mdDA Neurons Research Articles

Mechanical Nociception Assay in Drosophila Larvae.

The nervous system of animals can sense and respond to noxious stimuli, which include noxious thermal, chemical, or mechanical stimuli, through a process called nociception. Here, we describe a simple behavioral assay to measure mechanically induced nociceptive responses in Drosophila larvae. This assay tests larval mechanosensitivity to noxious force with calibrated von Frey filaments. First, we explain how to construct and calibrate the customizable von Frey filaments that can be used to deliver reproducible stimuli of a defined force or pressure. Next, we describe how to perform the mechanical nociception assay on third-instar larvae. Through comparison of the responses of genotypes of interest, this assay can be useful for investigation of molecular, cellular, and circuit mechanisms of mechanical nociception. At the molecular level, prior studies have identified the importance of sensory ion channels such as Pickpocket/Balboa, Piezo, dTRPA1, and Painless. At the cellular level, the class IV multidendritic arborizing (md-da) neurons are the main mechanical nociceptor neurons of the peripheral system, but class III and class II md-da have been found to also play a role. At the circuit level, studies have shown that mechanical nociception relies on interneurons of the abdominal ganglia that integrate inputs from these various md-da neuron classes.

Roles of Transcription Factors in the Development and Reprogramming of the Dopaminergic Neurons.

The meso-diencephalic dopaminergic (mdDA) neurons regulate various critical processes in the mammalian nervous system, including voluntary movement and a wide range of behaviors such as mood, reward, addiction, and stress. mdDA neuronal loss is linked with one of the most prominent human movement neurological disorders, Parkinson’s disease (PD). How these cells die and regenerate are two of the most hotly debated PD research topics. As for the latter, it has been long known that a series of transcription factors (TFs) involves the development of mdDA neurons, specifying cell types and controlling developmental patterns. In vitro and in vivo, TFs regulate the expression of tyrosine hydroxylase, a dopamine transporter, vesicular monoamine transporter 2, and L-aromatic amino acid decarboxylase, all of which are critical for dopamine synthesis and transport in dopaminergic neurons (DA neurons). In this review, we encapsulate the molecular mechanism of TFs underlying embryonic growth and maturation of mdDA neurons and update achievements on dopaminergic cell therapy dependent on knowledge of TFs in mdDA neuronal development. We believe that a deeper understanding of the extrinsic and intrinsic factors that influence DA neurons’ fate and development in the midbrain could lead to a better strategy for PD cell therapy.

Nkx2.9 Contributes to Mid-Hindbrain Patterning by Regulation of mdDA Neuronal Cell-Fate and Repression of a Hindbrain-Specific Cell-Fate.

Nkx2.9 is a member of the NK homeobox family and resembles Nkx2.2 both in homology and expression pattern. However, while Nkx2.2 is required for development of serotonergic neurons, the role of Nkx2.9 in the mid-hindbrain region is still ill-defined. We have previously shown that Nkx2.9 expression is downregulated upon loss of En1 during development. Here, we determined whether mdDA neurons require Nkx2.9 during their development. We show that Nkx2.9 is strongly expressed in the IsO and in the VZ and SVZ of the embryonic midbrain, and the majority of mdDA neurons expressed Nkx2.9 during their development. Although the expression of Dat and Cck are slightly affected during development, the overall development and cytoarchitecture of TH-expressing neurons is not affected in the adult Nkx2.9-depleted midbrain. Transcriptome analysis at E14.5 indicated that genes involved in mid- and hindbrain development are affected by Nkx2.9-ablation, such as Wnt8b and Tph2. Although the expression of Tph2 extends more rostral into the isthmic area in the Nkx2.9 mutants, the establishment of the IsO is not affected. Taken together, these data point to a minor role for Nkx2.9 in mid-hindbrain patterning by repressing a hindbrain-specific cell-fate in the IsO and by subtle regulation of mdDA neuronal subset specification.

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 Pitx3fl/fl/DATCreERT2 bigenic mice using a tamoxifen inducible CreERT2/loxp gene-targeting system. Pitx3fl/fl/DATCreERT2 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 Pitx3fl/fl/DATCreERT2 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.

Dose-Dependent and Subset-Specific Regulation of Midbrain Dopaminergic Neuron Differentiation by LEF1-Mediated WNT1/b-Catenin Signaling.

