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Topical Review |
1 Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands
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Abstract |
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 Top Abstract IntroductionReferences |
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(Received 9 May 2006;
accepted after revision 22 June 2006;
first published online 29 June 2006)
Corresponding author M. P. Smidt: Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. Email: m.p.smidt{at}med.uu.nl
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Introduction |
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TopAbstractIntroductionReferences |
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Strategies to unravel molecular cascades underlying mdDA system development
Identification of transcription factors. Before the availability of the complete mouse and human genome, many groups focused on cloning known classes of transcription factors (TFs) from CNS mRNA pools. From these studies the first molecular data on genotype–phenotype relations was established (for an extensive overview see Krieglstein, 2004; Perlmann & Wallén-Mackenzie, 2004; Prakash & Wurst, 2004; Roussa & Krieglstein, 2004; Simon et al. 2004; Smidt et al. 2004b; Smits et al. 2006). The method of cloning was mainly based on a PCR strategy that used degenerate primers to clone conserved regions within the target transcript (Smidt et al. 1997). After the initial gene description, spatiotemporal expression patterns gave the first insight into a putative role in specific CNS developmental processes (Smidt et al. 1997). This type of analysis formed the basis for the identification of molecular mechanisms involved in CNS development and maintenance. With the introduction of techniques for generating genetically modified mice, gene function can be analysed in even more detail.
Phenotypic analysis of knock-outs. Analysis of genetically modified mice has revealed several regulatory genes that are crucial during different phases of mdDA development. Analysis of null mutants of the engrailed genes En1 and En2, Lmx1B, Nurr1, TGF-a and TGF-b, Ngn2 and Pitx3 has revealed their essential roles in either mdDA specification, differentiation and/or maintenance (Poulsen et al. 1994; Krieglstein et al. 1995; Blum, 1998; Smidt et al. 2000, 2004b; Farkas et al. 2003; Perlmann & Wallén-Mackenzie, 2004; Simon et al. 2004; Andersson et al. 2006a) (Fig. 1). Depending on the onset of expression of the affected gene, the effect on the emergence of mdDA neurons can be direct through disturbance of mdDA differentiation and/or maintenance, or indirect through ablation of the region where mdDA neurons are located (ventral midbrain, P1, P2 and P3 (Marín et al. 2005; Smits et al. 2006).
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The Pitx3-deficient phenotype poses a problem of selective phenotypic penetrance in the mdDA system. Although Pitx3 is expressed in all mdDA neurons (Smidt et al. 2004a; Zhao et al. 2004) only a subset of Pitx3-expressing cells display a phenotype after Pitx3 ablation. A possible explanation for this phenomenon could be the fact that the mdDA neuronal group is not a homogeneous set of neurons. Depending on anterior/posterior and dorsal/ventral anatomical positions, specific molecular coding defines mdDA subgroups (Smits et al. 2006). In the case of Pitx3, subgroup-specific transcriptional activators might cooperate with Pitx3 to exert its function in only those neurons that coexpress both activators. However, other subgroup-specific intrinsic factors involved in mdDA development could underlie specific survival of mdDA subpopulations. Therefore, subgroup-specific gene expression profiling may provide further insights into these phenotypic differences. In addition, an essential step to clarify the specific phenotype induced by ablation of transcriptional regulators is to identify the downstream transcriptional cascades of individual TFs. Some useful strategies to solve these issues are suggested below.
Identification of molecular cascades in mdDA neuronal subgroups. (1) Comparative gene expression profiling. The existence of different neuronal subsets within the mdDA system, highlighted by the observed selective vulnerability in genetically modified mice and PD has made it most interesting to define the differences in genome-wide gene expression profile between these neuronal subgroups. Clarification of differences between mRNA pool composition of the SNc compared to the VTA may display molecular details underlying the selective vulnerability of the SNc. Initially, techniques like suppressive subtraction hybridization were used to study the differences in mRNA composition (Petersen & Petersen, 2003). Using this type of analysis, PLC-b4 was identified as being differentially expressed between SNc and VTA (Smits et al. 2005), proving that this method can be successfully applied to the identification of molecular markers of neuronal subgroups. An important aspect of this type of analysis is that it displays genome-wide differential expression, not limited by a selection of genes beforehand as was the case with early microarray chips containing only a proportion of known genes. However, the sensitivity of microarray analysis compared to subtractive hybridization is much higher, which allows for the detection of small differences in gene expression. The development of microarray chips containing an increasing number of genes, Expressed sequence tag (EST) and splice variants has made microarray analysis by far the most commonly used technique to analyse gene expression profiles.
