Direct repression of Nanog and Oct4 by OTX2 modulates the contribution of epiblast-derived cells to germline and somatic lineage.

In mammals, the pre-gastrula proximal epiblast gives rise to primordial germ cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work has shown that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here, we show that, in mice, OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and a deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs also occurs in mutant embryos. We propose that the OTX2-mediated repressive control of Oct4 and Nanog is the basis of the mechanism that determines epiblast contribution to germline and somatic lineage.

OTX2 restricts entry to the mouse germline.

The successful segregation of germ cells from somatic lineages is vital for sexual reproduction and species survival. In the mouse, primordial germ cells (PGCs), precursors of all germ cells, are induced from the post-implantation epiblast1. Induction requires BMP4 signalling to prospective PGCs2 and the intrinsic action of PGC transcription factors3-6. However, the molecular mechanisms that connect BMP4 to induction of the PGC transcription factors that are responsible for segregating PGCs from somatic lineages are unknown. Here we show that the transcription factor OTX2 is a key regulator of these processes. Downregulation of Otx2 precedes the initiation of the PGC programme both in vitro and in vivo. Deletion of Otx2 in vitro markedly increases the efficiency of PGC-like cell differentiation and prolongs the period of PGC competence. In the absence of Otx2 activity, differentiation of PGC-like cells becomes independent of the otherwise essential cytokine signals, with germline entry initiating even in the absence of the PGC transcription factor BLIMP1. Deletion of Otx2 in vivo increases PGC numbers. These data demonstrate that OTX2 functions repressively upstream of PGC transcription factors, acting as a roadblock to limit entry of epiblast cells to the germline to a small window in space and time, thereby ensuring correct numerical segregation of germline cells from the soma.

Developmental abnormalities in cortical GABAergic system in mice lacking mGlu3 metabotropic glutamate receptors.

Polymorphic variants of the gene encoding for metabotropic glutamate receptor 3 (mGlu3) are linked to schizophrenia. Because abnormalities of cortical GABAergic interneurons lie at the core of the pathophysiology of schizophrenia, we examined whether mGlu3 receptors influence the developmental trajectory of cortical GABAergic transmission in the postnatal life. mGlu3-/- mice showed robust changes in the expression of interneuron-related genes in the prefrontal cortex (PFC), including large reductions in the expression of parvalbumin (PV) and the GluN1 subunit of NMDA receptors. The number of cortical cells enwrapped by perineuronal nets was increased in mGlu3-/- mice, suggesting that mGlu3 receptors shape the temporal window of plasticity of PV+ interneurons. Electrophysiological measurements of GABAA receptor-mediated responses revealed a more depolarized reversal potential of GABA currents in the somata of PFC pyramidal neurons in mGlu3-/- mice at postnatal d 9 associated with a reduced expression of the K+/Cl- symporter. Finally, adult mGlu3-/- mice showed lower power in electroencephalographic rhythms at 1-45 Hz in quiet wakefulness as compared with their wild-type counterparts. These findings suggest that mGlu3 receptors have a strong impact on the development of cortical GABAergic transmission and cortical neural synchronization mechanisms corroborating the concept that genetic variants of mGlu3 receptors may predispose to psychiatric disorders.-Imbriglio, T., Verhaeghe, R., Martinello, K., Pascarelli, M. T., Chece, G., Bucci, D., Notartomaso, S., Quattromani, M., Mascio, G., Scalabrì, F., Simeone, A., Maccari, S., Del Percio, C., Wieloch, T., Fucile, S., Babiloni, C., Battaglia, G., Limatola, C., Nicoletti, F., Cannella, M. Developmental abnormalities in cortical GABAergic system in mice lacking mGlu3 metabotropic glutamate receptors.

Otx2 cell-autonomously determines dorsal mesencephalon versus cerebellum fate independently of isthmic organizing activity.

