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FP7

MDDANEURODEV Report Summary

Project ID: 222999
Funded under: FP7-HEALTH
Country: Netherlands

Final Report Summary - MDDANEURODEV (Molecular coding and subset specification of dopamine neurons generating the the meso-limbic and nigro-striatal system.)

Executive Summary:
Publishable summary of the FP7 mdDANeurodev (222999) project ‘Molecular coding and subset specification of dopamine neurons generating the meso-limbic and nigro-stratal system’.

Our project is aimed at identifying molecular and physiological mechanisms of the development and maintenance of mesodiencephalic dopaminergic (mdDA) neurons. The main results of the three research work packages achieved so far are:

WP1) Ventricular zone programming and early specification of the mdDA neuronal phenotype.
We were very successful in the analysis of the LEF/TCF signaling pathway and have identified cross talk to the Pitx3 pathway. Our data strongly suggest that Lef1 is not the critical nuclear effector of Wnt/B-catenin signaling in the murine VM, and that still an unknown nuclear effectors (TF's) of this pathway might function in mdDA neuron development. Further examination on the effect of B-catenin signaling has shown Lmx1a is an important target in mdDA neurons of this signaling. Moreover, we have completed the analysis of signaling pathway component expression in the proximity of dopamine neurons for the following pathways in zebrafish: WNT, SHH, TGF/Nodal, and Delta/Notch at the systems-biology level and molecular networks of DA development and function.

WP2) Mechanisms of migration and guidance of mdDA neurons.
Guidance analysis on early outgrowth, trajectory and targeting of different classes of mdDA axons has been performed and identified critical factors for this process as Frizzled3, Wnt5a and Wnt7b.
Migration analysis indicate that mdDA neurons change their mode of migration from radial to tangential as they adopt their final position and involve pathways that are regulated by Cxcl12/Cxcr4. In contrast, Slit/Robo signaling is only required for the guidance of mdDA axons.
We investigated the role of EN1 in translation regulation and identified En1/2 interactors through pull-down analysis as: factors involved in mRNA translation or mRNA transport and stability. Furthermore, experiments showed that the En1K313E mutant (unable to interact with eIF4E) is unable to up-regulate the translation of selected En1 targets and this mutant is unable to induce growth cone collapse.

WP3) Genetic programming, terminal differentiation and maintenance.
Within this part we have acquired the downstream targets of the critically important homeodomain transcription factor Pitx3 and identified the expression profile of some of these targets. The analysis have identified a Retinoic Acid (RA) dependent and independent pathway of target gene activation downstream of Pitx3. Some of these genes are critical TF's themselves as En1.
We have found that in the generation of dopamine neurons, Lmx1a and Lmx1b have both unique and redundant functions as in the progenitor domain possible through the regulation of Phox2a.
Finally, we discovered that Otx2 is the first transcription factor, with a proven role in mdDA neuron development, expressed selectively in a large fraction of VTA neurons.
Through analysis of Otx2 deleter and overexpression in mouse mutants during development we concluded that in the absence of Otx2, VTA progenitors undergoing post-mitotic transition and maturation are fated to generate a specific subpopulation of VTA neurons. Otx2 is a post-mitotic selector of the VTA neuronal subtype identity and may confer resistance to the MPTP neurotoxin. Interestingly, Otx2 function in the floor plate is distance dependent to the isthmic area, suggesting a interplay with fgf8 signaling.

The results and publications are in line with the expected output of the consortium and form the basis of a clear description of the mdDA neuronal population. These data will enable novel understanding and the design of drugs that might be applicable in dopamine neurodegenerative diseases like Parkinson's.

Project Context and Objectives:
This is the summary description of the project context and the main objectives of the FP7 mdDANeurodev (222999) project ‘Molecular coding and subset specification of dopamine neurons generating the meso-limbic and nigro-stratal system’ at the second period report.

Our project is aimed at identifying molecular and physiological mechanisms of the development and maintenance of mesodiencephalic dopaminergic neurons, the neuronal population affected in Parkinson's disease, in affective disorders as schizophrenia and autism, and in anxiety related disorders as depression. The project aims to combine three distinct levels of research, early patterning, differentiation and axon pathfinding. The acquired knowledge will be used to design new strategies to treat the above mentioned patients and provide new leads for drug targeting.

Recent work by the consortium members has led to the proposal of a molecular pathway that leads to the formation of mdDA neurons in a specific region spanning parts of the diencephalon and mesencephalon (reviewed by Smidt and Burbach, 2007). The data has suggested that specific subsets arise in terms of molecular signature and function (reviewed by Smits et al. 2006). Although the overall developmental pathway has been uncovered recently, many questions remain. Therefore, in this proposal we aim to:
-Identify the role of transcription factors in mdDA development
-Define mdDA subset-specification
-Investigate the relation between subset specification and functional units and axon pathfinding codes.

In order to achieve this we proposed the following key objectives which are handled through these three Work packages:
1) Ventricular zone programming and early specification of the mdDA neuronal phenotype
2) Mechanisms of migration and guidance of mdDA neurons
3) Genetic programming, terminal differentiation and maintenance

The project relies on a multidisciplinary genomics and molecular biology approach to unravel the fundamental biological process used to generate and maintain proper functioning mdDA neurons.

The “mdDANEURODEV” project is essential for identifying new fundamental data that may lead to the generation of new therapies for diseases such as Schizophrenia, Autism, depression and addiction. All knowledge obtained in this project will be published and shared by the consortium so the results can be quickly exploited by other research groups and companies that can build upon the data generated within the consortium.

The project accurately fulfilled the objectives of Health-2007-2.1.2.5 'System biology' call: Multidisciplinary fundamental genomics and molecular biology approaches to study basic biological processes relevant to health and disease.

Psychiatric diseases and neurodegenerative diseases as Parkinson's have a great impact on our society. With the increase of the complexity of our society and the increase in mean age, diseases that hamper the cognitive function are having more and more impact.
The current treatment paradigms are collectively based on suppressing symptoms. To make the step towards understanding the disease itself and treating the disease and its onset, new experimental data are crucial. Looking at the last decade enormous progress has been made by the consortium members and others to provide new data on molecular players that are involved in the generation and function of the neuronal cell groups that are directly and indirectly involved in the above-mentioned diseases. Also the possibility to generate new mesodiencephalic Dopamineergic (mdDA) neurons from human ES-cells to be used in a cell replacement paradigm depends on data describing the basic biological process that generates mdDA neurons.

All consortium members are absolute experts in this field and complement each other in order to expand the area of expertise essential to address the multi-disciplinary approach. This generated confidence that the key objectives described could be fulfilled by this consortium. Moreover, the consortium members are certain that the achievements from this project, as presented here, will fulfill the socio-economic need for basic knowledge of complex diseases to generate basic knowledge essential to perform translational biology in the process of the development of novel medication.

Overall strategy of the work plan:
The project aimed to elucidate the molecular programming of mdDA neurons on the following levels:
-The early patterning mechanisms leading to specific molecular coding of the ventricular zone along the A/P axis and D/V axis.
-The molecular mechanisms of migration and subset specific axonal outgrowth and guidance.
-The molecular mechanisms of subset specific terminal differentiation and overall genetic programming of mdDA neurons.

These three different levels of research provided the whole scope of processes in this complex biological system to gain understanding of the generation, function and pathology of this system.
The logical separation into these three levels is structured by the formation of the three accompanying work packages. Within these work packages the selected PI's have directed their approach to elucidate the tasks that are listed in the tables of the deliverables.

Objectives Work package 1: Ventricular zone programming and early specification of the mdDA neuronal phenotype (partner 3 and 6)
The main goal of this work package was to gather data that directs to the coding of the ventricular zone and to elucidate patterning issues relevant for the generation of mdDA neurons derived from di- and mesencephalic regions of the central nervous system.

-Early signaling events that depend on their spatial location and coding in other species was the focus within this work package.
During brain development, the cell-intrinsic prepattern reflected by transcription factors as well as extrinsic signals specify differentiation of dopaminergic neurons. -The goal of the second part of this work package was to identify the signaling input into specification of dopaminergic neurons with ascending projections in zebrafish.
In contrast to mammals, where the di/mesencephalic dopaminergic (DA) groups A9 and A10 provide ascending projections, zebrafish DA neurons with corresponding ascending projections are located in the more anterior diencephalic areas of posterior tuberculum and ventral thalamus (so-called ventral diencephalic groups 1 and 3).

-This work package identified which of the signals previously related to DA development in mammals and fish contribute to specification of dopaminergic neurons of ascending projection characteristics.
The comparison of zebrafish and mammals should enable the identification of the evolutionarily conserved core regulatory circuits for ascending DA systems.

Objectives Work package 2: Mechanisms of migration and Guidance of mdDA neurons (partner 1, 4 and 7):
This WP aimed at deciphering the molecular mechanisms that control neuronal migration and axon guidance in the developing mdDA system by:
1) determining the normal migration and formation of axonal connections by mdDA neurons using genetic and imaging tools
2) assessing the role of specific candidate molecules during mdDA neuron migration and axon guidance in genetic mouse models
3) assessing the role of homeoprotein transcription factors (En)1 and 2, Otx2 and Pax6 in mdDA neuron migration and axon guidance.
4) identifying new molecules required for the migration and guidance of mdDA neurons through micro-array analyzes

Objectives Work package 3: Genetic programming, terminal differentiation and maintenance (partner 1, 2 and 5):
The objective of this WP was to complement the work done in WP1 and 2 by analyzing the transcriptional cascade (in early and terminal differentiation) active during mdDA differentiation.
The focus in this WP was on the identification of the role of Pitx3, Lmx1a and b and Otx2.

Objectives Work package 4: Project management (partner 1):
The overall objective of this work package was to ensure the proper functioning of the project and to ensure communication within the project and to the EU.

Project Results:
The main scientific and technological results and foregrounds of the FP7 mdDANeurodev (222999) project ‘Molecular coding and subset specification of dopamine neurons generating the meso-limbic and nigro-stratal system’ are described here.

In this piece of the report we mention the actual results that are obtained within the project presented by the separate partners. Overall the project has been extremely successful on all aims and deliverables are achieved well within the project time.

