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First published online September 26, 2008
doi: 10.1242/10.1242/dev.027003

Development 135, 3459-3470 (2008)
Published by The Company of Biologists 2008
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Anterior-posterior graded response to Otx2 controls proliferation and differentiation of dopaminergic progenitors in the ventral mesencephalon

Daniela Omodei1,*, Dario Acampora1,2,*, Pietro Mancuso1, Nilima Prakash3, Luca Giovanni Di Giovannantonio1, Wolfgang Wurst3,{dagger} and Antonio Simeone1,2,{dagger}

1 CEINGE Biotecnologie Avanzate, via Comunale Margherita 482, 80145 Naples, Italy and SEMM European School of Molecular Medicine - Naples site, Italy.
2 Institute of Genetics and Biophysics `A. Buzzati-Traverso', CNR, Via P. Castellino 111, 80131 Naples, Italy.
3 Max-Planck-Insitute of Psychiatry, Molecular, Neurogenetics, Kraepelinstr. 2-16, 80804 Munich, Germany and Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Developmental Genetics, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.


Figure 1
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  		Fig. 1. Generation of the tOtx2bov mutant and Otx2 overexpression in ES cells and mutant embryos. (A) The genomic position at the chromosome 7 D2 region (upper line) where the tOtx2bov cassette (second line) is inserted allows the genotyping of mutant embryos by using two pairs of primers (open arrows in the upper line for the wild type and open arrows in the second line for the mutant). Cre-mediated removal of the Neo-triple polyA stop cassette generates the tOtx2ov allele (third line) and this event is monitored by the primers shown in the second and third line (black arrows). (B) The R26CreER/+; tOtx2bov/+ ES clone is treated with tamoxifen for 24 hours prior to monitoring the removal of the Neo-triple polyA cassette (upper panel), the Otx2 protein level (second panel) and the GFP activation (third panel). (C-K) Immunohistochemistry and in situ hybridization performed to detect the expression of Otx2 (C-E), En1-driven Cre recombinase (F-H) and GFP transcripts (I-K) in adjacent sagittal sections of E10.5 En1Cre/+, En1Cre/+; tOtx2ov/+ and En1Cre/+; tOtx2ov/ov embryos show that Otx2 is ectopically activated in the anterior hindbrain and cerebellum anlage (arrows in D,E) and, as revealed by GFP expression, overexpressed in the whole mesencephalon (J,K). Abbreviations: Mes, mesencephalon; Hb, hindbrain; MHB, midbrain-hindbrain border.

 

Figure 2
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  		Fig. 2. AP differential response to Otx2 overexpression generates dose-dependent AP-graded increase of mesDA neurons. (A-L) TH immunohistochemistry performed at four anatomical levels corresponding to the posterior pretectum (A,E,I), anterior (B,F,J), intermediate (C,G,K) and posterior (D,H,L) mesencephalon. (M-P) Graphic representation showing the percentage of the tOtx2bov/+ mesDA neurons detected in the two mutants at the anatomical levels corresponding to the histological sections shown. Abbreviations: SNpc, substantia nigra pars compacta; VTA, ventral tegmental area. The broken line demarcates the SNpc (A,E,I) or the VTA (B-D,F-H,J-L) territories analyzed for the cell-counting of TH+ cells.

 

Figure 3
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  		Fig. 3. Otx2 overexpression induces AP graded increase of mdDA neurons. (A-L'') Immunohistochemistry performed at six sequential anatomical levels corresponding to the posterior pretectum and anterior mesencephalon (A-B'',G-H''), the intermediate mesencephalon (C-D'',I-J'') and the posterior mesencephalon (E-F'',K-L'') of E12.5 wild-type, En1Cre/+; tOtx2ov/+ and En1Cre/+; tOtx2ov/ov embryos with Pitx3 and Nkx6.1 (A-F'') and Otx2 and Nkx2.2 (G-L'') shows that in Otx2-overexpressing embryos the number of Pitx3+ neurons gradually increases, moving from the anterior towards the posterior mesencephalon. This increase correlates with the copy number of the Otx2-overexpressing allele. Conversely, the Otx2 overexpression does not affect the extent of progenitor domains located dorsal to the mdDA domain; indeed, while the Nkx6.1--Nkx2.2- mdDA domain (ventral to the arrow) gradually expands, the Nkx6.1+-Nkx2.2- (between arrow and arrowhead) and the Nkx6.1+-Nkx2.2+ (dorsal to the arrowhead) domains retain a similar distribution among the three genotypes at the different anatomical levels. Abbreviation: Ant, anterior.

 

Figure 4
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  		Fig. 4. Selective expansion of the mesDA domain in Otx2-overexpressing embryos. (A-O) Immunohistochemistry and in situ hybridization performed on adjacent sections through the intermediate mesencephalon of E10.5 tOtx2bov/+, En1Cre/+; tOtx2ov/+ and En1Cre/+; tOtx2ov/ov embryos with Otx2 and Lmx1b (A,F,K), Nkx6.1 and Shh (B,G,L), Nkx2.2 and Shh (C,H,M), Nkx2.2 and Foxa2 (D,I,N) antibodies, and Wnt1 probe (E,J,O). The arrow indicates the dorsal border of Lmx1b expression, which is adjacent to the ventral border of Nkx6.1 and the arrowhead indicates the dorsal border of Shh, which is adjacent to or slightly overlapping the ventral border of Nkx2.2. Abbreviations: rp, roof plate.

