Wnt5a is a transcriptional target of Dlx homeogenes and promotes differentiation of interneuron progenitors in vitro and in vivo.

During brain development, neurogenesis, migration, and differentiation of neural progenitor cells are regulated by an interplay between intrinsic genetic programs and extrinsic cues. The Dlx homeogene transcription factors have been proposed to directly control the genesis and maturation of GABAergic interneurons of the olfactory bulb (OB), subpallium, and cortex. Here we provide evidence that Dlx genes promote differentiation of olfactory interneurons via the signaling molecule Wnt5a. Dlx2 and Dlx5 interact with homeodomain binding sequences within the Wnt5a locus and activate its transcription. Exogenously provided Wnt5a promotes GABAergic differentiation in dissociated OB neurons and in organ-type brain cultures. Finally, we show that the Dlx-mutant environment is unfavorable for GABA differentiation, in vivo and in vitro. We conclude that Dlx genes favor interneuron differentiation also in a non-cell-autonomous fashion, via expression of Wnt5a.

SOX2 silencing in glioblastoma tumor-initiating cells causes stop of proliferation and loss of tumorigenicity.

Glioblastoma, the most aggressive cerebral tumor, is invariably lethal. Glioblastoma cells express several genes typical of normal neural stem cells. One of them, SOX2, is a master gene involved in sustaining self-renewal of several stem cells, in particular neural stem cells. To investigate its role in the aberrant growth of glioblastoma, we silenced SOX2 in freshly derived glioblastoma tumor-initiating cells (TICs). Our results indicate that SOX2 silenced glioblastoma TICs, despite the many mutations they have accumulated, stop proliferating and lose tumorigenicity in immunodeficient mice. SOX2 is then also fundamental for maintenance of the self-renewal capacity of neural stem cells when they have acquired cancer properties. SOX2, or its immediate downstream effectors, would then be an ideal target for glioblastoma therapy.

DLX5 overexpression impairs osteogenic differentiation of human bone marrow stromal cells.

The transcription factor DLX5 belongs to a family of homeoproteins required for craniofacial morphogenesis and forebrain development. DLX5 is expressed during formation of several skeletal elements such as cartilage, teeth and bone, and its knockout causes severe craniofacial malformations with a delay in the ossification process. Bone marrow contains mesenchymal progenitor cells which may differentiate along multiple pathways, therefore representing an interesting in vitro and in vivo model to study the mesodermal lineage differentiation. Here we report the effect of DLX5 overexpression in ex vivo expanded human bone marrow stromal cells by retroviral infection on the osteogenic lineage differentiation. A reduced mineral deposition was observed in DLX5-transduced cells upon osteogenic induction in culture. When DLX5-transduced cells were implanted in immunodeficient mice, a 60% reduction in bone matrix deposition was observed, whereas the in vitro chondrogenic potential was unaffected. A quantitative gene expression study indicated that DLX5 overexpression does not affect the early osteogenic commitment of bone marrow stromal cells but prevents their terminal differentiation. This block may be mediated by the observed persistent expression of SOX2, a transcription factor known to inhibit osteogenic differentiation.

Glioma immunotherapy by IL-21 gene-modified cells or by recombinant IL-21 involves antibody responses.

Most tumors of the central nervous system, especially glioblastoma, are refractory to treatment and invariably lethal. The aim of this study was to assess the ability of different interleukins (IL), IL-2, IL-12 and IL-21, produced by transduced glioma cells to activate an immune response and trigger intracranial tumor rejection. Such experiments were performed by the use of a slow-growing clone of GL261 (GL D2-60) that was used as orthotopic glioma model. Using GL D2-60-transduced cells, all cytokines elicited an immune response against the tumor. Most notably 100% of the animals receiving a primary implant of IL-21-transduced cells rejected the implant, and 76% of these animals survived to a subsequent rechallenge with GL261 parental cells, while the other transduced cytokine genes were not as effective. Rejection responses were also obtained by admixing wild-type tumor cells with IL-21-producing GL D2-60 cells, indicating a local bystander effect of IL-21. More importantly, IL-21-secreting GL D2-60 cells or 1 microg of rIL-21 protein stereotactically injected into established GL D2-60 tumors were able to trigger glioblastoma rejection in 90 and 77% of mice, respectively. Again most of these mice survived to GL261 rechallenge. Immune mice showed antibody responses to glioma antigens, predominantly involving IgG2a and IgG2b isotypes, which mediated complement- or cell-dependent glioma cell lysis. Antibody responses were crucial for glioma immunotherapy by IL-21-secreting GL D2-60 cells, as immunotherapy was uneffective in syngeneic microMT B-cell-deficient mice. These results suggest that IL-21 should be considered as a suitable candidate for glioma immunotherapy by local delivery.

OTX1 and OTX2 expression correlates with the clinicopathologic classification of medulloblastomas.