WP 1, Partner 6 HMGU, Prof. Wurst:

During the funding period, we identified a Lef-mediated Dkk3/Wnt1/B-catenin signaling cascade that sequentially activates Lmx1a, Pitx3 and Bdnf expression in the Substantia nigra pars compacta (SNc) DA progenitors, precursors and/or mature neurons to promote their differentiation and survival (see reporting figure XX). This signaling cascade is initiated by the binding of the Wnt1 ligand (Prakash et al., Development 133, 89-98, 2006) to the Frizzled 3 (Fzd3) receptor (Stuebner et al., Dev. Dyn. 239, 246-260, 2010) and low density lipoprotein receptor-related protein 6 (Lrp6) co-receptor (Castelo-Branco et al., Dev. Dyn. 239, 211-221, 2010) expressed on mdDA progenitors, precursors and mature neurons, and the subsequent activation of the intracellular Wnt/B-catenin signaling pathway in these cells (Joksimovic et al., Nat. Neurosci. 12, 125-131, 2009; Tang et al., Development 136, 2027-2038, 2009; Tang et al., J Neurosci. 30, 9280-91, 2010; L’Episcopo et al., Mol. Neurodegener. 6, 49, 2011). Extra- and/or intracellular Dickkopf 3 (Dkk3) protein potentiates Wnt1/B-catenin signal transduction in SNc DA progenitors/precursors, leading to the activation of Lmx1a transcription in these cells (Fukusumi et al., in preparation). Although the Lmx1a promoter region is bound by lymphoid enhancer binding factor 1 (Lef1) and ectopic Wnt1/Lef1 expression leads to the ectopic activation of Lmx1a transcription in the rostral hindbrain of En1+/Wnt1 mice, the very restricted and non-nuclear Lef1 expression pattern in the murine ventral midbrain (VM) and the absence of an mdDA phenotype in Lef1-/- mice strongly suggest the existence of other but yet unknown nuclear effectors of Wnt/B-catenin signaling in mdDA progenitors, precursors and/or mature neurons (Fukusumi et al., in preparation).We are therefore profiling the transcriptome of Wnt-responsive mdDA progenitors/precursors and postmitotic neurons in the VM of a transgenic Wnt/B-catenin reporter (BAT-gal) mouse line. The homeodomain transcription factor (TF) Lmx1a in turn activates the Pitx3 promoter in postmitotic SNc DA precursors, thus ensuring their proper differentiation into Tyrosine hydroxylase (Th)- and Pitx3-positive SNc DA neurons (Fukusumi et al., in preparation). The paired-like homeodomain TF Pitx3 itself activates the promoter of the brain-derived neurotrophic factor (Bdnf) gene in the maturing SNc DA neuronal subset, thereby leading to their enhanced survival and neuroprotection against cytotoxic insults during development and probably also in adulthood (Peng et al., J Neurosci. 31, 12802-12815, 2011). Alternatively or in addition, Dkk3 acts as a pro-survival factor for SNc DA neurons via an unknown intracellular effector pathway or mechanism (Fukusumi et al., in preparation). Notably, glial cell line-derived neurotrophic factor (Gdnf), a well-established neurotrophic factor for mdDA neurons capable of activating Pitx3 expression in these neurons via the NF-?B pathway, looses its neuroprotective effect against cytotoxic insults in the absence of Pitx3, indicating that Pitx3-mediated activation of Bdnf expression is the crucial event downstream of Gdnf signaling for the neuroprotection of mdDA neurons (Peng et al., J Neurosci. 31, 12802-12815, 2011).
In addition to the SNc DA neuronal subset, Wnt1-mediated B-catenin signaling is also active in a caudomedial (ventral tegmental area, VTA) mdDA neuronal subset (Götz et al., unpublished), where it promotes the proliferation of the corresponding progenitors (Omodei et al., Development 135, 3459-3470, 2008). In this context, Wnt1-mediated signaling is engaged in a feedback loop reinforcing the transcription of the orthodenticle homolog 2 (Otx2) TF and, as indicated above, of Lmx1a and Msx1 expression in caudomedial (VTA) mdDA neurons (Omodei et al., Development 135, 3459-3470, 2008). Notably, expression of Otx2 in the adult mouse VM only persists in the VTA DA neuronal subset (Di Salvio et al., Int. J Dev. Biol. 54, 939-45, 2010). In collaboration with Prof. A. Simeone’s group (Partner 5), we showed that Otx2 represses the expression of the dopamine transporter (Dat) in these neurons (Di Salvio et al., Nat. Neurosci. 13, 1481-8, 2010). As the neurotoxic agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is taken up into mdDA neurons via Dat, the suppression of Dat expression by Otx2 in VTA DA neurons confers neuroprotective properties to these cells (Di Salvio et al., Nat. Neurosci. 13, 1481-8, 2010). In this regard, we have explored the restorative potential of ectopic DA neurons derived from the rostral hindbrain of En1+/Otx2 embryos (Brodski et al., J. Neurosci. 23, 4199-4207, 2003) in a rat model of Parkinson’s Disease (PD) (Hackl et al., Cell Transplant. 19, 1085-101, 2010). These Otx2-overexpressing DA neurons in fact survive and functionally re-innervate the DA-depleted striatum in PD rats.

Altogether, we have successfully identified and dissected two Wnt1/B-catenin-controlled gene regulatory networks conferring subtype-specific characteristics and/or properties to the mdDA neurons in the murine VM during the mdDANEURODEV funding period. This work has resulted in the publication of 7 peer-reviewed articles, and in 2 articles that are currently being prepared for submission.

WP 1, Partner 3 ALU-Fr, Prof. Driever:

Analysis of the effect of knockdown or activation of specific signals on neuronal DA differentiation
We have completed the analysis of signaling pathway component expression in the proximity of DA neurons for the pathways listed in the application. While most anatomically distinct zebrafish DA groups have been investigated, we placed an emphasis on the ventral diencephalic groups (vDC) termed DC2,4, 5 and 6, which are the only ones to extend ascending axonal projections into the subpallium / striatum in zebrafish.

Delta/Notch Signaling: Our analysis revealed that DeltaA, DeltaD, and Jagged2a are the Notch ligands expressed in proximity of DA neurons with ascending projections in zebrafish. We mapped the time windows of DA neurogenesis for all DA groups by application of the Notch Signaling inhibitor DAPT in consecutive time windows. We identified the Notch ligands that contribute to the neurogenic decision of DA precursors to be DeltaA and DeltaD. The predominant contribution appears to be by DeltaA, while loss of DeltaD gives a weak neurogenic phenotype only. dla is the ortholog of mammalian Dll1. While the role of Dll1 in DA development has not been functionally analyzed, it has been shown to be expressed in regions of mes-diencephalic DA neurogenesis, and its expression, together with Hes5, depends on Ngn2 activity (Kele et al., 2006). Thus a conserved Delta ortholog may control DA neurogenesis in all vertebrates. This work has been published: Mahler, M., Filippi, A., Driever, W., 2010. DeltaA/DeltaD regulate multiple and temporally distinct phases of Notch signaling during dopaminergic neurogenesis in zebrafish. J Neurosci. 30, 16621-635.

WNT Signaling: We investigated whether zebrafish DA progenitors during proliferative phases of development may potentially be affected by WNT signaling. To visualize canonical WNT signaling activity in the proximity of DA neurons, we analyzed GFP and TH expression in top:dGFP reporter embryos on the second and third day of development by confocal microscopy. The GFP WNT-reporter is coexpressed in early DA neurons and continues to be active in precursor territories medial to the ventral diencephalic DA groups 2 through 6. This demonstrated that DA progenitors and DA neurons are likely subject to WNT signaling. We investigated the expression pattern of all WNTs identified in the zebrafish genome with relation to spatial proximity of expression domains and dopaminergic neurons. The canonical WNT ligands Wnt3l/Wnt3a, and Wnt8b, the non-canonical WNT ligands Wnt4a, Wnt5a, and Wnt11r, as well as four non-classified Wnt ligands, Wnt7/Wntc, Wnt9a, Wnt9b, and Wnt16 are expressed in proximity of diencephalic DA neurons. Detailed analysis revealed Wnt8b as a strong candidate for regulation of DA expressed in the ventricular zone adjacent to the vDC 2 and 4 DA neurons which send ascending projections, as well as in the walls of and adjacent to the hypothalamic ventricle. Similar analyses were performed for Frizzled receptors, revealing that Fzd7a, Fzd7b, Fzd8a, Fzd8b, Fzd8c, and Frzd9 are expressed in regions of DA neurogenesis or in mature DA neurons. We also analyzed expression of a set of extracellular modulators of WNT activity, revealing that Sfrp1a and Sfrp5 are expressed in a broad domain of DA neurogenesis in the ventral diencephalon, while Dkk1 is expressed in bilateral small domains directly medial to the DA groups 2 and 4, which send ascending projections. A manuscript about these data is in preparation (working title: "Expression of WNT signaling components during dopaminergic neuron differentiation in zebrafish" by A. Filippi, E. Kastenhuber, P. Panza, B. Behncke, and W. Driever)
For functional analysis, we performed both gain-of-function and loss-of-function studies of WNT signaling. For the genetic manipulation of WNT signaling, we used heat shock promoter driven WNT effector lines which express dkk1-GFP (secreted inhibitor), Delta-TCF3 (dominant negative TCF3), Wnt5b (non-canonical WNT), or Wnt8a (canonical WNT). These data revealed a requirement for WNT signaling for the specification of Otp-dependent dopaminergic neurons with ascending projections (groups DC4,5,6). Wnt8a overexpression between 20 and 30 hpf causes an expansion of the number of DA neurons in these groups, while overexpression of Dkk may eliminate DC5 and 6 groups. We complemented the functional analysis of WNT signaling by using pharmacological inhibitors of WNT signaling: IWR-1 and XAV939, which stabilize Axin (and thus inhibit b-catenin mediated transcription). Confirming the results from the heat shock experiments, IWR-1 and XAV939 mediated repression of WNT signaling strongly reduced the number of vDC 5 and 6 DA neurons (working title: "Modulation of WNT signaling activity differentially affects dopaminergic development in zebrafish" by P. Panza, A. Filippi, and W. Driever)

FGF Signaling: FGF signals are not expressed in direct proximity of zebrafish DA neuron development; however FGF8 from the optic stalk may potentially reach preoptic, ventral thalamic or medial hypothalamic DA groups, and FGF from the anterior neural ridge the subpallial group. To judge a potential role of FGF in DA development, we analyzed the expression the FGF responding genes pea3 and erm, which both revealed no co-expression in DA neurons, with the exception of DC3 DA neurons expressing erm. In contrast, spry is expressed at low levels in the area of DA differentiation but not in differentiating DA neurons. We have mapped temporal requirements for FGF signaling using SU5402 as a selective inhibitor applied in eight different overlapping 12 hrs time windows starting at 10 hpf, and controlled effectiveness of FGF inhibition by analysis of expression of the FGF8 target pea3. Our data revealed that FGF signaling is required to specify pretectal and ventral thalamic (DC1) DA neurons during early specification phases (10 to 39 hpf), but also confirmed that posterior tuberal DA neurons with ascending projections do not strictly require FGF signaling. Our pharmacological analysis of FGF signaling revealed a contribution to DA differentiation in the caudal hypothalamic group. Expression database analysis identified FGF3 as candidate signal to mediate the effect on this DA group. We analyzed fgf3 liat24152 mutant embryos, and found a selective loss of DC7 caudal hypothalamic DA neurons. Thus our work identified selective requirements for FGF signaling in defined DA subgroups. A manuscript about these data is almost ready for submission (working title: "FGF3 signaling controls differentiation of caudal hypothalamic DA neurons in zebrafish" by P. Koch, H. Löhr, and W. Driever).

Identification of transcriptional profiles of DA precursor cells
In the original work plan we had specified use of neurogenin1:GFP fish to characterize DA precursors. Our experiences with the neurogenin1:GFP transgenic line however have made us change our plans for obtaining tissue samples of DA precursors and mature samples for DA neurons with ascending projections from zebrafish. The neurogenin1:GFP transgenic expression of GFP is too dynamic to reliably identify the DA precursor pools. We were able to show that Otpa and Otpb are two paralogous transcriptions factor that are expressed in and specify zebrafish DA precursor populations with ascending projections (Ryu et al., 2007). Therefore, we have focused on otp:GFP transgenic label to isolate DA neurons and precursors. The transgenic line used in our work contains an otpb regulatory element that selectively drives Otp expression in the posterior tubercular area containing vDC DA neurons with ascending projections, as well as in the preoptic area. We have isolated otp:GFP expressing cells by FACS sorting from dissociated embryonic brain, and established expression profiles using the Illumina RNAseq approach both for GFP positive Otp:GFP cells and for GFP negative control cell populations. Brain expressed transcription factors enriched in Otp:GFP cells include cited3, foxa/a1/a2/a3, foxe3, id2b, isl1, lhx1a, mnx1, nkx2.2a, nkx2.9, nkx6.1, olig2, otpa, phox2b, pitx2, vgll2b. Many of these transcription factors are typical for the ventral diencephalic region in which the vDC DA neurons differentiate, or have been previously linked to dopaminergic differentiation. The following signaling pathway components were enriched in otp:GFPcells: dkk1 (m), insb, shha, shhb, tgfb3, wnt4 (m), with (m for p<0.3). The detailed analysis of candidate genes from this experimental approach however revealed that many of the factors that we have previously characterized were not sufficiently enriched in the GFP positive versus the control sample, likely caused by the fact that many of the factors with specific functions in DA development are also broadly expressed in other regions of the embryo. We continue the analysis of some factors identified in this approach.