 

Figure 5
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  		Fig. 5. Otx2 overexpression does not affect the generation and identity of RN and OM neurons. (A-X) Immunohistochemistry performed on the intermediate mesencephalon of E12.5 and E18.5 tOtx2bov/+, En1Cre/+; tOtx2ov/+ and En1Cre/+; tOtx2ov/ov with Otx2 and Pitx3 (A,I,Q), AADC and Pou4f1 (B,J,R), Lmx1b and Isl1 (C,K,S), Pitx3 and Pou4f1 (D,L,T), Nurr1 and Lim1/2 (E,M,U), Pitx3 (F,N,V), Pou4f1 (G,O,W) and Isl1 (H,P,X). Abbreviations: OM, oculomotor nucleus; RN, red nucleus; VTA, ventral tegmental area.

 

Figure 6
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  		Fig. 6. The proliferating activity of mesDA progenitors is enhanced in Otx2 overexpressing mutants. (A-F) Representative adjacent sections through the intermediate mesencephalon of tOtx2bov/+ (A,B), En1Cre/+; tOtx2ov/+ (C,D) and En1Cre/+; tOtx2ov/ov (E,F) embryos pulsed with BrdU for 30 minutes at E10.5 or E11.5, are immunostained with BrdU and Nkx6.1, and stained with Hoechst to determine the LI of progenitors in the mesDA and Nkx6.1 domains. The arrow and the broken line indicate approximately the Nkx6.1+ domain analyzed. (G-J) Graphic representation of the LI detected along the mesDA (G,H) and the Nkx6.1+ (I,J) domains shows a selective dose-dependent increase in the proliferating activity of mesDA progenitors developing in the intermediate and posterior mesencephalon. Abbreviations: Ant., anterior; Int., intermediate; Post., posterior.

 

Figure 7
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  		Fig. 7. Otx2 controls proliferation and post-mitotic transition of mesDA progenitors. (A-H) Representative adjacent sections through the intermediate mesencephalon of E10.5 (A-D) and E11.5 (E-H) wild-type and En1Cre/+; Otx2flox/flox embryos immunostained with Nkx6.1 and BrdU (A,B,E,F), Nkx2.2 and BrdU (C,D,G,H), and then stained with Hoechst show that the number of BrdU+ cells is dramatically and selectively reduced in the mesDA domain of mutants. (I-L) Graphic representation showing the AP graded reduction of the LI in the mesDA domain of mutants (I,J), whereas the LI measured in the Nkx2.2+ domain of mutant embryos is similar to that detected in the Nkx6.1+ domain of control embryos (K,L). (M-P) Representative adjacent sections through the intermediate mesencephalon of E11.5 wild-type and En1Cre/+; Otx2flox/flox mutants immunostained with Ki67 and BrdU (M,N), and Nkx2.2 and Nkx6.1 (O,P) show that, in mutant embryos, Ki67 is switched off in the majority of mesDA progenitors and that most of the mesDA BrdU+ cells (labeled at E10.5) become post-mitotic (Ki67-) at E11.5. Conversely, in the Nkx2.2+ domain, most of the BrdU+ progenitors retain Ki67 expression as in the Nkx6.1+ domain of control embryos. (Q,R) Graphic representation of the Qfs in the mesDA (Q), Nkx6.1+ (in wild type) and Nkx2.2+ (in En1Cre/+; Otx2flox/flox embryos) domains (R) shows a selective increase in the number of mesDA progenitors quitting the cell-cycle in mutant embryos, whereas a mild reduction is detected in the Nkx2.2+ domain. The arrow and the broken line in (A-H,M-P) indicate the Nkx6.1+ or the Nkx2.2+ territories analyzed. Abbreviations: Ant., anterior; Int., intermediate; Post., posterior.

 

Figure 8
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  		Fig. 8. En1Cre/+; Otx2flox/flox and En1Cre/+; tOtx2ov/ov embryos exhibit complementary expression abnormalities in cell-cycle components. (A-L) Adjacent sections through the intermediate mesencephalon of E11.5 wild type, En1Cre/+; Otx2flox/flox and En1Cre/+; tOtx2ov/ov embryos pulsed with BrdU for 30 minutes and immunostained with BrdU and Nkx6.1 (A,C), Nkx2.2 and BrdU (B), Sox2 and p27kip1 (G-I) and BrdU and CycD1 (J-L) or hybridized with the Wnt1 probe (D-F).

 

Figure 9
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  		Fig. 9. Differentiation of mesDA neurons requires Otx2. (A-P'') Adjacent sections through the intermediate mesencephalon of E11.5 and E12.5 wild type, En1Cre/+; tOtx2ov/ov and En1Cre/+; Otx2flox/flox embryos immunostained with Nkx6.1 and Nkx2.2 (A-B''), Mash1 and Sox2 (G-H''), Ngn2 and Sox2 (I-J''), Nurr1 and Ngn2 (K-L''), Nurr1 and Pitx3 (M-N''), Lmx1b and Otx2 (O,O''), Lmx1b and 5-HT (P,P'') or hybridized with Lmx1a (C-D'') or Msx1 (E-F'') probes. In En1Cre/+; Otx2flox/flox mutants, Lmx1b is dorsally expanded within the Nkx2.2+ domain that generates 5-HT+ neurons, and is abundant at E12.5 in the Sox2+ mesDA progenitors (O'',P''). The arrow indicates the ventral border of Nkx6.1+ domain in wild-type and En1Cre/+; tOtx2ov/ov embryos and the ventral border of the Nkx2.2+ domain in En1Cre/+; Otx2flox/flox mutants.

 

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© The Company of Biologists Ltd 2008