Wnt signaling has a known and pivotal impact on mdDA neuron development (Figure ). To begin with, midbrain morphogenesis is regulated by Wnt signaling. Wnt1
mutant mice present an abnormal posterior midbrain, isthmus and rostral hindbrain, unveiling the essential role of Wnt signaling in MHB formation [194
]. Other studies support the central role of the canonical pathway in the patterning of the MHB region, where the direct inactivation of β-catenin in a specific manner in the MHB mimics the Wnt1
mutant phenotype [170
]. Furthermore, mutant mice for the Wnt receptor Lrp6 also phenocopy some of the Wnt1
mutant defects [160
] and Fzd3
double mutants show a severe impairment of midbrain morphogenesis [198
]. In addition, Wnt1 directly regulates the expression of Otx2, a factor involved in midbrain morphogenesis, in a Wnt1-Lmx1a autoregulatory loop during embryonic development [199
The proliferation and differentiation of midbrain dopaminergic neurons during ventral midbrain neurogenesis depend on Wnt signaling (reviewed in [31
]). Already during early midbrain development, several members of the Wnt family are expressed and seem to be tightly regulated in a spatiotemporal way [158
]. In particular, β-catenin transcriptional activity has been observed to take place in the developing mouse midbrain before the birth of Th-positive neurons (at E10.5), with stronger intensity in the Nurr1 expression domain [201
]. It seems that activation of the Wnt/β-catenin pathway contributes to increased DA neurogenesis during development: β-catenin promotes midbrain dopaminergic neurogenesis in vivo
] and the stabilization of β-catenin in ventral mesencephalic precursors, by GSK3β inhibition, leads to an increase in DA differentiation [204
]. In these two recent studies, a targeted deletion of β-catenin in Th-IRES-Cre;β-Ctnfl/fl
mutants resulted in reduced mdDA neurogenesis [205
]. More recently, the same group reported that the constitutive activation of Wnt/β-catenin signaling in the ventral midbrain of the Th-IRES-Cre;β-CtnEx3/+
mutants revealed a significant increase in the number of dopaminergic neurons as well as an increase in the number of committed progenitors, in line with their previous work [206
]. On the other hand, activation of β-catenin in Shh-Cre; β-CtnEx3/+
mutants led to the expansion of dopaminergic progenitors by reducing their exit from the cell cycle, with a concomitant reduction in the number of dopaminergic neurons more intensely in the SNc [206
]. This suggests an opposing role for Wnt/β-catenin signaling in early and late mdDA development. Another study showed that Wnt1 activation enhanced the differentiation of mouse embryonic stem cells to mdDA neurons [199
]. Wnt1 was also found to be required for the terminal differentiation of midbrain dopaminergic neurons at later stages of embryogenesis [207
]. In addition, Wnt2 was recently identified as a novel regulator of dopaminergic progenitors, necessary in their proliferation; Wnt2-
null mice therefore have decreased numbers of dopaminergic neurons [208
In the next two sections we focus on what is known about Wnt signaling in connection to two decisive transcription factors involved in the development of the mdDA neurons, Nurr1 and En1.
Wnt signaling and Nurr1
It has been shown so far that activation of the Wnt/β-catenin pathway contributes to increased mdDA neurogenesis during development, that is, that it regulates the proliferation and differentiation of ventral mesodiencephalic Nurr1 precursors in vivo
]. Taking into account the data as described, Kitagawa et al. [156
] tested the possibility of Wnt signaling regulating Nurr1 activity, and found a convergence between Nurr1 transcriptional regulation and Wnt signaling in cell culture. In short, Wnt signaling via β-catenin enhanced the transcriptional activity of Nurr1 in cells, at Nurr1 responsive elements (NREs), leading to TH
promoter activation (Figure ). In the absence of β-catenin, Nurr1 is associated with Lef1 in co-repressor complexes on NREs. After activation of Wnt signaling, β-catenin interacts with Nurr1 on NREs, competing with Lef1 for Nurr1 binding, resulting in the disruption of co-repressors from the Nurr1 complex and the concomitant recruitment of coactivators, such as CBP (Creb binding protein) [156
] (Figure ). β-catenin functions, so it seems, as a transcriptional cofactor for Nurr1. Small interfering RNAs targeting Nurr1 abolished CBP and β-catenin association with the NRE in the TH
]. On the other hand, Nurr1 was found to slightly modulate, in a negative way, the canonical Wnt signaling by being able to associate with the TCF/LEF region (Figure ). After Wnt stimulation, β-catenin would compete with Nurr1 for Lef1 binding on a TCF/LEF promoter site, such as the cyclin D1 promoter, and disrupt Nurr1 binding, promoting Wnt-target gene transcription [156
]. A model to describe this mechanism was proposed and is shown in Figure . Whether this model is valid for mdDA neuron differentiation and maintenance in vivo
remains to be investigated. The question arises: do Nurr1 and β-catenin interact in vivo
synergistically to drive Th expression? Previous studies strongly suggest that this is the case [204
]. Importantly, besides the study from Kitagawa et al
., synergistic interactions between β-catenin and several nuclear receptors have already been described [155
]. Quite likely, β-catenin is involved in mdDA neurogeneis, cooperating with the Nurr1 transcription complex.
