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. 2003 Oct 28;100(22):12747-52.
doi: 10.1073/pnas.1534900100. Epub 2003 Oct 13.

Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a

Gonçalo Castelo-Branco  1 , Joseph Wagner, Francisco J Rodriguez, Julianna Kele, Kyle Sousa, Nina Rawal, Hilda Amalia Pasolli, Elaine Fuchs, Jan Kitajewski, Ernest Arenas
Affiliations

Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a

Gonçalo Castelo-Branco et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A. 2004 Nov 16;101(46):16390

Abstract

The Wnts are a family of glycoproteins that regulate cell proliferation, fate decisions, and differentiation. In our study, we examined the contribution of Wnts to the development of ventral midbrain (VM) dopaminergic (DA) neurons. Our results show that beta-catenin is expressed in DA precursor cells and that beta-catenin signaling takes place in these cells, as assessed in TOPGAL [Tcf optimal-promoter beta-galactosidase] reporter mice. We also found that Wnt-1, -3a, and -5a expression is differentially regulated during development and that partially purified Wnts distinctively regulate VM development. Wnt-3a promoted the proliferation of precursor cells expressing the orphan nuclear receptor-related factor 1 (Nurr1) but did not increase the number of tyrosine hydroxylase-positive neurons. Instead, Wnt-1 and -5a increased the number of rat midbrain DA neurons in rat embryonic day 14.5 precursor cultures by two distinct mechanisms. Wnt-1 predominantly increased the proliferation of Nurr1+ precursors, up-regulated cyclins D1 and D3, and down-regulated p27 and p57 mRNAs. In contrast, Wnt-5a primarily increased the proportion of Nurr1+ precursors that acquired a neuronal DA phenotype and up-regulated the expression of Ptx3 and c-ret mRNA. Moreover, the soluble cysteine-rich domain of Frizzled-8 (a Wnt inhibitor) blocked endogenous Wnts and the effects of Wnt-1 and -5a on proliferation and the acquisition of a DA phenotype in precursor cultures. These findings indicate that Wnts are key regulators of proliferation and differentiation of DA precursors during VM neurogenesis and that different Wnts have specific and unique activity profiles.

