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. 2006 Jul 6;575(Pt 2):403–410. doi: 10.1113/jphysiol.2006.113464

Genetic networks controlling the development of midbrain dopaminergic neurons

Nilima Prakash 1, Wolfgang Wurst 1,2
PMCID: PMC1819467  PMID: 16825303

Abstract

Recent data have substantially advanced our understanding of midbrain dopaminergic neuron development. Firstly, a Wnt1-regulated genetic network, including Otx2 and Nkx2-2, and a Shh-controlled genetic cascade, including Lmx1a, Msx1 and Nkx6-1, have been unravelled, acting in parallel or sequentially to establish a territory competent for midbrain dopaminergic precursor production at relatively early stages of neural development. Secondly, the same factors (Wnt1 and Lmx1a/Msx1) appear to regulate midbrain dopaminergic and/or neuronal fate specification in the postmitotic progeny of these precursors by controlling the expression of midbrain dopaminergic-specific and/or general proneural factors at later stages of neural development. For the first time, early inductive events have thus been linked to later differentiation processes in midbrain dopaminergic neuron development. Given the pivotal importance of this neuronal population for normal function of the human brain and its involvement in severe neurological and psychiatric disorders such as Parkinson's Disease, these advances open new prospects for potential stem cell-based therapies. We will summarize these new findings in the overall context of midbrain dopaminergic neuron development in this review.

Neurons synthesizing the neurotransmitter dopamine (DA) are found at different positions in the vertebrate brain (reviewed by Prakash & Wurst, 2006). The best studied populations are the dopaminergic (DA) neurons located in two nuclei of the midbrain tegmentum in higher vertebrates: the substantia nigra (SN, also called the A9 group) and the ventral tegmental area (VTA, also called the A10 group). Additional neurons are found in the retrorubral field (RrF, also named the A8 group). The neurons of the SN and VTA project to the forebrain forming the nigrostriatal and mesocorticolimbic pathway, respectively (reviewed by Prakash & Wurst, 2006). The mammalian midbrain dopaminergic (mDA) population plays a fundamental role in several brain and body functions and behaviours. The mDA neurons have thus been the focus of clinical interest for a long time because of their involvement in severe human neurological and psychiatric illnesses. Besides a deeper understanding of their physiology, the development of better treatments for these disorders has promoted their investigation. One potential therapy for Parkinson's disease, for example, would be the restitution of the degenerating or lost DA neurons in the human SN by healthy DA neurons that have been generated either in vitro or in vivo through the directed differentiation of stem cells (Winkler et al. 2005). To accomplish this, a full understanding of the genetic cues controlling the differentiation of a pluripotent, uncommitted neuroepithelial stem cell into a mature mDA neuron is required. In this regard, substantial progress has been made in recent years through the analysis of the developmental programme governing the emergence of the mDA cell type during mammalian embryogenesis. This programme is inherited and can be subdivided into three distinct processes: (1) the induction of a progenitor field within the neuroepithelium competent to generate mDA precursors at early stages of neural development (approx. from embryonic day (E) 8.5 to E10.5 in the mouse); (2) the specification of a mDA neuronal fate in these precursors at intermediate stages (approx. from mouse E10.5 to E12.5); and (3) the acquisition of the mature phenotype or terminal differentiation of mDA neurons at relatively late stages of neural development (i.e. from E12.5 onwards). Cells that have been induced but not yet specified are still able to switch cell fate, but cells that have been specified are committed to a unique cell fate. This commitment includes the acquisition of a distinct phenotype by the cells, such as generic neuronal and special characteristics including neurotransmitter, electrophysiological and projection area identities. These processes may be envisioned as controlled by different genetic networks acting either sequentially or in parallel. We and others have recently reviewed the cell-biological and molecular aspects of mDA neuron development (Goridis & Rohrer, 2002; Smidt et al. 2003; Simeone, 2005; Prakash & Wurst, 2006). We will therefore restrict ourselves to a summary of the genetic cascades and networks controlling the development of this neuronal population in the mouse, emphasizing the most recent progress that has been made in the field.

Two important signalling centres of the mouse embryo control the generation of mDA neurons

The embryonic mouse neural tube is patterned along its antero-posterior (A-P) and dorso-ventral (D-V) axis by the action of several important signalling centres. Patterning along the D-V axis is accomplished through the ventral (floor plate, FP) and dorsal (roof plate, RP) midline of the neural tube (reviewed by Chizhikov & Millen, 2004b; Placzek & Briscoe, 2005), whereas the most anterior edge of the presumptive forebrain (the anterior neural ridge, ANR), the boundary between the prospective dorsal and ventral thalamus in the diencephalon (the zona limitans intrathalamica, ZLI) and the mid-/hindbrain boundary (MHB) or isthmic organiser (IsO) together control the patterning of the anterior neural tube along the A-P axis (reviewed in Liu & Joyner, 2001; Wurst & Bally Cuif, 2001; Echevarria et al. 2003; Prakash & Wurst, 2004; Kiecker & Lumsden, 2005). These signalling centres are characterized by the expression of different secreted and transcription factors controlling the establishment of the adjacent neural territories and the specification of the distinct neuronal populations for each territory. A key feature of any signalling centre is that it must provide a diffusible signal that can be received by a group of cells, which in turn may then be ‘primed’ through transcription of cell-specific genes. The FP, for example, secretes the lipid-modified glycoprotein Sonic hedgehog (Shh), which is known for its pivotal role in the specification of the different ventral populations in the hindbrain and spinal cord by controlling the expression of different transcriptional regulators (reviewed by Placzek & Briscoe, 2005). The secreted molecules Wnt1 and fibroblast growth factor (Fgf) 8 are expressed in the caudal midbrain or rostral hindbrain at the MHB, respectively, and transcription factors belonging to the homeodomain (HD) and paired-box families such as Pax2/5 and Engrailed (En) 1 and 2 are expressed across this boundary in both the caudal midbrain and the rostral hindbrain. Expression of all these factors in the neural tube initiates at roughly the same time of mouse embryonic development, i.e. between E8.0 and E8.5 (see Prakash & Wurst, 2004). The mDA neurons arise at around E10.5 from the ventral midline (FP and basal plate, BP) of the cephalic flexure close to the MHB (reviewed by Prakash & Wurst, 2006), suggesting that these two signalling centres must play an important role in their development. Indeed, we were able to demonstrate that the position of the MHB during embryonic development also determines the number and location of the mDA neurons and of another cell population specified in the rostral hindbrain adjacent to the MHB, the rostral hindbrain serotonergic (rh-5HT) neurons (Brodski et al. 2003). The ectopic induction of mDA neurons in these transgenic animals occurred only in close proximity to the FP thus, supporting further the hypothesis that both signalling centres are necessary for mDA neuron induction.

