Annemarie J. A. van der Linden

Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3

The mesencephalic dopamine (mesDA) system is involved in the control of movement and behavior. The expression of Pitx3 in the brain is restricted to the mesDA system and the gene is induced relatively late, at E11.5, a time when tyrosine hydroxylase (Th) gene expression is initiated. We show here that, in the Pitx3-deficient aphakia (ak) mouse mutant, the mesDA system is malformed. Owing to the developmental failure of mesDA neurons in the lateral field of the midbrain, mesDA neurons are not found in the SNc and the projections to the caudate putamen are selectively lost. However, Pitx3 is expressed in all mesDA neurons in control animals. Therefore, mesDA neurons react specifically to the loss of Pitx3. Defects of motor control where not seen in the ak mice, suggesting that other neuronal systems compensate for the absence of the nigrostriatal pathway. However, an overall lower activity was observed. The results suggest that Pitx3 is specifically required for the formation of the SNc subfield at the onset of dopaminergic neuron differentiation.

Pitx3 potentiates Nurr1 in dopamine neuron terminal differentiation through release of SMRT-mediated repression

In recent years, the meso-diencephalic dopaminergic (mdDA) neurons have been extensively studied for their association with Parkinsons disease. Thus far, specification of the dopaminergic phenotype of mdDA neurons is largely attributed to the orphan nuclear receptor Nurr1. In this study, we provide evidence for extensive interplay between Nurr1 and the homeobox transcription factor Pitx3 in vivo. Both Nurr1 and Pitx3 interact with the co-repressor PSF and occupy the promoters of Nurr1 target genes in concert. Moreover, in vivo expression analysis reveals that Nurr1 alone is not sufficient to drive the dopaminergic phenotype in mdDA neurons but requires Pitx3 for full activation of target gene expression. In the absence of Pitx3, Nurr1 is kept in a repressed state through interaction with the co-repressor SMRT. Highly resembling the effect of ligand activation of nuclear receptors, recruitment of Pitx3 modulates the Nurr1 transcriptional complex by decreasing the interaction with SMRT, which acts through HDACs to keep promoters in a repressed deacetylated state. Indeed, interference with HDAC-mediated repression in Pitx3-/- embryos efficiently reactivates the expression of Nurr1 target genes, bypassing the necessity for Pitx3. These data position Pitx3 as an essential potentiator of Nurr1 in specifying the dopaminergic phenotype, providing novel insights into mechanisms underlying development of mdDA neurons in vivo, and the programming of stem cells as a future cell replacement therapy for Parkinsons disease.

Retinoic acid counteracts developmental defects in the substantia nigra caused by Pitx3 deficiency.

Selective neuronal loss in the substantia nigra (SNc), as described for Parkinsons disease (PD) in humans and for Pitx3 deficiency in mice, highlights the existence of neuronal subpopulations. As yet unknown subset-specific gene cascades might underlie the observed differences in neuronal vulnerability. We identified a developmental cascade in mice in which Ahd2 (Aldh1a1) is under the transcriptional control of Pitx3. Interestingly, Ahd2 distribution is restricted to a subpopulation of the meso-diencephalic dopaminergic (mdDA) neurons that is affected by Pitx3 deficiency. Ahd2 is involved in the synthesis of retinoic acid (RA), which has a crucial role in neuronal patterning, differentiation and survival in the brain. Most intriguingly, restoring RA signaling in the embryonic mdDA area counteracts the developmental defects caused by Pitx3 deficiency. The number of tyrosine hydroxylase-positive (TH+) neurons was significantly increased after RA treatment in the rostral mdDA region of Pitx3-/- embryos. This effect was specific for the rostral part of the developing mdDA area, and was observed exclusively in Pitx3-/- embryos. The effect of RA treatment during the critical phase was preserved until later in development, and our data suggest that RA is required for the establishment of proper mdDA neuronal identity. This positions Pitx3 centrally in a mdDA developmental cascade linked to RA signaling. Here, we propose a novel mechanism in which RA is involved in mdDA neuronal development and maintenance, providing new insights into subset-specific vulnerability in PD.

Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons.

The orphan nuclear receptor Nurr1 is essential for the development of meso-diencephalic dopamine (mdDA) neurons and is required, together with the homeobox transcription factor Pitx3, for the expression of genes involved in dopamine metabolism. In order to elucidate the molecular mechanisms that underlie the neuronal deficits in Nurr1-/- mice, we performed combined gene expression microarrays and ChIP-on-chip analysis and thereby identified Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in vivo. In line with the previously described cooperativity between Nurr1 and Pitx3, we show that the expression of Ptpru and Klhl1 in mdDA neurons is also dependent on Pitx3. Furthermore, we demonstrate that Nurr1 interacts with the Ptpru promoter directly and requires Pitx3 for full expression of Ptpru in mdDA neurons. By contrast, the expression of Dlk1 is maintained in Pitx3-/- embryos and is even expanded into the rostral part of the mdDA area, suggesting a unique position of Dlk1 in the Nurr1 and Pitx3 transcriptional cascades. Expression analysis in Dlk1-/- embryos reveals that Dlk1 is required to prevent premature expression of Dat in mdDA neuronal precursors as part of the multifaceted process of mdDA neuronal differentiation driven by Nurr1 and Pitx3. Taken together, the involvement of Nurr1 and Pitx3 in the expression of novel target genes involved in important neuronal processes such as neuronal patterning, axon outgrowth and terminal differentiation, opens up new avenues to study the properties of mdDA neurons during development and in neuronal pathology as observed in Parkinsons disease.