OTX1 and OTX2 are transcription factors with an essential role in the development of the cerebellum. We previously described a high OTX2 expression in medulloblastoma. Here, we analyzed amplification and mRNA expression of OTX1 and OTX2 in a series of human medulloblastomas. In addition, OTX2 protein expression was analyzed on tissue arrays. The OTX2 gene was amplified in the medulloblastoma cell line D425 and mRNA and protein data showed expression in 114 of 152 medulloblastomas (75%), but not in postnatal cerebellum. Northern blot (n = 10) and reverse transcriptase-polymerase chain reaction (n = 45) analyses demonstrated that virtually all medulloblastomas expressed OTX1, OTX2, or both. OTX2 mRNA expression correlated with a classic medulloblastoma histology (29 of 34 cases), whereas expression of OTX1 mRNA only was correlated with a nodular/desmoplastic histology (9 of 11 cases). Immunohistochemical analysis of a series of classic medulloblastomas detected OTX2 protein expression in 83 of 107 (78%) cases. The OTX2-positive tumors of this series were preferentially localized in the vermis of the cerebellum, whereas OTX2-negative tumors more frequently occurred in the hemispheres of the cerebellum. In addition, OTX2-positive tumors were mainly found in children, but OTX2-negative tumors occurred in 2 patient groups: very young patients (<5 years) and adults (>20 years). Nodular/desmoplastic medulloblastomas are thought to arise from the external granular layer (EGL). However, it is unclear whether classic medulloblastomas also originate from the EGL or from the ventricular matrix. Analysis of human fetal brain showed OTX2 protein expression in a small number of presumptive neuronal precursor cells of the EGL, but not in precursor cells of the ventricular matrix. Combined with data from rodents, our results therefore suggest that both nodular/desmoplastic and at least part of the classic medulloblastomas originate from cells of the EGL, albeit from different regions.

Effects of Emx2 inactivation on the gene expression profile of neural precursors.

Emx2 plays a crucial role in the development of the diencephalon and dorsal telencephalon. Thus, Emx2-null mutants have abnormal cortical lamination and a reduction in size of the caudal and medial areas of the prosencephalon. Emx2 is expressed in neural precursors of the subventricular zone in vivo and in cultured neurospheres in vitro where it controls the size of the transit-amplifying population, affecting proliferation and clonal efficiency of neural stem cells. To identify the cellular processes mastered by Emx2, and possibly the molecular mechanisms by which the gene exerts its action, we compared the expression profile of cultured neurospheres derived from wild-type and Emx2-null mouse embryos. The differential expression of several genes was also confirmed by semiquantitative RT-PCR, real-time PCR and cytofluorimetric analysis in different preparations of neurospheres, and by in situ hybridization. The gene expression profile suggested a role for Emx2 in regulating the differentiation and migration properties of neural precursor cells. This involvement was confirmed in vitro, where the altered clonogenicity and impaired migration of Emx2-null cells were partially corrected by transduction of the Emx2 gene. Taken together, our results indicate that Emx2 is indeed involved in the transition between resident early progenitors (perhaps stem cells) and more mature precursors capable of migrating out of the ventricular zone, becoming postmitotic and differentiating into the appropriate cell type, and help explain the alterations observed in the brains of knock-out mice.

Regulatory genes controlling cell fate choice in embryonic and adult neural stem cells.

Neural stem cells are the most immature progenitor cells in the nervous system and are defined by their ability to self-renew by symmetric division as well as to give rise to more mature progenitors of all neural lineages by asymmetric division (multipotentiality). The interest in neural stem cells has been growing in the past few years following the demonstration of their presence also in the adult nervous system of several mammals, including humans. This observation implies that the brain, once thought to be entirely post-mitotic, must have at least a limited capacity for self-renewal. This raises the possibility that the adult nervous system may still have the necessary plasticity to undergo repair of inborn defects and acquired injuries, if ways can be found to exploit the potential of neural stem cells (either endogenous or derived from other sources) to replace damaged or defective cells. A full understanding of the molecular mechanisms regulating generation and maintenance of neural stem cells, their choice between different differentiation programmes and their migration properties is essential if these cells are to be used for therapeutic applications. Here, we summarize what is currently known of the genes and the signalling pathways involved in these mechanisms.

Defective neuronogenesis in the absence of Dlx5.

Dlx genes play an important role in the control of the development of the central nervous system (CNS). Single or compound inactivation of Dlx1, Dlx2, or Dlx5 in the mouse causes defects of neuronal migration and differentiation. Dlx5, in particular, is essential for the correct development of the olfactory system. Targeted inactivation of Dlx1 and Dlx2 in the mouse results in abnormal neuronal differentiation in the embryonic subcortical forebrain and is associated to the loss of Dlx5 and Dlx6 expression. So far, however, it has been impossible to investigate the role of Dlx genes on late neurogenesis, as their inactivation leads to perinatal death. We have now generated cultures of neural stem cells (NSCs) derived from embryonic and newborn Dlx5-null mice, and we have compared their capacity to differentiate in vitro to that of equivalent cells derived from normal littermates. We show here that in the absence of Dlx5, NSCs derived from newborn animals have a severely reduced capacity to generate neurons. This is not the case for cells derived from E12.5 embryos. Forced expression of Dlx5 in cultures of newborn mutant NSCs fully restores their neuronogenic potential. Our data suggest that Dlx5 is essential for secondary (postnatal) neuronogenesis.