The mesodiencephalic dopaminergic (mdDA) neurons, including the nigrostriatal subset that preferentially degenerates in Parkinson’s Disease (PD), strongly depend on an accurately balanced Wingless-type MMTV integration site family member 1 (WNT1)/beta-catenin signaling pathway during their development. Loss of this pathway abolishes the generation of these neurons, whereas excessive WNT1/b-catenin signaling prevents their correct differentiation. The identity of the cells responding to this pathway in the developing mammalian ventral midbrain (VM) as well as the precise progression of WNT/b-catenin action in these cells are still unknown. We show that strong WNT/b-catenin signaling inhibits the differentiation of WNT/b-catenin-responding mdDA progenitors into PITX3+ and TH+ mdDA neurons by repressing the Pitx3 gene in mice. This effect is mediated by RSPO2, a WNT/b-catenin agonist, and lymphoid enhancer binding factor 1 (LEF1), an essential nuclear effector of the WNT/b-catenin pathway, via conserved LEF1/T-cell factor binding sites in the Pitx3 promoter. LEF1 expression is restricted to a caudolateral mdDA progenitor subset that preferentially responds to WNT/b-catenin signaling and gives rise to a fraction of all mdDA neurons. Our data indicate that an attenuation of WNT/b-catenin signaling in mdDA progenitors is essential for their correct differentiation into specific mdDA neuron subsets. This is an important consideration for stem cell-based regenerative therapies and in vitro models of neuropsychiatric diseases.

Acquisition of the Midbrain Dopaminergic Neuronal Identity.

The mesodiencephalic dopaminergic (mdDA) group of neurons comprises molecularly distinct subgroups, of which the substantia nigra (SN) and ventral tegmental area (VTA) are the best known, due to the selective degeneration of the SN during Parkinson’s disease. However, although significant research has been conducted on the molecular build-up of these subsets, much is still unknown about how these subsets develop and which factors are involved in this process. In this review, we aim to describe the life of an mdDA neuron, from specification in the floor plate to differentiation into the different subsets. All mdDA neurons are born in the mesodiencephalic floor plate under the influence of both SHH-signaling, important for floor plate patterning, and WNT-signaling, involved in establishing the progenitor pool and the start of the specification of mdDA neurons. Furthermore, transcription factors, like Ngn2, Ascl1, Lmx1a, and En1, and epigenetic factors, like Ezh2, are important in the correct specification of dopamine (DA) progenitors. Later during development, mdDA neurons are further subdivided into different molecular subsets by, amongst others, Otx2, involved in the specification of subsets in the VTA, and En1, Pitx3, Lmx1a, and WNT-signaling, involved in the specification of subsets in the SN. Interestingly, factors involved in early specification in the floor plate can serve a dual function and can also be involved in subset specification. Besides the mdDA group of neurons, other systems in the embryo contain different subsets, like the immune system. Interestingly, many factors involved in the development of mdDA neurons are similarly involved in immune system development and vice versa. This indicates that similar mechanisms are used in the development of these systems, and that knowledge about the development of the immune system may hold clues for the factors involved in the development of mdDA neurons, which may be used in culture protocols for cell replacement therapies.

Lmx1b Influences Correct Post-mitotic Coding of Mesodiencephalic Dopaminergic Neurons.

The Lim Homeobox transcription factor 1 beta (LMX1b) has been identified as one of the transcription factors important for the development of mesodiencephalic dopaminergic (mdDA) neurons. During early development, Lmx1b is essential for induction and maintenance of the Isthmic Organizer (IsO), and genetic ablation results in the disruption of inductive activity from the IsO and loss of properly differentiated mdDA neurons. To study the downstream targets of Lmx1b without affecting the IsO, we generated a conditional model in which Lmx1b was selectively deleted in Pitx3-expressing cells from embryonic day (E)13 onward. Supporting previous data, no significant changes could be observed in general dopamine (DA) marks, like Th, Pitx3 and Vmat2 at E14.5. However, in depth analysis by means of RNA-sequencing revealed that Lmx1b is important for the mRNA expression level of survival factors En1 and En2 and for the repression of mdDA subset mark Ahd2 during (late) development. Interestingly, the regulation of Ahd2 by Lmx1b was found to be Pitx3 independent, since Pitx3 mRNA levels were not altered in Lmx1b conditional knock-outs (cKOs) and Ahd2 expression was also up-regulated in Lmx1b/Pitx3 double mutants compared to Pitx3 mutants. Further analysis of Lmx1b cKOs showed that post-mitotic deletion of Lmx1b additional leads to a loss of TH+ cells at 3 months age both in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Remarkably, different cell types were affected in the SNc and the VTA. While TH+AHD2+ cells were lost the SNc, TH+AHD2- neurons were affected in the VTA, reflected by a loss of Cck expression, indicating that Lmx1b is important for the survival of a sub-group of mdDA neurons.

