Identification and characterization of genes preferentially expressed in embryonic telencephalon and CNS stem cells

One of the major goals in developmental neurobiology is to unravel the molecular programs controlling telencephalic development and neural differentiation. The complexity of brain cell types and circuits is reflected in the complexity of gene expression patterns in the brain. It is believed that perhaps a third to half of all genes are largely or exclusively dedicated to directing development, maintenance and functioning of the brain. In mammals, formation of the complex brain structure occurs over the long period of prenatal development. During this period neural progenitor cells must be instructed to undergo proper proliferation, migration, differentiation and connectivity. The aim of my study was to identify genes, within a collection of novel genes preferentially expressed in the embryonic telencephalon, controlling such processed in the mammalian forebrain. To this aim, as a preliminary step, an EST sequencing approach has been undertaken to catalogue and array the repertoire of genes represented in a subtractive library optimized to select rate or unique cDNAs preferentially expressed in the E14.5 mouse telencephalon (named "Telencephalic Embryonic Subtracted Sequences" (Porteus et al., 1992)). The hypothesis driving the production of such a library was that genes preferentially expressed during embryogenesis are likely to be specifically involved in the development of the telencephalon and in the biology of the neural progenitor cells. The selected transcriptome of 1026 unique cDNAs has been used to generate a unique microarray, and to perform gene expression profiling experiments on: (i) mice mutant for transcription factors involved in forebrain development (D1x1/2, Nkx2.1, Pax6, Ngn1/2), (ii) in vitro cultured neural stem cells, committed neural progenitor cells (transient amplifying) and terminally differentiated neural cells. The analysis of the resulting expression profiles has allowed the identification of novel genes that are candidates for playing a major role in neurogenesis and telencephalic development. The differential expression identified with the Tess has been validated using RNA in situ hybridization on embryonic tissue. Two novel genes (corresponding to Tess 28.8E and Tess 31.5E) have been found to be specifically down regulated in D1x1/2-/- subpallium (-46,51 fold for 28.8E; -6,44 fold for 31.5E), and up regulated in Pax6-/- (4,34 for 28.8E; 3,15 for 31.5E) and Ngn1/2-/- (9,02 for 28.8e; 5,04 for 31.5E) pallium The microarray experiments on neural stem cells allowed the identification of a selection of genes putatively involved in the process of self-renewal, lineage commitment and differentiation. Some of these genes have been analysed by RNA in situ hybridizations and demonstrated interesting restricted expression patterns in the developing telencephalon

Human Pluripotent Stem Cell Differentiation into Authentic Striatal Projection Neurons

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Genome-wide definition of promoter and enhancer usage during neural induction of human embryonic stem cells

Genome-wide mapping of transcriptional regulatory elements is an essential tool for understanding the molecular events orchestrating self-renewal, commitment and differentiation of stem cells. We combined high-throughput identification of transcription start sites with genome-wide profiling of histones modifications to map active promoters and enhancers in embryonic stem cells (ESCs) induced to neuroepithelial-like stem cells (NESCs). Our analysis showed that most promoters are active in both cell types while approximately half of the enhancers are cell-specific and account for most of the epigenetic changes occurring during neural induction, and most likely for the modulation of the promoters to generate cell-specific gene expression programs. Interestingly, the majority of the promoters activated or up-regulated during neural induction have a "bivalent" histone modification signature in ESCs, suggesting that developmentally-regulated promoters are already poised for transcription in ESCs, which are apparently pre-committed to neuroectodermal differentiation. Overall, our study provides a collection of differentially used enhancers, promoters, transcription starts sites, protein-coding and non-coding RNAs in human ESCs and ESC-derived NESCs, and a broad, genome-wide description of promoter and enhancer usage and of gene expression programs characterizing the transition from a pluripotent to a neural-restricted cell fate

Sprouty2 mediated tuning of signalling is essential for somite myogenesis

Background: Negative regulators of signal transduction cascades play critical roles in controlling different aspects of normal embryonic development. Sprouty2 (Spry2) negatively regulates receptor tyrosine kinases (RTK) and FGF signalling and is important in differentiation, cell migration and proliferation. In vertebrate embryos, Spry2 is expressed in paraxial mesoderm and in forming somites. Expression is maintained in the myotome until late stages of somite differentiation. However, its role and mode of action during somite myogenesis is still unclear. Results: Here, we analysed chick Spry2 expression and showed that it overlaps with that of myogenic regulatory factors MyoD and Mgn. Targeted mis-expression of Spry2 led to inhibition of myogenesis, whilst its C-terminal domain led to an increased number of myogenic cells by stimulating cell proliferation. Conclusions: Spry2 is expressed in somite myotomes and its expression overlaps with myogenic regulatory factors. Overexpression and dominant-negative interference showed that Spry2 plays a crucial role in regulating chick myogenesis by fine tuning of FGF signaling through a negative feedback loop. We also propose that mir-23, mir-27 and mir-128 could be part of the negative feedback loop mechanism. Our analysis is the first to shed some light on in vivo Spry2 function during chick somite myogenesis

