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Nilima Prakash

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Nilima Prakash studied Biology at the University of Konstanz/Germany and received the Doctoral Degree from the University of Hamburg/Germany. After Postdoctoral Research stays at the Karolinska Institute, Sweden, and the Weizmann Institute of Science, Israel, she worked as a Research Scientist at the Max Planck Institute of Psychiatry in Munich/Germany. In 2004, she became a team leader at the Institute of Developmental Genetics, Helmholtz Center Munich. In June 2015, she joined the faculty at the Hamm-Lippstadt University of Applied Sciences. Dr. Prakash is an expert in neurodevelopmental biology. Her work has focused on the generation of dopaminergic neurons in the mouse embryo and from pluripotent stem cells in the culture dish, including the nigrostriatal dopamine neurons that are most affected in Parkinson’s Disease. She is recipient of the Science Prize Weihenstephan of the City of Freising 2013, and the Speaker Award of the Helmholtz Center Munich Science Day 2009.

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Papers by Nilima Prakash

Vasopressin mRNA localization in nerve cells: Characterization of cis-acting elements and trans-acting factors

Proceedings of the National Academy of Sciences, 2001

mRNA localization is a complex pathway. Besides mRNA sorting per se, this process includes aspect... more mRNA localization is a complex pathway. Besides mRNA sorting per se, this process includes aspects of regulated translation. It requires protein factors that interact with defined sequences (or sequence motifs) of the transcript, and the protein͞RNA complexes are finally guided along the cytoskeleton to their ultimate destinations. The mRNA encoding the vasopressin (VP) precursor protein is localized to the nerve cell processes in vivo and in primary cultured nerve cells. Sorting of VP transcripts to dendrites is mediated by the last 395 nucleotides of the mRNA, the dendritic localizer sequence, and it depends on intact microtubules.

Identification of Both General and Region-Specific Embryonic CNS Enhancer Elements in the Nestin Promoter

Experimental Cell Research, 1999

In this report, we investigate how nestin expression is controlled in neural progenitor cells of ... more In this report, we investigate how nestin expression is controlled in neural progenitor cells of the embryonic CNS. A 374-bp region in the second intron of the human nestin gene is sufficient, and a 120-bp sequence in this region is required, to express the lacZ reporter gene throughout the developing CNS of E9.5-10.5 transgenic mouse embryos. The 120-bp element region contains putative binding sites for nuclear hormone receptors and we show that TRs, RXR, RAR, and COUP-TF bind to these motifs. A separate enhancer, located most probably 5 to the 120-bp sequence in the second intron, controls midbrain expression at E10.5. In conclusion, our data show that the nestin enhancer in the second intron contains elements both for general and for region-specific CNS progenitor cell expression and suggest that nuclear hormone receptors play a role in the regulation of nestin expression in the early CNS.

Dendritic Localization of Rat Vasopressin mRNA: Ultrastructural Analysis and Mapping of Targeting Elements

European Journal of Neuroscience, 1997

Transcripts encoding the vasopressin precursor are located in axons and dendrites of rat hypothal... more Transcripts encoding the vasopressin precursor are located in axons and dendrites of rat hypothalamic magnocellular neurons. While the axonal vasopressin mRNA has been extensively characterized both at the biochemical and morphological level, little is known about those transcripts residing in dendrites of magnocellular neurons. As revealed by in situ hybridization at the electron microscopic level, the mRNA is located in proximal and distal dendritic segments and is exclusively confined to regions containing rough endoplasmic reticulum. These results suggest that dendrites of hypothalamic neurons may be capable of local precursor synthesis independent of that occurring in the cell somata. A heterologous system has been employed to define cis-acting elements within the vasopressin mRNA which may be involved in dendritic compartmentalization. Expression vector constructs consisting of the cytomegalovirus promoter coupled to the rat vasopressin cDNA have been injected into the cell nuclei of cultured neurons derived from embryonic rat superior cervical ganglia. Vector-encoded vasopressin transcripts were also sorted to dendrites of these neurons indicating that the molecular determinants of dendritic mRNA transport are not cell specific. Mapping of the targeting elements revealed two segments within the vasopressin mRNA that are able to confer dendritic compartmentalization to alpha-tubulin mRNA which is normally confined to the cell body.

Fgf15 regulates thalamic development by controlling the expression of proneural genes

Brain structure & function, Jan 27, 2015

The establishment of the brain structural complexity requires a precisely orchestrated interplay ... more The establishment of the brain structural complexity requires a precisely orchestrated interplay between extrinsic and intrinsic signals modulating cellular mechanisms to guide neuronal differentiation. However, little is known about the nature of these signals in the diencephalon, a complex brain region that processes and relays sensory and motor information to and from the cerebral cortex and subcortical structures. Morphogenetic signals from brain organizers regulate histogenetic processes such as cellular proliferation, migration, and differentiation. Sonic hedgehog (Shh) in the key signal of the ZLI, identified as the diencephalic organizer. Fgf15, the mouse gene orthologous of human, chick, and zebrafish Fgf19, is induced by Shh signal and expressed in the diencephalic alar plate progenitors during histogenetic developmental stages. This work investigates the role of Fgf15 signal in diencephalic development. In the absence of Fgf15, the complementary expression pattern of pron...