EZH2 Influences mdDA Neuronal Differentiation, Maintenance and Survival.

Over the last decade several components have been identified to be differentially expressed in subsets of mesodiencephalic dopaminergic (mdDA) neurons. These differences in molecular profile have been implied to be involved in the selective degeneration of the SNc neurons in Parkinson’s disease. The emergence and maintenance of individual subsets is dependent on different transcriptional programs already present during development. In addition to the influence of transcription factors, recent studies have led to the hypothesis that modifications of histones might also influence the developmental program of neurons. In this study we focus on the histone methyltransferase EZH2 and its role in the development and maintenance of mdDA neurons. We generated two different conditional knock out (cKO) mice; an En1Cre driven cKO, for deletion of Ezh2 in mdDA progenitors and a Pitx3Cre driven cKO, to study the effect of post-mitotic deletion of Ezh2 on mdDA neurons maturation and neuronal survival. During development Ezh2 was found to be important for the generation of the proper amount of TH+ neurons. The loss of neurons primarily affected a rostrolateral population, which is also reflected in the analysis of the subset marks, Ahd2 and Cck. In contrast to early genetic ablation, post-mitotic deletion of Ezh2 did not lead to major developmental defects at E14.5. However, in 6 months old animals Cck was found ectopically in the rostral domain of mdDA neurons and Ahd2 expression was reduced in more mediocaudal positioned cells. In addition, Pitx3Cre driven deletion of Ezh2 led to a progressive loss of TH+ cells in the VTA and these animals display reduced climbing behavior. Together, our data demonstrates that Ezh2 is important for the generation of mdDA neurons during development and that during adult stages Ezh2 is important for the preservation of proper neuronal subset identity and survival.

BMP/SMAD Pathway and the Development of Dopamine Substantia Nigra Neurons.

One of the major hallmarks of Parkinson's disease (PD) is the loss of dopamine release in the striatum resulting from progressive loss of mesodiencephalic dopamine-producing (mdDA) neurons. In PD, the most vulnerable neurons are in the ventral tier of the substantia nigra (SN); mdDA neurons in the

Tcf12 Is Involved in Early Cell-Fate Determination and Subset Specification of Midbrain Dopamine Neurons.

The basic helix-loop-helix (bHLH) protein family has previously been shown to be involved in the development of mesodiencephalic dopaminergic (mdDA) neurons in the murine midbrain. Specifically, Ngn2 and Mash1 are known to have a role in the specification of neural progenitors in the ventricular zone (VZ) of the midbrain towards an mdDA neuronal cell-fate. Furthermore, other members of the bHLH protein family, the E-box factors, are expressed in the developing midbrain and are thought to have a role in neuronal differentiation. Here we show that the E-box factor Tcf12 is implicated in early and late development of mdDA neurons. Tcf12 is expressed in the midbrain and in young TH-expressing mdDA neurons throughout development. Tcf12lox/lox;En1cre/+ embryos, that lose Tcf12 at ~embryonic day (E)9 throughout the En1 expression domain, have a changed spatial expression of Lmx1a and Nurr1 and a consistent loss of rostral TH expression. Expression of the subset marker Ahd2 is initially delayed, but recovers during development, eventually showing an ~10% increase in AHD2-expressing cells at postnatal day (P)30. Tcf12lox/lox;Pitx3cre/+ embryos, that lose Tcf12 at ~E12 in post-mitotic mdDA neurons, show no effect on the amount of TH-expressing neurons in the developing midbrain. However, similar as to Tcf12lox/lox;En1cre/+ embryos, subset specification is delayed during development. Taken together, we have identified Tcf12 as a novel factor in mdDA neuronal development. It serves a dual function; one in early cell-fate commitment of neural progenitors and one late in subset specification.