Stem Cell-Derived Human Striatal Progenitors Innervate Striatal Targets and Alleviate Sensorimotor Deficit in a Rat Model of Huntington Disease

Huntington disease (HD) is an inherited late-onset neurological disorder characterized by progressive neuronal loss and disruption of cortical and basal ganglia circuits. Cell replacement using human embryonic stem cells may offer the opportunity to repair the damaged circuits and significantly ameliorate disease conditions. Here, we showed that in-vitro-differentiated human striatal progenitors undergo maturation and integrate into host circuits upon intra-striatal transplantation in a rat model of HD. By combining graft-specific immunohistochemistry, rabies virus-mediated synaptic tracing, and ex vivo electrophysiology, we showed that grafts can extend projections to the appropriate target structures, including the globus pallidus, the subthalamic nucleus, and the substantia nigra, and receive synaptic contact from both host and graft cells with 6.6 ± 1.6 inputs cell per transplanted neuron. We have also shown that transplants elicited a significant improvement in sensory-motor tasks up to 2 months post-transplant further supporting the therapeutic potential of this approach

The coding and long noncoding single-cell atlas of the developing human fetal striatum.

Deciphering how the human striatum develops is necessary for understanding the diseases that affect this region. To decode the transcriptional modules that regulate this structure during development, we compiled a catalog of 1116 long intergenic noncoding RNAs (lincRNAs) identified de novo and then profiled 96,789 single cells from the early human fetal striatum. We found that D1 and D2 medium spiny neurons (D1- and D2-MSNs) arise from a common progenitor and that lineage commitment is established during the postmitotic transition, across a pre-MSN phase that exhibits a continuous spectrum of fate determinants. We then uncovered cell type-specific gene regulatory networks that we validated through in silico perturbation. Finally, we identified human-specific lincRNAs that contribute to the phylogenetic divergence of this structure in humans. This work delineates the cellular hierarchies governing MSN lineage commitment

The Level of the Transcription Factor Pax6 Is Essential for Controlling the Balance between Neural Stem Cell Self-Renewal and Neurogenesis

Neural stem cell self-renewal, neurogenesis, and cell fate determination are processes that control the generation of specific classes of neurons at the correct place and time. The transcription factor Pax6 is essential for neural stem cell proliferation, multipotency, and neurogenesis in many regions of the central nervous system, including the cerebral cortex. We used Pax6 as an entry point to define the cellular networks controlling neural stem cell self-renewal and neurogenesis in stem cells of the developing mouse cerebral cortex. We identified the genomic binding locations of Pax6 in neocortical stem cells during normal development and ascertained the functional significance of genes that we found to be regulated by Pax6, finding that Pax6 positively and directly regulates cohorts of genes that promote neural stem cell self-renewal, basal progenitor cell genesis, and neurogenesis. Notably, we defined a core network regulating neocortical stem cell decision-making in which Pax6 interacts with three other regulators of neurogenesis, Neurog2, Ascl1, and Hes1. Analyses of the biological function of Pax6 in neural stem cells through phenotypic analyses of Pax6 gain- and loss-of-function mutant cortices demonstrated that the Pax6-regulated networks operating in neural stem cells are highly dosage sensitive. Increasing Pax6 levels drives the system towards neurogenesis and basal progenitor cell genesis by increasing expression of a cohort of basal progenitor cell determinants, including the key transcription factor Eomes/Tbr2, and thus towards neurogenesis at the expense of self-renewal. Removing Pax6 reduces cortical stem cell self-renewal by decreasing expression of key cell cycle regulators, resulting in excess early neurogenesis. We find that the relative levels of Pax6, Hes1, and Neurog2 are key determinants of a dynamic network that controls whether neural stem cells self-renew, generate cortical neurons, or generate basal progenitor cells, a mechanism that has marked parallels with the transcriptional control of embryonic stem cell self-renewal

