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First published online 28 February 2007
doi: 10.1242/dev.02809

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Conditional activation of Pax6 in the developing cortex of transgenic mice causes progenitor apoptosis

¹ Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
² DFG, Center of Molecular Physiology of the Brain (CMPB), Göttingen, Germany.
³ Dulbecco Telethon Institute at IRCCS Fondazione Santa Lucia, Rome, Italy.
⁴ University of Colorado, Boulder, Colorado, USA.

View larger version (15K): [in this window] [in a new window]   Fig. 1. The construct for transgenic activation of Pax6. The construct pJoP6 contains the ß-actin/CMV fusion promotor and a loxP-flanked gfp-stop cassette placed in front of the Pax6 and lacZ coding sequences. After Cre-mediated recombination, the gfp-stop cassette is eliminated allowing simultaneous expression of transgenic Pax6 and lacZ via an IRES sequence from the resulting construct pJoP6rec. Arrows indicate the annealing sites of the primers JoP6F and JoP6R.

View larger version (83K): [in this window] [in a new window]   Fig. 2. Efficiency of recombination in JoP6 mice. (A) Genomic PCR was performed with the primers JoP6F and JoP6R on DNA isolated from the pJoP6 plasmid and cortices from wild-type (negative control), JoP6 and JoP6;Emx1IREScre embryos. The PCR amplifies a 1831 bp fragment from unrecombined DNA templates and from pJoP6 plasmid and JoP6 cortex, whereas an additional 261 bp DNA band is seen with the DNA sample from the JoP6;Emx1IREScre cortex. (B,B') Abundant GFP fluorescence is detected on coronal sections of E14.5 JoP6 cortex, whereas the majority of the cells in the VZ and SVZ of the JoP6;Emx1IREScre cortex are GFP-negative. Arrows indicate preserved GFP+ cells in the VZ. Note the preserved fluorescence in the thin CP and the striatum (str). (C) Whole-mount staining for ß-gal indicates specific expression of lacZ in the telencephalon (tel), olfactory bulb (ob) and scattered cells in the diencephalon (di) of E15.5 JoP6;Emx1IREScre cortex (bottom), whereas the mesencephalon (mes) and the metencephalon (mt), as well as the JoP6 control brain (top), are unstained. (D) Western blot confirms higher level of Pax6 in the E15.5 JoP6;Nex-Cre as compared with the JoP6 cortex. (E) E12.5 section of JoP6;Emx1IREScre cortex reveals weak ß-gal staining of individual or aggregated (arrows and inset) progenitors. (F) At E15.5, ß-gal+ cells are within LP/VP, IZ and the lower part of the CP. (G) Strong ß-gal staining is seen at E18.5 in late progenitors as well as postmitotic cells of the CP and (H) at P28 throughout the whole cortex. (I,I') Pax6 immunohistochemistry on sections of E18.5 JoP6 (I) and JoP6;Emx1IREScre (I') cortex, showing ectopic Pax6 expression in postmitotic cells in the IZ and the lower part of the CP. The magnifications of the framed area in I' demonstrate Hoechst and Pax6 co-labeling. (J,K) q-PCRs with RNA isolated from cortex of JoP6 control, JoP6;Emx1IREScre, and JoP6;Nex-Cre normalized to 18S RNA indicate upregulation of Pax6 in the double-transgenic cortices (J) and specific elevation of the level of Pax6 transcripts in the JoP6;Emx1IREScre cortex, whereas the level of Pax6-5a is in fact diminished, as compared with the controls (K). (L) SAOS2 cells were transiently co-transfected with a luciferase reporter construct containing either the HD (blue) or the PD (red) domain and either pJoP6 or pJoP6rec, which express Pax6. Luciferase activities are shown as relative values compared with the activity measured in lysates from cells co-transfected with the pJoP6 control plasmid. Co-transfection of pJoP6rec with either the HD- or the PD-containing reporter construct induced an increase in luciferase activity, indicating functionality of transgenic Pax6. Error bars indicate s.d.