Floor plate Shh controls mDA neuron induction

Shh is secreted from the ventral midbrain FP and BP and was identified as one important molecule acting on the neuroepithelial precursors of the mDA population to specify their neurotransmitter identity. It was indeed shown that Shh and Fgf8 together are necessary and sufficient for the generation of ectopic mDA neurons in rat embryo explant cultures (Ye et al. 1998). Ectopic expression of Shh as well as its downstream effector molecule Gli1 is able to induce ectopic mDA and 5-HT neurons in the dorsal mid-/hindbrain region (MHR), in areas where normally Wnt1 and Fgf8 are expressed (Hynes et al. 1997). In addition, in Shh null mutants mDA neurons are missing and by conditionally inactivating Smoothened (Smo) (a receptor of Shh signalling) at E9.0 mDA neurons are considerably reduced (Blaess et al. 2006). Thus, Shh is necessary for mDA progenitor induction. However, the question remains as to how two symmetric signals, Shh (which is expressed along almost the entire length of the neural tube) and Fgf8 (since the development of the rh-5HT neurons also depends on this molecule (Ye et al. 1998)) could provide sufficient information for conveying the mDA phenotype exclusively to cells residing in the ventral midbrain. The role of Fgf-signalling for mDA neuron development has been compromised by recent findings showing that the ablation of Fgf receptor (Fgfr) 1 in the MHR has no effect on the mDA neuronal population, but leads to a reduction of the most rostral hindbrain 5-HT neurons (Trokovic et al. 2003). Fgfr1 is the prominent receptor in the MHR, although its loss in the midbrain may be partly compensated by Fgfr2 (Blak et al. 2005). Furthermore, Fgf8 beads on Wnt1 homozygous mutant forebrain explant cultures are unable to induce ectopic mDA neurons in contrast to wild-type tissue (Prakash et al. 2006). Based on these observations we hypothesized that the secreted glycoprotein Wnt1 would be a good candidate to provide such an ‘asymmetric’ signal as Wnt1 expression is confined to a ring encircling the neural tube at the rostral border of the MHB (caudal midbrain), the RP of the mes- and diencephalon, and two stripes adjacent to the FP of the midbrain. The latter expression domain overlaps with the region where mDA progenitors are first specified (Prakash et al. 2006).

Ventral midbrain Wnt1 expression controls mDA neuron induction

Taking advantage of gain-of-function and loss-of-function Wnt1 mutant mouse lines, we were able to demonstrate a crucial role of Wnt1 in the development of mDA neurons (Prakash et al. 2006). At early stages of mouse neural development (i.e. between E9.5 and E12.5), Wnt1 is required for the maintenance of Otx2 expression in the region encompassing the FP and BP of the midbrain. Loss of Otx2 in this region of En1+/Cre; Otx2flox/flox mice leads to a ventral expansion of the midbrain Nkx2-2 expression domain, which is normally confined to a narrow stripe at the boundary between the BP and the alar plate of the mesencephalon. As a consequence, the Wnt1 expression domain in the ventral midbrain is lost and ectopic rh-5HT neurons are generated instead of mDA neurons in this region of the mutant brain (Puelles et al. 2004; Prakash et al. 2006). The loss of Wnt1 expression and of mDA neurons in the ventral midbrain of En1+/Cre; Otx2flox/flox mice is probably a direct consequence of the repressive effect of Nkx2-2, as removal of this gene on an En1+/Cre; Otx2flox/flox mutant background (En1+/Cre; Otx2flox/flox; Nkx2-2−/− triple mutants) rescues the ventral Wnt1 expression domain and the normal generation of mDA neurons (Prakash et al. 2006). In addition to this ‘early’ activity of Wnt1, it is also required at later stages of neural development (i.e. between E11.5 and E12.5) for the proper differentiation of mDA neurons in the mouse embryo (Prakash et al. 2006). In Wnt1−/− mice, few tyrosine hydroxylase (Th, the rate-limiting enzyme in DA biosynthesis)-expressing mDA precursors are still generated, but these cells fail to initiate expression of the HD transcription factor Pitx3. Furthermore, ectopic mDA neurons cannot be induced by Shh and Fgf8 in the absence of Wnt1 in mouse embryo explant cultures (Prakash et al. 2006). In support of our findings, fate-mapping of ventral Wnt1-expressing cells during mouse embryonic development showed that a great extent of these cells will generate Th-expressing mDA neurons throughout the crucial stages (i.e. from E9.5 to E11.5) (Zervas et al. 2004). Previous data obtained from in vitro experiments already suggested an important function of Wnt proteins in the generation of differentiated mDA neurons from cultured mDA precursors (Castelo-Branco et al. 2003). In these studies, however, it was suggested that Wnt1 mostly controls the proliferation of mDA precursors and only to a minor extent their differentiation into Th-expressing mDA neurons, whereas Wnt5a was reported to be a more potent factor for the differentiation of these precursors into mDA neurons, with little effect on their proliferation (Castelo-Branco et al. 2003).

