Desire, Disease, and the Origins of the Dopaminergic System

Abstract

The dopaminergic neurons in the midbrain region of the central nervous system project an extensive network of connections throughout the forebrain, including the neocortex. The midbrain-forebrain dopaminergic circuits are thought to regulate a diverse set of behaviors, from the control of movement to modulation of cognition and desire—because they relate to mood, attention, reward, and addiction. Defects in these pathways, including neurodegeneration, are implicated in a variety of psychiatric and neurological diseases, such as schizophrenia, attention-deficit/hyperactivity disorder, drug addiction, and Parkinson disease. Based on the importance of the midbrain dopaminergic neurons to normal and pathological brain function, there is considerable interest in the molecular mechanisms that regulate their development. The goal of this short review is to outline new methods and recent advances in identifying the molecular networks that regulate midbrain dopaminergic neuron differentiation and fate. Midbrain dopaminergic neurons are descended from progenitor cells located near the ventral midline of the neural tube floor plate around the cephalic flexure. It is now clear that their initial formation is dependent on interactions between the signaling molecules Sonic hedgehog, WINGLESS 1, and FIBROBLAST growth factor 8, but there is still an extensive wider network of molecular interactions that must be resolved before the complete picture of dopaminergic neuron development can be described.

Since the discoveries in the late 1950s and early 60s that administration of L-3,4 dihydroxy phenylalanine, a dopamine precursor, could treat Parkinson disease and antipsychotic agents blocked the receptors for the neurotransmitter dopamine,¹ there has been intense interest in the role of dopamine in normal behaviors and in a variety of neurological and psychiatric illnesses. Extensive electrophysiological, biochemical, and behavioral studies over the past 50 years have established that there is an extensive network of dopaminergic neurons involved in the control of movement and a broad array of behaviors related to mood, cognition, attention, reward, addiction, and stress.^2–5 Furthermore, the death of dopamine neurons is central to the pathophysiology of Parkinson disease, and disruptions in dopaminergic signaling may be associated with psychiatric illnesses such as schizophrenia, attention-deficit/hyperactivity disorder, and drug addiction. In the central nervous system (CNS), dopaminergic neurons are restricted to small populations in the olfactory bulb, the hypothalamus, and the mesencephalon (the midbrain; figure 1A) but these neurons establish connections with most brain regions throughout the CNS. The focus of this review is the mesencephalic dopamine neurons localized in the substantia nigra pars compacta and the ventral tegmental area in the midbrain region of the CNS. Substantia nigra dopaminergic neurons project primarily to the striatum to form the neostriatal pathway, and dopamine neurons in the ventral tegmental area project primarily to cortical (mesocortical pathway) and limbic (mesolimbic pathway) regions. The neostriatal dopaminergic pathway is thought to regulate motor control, while the mesocortical and mesolimbic pathways mediate the wide range of behavioral functions ascribed to the dopaminergic system.

Fig. 1.

Dopaminergic Neurons are Generated in the Ventral Midbrain Through a Complex, Dynamic Genetic Network. (A) Schematic of a sagittal section cut through the adult brain illustrating the primary central nervous system locations of dopamingeric neurons in the olfactory bulb (OB), hypothalamus (HY), substantia nigra (SN), and the ventral tegmental area (VTA). (B, C) Schematic of a transverse section cut through an embryonic brain illustrating the dynamic molecular character of the ventral midbrain domains that give rise to midbrain dopaminergic (mDA) neurons. Establishment of the mDA precursor domain (B) and terminal differentiation of mDA neurons (C) is achieved through a series of molecular interactions within specific domains along the dorsal/ventral and medial/lateral axes. The midline of the brain is indicated by the dotted line. Each domain is represented in a different color, and that color reflects the genetic makeup of that domain. The genes critical for each stage of mDA neuron generation are written below their respective domains, and they are written in the same colors as their domains. See table 1 for the full names and functions of each gene product. (D, E) Schematics illustrating side views of E9.5 (E) and E12.5 (F) brains showing the floor plate (expressing Shh) and the mid/hindbrain junction (isthmic organizer; expressing Wnt1 and Fgf8) cooperating during development of mDA neurons.