View larger version (73K): [in this window] [in a new window]   Fig. 3. Activation of transgenic Pax6 disturbs the mitotic cell cycle. (A,A') HE staining of P23 cross-sections of JoP6 control and JoP6;Emx1IREScre cortex (cx). The size of the JoP6;Emx1IREScre cortex is significantly diminished, but cortical layering is preserved. Scale bars: 0.5 mm. (B,B') Coronal 5 µm sections of E11.0 JoP6 control and JoP6;Emx1IREScre cortex immunostained for BrdU and DAPI after a 30-minute BrdU pulse. Estimated BrdU labeling indices reveal a significant inhibition of the proliferation rate in the MP and DP of the JoP6;Emx1IREScre cortex as compared with the JoP6 control. The frames indicate the equally sized areas used for determination of the BrdU labeling index of the genotypes compared. (C-D') Patterns of BrdU pulse labeling of cortical progenitors at stage E11.0 for 90 minutes (C,C') and 6 hours (D,D') in JoP6 control (C,D) and JoP6;Emx1IREScre mice (C',D'). The arrows in C point to nuclei in the control cortex that were labeled at the end of their S phase and underwent mitosis thereafter, as indicated by their diluted content of BrdU. Nuclei of such appearance are scarce in the JoP6;Emx1IREScrecortex (C'; arrowheads point to cell aggregates). Note the severe distortion of the interkinetic nuclear migration in the JoP6;Emx1IREScre cortex, in which, even after a 6-hour BrdU pulse, many nuclei are still in the basal VZ (arrows in D'). (E,E') Immunolabeling of pHH3 at stage E14.5 reveals significantly fewer mitotic cells at the apical surface of the VZ in JoP6;Emx1IREScre as compared with JoP6 mice.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Enhanced neurogenesis and altered adhesive properties after activation of transgenic Pax6 in developing cortex. (A,A') The E13.5 JoP6;Emx1IREScre VZ is significantly diminished as compared with JoP6, but the size of the Tuj1+ mantle zone (M) is unchanged. Also, premature differentiated Tuj1+ cells are found ectopically in the VZ of JoP6;Emx1IREScre at E14.5 (B,B') and E11.5 (C,C'). (D) q-PCR of total RNA from control and JoP6;Emx1IREScre cortices normalized to 18S RNA. N-CAM and R-cadherin are upregulated, whereas Pxn, Tnc, Itgb3 and Itga5 are downregulated. Error bars indicate s.d.

View larger version (66K): [in this window] [in a new window]   Fig. 5. Pax6 GOF at early corticogenesis causes apoptosis. (A-B') TUNEL assay on sections from JoP6 (A,B) and JoP6;Emx1IREScre (A',B') embryos at stages E11.5 (A,A' with higher magnification) and E14.5 (B,B') illustrates apoptosis of JoP6;Emx1IREScre cortical progenitors, more severe at E11.5. (C,C') Immunolabeling against activated Casp3 confirms enhanced apoptosis in the JoP6;Emx1IREScre cortex. (D-D'') Double immunostaining of E13.5 JoP6;Emx1IREScre cortex against nestin (D) and activated Casp3 (D') illustrates that many of the apoptotic cells are nestin+ progenitors (D'' and magnifications counterstained with Hoechst). (E) At stage E18.5, apoptosis is no longer detected in JoP6;Emx1IREScre. (F) In situ hybridization with ephrin A5 reveals that the apoptotic cell aggregates of the JoP6;Emx1IREScre cortex are positive for ephrin A5 (arrows).

