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First published online June 1, 2006
Stem Cells Vol. 24 No. 9 September 2006, pp. 2120 -2129
doi:10.1634/stemcells.2005-0514; www.StemCells.com
© 2006 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Transforming Growth Factor ß Is Required for Differentiation of Mouse Mesencephalic Progenitors into Dopaminergic Neurons In Vitro and In Vivo: Ectopic Induction in Dorsal Mesencephalon

Eleni Roussaa, Michael Wiehlea, Nicole Dünkera, Steffen Becker-Katinsb, Oliver Oehlkea, Kerstin Krieglsteina

aDepartment for Neuroanatomy, Georg-August-University, DFG Research Center of Molecular Physiology of the Brain, Göttingen, Germany;
bMedical Faculty, University of Saarland, Homburg/Saar, Germany

Key Words. Isthmus organizer • Floor plate • Mutant mice • Transforming growth factor ß

Correspondence: Eleni Roussa, D.D.S., Department for Neuroanatomy, Research Center of Molecular Physiology of the Brain, University of Göttingen, Kreuzbergring 36, D-37075 Göttingen, Germany. Telephone: +49-551-397051; Fax: +49-551-3914016; e-mail: eroussa{at}gwdg.de

Received October 14, 2005; accepted for publication May 22, 2006.
First published online in STEM CELLS EXPRESS   June 1, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Tissue engineering is a prerequisite for cell replacement as therapeutic strategy for degenerative diseases, such as Parkinson’s disease. In the present study, we investigated regional identity of mesencephalic neural progenitors and characterized their development toward ventral mesencephalic dopaminergic neurons. We show that neural progenitors from ventral and dorsal mouse embryonic day 12 mesencephalon exhibit regional identity in vitro. Treatment of ventral midbrain dissociated neurospheres with transforming growth factor ß (TGF-ß) increased the number of Nurr1- and tyrosine hydroxylase (TH)-immunoreactive cells, which can be further increased when the spheres are treated with TGF-ß in combination with sonic hedgehog (Shh) and fibroblast growth factor 8 (FGF8). TGF-ß differentiation signaling is TGF-ß receptor-mediated, involving the Smad pathway, as well as the p38 mitogen-activated protein kinase pathway. In vivo, TGF-ß2/TGF-ß3 double-knockout mouse embryos revealed significantly reduced numbers of TH labeled cells in ventral mesencephalon but not in locus coeruleus. TH reduction in Tgfß2–/–/Tgfß3+/– was higher than in Tgf-ß2+/–/Tgf-ß3–/–. Most importantly, TGF-ß may ectopically induce TH-immunopositive cells in dorsal mesencephalon in vitro, in a Shh- and FGF8-independent manner. Together, the results clearly demonstrate that TGF-ß2 and TGF-ß3 are essential signals for differentiation of midbrain progenitors toward neuronal fate and dopaminergic phenotype.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Tissue engineering has been a special focus in research, underlying cell replacement as therapeutic strategy for degenerative diseases, such as Parkinson’s disease [1]. Consequently, understanding the molecular mechanisms dictating generation of mesencephalic dopaminergic neurons from neural progenitors and identifying signals that control their induction and differentiation is of importance.

To investigate the mechanisms controlling cellular fate, many studies have been performed to assay for "cues" or activities that induce changes in this fate, and it is well-established that neural fate is controlled by intrinsic and extrinsic determinants [2]. However, experimental control of neural progenitor differentiation toward a particular neuronal or glial fate and understanding the interplay of intrinsic and environmental restrictions, together with temporal constraints in establishing cell fate, are challenging [3].

Dopaminergic progenitors are located in ventral mesencephalon, a brain area known to contain stem cells, in spatial proximity to the floor plate and the isthmus. This distinct location of dopaminergic precursors has implied that generation of midbrain dopaminergic neurons is likely being controlled by sonic hedgehog (Shh) and fibroblast growth factor 8 (FGF8) [4], the key signals for patterning along the dorsoventral and anteroposterior axis, respectively [5, 6]. The importance and interplay of Shh and FGF8 in the development of dopaminergic neurons has been highlighted in studies showing that individual factors can induce ectopic tyrosine hydroxylase (TH)-positive cells, provided that the other factors are endogenously present [7, 8].

However, an increasing number of recent in vivo and in vitro studies have provided convincing evidence that transforming growth factor ß (TGF-ß), a molecule that is also expressed in mesencephalon, is an additional essential mediator for the induction and maintenance of midbrain dopaminergic neurons [9–11]. In zebrafish mutations with affected TGF-ß/Nodal signaling, ventral dopaminergic neurons do not develop [10, 12]. In chick embryos, neutralization of endogenous expressed TGF-ß isoforms suppressed differentiation of midbrain dopaminergic neurons [9]. In an in vitro model using cells dissociated from rat embryonic day 12 (E12) ventral mesencephalon, neutralization of all TGF-ß isoforms completely abolished induction of dopaminergic neurons. However, the underlying molecular mechanism is still not fully elucidated.

