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PDF file - 213K, Gene signatures distinguishing the different MB CSC populations segregate distinct human MB molecular subgroups, with MB1_EXCLUSIVE gene signature distinguishing the WNT molecular subgroup

PDF file - 183K, Long term-cultured hindbrain- and forebrain-derived NSCs are Sonic Hedgehog (Shh) pathway-independent, as cyclopamine treatment negatively affects the survival/proliferation of short-term cultured but not of long term-cultured NSCs

PDF file - 155K, Enforced expression of Ebf3 in CB WM and SVZ NSCs results in premature neuronal differentiation, under proliferative and differentiative conditions

PDF file - 157K, MB CSC lines are Sonic Hedgehog (Shh) pathway-independent, as their survival/proliferation is not affected by cyclopamine treatment and Smo silencing

PDF file - 104K, Details on experimental and bioinformatics procedures.

PDF file - 134K, Table S1 A. Lineage-specific differentiation of region-specific clonally derived NSCs is summarized as frequency of neurons, astrocytes and oligodendrocytes. B. Electrophysiological characteristics of neurons derived from the differentiation of region-specific NSCs. C. Analysis of the engraftment of region-specific clonally derived NSCs after transplantation in the early postnatal mouse cerebellum. Table S2 - Statistical analysis of GSEA for gene signatures generated by DEGs up regulated in hindbrain (CB/IVv) or forebrain (SVZ) NSCs. Table S3 - Lineage-specific differentiation of clonally derived MB CSCs is summarized as frequency of neuron-, astrocyte- and oligodendrocyte-like cells. Table S4 - Statistical analysis of GSEA for gene signatures generated by DEGs up regulated in tumorigenic Ptch+/- p53-/- MB CSCs vs. hindbrain NSCs (MB1_EXCLUSIVE), in non-tumorigenic Ptch+/- p53+/+ MB CSCs vs. hindbrain NSCs (MB2_EXCLUSIVE), and commonly expressed in both tumorigenic and non-tumorigenic CSCs vs. hindbrain NSCs (MB1_MB2 COMMON). Table S5 - Expression of EBF proteins in a collection of 33 human MB specimens as determined by immunohistochemistry.

PDF file - 130K, EBF3 expression can be retrieved in silico in human MBs by exploiting publicly available human data sets

PDF file - 132K, The gene signature containing the 297 genes differentially expressed between hindbrain- vs. forebrain-derived NSCs distinguishes the SHH molecular subgroup of human MBs

Supplementary Figures 1-9, Tables 1-4, Methods and Materials from Epidermal Growth Factor Receptor Expression Identifies Functionally and Molecularly Distinct Tumor-Initiating Cells in Human Glioblastoma Multiforme and Is Required for Gliomagenesis

Tolerogenic dendritic cells (DCs) are key players in maintaining immunological homeostasis, dampening immune responses, and promoting tolerance. DC-10, a tolerogenic population of human IL-10-producing DCs characterized by the expression of HLA-G and ILT4, play a pivotal role in promoting tolerance via T regulatory type 1 (Tr1) cells. Thus far, the absence of markers that uniquely identify DC-10 has limited in vivo studies. By in vitro gene expression profiling of differentiated human DCs, we identified CD141 and CD163 as surface markers for DC-10. The coexpression of CD141 and CD163 in combination with CD14 and CD16 enables the ex vivo isolation of DC-10 from the peripheral blood. CD14+CD16+CD141+CD163+ cells isolated from the peripheral blood of healthy subjects (ex vivo DC-10) produced spontaneously and upon activation of IL-10 and limited levels of IL-12. Moreover, in vitro stimulation of allogeneic naive CD4+ T cells with ex vivo DC-10 induced the differentiation of alloantigen-specific CD49b+LAG-3+ Tr1 cells. Finally, ex vivo DC-10 and in vitro generated DC-10 exhibited a similar transcriptional profile, which are characterized by an anti-inflammatory and pro-tolerogenic signature. These results provide new insights into the phenotype and molecular signature of DC-10 and highlight the tolerogenic properties of circulating DC-10. These findings open the opportunity to track DC-10 in vivo and to define their role in physiological and pathological settings.

Allogeneic fetal-derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient-specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene-therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC-derived neural stem cells (NSCs) showing a reliable “NSC signature” is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS-NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a “gold standard” in a side-by-side comparison when validating the phenotype of hiPS-NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS-NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA-overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA-overexpressing iPSC-derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient-specific ARSA-overexpressing hiPS-NSCs may be used in autologous ex vivo gene therapy protocols to provide long-lasting enzymatic supply in MLD-affected brains.