View larger version (105K): [in this window] [in a new window]   Fig. 6. Activation of transgenic Pax6 leads to progenitor apoptosis depending on the endogenous Pax6 expression level. (A) Pax6 in situ hybridization on E11.0 wild-type section demonstrates extremely faint endogenous Pax6 expression in the MP and (B) TUNEL assay on E11.0 JoP6;Emx1IREScre reveals massive apoptosis within the same region (arrow). (C) ß-gal staining of section at E14.5 indicates successful recombination in the JoP6;hGFAP-cre cortex and (D,D') TUNEL assay on JoP6 and JoP6;hGFAP-cre shows extensive apoptosis in the Pax6+ radial glial progenitors. (E) ß-gal staining of an E11.5 JoP6;E1-Ngn2/Cre embryo indicates recombination in the LP and VP (arrow indicates corticostriatal border). (F,F') No significant difference in apoptosis is detected via TUNEL assay on sections of E11.5 JoP6 (F) and JoP6;E1-Ngn2/Cre (F') embryos. (G) E14.5 section of JoP6;Nex-Cre cortex stained for ß-gal indicates recombination in postmitotic neurons in the dorsal telencephalon. (H,H') TUNEL assay did not detect additional apoptosis in the cortex of the JoP6;Nex-Cre embryos (H') as compared with the control (H).

View larger version (23K): [in this window] [in a new window]   Fig. 7. Effect of Pax6 protein on activity of the P53 promoter. Pax6 protein (3 µg of pPax6) does not positively regulate activity of the human P53 promoter in transiently transfected SAOS2 cells, but upon overexpression (16 µg pPax6, indicated by ++) does inhibit the P53 promoter activity. By contrast, Pax6-5a shows no significant effect. Normalized luciferase activity in the presence or absence of functional pRb is indicated; error bars indicate s.d. The luciferase activity of the `empty' vector pCMV without pRb was given an arbitrary value of 100. Note that pRB slightly increases the Pax6 inhibitory effect on P53.

View larger version (107K): [in this window] [in a new window]   Fig. 8. Conditional transgenic activation of Pax6-5a via the JoP6-5a mouse line. (A,A') GFP fluorescence on sections of E14.5 cortex of JoP6-5a (A) is partially lost in JoP6-5a;Emx1IREScre (A'). (B) Two products generated by genomic PCR with the primers JoP6F and JoP65aR (1955 bp and 394 bp) confirm successful recombination in the JoP6-5a;Emx1IREScre cortex, whereas the JoP6 cortex and the pJoP6-5a plasmid show no recombination. (C) Whole-mount ß-gal staining of E15.5 JoP6-5a and JoP6-5a;Emx1IREScre brains shows strong activation of the lacZ reporter in the JoP6-5a;Emx1IREScre telencephalon and olfactory bulb. (D,D' with inset) Immunostaining for Pax6 on E14.5 JoP6-5a and JoP6-5a;Emx1IREScre cortices shows ectopic antibody staining indicating Cre-induced activation of Pax6-5a in the JoP6-5a;Emx1IREScre CP. (E) SAOS2 cells were transiently co-transfected with a luciferase reporter construct containing either the HD (blue) or the PD (red) domain and either pJoP65a or pJoP65arec, which express Pax6-5a. Luciferase activities were normalized to co-transfections with the pJoP65a control plasmid. Co-transfection of pJoP6rec with either the HD- or the PD-containing reporter construct induced an increase in luciferase activity, indicating functionality of transgenic Pax6-5a. (F) q-PCRs with RNA isolated from E12.5 cortex of JoP6-5a control and JoP6;Emx1IREScre cortex, normalized to 18S RNA, indicate only a slight enhancement of the Pax6-5a expression level in the JoP6-5a;Emx1IREScre telencephalon and olfactory bulb. Error bars indicate s.d.. (G,G') TUNEL assay on E11.5 brain sections of both genotypes reveals no significant difference in apoptosis (arrowheads point to endogenous apoptosis). (H,H') BrdU immunohistochemistry after a 30-minute BrdU pulse on cross-sections of E11.0 JoP6-5a and JoP6-5a;Emx1IREScre cortices reveals a significant inhibition of the proliferation rate in the JoP6-5a;Emx1IREScre DP.