In the present study, we have investigated regional identity of neurospheres derived from mouse E12 ventral and dorsal mesencephalon and studied the role of TGF-ß in progenitor cell differentiation toward dopaminergic neurons in vitro. In addition, the contextual importance of different TGF-ß isoforms in midbrain dopaminergic neuron differentiation was investigated in vivo. A highlight of the present study is the observation that TGF-ß may be capable of ectopically inducing TH-immuno positive cells in neurospheres derived from dorsal mesencephalon in an Shh-independent manner.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Recombinant human (rh) N-terminal Shh was kindly provided by Dr. A. Galdes (Biogen, Cambridge, MA, http://www.biogen.com). rh FGF8 was purchased from R&D Systems (Wiesbaden, Germany, http://www.rndsystems.com) and FGF2 and TGF-ß1 was obtained from Tebu (Offenbach, Germany, http://www.tebu-bio.com). Lyophylized factors were resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, http://www.invitrogen.com) with 0.25% bovine serum albumin (BSA) (cell culture grade; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 100 U/ml penicillin, 0.5 µg/ml streptomycin, and 100 µg/ml neomycin (all from Gibco) to give a final concentration of 1 µg/ml and stored in aliquots of 50–100 µl at –70°C until use. Lyophylized TGF-ß was resuspended in 1 mM HCl containing 0.25% BSA (Sigma-Aldrich) and stored in aliquots of 50 µl at –70°C until use. The neutralizing monoclonal mouse anti-TGF-ß antibody (MAB1835) recognizing all three isoforms was obtained from R&D Systems. To ensure that the neutralizing anti-TGF-ß antibody specifically recognizes only the TGF-ß isoforms and not other growth factors, immunoprecipitation, dot-blots, and Western blots were performed (not shown) [cf. refs. 13, 14]. Activity of this antibody was determined using the Mv1Lu mink lung epithelial cell system [14, 15]. The function-blocking anti-Shh antibody was obtained from Developmental Studies Hybridoma Bank (Iowa City, IA, http://www.uiowa.edu/~dshbwww), and the fibroblast growth factor receptor (FGFR)3a-IgG that neutralizes FGF8 was obtained from R&D Systems. SB431542, an ALK4, ALK5, and ALK7 inhibitor, was purchased from Tocris Bioscience (Bristol, U.K., http://www.tocris.com), and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 was obtained from Calbiochem (Schwalbach am Taunus, Germany, http://www.emdbiosciences.com).

Cell Culture of E12 Ventral and Dorsal Mesencephalon
Ventral and dorsal mesencephalon from mouse E12 was isolated as described earlier [16]. Briefly, pregnant Naval Medical Research Institute mice were sacrificed by cervical dislocation, and E12 embryos were collected in Ca2+-Mg2+-free Hanks’ balanced salt solution (Sigma-Aldrich). The day of vaginal plug identification was designated E1. Ventral and dorsal mesencephalon were dissected, freed from meninges, and incubated in 0.15% trypsin for 15 minutes at 37°C. Small pieces of ventral and dorsal mesencephalon were subsequently dissociated by gentle trituration using fire-polished Pasteur pipettes. Dissociated cells were resuspended in high glucose DMEM-Ham’s F-12 medium supplemented with 0.25% BSA, N1 additives, 100 U/ml penicillin, 0.5 µg/ml streptomycin, 100 µg/ml neomycin (Gibco). Cells in suspension were plated in noncoated culture dishes and cultured in the presence of 20 ng/ml FGF2. After 3 days in culture, formation of neurospheres was evident. Cells were allowed to expand for 7 days before the neurospheres were plated onto polyornithin- and laminin-coated 12-mm2 glass coverslips in 24-well plates at a density of 200,000 cells per coverslip. FGF2 was removed from the expansion medium, and cells were incubated in serum-free medium in a 95% air/5% CO2 atmosphere at 37°C.

One day after plating (day in vitro [DIV] 1), factors were applied at a final volume of 750 µl of medium at the following concentrations: TGF-ß1, 1 ng/ml; Shh, 1 nM; FGF8, 10 ng/ml; anti-TGF-ß, 10 µg/ml; anti-Shh, 2.5 µg/ml; FGFR3a IgG, 20 ng/ml. SB431542 and SB203580 were applied at 10 and 20 µM, respectively. At DIV 3, cells were fixed and processed for immunocytochemistry.

Immunocytochemistry
Immunocytochemistry on cultured cells was performed essentially as described earlier [13]. Cultures were fixed in 4% paraformaldehyde for 30 minutes at room temperature, permeabilized with acetone for 10 minutes at –20°C, and blocked with 10% normal goat serum (NGS) in phosphate-buffered saline (PBS). Subsequently, cells were incubated overnight at 4°C with primary antibodies diluted in PBS containing 10% NGS and 0.01% Triton X-100. Rabbit polyclonal anti-TH (1:1,000), anti-Pitx3 (1:400) (Chemicon, Hofheim, Germany, http://www.chemicon.com), anti-Nurr1 (1:200) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), and anti-Smad2 (Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com) and were used as primary antibodies. Donkey anti-rabbit or anti-mouse IgGs coupled to indocarbocyanin (CY3) were used as secondary antibodies. Nuclei were counterstained with 4',6'-diamidino-2-phenylindole dihydrochloride diluted 1:1,000 in PBS for 5 minutes, washed with PBS, and viewed with a fluorescence microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com). Control experiments were performed by omitting primary antibody, confirming that immunostaining was then absent.

Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from ventral and dorsal mesencephalic primary dissected tissue, as well as from ventral- and dorsal-derived neurospheres, using the Qiagen Rneasy kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) and following the manufacturer’s instructions. Total RNA (1.0 µg) was reverse-transcribed from an oligo(dT) primer using a Qiagen Omniscript kit. Samples (3 µl) of the reverse transcription reaction were used in PCR containing 2.5 U of Taq DNA polymerase, 0.2 µM specific primers, 1x Qiagen PCR buffer, 200 µM deoxynucleoside triphosphates, and 2 mM MgCl2. For amplification of cDNA encoding Nestin, ß-III-tubulin, Nurr1, Pitx3, TH, TGF-ß3, Shh, and FGF8, the following forward (F) and reverse (R) primer sequences were used:

Nestin F: 5'-CAG GCT TCT CTT GGC TTT CCT G-3'

Nestin R: 5'-GGT GAG GGT TGA GGG GTG G-3'

ß-III-tubulin F: 5'-GGA ACA TAG CCG TAA ACT GC-3'

ß-III-tubulin R: 5'-TCA CTG TGC CTG AAC TTA CC-3'

Nurr1 F: 5'-TGA AGA GAG CGG AGA AGG AGA TC-3'

Nurr1 R: 5'-TCT GGA GTT AAG AAA TCG GAG CTG-3'

Pitx3 F: 5'-ACG CAC TAG ACC TCC CTC CAT-3'

Pitx3 R: TAC GAG TAG CCC GGG TACA

TH F: 5'-TCC TGC ACT CCC TGT CAG AG-3'

TH R: 5'-CCA AGA GCA GCC CAT CAA AGG-3'

TGF-ß3 F: 5'-GGAAATCAAATTCAAAGGAGTGG-3'

TGF-ß3 R: 5'-AGTTGGCATAGTAACCCTTAGG-3'

FGF8 F: 5'-TTTACACAGCATGTGAGGGAG-3',

FGF8 R: 5'-GTAGTTGAGGAACTCGAAGCG-3',

SHH F: 5'-TGATGTGTGGGCCCGGCAGGGGGTTT-3'

SHH R: 5'-TCAGCCGCCGGATTTGGCCGCCACG-3'.

For detection of cDNAs encoding the above-mentioned proteins, the following protocol was used: denaturation at 95°C for 15 minutes, the optimum number of cycles (depending on the primer pair) of PCR amplification were performed under the following conditions: denaturation at 94°C for 1 minute, annealing at the appropriate temperature (depending on primer pair) for 1 minute, and elongation at 72°C for 1 minute. Final extension at 72°C for 10 minutes was terminated by rapid cooling at 4°C. PCR products were analyzed by agarose gel electrophoresis, and the size of the reaction products was determined after ethidium bromide staining.

Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling Assay
Dissociated mouse E12 ventral mesencephalic neurospheres were plated onto 12-mm coverslips at a density of 200,000 cells per well. Cultures were treated with TGF-ß at DIV 1. At DIV 1, 2, and 3, cells were fixed in 4% paraformaldehyde and subsequently washed. For detection of apoptotic cells, the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay kit from Roche Diagnostics (Basel, Switzerland, http://www.roche-applied-science.com) was used following the manufacturer’s instructions. Apoptotic cells were then visualized by fluorescence microscopy.

5-Bromo-2'-deoxyuridine Incorporation
Dissociated mouse E12 ventral mesencephalic neurospheres were plated onto 12-mm coverslips at a density of 200,000 cells per well. For 5-bromo-2'-deoxyuridine (BrdU) labeling and detection, the detection kit from Boehringer Mannheim (Mannheim, Germany, http://www.boehringer.com) was used. Briefly, dissociated cells were incubated at DIV 1, 2, and 3 with BrdU for 1 hour in a 95% air/5% CO2 atmosphere at 37°C, fixed with 70% ethanol in 50 mM glycin buffer (pH 2.0) for 20 minutes at –20°C and incubated with anti-BrdU antibody for 30 minutes at 37°C. Subsequently, coverslips were washed with buffer, incubated with mouse IgG coupled to fluorescein isothiocyanate for 30 minutes at 37°C, washed again with buffer, and mounted with Vectashield (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Finally, coverslips were viewed with an epifluorescence microscope.

Animals
Tgf-ß2+/– and Tgf-ß3+/– heterozygous mice were offspring from breeding pairs kindly provided by T. Doetschman (University of Cincinnati, Cincinnati, OH). The generation of these strains has been described elsewhere [17, 18]. Tgf-ß2–/–Tgf-ß3–/– double knockouts were generated by cross-breeding the two heterozygous TGF-ß strains and setting up matings between these double-heterozygous mice (Tgf-ß2+/–Tgf-ß3+/–x Tgf-ß2+/–Tgf-ß3+/–). For morphological comparison, in each case, Tgf-ß2+/+Tgf-ß3+/+ littermates of the respective mutants were used as wild-type controls. The morning of the day on which a vaginal plug was detected in mating females was designated gestation day 0.5. As Tgf-ß2–/–Tgf-ß3–/– double knockouts exhibit an early embryonic lethality, dying at approximately E15.5 [19], analyses were performed on 14.5-day-old embryos. Either heads were fixed in Bouin’s fixative (75% picric acid, 25% formaldehyde, and 5% glacial acetic acid) for several hours, dehydrated in an ascending series of ethanol, and embedded in paraffin wax, or embryos were perfused and then brains were removed and postfixed in 4% paraformaldehyde for cryosectioning.