Expression analyzes of early factors in midbrain differentiation programs

Mesodiencephalic dopaminergic (mdDA) neurons are born in the ventricular zone (VZ) of the midbrain between E10 and E12. Although these neurons all express specific DA markers like Th and Pitx3, they are subdivided into distinct subsets, each depending on a unique set of transcription factors and signaling cascades for their differentiation. How a neural progenitor commits to an mdDA neuronal cell-fate and how the specification into the different subsets is determined remains unclear.To gain more insight into the development and specification of these neurons we have previously conducted a genome-wide expression analysis, in which dissected midbrain material (E10.5-E13.5) was compared to the adult mdDA region (Chakrabarty et al., 2012). In the present study, we have compared the genome-wide expression analysis including PITX3-GFP sorted (E12.5-E15.5) neurons to available expression data to search for genes specifically expressed in the midbrain during early stages of mdDA differentiation. We have divided these genes into 3 groups: (I) genes upregulated throughout differentiation (Mest, NeuroD1, and Tcf12), (II) genes upregulated during early stages of differentiation (Hes5, and Tcf3), and (III) genes upregulated during late stages of differentiation (Enc1).Here, we show the expression profile of these genes in the embryonic midbrain during development and adult stage and compared that to the appearance of mdDA neurons via co-staining for TH. With this analysis we have identified 6 novel factors that may play a role during cell-fate commitment of neural progenitors or later during differentiation of the mdDA group of neurons.

Pitx3 and En1 determine the size and molecular programming of the dopaminergic neuronal pool.

Mesodiencephalic dopaminergic (mdDA) neurons are located in the ventral midbrain. These neurons form the substantia nigra (SNc) and the ventral tegmental area (VTA). Two transcription factors that play important roles in the process of terminal differentiation and subset-specification of mdDA neurons, are paired-like homeodomain transcription factor 3 (Pitx3), and homeobox transcription factor Engrailed 1 (En1). We previously investigated the single Pitx3KO and En1KO and observed important changes in the survival of mdDA neurons of the SNc and VTA as well as altered expression of pivotal rostral- and caudal-markers, Ahd2 and Cck, respectively. To refine our understanding of the regional-specific relationships between En1 and Pitx3 and their (combined) role in the programming mdDA neurons on the rostral-to-caudal axis, we created double En1tm1Alj/tm1Alj;Pitx3gfp/gfp (En1KO;Pitx3GFP/GFP) animals. Here we report, that in absence of En1 and Pitx3, only a limited number of mdDA neurons are present at E14.5. These mdDA neurons have a rudimentary dopaminergic cell fate, as they express Nurr1, Pbx3 and Otx2 but have lost their rostral or caudal subset identity. Furthermore, we report that the expression of Cck depends on En1 expression, while (in contrast) both Pitx3 and En1 are involved in the initiation of Ahd2 expression. Thus we reveal in this manuscript that regulated levels of Pitx3 and En1 control the size and rostral/caudal-identity of the mdDA neuronal population.

Mest/Peg1 Is Essential for the Development and Maintenance of a SNc Neuronal Subset.

Mesodiencephalic dopaminergic (mdDA) neurons originate at the floor plate and floor plate-basal plate boundary of the midbrain ventricular zone. During development mdDA neurons are specified by a unique set of transcription factors and signaling cascades, to form the different molecular subsets of the mdDA neuronal population. In a time series micro-array study performed previously, mesoderm specific transcript (Mest) was found to be one of the most upregulated genes during early mdDA neuronal development. Here, we show that Mest transcript is expressed in the midbrain throughout development and becomes restricted to the substantia nigra (SNc) at late stages. In Mest KO animals mdDA neurons are progressively lost in the adult, mostly affecting the SNc, reflected by a 50% decrease of TH protein and DA release in the striatum and a reduction of climbing behavior. Analysis of Lrp6 KO embryos suggest a subtle opposite phenotype to the Mest KO, hinting toward the possibility that specific loss of mdDA neurons in Mest ablated animals could be due to affected WNT-signaling. Interestingly, the mdDA neuronal region affected by the loss of Mest remains relatively unaffected in Pitx3 mutants, suggesting that both genes are essential for the development and/or maintenance of different mdDA neuronal subsets within the SNc. Overall, the neuroanatomical and phenotypical consequences detected upon the loss of Mest, resemble the loss of SNc neurons and loss of movement control as seen in Parkinson’s Disease (PD), suggesting that the Mest mouse model may be used as a model-system for PD.