There is considerable interest in deciphering the rules that regulate the differentiation of midbrain dopamine neurons, in part because of the hope that stem cells can be induced to differentiate into dopamine neurons to replace diseased cells.⁶ Just as Carlsson¹ credits technological advances in the ability to measure dopamine levels and visualize catecholamine neurons in the CNS with the identification of the importance of dopamine neurotransmission, advances in modern molecular biology techniques have provided key insights into the mechanisms that regulate the development of dopamine neurons,⁷ though the full story is still far from understood. The goal of this minireview is to briefly summarize recent advances in deciphering the network of molecular interactions that directs the differentiation of dopamine neurons and to highlight new technical approaches that are likely to drive the field forward.

Molecular Mechanisms in Embryonic Development

The development of all vertebrates involves the sequential segregation and differentiation of more specialized cell types from the pluripotent fertilized egg to the completed newborn. Following fertilization, one of the primary events in early embryonic development is gastrulation, the segregation of embryonic primordial cells into the 3 germ layers that will give rise to the complete organism. The inner and middle layers, or endoderm and mesoderm, respectively, give rise to internal organs, the musculoskeletal system, and the circulatory system. The outer layer, or ectoderm, gives rise to the skin and the nervous system—in essence, ectodermal cells that do not develop into skin cells become brain tissue. Elegant transplantation experiments in frog embryos and other simple experimental systems by the founders of modern embryology long ago established that the specification and differentiation of the CNS depends on interactions between different tissues and the exchange of signaling molecules.⁸ Modern molecular biology and genetic techniques have made it possible to identify complex molecular networks of signaling cascades that regulate embryonic development. These signaling cascades depend on complex interactions between intercellular signaling molecules, their receptors, intracellular second messenger systems, and transcription factors. In the development of the CNS, signaling molecules include a diverse array of differentiation factors (morphogens, growth factors, and mitogens) that are secreted by specialized cells in discrete regions of the developing nervous system (eg, Sonic hedgehog [SHH], nodal proteins, bone morphogenetic proteins, wingless homolog ligands [WNT], fibroblast growth factors [FGFs], Nerve growth factor, Brain-derived neurotrophic factor, Neurotrophin3, and Jagged1 [There is considerable overlap between the roles that secreted factors can play in proliferation and differentiation of CNS neurons depending on where and when the factors are expressed and the competence of target tissue to respond to the factors.]). Passive and active cellular mechanisms establish and maintain a concentration gradient of secreted factors within defined developing regions of the CNS. Target cells express specific receptors for secreted signaling factors, and binding of the signaling molecule with its receptor triggers a cascade of intracellular signals that leads to expression of transcription factors in the cell nucleus. Transcription factors are proteins that either activate or suppress target genes within a cell in order to initiate or regulate a defined genetic cascade. Ultimately, transcription factors can regulate the fate of target cells. The response of any particular target cell to a signaling molecule depends on a wide range of factors including the concentration of the signaling molecule, the presence or absence of other signaling molecules, and the competence of the responding cells. The competence of cells to respond to signaling factors is determined, in part, by the complement of receptors expressed by the cell, the second messenger signaling pathways linked to the receptor, and the pattern and function of genes expressed within the cell. A cell's ability to respond to a signaling factor is dynamic because its response can change depending on its developmental stage.

