The essential role of transcription factor Pitx3 in preventing mesodiencephalic dopaminergic neurodegeneration and maintaining neuronal subtype identities during aging

Pituitary homeobox 3 (Pitx3) is required for the terminal differentiation of nigrostriatal dopaminergic neurons during neuronal development. However, whether Pitx3 contributes to the normal physiological function and cell-type identity of adult neurons remains unknown. To explore the role of Pitx3 in maintaining mature neurons, we selectively deleted Pitx3 in the mesodiencephalic dopaminergic (mdDA) neurons of Pitx3^fl/fl/DAT^CreERT2 bigenic mice using a tamoxifen inducible Cre^ERT2/loxp gene-targeting system. Pitx3^fl/fl/DAT^CreERT2 mice developed age-dependent progressive motor deficits, concomitant with a rapid reduction of striatal dopamine (DA) content and a profound loss of mdDA neurons in the substantia nigra pars compacta (SNc) but not in the adjacent ventral tegmental area (VTA), recapitulating the canonical neuropathological features of Parkinson’s disease (PD). Mechanistic studies showed that Pitx3-deficiency significantly increased the number of cleaved caspase-3⁺ cells in SNc, which likely underwent neurodegeneration. Meanwhile, the vulnerability of SNc mdDA neurons was increased in Pitx3^fl/fl/DAT^CreERT2 mice, as indicated by an early decline in glial cell line-derived neurotrophic factor (GDNF) and aldehyde dehydrogenase 1a1 (Aldh1a1) levels. Noticeably, somatic accumulation of α-synuclein (α-syn) was also significantly increased in the Pitx3-deficient neurons. Together, our data demonstrate that the loss of Pitx3 in fully differentiated mdDA neurons results in progressive neurodegeneration, indicating the importance of the Pitx3 gene in adult neuronal survival. Our findings also suggest that distinct Pitx3-dependent pathways exist in SNc and VTA mdDA neurons, correlating with the differential vulnerability of SNc and VTA mdDA neurons in the absence of Pitx3.Subject terms: Neuroscience, Neurological disorders     

Spatial and temporal lineage analysis of a pitx3-driven cre-recombinase knock-in mouse model

Development and function of mesodiencephalic dopaminergic (mdDA) neurons has received a lot of scientific interest since these neurons are critically involved in neurological diseases as Parkinson and psychiatric diseases as schizophrenia, depression and attention deficit hyperactivity disorder (ADHD). The understanding of the molecular processes that lead to normal development and function of mdDA neurons has provided insight in the pathology and provided critical information on new treatment paradigms. In order to be able to study specific genetic ablation in mdDA neurons a new tools was developed that drives Cre-recombinase under the control of the Pitx3 locus. The Pitx3 gene is well known for its specific expression in mdDA neurons and is present at the onset of terminal differentiation. Analysis of newly generated Pitx3-Cre knock-in mice shows that Cre expression, measured through the activation of eYfp by removal of a "Stop" signal (LoxP-Stop-LoxP-eYfp reporter mouse), is present at the onset of terminal differentiation and mimics closely the native Pitx3 expression domain. In conclusion, we present here a new Cre-driver mouse model to be used in the restricted ablation of interesting genes in mdDA neurons in order to improve our understanding of the underlying molecular programming.     

Pitx3 is required for motor activity and for survival of a subset of midbrain dopaminergic neurons

Mesencephalic dopaminergic (MesDA) neurons play crucial roles in motor and behavioral processes; their loss in Parkinson's disease (PD) results in striatal dopamine (DA) deficiency and hypokinetic movement disorder. The Pitx3 homeobox gene is expressed in the MesDA system. We now show that only a subset of MesDA neurons express Pitx3 and that in Pitx3-deficient aphakia mice, this subset is progressively lost by apoptosis during fetal (substantia nigra, SN) and postnatal (ventral tegmental area) development, resulting in very low striatal DA and akinesia. Similar to human PD, dorsal SN neurons (which are Pitx3 negative) are spared in mutant mice. Thus, Pitx3 defines a pathway for survival of neurons that are implicated in PD and that are required for spontaneous locomotor activity.     

Mesodiencephalic Dopaminergic Neuronal Differentiation Does Not Involve GLI2A-Mediated SHH-Signaling and Is under the Direct Influence of Canonical WNT Signaling

Sonic Hedgehog (SHH) and WNT proteins are key regulators in many developmental processes, like embryonic patterning and brain development. In the brain, SHH is expressed in a gradient starting in the floor plate (FP) progressing ventrally in the midbrain, where it is thought to be involved in the development and specification of mesodiencephalic dopaminergic (mdDA) neurons. GLI2A-mediated SHH-signaling induces the expression of Gli1, which is inhibited when cells start expressing SHH themselves. To determine whether mdDA neurons receive GLI2A-mediated SHH-signaling during differentiation, we used a BAC-transgenic mouse model expressing eGFP under the control of the Gli1 promoter. This mouse-model allowed for mapping of GLI2A-mediated SHH-signaling temporal and spatial in the mouse midbrain. Since mdDA neurons are born from E10.5, peaking at E11.0–E12.0, we examined Gli1-eGFP embryos at E11.5, E12.5, and E13.5, indicating whether Gli1 was induced before or during mdDA development and differentiation. Our data indicate that GLI2A-mediated SHH-signaling is not involved in mdDA neuronal differentiation. However, it appears to be involved in the differentiation of neurons which make up a subset of the red nucleus (RN). In order to detect whether mdDA neuronal differentiation may be under the control of canonical WNT-signaling, we used a transgenic mouse-line expressing LacZ under the influence of stable β-catenin. Here, we show that TH⁺ neurons of the midbrain receive canonical WNT-signaling during differentiation. Therefore, we suggest that early SHH-signaling is indirectly involved in mdDA development through early patterning of the midbrain area, whereas canonical WNT-signaling is directly involved in the differentiation of the mdDA neuronal population.     