Wnt5a is another member of the mammalian Wnt family (comprising 19 proteins), which is expressed in the FP and BP of the neural tube including the ventral midbrain (Parr et al. 1993). Our own analysis of the Wnt5a−/− mutant mouse, however, does not confirm these findings in vivo (Minina et al. unpublished). The Wnt1-mediated signal transduction pathway (including receptors and intracellular effectors) regulating the generation of mDA neurons is unclear at present. However, based on in vitro data, it was suggested that the canonical Wnt/β-catenin pathway may be crucial in mediating the signal necessary for mDA development (Castelo-Branco et al. 2004). One can therefore envisage the distinct neuronal progenitor domains of the MHR being established within a cartesian grid system of diffusible signals along the A-P (Wnt1) and D-V (Shh) axes of the neural tube during early neural development (i.e. between E9.5 and E10.5).

Other secreted factors regulating mDA neuron development

The importance of other secreted factors and signalling pathways active in the FP and BP of the midbrain, such as transforming growth factors (Tgfs) α and β (Blum, 1998; Farkas et al. 2003), for mDA neuron development is less clear at present. They appear rather to provide trophic and/or mitogenic signals to the mDA progenitors, ensuring their proper survival and/or amplification. Although Tgfβ appears to be necessary for the induction of mDA neurons in the chicken embryo (Farkas et al. 2003), a similar requirement in the mouse embryo has not been reported so far. Nevertheless, both Tgfα and Tgfβ are required for the maintenance of mDA neurons in chicken and mice (Blum, 1998; Farkas et al. 2003).

But, how are the signals provided by these diffusible factors translated into a specific cell fate? Each of the secreted factors mentioned above has its specific receptors on the surface of the receiving cell. Binding of the corresponding secreted factor to its receptor activates the latter and elicits an intracellular signalling cascade that ultimately leads to protein modifications or changes in gene expression within the receiving cell (reviewed by Ciani & Salinas, 2005; Liu & Niswander, 2005; Thisse & Thisse, 2005; Huangfu & Anderson, 2006). Each of the aforementioned secreted factors can elicit different signalling cascades within the receiving cell, depending on the type and complement of transducing (signalling) factors it will find inside the cell. The details of these signalling pathways involved in mDA neuron development are still totally unknown. However, target genes of these pathways are being unravelled, and it turns out that many of them are transcription factors activating and/or repressing a genetic programme that finally leads to the specification of the mDA neuronal phenotype in the receiving cell.

Transcription factors acting downstream of Wnt1 controlling mDA neuron development

In view of our result that Pitx3 expression is not initiated in the absence of Wnt1, it remains to be shown whether Pitx3 can be directly activated through canonical Wnt1-signalling. Pitx3 seems to be regulated by Lmx1b, a member of the LIM-HD family. Lmx1b is expressed in the ventral midbrain and later confined to the postmitotic mDA progeny (Asbreuk et al. 2002). Lmx1b has previously been implicated in the development of mDA neurons in mice (Smidt et al. 2000). In the absence of Lmx1b, Th-positive mDA precursors fail to initiate expression of Pitx3 and later disappear, suggesting that Lmx1b is required for the induction of Pitx3 in mDA neurons (Smidt et al. 2000). Interestingly, Lmx1b is able to induce ectopic expression of Wnt1, but not vice versa (Adams et al. 2000; Matsunaga et al. 2002). This would be in line with our findings showing no Pitx3 expression in mDA precursors in the absence of Wnt1 (Prakash et al. 2006).

Another important finding in our studies was that the transcriptional repressor Otx2, which was previously thought of playing a crucial role in mDA neuron development (see Brodski et al. 2003; Puelles et al. 2003; Puelles et al. 2004; Vernay et al. 2005), does not appear to be required for the initial specification of mDA precursors and their differentiation into mDA neurons (Prakash et al. 2006). Deletion of Otx2 from the ventral midbrain at E9.5 did not affect the normal generation of mDA neurons as long as Nkx2-2 was also removed from the ventral midbrain, suggesting that Otx2 function in mDA neuron induction is to repress Nkx2-2 (Prakash et al. 2006).