This simplified summary of the molecular mechanisms that regulate CNS development should emphasize the concept that there are complex networks of interacting factors that regulate neuronal differentiation.^9–11 One notable example of the complex molecular mechanisms that regulate developmental programs is the hedgehog signaling pathway in vertebrate CNS development (for recent reviews, see Bertrand and Dahmane¹² and Fuccillo et al¹³). Hedgehog was first identified in the late 70s by Weischaus and Nusslein-Volhard¹⁴ as part of their seminal screen in fruit flies that identified genes controlling the pattern of body segments in the embryonic fly. Three homologous genes have been identified in mammals; desert hedgehog, Indian hedgehog, and SHH. Of these 3, SHH plays the most critical role in mammalian development and cellular homeostasis; it is involved in the regulation of neural induction (Neural induction is the process by which interactions between the primary 3 layers of embryonic tissue, ectoderm, mesoderm, and endoderm, cause a portion of the ectoderm to begin to differentiate into tissue that will develop into the nervous system.) (including dopamine neurons), neuronal proliferation, and axonal pathfinding in CNS development; the patterning of the anterior-posterior (AP) axis of developing limbs; the regulation of vasculogenesis/angiogenesis, bone and cartilage formation, and lung morphogenesis (see Fuccillo et al¹³ and Riobo and Manning¹⁵). Recent studies have also indicated that SHH is involved in the maintenance of adult stem cell populations^9–11 and induces carcinogenesis in a wide variety of tissues (brain¹⁶, skin¹⁷, prostate¹⁸, pancreas¹⁹).

In the developing vertebrate CNS, SHH is expressed from a wide variety of specialized cellular sources and its function varies depending on its pattern of temporal and spatial expression. In the embryonic spinal cord, it is expressed in a decreasing concentration gradient from the ventral floor plate (FP) to the dorsal half of the spinal cord. The concentration of SHH at different dorsal vs ventral locations in the spinal cord determines the identity of the motoneurons and interneurons that differentiate within this gradient.^20,21 The developing forebrain has several centers of SHH expression, including the medial ganglionic eminences, the preoptic area, the amygdala, the hypothalamus, and the zona limitans interthalamica (a signaling center between the border of the ventral and dorsal thalami). By the time SHH is expressed in the forebrain, the basic pattern of the forebrain has been established and SHH appears to be required to maintain the expression of other genes that mark ventral forebrain structures (eg, Nkx2.1 expression). The differentiation of distinct regions in the forebrain may, in fact, be due to the earlier expression of SHH in tissue underlying the forebrain primordium. In the cerebellum, SHH is first expressed in Purkinje cells several days before birth (in mice) and it diffuses to the overlying external granule cell layer to induce granule cell proliferation.^22–25 With respect to the differentiation of dopamine neurons, SHH expression in the ventral FP organizer at the midbrain-hindbrain junction interacts with FGF8 to establish a 2-dimensional Cartesian coordinate system that specifies mesencephalic progenitors, including dopamine neurons.⁶

SHH exerts its effects on gene transcription through a complex signaling system; SHH receptive cells express the SHH membrane receptor, Patched1 (PTCH1), a transmembrane protein that in the absence of SHH signaling represses the activity of another transmembrane protein, smoothened (SMO). The binding of SHH with PTCH1 releases the inhibition of SMO activity, leading to the activation or derepression of a number of target genes. In vertebrates, the response of presumptive neuronal cells to derepression of SMO activity may be mediated through G-protein activation of the GLI family of transcription factors, GLI1, GLI2, and GLI3.¹⁵ While the details of how the activity of GLI transcription factors are regulated are beyond the scope of this review, in general, GLI1 and GLI2 act as transcriptional activators, while GLI3 represses gene transcription.^13,15