Immunohistochemistry
For immunocytochemistry 10-µm paraffin sections were deparaffinized and heated for 5 minutes in citrate buffer (pH 6) in a microwave oven at 600 W to improve antigen retrieval. After destroying endogenous peroxidase activity by 5 minutes of treatment with 3% H2O2 in H2O, sections were preblocked with the Vector blocking kit (Linaris, Wertheim-Bettingen, Germany, http://www.linaris.de) to avoid nonspecific binding of the biotin/avidin system used for immunodetection (described below). Immunostaining was performed using a specific monoclonal mouse anti-tyrosine hydroxylase antibody at a dilution of 1:100 following the manufacturer’s instructions for the Vector M.O.M. peroxidase immunodection kit (Linaris). After 1 hour of incubation at room temperature, the reaction was visualized by a nickel-intensified 3,3'-diaminobenzidine (Kementec Copenhagen, Denmark, http://www.kem-en-tec.com) reaction, and sections were counterstained by nuclear fast red. As controls, PBS was substituted for the primary antisera to test for nonspecific labeling. No specific cellular staining was observed when the primary antiserum was omitted.

For double-labeling studies, 10-µm cryosections were preincubated with 10% NGS in PBS containing 0.3% Triton X-100 for 1 hour. Immunostaining was performed using a rabbit Nurr1 antibody at a dilution of 1:100. After visualization of the first primary antibody using a goat anti-rabbit Cy3-conjugated IgG at a dilution of 1:2,000 in 10% NGS/PBS/Triton X-100, the second primary antibody, mouse anti-tyrosine hydroxylase antibody (Chemicon), was applied at a dilution of 1:100 following the manufacturer’s instructions for the Vector M.O.M. fluorescein immunodetection kit (Linaris).

Numbers of TH-labeled neurons were counted on the complete series of 10-µm transverse sections. A neuron was designated TH-positive if it revealed a darkly labeled cytoplasm and a clearly visible, unstained nucleus. Only cells fulfilling these criteria were included in the cell counts. To avoid double counting the same cell on two sequential sections, only every fifth section was counted.

Statistics
Data are presented as the mean ± SEM. Statistical analysis was performed using Student’s double t test when one treated group was compared with the control. For multiple comparisons between treated groups statistical differences were compared using a one-way analysis of variance and Bonferroni post-hoc test for multiple comparisons. Differences were considered statistically significant at p < .05 (*), p < .01 (**), and p < .001 (***).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
Cellular Composition of Neurospheres Derived from Ventral Mesencephalon
Self-renewal capacity and multipotency of neurospheres was tested by their ability to generate secondary neurospheres. After dissociation of primary neurospheres into single-cell suspension and subsequent expansion, secondary neurospheres formation was detected (data not shown). Multipotency of neurospheres was assessed in dissociated and plated neurospheres by immunostaining with ß-III-tubulin, GFAP, and O4 (not shown), specific markers for neuronal, glial, and oligodendrocyte lineages, respectively, as previously described [16].

Figure 1A illustrates cellular composition of generated neurospheres (NS) and comparison with those of primary tissue (T). Mouse E12 ventral midbrain T and NS expressed the precursor cell marker nestin and the neuron-specific marker ß-III-tubulin. In addition, expression of the early and late markers of dopaminergic neuron development, the transcription factors Pitx3, Nurr1, and TH were also detected. However, whereas Pitx3 and Nurr1 expression levels were found to be considerably higher in NS, TH expression was more prominent in ventral T.


Figure 1
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  		Figure 1. Dissected primary ventral mesencephalic T and ventral midbrain-derived NS were processed for reverse transcription-polymerase chain reaction using specific primers. (A): Determination of nestin and ß-III-tubulin expression for neural precursor cells and neurons, respectively. Expression of early dopaminergic markers Nurr1 and Pitx3 is increased in neurospheres compared with primary tissue. The late dopaminergic marker TH is expressed in ventral mesencephalic primary tissue and less prominently in neurospheres. (B): Detection of endogenous expression of Shh, FGF8, and TGF-ß3 in mouse embryonic day 12 ventral mesencephalic primary dissected tissue and generated ventral neurospheres. Abbreviations: bp, base pair(s); FGF, fibroblast growth factor; NS, neurospheres; Shh, sonic hedgehog; T, tissue; TGF-ß3, transforming growth factor ß3’3b TH, tyrosine hydroxylase.

 
Figure 1B shows endogenous expression of Shh, FGF8, and TGF-ß3 in mouse E12 ventral T and NS. The levels of expression varied between the factors studied, Shh being more highly expressed in NS and TGF-ß3 and FGF8 being considerably more dominant in T.