Achaete-scute homolog 1 gene (ASCL1) is a gene classifier for the proneural (PN) transcriptional subgroup of glioblastoma (GBM) that has a relevant role in the neuronal-like differentiation of GBM cancer stem cells (CSCs) through the activation of a PN gene signature. Besides prototypical ASCL1 PN target genes, the molecular effectors mediating ASCL1 function in regulating GBM differentiation and, most relevantly, subgroup specification are currently unknown. Here we report that ASCL1 not only promotes the acquisition of a PN phenotype in CSCs by inducing a glial-to-neuronal lineage switch but also concomitantly represses mesenchymal (MES) features by directly downregulating the expression of N-Myc downstream-regulated gene 1 (NDRG1), which we propose as a novel gene classifier of MES GBMs. Increasing the expression of ASCL1 in PN CSCs results in suppression of self-renewal, promotion of differentiation and, most significantly, decrease in tumorigenesis, which is also reproduced by NDRG1 silencing. Conversely, both abrogation of ASCL1 expression in PN CSCs and enforcement of NDRG1 expression in either PN or MES CSCs induce proneural-to-mesenchymal transition (PMT) and enhanced mesenchymal features. Surprisingly, ASCL1 overexpression in MES CSCs increases malignant features and gives rise to a neuroendocrine-like secretory phenotype. Altogether, our results propose that the fine interplay between ASCL1 and its target NDRG1 might serve as potential subgroup-specific targetable vulnerability in GBM; enhancing ASCL1 expression in PN GBMs might reduce tumorigenesis, whereas repressing NDRG1 expression might be actionable to hamper the malignancy of GBM belonging to the MES subgroup.

CD4+ type 1 T regulatory (Tr1) cells are induced in the periphery and have a pivotal role in promoting and maintaining tolerance. The absence of surface markers that uniquely identify Tr1 cells has limited their study and clinical applications. By gene expression profiling of human Tr1 cell clones, we identified the surface markers CD49b and lymphocyte activation gene 3 (LAG-3) as being stably and selectively coexpressed on mouse and human Tr1 cells. We showed the specificity of these markers in mouse models of intestinal inflammation and helminth infection and in the peripheral blood of healthy volunteers. The coexpression of CD49b and LAG-3 enables the isolation of highly suppressive human Tr1 cells from in vitro anergized cultures and allows the tracking of Tr1 cells in the peripheral blood of subjects who developed tolerance after allogeneic hematopoietic stem cell transplantation. The use of these markers makes it feasible to track Tr1 cells in vivo and purify Tr1 cells for cell therapy to induce or restore tolerance in subjects with immune-mediated diseases. © 2013 Nature America, Inc. All rights reserved

The role of forkhead box P3 (FOXP3) is well-established in T-regulatory cells, but the function of transient FOXP3 expression in activated human conventional T (Tconv) cells is unknown. In the present study, we used 2 approaches to determine the role of FOXP3 in human Tconv cells. First, we obtained Tconv clones from a female subject who is hemizygous for a null mutation in FOXP3, allowing the comparison of autologous T-cell clones that do or do not express FOXP3. Second, we knocked down activation-induced FOXP3 in Tconv cells from healthy donors with small interfering RNAagainst FOXP3. We found that FOXP3-deficient Tconv cells proliferate more and produce more cytokines than wild-type Tconv cells and have differential expression of 274 genes. We also investigated the role of FOXP3 in Th1 and Th17 cells and found that the expression of activation-induced FOXP3 was higher and more sustained in Th17 cells compared with Th1 cells. Knocking down FOXP3 expression in Th17 cells significantly increased the production of IFN-γ and decreased the expression of CCR4, but had no effect on IL-17 expression. These data reveal a novel function of FOXP3 in Tconv cells and suggest that expression of this protein is important in the function of multiple CD4(+) T-cell lineages.

Transposons and gamma-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells and that hamper the identification of early cancer-driving events among bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVVs) by which we could efficiently induce hepatocellular carcinoma (HCC) in three different mouse models. By virtue of the LVV's replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of four previously unknown liver cancer-associated genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. The newly identified genes are likely to play a role in human cancer because they are upregulated, amplified and/or deleted in human HCCs and can predict clinical outcomes of patients.

While insulin-like growth factor-I (IGF-I) supports neuronal and glial differentiation in the CNS, it is largely unknown whether IGF-I also influences neuronal migration and positioning. We show here that the pattern of olfactory bulb (OB) layering is altered in Igf-I (-/-) mice. In these animals, Tbr1(+)-glutamatergic neurons are misplaced in the mitral cell layer (ML) and the external plexiform layer (EPL). In addition, there are fewer interneurons in the glomerular layer and the EPL of the Igf-I (-/-) mice, and fewer newborn neurons are incorporated into the OB from the forebrain subventricular zone (SVZ). Indeed, neuroblasts accumulate in the postnatal/adult SVZ of Igf-I (-/-) mice. Significantly, the positioning of Tbr1(+)-cells in a primitive ML is stimulated by IGF-I in cultured embryonic OB slices, an effect that is partially repressed by the phosphoinositide 3-kinase (PI3K) inhibitor. In OB cell cultures, IGF-I increases the phosphorylation of disabled1 (P-Dab1), an adaptor protein that is a target of Src family kinases (SFK) in the reelin signalling pathway, whereas reduced P-Dab1 levels were found in Igf-I (-/-) mice. Neuroblast migration from the rostral migratory stream (RMS) explants of postnatal Igf-I (-/-) was similar to that from Igf-I (+/+) explants. However, cell migration was significantly enhanced by IGF-I added to the explants, an effect that was repressed by PI3K and SFK inhibitors. These findings suggest that IGF-I promotes neuronal positioning in the OB and support a role for IGF-I in stimulating neuroblast exit from the SVZ into the RMS, thereby promoting the incorporation of newly formed neurons into the OB.