The Development of Mesencephalic Dopamine Neurons

One key requirement for deciphering the mechanisms that regulate the development of particular neuronal cell types is the development of methods to uniquely identify the neurons. In general, dopaminergic neurons can be characterized by a number of distinguishing features, their position in the CNS, the pattern of connections they make, their electrophysiological properties, and the unique expression patterns of a variety of proteins. Twelve clusters of catecholamine-containing neurons (labeled A1 through A12) were originally identified throughout the CNS based on formaldehyde histofluorescence.²⁶ These included 3 clusters of dopaminergic neurons in the mesencephalon (A8, A9, and A10), the region of the embryonic CNS caudal to the precursor region of the thalamus (diencephalon) and rostral to the hindbrain (metencephalon). The identification of dopaminergic neurons and their projection patterns was further refined with the introduction of immunolabeling for tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthetic pathway for dopamine and other catecholamines (norepinephrine [noradrenaline] and epinephrine [adrenaline]²⁷). Dopamine neurons also uniquely express the dopamine transporter that scavenges extracellular dopamine for transport back into dopamine neurons. In addition, a number of transcription factors have been identified that mark postmitotic dopamine neurons: NURR1, LMX1b, PITX3, EN1, and EN2.⁶ Although TH expression has been an extremely useful marker for DA neuron identity, the more recent identification of several transcription factors that are expressed at different stages of DA development have been indispensable in advancing our understanding of their developmental history.

The development of mesencephalic dopamine (mDA) neurons, similar to other specialized subsets of neurons in the CNS, requires that a specific genetically regulated developmental program be initiated early during brain development. This developmental program is inherited by a neuronal precursor pool that will generate neurons with the same genotype (and at least initially the same phenotype) and can be subdivided into 3 distinct stages: (1) induction of the neuroepithelium to generate mDA precursors at early stages of neural development (between embryonic day [E] 8.5 and E10.5 in the mouse); (2) specification of the precursors to adopt a mDA neuronal fate at midembryonic development (between E10.5 and E12.5 in mouse); and (3) terminal differentiation of postmitotic mDA neurons during mid-to-late embryonic development (from E12.5 onward; figures 1B and 1C). While cells that have been specified to a mDA neuron identity do so by committing to this unique fate, cells that have not been specified are competent to respond to signals in their environment and thus might still have the potential to adopt new fates. In the case of the mDA neuronal system, the commitment of cells to this fate includes the sequential acquisition of general neuronal characteristics, initiation of genetic cascades that influence more specific phenotypic characteristics such as neurotransmitter and ion channel expression, distinct anatomical characteristics, and functional circuitry including the recognition of their appropriate synaptic targets. Specialized regions of neuroepithelium called signaling centers orchestrate the stereotypic developmental program of mDA neuron generation. These signaling centers produce the necessary signals that pattern and influence the adjacent neuroepithelium to acquire specific cell types.

Signaling Centers Establish Regional Identity in the Developing Brain

Regional identity in the developing CNS arises from a relatively simple, homogenous neural tube that becomes patterned along the AP and dorsal-ventral (DV) axes into specialized regions. Patterning along the AP axis is achieved through signals derived from the isthmic organizer (ISO) located between the midbrain and hindbrain.^28–32 Patterning along the DV axis of the midbrain is accomplished through molecules produced by the FP, a specialized region of cells located at the ventral base of the neural tube.^21,33 mDA neurons seem to require the activity of both the FP and the ISO signaling centers for their development. At approximately E10.5, a morphological curve in the neural tube positions the FP close to the ISO at the midbrain-hindbrain boundary (MHB)³⁴ (figure 1D). This morphological arrangement allows for the interplay between the FP and ISO signaling centers that is critical for the differentiation of the appropriate number of mDA neurons in the appropriate position.³⁵ Although 3 distinct groups of mDA neurons have been defined by anatomical criteria (A8–A10), there is as yet no evidence that there are distinct lineage differences between the 3 groups of dopaminergic neurons. At the current state of knowledge, the mDA neurons are viewed as a continuum of cells where the differences in electrophysiological properties, anatomical position, and connectivity between A8, A9, and A10 mDA neurons may just represent subtle differences in their response to mediolateral and AP signaling cues during development.