Transcription factors acting downstream of Shh control mDA neuron development

In a screen for HD transcription factors specifically expressed in the ventral midbrain and involved in mDA neuron development Lmx1a, like Lmx1b a LIM-HD family member, and Msx1, an orthologue of Drosophila muscle segment homeobox (msh) (reviewed by Ramos & Robert, 2005) have been identified (Andersson et al. 2006b). The factor Lmx1a (previously implicated only in dorsal neural tube development (Millonig et al. 2000; Chizhikov & Millen, 2004a), was shown to be expressed in the ventral midbrain in a spatio-temporal pattern correlating with the onset of mDA neurogenesis (Andersson et al. 2006b). The induction of ventral Lmx1a expression appeared to be Shh dependent. Most importantly, ectopic or overexpression of Lmx1a in the neural tube or in embryonic stem cells was sufficient to induce mDA neurons, albeit only in the ventral midbrain or in the presence of Shh (Andersson et al. 2006b). Lmx1a also seems to be required for the normal generation of mDA neurons, as RNA-interference (RNAi) experiments in the ventral midbrain of chicken embryos resulted in a drastic reduction of Nr4a2/Nurr1-positive cells in this region (Andersson et al. 2006b). A similar requirement of Lmx1a in the mouse has not been reported. Lmx1a appeared to exert its effects in part through the activation of another homeobox gene, Msx1 (Andersson et al. 2006b). Its transcription in the ventral midbrain initiates somewhat later than Lmx1a (Andersson et al. 2006b). Expression of Msx1, in contrast to Lmx1a, is confined to proliferating precursors in the ventricular/subventricular zone (VZ/SVZ) of the ventral midline of the mesencephalon. Although Msx1 was not sufficient to induce mDA neurons after ectopic expression in the chick midbrain and does not appear to be necessary for their generation in mice, it seems to be required for the repression of another homeobox gene, Nkx6-1, which is broadly expressed in the VZ/SVZ of the ventral neural tube (Andersson et al. 2006b). Repression of the most ventral Nkx6-1 domain in the midbrain by Msx1 may therefore delimit the mDA progenitor domain from the more laterally located progenitors of motorneurons (Puelles et al. 2004; Andersson et al. 2006b). Whether Lmx1a and Msx1 are regulated by Gli Zn-finger transcription factors, which are mediators of Shh signalling and control early mDA neuron development (Blaess et al. 2006) needs to be determined. Furthermore, Msx1 induced the expression of the proneural transcription factor Neurogenin2 (Ngn2) in the mesencephalic ventral midline of transgenic mice (Andersson et al. 2006b).

Proneural genes control mDA neuron differentiation

The proneural factor Ngn2 belongs to the family of basic helix-loophelix (bHLH) transcriptional regulators and confers mostly generic neuronal but also subtype-specific properties to differentiating neuroepithelial cells (reviewed by Bertrand et al. 2002). Indeed, Ngn2 has also been implicated in mDA neuron development (Andersson et al. 2006a; Kele et al. 2006). Ngn2 is expressed mostly in the VZ/SVZ of the ventral midbrain and in very few postmitotic Nr4a2/Nurr1-positive cells (Andersson et al. 2006a; Kele et al. 2006). In the absence of Ngn2, the mDA neuronal population was initially reduced to less than 20% and recovered postnatally to only about 50–60% of the wild-type numbers (Andersson et al. 2006a; Kele et al. 2006). This recovery was probably due to the redundant activity of another proneural factor, Mash1, expressed in the same region as Ngn2, since removal of Mash1 on an Ngn2−/− mutant background led to a further decrease in mDA neuron numbers, and the Ngn2−/− mutant phenotype could be partially rescued by overexpression of Mash1 in Ngn2Mash1/Mash1 knock-in mice (Kele et al. 2006). The remaining mDA neurons, however, differentiated normally into the SN and VTA subpopulations and established proper connections with their target fields in the forebrain in the absence of Ngn2 (Andersson et al. 2006a; Kele et al. 2006). Overexpression of Ngn2 in the dorsal midbrain or in cell cultures derived from it did not promote the generation of mDA neurons although it enhanced overall neurogenesis (Andersson et al. 2006a; Kele et al. 2006), indicating that it cannot specify a mDA neuronal fate in neural precursors. Neurogenesis appeared to be generally perturbed in the ventral midline of the Ngn2−/− midbrain as evidenced by a notorious lack of neuronal cell bodies and an increased expression of radial glia cell markers, concomitant with an aberrant expression of proneural and neurogenic genes like Dll1 and Hes5 in this region of the mutant midbrain (Andersson et al. 2006a; Kele et al. 2006). Since neighbouring cell populations such as the ventral mesencephalic motorneurons were not affected in the Ngn2−/− mutants (Andersson et al. 2006a), Ngn2 appears to be specifically required by the mDA precursors for the acquisition of generic neuronal properties, but not for their terminal differentiation.

Different transcription factors control distinct genetic networks required for the generation of mDA neurons

One of the first nuclear effectors recognized as being necessary for the proper development of mDA neurons is the orphan nuclear receptor Nr4a2/Nurr1 (reviewed by Perlmann & Wallen-Mackenzie, 2004; Prakash & Wurst, 2006). In the absence of Nr4a2/Nurr1, postmitotic mDA precursors are initially born but later disappear probably because they are lost by apoptosis (reviewed by Perlmann & Wallen-Mackenzie, 2004; Prakash & Wurst, 2006). Most importantly, the transcription of genes involved in DA biosynthesis and neurotransmission, such as Th, vesicular monoamine transporter 2 (Vmat2/Slc18a2) and the dopamine transporter (Dat/Slc6a3), is never initiated in these precursors in Nr4a2/Nurr1 null mutant mice (reviewed in Perlmann & Wallen-Mackenzie, 2004). It was therefore suggested that Nr4a2/Nurr1 is required for the transcriptional activation of the genes encoding those proteins that confer DA neurotransmitter properties of mDA neurons (Sakurada et al. 1999). Another transcription factor playing a similar role in the differentiation of postmitotic mDA precursors into mDA neurons is the paired-like HD protein Pitx3. Pitx3 was initially characterized as a bicoid-related homologue to Pitx2 that is exclusively expressed in the mDA neuronal population (reviewed by Smidt et al. 2004; Prakash & Wurst, 2006). Pitx3 is required for the proper differentiation of a subset of mDA neurons, possibly by directly regulating Th expression in these cells (Maxwell et al. 2005). In the absence of Pitx3, the mDA neurons of the SN and around 50% of the VTA neurons are lost during development (Maxwell et al. 2005; reviewed by Smidt et al. 2004; Prakash & Wurst, 2006). Interestingly, the homologous gene Pitx2 was shown to be directly activated through β-catenin/lymphoid-enhancer-factor (Lef) 1-mediated canonical Wnt-signalling during heart development (Kioussi et al. 2002). Finally, the En1 and En2 homeobox transcription factors, which are also under the transcriptional control of Wnt1, are required for the maintenance of the mDA neuronal population in later stages of embryonic development, but not for their initial specification (Simon et al. 2001).

