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First published online 5 January 2006
doi: 10.1242/dev.02224

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Development of the mesencephalic dopaminergic neuron system is compromised in the absence of neurogenin 2

E. Andersson^1,*, J. B. Jensen^1,*, M. Parmar¹, F. Guillemot² and A. Björklund^1,

¹ Wallenberg Neuroscience Center, Department of Experimental Medical Science, and Lund Strategic Center for Stem Cell Biology and Cell Therapy, Lund University, BMC A11, SE-221 84 Lund, Sweden.
² National Institute for Medical Research, Division of Molecular Neurobiology, The Ridgeway, Mill Hill, London NW7 1AA, UK.

View larger version (61K): [in a new window]   Fig. 1. Impaired development of Th-positive mesDA neurons in Ngn2 mutant mice. (A-C) In the VM Ngn2 (red) was expressed in part of the VZ lying immediately above the Nurr1-expressing mesDA neuron precursors (green, A). A few single Ngn2-positive cells were seen to co-express Nurr1 (arrow, B,C). (D-I) At E11.5-E15.5, the number of Th-positive cells in the VM was dramatically reduced, by approx 90%, in the homozygous (–/–) Ngn2 mutants (E,G,I), compared with the wild-type (WT, +/+) mice (D,F,H). The remaining Th-positive cells were located at the lateral edges of the mesDA neuron domain, whereas the medial portion was almost completely devoid of Th-positive cells. (J-M) Parallel sections stained for Aadc and Vmat2 showed the same degree of cell loss, with positive cells remaining in the same lateral population. (N,O) At later embryonic stages (E17.5), more Th-positive neurons had been generated in the Ngn2 mutant and the difference between wild type and Ngn2 mutants with respect to both the number and distribution of Th-positive neurons was less pronounced. Scale bars: 50 µm in A; 100 µm in D-O.

View larger version (91K): [in a new window]   Fig. 2. Loss of mesDA neurons in postnatal Ngn2 mutant mice. (A-M) In postnatal Ngn2 mutants, the number of Th-positive neurons was markedly reduced in both the SN and the VTA (A,B,D,E), and a population of Th-positive cells located close to the midbrain aqueduct was completely missing (F,G). Parallel sections stained for Vmat2 showed that no other cells present in the SN-VTA area displayed a DA phenotype (H,I). The Th-positive innervation of striatal and limbic forebrain areas was normal, although the target areas were reduced in size (L,M). The Ngn2 mutants were similar in size to wild-type mice at birth but showed a reduced weight gain postnatally (C), and the size of their brain was reduced by about 20% (J,K). Stars indicate the rostrocaudal levels used for cell counting. (N,O) Cell counts at P0 and P18 revealed a more than 50% loss of Th-positive neurons in the SN and VTA of the Ngn2 mutants. *P0.01 relative to wild type (N), and in wild type adn heterozygous (O) (Student's t-test). Scale bars: 500 µm.

View larger version (133K): [in a new window]   Fig. 3. Correct specification of mesDA neurons in Ngn2 mutants. In wild-type mice, the two major mesDA neuron subtypes, the SN and VTA neurons, are characterized by their expression of Girk2 (in SN DA neurons) and calbindin (in VTA DA neurons). (A-L) In postnatal Ngn2 mutant mice (D,E), double-stained sections showed that the expression of calbindin (red, E,F) in the Th-positive VTA neurons (green) was similar to that seen in wild-type mice (B,C), and Girk2 (red, K,L) was expressed in Th-positive SN neurons (green) in a pattern indistinguishable from that of wild type (H,I). (M-R) The expression of Pitx3 (red) was maintained in all Th-positive neurons (green) in Ngn2 mutants (R). Scale bars: 100 µm.

View larger version (136K): [in a new window]   Fig. 4. Loss of medially located mesDA neuron precursors in the Ngn2 mutant. (A,D,G,J,M,P) At all stages of mesDA neurogenesis (E11.5-E15.5), Nurr1 staining showed a lack of Nurr1-positive mesDA neuron precursors in the medial region of the VM in Ngn2 mutants. (E,K,Q) Interestingly, GFP expressed from the Ngn2 locus revealed the presence of cells in the medial region of the Ngn2 mutants. (B,E,H,K,O,S) GFP staining; (C,F,I,L,O,R) merged images. Scale bars: 100 µm.

View larger version (87K): [in a new window]   Fig. 5. Absence of Ngn2 causes a reduction of the MZ and retention of cells within the VZ/IZ. (A-D,F-I) ß-III-tubulin staining at E13.5 revealed the presence of neurons within the MZ of the mesDA domain despite the almost complete lack of Th-positive neurons. (E,J) However, DAPI staining showed a reduction in total cell number in the MZ and an accumulation of retained cells in the VZ/IZ of the Ngn2 mutants in comparison to heterozygous mice. Diagrams on the right show the positioning of the VZ, IZ and MZ in the heterozygous and mutant mice.

View larger version (125K): [in a new window]   Fig. 6. Build-up of unspecified postmitotic precursor cells in the IZ of the Ngn2 mutants. (A-C,I-K) Lack of Ngn2 function caused an increase in the number of GFP/GLAST-positive neural progenitors with maintained contact to the ventricular surface compared with wild type (arrows). (D,E) BrdU labeling, however, did not show an increase in the number of proliferating cells or an expansion of the VZ in the Ngn2 mutants. (L,M,O,P) Staining for the neuronal determination factor Neurod1 (double staining with GFP) showed that these IZ cells, in addition to being Nurr1-negative, were Neurod1-negative, with the exception of a few laterally positioned precursors. (G,H) Nuclear DAPI stain of the Neurod1/GFP double-stained sections shows the distribution of cells in the IZ. (F,N) At E15.5, the accumulation of cells, as detected by DAPI, in the VZ/IZ has diminished and more cells are present in the MZ compared with at E13.5 (Fig. 3E,J).

View larger version (150K): [in a new window]   Fig. 7. The development of non-DA neurons in the VM is unaffected in the Ngn2 mutant. In the heterozygotes, the Isl1-positive oculomotor neurons show weak GFP-expression at E11.5, and are thus presumably derived from the Ngn2-expressing VZ progenitors cells. Nevertheless, the number of Isl1-positive cells was unaffected in the Ngn2 mutants, both at E11.5 (A,D) and at P0 (H,K). Similarly, the Brn3a-positive neurons of the red nucleus were not affected in the Ngn2 mutant, neither at E13.5 (G,J), nor at P0 (I,L). (B,E) GFP staining at E11.5; (C,F) Merged image of A,B and D,E, respectively.