Signals From the MHB Are Critical for Development of the Midbrain

The initial step in regionalization of the neuroepithelium includes the secretion of molecules such as morphogens and the expression of transcription factors from signaling centers. Morphogens are presumed to provide spatial information by forming concentration gradients that subdivide a field of cells by inducing or maintaining the expression of different target genes (including transcription factors) at distinct concentration thresholds. A large number of regulatory genes and morphogens (eg, FGF8, WNT1, Otx2, Gbx2 [A note about gene and protein naming conventions: genes are generally written in italics in lower case {eg, Fgf8} while the corresponding protein is written in all caps with no italics {eg, FGF8}.]) are expressed in precise spatial and temporal patterns around the ISO at the MHB, and they regulate the complex choreography leading to the development of midbrain and hindbrain structures. Of these morphogens, FGF8 is the primary organizer molecule expressed and secreted by the ISO.^36,37 WNT1 is expressed immediately anterior to the FGF8 domain in the mesencephalon. Unlike FGF8, however, WNT1 does not have inductive activity on its own, but it is essential for midbrain and cerebellum development, because Fgf8 expression appears to be dependent upon WNT1 expression.^36,37 Thus, among the earliest known molecules secreted in the isthmus, FGF8 has emerged as the key molecule for mediating the inductive activity of the ISO.

A complex transcriptional network that operates during midbrain and cerebellar development regulates the expression of Fgf8 and Wnt1. The network includes the homeobox genes Otx2, Gbx2, En1, En2, Pax2, and Pax5, which interact with one another to induce and maintain Fgf8 and Wnt1 expression. The combination of precise spatial and temporal activation of secreted molecules and transcription factors has proven necessary and sufficient for setting up the midbrain and cerebellar territories by midembryogenesis in mouse, although the full network of interactions has yet to be described. Otx2 and Gbx2, eg, are among the earliest genes expressed in the CNS and initially mark the anterior and posterior epiblast (outer layer of the blastula before gastrulation), respectively.^{28,29,38–41} By E8.5, the border of Otx2 and Gbx2 expression in the neural plate corresponds to the future posterior border of the mesencephalon. OTX2 and GBX2 act antagonistically on each other to demarcate the precise positions of the Fgf8 and Wnt1 expression domains, respectively. However, neither OTX2 nor GBX2 are required for the induction of either Fgf8 or Wnt1.^28,42–45 Likewise, En1 and En2 expression are both required for the maintenance but not for the initiation of Fgf8 or Wnt1 expression⁴⁶; Pax2, however, is necessary and sufficient for induction of Fgf8 in the anteriormost segment of the hindbrain.⁴⁷ Recent experiments demonstrate that the LIM homeodomain transcription factor LMX1b (see table 1) is also necessary for the initiation of Fgf8 expression and it also maintains the expression of several genes including Wnt1, En1, En2, Pax2, and Gbx2.^50–53 Considerable research is still needed to determine how the expression of each of these genes is regulated during development.

Table 1.

Guide to Selected Differentiation Factors Implicated in the Development of Mesencephalic Dopaminergic Neurons

Protein	Abbreviations	Function	Floor/Base Plate Expression	Mid/Hindbrain Expression
Sonic hedgehog	SHH	Secreted morphogen	√
Fibroblast growth factor 8	FGF8	Secreted morphogen		√
Wingless homologue 1	WNT1	Secreted morphogen	√	√
Engrailed 1 and 2	EN1, EN2	Transcription factors	√	√
LIM homeoboxa transcription factor 1 alpha, beta	LMX1a, b	Transcription factors	√	√
Paired-like homeodomain1 transcription factor 3	PITX3	Transcription factor	√
Orthodenticle homolog 2	OTX2	Transcription factor	√	√
Paired box transcription factors 2 and 5	PAX2, PAX5	Transcription factors		√
Gastrulation brain homeobox 2	GBX2	Transcription factor		√
Nuclear receptor subfamily 4	NR4a2/NURR1	Nuclear orphan receptor	√
MicroRNA 133bb	mir-133b	MicroRNA	mDA expression