An increasing number of eukaryotic and prokaryotic genes are being found to have natural antisense transcripts (NATs). There is also growing evidence to suggest that antisense transcription could play a key role in many human diseases. Consequently, there have been several recent attempts to set up computational procedures aimed at identifying novel NATs. Our group has developed the AntiHunter program for the identification of expressed sequence tag (EST) antisense transcripts from BLAST output. In order to perform an analysis, the program requires a genomic sequence plus an associated list of transcript names and coordinates of the genomic region. After masking the repeated regions, the program carries out a BLASTN search of this sequence in the selected EST database, reporting via email the EST entries that reveal an antisense transcript according to the user-supplied list. Here, we present the newly developed version 2.0 of the AntiHunter tool. Several improvements have been added to this version of the program in order to increase its ability to detect a larger number of antisense ESTs. As a result, AntiHunter can now detect, on average, >45% more antisense ESTs with little or no increase in the percentage of the false positives. We also raised the maximum query size to 3 Mb (previously 1 Mb). Moreover, we found that a reasonable trade-off between the program search sensitivity and the maximum allowed size of the input-query sequence could be obtained by querying the database with the MEGABLAST program, rather than by using the BLAST one. We now offer this new opportunity to users, i.e. if choosing the MEGABLAST option, users can input a query sequence up to 30 Mb long, thus considerably improving the possibility to analyze longer query regions. The AntiHunter tool is freely available at http://bioinfo.crs4.it/AH2.0.

The developing neocortex contains two types of progenitor cells for glutamatergic, pyramidal-projection neurons. The first type, radial glia, produce neurons and glia, divide at the ventricular surface, and express Pax6, a homeodomain transcription factor. The second type, intermediate progenitor cells, are derived from radial glia, produce only neurons, and divide away from the ventricular surface. Here we show that the transition from radial glia to intermediate progenitor cell is associated with upregulation of Tbr2, a T-domain transcription factor, and downregulation of Pax6. Accordingly, Tbr2 expression in progenitor compartments (the subventricular zone and ventricular zone) rises and falls with cortical plate neurogenesis. The subsequent transition from intermediate progenitor cell to postmitotic neuron is marked by downregulation of Tbr2 and upregulation of Tbr1, another T-domain transcription factor. These findings delineate the transcription factor sequence Pax6 → Tbr2 → Tbr1 in the differentiation of radial glia → intermediate progenitor cell → postmitotic projection neuron. This transcription factor sequence is modified in preplate neurons, in which Tbr2 is transiently coexpressed with Tbr1, and in the direct differentiation pathway from radial glia → postmitotic projection neuron, in which Tbr2 is expressed briefly or not at all.

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.

A novel type of DNA-binding domain, the ‘T-box’ domain, characterizes an increasingly large family of transcription factors (Trends Genet. 15 (1999) 154). We have identified and characterized the expression pattern of a new member of the Tbr1 subfamily of T-box genes; this gene has been recently named T-bet/Tbx21 (Genomics 70 (2000) 41; Cell 17 (2000) 655; Science 292 (2001) 1907; Science 295 (2002) 338). The sequence and expression of Tbr1 and eomesodermin/Tbr2 are closely related to T-bet/Tbx21. The expression of Tbr1 (Neuron 15 (1995) 63) and Tbr2 (Mech Dev 84 (1999) 133) have virtually identical onset, at around E10.5, and expression domains in the mouse telencephalon. While Tbr1 is expressed in postmitotic neurons, Tbr2 (which is also expressed during gastrulation is also expressed in neural progenitors. We have used in situ hybridization to determine the temporal and spatial distribution of T-bet/Tbx21 expression during mouse development. T-bet/Tbx21 expression is exclusively restricted to the olfactory bulb and the thymus. To assess the distribution of T-BET/TBX21 expression in the haematopoietic compartment we used reverse transcriptase-polymerase chain reaction and found its expression in several human blood cell lineages, including progenitors/stem cells, immature B cells and peripheral T cells.

Oral-facial-digital type 1 syndrome (OFD1 [MIM 311200]) is transmitted as an X-linked dominant condition with lethality in males and is characterized by malformations of the face, oral cavity, and digits, and by a highly variable expressivity even within the same family. Malformation of the brain and polycystic kidneys are commonly associated with this disorder. The locus for OFD1 was mapped by linkage analysis to a 12-Mb interval, flanked by markers DXS85 and DXS7105 in the Xp22 region. To identify the gene responsible for this syndrome, we analyzed several transcripts mapping to the region and found mutations in OFD1 (formerly named "Cxorf5/71-7a"), encoding a protein containing coiled-coil alpha-helical domains. Seven patients with OFD1, including three with familial and four with sporadic cases, were analyzed. Analysis of the familial cases revealed a missense mutation, a 19-bp deletion, and a single base-pair deletion leading to a frameshift. In the sporadic cases, we found a missense (de novo), a nonsense, a splice, and a frameshift mutation. RNA in situ studies on mouse embryo tissue sections show that Ofd1 is developmentally regulated and is expressed in all tissues affected in OFD1 syndrome. The involvement of OFD1 in oral-facial-digital type I syndrome demonstrates an important role of this gene in human development.