Despite these considerable advances, many questions remain unanswered. The upstream regulators of Nr4a2/Nurr1 and Lmx1a expression in the ventral midbrain, for example, are still unknown. Equally unknown are the targets conveying the mDA phenotype downstream of Lmx1a, although it is very likely that this pathway only operates in parallel with a pathway driven by Shh or another but yet unidentified ventralizing factor. The target genes of the HD factor Pitx3 in mDA neuron differentiation also await identification.

Summary

Recent advances have provided us for the first time with an insight into the genetic cascades active during the early and intermediate steps of mDA neuron development, including induction of the mDA progenitors and specification of the mDA neuronal fate. Firstly, a Wnt1-regulated network together with a Shh-controlled genetic cascade establishes the mDA progenitor domain at early stages of neural development. They do so by maintaining Otx2-expression in the ventral midbrain which in turn represses Nkx2-2 in this domain, and by induction of Lmx1a and concomitantly of Msx1 in the mesencephalic ventral midline, which in turn re-presses Nkx6-1 expression in this region. Thereby, a Wnt1+, Otx2+, Lmx1a+, Msx1+, Shh+ but Nkx2-2−, Nkx6-1− territory is established in the neuroepithelium of the ventral midbrain from which mDA precursors expressing the retinaldehyde dehydrogenase Aldh1a1 (reviewed by Prakash & Wurst, 2006) and Nr4a2/Nurr1 develop (Fig. 1). Failure to establish this transcriptional code results in a fate-switch of mDA progenitors into other identities such as rh-5HT neurons. It is not clear at present whether the Wnt1- and the Lmx1a-regulated cascades do act in parallel or also act sequentially, as an induction of Lmx1a by Wnt1 has not been reported so far. It should be noted, however, that Lmx1a is able to induce Wnt1 expression in the chicken dorsal spinal cord (Chizhikov & Millen, 2004a). The same appears to apply for Msx1, which was reported to induce Wnt1 transcription after ectopic expression in the chicken neural tube, although this could rather be an indirect effect (reviewed by Ramos & Robert, 2005). It is almost certain, however, that both pathways require the presence of Shh or a Shh-regulated factor, as neither Wnt1 nor Lmx1a was able to induce ectopic mDA neurons in dorsal domains of the neural tube (hindbrain or midbrain, respectively), although both factors were present (Andersson et al. 2006b; Prakash et al. 2006). Secondly, Wnt1-mediated signalling may directly or indirectly initiate or maintain expression of the homeobox genes Pitx3 and En1/2 in the progeny. This signalling cascade acts together with the Nr4a2/Nurr1-regulated pathway conferring DA neurotransmitter identity (initiation of Th and Dat/Slc6a3 expression), thereby specifying the mDA phenotype in mDA precursors at intermediate stages of neural development (Fig. 2). At this stage, an Lmx1a/Msx1-controlled genetic cascade probably acts independently of the Wnt1/Nr4a2/Nurr1-regulated network to confer both generic neuronal (through activation of the proneural Ngn2 gene) and mDA cell type-specific (through activation of as yet unknown target genes) properties to these precursors. Again, the interactions between the Wnt1-, and the Lmx1a-regulated networks remain unknown, but a requirement of Shh or a Shh-mediated signalling cascade for mDA neuron development at this stage may be excluded based or more recent evidence (Andersson et al. 2006b; Blaess et al. 2006; Prakash et al. 2006). Nevertheless, as pointed out before, many questions still remain open, and elucidation of their answers is currently the most important task in the field.

Figure 1. Establishment of the mDA progenitor domain during early stages of mouse neural development.

Figure 1

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A, cross-sections through the E9.5–10.5 mouse embryonic ventral midbrain depicting the expression domains of the secreted and transcription factors involved in the genetic networks required for the proper establishment of an Aldh1a1-positive (dark blue) mDA progenitor domain. B, the genetic networks required for the proper establishment of an Aldh1a1-positive mDA progenitor domain. Wnt1 (green) together with Shh (black) and Lmx1a (brown) are expressed in the ventral midline (FP) and/or in the adjacent BP of the mesencephalon. These factors positively control a genetic cascade including Otx2 (yellow), required for repression of Nkx2-2 (red) in the BP, and Msx1 (pink), required for repression of Nkx6-1 (turquoise) in the FP. Induction of Lmx1a in the ventral midbrain appears to require Shh. The possible interactions between Lmx1a and Wnt1 are not yet clarified (indicated by the broken arrows and question mark). Wnt1 and Otx2 mutually induce and/or maintain their expression in the ventral midbrain, although neither factor is necessary for it (indicated by the broken arrows). Wnt1 and Fgf8 (grey, not shown) are also engaged in a cross-regulation of their expression, but a strict requirement of Fgf8 signalling for the development of mDA neurons in vivo is uncertain. See text for details.