^aThe homeobox is a conserved sequence of 180 DNA base pairs that codes for a segment of 60 amino acids that will bind to specific DNA regions. Transcription factors that include this sequence of amino acids are called homeodomain proteins, and the 60 amino acid segment is referred to as the “homeodomain.” The homeodomain was first recognized in proteins that regulate the formation of body parts in fruit flies. Mutations in a homeobox genes could transform one body segment into a different body part. A classic example is a mutation in the antennapedia gene that converts antennae on the head of a fruit fly into legs.⁴⁸

^bMicroRNAs are a recently discovered class of RNA molecules that appear to play a critical role in the regulation of gene expression.⁴⁹ mDA, mesoencephalic dopamine.

Signals From the FP Generate Midbrain Dopaminergic Neurons

mDA neurons arise at around E10.5 from the FP and base plate (adjacent to the FP on both sides of neural tube) at the ventral midline around the cephalic flexure (figure 1B). The FP and notochord (a rod-shaped structure derived from the mesoderm and underlying the nervous system) secrete the lipid-modified glycoprotein SHH, while FGF8 is secreted by the ISO. The combination of SHH and FGF8 are necessary and sufficient for the generation of ectopic mDA neurons in embryonic explant cultures derived from rat brain.⁵⁴ In addition, ectopic expression of SHH is able to induce mDA neurons in the dorsal mid/hindbrain region in areas where FGF8 and WNT1 are normally expressed.⁵⁵ In a more recent set of mouse genetic experiments, mDA neurons were missing in Shh null mutants and conditional inactivation of Smoothened (Smo), the gene that encodes the SHH receptor, resulted in a substantially reduced population of mDA neurons after E9.0.⁵⁶ These studies demonstrate that SHH and FGF8 are critical determinants for the induction of mDA neuronal progenitors.

While SHH and FGF8 appear to be key for the induction of mDA neuronal progenitors, a number of molecules including differentiation factors, transcription factors, signaling molecules, and morphogens have been shown to be involved in subsequent development and survival of postmitotic mDA neurons (eg, EN1 and EN2, WNT1, PITX3, LMX1a, and NR4a2/NURR1; table 1; figures 1B–E). Many of the molecular signals necessary for the initial patterning of the midbrain are later required for the generation and differentiation of mDA neurons from a specialized neuroepithelium. Wnt1 expression, eg, appears to play a role both in the early proliferation of mDA neuron precursors and in the later differentiation of these neurons^34,57 (figures 1B and 1C). Other transcription factors are differentially expressed in mDA neurons, but their function is not yet completely understood. For example, while the EN1 transcription factor is expressed in all mDA neurons, EN2 is only expressed in a subset of the EN1–positive neurons.⁵⁸ This differential pattern of EN2 expression may indicate that it is involved in the differentiation of different subtypes of mDA neurons, but this has not yet been demonstrated. It has been shown that En1 and -2 expressions are necessary for mDA neuron survival in a dose-dependent manner, but the mechanisms are not yet understood.^58,59

Other factors have been identified that regulate specific aspects of mDA differentiation. The nuclear hormone receptor, NR4a2/NURR1, eg, has been shown to be critical for the transcriptional activation of genes required for dopamine biosynthesis and neurotransmitter expression (eg, TH; reviewed in Perlmann and Wallen-Mackenzie⁶⁰). This finding may be of particular relevance to the future development of stem cell therapies because NR4a2/NURR1 and the transcription factor, PITX3, have been shown to act cooperatively in vitro to promote the terminal maturation of mammalian embryonic stem cells into mDA neurons.⁶¹ Finally, there are likely to be discoveries of new regulatory mechanisms that control mDA neuron development. MicroRNAs (miRNAs) were recently identified as a new class of small RNA molecules that regulate gene expression by targeting mRNA degradation and blocking protein translation.⁴⁹ One of these miRNAs, mir-133b, has recently been shown in embryonic stem cell cultures to regulate the maturation and function of mDA neurons through a negative feedback loop that includes PITX3.⁶²

Clearly there is a complex network of regulatory factors that controls the diverse aspects of mDA neuron differentiation. Furthermore, it is still unclear how these molecules interact with each other and many of the key downstream effector molecules have not been determined. In the next section, we briefly describe new genetic strategies that may help define the molecules and interactions that regulate mDA neuron differentiation. These recent advances in mouse molecular genetic technology come at an opportune time when more sophisticated methods are required to unravel the complex signaling networks active during the generation of distinct neuronal populations that are susceptible to specific diseases.