Modeling Maintenance of the Mid/hindbrain Boundary

Objective: Shortly after gastrulation the vertebrate neural tube is patterned along the anterior-... more Objective: Shortly after gastrulation the vertebrate neural tube is patterned along the anterior-posterior axis into four regions, which continue to develop into forebrain, midbrain, hindbrain and spinal cord. This patterning is induced and determined by a well defined and locally restricted ex- pression of genes. A key player in this process is the isthmic organizer located at the mid-hindbrain boundary

Theoretical and Experimental Analysis of Gene Regulation at the Mid-Hindbrain Boundary

Shortly after gastrulation the vertebrate neural tube is patterned into forebrain, midbrain, hind... more Shortly after gastrulation the vertebrate neural tube is patterned into forebrain, midbrain, hindbrain and spinal cord. This patterning is determined by a well-defined and locally restricted expression of genes and the action of short and long range signaling centers. The development of the mid- and hindbrain, for example, is controlled by the activity of the isthmic organizer located at the mid-hindbrain boundary (MHB). We show that a Boolean analysis of the characteristic spatial gene expression pattern at the murine MHB allows prediction of key regulating interactions. Based on this analysis, we predict a maintaining rather than inducing effect of Fgf8 on Wnt1 expression, an issue that remained unclear from published in vitro and in vivo data. Using mouse anterior neural tube explant cultures, we provide experimental evidence that Fgf8 in fact only maintains but does not induce ectopic Wnt1 expression in these explants. In combination with previously validated interactions this f...

Limitations of In Vivo Reprogramming to Dopaminergic Neurons via a Tricistronic Strategy

by Nilima Prakash and Jingzhong Zhang

Human gene therapy methods, Jan 29, 2015

Parkinson's disease is one of the most common neurodegenerative disorders characterized by ce... more Parkinson's disease is one of the most common neurodegenerative disorders characterized by cell death of dopaminergic neurons in the substantia nigra. Recent research has focused on cellular replacement through lineage reprogramming as a potential therapeutic strategy. This study sought to use genetics to define somatic cell types in vivo amenable to reprogramming. To stimulate in vivo reprogramming to dopaminergic neurons, we generated a Rosa26 knock-in mouse line conditionally overexpressing Mash1, Lmx1a, and Nurr1. These proteins are characterized by their role in neuronal commitment and development of midbrain dopaminergic neurons and have previously been shown to convert fibroblasts to dopaminergic neurons in vitro. We show that a tricistronic construct containing these transcription factors can reprogram astrocytes and fibroblasts in vitro. However, cassette overexpression triggered cell death in vivo, in part through endoplasmic reticulum stress, while we also detected &q...

A WNT1-regulated developmental gene cascade prevents dopaminergic neurodegeneration in adult En1(+/-) mice

by Nilima Prakash and Jingzhong Zhang

Neurobiology of disease, Jan 3, 2015

The protracted and age-dependent degeneration of dopamine (DA)-producing neurons of the Substanti... more The protracted and age-dependent degeneration of dopamine (DA)-producing neurons of the Substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) in the mammalian midbrain is a hallmark of human Parkinson's Disease (PD) and of certain genetic mouse models of PD, such as mice heterozygous for the homeodomain transcription factor Engrailed 1 (En1(+/-) mice). Neurotoxin-based animal models of PD, in contrast, are characterized by the fast and partly reversible degeneration of the SNc and VTA DA neurons. The secreted protein WNT1 was previously shown to be strongly induced in the neurotoxin-injured adult ventral midbrain (VM), and to protect the SNc and VTA DA neurons from cell death in this context. We demonstrate here that the sustained and ectopic expression of Wnt1 in the SNc and VTA DA neurons of En1(+/Wnt1) mice also protected these genetically affected En1 heterozygote (En1(+/-)) neurons from their premature degeneration in the adult mouse VM. We identified a deve...

Wnt1-regulated genetic networks in midbrain dopaminergic neuron development

Journal of molecular cell biology, 2014

Neurons synthesizing the neurotransmitter dopamine exert crucial functions in the mammalian brain... more Neurons synthesizing the neurotransmitter dopamine exert crucial functions in the mammalian brain. The biggest and most important population of dopamine-synthesizing neurons is located in the mammalian ventral midbrain (VM), and controls and modulates the execution of motor, cognitive, affective, motivational, and rewarding behaviours. Degeneration of these neurons leads to motor deficits that are characteristic of Parkinson's disease, while their dysfunction is involved in the pathogenesis of psychiatric disorders including schizophrenia and addiction. Because the aetiology and therapeutic prospects for these diseases include neurodevelopmental aspects, substantial scientific interest has been focused on deciphering the mechanistic pathways that control the generation and survival of these neurons during embryonic development. Researches during the last decade revealed the pivotal role of the secreted Wnt1 ligand and its signalling cascade in the generation of the dopamine-synt...

Mouse IDGenes: a reference database for genetic interactions in the developing mouse brain

by Nilima Prakash, Jingzhong Zhang, and Dietrich Trümbach

Database : the journal of biological databases and curation, 2014

The study of developmental processes in the mouse and other vertebrates includes the understandin... more The study of developmental processes in the mouse and other vertebrates includes the understanding of patterning along the anterior-posterior, dorsal-ventral and medial- lateral axis. Specifically, neural development is also of great clinical relevance because several human neuropsychiatric disorders such as schizophrenia, autism disorders or drug addiction and also brain malformations are thought to have neurodevelopmental origins, i.e. pathogenesis initiates during childhood and adolescence. Impacts during early neurodevelopment might also predispose to late-onset neurodegenerative disorders, such as Parkinson's disease. The neural tube develops from its precursor tissue, the neural plate, in a patterning process that is determined by compartmentalization into morphogenetic units, the action of local signaling centers and a well-defined and locally restricted expression of genes and their interactions. While public databases provide gene expression data with spatio-temporal re...

Genetic networks controlling the development of midbrain dopaminergic neurons

The Journal of physiology, 2006

Recent data have substantially advanced our understanding of midbrain dopaminergic neuron develop... more Recent data have substantially advanced our understanding of midbrain dopaminergic neuron development. Firstly, a Wnt1-regulated genetic network, including Otx2 and Nkx2-2, and a Shh-controlled genetic cascade, including Lmx1a, Msx1 and Nkx6-1, have been unravelled, acting in parallel or sequentially to establish a territory competent for midbrain dopaminergic precursor production at relatively early stages of neural development. Secondly, the same factors (Wnt1 and Lmx1a/Msx1) appear to regulate midbrain dopaminergic and/or neuronal fate specification in the postmitotic progeny of these precursors by controlling the expression of midbrain dopaminergic-specific and/or general proneural factors at later stages of neural development. For the first time, early inductive events have thus been linked to later differentiation processes in midbrain dopaminergic neuron development. Given the pivotal importance of this neuronal population for normal function of the human brain and its involv...