Figure 2. Determination of the mDA-specific and generic neuronal cell fate in mDA precursors at intermediate stages of mouse neural development.

Figure 2

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A, cross-sections through the E12.5 mouse embryonic ventral midbrain depicting the expression domains of the secreted and transcription factors involved in the genetic networks required for the differentiation of mDA precursors into mature mDA neurons (dark blue). B, the genetic networks required for the differentiation of mDA precursors into mature mDA neurons. Wnt1 (green) and Lmx1a (brown) continue to be expressed in the ventral midline (FP) and/or in the adjacent BP of the mesencephalon, but Wnt1 expression is now restricted to the VZ/SVZ of the neuroepithelium, whereas Lmx1a is also expressed in the mantle zone containing the postmitotic progeny. At this stage, these factors positively control a genetic cascade including Msx1 (pink), required for expression of Ngn2 (turquoise) in the VZ/SVZ of the ventral midbrain, and probably also Pitx3 (dark blue), although this remains to be shown (indicated by the broken arrow and question mark). Wnt1 may also maintain the expression of En1/2 (dark blue) in postmitotic mDA neurons at these stages. A complementary pathway controlled by Nurr1/Nr4a2 induces the expression of DA biosynthetic enzymes or transporters (Th and Dat/Slc6a3, dark blue) in these cells. Expression of Nurr1/Nr4a2, Pitx3, Th, Dat/Slc6a3, and En1/2 together define the mDA-specific identity of the postmitotic neurons, whereas their generic neuronal properties are regulated by the expression of the proneural factor Ngn2. The possible interactions between Lmx1a and Wnt1 at this stage are unknown (indicated by the broken arrows and question mark). Neither Otx2 (yellow stripes) nor Shh (black stripes) are required for the generation of mature mDA neurons at this developmental stage. Red: Nkx2-2. See text for details.

Acknowledgments

We apologize to colleagues whose work could not be cited due to space constraints, and we refer readers to the quoted reviews for many older and/or primary references. Our work was funded by the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (NGFN), Förderkennzeichen 01GS0476 (W.W.), by the BMBF Förderkennzeichen 01 GN0512 (W.W.), and by the Deutsche Forschungsgemeinschaft (DFG) Geschäftszeichen WU 164/3-1 and WU 164/3-2 (W. W. and N. P.).