Differences in the spatiotemporal expression and epistatic gene regulation of the mesodiencephalic dopaminergic precursor marker PITX3 during chicken and mouse development.

Mesodiencephalic dopaminergic (mdDA) neurons are located in the ventral mesencephalon and caudal diencephalon of all tetrapod species studied so far. They are the most prominent DA neuronal population and are implicated in control and modulation of motor, cognitive and rewarding/affective behaviors. Their degeneration or dysfunction is intimately linked to several neurological and neuropsychiatric human diseases. To gain further insights into their generation, we studied spatiotemporal expression patterns and epistatic interactions in chick embryos of selected marker genes and signaling pathways associated with mdDA neuron development in mouse. We detected striking differences in the expression patterns of the chick orthologs of the mouse mdDA marker genes Pitx3 and Aldh1a1, which suggests important differences between the species in the generation/generating of these cells. We also discovered that the sonic hedgehog signaling pathway is both necessary and sufficient for the induction of ectopic PITX3 expression in chick mesencephalon downstream of WNT9A-induced LMX1a transcription. These aspects of early chicken development resemble the ontogeny of zebrafish diencephalic DA neuronal populations, and suggest a divergence between birds and mammals during evolution.

Otx2 Requires Lmx1b to Control the Development of Mesodiencephalic Dopaminergic Neurons.

Studying the development of mesodiencephalic dopaminergic (mdDA) neurons provides an important basis for better understanding dopamine-associated brain functions and disorders and is critical for establishing cell replacement therapy for Parkinson’s disease. The transcription factors Otx2 and Lmx1b play a key role in the development of mdDA neurons. However, little is known about the genes downstream of Otx2 and Lmx1b in the pathways controlling the formation of mdDA neurons in vivo. Here we report on our investigation of Lmx1b as downstream target of Otx2 in the formation of mdDA neurons. Mouse mutants expressing Otx2 under the control of the En1 promoter (En1 +/Otx2) showed increased Otx2 expression in the mid-hindbrain region, resulting in upregulation of Lmx1b and expansion of mdDA neurons there. In contrast, Lmx1b -/- mice showed decreased expression of Otx2 and impairments in several aspects of mdDA neuronal formation. To study the functional interaction between Otx2 and Lmx1b, we generated compound mutants in which Otx2 expression was restored in mice lacking Lmx1b (En1 +/Otx2;Lmx1b -/-). In these animals Otx2 was not sufficient to rescue any of the aberrations in the formation of mdDA neurons caused by the loss of Lmx1b, but rescued the loss of ocular motor neurons. Gene expression studies in Lmx1b -/- embryos indicated that in these mutants Wnt1, En1 and Fgf8 expression are induced but subsequently lost in the mdDA precursor domain and the mid-hindbrain organizer in a specific, spatio-temporal manner. In summary, we demonstrate that Otx2 critically depends on Lmx1b for the formation of mdDA neurons, but not for the generation of ocular motor neurons. Moreover, our data suggest that Lmx1b precisely maintains the expression pattern of Wnt1, Fgf8 and En1, which are essential for mid-hindbrain organizer function and the formation of mdDA neurons.

Dickkopf 3 Promotes the Differentiation of a Rostrolateral Midbrain Dopaminergic Neuronal Subset In Vivo and from Pluripotent Stem Cells In Vitro in the Mouse.