Mapping of the midbrain dopaminergic system during development of the chicken embryo reveals evolutionary differences between birds and mammals

International Journal of Developmental Neuroscience, 2006

FGF/FGFR2 Signaling Regulates the Generation and Correct Positioning of Bergmann Glia Cells in the Developing Mouse Cerebellum

by Nilima Prakash and D. Vogt

PLoS ONE, 2014

The normal cellular organization and layering of the vertebrate cerebellum is established during ... more The normal cellular organization and layering of the vertebrate cerebellum is established during embryonic and early postnatal development by the interplay of a complex array of genetic and signaling pathways. Disruption of these processes and of the proper layering of the cerebellum usually leads to ataxic behaviors. Here, we analyzed the relative contribution of Fibroblast growth factor receptor 2 (FGFR2)-mediated signaling to cerebellar development in conditional Fgfr2 single mutant mice. We show that during embryonic mouse development, Fgfr2 expression is higher in the anterior cerebellar primordium and excluded from the proliferative ventricular neuroepithelium. Consistent with this finding, conditional Fgfr2 single mutant mice display the most prominent defects in the anterior lobules of the adult cerebellum. In this context, FGFR2-mediated signaling is required for the proper generation of Bergmann glia cells and the correct positioning of these cells within the Purkinje cell layer, and for cell survival in the developing cerebellar primordium. Using cerebellar microexplant cultures treated with an FGFR agonist (FGF9) or antagonist (SU5402), we also show that FGF9/FGFR-mediated signaling inhibits the outward migration of radial glia and Bergmann glia precursors and cells, and might thus act as a positioning cue for these cells. Altogether, our findings reveal the specific functions of the FGFR2-mediated signaling pathway in the generation and positioning of Bergmann glia cells during cerebellar development in the mouse.

Wnt5a Regulates Ventral Midbrain Morphogenesis and the Development of A9–A10 Dopaminergic Cells In Vivo

PLoS ONE, 2008

Wnt5a is a morphogen that activates the Wnt/planar cell polarity (PCP) pathway and serves multipl... more Wnt5a is a morphogen that activates the Wnt/planar cell polarity (PCP) pathway and serves multiple functions during development. PCP signaling controls the orientation of cells within an epithelial plane as well as convergent extension (CE) movements. Wnt5a was previously reported to promote differentiation of A9-10 dopaminergic (DA) precursors in vitro. However, the signaling mechanism in DA cells and the function of Wnt5a during midbrain development in vivo remains unclear. We hereby report that Wnt5a activated the GTPase Rac1 in DA cells and that Rac1 inhibitors blocked the Wnt5ainduced DA neuron differentiation of ventral midbrain (VM) precursor cultures, linking Wnt5a-induced differentiation with a known effector of Wnt/PCP signaling. In vivo, Wnt5a was expressed throughout the VM at embryonic day (E)9.5, and was restricted to the VM floor and basal plate by E11.5-E13.5. Analysis of Wnt5a2/2 mice revealed a transient increase in progenitor proliferation at E11.5, and a precociously induced NR4A2+ (Nurr1) precursor pool at E12.5. The excess NR4A2+ precursors remained undifferentiated until E14.5, when a transient 25% increase in DA neurons was detected. Wnt5a2/2 mice also displayed a defect in (mid)brain morphogenesis, including an impairment in midbrain elongation and a rounded ventricular cavity. Interestingly, these alterations affected mostly cells in the DA lineage. The ventral Sonic hedgehogexpressing domain was broadened and flattened, a typical CE phenotype, and the domains occupied by Ngn2+ DA progenitors, NR4A2+ DA precursors and TH+ DA neurons were rostrocaudally reduced and laterally expanded. In summary, we hereby describe a Wnt5a regulation of Wnt/PCP signaling in the DA lineage and provide evidence for multiple functions of Wnt5a in the VM in vivo, including the regulation of VM morphogenesis, DA progenitor cell division, and differentiation of NR4A2+ DA precursors.

Spatial Analysis of Expression Patterns Predicts Genetic Interactions at the Mid-Hindbrain Boundary

PLoS Computational Biology, 2009

The isthmic organizer mediating differentiation of mid-and hindbrain during vertebrate developmen... more The isthmic organizer mediating differentiation of mid-and hindbrain during vertebrate development is characterized by a well-defined pattern of locally restricted gene expression domains around the mid-hindbrain boundary (MHB). This pattern is established and maintained by a regulatory network between several transcription and secreted factors that is not yet understood in full detail. In this contribution we show that a Boolean analysis of the characteristic spatial gene expression patterns at the murine MHB reveals key regulatory interactions in this network. Our analysis employs techniques from computational logic for the minimization of Boolean functions. This approach allows us to predict also the interplay of the various regulatory interactions. In particular, we predict a maintaining, rather than inducing, effect of Fgf8 on Wnt1 expression, an issue that remained unclear from published data. Using mouse anterior neural plate/tube explant cultures, we provide experimental evidence that Fgf8 in fact only maintains but does not induce ectopic Wnt1 expression in these explants. In combination with previously validated interactions, this finding allows for the construction of a regulatory network between key transcription and secreted factors at the MHB. Analyses of Boolean, differential equation and reactiondiffusion models of this network confirm that it is indeed able to explain the stable maintenance of the MHB as well as timecourses of expression patterns both under wild-type and various knock-out conditions. In conclusion, we demonstrate that similar to temporal also spatial expression patterns can be used to gain information about the structure of regulatory networks. We show, in particular, that the spatial gene expression patterns around the MHB help us to understand the maintenance of this boundary on a systems level.