During corticogenesis, early-born neurons of the preplate and layer 6 are important for guiding subsequent neuronal migrations and axonal projections. Tbr1 is a putative transcription factor that is highly expressed in glutamatergic early-born cortical neurons. In Tbr1-deficient mice, these early-born neurons had molecular and functional defects. Cajal-Retzius cells expressed decreased levels of Reelin, resulting in a reeler-like cortical migration disorder. Impaired subplate differentiation was associated with ectopic projection of thalamocortical fibers into the basal telencephalon. Layer 6 defects contributed to errors in the thalamocortical, corticothalamic, and callosal projections. These results show that Tbr1 is a common genetic determinant for the differentiation of early-born glutamatergic neocortical neurons and provide insights into the functions of these neurons as regulators of cortical development.

We report on the expression pattern of a novel EGF- containing gene named Maeg. RNA in situ studies indicate that Maeg is first activated during specification of the early lateral dermatome, and continues to be expressed in all the dermatome derivatives as the dermis of the trunk, the hair follicles, and the mesenchyme of the cranio-facial region.

We isolated a novel mouse gene, RP42, in a systematic search for genes expressed in proliferating neuroblasts whose human orthologs map to suspectibility loci for autism. This gene is intronless and encodes a putative 259-amino-acid protein that exhibits 30–36% overall sequence identity to a fission yeast and a nematode protein (GenPept Accession Nos. CAA17006 and CAB54261). Nevertheless, no homology to any known gene was found. RP42 has developmentally regulated expression, particularly in proliferating neuroblasts from which neocortical neurons originate. Its human ortholog is located in a cluster of embryonic neuronally expressed genes on the 6q16 chromosome, making it a positional candidate susceptibility gene for autism.

We have isolated a human and murine homologue of the Drosophila prune gene through dbEST searches. The gene is ubiquitously expressed in human adult tissues, while in mouse developing embryos a high level of expression is confined to the nervous system particularly in the dorsal root ganglia, cranial nerves, and neural retina. The gene is composed of eight exons and is located in the 1q21.3 chromosomal region. A pseudogene has been sequenced and mapped to chromosomal region 13q12. PRUNE protein retains the four characteristic domains of DHH phosphoesterases. The synergism between prune and awdK-pn in Drosophila has led various authors to propose an interaction between these genes. However, such an interaction has never been supported by biochemical data. By using interaction-mating and in vitro co-immunoprecipitation experiments, we show for the first time the ability of human PRUNE to interact with the human homologue of awd protein (nm23-H1). In contrast, PRUNE is impaired in its interaction with nm-23-H1-S120G mutant, a gain-of-function mutation associated with advanced neuroblastoma stages. Consistently, PRUNE and nm23-H1 proteins partially colocalize in the cytoplasm. The data presented are consistent with the view that PRUNE acts as a negative regulator of the nm23-H1 protein. We discuss how PRUNE regulates nm23-H1 protein and postulate possible implications of PRUNE in neuroblastoma progression.

The members of the T-box gene family share a highly conserved DNA binding domain named the T-domain, and important developmental functions. Here we report the cloning of chicken Tbr1and of murine and chickenTbr2 (orthologs of the Xenopus eomesodermin gene), the mapping of the murine Tbr2 to chromosome 9, and their pattern of expression during mouse and chick embryogenesis. Both Tbr 1 and 2 have a restricted and conserved domain of expression in the telencephalic pallium of the two species. Chick Tbr2 has a specific and dynamic expression in the gastrulating embryo.

We have isolated a family of four vertebrate genes homologous to eyes absent (eya), a key regulator of ocular development in Drosophila. Here we present the detailed characterization of the EYA4 gene in human and mouse. EYA4 encodes a 640 amino acid protein containing a highly conserved C-terminal domain of 271 amino acids which in Drosophila eya is known to mediate developmentally important protein-protein interactions. Human EYA4 maps to 6q23 and mouse Eya4 maps to the predicted homology region near the centromere of chromosome 10. In the developing mouse embryo, Eya4 is expressed primarily in the craniofacial mesenchyme, the dermamyotome and the limb. On the basis of map position and expression pattern, EYA4 is a candidate for oculo-dento-digital (ODD) syndrome, but no EYA4 mutations were found in a panel of ODD patients.