During embryonic development, the rostral neuroectoderm is regionalized into broad areas that are subsequently subdivided into progenitor compartments with specialized identity and fate. These events are controlled by signals emitted by organizing centers and interpreted by target progenitors, which activate superimposing waves of intrinsic factors restricting their identity and fate. The transcription factor Otx2 plays a crucial role in mesencephalic development by positioning the midbrain-hindbrain boundary (MHB) and its organizing activity. Here, we investigated whether Otx2 is cell-autonomously required to control identity and fate of dorsal mesencephalic progenitors. With this aim, we have inactivated Otx2 in the Pax7(+) dorsal mesencephalic domain, previously named m1, without affecting MHB integrity. We found that the Pax7(+) m1 domain can be further subdivided into a dorsal Zic1(+) m1a and a ventral Zic1(-) m1b sub-domain. Loss of Otx2 in the m1a (Pax7(+) Zic1(+)) sub-domain impairs the identity and fate of progenitors, which undergo a full switch into a coordinated cerebellum differentiation program. By contrast, in the m1b sub-domain (Pax7(+) Zic1(-)) Otx2 is prevalently required for post-mitotic transition of mesencephalic GABAergic precursors. Moreover, genetic cell fate, BrdU cell labeling and Otx2 conditional inactivation experiments indicate that in Otx2 mutants all ectopic cerebellar cell types, including external granule cell layer (EGL) precursors, originate from the m1a progenitor sub-domain and that reprogramming of mesencephalic precursors into EGL or cerebellar GABAergic progenitors depends on temporal sensitivity to Otx2 ablation. Together, these findings indicate that Otx2 intrinsically controls different aspects of dorsal mesencephalic neurogenesis. In this context, Otx2 is cell-autonomously required in the m1a sub-domain to suppress cerebellar fate and promote mesencephalic differentiation independently of the MHB organizing activity.

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

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

Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes.

In the human, mutations of OTX2 (Orthodenticle homeobox 2 transcription factor) translate into eye malformations of variable expressivity (even between the two eyes of the same individual) and incomplete penetrance, suggesting the existence of subtle thresholds in OTX2 activity. We have addressed this issue by analyzing retinal structure and function in six mutant mice with graded Otx2 activity: Otx2(+/+), Otx2(+/AA), Otx2(+/GFP), Otx2(AA/AA), Otx2(AA/GFP) and Otx2(GFP/GFP). Null mice (Otx2(GFP/GFP)) fail to develop the head and are embryonic lethal, and compound heterozygous Otx2(AA/GFP) mice show a truncated head and die at birth. All other genotypes develop until adulthood. We analyzed eye structure and visual physiology in the genotypes that develop until adulthood and report that phenotype severity parallels Otx2 activity. Otx2(+/AA) are only mildly affected whereas Otx2(+/GFP) are more affected than Otx2(+/AA) but less than Otx2(AA/AA) mice. Otx2(AA/AA) mice later manifest the most severe defects, with variable expressivity. Electrophysiological and histological analyses of the mouse retina revealed progressive death of bipolar cells and cone photoreceptors that is both Otx2 activity- and age-dependent with the same ranking of phenotypic severity. This study demonstrates the importance of gene dosage in the development of age-dependent pathologies and underscores the fact that small gene dosage differences can cause significant pathological states.

Otx2 is an intrinsic determinant of the embryonic stem cell state and is required for transition to a stable epiblast stem cell condition.

Mouse embryonic stem cells (ESCs) represent the naïve ground state of the preimplantation epiblast and epiblast stem cells (EpiSCs) represent the primed state of the postimplantation epiblast. Studies have revealed that the ESC state is maintained by a dynamic mechanism characterized by cell-to-cell spontaneous and reversible differences in sensitivity to self-renewal and susceptibility to differentiation. This metastable condition ensures indefinite self-renewal and, at the same time, predisposes ESCs for differentiation to EpiSCs. Despite considerable advances, the molecular mechanism controlling the ESC state and pluripotency transition from ESCs to EpiSCs have not been fully elucidated. Here we show that Otx2, a transcription factor essential for brain development, plays a crucial role in ESCs and EpiSCs. Otx2 is required to maintain the ESC metastable state by antagonizing ground state pluripotency and promoting commitment to differentiation. Furthermore, Otx2 is required for ESC transition into EpiSCs and, subsequently, to stabilize the EpiSC state by suppressing, in pluripotent cells, the mesendoderm-to-neural fate switch in cooperation with BMP4 and Fgf2. However, according to its central role in neural development and differentiation, Otx2 is crucially required for the specification of ESC-derived neural precursors fated to generate telencephalic and mesencephalic neurons. We propose that Otx2 is a novel intrinsic determinant controlling the functional integrity of ESCs and EpiSCs.

The transcription factor Otx2 regulates choroid plexus development and function.