Distinct but redundant expression of the Frizzled Wnt receptor genes at signaling centers of the developing mouse brain

Neuroscience, 2007

The establishment of the regional subdivisions of the vertebrate CNS is accomplished through the ... more The establishment of the regional subdivisions of the vertebrate CNS is accomplished through the activity of different neuroepithelial organizing centers. The wingless/int (Wnt) family of secreted glycoproteins, among other factors, plays a crucial role in signaling from these centers. Wnt1 secreted from the boundary between the mid- and hindbrain, for instance, controls the development of this brain region and of associated neuronal populations. Different Wnts secreted from the caudomedial pallium, the cortical hem, pattern the adjacent hippocampal field. The first step in Wnt signal transduction is binding of the Wnt ligand to its receptors, the seven-pass transmembrane Frizzled proteins. Inactivation of different Frizzled genes in mice have revealed an extensive functional redundancy between these receptors. In order to discriminate between a possible participation of different Frizzled receptors in the transduction of Wnt signals at the mid-/hindbrain boundary and the cortical hem, we have performed a detailed expression study of the 10 known murine Frizzled genes at crucial stages of mouse embryonic development. Our analysis reveals a highly dynamic yet distinct expression pattern of individual Frizzled genes in the anterior neural tube of the developing mouse embryo. The overlapping spatio-temporal expression of at least two and up to six Frizzled genes in any region of the developing mouse brain, however, also suggests a vast functional redundancy of the murine Frizzled receptors. This redundancy has to be taken into consideration for future analyses of Frizzled receptor function at these signaling centers in the mouse.

A Wnt Signal Regulates Stem Cell Fate and Differentiation in vivo

Neurodegenerative Diseases, 2007

Our knowledge about the normal generation of midbrain dopaminergic neurons in vivois still rudime... more Our knowledge about the normal generation of midbrain dopaminergic neurons in vivois still rudimentary, despite many attempts to recapitulate the underlying events in vitro. Because the loss of these neurons is implicated in Parkinson&amp;amp;#39;s disease, this lack of information is one of the major drawbacks in the development of better therapies for this severe human neurological disorder. Recently, substantial advances have been made by demonstrating that the secreted molecule Wnt1 regulates a genetic network, including the transcription factors Otx2 and Nkx2-2, for the initial establishment of the dopaminergic progenitor domain in the mammalian ventral midbrain. In addition, Wnt1 appears to regulate the differentiation of the postmitotic progeny of these precursors by initiating the expression of midbrain dopaminergic-specific transcription factors. A genetic cascade controlled by the secreted molecule Sonic hedgehog, including the transcription factors Lmx1a, Msx1 and Nkx6-1, acts in parallel with the Wnt1-regulated network to establish the midbrain dopaminergic progenitor domain. The Sonic-hedgehog-controlled cascade may diverge from the Wnt1-regulated network at later stages of neural development through induction of proneural transcription factors required for the acquisition of generic neuronal properties by the midbrain dopaminergic progeny. Here we provide a brief overview of these regulatory gene networks.

Otx2 controls neuron subtype identity in ventral tegmental area and antagonizes vulnerability to MPTP

Nature Neuroscience, 2010

Mouse Notch 3 Expression in the Pre- and Postnatal Brain: Relationship to the Stroke and Dementia Syndrome CADASIL

by Nilima Prakash and U. Lendahl

Experimental Cell Research, 2002

Mutations in the human Notch 3 gene cause the vascular stroke and dementia syndrome CADASIL (Cere... more Mutations in the human Notch 3 gene cause the vascular stroke and dementia syndrome CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) characterized by degeneration of vascular smooth muscle cells and multiple small infarcts in the white and deep gray matter of the brain. Here we have analyzed the expression pattern of the Notch 3 gene in the pre-and postnatal mouse brain. Prenatal Notch 3 expression is restricted to a scattered population of cells within the vessel wall of all major blood vessels in the developing embryo, including those that form the perineural vascular plexus. Expression in the postnatal brain is confined to a scattered cell population within the vessel wall of small to medium-sized penetrating arteries, which are the vessel type primarily affected in CADASIL patients. In contrast, no expression was observed in capillaries and veins. Notch 3 is most likely expressed in a subset of vascular smooth muscle cells, and the expression pattern of one of the Notch ligands, Serrate 1, was very similar to that observed for Notch 3. The Notch 3 expressing pattern was not significantly altered in platelet-derived growth factor B-(PDGF-B) deficient mouse embryos, demonstrating that Notch 3 expression is not under direct control of PDGF-B. These data show that Notch 3 expression is conserved between mouse and human and suggest that the mouse is a valid system for analysis of CADASIL.

Otx2 selectively controls the neurogenesis of specific neuronal subtypes of the ventral tegmental area and compensates En1-dependent neuronal loss and MPTP vulnerability

Developmental Biology, 2013

Understanding the molecular basis underlying the neurogenesis of mesencephalic-diencephalic Dopam... more Understanding the molecular basis underlying the neurogenesis of mesencephalic-diencephalic Dopaminergic (mdDA) neurons is a major task fueled by their relevance in controlling locomotor activity and emotion and their involvement in neurodegenerative and psychiatric diseases. Increasing evidence suggests that mdDA neurons of the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) represent two main distinct neuronal populations, which, in turn, include specific neuronal subsets. Relevant studies provided important results on mdDA neurogenesis, but, nevertheless, have not yet clarified how the identity of mdDA neuronal subtypes is established and, in particular, whether neurogenic factors may direct progenitors towards the differentiation of specific mdDA neuronal subclasses. The transcription factor Otx2 is required for the neurogenesis of mesencephalic DA (mesDA) neurons and to control neuron subtype identity and sensitivity to the MPTP neurotoxin in the adult VTA. Here we studied whether Otx2 is required in mdDA progenitors for the generation of specific mdDA neuronal subtypes. We found that although expressed in virtually all mdDA progenitors, Otx2 is required selectively for the differentiation of VTA neuronal subtypes expressing Ahd2 and/or Calb but not for those co-expressing Girk2 and glyco-Dat. Moreover, mild over-expression of Otx2 in SNpc progenitors and neurons is sufficient to rescue En1 haploinsufficiency-dependent defects, such as progressive loss and increased MPTP sensitivity of SNpc neurons. Collectively, these data suggest that mdDA progenitors exhibit differential sensitivity to Otx2, which selectively influences the generation of a large and specific subset of VTA neurons. In addition, these data suggest that Otx2 and En1 may share similar properties and control survival and vulnerability to MPTP neurotoxin respectively in VTA and SNpc.