The genetic background of donor and recipient is an important factor determining the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We applied a whole genome analysis to investigate genetic variants - other than HLA class I and II - associated with negative outcome after HLA-identical sibling allo-HSCT in a cohort of 110 β-Thalassemic patients. We identified two single nucleotide polymorphisms in BAT2 (A/G) and BAT3 (T/C) genes, SNP rs11538264 and SNP rs10484558, both located in the HLA class III region, in strong Linkage Disequilibrium between each other (R2=0.92). When considered as single SNP, none of them reached a significant association with graft rejection (nominal P < 0.00001 for BAT2 SNP rs11538264, and P < 0.0001 for BAT3 SNP rs10484558). Whereas, the BAT2/BAT3 A/C haplotype was present at significantly higher frequency in patients who rejected as compared to those with functional graft (30.0% vs. 2.6%, nominal P = 1.15×10−8; and adjusted P = 0.0071). The BAT2/BAT3 polymorphisms and specifically the A/C haplotype may represent novel immunogenetic factor associated with graft rejection in patients undergoing allo-HSCT.

The relevant social and economic impact of prostate adenocarcinoma, one of the leading causes of death in men, urges critical improvements in knowledge of the pathogenesis and cure of this disease. These can also be achieved by implementing in vitro and in vivo preclinical models by taking advantage of prostate cancer stem cells (PCSCs). The best-characterized mouse model of prostate cancer is the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. TRAMP mice develop a progressive lesion called prostatic intraepithelial neoplasia that evolves into adenocarcinoma (AD) between 24 and 30 weeks of age. ADs often metastasize to lymph nodes, lung, bones, and kidneys. Eventually, approximately 5% of the mice develop an androgen-independent neuroendocrine adenocarcinoma. Here we report the establishment of long-term self-renewing PCSC lines from the different stages of TRAMP progression by application of the neurosphere assay. Stage-specific prostate cell lines were endowed with the critical features expected from malignant bona fide cancer stem cells, namely, self-renewal, multipotency, and tumorigenicity. Notably, transcriptome analysis of stage-specific PCSCs resulted in the generation of well-defined, meaningful gene signatures, which identify distinct stages of human tumor progression. As such, TRAMP-derived PCSCs represent a novel and valuable preclinical model for elucidating the pathogenetic mechanisms leading to prostate adenocarcinoma and for the identification of molecular mediators to be pursued as therapeutic targets.

Genetic analysis of the development and evolution of the vertebrate head is at a primitive stage. Many homeo box genes, including the Distal-less family, are potential regulators of head development. To determine the function of Dlx-2, we generated a null mutation in mice using gene targeting. In homozygous mutants, differentiation within the forebrain is abnormal and the fate of a subset of cranial neural crest cells is respecified. The latter causes abnormal morphogenesis of the skeletal elements derived from the proximal parts of the first and second branchial arches. We hypothesize that the affected skull bones from the first arch have undergone a transformation into structures similar to those found in reptiles. These results show that Dlx-2 controls development of the branchial arches and the forebrain and suggests its role in craniofacial evolution.

The mechanisms that regulate regional specification and evolution of the cerebral cortex are obscure. To this end, we have identified and characterized a novel murine and human gene encoding a putative transcription factor related to the Brachyury (T) gene that is expressed only in postmitotic cells. T-brain-1 (Tbr-1) mRNA is largely restricted to the cerebral cortex, where during embryogenesis it distinguishes domains that we propose may give rise to paleocortex, limbic cortex, and neocortex. Tbr-1 and Id-2 expression in the neocortex have discontinuities that define molecularly distinct neocortical areas. Tbr-1 expression is analyzed in the context of the prosomeric model. Topological maps are proposed for the organization of the dorsal telencephalon.

Recently, the Dlx family of homeobox genes have been identified as candidates for regulating patterning and differentiation of the forebrain. We have made a polyclonal antiserum to the protein product of the Dlx-2 gene. Using this antiserum, we have characterized the spatial and temporal pattern of DLX-2 protein expression during murine development and in the adult mouse brain. These studies demonstrate that, like the mRNA from the Dlx-2 gene, DLX-2 protein is expressed in mouse embryonic forebrain, limbs, tail, genital tubercle, and branchial arches. Within the embryonic forebrain, DLX-2 protein is expressed within specific transverse and longitudinal domains. Analysis of expression within the wall of the forebrain shows that DLX-2 is expressed in proliferative regions including the ventricular and subventricular zones. DLX-2 is expressed in the same cells as MASH-1, a marker of relatively undifferentiated cells, but in a reciprocal fashion to MAP-2, a marker of terminal neuronal differentiation. A number of DLX-2-expressing cells, but not all, can be labeled with bromodeoxyuridine (BrdU). Using the patterns of DLX-2, MASH-1, MAP-2 expression, and bromodeoxyuridine incorporation, we identify four molecularly distinct populations of cells that may correspond to different stages of neuronal differentiation in the mouse basal forebrain, in which DLX-2 is expressed at the transition from proliferation to terminal differentiation.