The choroid plexuses (ChPs) are the main regulators of cerebrospinal fluid (CSF) composition and thereby also control the composition of a principal source of signaling molecules that is in direct contact with neural stem cells in the developing brain. The regulators of ChP development mediating the acquisition of a fate that differs from the neighboring neuroepithelial cells are poorly understood. Here, we demonstrate in mice a crucial role for the transcription factor Otx2 in the development and maintenance of ChP cells. Deletion of Otx2 by the Otx2-CreERT2 driver line at E9 resulted in a lack of all ChPs, whereas deletion by the Gdf7-Cre driver line affected predominately the hindbrain ChP, which was reduced in size, primarily owing to an increase in apoptosis upon Otx2 deletion. Strikingly, Otx2 was still required for the maintenance of hindbrain ChP cells at later stages when Otx2 deletion was induced at E15, demonstrating a central role of Otx2 in ChP development and maintenance. Moreover, the predominant defects in the hindbrain ChP mediated by Gdf7-Cre deletion of Otx2 revealed its key role in regulating early CSF composition, which was altered in protein content, including the levels of Wnt4 and the Wnt modulator Tgm2. Accordingly, proliferation and Wnt signaling levels were increased in the distant cerebral cortex, suggesting a role of the hindbrain ChP in regulating CSF composition, including key signaling molecules. Thus, Otx2 acts as a master regulator of ChP development, thereby influencing one of the principal sources of signaling in the developing brain, the CSF.

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

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 developmental gene cascade that is up-regulated in the adult En1(+/Wnt1) VM, including the direct WNT1/ß-catenin signaling targets Lef1, Lmx1a, Fgf20 and Dkk3, as well as the indirect targets Pitx3 (activated by LMX1A) and Bdnf (activated by PITX3). We also show that the secreted neurotrophin BDNF and the secreted WNT modulator DKK3, but not the secreted growth factor FGF20, increased the survival of En1 mutant dopaminergic neurons in vitro. The WNT1-mediated signaling pathway and its downstream targets BDNF and DKK3 might thus provide a useful means to treat certain genetic and environmental (neurotoxic) forms of human PD.

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

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.

Embryonic defects and growth alteration in mice with homozygous disruption of the Patz1 gene.

PATZ1 is an emerging cancer-related gene coding for a POZ/AT-hook/kruppel Zinc finger transcription factor, which is lost or misexpressed in human neoplasias. Here, we investigated its role in development exploring wild-type and Patz1-knockout mice during embryogenesis. We report that the Patz1 gene is ubiquitously expressed at early stages of development and becomes more restricted at later stages, with high levels of expression in actively proliferating neuroblasts belonging to the ventricular zones of the central nervous system (CNS). The analysis of embryos in which Patz1 was disrupted revealed the presence of severe defects in the CNS and in the cardiac outflow tract, which eventually lead to a pre-mature in utero death during late gestation or soon after birth. Moreover, the Patz1-null mice showed a general growth retardation, which was consistent with the slower growth rate and the increased susceptibility to senescence of Patz1(-/-) mouse embryonic fibroblasts (MEFs) compared to wild-type controls. Therefore, these results indicate a critical role of PATZ1 in the control of cell growth and embryonic development.

Cellular heterogeneity during embryonic stem cell differentiation to epiblast stem cells is revealed by the ShcD/RaLP adaptor protein.

The Shc family of adaptor proteins are crucial mediators of a plethora of receptors such as the tyrosine kinase receptors, cytokine receptors, and integrins that drive signaling pathways governing proliferation, differentiation, and migration. Here, we report the role of the newly identified family member, ShcD/RaLP, whose expression in vitro and in vivo suggests a function in embryonic stem cell (ESC) to epiblast stem cells (EpiSCs) transition. The transition from the naïve (ESC) to the primed (EpiSC) pluripotent state is the initial important step for ESCs to commit to differentiation and the mechanisms underlying this process are still largely unknown. Using a novel approach to simultaneously assess pluripotency, apoptosis, and proliferation by multiparameter flow cytometry, we show that ESC to EpiSC transition is a process involving a tight coordination between the modulation of the Oct4 expression, cell cycle progression, and cell death. We also describe, by high-content immunofluorescence analysis and time-lapse microscopy, the emergence of cells expressing caudal-related homeobox 2 (Cdx2) transcription factor during ESC to EpiSC transition. The use of the ShcD knockout ESCs allowed the unmasking of this process as they presented deregulated Oct4 modulation and an enrichment in Oct4-negative Cdx2-positive cells with increased MAPK/extracellular-regulated kinases 1/2 activation, within the differentiating population. Collectively, our data reveal ShcD as an important modulator in the switch of key pathway(s) involved in determining EpiSC identity.