Vasopressin mRNA localization in nerve cells: Characterization of cis-acting elements and trans-acting factors

Proceedings of the National Academy of Sciences, 2001

mRNA localization is a complex pathway. Besides mRNA sorting per se, this process includes aspect... more mRNA localization is a complex pathway. Besides mRNA sorting per se, this process includes aspects of regulated translation. It requires protein factors that interact with defined sequences (or sequence motifs) of the transcript, and the protein͞RNA complexes are finally guided along the cytoskeleton to their ultimate destinations. The mRNA encoding the vasopressin (VP) precursor protein is localized to the nerve cell processes in vivo and in primary cultured nerve cells. Sorting of VP transcripts to dendrites is mediated by the last 395 nucleotides of the mRNA, the dendritic localizer sequence, and it depends on intact microtubules.

Identification of Both General and Region-Specific Embryonic CNS Enhancer Elements in the Nestin Promoter

Experimental Cell Research, 1999

In this report, we investigate how nestin expression is controlled in neural progenitor cells of ... more In this report, we investigate how nestin expression is controlled in neural progenitor cells of the embryonic CNS. A 374-bp region in the second intron of the human nestin gene is sufficient, and a 120-bp sequence in this region is required, to express the lacZ reporter gene throughout the developing CNS of E9.5-10.5 transgenic mouse embryos. The 120-bp element region contains putative binding sites for nuclear hormone receptors and we show that TRs, RXR, RAR, and COUP-TF bind to these motifs. A separate enhancer, located most probably 5 to the 120-bp sequence in the second intron, controls midbrain expression at E10.5. In conclusion, our data show that the nestin enhancer in the second intron contains elements both for general and for region-specific CNS progenitor cell expression and suggest that nuclear hormone receptors play a role in the regulation of nestin expression in the early CNS.

Dendritic Localization of Rat Vasopressin mRNA: Ultrastructural Analysis and Mapping of Targeting Elements

European Journal of Neuroscience, 1997

Transcripts encoding the vasopressin precursor are located in axons and dendrites of rat hypothal... more Transcripts encoding the vasopressin precursor are located in axons and dendrites of rat hypothalamic magnocellular neurons. While the axonal vasopressin mRNA has been extensively characterized both at the biochemical and morphological level, little is known about those transcripts residing in dendrites of magnocellular neurons. As revealed by in situ hybridization at the electron microscopic level, the mRNA is located in proximal and distal dendritic segments and is exclusively confined to regions containing rough endoplasmic reticulum. These results suggest that dendrites of hypothalamic neurons may be capable of local precursor synthesis independent of that occurring in the cell somata. A heterologous system has been employed to define cis-acting elements within the vasopressin mRNA which may be involved in dendritic compartmentalization. Expression vector constructs consisting of the cytomegalovirus promoter coupled to the rat vasopressin cDNA have been injected into the cell nuclei of cultured neurons derived from embryonic rat superior cervical ganglia. Vector-encoded vasopressin transcripts were also sorted to dendrites of these neurons indicating that the molecular determinants of dendritic mRNA transport are not cell specific. Mapping of the targeting elements revealed two segments within the vasopressin mRNA that are able to confer dendritic compartmentalization to alpha-tubulin mRNA which is normally confined to the cell body.

Fgf15 regulates thalamic development by controlling the expression of proneural genes

Brain structure & function, Jan 27, 2015

The establishment of the brain structural complexity requires a precisely orchestrated interplay ... more The establishment of the brain structural complexity requires a precisely orchestrated interplay between extrinsic and intrinsic signals modulating cellular mechanisms to guide neuronal differentiation. However, little is known about the nature of these signals in the diencephalon, a complex brain region that processes and relays sensory and motor information to and from the cerebral cortex and subcortical structures. Morphogenetic signals from brain organizers regulate histogenetic processes such as cellular proliferation, migration, and differentiation. Sonic hedgehog (Shh) in the key signal of the ZLI, identified as the diencephalic organizer. Fgf15, the mouse gene orthologous of human, chick, and zebrafish Fgf19, is induced by Shh signal and expressed in the diencephalic alar plate progenitors during histogenetic developmental stages. This work investigates the role of Fgf15 signal in diencephalic development. In the absence of Fgf15, the complementary expression pattern of pron...

Modeling Maintenance of the Mid/hindbrain Boundary

Objective: Shortly after gastrulation the vertebrate neural tube is patterned along the anterior-... more Objective: Shortly after gastrulation the vertebrate neural tube is patterned along the anterior-posterior axis into four regions, which continue to develop into forebrain, midbrain, hindbrain and spinal cord. This patterning is induced and determined by a well defined and locally restricted ex- pression of genes. A key player in this process is the isthmic organizer located at the mid-hindbrain boundary

Theoretical and Experimental Analysis of Gene Regulation at the Mid-Hindbrain Boundary

Shortly after gastrulation the vertebrate neural tube is patterned into forebrain, midbrain, hind... more Shortly after gastrulation the vertebrate neural tube is patterned into forebrain, midbrain, hindbrain and spinal cord. This patterning is determined by a well-defined and locally restricted expression of genes and the action of short and long range signaling centers. The development of the mid- and hindbrain, for example, is controlled by the activity of the isthmic organizer located at the mid-hindbrain boundary (MHB). We show that a Boolean analysis of the characteristic spatial gene expression pattern at the murine MHB allows prediction of key regulating interactions. Based on this analysis, we predict a maintaining rather than inducing effect of Fgf8 on Wnt1 expression, an issue that remained unclear from published in vitro and in vivo data. Using mouse anterior neural tube explant cultures, we provide experimental evidence that Fgf8 in fact only maintains but does not induce ectopic Wnt1 expression in these explants. In combination with previously validated interactions this f...