Systems Biology level analysis of input of signaling pathways in DA differentiation.
Systems level understanding of DA neuronal groups demanded a clear definition of DA groups and features that may distinguish specific subtypes in each group. We undertook an endeavor to characterize the complete "projectome" of each anatomically defined DA group in order to establish a solid framework for DA subtype analysis. The most extensive DA projections are established by posterior tubercular Otp-dependent vDC neurons, with individual somata integrating the ascending DA system, the descending diencephalospinal, as well as the endohypothalamic circuitry. These findings suggest a major role in the modulation of physiology and behaviour for otp-dependent DA neurons, which correlate with the mammalian A11 group. Based on the fact that these vDC DA neurons are the only DA neurons with ascending projections in the zebrafish larvae, we postulate that they indeed provide similar DA neuromodulatory activity to the subpallium / striatum as compared to mammalian mdDA neurons. We further identified an endogenous subpallial DA system that not only provides most of the local DA projections, but also connects to the ventral diencephalon. This striatal dopaminergic system promises to become an interesting model for differentiation of DA precursors in the striatium, which is the basis of current initiatives in many labs to establish neuro-restorative therapies for Parkinson's disease. Our work established the first systems level complete analysis of zebrafish larval DA systems. This work has been published: Tay, T.L., Ronneberger, O., Ryu, S., Nitschke, R., Driever, W., 2011. Comprehensive catecholaminergic projectome analysis reveals single-neuron integration of zebrafish ascending and descending dopaminergic systems. Nature Communications. 2, 171.
In a second systems level approach, we aimed at providing a solution to the problem that a detailed understanding of the transcription and signaling networks controlling DA differentiation needs to implement a true 3-dimensional anatomical aspect to map the expression domains of transcription factors or signals to each DA group, ideally at cellular resolution to identify neuronal subtypes that may differ in transcription factor contents. We have established a systems level analysis tool to map expression domains of a large number of genes at high resolution to dopaminergic group. We name the system VIBE-Z (virtual brain explorer for zebrafish). We have utilized this systems biology tool to identify subtypes of the vDC DA groups that differ in expression of the transcription factor Dlx5a.
This work has just been accepted for publication by the journal Nature Methods: O. Ronneberger, K. Liu, M. Rath, D. Rueß, T. Mueller, H. Skibbe, B. Drayer, T. Schmidt, A. Filippi, R. Nitschke, T. Brox, H. Burkhardt, and W. Driever: Virtual Brain Explorer: An Anatomical Atlas and Imaging Framework for 3D High Resolution Coexpression Analysis in the Larval Zebrafish Brain Nature Methods, in press.

Testing of hypothesis by knockdown of crucial systems components
One of the fundamental open questions towards understanding of DA system development is the integration of transcriptional codes and signaling mechanisms to control the projection phenotype of DA neurons. We have previously shown that the ascending DA system in zebrafish is dependent on Otp, Sim1 and Arnt transcription factors. Here, we investigated whether and how these factors control the projection DA phenotype. Precise spatio-temporal control of axon guidance factor expression is a prerequisite for formation of functional neuronal connections. While Netrin/DCC and Robo/Slit mediated attractive and repulsive guidance of commissural axons have been extensively studied, little is known about mechanisms controlling mediolateral positioning of longitudinal axons in vertebrates. Here, we use a genetic approach in zebrafish embryos to study pathfinding mechanisms of dopaminergic longitudinal axons projecting from the vDC/hypothalamus into hindbrain and spinal cord. We show that both Sim1a and Arnt2 control aspects of axon guidance: Sim1a or Arnt2 depletion results in displacement of hypothalamo-spinal longitudinal axons towards the midline. This phenotype is suppressed in robo3 guidance receptor mutant embryos. In absence of Sim1a and Arnt2, expression of the robo3 splice isoform robo3a.1 is increased in the hypothalamus, indicating negative control of robo3a.1 transcription by these factors. We further provide evidence that increased Robo3a.1 levels interfere with Robo2 mediated repulsive axon guidance. Finally, we show that the N-terminal domain unique to Robo3a.1 mediates the block of Robo2 repulsive activity. Therefore, Sim1a and Arnt2 contribute to control of lateral positioning of longitudinal hypothalamic-spinal axons by negative regulation of robo3a.1 expression, which in turn attenuates the repulsive activity of Robo2. Our data demonstrate that the transcriptional networks involved in vDC DA differentiation also control the projection phenotype of these neurons.
A manuscript about these data is currently under review (title: "Sim1/Arnt2 Transcription Factors Contribute to Hypothalamo-Spinal Axon Guidance by Regulating Robo2 Activity via a Robo3 Dependent Mechanism " by J. Schweitzer, H. Löhr, J.L. Bonkowsky, K. Hübscher, and W. Driever)

Significant results:
-Identification of WNT signaling mechanisms contributing to zebrafish vDCA DA development
-Detailed molecular and developmental understanding of of Delta/Notch signaling in zebrafish DA neuron development
-Transcriptional profile of Otp-positive vDC DA precursors
- Systems level analysis of the complete DA projectome in zebrafish larvae, leading to identification of DA neuronal group subtypes
-Systems level virtual environment for high resolution analysis of transcription factors and signaling pathway components in the proper 3D anatomical context
-Identification of transcriptional nodes in the vDC DA transcriptional specification network that control signaling pathway components regulating the projection behavior of DA axons.

WP2, Partner 1 UMC Utrecht, (Prof. Smidt and) Pasterkamp, PhD:

We have explored the normal patterns of mdDA pathway development in the mouse using TH immunohistochemistry, lipophilic Dye tracing and pitX3-GFP mice (Kolk et al., 2009). This work has formed a framework for the study of the axon growth and guidance proteins described below. Several different mouse mutant strains of specific axon guidance proteins and receptors have been analyzed for defects in mdDA pathway formation. Thus far we have assessed the role of Semaphorin3F and its receptor neuropilin-2 in the formation of mdDA pathways. This work has shown that Sema3F-Npn-2 signaling is required for various aspects of mdDA pathway development, including the rostral oriented growth of mdDA axons and the targeting of mesoprefrontal axons at the level of the cortex (Kolk et al., 2009). We have also studied the role of Frizzled3 and its Wnt ligands and have reported that Wnt-Frizzled3 signaling is required for the rostral growth of mdDA axons at the level of the midbrain (Fenstermaker et al., 2010). Fiannly, we have shown that Semaphorin7A directs migrating GnRH neurons to the hypothalamus during development (Messina et al., 2011).

Significant results: We have dissected the anatomical organization of different mdDA axon tracts (mesolimbic, mesostriatal and mesoprefrontal) during mid-embryonic development and have obtained detailed information about the early outgrowth, trajectory and targeting of different classes of mdDA axons. Furthermore, we have observed a prominent role for Sema3F and its receptor in different aspects of the formation mdDA projections, e.g. in the targeting of axons in the prefrontal cortex. Finally, we have discovered a role for Frizzled3 and Wnt5a and Wnt7b in the rostrally oriented growth of mdDA axons. Finally, in collaboration with the group of Paolo Giacobini we have identified a role for Semaphorin7A in the migration of neurons in the embryonic brain.

Kolk SM, Gunput RA, Tran TS, van den Heuvel DM, Prasad AA, Hellemons AJ, Adolfs Y, Ginty DD, Kolodkin AL, Burbach JP, Smidt MP, Pasterkamp RJ. Semaphorin 3F is a bifunctional guidance cue for dopaminergic axons and controls their fasciculation, channelling, rostral growth, and intracortical targeting. J Neurosci. 2009 Oct 7;29(40):12542-57. PubMed PMID: 19812329.

Fenstermaker AG, Prasad AA, Bechara A, Adolfs Y, Tissir F, Goffinet A, Zou Y, Pasterkamp RJ. Wnt/planar cell polarity signaling controls the anterior-posterior organization of monoaminergic axons in the brainstem. J Neurosci. 2010 Nov 24;30(47):16053-64.

Messina A, Ferraris N, Wray S, Cagnoni G, Donohue DE, Casoni F, Kramer PR, Derijck AA, Adolfs Y, Fasolo A, Pasterkamp RJ, Giacobini P. Dysregulation of Semaphorin7A/beta1 integrin signaling leads to defective GnRH-1 cell migration, abnormal gonadal development and altered fertility. Hum Mol Genet. 2011 Dec 15;20(24):4759-74.

WP2, Partner 4 UMH, Prof. Marin:

During this project, we have investigated the migration of mdDA neurons during early phases of their development, with the idea of gaining insights into the possible cellular and molecular mechanisms controlling this process. To this end, we have used real-time videomicroscopy in slices obtained from mouse embryos in which mdDA neurons were labeled with a very bright red fluorescent protein (Shh-Cre;R26R-tdTomato). The results of this analysis suggest that many mdDA neurons change their mode of migration from radial to tangential as they move towards their final destination. These results are relevant from the perspective of understanding the mechanisms controlling the guidance of these neurons, because they indicate that different factors might be involved in controlling each of the different steps in the guidance of mdDA neurons.

Another important goal of the project was to identify the function of several signaling pathways that were thought to be involved in regulating the guidance of mdDA neurons. In particular, we studied the role of three signaling pathways in this process: Cxcl12/Cxcr4, Nrg1/ErbB4 and Slit/Robo. Our results indicate that signaling through the Cxcl12/Cxcr4 and Slit/Robo pathways is directly involved in regulating the guidance of mdDA neurons.

Cxcl12 (also known as Stromal cell-derived factor-1/SDF-1) is a member of the chemokine family, and together with its cognate receptor, Cxcr4, they represent the best-known chemokine ligand/receptor pair. Cxcl12/Cxcr4 signaling has been reported to regulate three essential processes for the establishment of neural networks in different neuronal systems: migration, cell positioning and axon wiring. We have found that Cxcr4 is expressed in Nurr1+ DA precursors and DA neurons, while Cxcl12 is expressed in the meninges surrounding the midbrain. We also have evidence that suggests that meningeal Cxcl12 promotes neuritogenesis in TH+ cells from ventral midbrain explant cultures, an effect that is blocked by the specific Cxcr4 antagonist, AMD3100. Moreover, administration of Cxcl12 in organotypic cultures as well as expression of Cxcl12 by electroporation in vivo enhance neuritogenesis and promote the migration of TH+ DA neurons. Additional gain and loss of function experiments in utero suggest that Cxcl12/Cxcr4 signaling controls the proper allocation of mdDA neurons. Furthermore, we have found that TH+ cells do not complete their radial migration and their cell processes are disoriented in Cxcr4 null mice. Altogether, our findings suggest that Cxcl12/Cxcr4 signaling modulates the initial migration of A9-A10 DA neurons, but it is not required for dopaminergic axons to reach the telencephalon.

The Slit/Robo signaling pathway has been involved in the guidance of multiple populations in the CNS. We observed that mdDA neurons reach their position in mice lacking the Slit receptors Robo1 and Robo2, but their projections to the telencephalon are largely misguided. Specifically, we found that the cerebral cortex of Robo1/2 double mutants only contained a small fraction of the normal complement of dopaminergic axons. We carried out experiments in slice cultures to determine whether the defects observed in Robo1/2 mutants were cell- or non cell-autonomous. To this end, we co-culture midbrain explants from wild type or Robo1/2 double mutants with telencephalic slices from the two phenotypes. We found that the defects observed in Robo1/2 mutants likely arise through a cell-autonomous mechanism. In sum, our experiments suggest that this signaling system is not required for the normal allocation of mdDA neurons, but it is critical for the normal guidance of TH+ axons toward the cortex.

In summary, during this project we have advanced our knowledge on the mechanisms controlling the(Operon, Mouse V2 AROS) final allocation of mdDA neurons as well as their projections to the telencephalon.