SummaryTuberous Sclerosis Complex (TSC) is a multisystem genetic disorder characterized by hamartomatous neurological lesions that exhibit abnormal cell proliferation and differentiation. Hyperactivation of mTOR pathway by mutations in either the Tsc1 or Tsc2 gene underlies TSC pathogenesis, but involvement of specific neural cell populations in the formation of TSC-associated neurological lesions remains unclear. We deleted Tsc1 in Emx1-expressing embryonic telencephalic neural stem cells (NSCs) and found that mutant mice faithfully recapitulated TSC neuropathological lesions, such as cortical lamination defects and subependymal nodules (SENs). These alterations were caused by enhanced generation of SVZ neural progeny, followed by their premature differentiation and impaired maturation during both embryonic and postnatal development. Notably, mTORC1-dependent Akt inhibition and STAT3 activation were involved in the reduced self-renewal and earlier neuronal and astroglial differentiation of mutant NSCs. Thus, finely tuned mTOR activation in embryonic NSCs may be critical to prevent development of TSC-associated brain lesions.

The pattern of RNA expression of the murine Dlx-2 (Tes-1) homeobox gene is described in embryos ranging in age from E8.5 through E11.5. Dlx-2 is a vertebrate homologue of the Drosophila Distal-less (Dll) gene. Dll expression in the Drosophila embryo is principally limited to the primordia of the brain, head and limbs. Dlx-2 is also expressed principally in the primordia of the forebrain, head and limbs. Within these regions it is expressed in spatially restricted domains. These include two discontinuous regions of the forebrain (basal telencephalon and ventral diencephalon), the branchial arches, facial ectoderm, cranial ganglia and limb ectoderm. Several mouse and human disorders have phenotypes which potentially are the result of mutations in the Dlx genes.

Epidermal growth factor receptor (EGFR) is a known diagnostic and, although controversial, prognostic marker of human glioblastoma multiforme (GBM). However, its functional role and biological significance in GBM remain elusive. Here, we show that multiple GBM cell subpopulations could be purified from the specimens of patients with GBM and from cancer stem cell (CSC) lines based on the expression of EGFR and of other putative CSC markers. All these subpopulations are molecularly and functionally distinct, are tumorigenic, and need to express EGFR to promote experimental tumorigenesis. Among them, EGFR-expressing tumor-initiating cells (TIC) display the most malignant functional and molecular phenotype. Accordingly, modulation of EGFR expression by gain-of-function and loss-of-function strategies in GBM CSC lines enhances and reduces their tumorigenic ability, respectively, suggesting that EGFR plays a fundamental role in gliomagenesis. These findings open up the possibility of new therapeutically relevant scenarios, as the presence of functionally heterogeneous EGFRpos and EGFRneg TIC subpopulations within the same tumor might affect clinical response to treatment. Cancer Res; 70(19); 7500–13. ©2010 AACR.

The T-box brain 1 (Tbr1) gene encodes a transcription factor necessary for the maintenance and/or differentiation of glutamatergic cells in the olfactory bulb (OB) and cortex, although its precise function in the development of glutamatergic neurons is not known. Furthermore, Tbr1 has not been reported to regulate the formation of glial cells. We show that Tbr1 is expressed during the initial stages in the generation of glutamatergic mitral neurons from dividing progenitors in the E12.5 mouse OB. Retroviral-mediated overexpression of Tbr1 in cultured embryonic and adult OB stem cells (OBSC) produces a marked increase in the number of TuJ1+ neurons (including VGLUT1+ glutamatergic and GABA+ neurons) and O4+ oligodendrocytes. Moreover, transduction of Tbr1 inhibits the production of GFAP+ astrocytes from both cultured OBSC and dividing progenitor cells in vivo. These results show that the expression of Tbr1 in neural stem and progenitor cells prevents them from following an astrocyte fate during OB development. Our findings suggest that the transduction of Tbr1 into neural stem cells could be useful to increase the production of neurons and oligodendrocytes in studies of neuroregeneration. © 2010.

The vertebrate telencephalon is composed of many architectonically and functionally distinct areas and structures, with billions of neurons that are precisely connected. This complexity is fine-tuned during development by numerous genes. To identify genes involved in the regulation of telencephalic development, a specific subset of differentially expressed genes was characterized. Here, we describe a set of cDNAs encoded by genes preferentially expressed during development of the mouse telencephalon that was identified through a functional genomics approach. Of 832 distinct transcripts found, 223 (27%) are known genes. Of the remaining, 228 (27%) correspond to expressed sequence tags of unknown function, 58 (7%) are homologs or orthologs of known genes, and 323 (39%) correspond to novel rare transcripts, including 48 (14%) new putative noncoding RNAs. As an example of this latter group of novel precursor transcripts of micro-RNAs, telencephalic embryonic subtractive sequence (TESS) 24.E3 was functionally characterized, and one of its targets was identified: the zinc finger transcription factorZFP9. The TESS transcriptome has been annotated, mapped for chromosome loci, and arrayed for its gene expression profiles during neural development and differentiation (in Neuro2a and neural stem cells). Within this collection, 188 genes were also characterized on embryonic and postnatal tissue byin situhybridization, demonstrating that most are specifically expressed in the embryonic CNS. The full information has been organized into a searchable database linked to other genomic resources, allowing easy access to those who are interested in the dissection of the molecular basis of telencephalic development.