Limitations of In Vivo Reprogramming to Dopaminergic Neurons via a Tricistronic Strategy

by Nilima Prakash and Jingzhong Zhang

Human gene therapy methods, Jan 29, 2015

Parkinson's disease is one of the most common neurodegenerative disorders characterized by ce... more Parkinson's disease is one of the most common neurodegenerative disorders characterized by cell death of dopaminergic neurons in the substantia nigra. Recent research has focused on cellular replacement through lineage reprogramming as a potential therapeutic strategy. This study sought to use genetics to define somatic cell types in vivo amenable to reprogramming. To stimulate in vivo reprogramming to dopaminergic neurons, we generated a Rosa26 knock-in mouse line conditionally overexpressing Mash1, Lmx1a, and Nurr1. These proteins are characterized by their role in neuronal commitment and development of midbrain dopaminergic neurons and have previously been shown to convert fibroblasts to dopaminergic neurons in vitro. We show that a tricistronic construct containing these transcription factors can reprogram astrocytes and fibroblasts in vitro. However, cassette overexpression triggered cell death in vivo, in part through endoplasmic reticulum stress, while we also detected &q...

A WNT1-regulated developmental gene cascade prevents dopaminergic neurodegeneration in adult En1(+/-) mice

by Nilima Prakash and Jingzhong Zhang

Neurobiology of disease, Jan 3, 2015

The protracted and age-dependent degeneration of dopamine (DA)-producing neurons of the Substanti... more The protracted and age-dependent degeneration of dopamine (DA)-producing neurons of the Substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) in the mammalian midbrain is a hallmark of human Parkinson's Disease (PD) and of certain genetic mouse models of PD, such as mice heterozygous for the homeodomain transcription factor Engrailed 1 (En1(+/-) mice). Neurotoxin-based animal models of PD, in contrast, are characterized by the fast and partly reversible degeneration of the SNc and VTA DA neurons. The secreted protein WNT1 was previously shown to be strongly induced in the neurotoxin-injured adult ventral midbrain (VM), and to protect the SNc and VTA DA neurons from cell death in this context. We demonstrate here that the sustained and ectopic expression of Wnt1 in the SNc and VTA DA neurons of En1(+/Wnt1) mice also protected these genetically affected En1 heterozygote (En1(+/-)) neurons from their premature degeneration in the adult mouse VM. We identified a deve...

Wnt1-regulated genetic networks in midbrain dopaminergic neuron development

Journal of molecular cell biology, 2014

Neurons synthesizing the neurotransmitter dopamine exert crucial functions in the mammalian brain... more Neurons synthesizing the neurotransmitter dopamine exert crucial functions in the mammalian brain. The biggest and most important population of dopamine-synthesizing neurons is located in the mammalian ventral midbrain (VM), and controls and modulates the execution of motor, cognitive, affective, motivational, and rewarding behaviours. Degeneration of these neurons leads to motor deficits that are characteristic of Parkinson's disease, while their dysfunction is involved in the pathogenesis of psychiatric disorders including schizophrenia and addiction. Because the aetiology and therapeutic prospects for these diseases include neurodevelopmental aspects, substantial scientific interest has been focused on deciphering the mechanistic pathways that control the generation and survival of these neurons during embryonic development. Researches during the last decade revealed the pivotal role of the secreted Wnt1 ligand and its signalling cascade in the generation of the dopamine-synt...

Mouse IDGenes: a reference database for genetic interactions in the developing mouse brain

by Nilima Prakash, Jingzhong Zhang, and Dietrich Trümbach

Database : the journal of biological databases and curation, 2014

The study of developmental processes in the mouse and other vertebrates includes the understandin... more The study of developmental processes in the mouse and other vertebrates includes the understanding of patterning along the anterior-posterior, dorsal-ventral and medial- lateral axis. Specifically, neural development is also of great clinical relevance because several human neuropsychiatric disorders such as schizophrenia, autism disorders or drug addiction and also brain malformations are thought to have neurodevelopmental origins, i.e. pathogenesis initiates during childhood and adolescence. Impacts during early neurodevelopment might also predispose to late-onset neurodegenerative disorders, such as Parkinson's disease. The neural tube develops from its precursor tissue, the neural plate, in a patterning process that is determined by compartmentalization into morphogenetic units, the action of local signaling centers and a well-defined and locally restricted expression of genes and their interactions. While public databases provide gene expression data with spatio-temporal re...

Genetic networks controlling the development of midbrain dopaminergic neurons

The Journal of physiology, 2006

Recent data have substantially advanced our understanding of midbrain dopaminergic neuron develop... more Recent data have substantially advanced our understanding of midbrain dopaminergic neuron development. Firstly, a Wnt1-regulated genetic network, including Otx2 and Nkx2-2, and a Shh-controlled genetic cascade, including Lmx1a, Msx1 and Nkx6-1, have been unravelled, acting in parallel or sequentially to establish a territory competent for midbrain dopaminergic precursor production at relatively early stages of neural development. Secondly, the same factors (Wnt1 and Lmx1a/Msx1) appear to regulate midbrain dopaminergic and/or neuronal fate specification in the postmitotic progeny of these precursors by controlling the expression of midbrain dopaminergic-specific and/or general proneural factors at later stages of neural development. For the first time, early inductive events have thus been linked to later differentiation processes in midbrain dopaminergic neuron development. Given the pivotal importance of this neuronal population for normal function of the human brain and its involv...