WP 2, Partner 7 CNRS, Prof. Prochiantz

Previous research from the laboratory has shown that homeoprotein transcription factors can be secreted and internalized by live cells, thanks to the presence of secretion and internalization sequences, suggesting a new non-cell autonomous mode of action of homeoproteins (Joliot & Prochiantz, Nat. Cell Biol. 6: 189-196, 2004). The biological significance of the intercellular transfer of certain homeoproteins, i.e. Engrailed-1/Engrailed-2 (collectively En1/2), Pax6 and Otx2, has now been demonstrated in axon guidance (Brunet et al., Nature 438: 94-98, 2007), growth cone collapse and retino-tectal patterning (Weizenmann et al., Neuron 64: 355-366, 2009), survival of adult mDA neurons (Sonnier et al., J. Neurosci. 27: 1063-1071, 2007), eye development (Lesaffre et al., Neural Dev. 2 : 1-12, 2007) or visual cortex plasticity (Sugiyama et al., Cell 134: 508-520, 2008). In addition, we have now shown (see ‘Significant results’ below) that exogenous En1/2 is able to protect mDA neurons from cell death induced by MPTP/MPP+ toxins, 6OH-DA and mutated alpha-synuclein, three models of toxicity widely used to generate experimental Parkinson’s disease (PD) models, both in vitro and in vivo (D. Alvarez Fisher, J. Fuchs et al., Nature Neurosci. 14: 1260-1266, 2011).
The major objective of our project in the mdDANeurodev consortium is to understand the mechanisms and pathways involved in the biological activities of homeoproteins. In this context, it is important to recognize that homeoproteins are not only transcription factors but can also regulate mRNA translation. This regulation, first observed for En1/2 action on growth cones guidance (Brunet et al. Nature 438 : 94-98, 2007), probably engages the mTOR pathway as it is accompanied by the phosphorylation of eIF4E (a translation initiation factor) and eIF4E-BP1, two key regulators of protein translation initiation. However, translational target mRNAs remain to be identified. The ability to regulate translation may be a common property of several homeoproteins since many of them, including En1/2, are known to bind eIF4E.

The project, mainly focused on the study of the biological significance of En1/2-mediated regulation of local translation, has 4 objectives in WP2:
1. The mTOR pathway: generation of an En1 translation regulation-deficient mutant
We have extensively worked to generate a mutant En1 protein which would be unable to interact with eIF4E (and thus would loose its ability to regulate translation), but would retain its transcriptional activity ("Transcription Only" mutant).
2. Identification of En1/2 co-factors
We have also attempted to identify potential En1/2 protein partners (other than eIF4E) which could be involved in En1/2 activity by performing GST-En1/2 pull down experiments using midbrain protein extracts. In another context we have shown that En1 works in synergy with EphrinA5, Adenosine and BMP signaling.
3. Search for En1/2 target mRNAs
We have undertaken the identification of potential En1/2 translational targets in growth cones (GC) or midbrain synaptoneurosomes (SNS). Interestingly, a large majority of these targets corresponds to nuclear-encoded mitochondrial genes. The involvement of selected targets in En1/2 activity on mDA neuron survival has been examined.
4. Other aspects: mechanisms of action of other homeoptoteins
In addition, certain aspects concerning non-cell autonomous roles of Otx2 and Pax6 are also being explored, in relation to mDA neuron development and function or cell patterning and migration.

Significant results
1.) We produced several En1 proteins in which putative motifs reminiscent of the canonical eIF4E binding site (YXXXX**? where * is hydrophobic) and non-canonical (DKR) were mutated by site-directed mutagenesis or in which large protein domains (N-ter, middle part or C-ter) were deleted. The interaction of these various mutant En1 proteins with eIF4E was checked using GST-eIF4E pull-down experiments. One of the mutant En1 carrying K313E mutation located at the N-ter part of the homeodomain displayed reduced interaction with eIF4E. Preliminary experiments showed that the En1K313E mutant is unable to up-regulate the translation of selected En1 targets in primary midbrain neuron cultures. Importantly, the En1K313E mutant seems to have retained its transcriptional activity as judged by its ability to inhibit the MAP1b promoter in cotransfected NIH-3T3 cells using a MAP1b-luc reporter construct. Such "Transcription Only" En1 mutant protein represents a unique tool to dissect the specific contribution of En1/2-mediated regulation of translation in axon guidance, growth cone collapse or mDA cell survival. We have now shown that En1K313E mutant is unable to induce growth cone collapse as compared to En1, using mouse and chick retinal explant cultures (Stettler et al., Development 139:215-224, 2112). Although this will not be realized during this period of funding, we will attempt to dissect the phenotypic alterations that such mutant En1 may induce in vivo by knocking En1K313E into the En1 locus (collaboration with Wolfgang Wurst).

2.) We performed GST-En1/2 pull-down experiments using midbrain protein extracts followed by 1D-PAGE analysis and identification of protein bands by mass spectroscopy. The first results obtained by this approach revealed that En1/2 can bind, in addition to eIF4E, several other proteins (i.e., PABP, eEF1a, eEF1Bgamma, mortalin, Hsc70, Elav-like proteins, nucleolin, several ribosomal protein subunits, CKII-alpha, cytoskeleton proteins, ATP synthase beta). Like eIF4E, many of these proteins are involved in the control of mRNA translation or mRNA transport and stability. This is in agreement with the fact that En1/2 regulates translation. Some of these factors such as eEF1a have also been shown to function outside the ribosome in various cellular processes, i.e. in protection against oxidative stress and cell survival. Another interesting En1/2 binding partner is Hsc70 which is suspected to play an important role in the regulation of autophagy in Parkinson’s disease. Interestingly, the interaction of En1/2 with Hsc70 seems to be dependent on the presence of ATP. The specific role of such binding partners in the biological activities of En1/2 are being assessed.
We have also looked at the interaction between En1/2 signaling pathways and other pathways. This has led to the following conclusions. Firstly, in growth cones from retinal ganglion cells (RGCs), En1 synergizes with EphrinA5 and this synergy requires (i) the En1-dependent ATP synthesis, (ii) ATP release and degradation in Adenosine, (iii) the activation of Adenosine receptor 1(A1R). the entire processes is protein-synthesis dependent and takes place in less than 90 seconds ((Stettler et al., Development 139:215-224, 2112). Secondly, in the fly, Engrailed signaling synergizes with the DPP ("fly BMP") signaling pathway (Layalle et al., Development 138: 2315-2323, 2011).

3.) We undertook the search for En1/2 target mRNAs by two different approaches. The first approach consists in stimulating GC or SNS preparations by En1/2 followed by microarray analysis using RNAs extracted from isolated polysomes. The second strategy consists in performing metabolic labeling of GC or SNS fractions in the presence of En1/2 followed by 2D-PAGE analysis and mass spectroscopy. A large majority of En1/2 target mRNAs identified by the second strategy correspond to nuclear-encoded mitochondrial proteins, including mitochondrial complex I subunits, namely Ndufs1 and Ndufs3. We thus examined whether the neuroprotective activity of Engrailed could involve such translational targets, an idea supported by the presence of En1 protein and of its mRNA in their dendrites where no transcription takes place (Di Nardo et al., Mol. Cell. Neurosci. 35 : 230-236, 2007).
Mitochondrial complex I impairment plays a central role in PD pathogenesis. In this respect, complex I subunits were particularly attractive En1/2 translational targets since we showed in a parallel study that exogenous En1/2 protein can protect mDA neurons against cell death induced by MPTP (complex I inhibitor), both in vitro and in vivo. We found that En1/2 can also protect neurons against rotenone, another complex I toxin, but not against complex II inhibitor, 3-NP. This protection by En1/2 was extended to two other mouse models of PD, 6OH-DA and the overexpression of A30P-alpha-synuclein.
We have confirmed that translational upregulation of Ndufs1 by En1/2 is necessary for En1/2-mediated mDA neuron protection in vivo against MPTP by loss-of-function experiment using a selective siRNA. We have also shown that expression of Ndufs1 and Ndufs3 is decreased in mDA neurons of old En1 heterozygous mice, suggesting a possible link between En1/2, complex I activity and mDA neuronal degeneration that has been described in these mice.
In addition, and much to our surprise, we found that Engrailed infused in the midbrain enhances the levels of striatal dopamine and the associated motor activity, establishing, for the first time, an important adult physiological function for a homeoprotein transcription factor in the adult brain. Thus, if Engrailed were to be considered as a putative therapeutic protein, these results suggest that it could not only protect neurons that otherwise would undergo degeneration but might also increase the functional output of the remaining neurons. The work described in this section has been published ((D. Alvarez Fisher, J. Fuchs et al., Nature Neurosci. 14: 1260-1266, 2011).
But En1 is also a transcription factor and we have identified a list of 16 genes regulated in En1 gain of function experiments (infusion) and (in the other direction) in the En1+/- mutant (genetic loss of function). Among these targets we are presently verifying if the down regulation (with siRNAs) of C1q or TNF-R2 antagonizes the pro-survival activity of En1.
We plan to pursue these studies and other targets will be validated and their involvement in En1/2-mediated biological effects (axon guidance, growth cone collapse or mDA neuron survival or physiology) will be examined. We will also attempt to extend our studies concerning En1/2 to examine possible involvement of translational regulation by other homeoproteins in their mechanism of action.

4.) In addition to this work, certain aspects concerning non-cell autonomous roles of Otx2 and Pax6 are also being explored in the laboratory.
For Otx2, recent work in the team has shown that intercellular transfer of Otx2 in the visual cortex requires the presence of perineuronal nets. A consensus GAG-binding motif containing an arginine-lysine (RK) doublet has been identified within Otx2 primary sequence. The RK doublet was thus replaced by AA to generate a mutant protein (Otx2-AA). It was shown that the mutant protein is looses its specificity of entry into its target cells (PV interneurones) in the visual cortex (Beurdeley et al., submitted). The corresponding cDNA was used to generate a Otx2-AA knock-in mouse in collaboration with A. Simeone (within mdDANeurodev consortium). These mice represent an interesting model not only for the study of the role of Otx2 in the visual cortex plasticity but also to examine whether Otx2-AA mice display abnormal development or function of mDA neurons in the VTA or the Substantia nigra. Indeed, it was recently shown that Otx2 can play an important role in selective neuronal vulnerability of mDA neurons. Part of this work is done in collaboration with the group of Antonio Simeone and will soon be submitted for publication.
Finally, we have tested the hypothesis that Pax6 could be a guidance factor on a model simpler than mDA cells, namely the oligodendrocyte precursor cells (OPCs) in the ventral part of the chick neural tube. To that end we have developed in vitro and in vivo models of extracellular Pax6 gain and loss of function and demonstrated tat Pax6 enhances OPC migration (E. Di Lullo, C. Haton et al., Development, 138:4991-5001, 2011). This model will now be applied to mDA growth cones that, during development cross the Pax6-positive diencephalon.

WP 3, Partner 1 UMC Utrecht, Prof. Smidt:

Expression profile of potential Pitx3 downstream targets.
A micro-array analysis of Pitx3 mutants was performed. RNA was isolated from dissected ventral midbrains of E14.5 Pitx3-/- and Pitx3+/+ embryos in a Black6 background. The resulting data provided us with a list of genes that were up or down regulated by the transcription factor Pitx3. The data are part of a larger data set and a manuscript is ready for submission.
The genes that are regulated by Pitx3 have an expression pattern that is restricted to the mdDA system (as hypothesized). Some of the targets are present in sub-sets of mdDA neurons, suggesting that other modulating signals are present that influence the full spectrum of expression in the mdDA system. An important co-regulator was identified as part of parallel research efforts, which is Nurr1. We published on this co-regulation and the activity of Nurr1 in two important papers, closely related to this work but not a direct result of the work performed in the consortium.