Common and Rare Variants in TMEM175 Gene Concur to the Pathogenesis of Parkinson's Disease in Italian Patients.

Parkinson's disease (PD) represents the most common neurodegenerative movement disorder. We recently identified 16 novel genes associated with PD. In this study, we focused the attention on the common and rare variants identified in the lysosomal K+ channel TMEM175. The study includes a detailed clinical and genetic analysis of 400 cases and 300 controls. Molecular studies were performed on patient-derived fibroblasts. The functional properties of the mutant channels were assessed by patch-clamp technique and co-immunoprecipitation. We have found that TMEM175 was highly expressed in dopaminergic neurons of the substantia nigra pars compacta and in microglia of the cerebral cortex of the human brain. Four common variants were associated with PD, including two novel variants rs2290402 (c.-10C > T) and rs80114247 (c.T1022C, p.M341T), located in the Kozak consensus sequence and TM3II domain, respectively. We also disclosed 13 novel highly penetrant detrimental mutations in the TMEM175 gene associated with PD. At least nine of these mutations (p.R35C, p. R183X, p.A270T, p.P308L, p.S348L, p. L405V, p.R414W, p.P427fs, p.R481W) may be sufficient to cause the disease, and the presence of mutations of other genes correlated with an earlier disease onset. In vitro functional analysis of the ion channel encoded by the mutated TMEM175 gene revealed a loss of the K+ conductance and a reduced channel affinity for Akt. Moreover, we observed an impaired autophagic/lysosomal proteolytic flux and an increase expression of unfolded protein response markers in patient-derived fibroblasts. These data suggest that mutations in TMEM175 gene may contribute to the pathophysiology of PD.

Fgf15-mediated control of neurogenic and proneural gene expression regulates dorsal midbrain neurogenesis.

The balanced proliferation and cell cycle exit of neural progenitors, by generating the appropriate amount of postmitotic progeny at the correct time and in the proper location, is required for the establishment of the highly ordered structure of the adult brain. Little is known about the extrinsic signals regulating these processes, particularly in the midbrain. Fibroblast growth factor (Fgf) 15, the mouse ortholog of FGF19 and member of an atypical Fgf subfamily, is prominently expressed in the dorsolateral midbrain of the midgestational mouse embryo. In the absence of Fgf15, dorsal midbrain neural progenitors fail to exit the cell cycle and to generate the proper amount of postmitotic neurons. We show here that this is due to the altered expression of inhibitory/neurogenic and proneural/neuronal differentiation helix-loop-helix transcription factor (TF) genes. The expression of Id1, Id3, and Hes5 was strongly increased and ectopically expanded, whereas the expression of Ascl1 (Mash1), Neurog1 (Ngn1) and Neurog2 (Ngn2) was strongly decreased and transcription of Neurod1 (NeuroD) was completely abolished in the dorsolateral midbrain of Fgf15(-/-) mice. These abnormalities were not caused by the mis-expression of cell cycle regulatory proteins such as cyclin-dependent kinase inhibitors or retinoblastoma proteins. Furthermore, human FGF19 promotes cell cycle exit of murine dorsal neural progenitors in vitro. Therefore, our data suggest that Fgf15 is a crucial signaling molecule regulating the postmitotic transition of dorsal neural progenitors and thus the initiation and proper progression of dorsal midbrain neurogenesis in the mouse, by controlling the expression of neurogenic and proneural TFs.

The transcription factor orthodenticle homeobox 2 influences axonal projections and vulnerability of midbrain dopaminergic neurons.

