En1 and Wnt signaling in midbrain dopaminergic neuronal development

Abstract

Dopaminergic neurons of the ventral mesodiencephalon are affected in significant health disorders such as Parkinson's disease, schizophrenia, and addiction. The ultimate goal of current research endeavors is to improve the clinical treatment of such disorders, such as providing a protocol for cell replacement therapy in Parkinson's disease that will successfully promote the specific differentiation of a stem cell into a dopaminergic neuronal phenotype. Decades of research on the developmental mechanisms of the mesodiencephalic dopaminergic (mdDA) system have led to the identification of many signaling pathways and transcription factors critical in its development. The unraveling of these pathways will help fill in the pieces of the puzzle that today dominates neurodevelopment research: how to make and maintain a mdDA neuron. In the present review, we provide an overview of the mdDA system, the processes and signaling molecules involved in its genesis, with a focus on the transcription factor En1 and the canonical Wnt pathway, highlighting recent findings on their relevance--and interplay--in the development and maintenance of the mdDA system.

Figure 1

Spatial and temporal developmental stages leading to mesodiencephalic dopaminergic neurogenesis. (A) Sagittal and coronal schematic sections showing the region in the developing central nervous system where mesodiencephalic dopaminergic (mdDA) neurons are born. Anterior-posterior patterning leads to the genesis of morphogenetic domains: telencephalon (Tel), rostral diencephalon (RD), midbrain (M) and hindbrain (H), whereas dorsal-ventral patterning results in crosswise subdivisions in the brain: floor plate (FP), basal plate (BP), alar plate (AP) and roof plate. The mdDA area encompasses the midbrain and prosomeres (P) 1 to 3. The floor plate is divided in three main areas: the ventricular zone (VZ), the intermediate zone (IZ) and the marginal zone (MZ). (B) Molecular cascades leading to mdDA neurogenesis, illustrated by three different stages from top to bottom. The key genes driving mdDA development are represented. En1 and Wnt signaling are required already in early development, being essential throughout mdDA development, from early patterning up to the induction of mdDA neurons. Although we placed En1 in all these developmental stages, a molecular characterization of how En1 contributes to each of these has not yet been performed. It remains to be seen as well whether Wnt signaling is active in a settled mdDA neuron (after embryonic day (E)14). The progenitor pool is located in the VZ and its progeny migrates to the IZ, where it differentiates into post-mitotic mdDA precursors, expressing Nurr1 and L-aromatic amino acid decarboxylase (Aadc). Later on, after E12, mdDA neurons start to differentiate, expressing mdDA key identity genes like Pitx3, Th and Vmat2. The differentiated settled mdDA neurons localize in the MZ.

Figure 2

The impact of the engrailed genes in the development of the central nervous system and the mesodiencephalic dopaminergic system. (A) Engrailed proteins are key players in diverse processes during embryonic development of the central nervous system (CNS), including patterning, axonal guidance and neuron specification. (B) Engrailed proteins are essential in mesodiencephalic dopaminergic (mdDA) neuron development from an early stage, where they are involved in morphogenesis and mdDA neurogenesis, and in the adult, where they play a role in mdDA neuron maintenance E, embryonic day.

Figure 3

Canonical Wnt signaling mechanism. (1) Wnts bind to Frizzled (Fz) transmembrane receptors and low-density lipoprotein receptor-related protein (Lrp) co-receptors (2) triggering the dissolution of the 'β-catenin destruction complex', resulting in (3) β-catenin not being marked for degradation (asterisk) thereby accumulating in the cytoplasm and (4) translocating to the nucleus. (5) Once in the nucleus, β-catenin binds to the T cell factor/lymphoid enhancer factor (Tcf/Lef) family of DNA-binding factors to form a transcriptional complex that binds target promoter sequences via a specific DNA-binding domain in TCFs, mediating Wnt target gene expression. APC, adenomatous polyposis coli.

Figure 4

Wnt signaling during the central nervous system and mesodiencephalic dopaminergic neuron development. (A) Wnt signaling is critical in embryonic development, controlling diverse processes, such as cell proliferation and cell polarity. It is involved during early central nervous system (CNS) development in gastrulation, early pattern formation, morphogenesis and precursor proliferation, in late CNS development in processes such as neuronal differentiation and migration, and in adult organisms, where it plays a central role in the maintenance of tissue homeostasis and stem cell regulation. Wnt signaling controls diverse processes, such as cell proliferation, cell polarity, cell death and cell fate specification Wnts can also function as morphogens in both short- and long-range signaling, modulating target cells in a dose- and distance-dependent manner. (B) Wnt signaling is involved in mesodiencephalic dopaminergic (mdDA) neuron development from early on, where it is involved in morphogenesis, and later on as well in mdDA differentiation.

Figure 5

Interplay between Wnt, Nurr1 and En1 signaling in vitro and in vivo. (A) Model adapted from Kitagawa et al. [156]: Wnt signaling via β-catenin enhances the transcriptional activity of Nurr1 in cells at Nurr1 responsive elements (NREs). In the absence of β-catenin, Nurr1 associates with T cell factor/lymphoid enhancer factor (TCF/LEF) in co-repressor complexes on NREs. After activation of Wnt signaling, β-catenin interacts with Nurr1 on NREs, competing with TCF/LEF for Nurr1 binding, resulting in the disruption of the co-repressors from the Nurr1 complex and the concomitant recruitment of coactivators. (B) Model adapted from Kitagawa et al. [156]: on the other hand, Nurr1 was observed to slightly modulate, in a negative way, the canonical Wnt signaling through association with the TCF/LEF region. After Wnt stimulation, β-catenin competed with Nurr1 for Lef binding on the TCF/LEF promoter site and disrupted Nurr1 binding, promoting Wnt-target gene transcription. (C) Several studies in Drosophila and chick embryos have described interactions between En1/engrailed (en) and the Wnt/wg signaling pathway whereby engrailed expression is dependent on Wnt/wg signaling and vice versa. However, in Drosophila, engrailed expressing cells did not have active wg signaling. From mice studies it is known that Wnt signaling regulates En1 expression early in midbrain development. Whether the reverse happens in the mouse midbrain is not known. (D) In one cell culture study [216], it was observed that En1 can function as a negative regulator of β-catenin transcriptional activity in a post-translational manner (that is, by affecting β-catenin protein levels only). (E) Three questions remain currently unsolved: first, whether En1 cooperates with Nurr1 during mdDA development; second, whether Nurr1, En1 and canonical Wnt signaling cooperate in later stages of mdDA neuron development, such as in mdDA neuron specification; and third, whether Nurr1 and/or En1 regulate canonical Wnt signaling during mdDA neuron development.