By using a custom-made array containing cDNAs preferentially expressed in the mouse embryonic telencephalon (Porteus et al. [1992] Brain Res Mol Brain Res 12:7-22; and Alessandro Bulfone, unpublished data), we studied the gene expression profile of the Dlx1/Dlx2(-/-) subpallium and Pax6(-/-) pallium. We identified a transcript corresponding to Unigene Cluster Mm.94021 and rat Evf-1, which is down-regulated in the Dlx1/Dlx2(-/-) subpallium and up-regulated in the Pax6(-/-) pallium. Here, we report the expression pattern of this transcript, designated mouse Evf1 (mEvf1), in the prenatal forebrain of wild-type, Dlx1/Dlx2(-/-) and Pax6(-/-) mice using RNA in situ hybridization and reverse transcriptase-polymerase chain reaction. In the wild-type forebrain mEvf1 expression is restricted to the ventral thalamus, hypothalamus, and subpallial telencephalon (caudal, lateral, and medial ganglionic eminences and septal primordia), whereas it is down-regulated in the Dlx1/Dlx2(-/-) subpallium (mainly in caudal, lateral, and medial ganglionic eminences), and up-regulated in the Pax6(-/-) lateral and ventral pallium at embryonic day 12.5 and in the dorsal, lateral, and ventral pallium at embryonic day 14.5.

Corrigendum: Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells

The signal for somatic sex determination in mammals, Caenorhabditis elegans and Drosophila melanogaster is chromosomal, but the overall mechanisms do not appear to be conserved between the phyla. However it has been found quite recently that the C. elegans sex-determining gene Mab-3 contains a domain highly homologous to the Drosophila sex-determining gene doublesex (dsx) and shares a similar role. These data suggest that at least some aspects of the regulation of sex determination might be conserved. In humans, a doublesex-related gene (DMRT1) was identified at less than 30 kb from the critical region for sex reversal on chromosome 9p24 (TD9). In order to get insights into the role of DMRT1 in sex determination/differentiation, we have isolated DMRT1 mouse homologue (Dmrt1) and analysed its expression pattern. The gene is expressed in the genital ridges of both sexes during the sex-determining switch and it shows male/female dimorphism at late stages of sex differentiation.

We identified in the EST database murine and human sequences similar, but not identical, to the members of the PC3/BTG/TOB family of cell cycle inhibitors. A conserved domain (aa 50–68) of the PC3 protein, the prototype member of the family, was used as a query. That domain has been shown by us to be necessary for the antiproliferative activity of PC3. A murine EST clone and a highly homologous human EST clone, containing the entire ORF, were chosen for sequencing. Comparison to databases and a phylogenetic tree analysis indicated that these EST clones are the mouse and human homologues of a gene that represents a novel member of the PC3/BTG/TOB family. This gene, named PC3B, is endowed with marked antiproliferative activity, being able to induce G1 arrest, and is highly expressed in testis, in oocyte, and in preimplantation embryos. Analysis of its expression during murine development indicated a specific localization in the olfactory epithelium at midgestation, suggesting that PC3B might be involved in the differentiation of this neuronal structure. Human PC3B mapped to chromosome 11q23, as indicated by radiation hybrid analysis.

Using a bioinformatic approach, we have identified a new transcript, SLC7A8, mapping to 14q11.2, within the lysinuric protein intolerance (LPI) critical region. This gene is highly expressed in skeletal muscle, intestine, kidney, and placenta and encodes a predicted protein of 535 amino acids, homologous to the amino acid permease CD98 light chain and cationic amino acid transporters. RNA in situ hybridization data on mouse embryos confirm the expression in kidney and intestine and, interestingly, reveal that SLC7A8 is also highly expressed in eye, in retinal pigmented epithelium, and in tooth buds at day 16.5 of gestation. Mutational analysis excluded any direct involvement of the SLC7A8 gene product in LPI disease. The homology data and the expression pattern are in agreement with the hypothesis that SLC7A8 represents a novel light chain interacting with the 4F2 heavy chain in the multimeric complex mediating neutral and/or cationic amino acid transport and cystine/glutamate exchange.

Herein, the genetic control of regionalization and connectivity of the neocortex are reviewed. Evidence is accumulating which suggests that intrinsic mechanisms have a central role in controlling cortical regional specification and differentiation. Expression patterns of several genes (Id-2, Tbr-1, cadherin-6, cadherin-8, neuropilin-2, Wnt-7b, Eph-A7 and RZR-beta) are described; the expressions of these genes have regional boundaries which demarcate distinct functional areas of the cerebral cortex in neonatal mice.