Mapping of the midbrain dopaminergic system during development of the chicken embryo reveals evolutionary differences between birds and mammals

International Journal of Developmental Neuroscience, 2006

FGF/FGFR2 Signaling Regulates the Generation and Correct Positioning of Bergmann Glia Cells in the Developing Mouse Cerebellum

by Nilima Prakash and D. Vogt

PLoS ONE, 2014

The normal cellular organization and layering of the vertebrate cerebellum is established during ... more The normal cellular organization and layering of the vertebrate cerebellum is established during embryonic and early postnatal development by the interplay of a complex array of genetic and signaling pathways. Disruption of these processes and of the proper layering of the cerebellum usually leads to ataxic behaviors. Here, we analyzed the relative contribution of Fibroblast growth factor receptor 2 (FGFR2)-mediated signaling to cerebellar development in conditional Fgfr2 single mutant mice. We show that during embryonic mouse development, Fgfr2 expression is higher in the anterior cerebellar primordium and excluded from the proliferative ventricular neuroepithelium. Consistent with this finding, conditional Fgfr2 single mutant mice display the most prominent defects in the anterior lobules of the adult cerebellum. In this context, FGFR2-mediated signaling is required for the proper generation of Bergmann glia cells and the correct positioning of these cells within the Purkinje cell layer, and for cell survival in the developing cerebellar primordium. Using cerebellar microexplant cultures treated with an FGFR agonist (FGF9) or antagonist (SU5402), we also show that FGF9/FGFR-mediated signaling inhibits the outward migration of radial glia and Bergmann glia precursors and cells, and might thus act as a positioning cue for these cells. Altogether, our findings reveal the specific functions of the FGFR2-mediated signaling pathway in the generation and positioning of Bergmann glia cells during cerebellar development in the mouse.

Wnt5a Regulates Ventral Midbrain Morphogenesis and the Development of A9–A10 Dopaminergic Cells In Vivo

PLoS ONE, 2008

Wnt5a is a morphogen that activates the Wnt/planar cell polarity (PCP) pathway and serves multipl... more Wnt5a is a morphogen that activates the Wnt/planar cell polarity (PCP) pathway and serves multiple functions during development. PCP signaling controls the orientation of cells within an epithelial plane as well as convergent extension (CE) movements. Wnt5a was previously reported to promote differentiation of A9-10 dopaminergic (DA) precursors in vitro. However, the signaling mechanism in DA cells and the function of Wnt5a during midbrain development in vivo remains unclear. We hereby report that Wnt5a activated the GTPase Rac1 in DA cells and that Rac1 inhibitors blocked the Wnt5ainduced DA neuron differentiation of ventral midbrain (VM) precursor cultures, linking Wnt5a-induced differentiation with a known effector of Wnt/PCP signaling. In vivo, Wnt5a was expressed throughout the VM at embryonic day (E)9.5, and was restricted to the VM floor and basal plate by E11.5-E13.5. Analysis of Wnt5a2/2 mice revealed a transient increase in progenitor proliferation at E11.5, and a precociously induced NR4A2+ (Nurr1) precursor pool at E12.5. The excess NR4A2+ precursors remained undifferentiated until E14.5, when a transient 25% increase in DA neurons was detected. Wnt5a2/2 mice also displayed a defect in (mid)brain morphogenesis, including an impairment in midbrain elongation and a rounded ventricular cavity. Interestingly, these alterations affected mostly cells in the DA lineage. The ventral Sonic hedgehogexpressing domain was broadened and flattened, a typical CE phenotype, and the domains occupied by Ngn2+ DA progenitors, NR4A2+ DA precursors and TH+ DA neurons were rostrocaudally reduced and laterally expanded. In summary, we hereby describe a Wnt5a regulation of Wnt/PCP signaling in the DA lineage and provide evidence for multiple functions of Wnt5a in the VM in vivo, including the regulation of VM morphogenesis, DA progenitor cell division, and differentiation of NR4A2+ DA precursors.

Spatial Analysis of Expression Patterns Predicts Genetic Interactions at the Mid-Hindbrain Boundary

PLoS Computational Biology, 2009

The isthmic organizer mediating differentiation of mid-and hindbrain during vertebrate developmen... more The isthmic organizer mediating differentiation of mid-and hindbrain during vertebrate development is characterized by a well-defined pattern of locally restricted gene expression domains around the mid-hindbrain boundary (MHB). This pattern is established and maintained by a regulatory network between several transcription and secreted factors that is not yet understood in full detail. In this contribution we show that a Boolean analysis of the characteristic spatial gene expression patterns at the murine MHB reveals key regulatory interactions in this network. Our analysis employs techniques from computational logic for the minimization of Boolean functions. This approach allows us to predict also the interplay of the various regulatory interactions. In particular, we predict a maintaining, rather than inducing, effect of Fgf8 on Wnt1 expression, an issue that remained unclear from published data. Using mouse anterior neural plate/tube explant cultures, we provide experimental evidence that Fgf8 in fact only maintains but does not induce ectopic Wnt1 expression in these explants. In combination with previously validated interactions, this finding allows for the construction of a regulatory network between key transcription and secreted factors at the MHB. Analyses of Boolean, differential equation and reactiondiffusion models of this network confirm that it is indeed able to explain the stable maintenance of the MHB as well as timecourses of expression patterns both under wild-type and various knock-out conditions. In conclusion, we demonstrate that similar to temporal also spatial expression patterns can be used to gain information about the structure of regulatory networks. We show, in particular, that the spatial gene expression patterns around the MHB help us to understand the maintenance of this boundary on a systems level.