Analyze identified Pitx3 targets.
In order to obtain an elaborate view on the molecular alterations caused by Pitx3-deficiency, we performed gene expression analysis on dissected ventral midbrains of E14.5 Pitx3-/- and Pitx3+/+ embryos.
Non-background corrected MAANOVA analysis of microarray data identified 46 transcripts that were differentially expressed between Pitx3+/+ and Pitx3-/- littermate embryos of which 24 and 21 were down- and up-regulated respectively. Background corrected MAANOVA analysis revealed three additional regulated transcripts, two up-regulated and one down-regulated.

The majority of previously described Pitx3 regulated genes like Ahd2(Aldh1a1), Dat(Slc6a3) and Vmat2(Slc18a2) were in the down-regulated list, confirming the validity of the approach. Interestingly, an overlay of the Pitx3 expression array with our previously published Nurr1 data (Jacobs et al., 2009a; Jacobs et al., 2009b) identified Dat, Vmat2 and Dlk1 as target of both Pitx3 and Nurr1. Whereas Dlk1 is down-regulated in Nurr1-deficient embryos, Dlk1 was significantly up-regulated in Pitx3-deficient embryos, in agreement to what was suggested by in situ hybridization before in our lab. Other midbrain expressed genes like En1, En2 and Cck were in the top ten of most up-regulated genes.
To validate our micro-array data and to verify whether the genes were in fact expressed in DA neurons, we FAC-sorted Pitx3gfp/+ and Pitx3gfp/- neurons, isolated RNA and subjected the samples to qPCR analysis. We confirmed down-regulation of Vmat2, Dat and Ahd2. Moreover, we analyzed expression of Th because it was a previously identified Pitx3 / RA target gene and D2R because it was previously described as a target gene of both Nurr1 and Pitx3 and it is known to be regulated by retinoic acid in the mouse striatum. Here, we show an almost 40% down-regulation of Th and an almost threefold down-regulation of D2R transcript levels. Furthermore, we confirmed that Dlk1, Cck, En1 and En2 are up-regulated in Pitx3-deficient dopaminergic neurons.
The direct interaction of Pitx3 with putative promoter elements was studied through CHIP-on-Chip experiments in mn9d cells and in-vivo E14.5 material. The initial IP's were performed by using our in house generated Pitx3 antibody. The data of the CHIP-on-Chip were analyzed through a bioinfomatics package and resulted in the identification of binding positions on the Vmat2 promoter and VIP promoter as well as suspected bindings sites in the Ahd2, Dat, TH and D2R promoter. The binding positions of our earlier identified Ahd2 binding position (Jacobs et al., Development 2007) is not present on the promoter chip used and was therefore not confirmed. The results of the binding experiment were less useful than the initial micro array analysis, but did indicate that direct interaction of Pitx3 to the Vmat2 promoter, in addition to the Ahd2 promoter, may be important for the regulatory effect Pitx3 has on Vmat2.
One of the Pitx3 targets that was up-regulated was En1. We have acquired the En1 ko and are analyzing this animal to study the possible functional interaction of Pitx3 and En1 and to investigate the possible shared target genes between these two homeodomains. Moreover, it is apparent that the up-regulation of CCK , as observed in the Pitx3 mutant, is connected to En1 up-regulation, since we have evidence that En1 is important for the activation of the Cck gene in mdDA neurons.
One other regulated gene was of interest, Dlk1, since this gene seems to be subset specific regulated through Pitx3/RA and we have identified DLK1 in a parallel experiment as being important for the regulation of DAT expression in mdDA neurons. Therefore we are currently investigating the role of this protein in more detail.

RA signaling in the terminal differentiation of mdDA neurons.
Since we have evidence that Pitx3 activates Ahd2 and therefore RA production, most Pitx3 target might in reality be activated through RA signaling. Therefore we compared the expression of Dlk1 in E14.5 Pitx3+/+ and Pitx3-/- embryos to Dlk1 expression in Pitx3-/- embryos supplemented with RA from E11.75-E13.75. In agreement to what was observed in saggital sections of Pitx3-deficient embryos, ISH on coronal sections revealed a rostral expansion of the Dlk1 expression domain in control-treated Pitx3-/- embryos. Strikingly, embryonic RA treatment of Pitx3-/- embryos clearly restricted the expression domain of Dlk1 in the rostral part of the mdDA area (Fig 4C), to a pattern highly similar to WT embryos. To verify whether the Pitx3 / RA-mediated regulation of Dlk1 transcript was followed by a change in level of Dlk1 protein, we performed immunofluorescence on saggital sections of Pitx3gfp/+, Pitx3gfp/gfp and RA-treated Pitx3gfp/gfp embryos. We find that in the absence of Pitx3, Dlk1 protein is up-regulated in the whole mdDA system in Pitx3gfp/gfp embryos and observed a rostral expansion of Pitx3/Dlk1 co-localization in Pitx3gfp/gfp embryos. In agreement with our ISH data, embryonic RA treatment of Pitx3-deficient embryos clearly suppressed the up-regulation of Dlk1 within dopaminergic neurons to a level similar to WT embryos, decreasing expression in the caudal mdDA and restricting the expression domain of Dlk1 in the rostral mdDA area.
Thus, the previously observed RA-induced upregulation of Th expression in the rostral population of Pitx3-deficient mdDA neurons is paralleled by a RA-induced reciprocal downregulation of Dlk1 expression within the same subset of dopaminergic cells, destined to form the SNc. These data are highly suggestive of an important relationship between RA-signaling, Dlk1 and differentiation of SNc neurons into Th-positive neurons. A high level of Dlk1 expression in DA neurons of the SNc is correlated to deficits in terminal differentiation and consequential Th expression, which is in agreement to the suppressive role of Dlk1 in differentiation of neuronal and non-neuronal cell types such as neuroblastoma cells, adipocytes and osteoblasts (Kim, 2010; Enomoto et al., 2004; Abdallah et al., 2004). Altogether, these data highly suggest that in addition to Th, the expression of Dlk1 in mdDA neurons of the SNc is modulated by endogenous Ahd2-mediated RA signaling, and is therefore only indirectly regulated by Pitx3.
In addition to Dlk1, one of the up-regulated transcripts in Pitx3-deficient midbrains is Cck. To determine if Cck expression is, also dependent on Nurr1 we FAC-sorted Nurr1+ /Pitx3(gfp/+) and Nurr1-/Pitx3(gfp/+) neurons, isolated RNA and subjected the samples to qPCR. We found that Cck is massively downregulated in the absence of Nurr1, establishing Cck as a novel Nurr1 target. Thus, as was observed for a number of other Pitx3 target genes (Jacobs et al 2009b), Cck is regulated through the combinatorial action of Nurr1 and Pitx3. Further analysis of the Cck expression domain in E13.5 embryos revealed that Cck is restricted to the most caudal part of the mdDA neuronal population. In Pitx3-deficient embryos, we observed an overall increase of Cck expression and a rostral expansion of the Cck-positive domain, making it to overlap with the Th expression domain. Upregulation of Cck in Pitx3-deficient embryo's is most prominent in the more rostrolateral part of the mdDA system, where Cck is normally not expressed. Importantly, this area of ectopic Cck expression corresponds to the part of the mdDA system where Th-expression is lost in Pitx3-deficient embryo's.
Because the rostral expansion of the Cck domain is strikingly similar to the rostral expansion of the Dlk1 expression domain, we tested if the upregulation of Cck in Pitx3-deficient embryos could be suppressed by RA treatment. In coronal sections of Pitx3-deficient E14.5 embryos the rostral expansion of the Cck expression domain is evident. However, in contrast to what was observed for Dlk1, this expansion could not be suppressed by RA supplementation. To provide a second line of evidence that RA does not play a role in the regulation of Cck we performed qPCR on dissected ventral midbrains cultured with and without RA for Cck and Th as a control. This experiment confirmed earlier data that Th is significantly upregulated by RA treatment, and that Cck is not regulated by RA but is kept restricted to the caudal part of the mdDA area by the actions of Pitx3 through an unknown but RA-independent mechanism.
After our finding that Dlk1 is indirectly suppressed through the Pitx3 /Ahd2/ RA pathway, whereas Cck is not, we continued to analyze if the two highest upregulated genes, En1 and En2, can also be suppressed by RA supplementation. The intense upregulation of the critical mdDA developmental important genes En1 and En2 might hint towards a compensatory effect towards the loss of Pitx3 or an indication of crosstalk between the two homeodomain transcription factors. To analyze the upregulation of En1 and En2 in more detail and study the potential role of RA in their regulation, we performed ISH on E14.5 coronal sections and found that in Pitx3-deficient embryos En1 and En2 are heavily upregulated in the rostral part of the mdDA. However, RA supplementation did not suppress the En1/2 upregulation in Pitx3-deficient embryos.
Dat, Vmat2 and Ahd2 are dopaminergic genes that are down-regulated in Pitx3-deficient embryo's and co-regulated by Nurr1 and Pitx3. In order to test these genes for their potential to be regulated by RA we performed ISH analysis on in vivo RA treated Pitx3-deficient mice, control-treated Pitx3-deficient mice and their WT littermates (Jacobs et al., 2007). Our data confirm that Dat, Vmat2 and Ahd2 are down-regulated in the rostral mdDA area in E14.5 Pitx3-deficient embryos compared to WT embryos. Interestingly, RA treatment was not able to restore Dat, Vmat2 and Ahd2 expression, whereas Th expression was rescued in RA-treated Pitx3-/- embryos compared to age-matched controls.
Whereas the expression of Cck, En1, En2, Dat, Ahd2 and Vmat2 in RA-treated Pitx3-/- embryos was almost indistinguishable from control-treated Pitx3-/- embryos, the expression of D2R was slightly increased in the rostral mdDA area after RA treatment. To validate and quantify the increase in D2R expression, we micro-dissected E13.5 Pitx3gfp/- ventral midbrains, cultured them for 48 hours with or without RA, FAC-sorted gfp-positive mdDA neurons, isolated RNA and subjected the samples to qPCR. These qPCR data validate our ISH data and show that indeed, the expression of D2R expression in Pitx3-deficient mdDA neurons is partly restored by RA treatment . This positions D2R next to Th and Dlk1 as genes that are affected in Pitx3-deficient mdDA neurons and whose expression pattern is partly restored by resupplying RA-signaling to effectively compensate for the loss of Pitx3.
We are in the process to prepare another manuscript that will describe in more detail the mechanistic aspects of RA signaling in mdDA neurons.

Jacobs FM, van der Linden AJ, Wang Y, von Oerthel L, Sul HS, Burbach JP, Smidt MP. Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons. Development. 2009 Jul;136(14):2363-73.

Jacobs FM, van Erp S, van der Linden AJ, von Oerthel L, Burbach JP, Smidt MP. Pitx3 potentiates Nurr1 in dopamine neuron terminal differentiation through release of SMRT-mediated repression. Development. 2009 Feb;136(4):531-40.

Jacobs FM, Veenvliet JV, Almirza WH, Hoekstra EJ, von Oerthel L, van der Linden AJ, Neijts R, Koerkamp MG, van Leenen D, Holstege FC, Burbach JP, Smidt MP. Retinoic acid-dependent and -independent gene-regulatory pathways of Pitx3 in meso-diencephalic dopaminergic neurons. Development. 2011 Dec;138(23):5213-22.PMID:22069189
Koushik Chakrabarty*, Lars Von Oerthel*, Anita Hellemons, Frédéric Clotman, Marian Groot Koerkamp, Frank C.P. Holstege, R. Jeroen Pasterkamp* and Marten P. Smidt* Genome wide expression profiling of the mesodiencephalic region identifies novel factors involved in early and late dopaminergic development. Biology Open in press.

