Every year, a significant numbers of Canadians are affected by psychiatric and neurological disorders such as depression, autism and schizophrenia. The possibility of promoting neurogenesis following the onset of these diseases is an extremely appealing therapeutic option. The functionally intertwined Retinoblastoma (pRb) and E2F protein families are well known for their essential roles in regulating cell cycle entry; however we have described expanded roles for these proteins in regulating multiple aspects of neural development. Determining the mechanism that regulates these processes is essential for our understanding of how neural development proceeds. We show that the absence of the cell cycle regulatory protein E2F4 leads to a deficit in neural stem cell numbers and a severe impairment of self renewal. Additionally, E2F4 deficiency results in a loss of ventral telencephalic structures, a phenotype with striking similarity to animals lacking the Sonic Hedgehog (Shh) gene. We have previously shown that loss of both Shh expression and ventral telencephalic structures are rescued by interbreeding E2F4 s mutant with mice heterozygous for Gli3, a negative regulator of the Shh pathway. Preliminary data using ChIP-on Chip has identified Gli3 as a regulatory target for the E2F4 transcription factor. These findings suggest that E2F4 negatively regulates Gli3 expression and that in the absence of E2F4, Gli3 aberrantly represses Shh expression, consequently impeding ventral telencephalic patterning. In conclusion, these findings suggest that E2F4 is an essential regulator of neural stem cell renewal and telencephalic patterning by regulating the activity of the Shh pathway through repression of Gli3 expression.In order to effectively harness the potential of endogenous neural stem cells for therapeutic application the mechanisms which govern their proliferation, fate decisions and integration into their proper neural circuit need to be fully elucidated. Appreciated for its role in regulating cell cycle progression, it is now emerging that the pRb/E2F pathway is capable of mediating events in neural development beyond cell cycle regulation. The exact molecular mechanisms through which these roles are achieved remain unknown. We have previously described a requirement for the interaction between pRb and E2F3 to mediate neuronal migration during nervous system development, and identified neogenin as specifically deregulated in the absence of pRb. Through in-vitro explant cultures and ex-vivo electroporation, we observe a novel role for neogenin and its netrin ligand, in mediating migration from the ganglionic eminence. Conditional pRb neural precursors cells exhibit an increased propensity to adhere to substrate bound netrin-1 suggesting aberrant adhesion as the defective process in tangential migration. We also display an interaction between E2F3 and a conserved nonconsensus E2F motif in the promoter region of neogenin. By identifying a receptor ligand pathway disrupted in pRb deficiency, these results contribute to our h...

Recent evidence suggests that cell cycle proteins may have novel functions beyond the control of cell division. We have investigated the role of Rb/E2F pathway in the regulation of neuronal differentiation and migration during late embryonic development.We show that loss of Rb leads to terminal differentiation and radial migration defects as well as loss of specific interneuron subtypes in the developing forebrain and olfactory bulb. This phenotype is linked to a dramatic reduction in the levels of Dlx homeodomain genes that regulate ventral telencephalic development, most significantly Dlx2. To ask if Rb plays a direct role in controlling the induction of Dlx2, we examined the regulatory regions of the Dlx1/Dlx2 locus. Using chromatin immunoprecipitation experiments, we show that Rb modulates Dlx gene expression through interaction with the Dlx forebrain-specific enhancer, I12b, the Dlx2 proximal promoter and 3'UTR region in vivo. This interaction is mediated by E2F functional sites located in I12b that act as repressor sites. Deletion of E2F consensus sites on the I12b-Dlx1/Dlx2 enhancer results in increased reporter activity in the subventricular zone of the developing brain. We demonstrate that in the absence of Rb, E2F7, an Rb-independent repressor, is upregulated in the brain and could ectopically repress the I12b activity and Dlx2 transcription.In conclusion, our data provides the first evidence that cell cycle proteins such as Rb play an essential role to coordinate the transition from proliferation to differentiation and maintain terminal differentiation by regulating the levels of key transcription factors such as Dlx2 during neurogenesis. This work was supported by a grant from CIHR.The specification of unique neuronal and glial sub-types relies upon the restricted expression of cell fate determinants, acting primarily in 'combinatorial codes'. The expression of the proper combinatorial code in each sub-type in turn relies upon the combinatorial action of upstream regulators, acting in a positional and temporal manner. While many players acting at these different levels have been identified, the complex multi-step regulatory flow acting to dictate any one unique cell fate in the nervous system has not been deciphered. Moreover, because each sub-type is generated in distinct numbers, such in-depth decoding of the neural diversification process also requires addressing how upstream and downstream combinatorial codes intersect with the cell cycle and cell death machineries.To address these issues, we are using a specific Drosophila CNS progenitor cell, the neuroblast 5-6, as a model. This neuroblast is presented in all 18 segments of the developing fly CNS, but generates a unique group of neurons, the Apterous cluster of four neurons, only in the three thoracic segments. Two of the Ap cluster cells are furthermore uniquely identifiable by their selective expression of the FMRFa and Nplp1 neuropeptide genes. Specification of Ap neurons requires a complex interplay between positional and temporal cues, whic...

