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First published online June 25, 2007
doi: 10.1242/10.1242/dev.02865

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Retinoic acid counteracts developmental defects in the substantia nigra caused by Pitx3 deficiency

Frank M. J. Jacobs^*, Simone M. Smits^*, Cornelle W. Noorlander, Lars von Oerthel, Annemarie J. A. van der Linden, J. Peter H. Burbach and Marten P. Smidt

Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.

View larger version (101K): [in this window] [in a new window]   Fig. 1. Ahd2 expression marks a specific mdDA subpopulation in the adult brain. (A) Coronal sections from the rostral (R) to caudal (C) mdDA area containing neurons in the SNc (A-D) and VTA (E-L). (Left panels) The cell bodies of these mdDA neurons were identified by Th mRNA expression. (Right panels) Analysis of Ahd2 expression on adjacent sections. (B) Combined in situ hybridization for Ahd2 mRNA (purple) and an immunohistochemical staining for TH protein (brown) on adult coronal sections. The boxed areas indicated in A and D are displayed at a higher magnification in the panels B and C, and E and F, respectively. (C) Adult coronal sections of the SNc (A-C,G-I) and VTA (D-F,J-L). (B,E) Ahd2 protein distribution (red). MdDA neurons were identified by the expression of TH protein (green; A,D). Overlays demonstrate the co-expression of the TH and Ahd2 proteins in a subpopulation of mdDA neurons located in the SNc and VTA (C,F-L). The boxed areas indicated in G and J are displayed at a higher magnification in H and I, and K and L, respectively. mdDA, meso-diencephalic dopaminergic; SNc. substantia nigra pars compacta; VTA, ventral tegmental area.

View larger version (53K): [in this window] [in a new window]   Fig. 2. Ahd2 expression is restricted to the lateral part of the mdDA during the late differentiation phase. Distribution of Ahd2 mRNA in sagittal E13.5 sections from the lateral to medial part of the meso-diencephalic dopaminergic (mdDA) area containing neurons in the presumptive substantia nigra pars compacta (SNc) and ventral tegmental area (VTA), respectively. The cell bodies of the fully differentiated mdDA neurons were identified by Th mRNA expression, and Ahd2 expression was analyzed on the adjacent sections. Expression of Aadc on the adjacent sections identified, in addition to the fully differentiated mdDA neurons, young migrating DA neurons located more dorsally. 3V, third ventricle; 4V, fourth ventricle; Aq, aqueduct; MF, mesencephalic flexure.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Ahd2 expression is largely lost in the mdDA system of Pitx3-deficient mice. (A) Distribution of Ahd2 in adult coronal sections of Pitx3-deficient mice from the rostral (R) to the caudal (C) mdDA area containing neurons in the SNc and VTA. The cell bodies of these mdDA neurons were identified by Th mRNA expression (A,C,E), and Ahd2 expression was analyzed on adjacent sections (B,D,F). (B) Distribution of Ahd2 mRNA in sagittal E13.5 sections from the lateral (L) to medial (M) part of the mdDA area of Pitx3-deficient mice. The cell bodies of the fully differentiated mdDA neurons were identified by Th mRNA expression, and Ahd2 expression was analyzed on adjacent sections. Expression of Aadc on adjacent sections identified, in addition to the fully differentiated mdDA neurons, young migrating DA neurons located more dorsally. 3V, third ventricle; 4V, fourth ventricle; Aq, aqueduct; mdDA, meso-diencephalic dopaminergic; MF, mesencephalic flexure; SNc. substantia nigra pars compacta; VTA, ventral tegmental area.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Expression of Ahd2 is decreased in SNc mdDA neurons of Pitx3+/- mice. (A) Quantitative in situ hybridization analysis of the Ahd2, Th, Vmat2 and a-synuclein mRNA expression levels in the substantia nigra pars compacta (SNc) of Pitx3+/+ (wt, white bars, n=8) and Pitx3+/- (black bars, n=5) mice. The graphs represent the average labeling expressed as percentage of levels in Pitx3+/+ mice ±s.e.m. and were analyzed by using the Student's t-test (*, P<0.05). (B) Typical examples of individual autoradiographic film labeling from coronal sections through the meso-diencephalic dopaminergic (mdDA) area containing the SNc from adult wild-type mice, which were used for determination of mRNA levels.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Effect of Pitx3 overexpression on endogenous Ahd2 transcript levels and other mdDA marker genes. (A) Overexpression of Pitx3 in MN9D cells leads to an increase of endogenous Ahd2 transcript levels. Expression of Th, Aadc, Vmat2, Snca and Tbp were unaffected. (B) Densitometer-based quantification showing the fold change of endogenous transcripts between cells transfected with empty expression vector (+ Control, white bars) and Pitx3 transfected cells (+ Pitx3, black bars).