WP 3, Partner 2 Karolinska Institutet, Prof. Perlmann:

Progress towards objectives:
We have generated two mouse strains containing loss of function alleles of Lmx1a. The first strain is knockout with the EGFP coding sequence inserted in the Lmx1a locus. The second strain is designed to allow for conditional Lmx1a gene targeting by inserting LoxP sites flanking essential exons in the Lmx1a gene. Both strains have been generated. The consequences of knockout during embryonic development and also after conditional ablation at late development and at postnatal stages have been analyzed. These analyses have also included work on the Dreher mouse, a natural mouse mutant harboring a Lmx1a null mutation and knockout mice derived by R. Johnson. Individual knockout embryos and embryos with a combined knockout of both Lmx1a and Lmx1b have been analyzed. Various stages of mouse embryogenesis have been analyzed by investigating the expression of known markers, the morphology of the ventral midbrain and cell proliferation.
We have performed gene arrays from ES cells overexpressing Lmx1a under the Nestin promoter. In these experiments mRNA has been collected at different time-points and compared to wildtype ES cells differentiation. We have also generated cell lines expressing other transcription factors allowing the efficient generation of viceral motor neurons, somatic motor neurons and serotonergic neurons in addition to dopamine neurons. These cell types have also been included in gene expression analyses by gene arrays. The gene array data has been validated by comprehensive in situ hybridization in mouse embryos. More specifically, gene lists representing genes that are scored as expressed specifically in distinct neuronal lineages were derived from bioinformatic analysis of the gene array data. For the validation, random genes were selected for generation of riboprobes and expression patterns were analyzed. Over 400 probes in total were tested in this analysis. Several of the genes that were shown to be expressed selectively in dopamine neurons have been further analyzed in several ways. We have investigated how genes are regulated in different mouse models, including Lmx1a- and Nurr1 knockout mice. We have also selected two of the genes for further functional analysis since they are likely to play roles in dopamine neuron development downstream of Lmx1a.

Target gene analysis:
The ES cells with Nestin-driven Lmx1a are being used to search for target genes by chromatin immunoprecipitation (ChIP) experiments. Antibodies recognizing Lmx1a and Lmx1b have been generated and found suitable for ChIP studies. We are now beginning to use them in dopamine neurons derived from Nestin-Lmx1a ES cells. These studies are currently in progress.

Significant results:
Lmx1a-EGFP mice have been verified and found to express EGFP in the expected pattern in vivo. Fluorescence is very strong in the ventral midbrain and in neurons extending from the mesencephalic dopamine neurons. Comprehensive phenotypic analysis has been completed. Floxed Lmx1a mice have been verified by 1) making sure that the homozygous non-floxed animals are viable and appear normal and 2) by crossing to deleter strain thus generating null embryos. These studies have verified that knockout embryos display the expected phenotype resembling that of Dreher in the cerebellum and dorsal neural tube. We have found that in the generation of dopamine neurons, Lmx1a and Lmx1b have both unique and redundant functions. It has not been possible to combine Lmx1a knockout in a null Lmx1b knockout background as these latter embryos are too severely affected also in regions outside of the domain from dopamine neurons are generated. However, we have noted that in Lmx1b heterozygous embryos, in which dopamine neuron generation is not affected, knockout of either one or two Lmx1a alleles leads to a greately diminished generation of dopamine neurons. Thus, we have concluded that Lmx1a and Lmx1b are redundant and together essential for the specification of all dopamine neurons. These results have been published (Deng et al., 2011, Development, 138, 3399). In addition, we also demonstrated that Lmx1a and Lmx1b have unique functions in dopamine neuron progenitors. Loss of Lmx1a leads to a loss of more medial early dopamine neurons while Lmx1b knockout seems to affect early generation of more lateral dopamine neurons. These defects seem to be compensated for at later stages of development but a slight decrease in medial dopamine neurons was noted also in postnatal stages in Lmx1a knockout mice. Our data indicates that Lmx1a is required for inducing midbrain floorplate cells to become neurogenic progenitors, in part by regulating the Notch signaling pathway (Deng et al., 2011, Development, 138, 3399). An important outcome of these studies were also results that further explain the patterning of the ventral midbrain, i.e. the region from which dopamine neurons are generated. We found that Lmx1b, in addition to its role in dopamine neuron progenitors is also important for regulating the temporal switch in generation of first viceral motor neurons and then red nucleus neurons. Importantly, we could demonstrate that this regulatory switch depended on the ability of Lmx1b to control the expression of Phox2a in viceral motor neuron/red nucleus neuron progenitors (Deng et al., 2011, Development, 138, 3399).

The transcriptome analyses from ES cells differentiating into dopamine neurons and other cell types resulted in the identification of a large number of genes with putative functions in dopamine neurons. The gene arrays and in situ analysis has now been reported in Panman et al., 2011 (Cell Stem Cell, 2011, 8, 663). The collected data has provided a rich source of new dopamine neuron markers expressed in early developing neurons (Panman et al., Cell Stem Cell, 2011, 8, 663). Moreover, this study has also resulted in a generalized strategy for the generation of specific neuron types from ES cells (Panman et al., Cell Stem Cell, 2011, 8, 663). Some of the genes have been studied in further experiments. We have identified transcription factor expression patterns, including that of HMG-domain transcription factor. We are currently investigating this gene as a new regulatory component in the dopamine neuron transcription factor network and we believe it has important functions in the generation of dopamine neuron subpopulations. These analysis also include studies of the HMG-domain transcription factor knockout embryos. In our proposal, we also suggested to use Lmx1a-egfp mouse knockout embryos to look for target genes. These studies are ongoing and we anticipate that we will soon be able to analyze gene expression patterns from these animals (see also below, reasons for deviation).

WP 3, Partner 5 CEINGE, Prof. Simeone:

Otx2 expression in mdDA progenitors and neurons
Otx2 is expressed in all mdDA progenitors from their initial specification at around E8. This is based on detailed immunohistochemistry experiments and cell fate analysis by using the Otx2CreER deleter strain we generated to this aim and analyzed in the second part of the project. Collectively, our data have revealed that i) Otx2 is expressed in all mdDA progenitors by fully co-localizing with the expression of Lmx1a, Shh, Lmx1b; ii) in adult mdDA neurons Otx2 is expressed only in a large fraction of VTA neurons being excluded from those of the SNpc; iii) in the VTA Otx2 is prevalently expressed in TH+ neurons co-expressing Calbindin (Calb) and/or Ahd2 and excluded from most of those expressing Girk2 and high levels of the glycosilated form of the Dopamine Transporter (glyco-Dat), iv) cell-fate analysis by using the Otx2CreER deleter allele confirmed that Otx2 is expressed in all mdDA progenitors: including those fated to generate VTA neurons expressing G irk2 and those generating SNpc neurons. This has suggested the existence of a regulatory control excluding Otx2 from post-mitotic neurons of the SNpc and the Girk2+ neuronal subtype of the VTA.

The role of Otx2 in mdDA progenitors and their post-mitotic progeny
Otx2 expression analysis indicated that Otx2 is expressed in all mdDA progenitors. Cell fate experiments have confirmed this finding showing that at E8.0, E9.0 and E10, Otx2+ mdDA progenitors were fated to generate all the VTA and SNpc neuronal subtypes. We have studied a mouse model where Otx2 was inactivated by a ShhCre deleter and not by a ShhCreER allele (see Reasons for deviations). This analysis has confirmed and reinforced previous findings indicating that Otx2 play a crucial role in the activation / maintenance of the molecular patway controlling mesencephalic DA (mesDA) neurogenesis. Indeed in ShhCre/+; Otx2flox/flox mutant embryos, Lmx1a, Msx1 and Ngn2 are down-regulated; furthermore late progenitors (Nurr1+) and early post-mitotic neurons (Nurr1+-Pitx3+) were heavily diminished in number. Analysis of the proliferating activity indicated a severe reduction in the number of BrdU+ progenitors. Similarly we also analyzed apoptotic cell-death by using the activate Caspase3 antibody. Compared to control embryos, no significant variations were observed in mutants.
A further aspect based on the Otx2 expression analysis and cell fate experiments, was to understand whether Otx2 is required in mdDA progenitors for the generation of all VTA neuronal subtypes and SNpc neurons.
In particular, we asked whether the strong impairment observed in mdDA progenitors, particularly in those located in the mesencephalon, affected the generation of all mdDA neuronal subtypes or a specific subset.
We found that in En1Cre/+; Otx2flox/flox adult mice the number of SNpc and VTA neurons is severely affected (mainly VTA neurons). However while the expression code 1-58of SNpc neurons analyzed with Girk2, glyco-Dat, Ahd2 and Calb revealed no significant variations in the percentage of TH+ neurons expressing these markers, virtually all VTA neurons expressed Girk2 and glyco-Dat and very few of them Calb and/or Ahd2. Furthermore, Girk2+ neurons were detected approximately in the same position where they are normally located.
Analysis of E13.5 En1Cre/+; Otx2GFP/flox showed that only few of the Pitx3+ mdDA post-mitotic neurons were GFP+ (Otx2), thus suggesting that Otx2+ progenitors fated to generate TH+-Girk2--Otx2+ VTA neurons were selectively impaired in their neurogenesis. In sum these results suggest that Otx2, although expressed in all mesDa progenitors, is prevalently required for the generation of TH+ neuronal subtypes expressing Calb and/or Ahd2.

The role of Otx2 in mdDA neurons
As above reported Otx2 is expressed selectively in a large fraction of VTA neurons. In particular, we discovered that Otx2 was expressed in TH+ neurons that were Ahd2+-Calbindin+ or in those only Calbindin+, while it is excluded from most of those expressing Girk2 in the dorsal-lateral VTA (Di Salvio et al., 2010).
Based on these data, we studied the consequence of Otx2 inactivation and over-expression in progenitors and post-mitotic mdDA neurons.
To asses the role of Otx2 in adult mdDA neurons, we inactivated or over-expressed Otx2 selectively in mature mdDA neurons by using the DatIresCre deleter strain.
Lack of Otx2 induces in neurons of the central VTA the expression of Girk2, which is prevalently confined to neurons of the dorsal-lateral VTA. Consistent with this finding, robust activation of Otx2 also in the Otx2- fraction of VTA neurons generates a relevant reduction in the number of TH+ neurons expressing high level of Girk2. This suggests that Otx2 is cell-autonomously required in neurons of the central VTA to antagonize identity features of the dorsal-lateral VTA. Noteworthy, a major role for Otx2 in embryogenesis is to define the border between adjacent territories or compartments as in the case of the midbrain-hindbrain border or in the ventral midbrain between progenitors of the mesDA and red nucleus compartments. In these processes, Otx2 operates as a repressing factor by interacting with co-repressing molecules such as Grg4. Based also on these previous findings, our data are not in contrast with the possibility that VTA neurons are distributed in compartment-like subregions whose identity is maintained by a molecular code of transcription factors together with positive and negative interacting co-factors. Our data supports the possibility that Otx2 may represent one of these factors whose competence appeared limited to define the identity of central VTA and/or presumptive border between central and dorsal-lateral VTA. Based on this first evidence, we analyzed whether Otx2 may also be involved in controlling relevant aspects determining the functioning of VTA neurons. To this aim we analyzed the distribution of glyco-Dat, which is required for the uptake of both DA and DA analogue toxin MPTP. Interestingly, a rather extensive complementary expression between Otx2 and glyco-Dat was observed in wild-type mice; in contrast, lack of Otx2 resulted in an increase of GFP+ neurons expressing high level of glyco-Dat. This suggests that Otx2 may have a relevant role on DA signalling by suppressing excessive DA uptake in Otx2+ neurons of the central VTA, thus confining those with efficient DA uptake to the dorsal-lateral VTA. Through this control, Otx2 might likely contribute to define the VTA map of DA uptake. Consistent with this interpretation, ectopic expression of Otx2 in dorsal VTA and SNpc generates a severe reduction in the number of neurons expressing high level of glyco-Dat. At the molecular level, our data indicate that Otx2 modulates glyco-Dat levels by controlling negatively the level of Dat mRNA rather than the glycosylation process.
A relevant consequence of Otx2 ablation in the VTA is an increased vulnerability to the MPTP toxin likely due to a higher level of glyco-Dat and consequent increase of MPTP internalization. Consistent with this, mice lacking Otx2 and exposed to MPTP treatment show a significant and specific loss of GFP+-TH+ neurons, which in control mice exhibit high resistance to MPTP toxicity. In contrast, the Otx2- population of VTA neurons shows in control and mutant mice a similar and more pronounced sensitivity to MPTP. These findings suggest that also the vulnerability to MPTP, a third phenotypic character, converges towards the existence of at least two major functionally distinct subpopulations of VTA neurons. The molecular basis controlling SNpc and VTA differential vulnerability to MPTP and Parkinsonian neurodegeneration represents a yet unsolved key issue. Interestingly, the MPTP vulnerability exhibited by VTA neurons of mice lacking Otx2 is comparable to that of normal SNpc neurons, and robust ectopic expression of Otx2 in SNpc generates high resistance of these neurons to MPTP. Our findings suggest that Otx2 is responsible, at least in part, for SNpc and VTA differential vulnerability to MPTP-induced neurodegeneration and indicate that Otx2 may cell-autonomously provide SNpc neurons with neuroprotection to MPTP.
Our findings also suggest the possibility that factors other than Otx2 are required for maintenance and/or induction of SNpc neuronal identity. In this context, the fact that SNpc neurons are not responsive to Otx2 is similar to findings previously reported for pretectal SNpc and mesencephalic VTA progenitors both expressing Otx2 but with only those of the VTA responding to gain or loss of Otx2.
In summary we have shown that Otx2 is a post-mitotic selector of the VTA neuronal subtype identity; may limit the number of VTA neurons with efficient dopamine uptake, and may confer resistance to the MPTP neurotoxin if over-expressed ubiquitously in the VTA or ectopically in the SNpc (Di Salvio et al., 2010b).