Two adjacent groups of midbrain dopaminergic neurons, A9 (substantia nigra pars compacta) and A10 (ventral tegmental area), have distinct projections and exhibit differential vulnerability in Parkinson's disease. Little is known about transcription factors that influence midbrain dopaminergic subgroup phenotypes or their potential role in disease. Here, we demonstrate elevated expression of the transcription factor orthodenticle homeobox 2 in A10 dopaminergic neurons of embryonic and adult mouse, primate and human midbrain. Overexpression of orthodenticle homeobox 2 using lentivirus increased levels of known A10 elevated genes, including neuropilin 1, neuropilin 2, slit2 and adenylyl cyclase-activating peptide in both MN9D cells and ventral mesencephalic cultures, whereas knockdown of endogenous orthodenticle homeobox 2 levels via short hairpin RNA reduced expression of these genes in ventral mesencephalic cultures. Lack of orthodenticle homeobox 2 in the ventral mesencephalon of orthodenticle homeobox 2 conditional knockout mice caused a reduction of midbrain dopaminergic neurons and selective loss of A10 dopaminergic projections. Orthodenticle homeobox 2 overexpression protected dopaminergic neurons in ventral mesencephalic cultures from Parkinson's disease-relevant toxin, 1-methyl-4-phenylpyridinium, whereas downregulation of orthodenticle homeobox 2 using short hairpin RNA increased their susceptibility. These results show that orthodenticle homeobox 2 is important for establishing subgroup phenotypes of post-mitotic midbrain dopaminergic neurons and may alter neuronal vulnerability.

Bmp5/7 in concert with the mid-hindbrain organizer control development of noradrenergic locus coeruleus neurons.

The locus coeruleus (LC) which is the major noradrenergic nucleus in the brain develops under the influence of Bmps secreted by the roof plate and Fgf8 emitted from the mid-hindbrain organizer. We studied the development of the LC in different Bmp mouse mutants and report the absence of this nucleus in Bmp5(-/-);Bmp7(-/-) double knockouts. Notably, genes marking organizers and neuronal populations adjacent to the LC precursor field are unperturbed in Bmp5(-/-);Bmp7(-/-) animals. In addition, we found that in En1(+/Otx2) mutants in which the caudal Otx2 expression domain and thereby the mid-hindbrain organizer are shifted caudally, LC neurons are concomitantly reduced along with Bmp5/7. Complementing these results, Otx1(-/-);Otx2(+/-) mutants, in which the mid-hinbrain organizer is shifted rostrally, show a rostrally extended Bmp5 expression area and an increase in LC neurons. Taken together, our data indicate that LC development requires either Bmp5 or Bmp7, and one is able to compensate for the loss of the other. In addition, we conclude that the position of the mid-hindbrain organizer determines the size of the LC and propose that Bmp5/7 play an important role in mediating this organizer function.

Identification of sixteen novel candidate genes for late onset Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative movement disorder affecting 1-5% of the general population for which neither effective cure nor early diagnostic tools are available that could tackle the pathology in the early phase. Here we report a multi-stage procedure to identify candidate genes likely involved in the etiopathogenesis of PD. METHODS: The study includes a discovery stage based on the analysis of whole exome data from 26 dominant late onset PD families, a validation analysis performed on 1542 independent PD patients and 706 controls from different cohorts and the assessment of polygenic variants load in the Italian cohort (394 unrelated patients and 203 controls). RESULTS: Family-based approach identified 28 disrupting variants in 26 candidate genes for PD including PARK2, PINK1, DJ-1(PARK7), LRRK2, HTRA2, FBXO7, EIF4G1, DNAJC6, DNAJC13, SNCAIP, AIMP2, CHMP1A, GIPC1, HMOX2, HSPA8, IMMT, KIF21B, KIF24, MAN2C1, RHOT2, SLC25A39, SPTBN1, TMEM175, TOMM22, TVP23A and ZSCAN21. Sixteen of them have not been associated to PD before, were expressed in mesencephalon and were involved in pathways potentially deregulated in PD. Mutation analysis in independent cohorts disclosed a significant excess of highly deleterious variants in cases (p = 0.0001), supporting their role in PD. Moreover, we demonstrated that the co-inheritance of multiple rare variants (≥ 2) in the 26 genes may predict PD occurrence in about 20% of patients, both familial and sporadic cases, with high specificity (> 93%; p = 4.4 × 10- 5). Moreover, our data highlight the fact that the genetic landmarks of late onset PD does not systematically differ between sporadic and familial forms, especially in the case of small nuclear families and underline the importance of rare variants in the genetics of sporadic PD. Furthermore, patients carrying multiple rare variants showed higher risk of manifesting dyskinesia induced by levodopa treatment. CONCLUSIONS: Besides confirming the extreme genetic heterogeneity of PD, these data provide novel insights into the genetic of the disease and may be relevant for its prediction, diagnosis and treatment.

