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First published online February 18, 2004
doi: 10.1242/10.1242/dev.01022

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Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3

¹ Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands
² GSF, Institute of Developmental Genetics, Ingolstaedter Landstrasse 1, D-85764 München, Germany

View larger version (97K): [in a new window]   Fig. 1. Expression of Pitx3 in wild-type (A,C,E) and ak mutants (B,D,F) in the adult brain (A-D) and at stage E12.5 of brain development (E,F). Pitx3 was not expressed in the brains of ak mice. SNc, substantia nigra compacta; VTA, ventral tegmental area; MF, mesencephalic flexure.

View larger version (57K): [in a new window]   Fig. 2. Th-positive neurons in the mesencephalon at embryonic stages E11.5 to E14.5. (A) Coronal sections from the posterior to anterior mesencephalon. (B) Enlargement of sections displaying the aberrant development in the SNc region (arrow) at stage E12.5 and E14.5. The arrows indicate the anatomical positions where mesDA neurons are missing. The asterisk indicates the medial position where mesDA neurons are ectopically present in the ak.

View larger version (77K): [in a new window]   Fig. 3. Aadc and Pitx3 expression in the mesencephalic neuronal field at E12.5. (A) Aadc expression in a sagittal section showing the mesencephalic dopaminergic (mesDA) field and additional positive neurons located dorsally in the midbrain and serotonergic neurons positioned caudal to the midbrain/hindbrain border (MHB). (B) Tyrosine hydroxylase (Th) expression in an adjacent section to A. (C) Schematic representation of a sagittal section showing the position of the coronal sections shown in D-I. (D-F) Aadc expression in coronal sections at three different positions as indicated in C. (G-H) Pitx3 expression in adjacent sections to D-F. Aq, aqueduct.

View larger version (54K): [in a new window]   Fig. 4. Level of tyrosine hydroxylase (Th) protein measured by immunohistochemistry in wild-type and ak brain sections. (A) Coronal section of the mesencephalic dopaminergic (mesDA) neuronal region. The normal architecture of the system is indicated in the wild-type (arrows). Similar sections from the ak brain are paired to match the wild-type level. (B) The left two panels indicate the neurons of the mesDA system and their architecture, the projecting axons and the target area in the striatum in sagittal sections. The right two panels show similar sections in the ak brain with the altered mesDA neuron architecture and the missing projection area in the caudate putamen indicated by the asterisk. (C) Th expression in coronal sections of the striatal area. CPu, caudate putamen; MFB, main forebrain bundle; VTA, ventral tegmental area; SNc, substantia nigra compacta; Acb, nucleus accumbens; Tu, tubercle olfactorium.

View larger version (108K): [in a new window]   Fig. 5. Colocalization of Pitx3 and tyrosine hydroxylase (Th) in adult coronal sections in the mesencephalic dopaminergic (mesDA) neuronal field. Colocalization of Pitx3 mRNA (blue) and Th protein (brown) in adult SNc (A-A'') and VTA (B-B'') neurons of wild-type mice. Colocalization of Th mRNA (blue) and Pitx3 protein (brown) in adult SNc (C-C'') and VTA (D-D'') neurons of wild-type mice. The boxed areas indicated in A-D are given in a higher magnification in the panels on the right. SNc, substantia nigra compacta; VTA, ventral tegmental area.

View larger version (132K): [in a new window]   Fig. 6. Coronal sections showing Nissl-staining (A) of the wild-type and ak adult mesDA neuronal field compared to adjacent sections stained for Th (B). Coronal sections of lateral SNc neurons (a,a',b,b'), medial SNc neurons (c,c',d,d') and neurons of the VTA (e,e',f,f') are depicted. Boxed areas shown in A-F are shown at a higher magnification in the figures below (a'-f'). (g,h) High magnification of mesDA neurons in the VTA area, showing the altered morphology (arrows) in the ak compared with the wild-type mice.

View larger version (69K): [in a new window]   Fig. 7. Retrograde tracing in the mesencephalic dopaminergic (mesDA) system and double labeling by Pitx3 immunohistochemistry. (A) Retrograde tracing in the control brain. The left two panels (FG) show the fluorogold label in the traced neurons in the mesDA system. The middle two panels (FG/Pitx3) show the double labeling. The arrow indicates one of the double labeled mesDA neurons. The right two panels (Pitx3) show the labeling by Pitx3 alone. The lower two panels show the injection site (right) and a schematic representation indicating the injection site (arrows). (B) Retrograde tracing in the ak mutant brain. The left panel represents the injection position. The right panel shows a neuron (arrow) in the VTA region that was traced by injection in the dorsal striatum.

View larger version (76K): [in a new window]   Fig. 8. Analysis of mesencephalic dopaminergic (mesDA)-associated gene expression in SNc (A) and VTA (B) of ak mice compared with wild-type mice. Coronal sections of Th, Aadc, Vmat2, D2r, Dat, Cck, Ntr1 and alpha-synuclein expression in mesDA neurons are depicted. SNc, substantia nigra compacta; VTA, ventral tegmental area; EW, Edinger-Westphal nucleus; DG, dentate gyrus; SuM, supra mammilary nucleus.

View larger version (19K): [in a new window]   Fig. 9. Behavioral analysis of wild-type (bl6), blind wild-type (bl6b) and ak mice. (A) Level of climbing behavior (n=10, all males). The y-axis represents the average score of 10 animals in each group. The error bars indicate the standard error of the mean within the groups. The groups were significantly different from each other (Krukal-Wallis test, P<0.01). Wild-type mice/blind mice (P<0.01); wild-type mice/ak (P<0.01); blind mice/ak (P=0.124). B) Horizontal movement in an open field. Movement was recorded by automatic analysis software (Ethovision). All groups were significantly different from each other (One-way ANOVA P<0.01), as well as within groups (P<0.01, for all combinations).

View larger version (68K): [in a new window]   Fig. 10. Graphical representation of the pattern of cell genesis and migration in the midbrain. (A) Reported models for development of the mesencephalic dopaminergic (mesDA) neuronal field combined with tyrosine hydroxylase (Th) staining in coronal sections of E12.5 midbrain. The neuroepithelium is marked by a double line. Model 1 represents vertical migration followed by lateral migration. Model 2 represents perpendicular migration of SN precursors and ventral migration of VTA precursors. The direction of the migration in both models is indicated by arrows. (B) Schematic drawing representing the molecular events of mesDA development, in line with model 2. Aq, aqueduct.

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