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Figures

Fig. 5.
Fig. 5.
Wnts differentially control the development of DA neurons by regulating precursor proliferation and the acquisition of a DA phenotype. Wnt-5a, but not Wnt-1, up-regulated the expression of Ptx3 mRNA (A) and c-ret mRNA (B) and maintained the expression of GDNF family receptor α1 (GFRα1) mRNA (C) and NCAM mRNA (D) at 3 days in vitro, as assessed by real-time RT-PCR in E14.5 rat VM precursor cultures. (E and F) Double immunocytochemistry revealed that Wnt-5a increased the percentage of TH+/Nurr1+ cells from 50% to 90%. Wnt-1 was less efficient than Wnt-5a, and Wnt-3a actually decreased the proportion of TH+ cells from 50% to 30%. (G and I) Fz8-CRD decreased, in a dose-dependent manner, the proportion of Nurr1+ cells that acquired TH expression in E14.5 VM precursor cultures, indicating that Wnt signaling is required for the acquisition of a DA phenotype. (H and I) Treatment of rat E14.5 VM precursor cultures with Fz8-CRD decreased the percentage of TH+/Nurr1+ cells after treatment with Wnt-5a. Statistical analysis and concentrations as in Fig. 4. (J) Model of the mechanisms by which Wnt-1, -3a, and -5a regulate the development of VM DA neurons. Wnt-3a, which is mainly expressed in the dorsal midbrain, enhances the proliferation of Nurr1-expressing precursors and decreases the proportion of neurons that acquire a DA phenotype. Wnt1, probably derived from the midbrain–hindbrain organizer, controls the proliferation of Nurr1-expressing precursors and increases the number of VM neurons. Finally, Wnt-5a increases the number of VM DA neurons specifically by regulating the acquisition of a DA phenotype in Nurr1-expressing precursors. Note that the size of the arrows correlates with the intensity of the effects.
Fig. 1.
Fig. 1.
Detection of β-catenin transcriptional activity (A and B) and Wnt expression (C–J) in the developing midbrain in vivo. (A) Sagittal sections through the developing VM revealed that β-catenin and Nurr1 are expressed in the same domain at E10.5. (B) Analysis of the Nurr1+ domain in TOPGAL reporter mice at E10.5 revealed the presence of β-galactosidase-positive cells, indicating that the Wnt canonical signaling pathway is active in the developing VM before the birth of TH+ neurons. (C–F) In situ hybridization on sagittal brain sections including forebrain (fb), dorsal midbrain (dm), VM (vm), and hindbrain (hb). Wnt-1 (E) and Wnt-5a (F) mRNA expression domains coincide with those of Nurr1+ (D) and TH+ (C) cells. (G–J) Real-time RT-PCR analysis revealed that Wnt-1 is expressed at high levels in the VM and dorsal midbrain, whereas Wnt-3a expression predominates in the dorsal midbrain and Wnt-5a predominates in the VM. *, P < 0.05; **, P < 0.001; and ***, P < 0.0001 (compared with the first stage analyzed for every brain region by one-way ANOVA with Fisher's post hoc test).
Fig. 2.
Fig. 2.
Wnt-1 and -5a, but not Wnt-3a, increase the number of DA neurons (A–C) and proliferating cell clusters containing DA neurons (D–F) in rat E14.5 VM precursor cultures. Partially purified Wnts were normalized to each other based on the density of Western blot product bands and expressed in arbitrary units, equivalent to 1 μl of normalized partially purified product. Control partially purified medium (CP) was diluted to achieve the same protein concentration as the partially purified Wnts. (A and D) Dose dependency at 3 and 7 days in vitro, respectively. (B and E) Time-course analysis (10 units). (C and F) Comparison of the effect of 10 units of Wnts with CP. TH-immunostained cultures show that Wnt-1 and -5a induce a very dramatic increase in the number of DA neurons and TH+ cell clusters. *, P < 0.01 [compared with CP, by one-way ANOVA with Fisher's post hoc test (n = 4–6)]. A field is 3.14 mm2 and a well is 4 cm2.
Fig. 3.
Fig. 3.
Wnts promote neurogenesis and increase the number of midbrain DA neurons in E14.5 rat VM precursor cultures by two different mechanisms. (A and B) Wnt-1, but not Wnt-3a or -5a, increased the number of TuJ1+ neurons outside cell clusters in VM cultures. A field is 3.14 mm2.(C) Wnt-1 and -3a, but not Wnt-5a, increased the number of proliferating clusters that contained TuJ1+ neurons. A well is 4 cm2.(D) Despite the increase in neurons outside the clusters, Wnt-1 did not increase the proportion of DA neurons. Instead, treatments that did not change the numbers of Tuj1+ neurons either decreased (Wnt-3a) or increased (Wnt-5a) the proportion of DA neurons. (E) The proportion of DA neurons in proliferating clusters containing neurons did not change after Wnt-1 treatment, was decreased by Wnt-3a, and was increased by treatment with Wnt-5a. Concentrations were 10 units/μl partially purified media. *, P < 0.01; **, P < 0.001; and ***, P < 0.0001 [compared with CP, by one-way ANOVA with Fisher's post hoc test (n = 3–5)].
Fig. 4.
Fig. 4.
Wnt-1 regulated the expression of cyclin D1 and the cell cycle inhibitors p27 and p57 and increased the proliferation of VM precursors. Real-time RT-PCR showed that Wnt-1, but not Wnt-5a, increased the expression of cyclin D1 mRNA (A) and decreased the expression of the cell cycle inhibitors p27 (B) and p57 (C) at 3 days in vitro. (D) Wnt-1 and -3a, but not Wnt-5a, increased the proliferation of VM precursors at 3 days in vitro.(E and F) Wnt-5a was less efficient than Wnt-1 and -3a at increasing BrdUrd incorporation in VM Nurr1+ precursor cells after 1 day in vitro.(G) Increasing units of partially purified Fz8-CRD, a Wnt blocking reagent, reduced the number of TH+ neurons in VM cultures dose-dependently after 3 days in vitro, indicating that endogenous Wnts are required for DA development. (H and I) The increase in the number of TH+ neurons by Wnt-1 was partially blocked by Fz8-CRD, suggesting that the effects of Wnts are specific. •, P < 0.05; *, P < 0.01; **, P < 0.001; ***, P < 0.0001, compared with control; and ##, P < 0.0001 [compared Wnt treatment alone, by one-way ANOVA with Fisher's post hoc test (n = 3–5; for Fz8-CRD, n = 2 or 3)]. Concentrations are 10 units/μl Wnts or CP and 5 units/μl Fz8-CRD. A field (in D and H) is 3.14 mm2.
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