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.     

Differences in neurogenic potential in floor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells

Directed differentiation and purification of mesencephalic dopaminergic (mesDA) neurons from stem cells are crucial issues for realizing safe and efficient cell transplantation therapies for Parkinson's disease. Although recent studies have identified the factors that regulate mesDA neuron development, the mechanisms underlying mesDA neuron specification are not fully understood. Recently, it has been suggested that mesencephalic floor plate (FP) cells acquire neural progenitor characteristics to generate mesDA neurons. Here, we directly examined this in a fate mapping experiment using fluorescence-activated cell sorting (FACS) with an FP cell-specific surface marker, and demonstrate that mesencephalic FP cells have neurogenic activity and generate mesDA neurons in vitro. By contrast, sorted caudal FP cells have no neurogenic potential, as previously thought. Analysis of dreher mutant mice carrying a mutation in the Lmx1a locus and transgenic mice ectopically expressing Otx2 in caudal FP cells demonstrated that Otx2 determines anterior identity that confers neurogenic activity to FP cells and specifies a mesDA fate, at least in part through the induction of Lmx1a. We further show that FACS can isolate mesDA progenitors, a suitable transplantation material, from embryonic stem cell-derived neural cells. Our data provide insights into the mechanisms of specification and generation of mesDA neurons, and illustrate a useful cell replacement approach for Parkinson's disease.     

Ngn2 and Nurr1 act in synergy to induce midbrain dopaminergic neurons from expanded neural stem and progenitor cells

Parkinson's Disease (PD) is a debilitating motor function disorder due primarily to a loss of midbrain dopaminergic neurons and a subsequent reduction in dopaminergic innervation of the striatum. Several attempts have been made to generate dopaminergic neurons from progenitor cell populations in vitro for potential use in cell replacement therapy for PD. However, expanding cells from fetal brain with retained potential for dopaminergic differentiation has proven to be difficult. In this study, we sought to generate mesencephalic dopaminergic (mesDA) neurons from an expanded population of fetal mouse ventral midbrain (VM) progenitors through the use of retroviral gene delivery. We over-expressed Ngn2 and Nurr1, two genes present in the ventral midbrain and important for normal development of mesDA neurons, in multi-passaged neurosphere-expanded midbrain progenitors. We show that over-expression of Ngn2 in these progenitors results in increased neuronal differentiation but does not promote mesDA formation. We also show that over-expression of Nurr1 alone is sufficient to generate tyrosine hydroxylase (TH) expressing cells with an immature morphology, however the cells do not express any additional markers of mesDA neurons. Over-expression of Nurr1 and Ngn2 in combination generates morphologically mature TH-expressing neurons that also express additional mesencephalic markers.     

Development of the mesencephalic dopaminergic neuron system is compromised in the absence of neurogenin 2

Neurogenin 2 (Ngn2) is a proneural gene involved in neuronal differentiation and subtype specification in various regions of the nervous system. In the ventral midbrain, Ngn2 is expressed in a spatiotemporal pattern that correlates with the generation of mesencephalic dopaminergic (mesDA) neurons. We show here that lack of Ngn2 impairs the development of mesDA neurons, such that less than half of the normal mesDA neuron number remain in Ngn2 mutant mice at postnatal stages. Analysis of Ngn2 mutant mice during mesDA neurogenesis show that medially located precursors are formed but are arrested in their differentiation at a stage when they have not yet acquired the characteristics of mesDA neuron precursors. Loss of Ngn2 function appears to specifically affect the generation of DA neurons, as the development of other types of neurons within the ventral midbrain is unaltered. Ngn2 is the first example of a gene expressed in progenitors in the ventricular zone of the mesDA neuron domain that is essential for proper mesDA neuron differentiation, and whose loss of function causes impaired mesDA neurogenesis without other major abnormalities in the ventral midbrain.     

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.     

中脑多巴胺能神经元的轴突导向及其诱向因子

中脑多巴胺能神经元（mesodiencephalic dopamine,mdDA,neurons）由于涉及帕金森病、精神分裂症和药物成瘾等多种神经疾病的病理过程而历来受到人们的重视。研究中脑多巴胺能神经元的发育机制将给这些疾病的治疗带来希望。近来的研究表明多巴胺能神经元轴突的导向由各种诱向因子决定,诱向因子主要由相应投射部位的细胞所分泌,其中研究得最多的是ephrins,netrins,semaphorins,Slits及它们各自的受体。介绍胚胎期中脑多巴胺能神经元轴突导向过程及其主要诱向因子。     

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.     

A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo

Midbrain neurons synthesizing the neurotransmitter dopamine play a central role in the modulation of different brain functions and are associated with major neurological and psychiatric disorders. Despite the importance of these cells, the molecular mechanisms controlling their development are still poorly understood. The secreted glycoprotein Wnt1 is expressed in close vicinity to developing midbrain dopaminergic neurons. Here, we show that Wnt1 regulates the genetic network, including Otx2 and Nkx2-2, that is required for the establishment of the midbrain dopaminergic progenitor domain during embryonic development. In addition, Wnt1 is required for the terminal differentiation of midbrain dopaminergic neurons at later stages of embryogenesis. These results identify Wnt1 as a key molecule in the development of midbrain dopaminergic neurons in vivo. They also suggest the Wnt1-controlled signaling pathway as a promising target for new therapeutic strategies in the treatment of Parkinson's disease.     

Hes1 regulates the number and anterior-posterior patterning of mesencephalic dopaminergic neurons at the mid/hindbrain boundary (isthmus)

The lack of the Hes1 gene leads to the failure of cranial neurulation due to the premature onset of neural differentiation. Hes1 homozygous null mutant mice displayed a neural tube closure defect, and exencephaly was induced at the mid/hindbrain boundary. In the mutant mesencephalon, the roof plate was not formed and therefore the ventricular zone showing cell proliferation was displaced to the brain surface. Furthermore, the telencephalon and ventral diencephalon were defective. Despite the severe defects of neurogenesis in null mutants, the mesencephalic dopaminergic (mesDA) neurons were specified at the midline of the ventral mesencephalon in close proximity to two important signal centers — floor plate and mid/hindbrain boundary (i.e., the isthmic organizer). Using mesDA neuronal markers, tyrosine hydroxylase (TH) and Pitx3, the development of mesDA neurons was studied in Hes1 null mice and compared with that in the wild type. At early stages, between embryonic day (E) 11.5 and E12.5, mesDA neurons were more numerous in null mutants than in the wild type. From E13.5 onward, however, the cell number and fiber density of mesDA neurons were decreased in the mutants. Their distribution pattern was also different from that of the wild type. In particular, mesDA neurons grew dorsally and invaded the rostral hindbrain. 5-HT neurons were also ectopically located in the mutant midbrain. Thus, the loss of Hes1 resulted in disturbances in the inductive and repulsive activities of the isthmic organizer. It is proposed that Hes1 plays a role in regulating the location and density of mesDA neurons.     

Isolation,Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson’s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.     

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     

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.