Pcp4l1, a novel gene encoding a Pcp4-like polypeptide, is expressed in specific domains of the developing brain

We report the cloning of a novel mouse gene (Pcp4l1) that encodes a polypeptide with significant sequence similarity to the Purkinje cell protein 4 gene (Pcp4) and describe its expression pattern during mouse development. Similar to Pcp4, the Pc4l1 gene product is characterized by the presence of an IQ domain and is highly conserved across evolution. RNA in situ hybridization reveals instead that Pcp4l1 has a distinct pattern of expression: it is only expressed in the central nervous system (CNS), and is first detected at E9.5 in the mesencephalic and metencephalic roof plate as well as in the isthmus, in a region that overlaps the expression domains of Pax2, Fgf8 and Wnt1. Thus, the early Pcp4l1 expression pattern coincides with the regional expression of well-characterized patterning molecules in the organizing centers of the developing brain. Starting at midgestation, Pcp4l1 is mainly expressed in the structures of the circumventricular organs, including the subcommissural organ, the rhombencephalic and telencephalic choroid plexi, and the pineal gland. In the adult brain, this transcript is also detected in laminar as well as in several nuclear structures of the CNS.This work was supported by the Italian Telethon Foundation, by the European Community (grant nr. QLRT-1999-00793) and by the Fondazione Agarini.Peer reviewe

Long-term follow-up of custom-made porous hydroxyapatite cranioplasty in adult patients: a multicenter European study. Can we trust self-reported complications?

BackgroundCranioplasty is a surgical intervention aiming to re-establish the integrity of skull defects. Autologous bone and different heterologous materials are used for this purpose, with various reported related complications. The aim of the study was to evaluate the complication rate in a multicentric cohort of patients underwent porous hydroxyapatite (PHA) cranioplasty implantation and to assess the validity of company post-market clinical analysis.MethodsAuthors analyzed a company based register of 6279 PHA cranioplasty implanted all over the world. In these adult patients only self-reported complications were available. We then obtained the data of adult patients treated with custom-made porous HA prostheses (CustomBone Service) in 20 institutions from different European countries through an on-site interview with the physicians in charge of the patients (494 patients). The endpoints were the incidence of adverse events and of related implant removal.ResultsThe groups of patients had similar demographics characteristics. The average follow-up was 26.7 months. A significantly higher number of complications was recorded in the group of patients underwent onsite interview. Thirty-nine complications were reported (7.89%) with an explantation rate of 4.25% (21 cases) in the series, compared to the data reported from the Company (complications rate of 3.3% and explantation rate of 3.1%). The most common complications were infection (4.86%), hematomas (1.22%), fractures (1.01%), mobilization (0.4%) and scar retraction (0.4%).ConclusionsOur data confirm that porous HA cranioplasty is at least as effective as other heterologous materials to repair cranial defects. Another interesting finding is that self-reporting complicantions by surgeons does not give a precise picture of the real rate of complications of the devices. These data in future studies need to be re-confirmed with on-site interviews

Sp8 and COUP-TF1 Reciprocally Regulate Patterning and Fgf Signaling in Cortical Progenitors.

International audienceTo gain new insights into the transcriptional regulation of cortical development, we examined the role of the transcription factor Sp8, which is downstream of Fgf8 signaling and known to promote rostral cortical development. We have used a binary transgenic system to express Sp8 throughout the mouse telencephalon in a temporally restricted manner. Our results show that misexpression of Sp8 throughout the telencephalon, at early but not late embryonic stages, results in cortical hypoplasia, which is accompanied by increased cell death, reduced proliferation, and precocious neuronal differentiation. Misexpression of Sp8 at early developmental stages represses COUP-TF1 expression, a negative effector of Fgf signaling and a key promoter of posterior cortical identity, while ablation of Sp8 has the opposite effect. In addition, transgenic misexpression of COUP-TF1 resulted in downregulation of Sp8, indicating a reciprocal cross-regulation between these 2 transcription factors. Although Sp8 has been suggested to induce and/or maintain Fgf8 expression in the embryonic telencephalon, neither Fgf8 nor Fgf15 was upregulated using our gain-of-function approach. However, misexpression of Sp8 greatly increased the expression of Fgf target molecules, suggesting enhanced Fgf signaling. Thus, we propose that Sp8 promotes rostral and dorsomedial cortical development by repressing COUP-TF1 and promoting Fgf signaling in pallial progenitors