With the introduction of microarray technology, the analysis of differences between two neuronal mRNA pools has become increasingly easy (Hoheisel, 2006). Differential hybridization of mRNAs to an array of oligonucleotides corresponding to known transcripts directly represents differences in gene expression. Micro-dissection and laser capture techniques are applied to select for neuronal tissue samples containing DA neurons of either the SNc or the VTA. Microarray analysis has revealed that many genes are differentially expressed between these mdDA neuronal subsets (Grimm et al. 2004; Greene et al. 2005; Chung et al. 2005). However, the overlap between different data sets is very limited and some genes, known to be restricted to either of the neuronal subsets, are not present in the data sets. These omissions may be due to the use of different arrays or different isolation procedures for selection of the neuronal material. However, in-depth analysis of the set of genes that is present in all data sets could give a very solid clue to the definition of molecular subsets of mdDA neurons and provide new ideas about the cause of selective vulnerability of DA neurons of the SNc.
Similarly, some studies have performed microarray analysis on post mortem SNc mRNA of PD patients and controls (Grünblatt et al. 2004; Hauser et al. 2005). Interpretation of these data sets is treacherous because the most interesting neuronal population – the set that shows increased vulnerability – has already degenerated, at least to some extent, in PD patients. To circumvent this issue, many groups have focused on animal models for PD. The specific vulnerability of the SNc for neurotoxins such as MPTP and 6-OHDA provides a tool for studying time-dependent changes in cellular processes ultimately leading to selective cell death. Application of microarray analysis to compare the expression profile at different time-points in the process of toxin-induced cell death might uncover molecular cascades associated with this selective vulnerability (Mandel et al. 2002; Miller et al. 2004; Miller & Federoff, 2005).
Finally, microarray analysis is used to define the effect of individual TFs on in vitro DA cell models to elucidate the molecular cascades in which a specific TF is involved (Yoo et al. 2004; Hermanson et al. 2006). Such approaches can be used efficiently to explain specific phenotypes in the mdDA system after ablation of specific TFs.
(2) Identification of targets of essential transcription factors. Fundamental to the development of functionally divergent structures in the brain is the spatiotemporal expression of gene-specific TFs and their activation of transcriptional cascades. Gene expression profiling as described in the previous section could define the involvement of a TF in specific molecular cascades. However, due to altered cellular state, a large set of genes can be expected to be differentially expressed in the presence of a single TF. Therefore, additional analysis is necessary to distinguish between the actual function of a TF and its indirect effects on cellular balance. Determination of the set of direct target genes of a TF could help to get a more detailed picture of the molecular cascade in which it is involved.
A direct analysis of the interaction of a TF with genomic DNA elements can be performed through a chromatin immunoprecipitation (ChIP) assay (Das et al. 2004; Hearnes et al. 2005). With this experimental design it is possible to identify genomic binding sites through the specific interaction of antibodies to TF– DNA complexes. Immunoprecipitated DNA can be cloned directly to create a library of genomic TF binding sites (Hearnes et al. 2005). It should be noted, however, that non-specific background is a common problem in ChIP assays, which makes the PCR-based analysis of putative TF binding sites in an independent ChIP assay a crucial step to validate enrichment of the target DNA. Database analysis reveals the location of the set of TF binding sites on the genome, and genes in the proximity of TF binding sites can be selected for further analysis. Genes of interest should be analysed for coexpression with the TF in vivo, and TF-mediated regulation of the gene of interest can be analysed in an appropriate in vitro cell model. Alteration of gene expression, in combination with specific binding to its promoter, strongly suggests a direct regulatory relation between the TF and the gene of interest.