References

  1. Adams KA, Maida JM, Golden JA, Riddle RD. The transcription factor Lmx1b maintains Wnt1 expression within the isthmic organizer. Development. 2000;127:1857–1867. doi: 10.1242/dev.127.9.1857. [DOI] [PubMed] [Google Scholar]
  2. Andersson E, Jensen JB, Parmar M, Guillemot F, Bjorklund A. Development of the mesencephalic dopaminergic neuron system is compromised in the absence of neurogenin 2. Development. 2006a;133:507–516. doi: 10.1242/dev.02224. [DOI] [PubMed] [Google Scholar]
  3. Andersson E, Tryggvason U, Deng Q, Friling S, Alekseenko Z, Robert B, Perlmann T, Ericson J. Identification of intrinsic determinants of midbrain dopamine neurons. Cell. 2006b;124:393–405. doi: 10.1016/j.cell.2005.10.037. [DOI] [PubMed] [Google Scholar]
  4. Asbreuk CHJ, Vogelaar CF, Hellemons A, Smidt MP, Burbach JPH. CNS expression pattern of Lmx1b and coexpression with Ptx genes suggest functional cooperativity in the development of forebrain motor control systems. Mol Cell Neurosci. 2002;21:410–420. doi: 10.1006/mcne.2002.1182. [DOI] [PubMed] [Google Scholar]
  5. Bertrand N, Castro DS, Guillemot F. Proneural genes and the specification of neural cell types. Nat Rev Neurosci. 2002;3:517–530. doi: 10.1038/nrn874. [DOI] [PubMed] [Google Scholar]
  6. Blaess S, Corrales JD, Joyner AL. Sonic hedgehog regulates Gli activator and repressor functions with spatial and temporal precision in the mid/hindbrain region. Development. 2006;133:1799–1809. doi: 10.1242/dev.02339. [DOI] [PubMed] [Google Scholar]
  7. Blak AA, Naserke T, Vogt Weisenhorn DM, Prakash N, Partanen J, Wurst W. Expression of Fgf receptors 1, 2, and 3 in the developing mid- and hindbrain of the mouse. Dev Dyn. 2005;233:1023–1030. doi: 10.1002/dvdy.20386. [DOI] [PubMed] [Google Scholar]
  8. Blum M. A null mutation in TGF-α leads to a reduction in midbrain dopaminergic neurons in the substantia nigra. Nat Neurosci. 1998;1:374–377. doi: 10.1038/1584. [DOI] [PubMed] [Google Scholar]
  9. Brodski C, Weisenhorn DMV, Signore M, Sillaber I, Oesterheld M, Broccoli V, Acampora D, Simeone A, Wurst W. Location and size of dopaminergic and serotonergic cell populations are controlled by the position of the midbrain-hindbrain organizer. J Neurosci. 2003;23:4199–4207. doi: 10.1523/JNEUROSCI.23-10-04199.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Castelo-Branco G, Rawal N, Arenas E. GSK-3β inhibition/β-catenin stabilization in ventral midbrain precursors increases differentiation into dopamine neurons. J Cell Sci. 2004;117:5731–5737. doi: 10.1242/jcs.01505. [DOI] [PubMed] [Google Scholar]
  11. Castelo-Branco G, Wagner J, Rodriguez FJ, Kele J, Sousa K, Rawal N, Pasolli HA, Fuchs E, Kitajewski J, Arenas E. Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a. Proc Natl Acad Sci U S A. 2003;100:12747–12752. doi: 10.1073/pnas.1534900100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chizhikov VV, Millen KJ. Control of roof plate formation by Lmx1a in the developing spinal cord. Development. 2004a;131:2693–2705. doi: 10.1242/dev.01139. [DOI] [PubMed] [Google Scholar]
  13. Chizhikov VV, Millen KJ. Mechanisms of roof plate formation in the vertebrate CNS. Nat Rev Neurosci. 2004b;5:808–812. doi: 10.1038/nrn1520. [DOI] [PubMed] [Google Scholar]
  14. Ciani L, Salinas PC. WNTs in the vertebrate nervous system: from patterning to neuronal connectivity. Nat Rev Neurosci. 2005;6:351–362. doi: 10.1038/nrn1665. [DOI] [PubMed] [Google Scholar]
  15. Echevarria D, Vieira C, Gimeno L, Martinez S. Neuroepithelial secondary organizers and cell fate specification in the developing brain. Brain Res Brain Res Rev. 2003;43:179–191. doi: 10.1016/j.brainresrev.2003.08.002. [DOI] [PubMed] [Google Scholar]
  16. Farkas LM, Dunker N, Roussa E, Unsicker K, Krieglstein K. Transforming growth factor-βs are essential for the development of midbrain dopaminergic neurons in vitro and in vivo. J Neurosci. 2003;23:5178–5186. doi: 10.1523/JNEUROSCI.23-12-05178.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Goridis C, Rohrer H. Specification of catecholaminergic and serotonergic neurons. Nat Rev Neurosci. 2002;3:531–541. doi: 10.1038/nrn871. [DOI] [PubMed] [Google Scholar]
  18. Huangfu D, Anderson KV. Signaling from Smo to Ci/Gli: conservation and divergence of Hedgehog pathways from Drosophila to vertebrates. Development. 2006;133:3–14. doi: 10.1242/dev.02169. [DOI] [PubMed] [Google Scholar]
  19. Hynes M, Stone DM, Dowd M, Pitts-Meek S, Goddard A, Gurney A, Rosenthal A. Control of cell pattern in the neural tube by the zinc finger transcription factor and oncogene Gli-1. Neuron. 1997;19:15–26. doi: 10.1016/s0896-6273(00)80344-x. [DOI] [PubMed] [Google Scholar]
  20. Kele J, Simplicio N, Ferri ALM, Mira H, Guillemot F, Arenas E, Ang S-L. Neurogenin 2 is required for the development of ventral midbrain dopaminergic neurons. Development. 2006;133:495–505. doi: 10.1242/dev.02223. [DOI] [PubMed] [Google Scholar]
  21. Kiecker C, Lumsden A. Compartments and their boundaries in vertebrate brain development. Nat Rev Neurosci. 2005;6:553–564. doi: 10.1038/nrn1702. [DOI] [PubMed] [Google Scholar]
  22. Kioussi C, Briata P, Baek SH, Rose DW, Hamblet NS, Herman T, Ohgi KA, Lin C, Gleiberman A, Wang J. Identification of a Wnt/Dvl/β-catenin → Pitx2 pathway mediating cell-type-specific proliferation during development. Cell. 2002;111:673–685. doi: 10.1016/s0092-8674(02)01084-x. [DOI] [PubMed] [Google Scholar]
  23. Liu A, Joyner A. Early anterior/posterior patterning of the midbrain and cerebellum. Annu Rev Neurosci. 2001;24:869–896. doi: 10.1146/annurev.neuro.24.1.869. [DOI] [PubMed] [Google Scholar]
  24. Liu A, Niswander LA. Bone morphogenetic protein signalling and vertebrate nervous system development. Nat Rev Neurosci. 2005;6:945–954. doi: 10.1038/nrn1805. [DOI] [PubMed] [Google Scholar]