TGF-ß Triggers Differentiation of Precursor Cells Toward Dopaminergic Neurons In Vitro
Figure 2 demonstrates differentiation potential of mouse E12 ventral midbrain-derived neurospheres, after treatment with TGF-ß alone, in combination with Shh and FGF8, and after neutralization of individual endogenous expressed factor using function blocking antibodies. The molecular identity of differentiated neurons is shown in Figure 2A, and quantitative analysis is shown in Figure 2B. Untreated ventral mesencephalic neurospheres exhibited Nurr1, Pitx3, and TH immunoreactivity, observations that match the reverse transcription-polymerase chain reaction (RT-PCR) data (Fig. 1A). Single treatment of the neurospheres with TGF-ß significantly increased the number of Nurr1- and TH-positive cells compared with controls, whereas application of TGF-ß, Shh, and FGF8 added together induced a further significant increase in the number of Nurr1- and TH-immunoreactive cells compared with treatment of the spheres with TGF-ß alone. In contrast, none of the treatments used had an effect in the number of Pitx3-immunoreactive cells compared with control spheres. Neutralizing antibodies against all three TGF-ß isoforms (10 µg/ml) [14] or neutralization of endogenous Shh (anti-Shh) or FGF8 (FGFR3a) significantly reduced the number of Nurr1-, Pitx3-, and TH-positive neurons compared with the untreated controls (Fig. 2B).


Figure 2
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  		Figure 2. Differentiation potential of neurospheres derived from mouse embryonic day 12 ventral midbrain after factor treatment. (A): Protein expression of dopaminergic phenotype markers Nurr1 (left column), Pitx3 (middle column), and TH (right column) was assessed by immunofluorescence light microscopy. Scale bar = 50 µm, except * = 100 µm. (B): Quantification: TGF-ß treatment of the cultures significantly increased the number of Nurr1- and TH-positive neurons, but not Pitx3-positive cells, compared with untreated controls. Treatment of the neurospheres with TGF-ß in combination with Shh and FGF8 resulted to additional increase in the number of Nurr1- and TH-positive cells compared with single-factor treatment. Data are given as mean ± SEM (n = 3); p values derived from Student’s t test are as follows: *, p < .05 for increased numbers of immunoreactive cells after factor treatment as compared with untreated controls; +, p < .05, derived from one-way analysis of variance and Bonferroni post hoc test for increased numbers of immunoreactive cells as compared with single-factor treatment. (C): Neutralization of endogenous TGF-ß, Shh, or FGF8 (FGFR3a) significantly decreased the number of Nurr1-, Ptx3-,and TH-immunoreactive cells. Data are given as mean ± SEM (n = 3); p values derived from Student’s t test are as follows: *, p < .05; **, p < .01 for decreased numbers of immunoreactive cells after factor treatment as compared with untreated controls. Abbreviations: FGF8, fibroblast growth factor 8; FGFR, fibroblast growth factor receptor; Shh, sonic hedgehog; TGF, transforming growth factor; TH, tyrosine hydroxylase.

 
TGF-ß-Induced Dopaminergic Differentiation Is Dependent on the TGF-ß Receptor Complex and Mediated via the Smad and p38 MAPK Pathways
TGF-ß signaling is initiated via binding to the TGF-ß receptor complex composed of the ligand binding type II and a type I receptor, also known as ALK-5. Upon receptor activation, receptor-associated Smads (Smad2/Smad3) become phosphorylated, associate with Smad4, and translocate into the nucleus to regulate transcription [20]. Furthermore, it has been shown that Smad-mediated signaling may be modulated by p38 MAP kinase [21].

To elaborate the signaling pathway involved in TGF-ß-dependent differentiation of mesencephalic progenitors toward a dopmainergic phenotype, we performed the following experiments. We documented that TGF-ß is signaling via its specific receptor complex by using SB431542, which potently inhibits Alk5, as well as Alk4 and Alk7 [22]. As shown in Figure 3, 10 µM SB431542 abolished the TGF-ß-mediated Smad translocation, as well as the TGF-ß induced increase in numbers of TH-positive cells. The precursor cells respond by TGF-ß-dependent Smad translocation to the nucleus (Fig. 3A, arrows), which is blocked by application of Alk5 inhibitor SB431542 (Fig. 3A). Furthermore, to test whether TGF-ß-dependent signaling is modulated by p38 MAP kinase, SB203580, a potent p38 MAPK pathway inhibitor, was used. As shown in Figure 3B, the application of 20 µM SB203580 together with exogenous TGF-ß significantly reduced number of TH-positive cells to control numbers, suggesting that p38 MAP kinase pathway is also required for TGF-ß-mediated differentiation toward a dopaminergic phenotype. Taken together, the results show that the effects of TGF-ß on differentiation of midbrain progenitors are a receptor-mediated process that involves both the Smad and the p38 MAPK pathway.