factor 8 (Fgf8), Sonic hedgehog (Shh), bone morphogenetic proteins (BMPs)2,4,6 and 7, and Wnts 2b,3a,5a and 7a control the regional expression of specific transcription factors. Some of these secreted factors are expressed at the region of the ZLI, being Shh the main factor studied controlling proliferation, regionalization and polarity in the diencephalic segments. Nevertheless, despite the importance of the ZLI, the mechanism of its formation remains poorly understood.In this work, we have studied the molecular mechanisms for generating a permissive territory for Shh expression in the ZLI, by using experimental embryology techniques: insertion of microbarriers and protein soaked beads in the diencephalic neuroepithelium. We have analyzed the molecular interactions between genes expressed in the diencephalon such as Shh, Glis, Wnts, Fgf and L-fng, among others, by in situ hybridization. Our results let us to describe new molecular processes regulating the establishment of the ZLI compartment.

Every year, a significant numbers of Canadians are affected by psychiatric and neurological disorders such as depression, autism and schizophrenia. The possibility of promoting neurogenesis following the onset of these diseases is an extremely appealing therapeutic option. The functionally intertwined Retinoblastoma (pRb) and E2F protein families are well known for their essential roles in regulating cell cycle entry; however we have described expanded roles for these proteins in regulating multiple aspects of neural development. Determining the mechanism that regulates these processes is essential for our understanding of how neural development proceeds. We show that the absence of the cell cycle regulatory protein E2F4 leads to a deficit in neural stem cell numbers and a severe impairment of self renewal. Additionally, E2F4 deficiency results in a loss of ventral telencephalic structures, a phenotype with striking similarity to animals lacking the Sonic Hedgehog (Shh) gene. We have previously shown that loss of both Shh expression and ventral telencephalic structures are rescued by interbreeding E2F4 s mutant with mice heterozygous for Gli3, a negative regulator of the Shh pathway. Preliminary data using ChIP-on Chip has identified Gli3 as a regulatory target for the E2F4 transcription factor. These findings suggest that E2F4 negatively regulates Gli3 expression and that in the absence of E2F4, Gli3 aberrantly represses Shh expression, consequently impeding ventral telencephalic patterning. In conclusion, these findings suggest that E2F4 is an essential regulator of neural stem cell renewal and telencephalic patterning by regulating the activity of the Shh pathway through repression of Gli3 expression.In order to effectively harness the potential of endogenous neural stem cells for therapeutic application the mechanisms which govern their proliferation, fate decisions and integration into their proper neural circuit need to be fully elucidated. Appreciated for its role in regulating cell cycle progression, it is now emerging that the pRb/E2F pathway is capable of mediating events in neural development beyond cell cycle regulation. The exact molecular mechanisms through which these roles are achieved remain unknown. We have previously described a requirement for the interaction between pRb and E2F3 to mediate neuronal migration during nervous system development, and identified neogenin as specifically deregulated in the absence of pRb. Through in-vitro explant cultures and ex-vivo electroporation, we observe a novel role for neogenin and its netrin ligand, in mediating migration from the ganglionic eminence. Conditional pRb neural precursors cells exhibit an increased propensity to adhere to substrate bound netrin-1 suggesting aberrant adhesion as the defective process in tangential migration. We also display an interaction between E2F3 and a conserved nonconsensus E2F motif in the promoter region of neogenin. By identifying a receptor ligand pathway disrupted in pRb deficiency, these results contribute to our h...

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