Figure 5 Interplay between Wnt, Nurr1 and En1 signaling in vitro and in vivo. (A) Model adapted from Kitagawa et al. : Wnt signaling via β-catenin enhances the transcriptional activity of Nurr1 in cells at Nurr1 responsive elements (NREs). In the (more ...)
Wnt signaling and En1
and chick embryos, interactions between engrailed (en) and Wnt/wg signaling pathways have been described whereby engrailed expression is dependent on Wnt/wg signaling and vice versa
] (Figure ). However, in Drosophila
, engrailed expressing cells did not have active wg signaling [209
]. A modulation of Fz receptor expression by engrailed was shown in Drosophila
wherein the expression of Fz is lower in engrailed-positive domains and, in the engrailed
null mutant, the usual striped expression of Fz is disturbed, spreading everywhere in a non-segmental pattern [212
]. Later, by means of chromatin immunoprecipitation (ChIP) assays, engrailed was established to be a direct repressor of Fz2 expression in vivo
]. In mice, Wnt1 expression was found to overlap with En1
gene expression in the midbrain at 8.5 days post-coitus [65
], but while Wilkinson et al.
] found Wnt1 expression in the midbrain after 12 days post-coitus, Davis and Joyner [65
] did not observe overlapping expression domains between En1 and Wnt1 within the midbrain after this time point. As an explanation for this discrepancy they advance the fact that Wnt1 expression is punctuated, making it hard to get brain slices containing visible expression. So, a more detailed analysis of the Wnt/β-catenin signaling in the mdDA system is needed. As mentioned above, inactivation of the Wnt1
gene leads to the deletion of the midbrain-hindbrain area with concomitant loss of En1 (its expression in the MHB region is initiated normally but is subsequently lost) [194
]. Furthermore, the expression of En1 under the Wnt1 promoter rescues most of the Wnt1 phenotype, suggesting that En1 is a downstream target of Wnt1 [91
] (Figure ). In conclusion, both Wnt1 and En1 cooperate in the patterning of the MHB region during early development.
In cell culture studies, it was observed that En1 can function as a negative regulator of β-catenin transcriptional activity in a Gro/TLE-independent manner (TLE: transducin-like enhancer of split 1) [216
] (Figure ). Silencing En1 expression using small interfering RNA stimulated β-catenin transcriptional activity, measured by luciferase reporter assays. By Northern analysis and cycloheximide assays, Bachar-Dahan et al.
] observed that En1 affects the level of a constitutively active form of β-catenin at a post-translational level only. They suggest that En1 acts by destabilizing β-catenin via a proteasomal degradation pathway that is GSK3β-independent [216
As we mentioned above, there might be a link between En1 depletion and the onset of neurological disorders such as PD. A direct interaction between the PD-associated protein parkin and β-catenin has recently been observed [217
]. In this study, increased levels of β-catenin activity were found in parkin
mutant mice. This increase in Wnt-β-catenin signaling led to an increase in dopaminergic neuron proliferation and death [217
], which is in contrast to the positive role Wnt-β-catenin signaling plays during mdDA neuron development. This might be due to the different needs in Wnt signaling activity in morbid adult dopaminergic midbrain tissue when compared to healthy one [158
]. It is currently unknown whether En1 and canonical Wnt signaling cooperate in later stages of mdDA neuron development, such as in mdDA neuron specification and maintenance (Figure ).
Conclusions and future perspectives
The vertebrate mdDA system has been intensively studied in the past decades and an enormous wealth of information on the molecular cues controlling its development has been gathered. Our future challenge is to unravel in depth the gene cascades linking early induction to the differentiation and maintenance of mdDA neurons, eventually obtaining a complete picture of mdDA development (including the developmental origin and the molecular coding characterizing various mdDA subsets). Once this is accomplished, effective clinical treatments for mdDA-associated neurological disorders, such as PD, can be generated. As described in this review, current evidence strengthens the central roles that En1 and Wnt signaling might play in the advancement of these therapies, especially for PD. However, a detailed molecular characterization of the En1 mutant is lacking. Furthermore, the precise function of En1 in some mdDA developmental processes is also not known, and questions such as whether En1 is essential in the differentiation of the mdDA system and is part of key transcriptional complexes mediating such processes (such as the Nurr1 complex) need to be investigated.
Concerning Wnt signaling, detailed knowledge about which developmental processes it regulates within a particular dopaminergic neuron, as well as which key players are involved, is still incipient. Furthermore, a more detailed characterization of Wnt/β-catenin activity during ventral midbrain development is essential. Wnt signaling overlaps with that of En1 in time and space during CNS development and these two pathways interact functionally at least at one time point during embryonic mdDA development. It is now known that canonical Wnt signaling and En1 cooperate in the genesis of a competent mdDA field during early development. However, whether Wnt signaling and En1 might cooperate in later midbrain developmental stages, such as in the differentiation of mdDA neurons, is still not known. Future research will have to focus on disclosing the En1 mutant phenotype and its relevance and eventual interplay with Wnt signaling during mdDA differentiation. Recent improvements in techniques, such as transcript expression profiling, ChIP-seq, proteomics, and mdDA neuronal cell isolation and culture, will certainly help unveil the molecular repertoire necessary to generate a mdDA neuron.