Figure 3
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  		Figure 3. TGF-ß differentiation signaling pathway. (A): Smad2 localization in untreated controls, in TGF-ß-treated cells, or after application of the potent ALK4, ALK5, and ALK7 inhibitor SB431542 (10 µM) in the presence of exogenous TGF-ß for 1 h. TGF-ß treatment resulted in Smad2 translocation into the nucleus (arrows). (B): The number of TH-immunopositive cells was significantly reduced after treatment of the cultures with exogenous TGF-ß in the presence of SB431542 or SB203580 (20 µM; p38 mitogen-activated protein kinase pathway inhibitor) compared with TGF-ß treatment alone. In controls, application of SB431542 or SB203580 had no effect on the number of TH-positive cells. Data are given as mean ± SEM (n = 3); p values derived from Student’s t test are as follows: ++, p < .01; +++, p < .001, derived from one-way analysis of variance and Bonferroni post hoc test for decreased numbers of immunoreactive cells as compared with TGF-ß treatment. Abbreviations: DAPI, 4',6'-diamidino-2-phenylindole dihydrochloride; TGF-ß, transforming growth factor ß; TH, tyrosine hydroxylase.

 
TGF-ß Isoforms (TGF-ß2 and TGF-ß3) Are Required for Differentiation of Dopaminergic Neurons In Vivo
Figure 4A illustrates midbrain TH-immunoreactive neurons as well as TH/Nurr1 double-labeled neurons in E14.5 wild-type (wt) and in Tgfß2–/–/Tgfß3–/– (double knockout [dko]) mouse embryos. The number of ventral mesencephalic TH positive cells, as well as the number of TH/Nurr1 double-labeled cells, was considerable decreased in TGF-ß double-knockout mice compared with wild-type. Quantification of the data (Fig. 4B, Fig. 4C) revealed significant differences between wt and dko. The relative importance of the two TGF-ß isoforms expressed in brain (i.e. TGF-ß2 and TGF-ß3) in differentiation of midbrain dopaminergic neurons is shown in Figure 4B through analysis of embryos carrying one allele of TGF-ß2 or TGF-ß3. Although both Tgf-ß2+/–/Tgf-ß3–/– and Tgf-ß2–/–/Tgf-ß3+/– embryos exhibited significantly reduced number of midbrain TH-positive cells compared with wt (Fig. 4B), the reduction caused by absence of both Tgf-ß2 alleles was higher than that observed by absence of both Tgf-ß3 alleles.


Figure 4
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  		Figure 4. TGF-ß isoforms are necessary for dopaminergic neuron induction and differentiation in vivo. (A): Mouse embryonic day 14.5 (E14.5) wt or TGF-ß dko (Tgf-ß2–/–/Tgf-ß3–/–) mesencephalon immunostained against TH (upper row) or double-labeled with TH and Nurr1 (lower row). (B): The number of TH-immunopositive cells was significantly reduced in TGF-ß double-knockout mouse embryos, as well as in Tgf-ß2–/–/Tgf-ß3+/– and Tgfß2+/–/Tgf-ß3–/– mouse E14.5 midbrain. Data are given as mean ± SEM; p values derived from Student’s t test are as follows: *, p < .05; **, p < .01; ***, p < .001 for decreased number of dopaminergic neurons compared with wild-type. (C): TGF-ß dko exhibits reduced number of TH/Nurr1 double-labeled cells in mesencephalon at E14.5 compared with wt. (D): Tgf-ß2–/–/Tgf-ß3+/– (ß2–/–/ß3+/–), Tgfß2+/–/Tgf-ß3–/– (ß2+/–/ß3–/–), and double knockouts (ß2–/–/ß3–/–) revealed no differences in the number of TH-positive cells in locus coeruleus compared with wild-type. Abbreviations: dko, double knockout; TH, tyrosine hydroxylase; wt, wild-type.

 
In contrast, as shown in Figure 4D, the number of TH-immunoreactive cells in locus coeruleus remained unaffected in Tgf-ß2–/–/Tgf-ß3+/–, Tgfß2+/–/Tgf-ß3–/–, and TGF-ß2/TGF-ß3 double-knockout mice compared with wild-type.

Dorsal Mesencephalic Progenitors Exhibit Regional Identity
Neurospheres capable of self-renewal, secondary neurosphere formation, and multilineage differentiation were also generated from dorsal mesencephalon (not shown). As illustrated in Figure 5A, dorsal mesencephalic primary tissue and dorsal mesencephalon-derived NS expressed nestin and ß-III-tubulin. Nestin expression was considerably higher in NS compared with T, whereas ß-III-tubulin expression was higher in T compared with NS. Notably, expression of the dopaminergic neuron markers Pitx3 and TH was absent in both mouse E12 dorsal mesencephalon primary tissue and in dorsal midbrain-derived neurospheres. As positive control for TH expression, E12 mouse ventral midbrain primary tissue was used. In contrast, Nurr1 expression was detectable, although weak, in both dorsal primary tissue and generated neurospheres.


Figure 5
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  		Figure 5. Dorsal mesencephalon progenitor cells exhibit regional identity. Dissected primary dorsal mesencephalic T and dorsal midbrain-derived NS were processed for reverse transcription-polymerase chain reaction (RT-PCR) using specific primers. (A): Determination of nestin and ß-III-tubulin expression for neural precursor cells and neurons, respectively. Lack of Pitx3 and TH expression in mouse embryonic day 12 (E12) dorsal mesencephalon primary tissue, as well as in dorsal-derived neurospheres. Nurr1 was detected in primary dorsal tissue and generated neurospheres. Right lane represents TH expression in ventral mesencephalon, which has been used as positive control. (B): RT-PCR was performed in primary dissected dorsal mesencephalic tissue and dorsal derived neurospheres using specific primer pairs for TGF-ß3, Shh, and FGF8. Endogenous expression of TGF-ß3 and FGF8 was evident in mouse E12 dorsal mesencephalic primary dissected tissue. TGF-ß3, but not FGF8, was expressed in dorsal neurospheres as well. In contrast, primary dorsal midbrain tissue and dorsal neurospheres were devoid of Shh expression. Abbreviations: bp, base pair(s); FGF8, fibroblast growth factor 8; NS, neurospheres; Shh, sonic hedgehog; T, tissue; TGF-ß, transforming growth factor ß; TH, tyrosine hydroxylase.