Wingless-related MMTV integration site 1 (WNT1)/β-catenin signaling plays a crucial role in the generation of mesodiencephalic dopaminergic (mdDA) neurons, including the substantia nigra pars compacta (SNc) subpopulation that preferentially degenerates in Parkinson's disease (PD). However, the precise functions of WNT1/β-catenin signaling in this context remain unknown. Stem cell-based regenerative (transplantation) therapies for PD have not been implemented widely in the clinical context, among other reasons because of the heterogeneity and incomplete differentiation of the transplanted cells. This might result in tumor formation and poor integration of the transplanted cells into the dopaminergic circuitry of the brain. Dickkopf 3 (DKK3) is a secreted glycoprotein implicated in the modulation of WNT/β-catenin signaling. Using mutant mice, primary ventral midbrain cells, and pluripotent stem cells, we show that DKK3 is necessary and sufficient for the correct differentiation of a rostrolateral mdDA neuron subset. Dkk3 transcription in the murine ventral midbrain coincides with the onset of mdDA neurogenesis and is required for the activation and/or maintenance of LMX1A (LIM homeobox transcription factor 1α) and PITX3 (paired-like homeodomain transcription factor 3) expression in the corresponding mdDA precursor subset, without affecting the proliferation or specification of their progenitors. Notably, the treatment of differentiating pluripotent stem cells with recombinant DKK3 and WNT1 proteins also increases the proportion of mdDA neurons with molecular SNc DA cell characteristics in these cultures. The specific effects of DKK3 on the differentiation of rostrolateral mdDA neurons in the murine ventral midbrain, together with its known prosurvival and anti-tumorigenic properties, make it a good candidate for the improvement of regenerative and neuroprotective strategies in the treatment of PD. Significance statement: We show here that Dickkopf 3 (DKK3), a secreted modulator of WNT (Wingless-related MMTV integration site)/β-catenin signaling, is both necessary and sufficient for the proper differentiation and survival of a rostrolateral (parabrachial pigmented nucleus and dorsomedial substantia nigra pars compacta) mesodiencephalic dopaminergic neuron subset, using Dkk3 mutant mice and murine primary ventral midbrain and pluripotent stem cells. The progressive loss of these dopamine-producing mesodiencephalic neurons is a hallmark of human Parkinson's disease, which can up to now not be halted by clinical treatments of this disease. Thus, the soluble DKK3 protein might be a promising new agent for the improvement of current protocols for the directed differentiation of pluripotent and multipotent stem cells into mesodiencephalic dopaminergic neurons and for the promotion of their survival in situ.

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.

Detailed Analysis of the Genetic and Epigenetic Signatures of iPSC-Derived Mesodiencephalic Dopaminergic Neurons

SummaryInduced pluripotent stem cells (iPSCs) hold great promise for in vitro generation of disease-relevant cell types, such as mesodiencephalic dopaminergic (mdDA) neurons involved in Parkinson’s disease. Although iPSC-derived midbrain DA neurons have been generated, detailed genetic and epigenetic characterizations of such neurons are lacking. The goal of this study was to examine the authenticity of iPSC-derived DA neurons obtained by established protocols. We FACS purified mdDA (Pitx3Gfp/+) neurons derived from mouse iPSCs and primary mdDA (Pitx3Gfp/+) neurons to analyze and compare their genetic and epigenetic features. Although iPSC-derived DA neurons largely adopted characteristics of their in vivo counterparts, relevant deviations in global gene expression and DNA methylation were found. Hypermethylated genes, mainly involved in neurodevelopment and basic neuronal functions, consequently showed reduced expression levels. Such abnormalities should be addressed because they might affect unambiguous long-term functionality and hamper the potential of iPSC-derived DA neurons for in vitro disease modeling or cell-based therapy.

Molecular Marker Differences Relate to Developmental Position and Subsets of Mesodiencephalic Dopaminergic Neurons

The development of mesodiencephalic dopaminergic (mdDA) neurons located in the substantia nigra compacta (SNc) and ventral tegmental area (VTA) follow a number of stages marked by distinct events. After preparation of the region by signals that provide induction and patterning, several transcription factors have been identified, which are involved in specifying the neuronal fate of these cells. The specific vulnerability of SNc neurons is thought to root in these specific developmental programs. The present study examines the positions of young postmitotic mdDA neurons to relate developmental position to mdDA subset specific markers. MdDA neurons were mapped relative to the neuromeric domains (prosomeres 1-3 (P1-3), midbrain, and hindbrain) as well as the longitudinal subdivisions (floor plate, basal plate, alar plate), as proposed by the prosomeric model. We found that postmitotic mdDA neurons are located mainly in the floorplate domain and very few in slightly more lateral domains. Moreover, mdDA neurons are present along a large proportion of the anterior/posterior axis extending from the midbrain to P3 in the diencephalon. The specific positions relate to some extent to the presence of specific subset markers as Ahd2. In the adult stage more of such subsets specific expressed genes are present and may represent a molecular map defining molecularly distinct groups 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.