View larger version (37K): [in this window] [in a new window]   Fig. 6. Pitx3 specifically interacts with a highly conserved region in proximity of the Ahd2 transcription start site. (A) Genomic positions of a selection (regions 2, 4 and 5) of the conserved putative Pitx3-binding sites that were selected for PCR-based analysis in a ChIP assay. (B) ChIP assay with either Pitx3 antibody or control rabbit immunoglobulins (IgG), with input chromatin as positive control. Genomic region 5 is specifically amplified from the Pitx3 ChIP sample. No amplification was observed for the previously described Pitx3-binding site (region 2) (Chung et al., 2005) or genomic region 4. Also, a randomly chosen genomic region (Control) showed no amplification. (C) Comparison of genomic region 5 between mouse, rat and human, showing high sequence conservation (gray) and a fully conserved non-consensus putative Pitx3-binding site amongst species (black).

View larger version (65K): [in this window] [in a new window]   Fig. 7. Retinoic acid treatment counteracts the developmental mdDA defects in Pitx3 deficiency. (A) Schematic representation of an E14.5 sagittal section showing the position of the caudal (I) and rostral (II) sections shown in B and C. The position of the meso-diencephalic dopaminergic (mdDA) neurons are depicted in gray. (B) E14.5 coronal sections of the caudal (A-C) and rostral (D-F) part of the mdDA area. (B,E) Ahd2 protein distribution (red). MdDA neurons were identified by the expression of TH protein (green; A,D). Overlays demonstrate the co-expression of the TH and Ahd2 proteins in a specific subpopulation of mdDA neurons located in rostro-lateral positions and defining the developing substantia nigra pars compacta (SNc; C,F). (C) Distribution of TH-immunoreactive (TH-IR) neurons in E14.5 coronal brain sections of retinoic acid (RA)-treated Pitx3-/- embryos (B,F), RA-treated Pitx3+/+ embryos (D,H), untreated Pitx3-/- embryos (A,E) and untreated Pitx3+/+ embryos (C,G) at the caudal (A-D) and rostral (E-H) level of the mdDA area. (D) Quantitative analysis of TH-IR neurons in the caudal and rostral part of the mdDA area in RA-treated (black bars) and untreated (white bars) embryos. The average number of TH-IR neurons per section are expressed as a percentage of the number of Pitx3+/+ embryos ±s.e.m. (n=3; Student's t-test; *P<0.01). F, forebrain; H, hindbrain; M, midbrain; MHB, mid/hindbrain border.

View larger version (36K): [in this window] [in a new window]   Fig. 8. The effect of temporary retinoic acid treatment in Pitx3-/- embryos is maintained at later developmental stages. (A) Distribution of TH-immunoreactive (TH-IR) neurons in E18.5 coronal brain sections at the level of the substantia nigra pars compacta (SNc) of untreated Pitx3+/+ embryos (A), untreated Pitx3-/- embryos (B), retinoic acid (RA)-treated Pitx3+/+ embryos (C) and RA-treated Pitx3-/- embryos (D). (B) Quantitative analysis of TH-IR neurons in the SNc of E18.5 RA-treated (black bars) and untreated (white bars) embryos. The average number of TH-IR neurons per section are expressed as percentage of the number of Pitx3+/+ embryos ±s.e.m. (n=3 for untreated Pitx3-/-, RA treated Pitx3-/- and RA-treated Pitx3+/+ embryos; n=2 for untreated Pitx3+/+ embryos; Student's t-test; *P0.01). (C) Schematic representation of an E18.5 sagittal section showing the position of the striatal regions (I and II) demonstrated in D. (D) TH-immunoreactivity at two levels of the striatum of E18.5 embryos. The arrows mark the dorsal striatal areas in which an increase of TH-immunoreactivity is observed.