Specification of dopaminergic subsets involves interplay of En1 and Pitx3

Mesodiencephalic dopaminergic (mdDA) neurons control locomotion and emotion and are affected in multiple psychiatric and neurodegenerative diseases, including Parkinson’s disease (PD). The homeodomain transcription factor Pitx3 is pivotal in mdDA neuron development and loss of Pitx3 results in programming deficits in a rostrolateral subpopulation of mdDA neurons destined to form the substantia nigra pars compacta (SNc), reminiscent of the specific cell loss observed in PD. We show here that in adult mice in which the gene encoding a second homeoprotein, engrailed 1 (En1), has been deleted, dramatic loss of mdDA neurons and striatal innervation defects were observed, partially reminiscent of defects observed in Pitx3-/- mice. We then continue to reveal developmental crosstalk between En1 and Pitx3 through genome-wide expression analysis. During development, both En1 and Pitx3 are required to induce expression of mdDA genes in the rostrolateral subset destined to form the SNc. By contrast, Pitx3 and En1 reciprocally regulate a separate gene cluster, which includes Cck, demarcating a caudal mdDA subset in wild-type embryos. Whereas En1 is crucial for induction of this caudal phenotype, Pitx3 antagonizes it rostrolaterally. The combinatorial action of En1 and Pitx3 is potentially realized through at least three levels of molecular interaction: (1) influencing each other’s expression level, (2) releasing histone deacetylase-mediated repression of Nurr1 target genes and (3) modulating En1 activity through Pitx3-driven activation of En1 modulatory proteins. These findings show how two crucial mediators of mdDA neuronal development, En1 and Pitx3, interact in dopaminergic subset specification, the importance of which is exemplified by the specific vulnerability of the SNc found in PD.

Retinoic acid-dependent and -independent gene-regulatory pathways of Pitx3 in meso-diencephalic dopaminergic neurons

Development of meso-diencephalic dopamine (mdDA) neurons requires the combined actions of the orphan nuclear receptor Nurr1 and the paired-like homeobox transcription factor Pitx3. Whereas all mdDA neurons require Nurr1 for expression of Th and survival, dependence on Pitx3 is displayed only by the mdDA subpopulation that will form the substantia nigra (SNc). Previously, we have demonstrated that Pitx3–/– embryos lack the expression of the retinoic acid (RA)-generating enzyme Ahd2, which is normally selectively expressed in the Pitx3-dependent DA neurons of the SNc. Restoring RA signaling in Pitx3–/– embryos revealed a selective dependence of SNc neurons on the presence of RA for differentiation into Th-positive neurons and maintenance throughout embryonic development. Whereas these data are suggestive of an important developmental role for RA in neurons of the SNc, it remained unclear whether other Nurr1 and Pitx3 target genes depend on RA signaling in a manner similar to Th. In the search for genes that were affected in Pitx3-deficient mdDA neurons and restored upon embryonic RA treatment, we provide evidence that Delta-like 1, D2R (Drd2) and Th are regulated by Pitx3 and RA signaling, which influences the mdDA terminal differentiated phenotype. Furthermore, we show that regulation of Ahd2-mediated RA signaling represents only one aspect of the Pitx3 downstream cascade, as Vmat2, Dat, Ahd2 (Aldh1a1), En1, En2 and Cck were unaffected by RA treatment and are (subset) specifically modulated by Pitx3. In conclusion, our data reveal several RA-dependent and -independent aspects of the Pitx3-regulated gene cascade, suggesting that Pitx3 acts on multiple levels in the molecular subset-specification of mdDA neurons.