Retinoic acid signaling in perioptic mesenchyme represses Wnt signaling via induction of Pitx2 and Dkk2

Morphogenesis during eye development requires retinoic acid (RA) receptors plus RA-synthesizing enzymes, and loss of RA signaling leads to ocular disorders associated with loss of Pitx2 expression in perioptic mesenchyme. Several Wnt signaling components are expressed in ocular tissues during eye development including Dkk2, encoding an inhibitor of Wnt/β-catenin signaling, which was previously shown to be induced by Pitx2 in the perioptic mesenchyme. Here, we investigated potential cross-talk between RA and Wnt signaling during ocular development. Genetic studies using Raldh1/Raldh3 double null mice deficient for ocular RA synthesis demonstrated that Pitx2 and Dkk2 were both down-regulated in perioptic mesenchyme. Chromatin immunoprecipitation and gel mobility shift studies demonstrated the existence of a DR5 RA response element upstream of Pitx2 that binds all three RA receptors in embryonic eye. Axin2, an endogenous readout of Wnt/β-catenin signaling, was up-regulated in cornea and perioptic mesenchyme of RA deficient embryos. Also, expression of Wnt5a was expanded in perioptic mesenchyme of RA deficient eyes. Our findings demonstrate excessive activation of Wnt signaling in the perioptic mesenchyme of RA deficient mice which may be responsible for abnormal development leading to defective optic cup, cornea, and eyelid morphogenesis.     

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.     

Anterior-posterior graded response to Otx2 controls proliferation and differentiation of dopaminergic progenitors in the ventral mesencephalon

Meso-diencephalic dopaminergic (mdDA) neurons control voluntary movement, cognition and the reward response, and their degeneration is associated with Parkinson's disease (PD). Prospective cell transplantation therapies for PD require full knowledge of the developmental pathways that control mdDA neurogenesis. We have previously shown that Otx2 is required for the establishment of the mesencephalic field and molecular code of the entire ventral mesencephalon (VM). Here, we investigate whether Otx2 is a specific determinant of mesencephalic dopaminergic (mesDA) neurogenesis by studying mouse mutants that conditionally overexpress or lack Otx2. Our data show that Otx2 overexpression in the VM causes a dose-dependent and selective increase in both mesDA progenitors and neurons, which correlates with a remarkable and specific enhancement in the proliferating activity of mesDA progenitors. Consistently, lack of Otx2 in the VM specifically affects the proliferation of Sox2+ mesDA progenitors and causes their premature post-mitotic transition. Analysis of the developmental pathway that controls the differentiation of mesDA neurons shows that, in the absence of Otx2, the expression of Lmx1a and Msx1, and the proneural genes Ngn2 and Mash1 is not activated in Sox2+ mesDA progenitors, which largely fail to differentiate into Nurr1+ mesDA precursors. Furthermore, proliferation and differentiation abnormalities exhibit increasing severity along the anterior-posterior (AP) axis of the VM. These findings demonstrate that Otx2, through an AP graded effect, is intrinsically required to control proliferation and differentiation of mesDA progenitors. Thus, our data provide new insights into the mechanism of mesDA neuron specification and suggest Otx2 as a potential target for cell replacement-based therapeutic approaches in PD.     

Beyond the neckless phenotype: influence of reduced retinoic acid signaling on motor neuron development in the zebrafish hindbrain

Retinoic acid (RA) has been identified as a key signal involved in the posteriorization of vertebrate neural ectoderm. The main biosynthetic enzyme responsible for RA signaling in the hindbrain and spinal cord is Raldh2. However, neckless/raldh2-mutant (nls) zebrafish exhibit only mild degrees of anteriorization in the neural ectoderm, compared to full vitamin A deficiency in amniotes and the Raldh2-/- mouse. Here we investigated the role of RA during neuronal development in the zebrafish hindbrain and anterior spinal cord using DEAB, an inhibitor of retinaldehyde dehydrogenases. We show that the nls hindbrain and spinal cord are not fully devoid of RA, since blocking Raldh-mediated RA signaling leads to a more severe hindbrain phenotype than in nls. The anteroposterior distribution of branchiomotor neurons in the facial and more posterior nuclei depends on full RA signaling throughout early and late gastrula stages. In contrast, inhibition of RA synthesis after gastrulation reduces the number of branchiomotor neurons in the vagal nucleus, but has no effect on anteroposterior cell fates. In addition, blockage of RA-mediated signaling not only interferes with the differentiation of branchiomotor neurons and their axons in the hindbrain, but also affects the development of the posterior lateral line nerve.