Otx2 ectopic expression in the floor plate region of hindbrain and spinal cord.
Previous data have indicated that i) Wnt1 ectopic expression in the floor plate region posterior to the isthmic organizer was sufficient to induce Otx2 and mdDA neurogenesis (Prakash et al., 2006); and ii) Otx2 ectopic activation was sufficient to confer mdDA neurogenic activity to the floor plate region posterior to the isthmic organizer (Ono et al., 2007). We aim at assessing how far from the isthmic organizer the Otx2 ectopic activation was sufficient to confer mdDA neurogenic activity to the floor plate region. By using the ShhCre mutant allele, we found that Otx2 was correctly and quite efficiently activated along all the hindbrain and spinal cord floor plate. Analysis with markers for mdDA progenitors (Lmx1a and Lmx1b), markers mdDA late progenitors (Ngn2 and Nurr1), and early and mature mdDA neurons (Nurr1, Pitx3 and TH) has revaled that Otx2-mediated mdDA neurogenic activity extends in E12.5 embryos up to the anterior region of the presumptive bulb and therefore includes all the pons. More posteriorly and along the spinal cord Otx2 activation was not sufficient to induce mdDA neurogenesis. Thus these data suggest that the responding ability of the floor plate region posterior to the isthmic organizer is not uniform but depends on the distance from the isthmic organizer, suggesting a distance-dependent effect of its mdDA inducing activity or, alternatively, a decreasing competence of the floor plate region in responding to Otx2 activation.

Potential Impact:
The potential impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results of the FP7 mdDANeurodev (222999) project ‘Molecular coding and subset specification of dopamine neurons generating the meso-limbic and nigro-stratal system’.

Psychiatric diseases and neurodegenerative diseases as Parkinson's have a great impact on our society. With the increase of the complexity of our society and the increase in mean age, diseases that hamper the cognitive function are having more and more impact.
The current treatment paradigms are collectively based on suppressing symptoms. To make the step towards understanding the disease itself and treating the disease and its onset, new experimental data are crucial. Looking at the last decade enormous progress has been made by the consortium members and others to provide new data on molecular players that are involved in the generation and function of the neuronal cell groups that are directly and indirectly involved in the above-mentioned diseases. All consortium members are experts in this field and complement each other in order to expand the area of expertise essential to address the multidisciplinary approach.

Overall the project has been extremely successful on all aims and deliverables are achieved within the project time. Moreover, the consortium members are certain that the achievements from this project will fulfill the socio-economic need for basic knowledge of complex diseases to generate basic knowledge essential to perform translational biology in the process of the development of novel medication.

The work of the mdDANeurodev consortium has been disseminated towards the general public, the scientific community as well as specific patients groups, respectively through the world wide web, scientific publications, (inter)national scientific meetings as well as specialized meetings with the German and UK Parkinson's patient community.

Specific project actions presented by the separate partners:

WP 1, Partner 6 HMGU, Prof. Wurst:

We have successfully identified and dissected two Wnt1/B-catenin-controlled gene regulatory networks conferring subtype-specific characteristics and/or properties to the mdDA neurons in the murine VM during the mdDANEURODEV funding period. This work has resulted in the publication of 7 peer-reviewed articles, and in 2 articles that are currently being prepared for submission.

All PIs presented their work related to the mdDANeurodev consortium at national and international scientific meetings. See for their dissemination activities the dissemination table of the final report. Also, a close HMGU cooperation exists with the following national and international projects:
-EUCOMM - The European Conditional Mouse Mutagenesis Program
-EuTRACC – The European Transcriptome, Regulome and Cellular Commitment Consortium
-NGFNPLUS Consortium “Functional Genomics of Parkinson’s Disease”
-NGFNPLUS Consortium “Disease Genes to Protein Pathways (DiGtoP)”

Pitx3 Is a Critical Mediator of GDNF-Induced BDNF Expression in Nigrostriatal Dopaminergic Neurons.
Peng C, Aron L, Klein R, Li M, Wurst W, Prakash N, Le W.
J Neurosci. 2011 Sep 7;31(36):12802-12815. PMID: 21900559

Fgf15-mediated control of neurogenic and proneural gene expression regulates dorsal midbrain neurogenesis.
Fischer T, Faus-Kessler T, Welzl G, Simeone A, Wurst W, Prakash N.
Dev Biol. 2011 Feb 15;350(2):496-510. Epub 2010 Dec 21. PMID: 21172336

Otx2 controls neuron subtype identity in ventral tegmental area and antagonizes vulnerability to MPTP.
Di Salvio M, Di Giovannantonio LG, Acampora D, Prosperi R, Omodei D, Prakash N, Wurst W, Simeone A.
Nat Neurosci. 2010 Dec;13(12):1481-8. Epub 2010 Nov 7. PMID: 21057506

Ectopic dopaminergic progenitor cells from En1(+/Otx2lacZ) transgenic mice survive and functionally reinnervate the striatum following transplantation in a rat model of Parkinson's disease.
Hackl C, Papazoglou A, Ganser C, Klein A, Prakash N, Wurst W, Nikkhah G.
Cell Transplant. 2010 May 4. [Epub ahead of print]. PMID: 20447348

Spatial analysis of expression patterns predicts genetic interactions at the mid-hindbrain boundary.
Wittmann DM, Blöchl F, Trümbach D, Wurst W, Prakash N, Theis FJ.
PLoS Comput Biol. 2009 Nov;5(11):e1000569. Epub 2009 Nov 20. PMID: 19936059

Fzd3 and Fzd6 deficiency results in a severe midbrain morphogenesis defect.
Stuebner S, Faus-Kessler T, Fischer T, Wurst W, Prakash N.
Dev Dyn. 2010 Jan;239(1):246-60. PMID: 19842188

Delayed dopaminergic neuron differentiation in Lrp6 mutant mice.
Castelo-Branco G, Andersson ER, Minina E, Sousa KM, Ribeiro D, Kokubu C, Imai K, Prakash N, Wurst W, Arenas E.
Dev Dyn. 2010 Jan;239(1):211-21. PMID: 19795519

WP 1, Partner 3 ALU-Fr, Prof. Driever:

The results of the project have identified evolutionary conserved molecular mechanisms of dopaminergic neuron specification and differentiation. The data will motivate new experimental approaches in mammalian model systems, and give new impulses for the control of dopaminergic differentiation as well as for the integration of dopaminergic neurons in the striatum. The results have been published in three high ranking publications, and four additional manuscripts are under review or in preparation.

Publications with reference to the contract:
DeltaA/DeltaD regulate multiple and temporally distinct phases of Notch signaling during dopaminergic neurogenesis in zebrafish.
Mahler, M., Filippi, A., Driever, W., 2010. J Neurosci. 30, 16621-635.

Comprehensive catecholaminergic projectome analysis reveals single-neuron integration of zebrafish ascending and descending dopaminergic systems.
Tay, T.L., Ronneberger, O., Ryu, S., Nitschke, R., Driever, W., 2011. Nature Communications. 2, 171.

Virtual Brain Explorer: An Anatomical Atlas and Imaging Framework for 3D High Resolution Coexpression Analysis in the Larval Zebrafish Brain.
O. Ronneberger, K. Liu, M. Rath, D. Rueß, T. Mueller, H. Skibbe, B. Drayer, T. Schmidt, A. Filippi, R. Nitschke, T. Brox, H. Burkhardt, and W. Driever: Nature Methods, in press.

Dissemination at conferences:
-Ringberg Symposium on Neurodegeneration in Zebrafish, Ringberg/Tegernsee, Germany, February 21-24, 2010
-Systems Biology of Development EMBO Workshop, Ascona, Switzerland, August 16-20, 2010
-FRIAS LIFENET Workshop "Perspectives in Developmental Neurosciences"
October 5 - 6, 2010, Freiburg (Joint Workshop of ALU/FRIAS and HU Jerusalem)
-Imaging Structure and Function in the Zebrafish Brain (Gulbenkian Foundation),
Lisbon, Portugal, December 13-15, 2010
-EU FP7 mdDAneurodev Conference: "Systems Biology of Dopaminergic Neurons",
April 28, 2011, Freiburg.
-Janelia Conference: Control of neuronal Identity (HHMI/Janelia Farms) Ashburn,
USA, October 9-12, 2011
-Neurex Workshop "Plasticity in the dopaminergic system and in Parkinson's disease"
Freiburg University Hospital, October 18, 2011
-Neurological and psychiatric diseases: a developmental perspective. College de France, Paris. March 15-16, 2012
-2nd EZPM, Karlsruhe Institute of Technology, Karlsruhe, March 21-23, 2012

Dissemination at seminar presentations:
-Instituto de Neurociencias de Alicante, Alicante, Spain 29. February 2009
-University of Helsinki Medical School, June 9, 2009
-Institut de Neurobiologie A. Fessard, CNRS, Gif-sur-Yvette / Paris, April 1, 2011
-SFB 629 University of Münster, Münster, Germany, 30. June 3011
-University Medical Center, Utrecht, NL, March 29, 2012

WP2, Partner 1 UMC Utrecht, Pasterkamp, PhD:

Knowledge of the normal development of mdDA pathways is important for understanding situations of perturbed connectivity (e.g. Parkinson's disease, schizophrenia). Furthermore, the molecular cues identified (semaphorins, Wnts) will help in reconstructing mdDA circuits for example in cell transplantation approaches in Parkinson's disease. The work has been presented at several consortium meetings as well as published in peer review journals. In addition, the work has been presented towards patient organizations as the German PD foundation.