 
Endogenous expression of TGF-ß3, Shh, and FGF8 in dorsal mesencephalic primary tissue and dorsal neurospheres, as determined by RT-PCR, is shown in Figure 5B. Shh expression was absent in both dorsal primary tissue and generated neurospheres. In contrast, low expression levels for TGF-ß3 and FGF8 were detected in dorsal primary tissue. In dorsal neurospheres, TGF-ß3 but not FGF8 expression could be found. These data indicate that mesencephalic regions likely exhibit intrinsic regional identity in vitro and in vivo.

TGF-ß Promotes Ectopic Induction of Dopaminergic Neurons
Provided that cells within mesencephalic dorsal neurospheres arise from regionally specified progenitors, the effects of factors that have been shown to promote differentiation of ventral neurospheres toward dopaminergic neurons were tested in dorsal neurospheres. Untreated dorsal neurospheres were devoid of TH (Fig. 6A), Pitx3, and En-1 (not shown) immunoreactivity. However, single treatment of dorsal neurospheres with TGF-ß1 (Fig. 6A) induced TH immunoreactivity in colocalization with Pitx3 and En-1 in some cells. Application of Shh or FGF8 (not shown) in dorsal neurospheres also resulted in TH-immunopositive cells, confirming previous observations in other tissues [7, 23]. Since neutralization of Shh had no effect on the number of TH-immunoreactive cells in dorsal neurospheres, these results imply that TGF-ß-induced TH expression in dorsal mesencephalic neurospheres in vitro is likely Shh-independent.


Figure 6
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  		Figure 6. TGF-ß induces Shh-independent ectopic TH expression in dorsal neurospheres. (A): TH immunocytochemistry in dorsal neurospheres. (B): Treatment of the neurospheres with TGF-ß or Shh significantly increased the number of TH-immunoreactive neurons compared with the untreated controls. Neutralization of Shh in the presence of exogenous TGF-ß had no effect on the number of TH-positive cells compared with TGF-ß treatment alone. Data are given as mean ± SEM (n = 3); p values derived from Student’s t test are as follows: *, p < .05 for increased numbers of immunoreactive cells after factor treatment as compared with untreated controls. Abbreviations: Shh, sonic hedgehog; TGF, transforming growth factor; TH, tyrosine hydroxylase.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
Experimental control of neural stem and progenitor cell differentiation toward neuronal lineage and neurotransmitter phenotype is a valuable tool for studying and understanding brain development and for evaluation of neural stem cells regarding their therapeutic potential in neurodegenerative diseases [24]. In the present study, we have investigated the role of TGF-ß in the differentiation of mesencephalic progenitors toward dopaminergic neurons in vitro and in vivo.

Neurospheres from ventral and dorsal midbrain were generated. Great caution was taken during primary tissue dissection to avoid contamination of ventral tissue with dorsal and vice versa. Both ventral and dorsal mesencephalon-derived neurospheres exhibited self-renewal capacity and multilineage differentiation, consistent with studies comparing distinct rodent and human neural compartments [25–28]. However, regional differences were observed between ventral and dorsal midbrain, reflected by the molecular identity of generated neurons and by differential expression of key molecules involved in patterning of the neural tube: FGF8 [29] was not detectable in dorsal neurospheres in vitro, and Shh [5] expression was absent from both dorsal primary tissue and neurospheres, consistent with the notion that Shh acts as ventralizing factor in patterning of the neural tube. In contrast, TGF-ß3 [30–32] was expressed in both ventral and dorsal tissue and neurospheres (Fig. 1, 5). Ventral neurospheres apparently contained dopaminergic progenitors represented by expression of the dopaminergic lineage markers Nurr1 and Pitx3 [33–37]. TH expression was downregulated in neurospheres compared with primary tissue, indicating more progenitor cells in neurospheres than in primary tissue. Notably, dorsal primary tissue and generated neurospheres expressed Nurr1 but not Pitx3 or TH.