Orthopedia homeodomain protein is essential for diencephalic dopaminergic neuron development.

Neurons that produce dopamine as a neurotransmitter constitute a heterogeneous group involved in the control of various behaviors and physiology. In mammals, dopaminergic neurons are found in distinct clusters mainly located in the ventral midbrain and the caudal forebrain [1]. Although much is known about midbrain dopaminergic neurons, development of diencephalic dopaminergic neurons is poorly understood. Here we demonstrate that Orthopedia (Otp) homeodomain protein is essential for the development of specific subsets of diencephalic dopaminergic neurons. Zebrafish embryos lacking Otp activity are devoid of dopaminergic neurons in the hypothalamus and the posterior tuberculum. Similarly, Otp-/- mouse [2, 3] embryos lack diencephalic dopaminergic neurons of the A11 group, which constitutes the diencephalospinal dopaminergic system. In both systems, Otp is expressed in the affected dopaminergic neurons as well as in potential precursor populations, and it might contribute to dopaminergic cell specification and differentiation. In fish, overexpression of Otp can induce ectopic tyrosine hydroxylase and dopamine transporter expression, indicating that Otp can specify aspects of dopaminergic identity. Thus, Otp is one of the few known transcription factors that can determine aspects of the dopaminergic phenotype and the first known factor to control the development of the diencephalospinal dopaminergic system.

Expression of the brain transcription factor OTX1 occurs in a subset of normal germinal-center B cells and in aggressive Non-Hodgkin Lymphoma.

The roles in brain development. Previous studies have shown the association between OTX2 and OTX1 with anaplastic and desmoplastic medulloblastomas, respectively. Here, we investigated OTX1 and OTX2 expression in Non-Hodgkin Lymphoma (NHL) and multiple myeloma. A combination of semiquantitative RT-PCR, Western blot, and immunohistochemical analyses was used to measure OTX1 and OTX2 levels in normal lymphoid tissues and in 184 tumor specimens representative of various forms of NHL and multiple myeloma. OTX1 expression was activated in 94% of diffuse large B-cell lymphomas, in all Burkitt lymphomas, and in 90% of high-grade follicular lymphomas. OTX1 was undetectable in precursor-B lymphoblastic lymphoma, chronic lymphocytic leukemia, and in most marginal zone and mantle cell lymphomas and multiple myeloma. OTX2 was undetectable in all analyzed malignancies. Analysis of OTX1 expression in normal lymphoid tissues identified a subset of resting germinal center (GC) B cells lacking PAX5 and BCL6 and expressing cytoplasmic IgG and syndecan. About 50% of OTX1(+) GC B cells co-expressed CD10 and CD20. This study identifies OTX1 as a molecular marker for high-grade GC-derived NHL and suggests an involvement of this transcription factor in B-cell lymphomagenesis. Furthermore, OTX1 expression in a subset of normal GC B cells carrying plasma cell markers suggests its possible contribution to terminal B-cell differentiation.

Gbx2 and Otx2 interact with the WD40 domain of Groucho/Tle corepressors.

One of the earliest organizational decisions in the development of the vertebrate brain is the division of the neural plate into Otx2-positive anterior and Gbx2-positive posterior territories. At the junction of these two expression domains, a local signaling center is formed, known as the midbrain-hindbrain boundary (MHB). This tissue coordinates or "organizes" the development of neighboring brain structures, such as the midbrain and cerebellum. Correct positioning of the MHB is thought to depend on mutual repression involving these two homeobox genes. Using a cell culture colocalization assay and coimmunoprecipitation experiments, we show that engrailed homology region 1 (eh1)-like motifs of both transcription factors physically interact with the WD40 domain of Groucho/Tle corepressor proteins. In addition, heat shock-induced expression of wild-type and mutant Otx2 and Gbx2 in medaka embryos demonstrates that Groucho is required for the repression of Otx2 by Gbx2. On the other hand, the repressive functions of Otx2 on Gbx2 do not appear to be dependent on corepressor interaction. Interestingly, the association of Groucho with Otx2 is also required for the repression of Fgf8 in the MHB. Therefore Groucho/Tle family members appear to regulate key aspects in the MHB development of the vertebrate brain.