Dysregulation of Semaphorin7A/{beta}1-integrin signaling leads to defective GnRH-1 cell migration, abnormal gonadal development and altered fertility.
Messina A, Ferraris N, Wray S, Cagnoni G, Donohue DE, Casoni F, Kramer PR, Derijck AA, Adolfs Y, Fasolo A, Pasterkamp RJ, Giacobini P.
Hum Mol Genet. 2011 Sep 16. 20(24):4759-74.PMID: 21903667

Wnt/planar cell polarity signaling controls the anterior-posterior organization of monoaminergic axons in the brainstem.
Fenstermaker AG, Prasad AA, Bechara A, Adolfs Y, Tissir F, Goffinet A, Zou Y, Pasterkamp RJ.
J Neurosci. 2010 Nov 24;30(47):16053-64. PMID: 21106844

WP2, Partner 4 UMH, Prof. Marin:

The results of this project will lead to the publication of three manuscripts, which are currently being finalized and prepared for submission. One of the manuscripts will describe the normal migration of mdDA neurons during embryonic development, using real-time videomicroscopy in slice cultures. The other manuscripts will report the function of Cxcl12/Cxcr4 and Slit/Robo signaling pathways in the guidance of mdDA neurons.

We have also reported preliminary versions of our results at seminars in different Universities and Research Institutions across Europe, including the San Raffaele Scientific Institute (17/1/2011), the Gordon Research Conference of Neurotrophic Factors (Salve Regina University, 5/6/2011), the University of Hannover (25/1/2012) and the University of Navarra - CIMA (10/2/2012). We will also disseminate our findings through our web page (http://in.umh.es/marinlab/).

WP 2, Partner 7 CNRS, Prof. Prochiantz:

Articles with references to the contract:
Engrailed homeoprotein recruits the adenosine A1 receptor to potentiate ephrin A5 function in retinal growth cones.
Stettler O, Joshi RL, Wizenmann A, Reingruber J, Holcman D, Bouillot C, Castagner F, Prochiantz A, Moya KL. Development. 2012 Jan;139(1):215-224.

Engrailed proteins protect mouse midbrain dopaminergic neurons against mitochondrial complex I insults and regulate their physiology.
D. Alvarez-Fisher (co-first), J. Fuchs (co-first), F. Castagner, O. Stettler, O. Massiani-Beaudoin, K.L. Moya, C. Bouillot, W.H. Oertel, A. Lombès, W. Faigle, R.L. Joshi*, A. Hartmann* & A. Prochiantz* (2011). Nature Neurosci., 14, 1260-1266.

Cell non-autonomousfunctions of homeoproteins in neuroprotection in the brain.
R.L. Joshi, R. Torero-Ibad, J. Rheey, F. Castagner, A. Prochiantz, K. Moya. FEBS Lett. 2011, 585(11):1573-8

Engrailed signaling in axon guidance and neuron survival.
J. Fuchs, O. Stettler, D. Alvarez-Fischer, A. Prochiantz, K. L. Moya and R. L. Joshi. Eu. J. N. In press.

Conferences where the work was presented
-International Conference on Innovative Research in Autism. April 15-17, 2009. Tours, France. Keynote lecture.
-3th Intracellular Delivery of Therapeutic Molecules, from Bench to Bedside. August 31th - September 2nd 2009, Montpellier, France. Keynote lecture.
-ARVO International Society for Ocular Cell Biology, September 9-12 2009, Ericeira, Portugal. Keynote lecture.
-How to build the Dopamine System. December 2nd 2009, Utrecht, NL.
-Neurobiology, from molecules to systems. February 8-9 2010, Montpellier, France.
-The 1st Symposium for the Global Research Laboratory (GRL) Program of Korea. February 23rd 2010, Seoul, South-Korea.
-WE-Heraeus Seminar on Biophysics of Membrane-Active Peptides. April 11-14, 2010, Bad Honneff, Germany, Keynote lecture.
-Peptide Vectors and Delivery of Therapeutics. May 19-21, 2011, Tallinn, Lituania.
-Systems Biology of Dopaminergic Neurons. April 28-20, 2011, Freiburg, DE.
-The Plastic Brain. June 8-9, 2011, Basle, CH, Keynote lecture.
-36th FEBS Congress. Biochemistry for Tomorrow’s Medicine, Turin, Italy.
-Chemistry and Biology of Peptides. July 27, Oxford, UK. Keynote lecture.
-HOX and TALES Transcription Factors in Development and Diseases. September 28th-October 1st, 2011, Carry Le Rouet, France, Keynote lecture.

WP 3, Partner 1, Coordinator, UMC Utrecht, Prof. Smidt:

The data uncovered the role of Pitx3 in terminal differentiated neurons and showed that many components of the mdDA transcriptional profile are regulated through a small molecule as retinoic acid (RA).
The data of the work has been published and another paper is in preparation that discusses in more detail the mechanistic aspects of RA signaling in mdDA neurons. All the data is significant to the scientific community, as has been exemplified by the high level of the published manuscript and the high attention it has raised in the field.

Published papers related to the work of the consortium and as a direct result of the consortium:
Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons.
Jacobs FM, van der Linden AJ, Wang Y, von Oerthel L, Sul HS, Burbach JP, Smidt MP. Development. 2009 Jul;136(14):2363-73.

Pitx3 potentiates Nurr1 in dopamine neuron terminal differentiation through release of SMRT-mediated repression.
Jacobs FM, van Erp S, van der Linden AJ, von Oerthel L, Burbach JP, Smidt MP. Development. 2009 Feb;136(4):531-40.

Retinoic acid-dependent and -independent gene-regulatory pathways of Pitx3 in meso-diencephalic dopaminergic neurons.
Jacobs FM, Veenvliet JV, Almirza WH, Hoekstra EJ, von Oerthel L, van der Linden AJ, Neijts R, Koerkamp MG, van Leenen D, Holstege FC, Burbach JP, Smidt MP. Development. 2011 Dec;138(23):5213-22.PMID:22069189

Genome wide expression profiling of the mesodiencephalic region identifies novel factors involved in early and late dopaminergic development.
Koushik Chakrabarty*, Lars Von Oerthel*, Anita Hellemons, Frédéric Clotman, Marian Groot Koerkamp, Frank C.P. Holstege, R. Jeroen Pasterkamp* and Marten P. Smidt* Biology Open in press.

The RA signaling might be of specific interest since it may provide a tool to interfere with mdDA transcriptional profile also in disease states. This is important in terms of the development of new treatment paradigm in the case of dopaminergic dysfunction as is the case with Parkinson's disease.
The work described here may have a significant socioeconomic impact if the follow-up in the direction of drug discovery will be pursued, next to basic mechanistic studies into the cross talk between identified homeodomain proteins as Pitx3 and En1, that together with the orphan nuclear hormone receptor Nurr1 form the basis of subset specific expression of mdDA markers and in that way define subsets of neurons with their own specific vulnerability as described for SNc neurons.

The work as described has been disseminated towards the general public, the scientific community as well as specific patients groups, respectively through the world wide web, scientific publications and specialized meeting with the German Parkinson's patient community as well as the UK Parkinson's community.

WP 3, Partner 2 Karolinska Institutet, Prof. Perlmann:

The results have been published in two research papers: Deng et al., 2011, Development,138, 3399 and in Panman et al., Cell Stem Cell, 2011, 8, 663. The results have also been presented at various scientific conferences and at seminars. The results are currently exploited in further research that is anticipated to help to refine methods for stem cell engineering of dopamine- and other types of clinically important neurons. Efforts are made to develop stem cell-derived neurons as models for disease in vitro.

Specific and integrated roles of Lmx1a, Lmx1b and Phox2a in ventral midbrain development.
Deng Q, Andersson E, Hedlund E, Alekseenko Z, Coppola E, Panman L, Millonig JH, Brunet JF, Ericson J, Perlmann T.
Development. 2011 Aug;138(16):3399-408. Epub 2011 Jul 13. PMID: 21752929

Transcription factor-induced lineage selection of stem-cell-derived neural progenitor cells.
Panman L, Andersson E, Alekseenko Z, Hedlund E, Kee N, Mong J, Uhde CW, Deng Q, Sandberg R, Stanton LW, Ericson J, Perlmann T.
Cell Stem Cell. 2011 Jun 3;8(6):663-75. PMID: 21624811

WP 3, Partner 5 CEINGE, Prof. Simeone:

In this project Partner 5 has studied through the generation of several mouse models the role of the transcription factor Otx2. The main results have indicated that this factor is required for the neurogenesis of mdDA neurons, and that in adult mice it controls neuron subtype identity in the VTA and may confer resistance to the neurotoxin MPTT, which mimics Parkinsonian neurodegeneration to both VTA and SNpc neurons.
These findings have been disseminated by 5 publications on peer reviewed Journals (see Table A1), and several contributions to national and international Congresses, Workshops and invited Seminars (see Table A2). Furthermore these findings have been recognized with the award of an international price (see Table A2). These findings however have a limited exploitation as general advancement of knowledge and require further studies for potential therapeutic approaches.

Otx genes in neurogenesis of mesencephalic dopaminergic neurons.
Simeone A, Puelles E, Omodei D, Acampora D, Di Giovannantonio LG, Di Salvio M, Mancuso P, Tomasetti C.
Dev Neurobiol. 2011 Aug;71(8):665-79. doi: 10.1002/dneu.20877. PMID: 21309083

The role of otx2 in adult mesencephalic-diencephalic dopaminergic neurons.
Simeone A, Di Salvio M, Di Giovannantonio LG, Acampora D, Omodei D, Tomasetti C.
Mol Neurobiol. 2011 Apr;43(2):107-13. Epub 2010 Nov 18. Review.

Fgf15-mediated control of neurogenic and proneural gene expression regulates dorsal midbrain neurogenesis.
Fischer T, Faus-Kessler T, Welzl G, Simeone A, Wurst W, Prakash N.
Dev Biol. 2011 Feb 15;350(2):496-510. Epub 2010 Dec 21. PMID: 21172336

Otx2 controls neuron subtype identity in ventral tegmental area and antagonizes vulnerability to MPTP.
Di Salvio M, Di Giovannantonio LG, Acampora D, Prosperi R, Omodei D, Prakash N, Wurst W, Simeone A.
Nat Neurosci. 2010 Dec;13(12):1481-8. Epub 2010 Nov 7. PMID: 21057506

Otx2 expression is restricted to dopaminergic neurons of the ventral tegmental area in the adult brain.
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List of Websites:
This is the public website address of the FP7 mdDANeurodev (222999) project ‘Molecular coding and subset specification of dopamine neurons generating the meso-limbic and nigro-stratal system’:

www.mddaneurodev.eu

These are the relevant contact details of the FP7 mdDANeurodev (222999) project ‘Molecular coding and subset specification of dopamine neurons generating the meso-limbic and nigro-stratal system’:

Beneficiary number; name; short name; Country; Contact Name:
1) Universitair Medisch Centrum Utrecht; UMCU; NL; Prof. Dr. M.P. Smidt
2) Karolinska Institutet; KI; SE; Prof. Dr. T. Perlmann
3) Albert-Ludwigs-Universitaet Freiburg; Alu-Fr; DE; Prof. Dr. W. Driever
4) Universida Miguel Hernandez de Elche; UMH; SP; Prof. Dr. O. Marin
5) University of Naples; CEINGE; IT; Prof. Dr. A. Simeone
6) Helmholtz Zentrum München Deutsches Forschungszentrum fuer Gesundheit und Umwelt GMBH; HMGU; DE; Prof. Dr. W. Wurst
7) Centre Nationale de la Recherche Scientifique; CNRS; FR; Prof. Dr. A. Prochiantz

Contact details of the Coordinator Beneficiary:
UMC Utrecht, Division of Neuroscience, Department of Neuroscience and Pharmacology.

Contact details of the Research Coordinator, since 2012:
Prof. Dr. Marten P. Smidt, m.p.smidt@uva.nl

Related information

Contact

Marten P. Smidt, (Professor Developmental Neuroscience)
Tel.: +31 88 75 68810
Fax: +31 88 75 69032
E-mail
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