Lmx1a Encodes a Rostral Set of Mesodiencephalic Dopaminergic Neurons Marked by the Wnt/B-Catenin Signaling Activator R-spondin 2

Recent developments in molecular programming of mesodiencephalic dopaminergic (mdDA) neurons have led to the identification of many transcription factors playing a role in mdDA specification. LIM homeodomain transcription factor Lmx1a is essential for chick mdDA development, and for the efficient differentiation of ES-cells towards a dopaminergic phenotype. In this study, we aimed towards a more detailed understanding of the subtle phenotype in Lmx1a-deficient (dreher) mice, by means of gene expression profiling. Transcriptome analysis was performed, to elucidate the exact molecular programming underlying the neuronal deficits after loss of Lmx1a. Subsequent expression analysis on brain sections, confirmed that Nurr1 is regulated by Lmx1a, and additional downstream targets were identified, like Pou4f1, Pbx1, Pitx2, C130021l20Rik, Calb2 and Rspo2. In line with a specific, rostral-lateral (prosomer 2/3) loss of expression of most of these genes during development, Nurr1 and C130021l20Rik were affected in the SNc of the mature mdDA system. Interestingly, this deficit was marked by the complete loss of the Wnt/b-catenin signaling activator Rspo2 in this domain. Subsequent analysis of Rspo2−/− embryos revealed affected mdDA neurons, partially phenocopying the Lmx1a mutant. To conclude, our study revealed that Lmx1a is essential for a rostral-lateral subset of the mdDA neuronal field, where it might serve a critical function in modulating proliferation and differentiation of mdDA progenitors through the regulation of the Wnt activator Rspo2.

Phenotypic segregation of aphakia and Pitx3-null mutants reveals that Pitx3 deficiency increases consolidation of specific movement components

Deficiency of the meso-diencephalic dopamine (mdDA) neuron specific transcription factor Pitx3 in aphakia (ak) mice results in the loss of the substantia nigra compacta (SNc). Concomitantly, reduced spontaneous locomotor behavior, symptoms reminiscent to those in Parkinsons disease, has been reported. However, the ak mouse line originates from the 1960s and has been compared to C57BL/6J inbred controls. Therefore, to define Pitx3 gene function in baseline and novelty-induced locomotor behavior and mdDA neuronal activity, we analyzed Pitx3-deficiency in a controlled genetic and epigenetic background. The analysis implicated that, in contrast to the controversial and previously reported hypo-activity in ak mice, Pitx3-/- mice showed normal dark phase motor activity levels. Our data also revealed that ak and Pitx3-/- mice both display a similar neuro-anatomical and physiological phenotype, and, interestingly, showed increased spontaneous home cage activity levels during their habitual sleep phase. Further behavioral analysis revealed that both ak and Pitx3-/- mice have reduced transitions but increased consolidation of specific locomotor behaviors, such as rearing and horizontal movement. Thus, Pitx3 is not involved in the expression of nighttime motor activity levels, but is critical for selective mdDA neuronal activity and associated with increased consolidation of movement.

Lmx1a is an activator of Rgs4 and Grb10 and is responsible for the correct specification of rostral and medial mdDA neurons

The LIM homeodomain transcription factor Lmx1a is a very potent inducer of stem cells towards dopaminergic neurons. Despite several studies on the function of this gene, the exact in vivo role of Lmx1a in mesodiencephalic dopamine (mdDA) neuronal specification is still not understood. To analyse the genes functioning downstream of Lmx1a, we performed expression microarray analysis of LMX1A‐overexpressing MN9D dopaminergic cells. Several interesting regulated genes were identified, based on their regulation in other previously generated expression arrays and on their expression pattern in the developing mdDA neuronal field. Post analysis through in vivo expression analysis in Lmx1a mouse mutant (dr/dr) embryos demonstrated a clear decrease in expression of the genes Grb10 and Rgs4, in and adjacent to the rostral and dorsal mdDA neuronal field and within the Lmx1a expression domain. Interestingly, the DA marker Vmat2 was significantly up‐regulated as a consequence of increased LMX1A dose, and subsequent analysis on Lmx1a‐mutant E14.5 and adult tissue revealed a significant decrease in Vmat2 expression in mdDA neurons. Taken together, microarray analysis of an LMX1A‐overexpression cell system resulted in the identification of novel direct or indirect downstream targets of Lmx1a in mdDA neurons: Grb10, Rgs4 and Vmat2.

Phox2b influences the development of a caudal dopaminergic subset.

The developing mesodiencephalic dopaminergic (mdDA) neuronal field can be subdivided into several molecularly distinct domains that arise due to spatiotemporally distinct origins of the neurons and distinct transcriptional pathways controlling these neuronal subsets. Two large anatomically and functionally different subdomains are formed that eventually give rise to the SNc and VTA, but more subsets exist which require detailed characterization in order to better understand the development of the functionally different mdDA subsets, and subset-specific vulnerability. In this study, we aimed to characterize the role of transcription factor Phox2b in the development of mdDA neurons. We provide evidence that Phox2b is co-expressed with TH in a dorsal-caudal subset of neurons in the mdDA neuronal field during embryonic development. Moreover, Phox2b transcripts were identified in FAC-sorted Pitx3 positive neurons. Subsequent analysis of Phox2b mutant embryos revealed that in the absence of Phox2b, a decrease of TH expression occurred specifically in the midbrain neuronal subset that normally co-expresses Phox2b with TH. Our data suggest that Phox2b is, next to the known role in the development of the oculomotor complex, involved in the development of a specific caudal mdDA neuronal subset.