We have identified a transcription factor specifically expressed in the developing vertebrate eye. We named this gene vax2 because of the high degree of sequence similarity to the recently described vax1 . Both in the human and mouse genomes, vax2 is localized in the vicinity of the emx1 gene. This mapping assignment, together with the previously reported colocalization of Vax1 and Emx2 in mouse, indicates that the vax and the emx genes may be organized in clusters. vax2 has a remarkable expression domain confined to the ventral portion of the prospective neural retina in mouse, human, and Xenopus . The overexpression of either the frog Xvax2 or the human VAX2 in Xenopus embryos leads to an aberrant eye phenotype and, in particular, determines a ventralizing effect on the developing eye. The expression domain of the transcription factor Xpax2 , normally confined to the ventral developing retina, extends to the dorsal region of the retina after overexpression of vax2 . On the other hand, the expression of Xvent2 , a molecular marker of the dorsal retina, is strongly reduced. Furthermore, vax2 overexpression induces a striking expansion of the optic stalk, a structure deriving from the ventralmost region of the eye vesicle. Altogether, these data indicate that vax2 plays a crucial role in eye development and, in particular, in the specification of the ventral optic vesicle.

We describe the identification and characterization of a new gene deleted in the AMME contiguous gene syndrome. This gene is predominantly expressed in heart, skeletal muscle, spinal cord, and brain. Screening of placenta and NT2 cDNA libraries enabled us to obtain the 1.5-kb full-length transcript, which shows a 426-bp open reading frame. Since the resulting 142-amino-acid peptide has a single putative transmembrane domain and a weak but suggestive homology with KCNE1 ( minK ), a protein associated with the KCNQ1 potassium channel ( KVLQT1 ), we named this new gene KCNE1 -like ( KCNE1L ). To obtain greater insight into this new member of an apparently distinct protein family, we have identified and characterized the homologous mouse gene ( Kcne1l ), which encodes a peptide of 143 amino acids with 91% homology and 80% identity. The expression pattern of mouse Kcne1l in the developing embryo revealed strong signal in ganglia, in the migrating neural crest cells of cranial nerves, in the somites, and in the myoepicardial layer of the heart. The specific distribution in adult tissues, the putative channel function, and the expression pattern in the developing mouse embryo suggest that KCNE1L could be involved in the development of the cardiac abnormalities as well as of some neurological signs observed in patients with AMME contiguous gene syndrome.

The general strategies of phototransduction in vertebrates and invertebrates share many similarities, but differ significantly in their underlying molecular machinery. The CDS gene encodes the CDP-diacylglycerol synthase (CDS) enzyme and is required for phototransduction in Drosophila. Using a bioinformatic approach, we have identified two novel transcripts (CDS1 and CDS2) highly homologous to the Drosophila CDS gene. We isolated and sequenced the CDS2 full-length cDNA and mapped the two genes to human chromosomes 20p13 (CDS2) and 4q21.1 (CDS1). Sequence analysis revealed that both genes are highly homologous to the Drosophila protein (64.4 and 58. 6% identity at the protein level between CDS and CDS2 and between CDS and CDS1, respectively). The mouse homologs for both genes were isolated and used in RNA in situ hybridization studies on adult and embryonic mouse tissue sections. These studies showed that Cds2 is highly expressed in the differentiating neuroblasts of the neural retina and in the central nervous system during embryonic development, while it was not detected in adult retina. Cds1, on the other hand, shows a high level of expression in the photoreceptor layer of adult retina, which strongly suggests a role for Cds1 in phototransduction. Knowledge of the expression pattern of these genes in mammals may shed light on the evolution of vision mechanisms and help in the evaluation of candidate genes for human retinopathies.

Nature often utilizes the same metabolic 'core groups' of interacting genes or 'pathways' in completely different organs, tissues and cellular compartments. Deciphering the physiological role of a particular gene in a living organism is therefore critical to understanding not only how a gene/protein works, but also where (in which tissue/organ) and when (at what developmental stage) it functions. We have performed systematic RNA in situ hybridization on a subset of murine genes homologous to Drosophila mutant genes, called Drosophila -related expressed sequences (Dres). This approach combines functional information derived from cross-species sequence comparisons and biochemical, physiological and pathological studies performed in the fly with knowledge of the spatial and temporal distribution of gene expression. Forty murine Dres were tested by RNA in situ hybridization on sagittal, coronal and transverse sections at three developmental stages, E10.5, E12.5 and E17.5. For some of them, whole mount in situ hybridization was performed at earlier stages. These data are valuable for establishing how the function of these genes and the genetic programs underlying the development of a particular tissue or organ have evolved during evolution. For example, six Dres genes showed restricted expression domains within the murine retina, suggesting a different role for each of these genes in eye development and functioning. Furthermore, the information derived from this combined approach will be instrumental in predicting the phenotypic consequences of gene dysfunction in both mouse mutants and human genetic diseases.

The study of model organisms has been instrumental towards the elucidation of the basic mechanisms of human biology. Drosophila melanogaster has been the target of extensive genetic analyses over the past 90 years and a notable amount of information is known about its gene structure, gene regulation and gene function. The vast gene resource generated by the expressed sequence tags (ESTs) efforts was exploited to identify, using a bioinformatic approach, novel human and murine gene transcripts homologous to Drosophila mutant genes. A systematic characterization of these genes, named Drosophila-related expressed sequences (DRES), was performed including genomic mapping in human and mouse and detailed study of their expression pattern by RNA in situ hybridization experiments. Comparison between DRES genes and their putative partners in Drosophila contributes to the understanding of their function in mammals and to the discovery of their possible role in disease.