders between adjacent cortical layers were identified based on differences in cytoarchitecture. (C-E) Thickness of cortical layers I, II-IV (combined), V and VI in the (C) rostral, (D) central and (E) caudal cortex of wild-type and PAX77 mice at P7. The thickness of layers II-IV is significantly decreased in the rostral and central cortex of PAX77 mice compared with wild type. Scale bar: 300 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Predicted shifts in the cortex of PAX77 mice. (A) Pax6 levels are about three times higher in the anterior cortex than in the posterior cortex. In PAX77 mice, Pax6 expression is still graded, but is increased by 1.5- to 3-fold (shown here as an average of approximately 2-fold). Thus, in PAX77 embryos, cells in posterior regions of the cortex express levels of Pax6 that are normally found in anterior regions. (B-D) Dorsal views of mouse neocortex. (B) In the wild-type, Pax6 is expressed in a rostro-lateralhigh to caudo-mediallow gradient, while (C) Emx2 is expressed in an opposite gradient. We predicted that the overexpression of Pax6 would result in a downregulation of Emx2 expression and (D) in a caudal shift of rostral areas at the expense of caudal areas. M, motor area; S, somatosensory area; V, visual area.

View larger version (54K): [in this window] [in a new window]   Fig. 7. Pax6 overexpression does not alter the expression profile of cortical regionalization markers. (A,B) Immunohistochemistry on sagittal sections through the cortex of (A) wild-type and (B) PAX77+ E12.5 embryos showing the expression of Emx2. The gradient of Emx2 expression is conserved in the cortex of PAX77 mice and overall levels are not reduced. (C,D) Immunohistochemistry on horizontal sections through the cortex of (C) wild-type and (D) PAX77+ E16.5 embryos showing the expression of Ephrin B2 in the right hemisphere. In the wild-type embryo, Ephrin B2 is strongly expressed in a caudal domain (arrowhead shows the anterior limit of the Ephrin B2 caudal expression domain). This caudal domain is present and does not appear contracted in the PAX77 cortex. (E,F) In situ hybridizations on sagittal sections through the forebrain of (E) wild-type and (F) PAX77+/+ E18.5 embryos showing the expression of Id2 mRNA. In the wild-type embryo, Id2 is strongly expressed in layers 2 and 3 in an anterior domain (black arrow shows the posterior limit of this domain), and in layer 5 in a caudal domain (white arrowhead shows the anterior limit of this domain). The expression of Id2 appears unaltered in the PAX77 cortex. Scale bars: 200 µm in A-C; 0.5 mm in D-H.

View larger version (60K): [in this window] [in a new window]   Fig. 8. The position of the postero-medial barrel subfield (PMBSF) is not shifted in PAX77 cortex but its size is reduced. (A) Tangential sections through one cortical hemisphere of P7 wild-type and PAX77+/+ mice stained with anti-serotonin transporter. The primary somatosensory (S1), auditory (A1) and visual (V1) areas are revealed. (B) Measurements of the ratio between the distance of barrel c4 from the rostral pole of the cortex (R, black arrow) and the total length of the cortex (T, white arrow). No significant difference was found between wild-type and PAX77 mice (Student's t-test, n=16). (C) Measurement of the ratio between the distance of barrel c4 from the caudal pole of the cortex (C, black arrow) and the total length of the cortex (T, white arrow). No significant difference was found between wild-type and PAX77 mice (Student's t-test, n=16). (D) Measurement of the ratio between the area of the PMBSF (Pa) and the total area of the cortex (Ca). **Area of PMBSF relative to the total cortical area was significantly reduced in the PAX77 brain compared with wild type (Student's t-test, P<0.01, n=13 wild-type brains and 8 PAX77+/+ brains). Scale bars: 1 mm.