Distinct but redundant expression of the Frizzled Wnt receptor genes at signaling centers of the developing mouse brain

Neuroscience, 2007

The establishment of the regional subdivisions of the vertebrate CNS is accomplished through the ... more The establishment of the regional subdivisions of the vertebrate CNS is accomplished through the activity of different neuroepithelial organizing centers. The wingless/int (Wnt) family of secreted glycoproteins, among other factors, plays a crucial role in signaling from these centers. Wnt1 secreted from the boundary between the mid- and hindbrain, for instance, controls the development of this brain region and of associated neuronal populations. Different Wnts secreted from the caudomedial pallium, the cortical hem, pattern the adjacent hippocampal field. The first step in Wnt signal transduction is binding of the Wnt ligand to its receptors, the seven-pass transmembrane Frizzled proteins. Inactivation of different Frizzled genes in mice have revealed an extensive functional redundancy between these receptors. In order to discriminate between a possible participation of different Frizzled receptors in the transduction of Wnt signals at the mid-/hindbrain boundary and the cortical hem, we have performed a detailed expression study of the 10 known murine Frizzled genes at crucial stages of mouse embryonic development. Our analysis reveals a highly dynamic yet distinct expression pattern of individual Frizzled genes in the anterior neural tube of the developing mouse embryo. The overlapping spatio-temporal expression of at least two and up to six Frizzled genes in any region of the developing mouse brain, however, also suggests a vast functional redundancy of the murine Frizzled receptors. This redundancy has to be taken into consideration for future analyses of Frizzled receptor function at these signaling centers in the mouse.

A Wnt Signal Regulates Stem Cell Fate and Differentiation in vivo

Neurodegenerative Diseases, 2007

Our knowledge about the normal generation of midbrain dopaminergic neurons in vivois still rudime... more Our knowledge about the normal generation of midbrain dopaminergic neurons in vivois still rudimentary, despite many attempts to recapitulate the underlying events in vitro. Because the loss of these neurons is implicated in Parkinson&amp;amp;#39;s disease, this lack of information is one of the major drawbacks in the development of better therapies for this severe human neurological disorder. Recently, substantial advances have been made by demonstrating that the secreted molecule Wnt1 regulates a genetic network, including the transcription factors Otx2 and Nkx2-2, for the initial establishment of the dopaminergic progenitor domain in the mammalian ventral midbrain. In addition, Wnt1 appears to regulate the differentiation of the postmitotic progeny of these precursors by initiating the expression of midbrain dopaminergic-specific transcription factors. A genetic cascade controlled by the secreted molecule Sonic hedgehog, including the transcription factors Lmx1a, Msx1 and Nkx6-1, acts in parallel with the Wnt1-regulated network to establish the midbrain dopaminergic progenitor domain. The Sonic-hedgehog-controlled cascade may diverge from the Wnt1-regulated network at later stages of neural development through induction of proneural transcription factors required for the acquisition of generic neuronal properties by the midbrain dopaminergic progeny. Here we provide a brief overview of these regulatory gene networks.

Otx2 controls neuron subtype identity in ventral tegmental area and antagonizes vulnerability to MPTP

Nature Neuroscience, 2010

Mouse Notch 3 Expression in the Pre- and Postnatal Brain: Relationship to the Stroke and Dementia Syndrome CADASIL

by Nilima Prakash and U. Lendahl

Experimental Cell Research, 2002

Mutations in the human Notch 3 gene cause the vascular stroke and dementia syndrome CADASIL (Cere... more Mutations in the human Notch 3 gene cause the vascular stroke and dementia syndrome CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) characterized by degeneration of vascular smooth muscle cells and multiple small infarcts in the white and deep gray matter of the brain. Here we have analyzed the expression pattern of the Notch 3 gene in the pre-and postnatal mouse brain. Prenatal Notch 3 expression is restricted to a scattered population of cells within the vessel wall of all major blood vessels in the developing embryo, including those that form the perineural vascular plexus. Expression in the postnatal brain is confined to a scattered cell population within the vessel wall of small to medium-sized penetrating arteries, which are the vessel type primarily affected in CADASIL patients. In contrast, no expression was observed in capillaries and veins. Notch 3 is most likely expressed in a subset of vascular smooth muscle cells, and the expression pattern of one of the Notch ligands, Serrate 1, was very similar to that observed for Notch 3. The Notch 3 expressing pattern was not significantly altered in platelet-derived growth factor B-(PDGF-B) deficient mouse embryos, demonstrating that Notch 3 expression is not under direct control of PDGF-B. These data show that Notch 3 expression is conserved between mouse and human and suggest that the mouse is a valid system for analysis of CADASIL.

Otx2 selectively controls the neurogenesis of specific neuronal subtypes of the ventral tegmental area and compensates En1-dependent neuronal loss and MPTP vulnerability

Developmental Biology, 2013

Understanding the molecular basis underlying the neurogenesis of mesencephalic-diencephalic Dopam... more Understanding the molecular basis underlying the neurogenesis of mesencephalic-diencephalic Dopaminergic (mdDA) neurons is a major task fueled by their relevance in controlling locomotor activity and emotion and their involvement in neurodegenerative and psychiatric diseases. Increasing evidence suggests that mdDA neurons of the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) represent two main distinct neuronal populations, which, in turn, include specific neuronal subsets. Relevant studies provided important results on mdDA neurogenesis, but, nevertheless, have not yet clarified how the identity of mdDA neuronal subtypes is established and, in particular, whether neurogenic factors may direct progenitors towards the differentiation of specific mdDA neuronal subclasses. The transcription factor Otx2 is required for the neurogenesis of mesencephalic DA (mesDA) neurons and to control neuron subtype identity and sensitivity to the MPTP neurotoxin in the adult VTA. Here we studied whether Otx2 is required in mdDA progenitors for the generation of specific mdDA neuronal subtypes. We found that although expressed in virtually all mdDA progenitors, Otx2 is required selectively for the differentiation of VTA neuronal subtypes expressing Ahd2 and/or Calb but not for those co-expressing Girk2 and glyco-Dat. Moreover, mild over-expression of Otx2 in SNpc progenitors and neurons is sufficient to rescue En1 haploinsufficiency-dependent defects, such as progressive loss and increased MPTP sensitivity of SNpc neurons. Collectively, these data suggest that mdDA progenitors exhibit differential sensitivity to Otx2, which selectively influences the generation of a large and specific subset of VTA neurons. In addition, these data suggest that Otx2 and En1 may share similar properties and control survival and vulnerability to MPTP neurotoxin respectively in VTA and SNpc.