Using Mouse Genetic Techniques to Study Neural Development and Function

The use of knockout/in mutant mice where specific genes are deleted or missexpressed has allowed great leaps to be made in our understanding of the molecular genetic regulation of mDA neuron development. However, we have now come to a point where the basic outline of mDA neuron development has been sketched in and the more difficult questions are ready to be tackled. Fortunately, recent advances in genetic methodology give us a powerful avenue with which to start to answer more difficult questions.^63,64 Although conventional cloning strategies remain powerful approaches for introducing manipulated DNA into mice, the development of transgenic mice made from bacterial artificial chromosomes (BACs) now allows investigators to probe expression patterns and function of genes without the laborious task of carefully characterizing promoter/enhancer regions.⁶⁵ In addition, using BAC DNA allows users to choose from multiple commercially available BAC libraries for large fragments of DNA that contain their gene of interest which should make it easier and quicker to study novel candidate regulators of mDA development.

Perhaps the most exciting, novel genetic tools recently introduced for use in mice are the inducible genetic systems. Based on the already widely used Cre and Flpe recombinase systems,⁶⁴ the fusion of a mutated version of the human estrogen receptor (ER) to Cre and Flpe allows recombinases to become active only when the ER is activated using exogenously supplied 4-hydroxytamoxifen.⁶³ By driving CreER and FlpeER using the enhancers/promoters of specific genes of interest, one can gain tight spatial and temporal control over alleles that can be either conditionally ablated (eg, by flanking a gene of interest with loxP sites that are recognized and recombined by CreER) or alleles that contain conditionally activated reporter gene products such as green fluorescent protein or β-galactosidase, encoded by the bacterial gene lacZ.⁶⁶ Using these strategies, the function of genes within specific cell populations can be studied in detail, and/or defined cell populations and their progeny can be permanently marked by reporter genes and followed through development and also into adulthood. This method is called genetic inducible fate-mapping and it has been elegantly used to gain insight into the developmental origins of mDA neurons derived from Wnt1 expressing and Shh responding cells in vivo.⁶⁶

Because it is now routinely possible to use different cell markers for reporter gene expression, inducible activation of neural circuit tracers such as wheat germ agglutinin horse radish peroxidase and C-terminal fragment of tetanus toxin can be used to understand the links between genes expressed during mDA development and the neuronal circuits that they contribute to during different stages of development.^63,64 In addition, because the genetic tracers will be stably inherited, one can potentially trace neuronal circuits of mDA neuronal into adulthood and assess how different diseases affect the circuitry of genetically defined neural cell groups. Similarly, using the enhancers/promoters of genes expressed during mDA development, investigators can use inducible systems to drive the expression of neuronal activity modulators such as ion channels and molecules that ablate neurons such as dipthera toxin.^63,64 In essence, we can now selectively manipulate the expression of genes in mDA neurons, the functional properties of distinct cell populations that are programmed to differentiate into mDA neurons, and we can also manipulate the surrounding nuclei that interact with mDA neurons. These manipulations can be performed selectively in mDA neurons at different stages of development and in adulthood. Importantly, the mDA neuron systems can be systematically analyzed using sophisticated genetic techniques that will prove indispensable when combined with mouse models of Parkinson disease and schizophrenia.

Funding

Alberta Heritage Foundation For Medical Research (to R.V.S.); National Institute of Mental Health (MH 076854 to M.W.V.).