Furthermore, ChIP assay is widely used to validate binding of TFs to promoter regions of known or expected target genes. With the use of PCR, specific promoter regions can be analysed for relative enrichment in immunoprecipitated genomic DNA samples. The opportunity to analyse time- or cellular condition-dependent interaction of TFs with target DNA has made this approach a powerful tool for studying TF function.
ChIP-on-chip combines chromatin immunoprecipitation with microarray analysis using a CpG island microarray containing enhancer and promoter regions of genes. This approach ensures a high throughput screen of TF–target DNA interaction under variable conditions (Weinmann et al. 2002; Kirmizis & Farnham, 2004). Although whole genome arrays are available for small genomes like yeast (Ren et al. 2000), they are not yet applicable for mammalian genomes due to their large sizes. However, progress has been made to improve genome coverage and resolution and it will only be a matter of time before ChIP-on-chip-based whole genome analysis of TF binding sites is possible for mammalian genomes as well (Kapranov et al. 2002; Kim et al. 2005a,b). Altogether, ChIP is an effective method with which to define a molecular cascade in great detail, focusing on the core function of the TF of interest.
In vivo analysis of defined molecular cascades
When genes have been identified as being part of a transcriptional cascade, the nature of their functional role should be analysed in vivo in order to obtain a better insight into the implications for brain development and function. To date, many in vivo techniques have been successfully applied to validate and explore molecular cascades in vivo. Some relatively new and promising techniques used to research mdDA development will be discussed here.
In vivo gene transfer. The time- and region-specific introduction of DNA by means of in vivo DNA electroporation has been shown to be an effective tool for studying the function of individual genes in development. Both down-regulation, through application of small interfering RNA (si-RNA), and overexpression of genes provide insights into their role in neuronal systems of interest. Recently, an elegant paper was published that describes the essential role of Lmx1a in the early developmental programming of mdDA neurons (Andersson et al. 2006b). Most of the functional analysis on this gene was done by gain and loss of function experiments in the developing chick through the application of in ovo DNA electroporation (Yasugi & Nakamura, 2000). The application of DNA transfer into mouse embryos by means of in utero electroporation was shown to be as effective as in ovo (Tabata & Nakajima, 2001). In addition to gene function analysis, gene transfer by in utero electroporation can be performed to validate the association of potential downstream targets of TFs in specific molecular cascades.
Knock-in transgenics. The ability to generate genetically modified mice has provided an enormous leap forward in the analysis of gene function during the development and function of the CNS. In the last 5 years many studies have been published that describe the ectopic expression of genes of interest under the control of region-specific promoters, like Wnt1 expression in the En1 domain (Puelles et al. 2003, 2004; Vernay et al. 2005; Smits et al. 2006; Prakash et al. 2006). This approach could provide clues to elucidate the effects of changing specific transcriptional patterning during mdDA development. An excellent tool for manipulating gene expression during the terminal differentiation phase of mdDA neurons was provided by a group that generated the TH-knock-in (Althini et al. 2003). In addition, this construct can be used to introduce potential downstream target genes of TFs such as Pitx3 into the mdDA neuronal population. Introduction of these genes into the background of the Pitx3 null-mutant, could result in a rescue of the phenotype in the mdDA system, validating the association of genes within the Pitx3-mediated transcriptional cascade.
Concluding remarks
Recent developments in molecular tools have enabled us to gain an insight into an immensely complex organ, the vertebrate CNS. Through the focus of a defined neuronal group, i.e. the mdDA neurons, it is possible to dissect the molecular programming of such neurons and to use that knowledge in CNS pathology and drug targeting. As the in vivo gain and loss of function experiments become more accessible and quicker, an increasing array of transcriptional cascades will be identified for specified neuronal groups. Intervention in these processes will eventually become available as a new asset in clinical applications. For the mdDA system some essential processes that lead to early programming, early and late differentiation and maintenance have been elucidated and many new potential drug targets are being investigated to solve issues related to PD and schizophrenia. Especially with the availability of the complete mouse and human genome, gene functional studies will become more and more important in providing the final step to understand how the brain is built.
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