  25. Matsunaga E, Katahira T, Nakamura H. Role of Lmx1b and Wnt1 in mesencephalon and metencephalon development. Development. 2002;129:5269–5277. doi: 10.1242/dev.129.22.5269. [DOI] [PubMed] [Google Scholar]
  26. Maxwell SL, Ho H-Y, Kuehner E, Zhao S, Li M. Pitx3 regulates tyrosine hydroxylase expression in the substantia nigra and identifies a subgroup of mesencephalic dopaminergic progenitor neurons during mouse development. Dev Biol. 2005;282:467–479. doi: 10.1016/j.ydbio.2005.03.028. [DOI] [PubMed] [Google Scholar]
  27. Millonig JH, Millen KJ, Hatten ME. The mouse Dreher gene Lmx1a controls formation of the roof plate in the vertebrate CNS. Nature. 2000;403:764–769. doi: 10.1038/35001573. [DOI] [PubMed] [Google Scholar]
  28. Parr BA, Shea MJ, Vassileva G, McMahon AP. Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds. Development. 1993;119:247–261. doi: 10.1242/dev.119.1.247. [DOI] [PubMed] [Google Scholar]
  29. Perlmann T, Wallen-Mackenzie A. Nurr1, an orphan nuclear receptor with essential functions in developing dopamine cells. Cell Tissue Res. 2004;318:45–52. doi: 10.1007/s00441-004-0974-7. [DOI] [PubMed] [Google Scholar]
  30. Placzek M, Briscoe J. The floor plate: multiple cells, multiple signals. Nat Rev Neurosci. 2005;6:230–240. doi: 10.1038/nrn1628. [DOI] [PubMed] [Google Scholar]
  31. Prakash N, Brodski C, Naserke T, Puelles E, Gogoi R, Hall A, Panhuysen M, Echevarria D, Sussel L, Weisenhorn DMV, Martinez S, Arenas E, Simeone A, Wurst W. A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo. Development. 2006;133:89–98. doi: 10.1242/dev.02181. [DOI] [PubMed] [Google Scholar]
  32. Prakash N, Wurst W. Specification of midbrain territory. Cell Tissue Res. 2004;318:5–14. doi: 10.1007/s00441-004-0955-x. [DOI] [PubMed] [Google Scholar]
  33. Prakash N, Wurst W. Development of dopaminergic neurons in the mammalian brain. Cell Mol Life Sci. 2006;63:187–206. doi: 10.1007/s00018-005-5387-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Puelles E, Acampora D, Lacroix E, Signore M, Annino A, Tuorto F, Filosa S, Corte G, Wurst W, Ang SL, Simeone A. Otx dose-dependent integrated control of antero-posterior and dorso-ventral patterning of midbrain. Nat Neurosci. 2003;6:453–460. doi: 10.1038/nn1037. [DOI] [PubMed] [Google Scholar]
  35. Puelles E, Annino A, Tuorto F, Usiello A, Acampora D, Czerny T, Brodski C, Ang SL, Wurst W, Simeone A. Otx2 regulates the extent, identity and fate of neuronal progenitor domains in the ventral midbrain. Development. 2004;131:2037–2048. doi: 10.1242/dev.01107. [DOI] [PubMed] [Google Scholar]
  36. Ramos C, Robert B. msh/Msx gene family in neural development. Trends Genet. 2005;21:624–632. doi: 10.1016/j.tig.2005.09.001. [DOI] [PubMed] [Google Scholar]
  37. Sakurada K, Ohshima-Sakurada M, Palmer TD, Gage FH. Nurr1, an orphan nuclear receptor, is a transcriptional activator of endogenous tyrosine hydroxylase in neural progenitor cells derived from the adult brain. Development. 1999;126:4017–4026. doi: 10.1242/dev.126.18.4017. [DOI] [PubMed] [Google Scholar]
  38. Simeone A. Genetic control of dopaminergic neuron differentiation. Trends Neurosci. 2005;28:62–65. doi: 10.1016/j.tins.2004.11.007. [DOI] [PubMed] [Google Scholar]
  39. Simon HH, Saueressig H, Wurst W, Goulding MD, O'Leary DD. Fate of midbrain dopaminergic neurons controlled by the engrailed genes. J Neurosci. 2001;21:3126–3134. doi: 10.1523/JNEUROSCI.21-09-03126.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Smidt MP, Asbreuk CH, Cox JJ, Chen H, Johnson RL, Burbach JP. A second independent pathway for development of mesencephalic dopaminergic neurons requires Lmx1b. Nat Neurosci. 2000;3:337–341. doi: 10.1038/73902. [DOI] [PubMed] [Google Scholar]
  41. Smidt MP, Smits SM, Burbach JPH. Molecular mechanisms underlying midbrain dopamine neuron development and function. Eur J Pharmacol. 2003;480:75–88. doi: 10.1016/j.ejphar.2003.08.094. [DOI] [PubMed] [Google Scholar]
  42. Smidt MP, Smits SM, Burbach JPH. Homeobox gene Pitx3 and its role in the development of dopamine neurons of the substantia nigra. Cell Tissue Res. 2004;318:35–43. doi: 10.1007/s00441-004-0943-1. [DOI] [PubMed] [Google Scholar]
  43. Thisse B, Thisse C. Functions and regulations of fibroblast growth factor signaling during embryonic development. Dev Biol. 2005;287:390–402. doi: 10.1016/j.ydbio.2005.09.011. [DOI] [PubMed] [Google Scholar]
  44. Trokovic R, Trokovic N, Hernesniemi S, Pirvola U, Vogt Weisenhorn DM, Rossant J, McMahon AP, Wurst W, Partanen J. FGFR1 is independently required in both developing mid- and hindbrain for sustained response to isthmic signals. EMBO J. 2003;22:1811–1823. doi: 10.1093/emboj/cdg169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vernay B, Koch M, Vaccarino F, Briscoe J, Simeone A, Kageyama R, Ang S-L. Otx2 regulates subtype specification and neurogenesis in the midbrain. J Neurosci. 2005;25:4856–4867. doi: 10.1523/JNEUROSCI.5158-04.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Winkler C, Kirik D, Bjorklund A. Cell transplantation in Parkinson's disease: how can we make it work? Trends Neurosci. 2005;28:86–92. doi: 10.1016/j.tins.2004.12.006. [DOI] [PubMed] [Google Scholar]
  47. Wurst W, Bally-Cuif L. Neural plate patterning: upstream and downstream of the isthmic organizer. Nat Rev Neurosci. 2001;2:99–108. doi: 10.1038/35053516. [DOI] [PubMed] [Google Scholar]
  48. Ye W, Shimamura K, Rubenstein JL, Hynes MA, Rosenthal A. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell. 1998;93:755–766. doi: 10.1016/s0092-8674(00)81437-3. [DOI] [PubMed] [Google Scholar]
  49. Zervas M, Millet S, Ahn S, Joyner AL. Cell behaviors and genetic lineages of the mesencephalon and rhombomere 1. Neuron. 2004;43:345–357. doi: 10.1016/j.neuron.2004.07.010. [DOI] [PubMed] [Google Scholar]

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