Experimental control of dopaminergic neuron differentiation was addressed by treating ventral midbrain dissociated neurospheres with TGF-ß, Shh, and FGF8, factors identified to promote dopaminergic neuron induction [reviewed in 38–40], or with function blocking antibodies for individual factors. Although Shh and FGF8 are established players in midbrain dopaminergic neuron development, the importance of TGF-ß in this process, in addition to its effect on promoting survival of dopaminergic neurons [11, 13, 41–43], has only recently been appreciated. In zebrafish mutations with affected TGF-ß/Nodal signaling, the corresponding ventral dopaminergic neuron population does not develop [10]. In the present study (Fig. 2), treatment of the cultures with TGF-ß significantly increased the number of Nurr1- and TH-positive cells compared with untreated controls, but it had no effect on the number of Pitx3-immunoreactive cells, thus consistent with the presence of two independent cascades in mesencephalic dopaminergic neuron development, one involving Nurr1 and the other involving Lmx1b and Pitx3 [44]. Application of TGF-ß, Shh, and FGF8 added together resulted in a further increase in the number of Nurr1- and TH-immunoreactive cells, without affecting Pitx3-positive cells. Neutralization of individual factor, however, implies that TGF-ß, Shh, and FGF8 are necessary for the maintenance of Pitx3 expression. The interaction between Nurr1 and Pitx3 is not clear yet. Nurr1 [32], originally identified as required for terminal differentiation of late mesencephalic precursor neurons into a full dopaminergic phenotype, is likely not a prerequisite for the induction of all dopaminergic genes (reviewed in [45]). With regard to Pitx3 [46], inactivation of this homeodomain gene leads to a selective loss of midbrain TH-immunoreactive neurons at E12.5 [35, 47–49].

Increased numbers of TH-immunopositive cells after TGF-ß treatment, together with the observation that the number of TUNEL- and BrdU-positive cells remained unchanged between controls and TGF-ß-treated cultures (data not shown), imply that TGF-ß triggers differentiation of neural precursor cells toward dopaminergic phenotype, rather than promoting survival. Which could be the differentiation pathway underlying TGF-ß effects? The signaling of the TGF-ß family members is mediated by a heteromeric complex of two types of transmembrane serine/threonine kinase receptors. The binding of ligand to the receptor complex leads the type II receptor kinase tophosphorylate and thereby activate type I receptor kinase (also called ALK5). The well-established canonical Smad-mediated TGF-ß signaling pathway implies phosphorylation of Smad2 and Smad3 proteins, formation of a complex with Smad4, and translocation into the nucleus, where, in association with other transcription factors, it activates transcription of target genes [20]. However, in addition to the Smad-mediated canonical TGF-ß signaling pathway, evidence over the last years suggests that TGF-ß may signal through other pathways, such as through activation of MAPKs, including p38 kinases [21]. In the present study, Smad2 translocation into the nucleus after TGF-ß treatment, together with decreased number of TH-immunopositive cells after application of SB203580, a specific p38 MAPK inhibitor, or with SB431542, a potent ALK4, ALK5, and ALK7 inhibitor, suggests that TGF-ß effect on differentiation of dopaminergic progenitors is a receptor-mediated event, employing a Smad-dependent mechanism, presumably modulated by the p38 MAPK signaling pathway. The notion that TGF-ß receptors can regulate gene transcription directly by activating the Smad transcription factors, indirectly via p38 MAPK, or by combination of both pathways, has been repeatedly demonstrated [21].

The requirement for TGF-ß isoforms in midbrain dopaminergic neuron induction and differentiation, as well as the relative importance of the two TGF-ß isoforms expressed in the brain, was further verified in vivo (Fig. 4). In TGF-ß double-knockout (Tgfß2–/–/Tgfß3–/–) mouse embryos, the number of midbrain TH-immunoreactive cells, as well as TH/Nurr1 double-labeled cells, was significantly decreased compared with the wild-type littermates, thus matching the in vitro data. In mice carrying one allele of TGF-ß2 (Tgf-ß2+/–/Tgf-ß3–/–) or TGF-ß3 (Tgfß2–/–/Tgfß3+/–), determination of TH-positive cells revealed that TGF-ß2 is, specifically for the midbrain, relatively more important than TGF-ß3. These observations confirm and extend previous data obtained in chick embryos following neutralization of TGF-ß between E6 and E10 [9].

Since TGF-ß is apparently required for ventral midbrain dopaminergic neuron development, we next tested the hypothesis that TGF-ß may ectopically induce TH-positive cells. Because dorsal neurospheres lack endogenous Shh and FGF8 expression, these results may imply that TGF-ß-induced TH immunoreactivity was Shh- and FGF8-independent. This assumption was further strengthened by demonstrating that treatment of dorsal neurospheres with anti-Shh or FGFR3a in the presence of TGF-ß had no effect in the number of TH-positive cells compared with single TGF-ß treatment. On the other hand, Shh—originally identified as a key signal in the specification of ventral cell fates along the neural tube (reviewed in [23])—has recently been proposed to likely control midbrain and caudal forebrain growth by promoting cell proliferation and survival rather than acting as a patterning molecule [50, 51]. The molecular mechanism underlying TGF-ß-induced TH expression in dorsal mesencephalon is not clear and is currently under investigation.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
References
 
We thank Dr. A. Galdes (Biogen) for providing Shh and S. Heidrich for excellent technical assistance. The monoclonal antibody against Shh developed by Thomas Jessell was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development, NIH, and maintained by The University of Iowa, Department of Biological sciences (Iowa City, IA). This work was supported by grants from the Deutsche Forschungsgemeinschaft (K.K.) and by Deutsche Forschungsgemeinschaft through the DFG-Research Center for Molecular Physiology of the Brain (E.R.). N.D. is currently affiliated with the Institute for Anatomy-Neuroanatomy, University of Essen, Essen, Germany.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

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