Expression of Drosophila MAGE gene encoding a necdin homologous protein in postembryonic neurogenesis

The MAGE (melanoma antigen) family is characterized by a large conserved domain termed MAGE homology domain. Originally identified MAGE genes encoding tumor rejection antigens are expressed only in cancers and male germ cells. Necdin, which contains the MAGE homology domain, is highly expressed in postmitotic cells such as neurons and skeletal muscle cells. The human necdin gene NDN is transcribed only from the paternal allele through genomic imprinting, and its deficiency is implicated in the pathogenesis of the neurodevelopmental disorder Prader-Willi syndrome. Although over 30 MAGE genes have been identified in humans, fruit fly (Drosophila melanogaster) has only a single MAGE gene that encodes a protein similar to necdin homologous MAGE proteins. In this study, we analyzed the spatiotemporal expression patterns of MAGE mRNA and the encoded protein during fly development. Whole-mount embryo in situ hybridization analysis revealed that MAGE mRNA was highly expressed at the syncytial blastoderm stage and in the ventral and procephalic neurogenic regions of the ectoderm during gastrulation. In contrast, MAGE expression was nearly undetectable in postmitotic neurons of the central nervous system at late embryonic stages. During postembryonic neurogenesis, MAGE was highly expressed in neural stem cells (neuroblasts) and their progeny (ganglion mother cells and postmitotic neurons) at larval and pupal stages. MAGE was also expressed in postmitotic neurons including mushroom body neurons and retinal photoreceptors in adulthood. These results indicate that MAGE expression lasts throughout the postembryonic neurogenesis in Drosophila.     

Combinatorial expression patterns of individual TLE proteins during cell determination and differentiation suggest non-redundant functions for mammalian homologs of Drosophila Groucho

The Drosophila protein Groucho is involved in the regulation of cell-determination events during insect neurogenesis and segmentation. A group of mammalian proteins, referred to as transducin-like Enhancer of split (TLE) 1 through 4, share with Groucho identical structures and molecular properties. The aim was to determine whether individual TLE proteins participate in the regulation of cell determination in mammals like their Drosophila counterpart. It is here reported that TLE family members are expressed in combinatorial ways during the in vitro differentiation of mouse P19 embryonic carcinoma cells (a model for neural determination) and rat CFK2 cells (a model for chondrocytic determination). TLE1 is up-regulated and TLE2 and TLE4 are down-regulated to different extents during early stages of differentiation. In contrast, later stages correlate with up-regulation of TLE2 and TLE4, and decreased expression of TLE1. Individual TLE proteins are also expressed in combinatorial as well as complementary patterns during the development of the cerebral cortex and spinal cord of mouse embryos. In particular, TLE1 is robustly expressed in both neural progenitor cells and postmitotic neurons of the outer layers of the cortical plate, whereas TLE4 expression marks preferentially postmitotic neurons of the inner layers. Taken together, these results strongly suggest non-redundant roles for individual TLE proteins during both cell-determination and cell-differentiation events.     

Mouse Prickle1 and Prickle2 are expressed in postmitotic neurons and promote neurite outgrowth

The Drosophila planar cell polarity (PCP) gene prickle has been previously indicated as one of the regulators of gastrulation in the early embryonic stage. However, the functional role of prickle in the brain in particular is not known. We first indicated that mouse Prickle1 and Prickle2 are continually expressed in the brain throughout the embryonic stages and are observed to be specifically expressed in the postmitotic neurons. Furthermore, Prickle1 or Prickle2 depletion effectively decreases the neurite outgrowth levels of mouse neuroblastoma Neuro2a cells. These results indicate that mouse Prickle1 and Prickle2 possibly regulate positive neurite formation during brain development.     

Life without huntingtin: normal differentiation into functional neurons

Huntington disease (HD) is a neurodegenerative disorder associated with polyglutamine expansion in a recently identified protein, huntingtin. Huntingtin is widely expressed and plays a crucial role in development, because gene-targeted HD-/- mouse embryos die early in embryogenesis. To analyze the function of normal huntingtin, we have generated HD-/- embryonic stem (ES) cells and used an in vitro model of ES cell differentiation to analyze their ability to develop into neuronal cells. Expression analysis of wild-type ES cells revealed that huntingtin is expressed at all stages during ES cell differentiation with high expression in neurons. Expression levels increased with the maturation of differentiating neurons, demonstrating that expression of huntingtin is developmentally regulated in cell culture and resembles the pattern of expression observed in differentiating neurons in the mouse brain. It is interesting that HD-/- ES cells could differentiate into mature postmitotic neurons that expressed functional voltage- and neurotransmitter-gated ion channels. Moreover, both excitatory and inhibitory spontaneous postsynaptic currents were observed, indicating the establishment of functional synapses in the absence of huntingtin. These results demonstrate that huntingtin is not required for the generation of functional neurons with features characteristic of postmitotic neurons in the developing mouse brain.     

Identification of genes up-regulated by retinoic-acid-induced differentiation of the human neuronal precursor cell line NTERA-2 cl.D1

The human teratocarcinoma cell line NTERA-2 cl.D1 (NT2 cells) can be induced with retinoic acid and cell aggregation to yield postmitotic neurones. This seems to model the in vivo situation, as high concentrations of retinoic acid, retinoic acid binding proteins, and receptors have been detected in the embryonic CNS and the developing spinal cord suggesting a role for retinoic acid in neurogenesis. Suppression subtractive hybridization was used to detect genes up-regulated by this paradigm of neuronal differentiation. Microfibril-associated glycoprotein 2 was found to be drastically up-regulated and has not been implicated in neuronal differentiation before. Suppression subtractive hybridization also identified DYRK4, a homologue of the Drosophila gene minibrain. Minibrain mutations result in specific defects in the development of the fly central nervous system. In adult rats, DYRK4 is only expressed in testis, but our results suggest an additional role for DYRK4 in neuronal differentiation. We have shown that suppression subtractive hybridization in conjunction with an efficient screening procedure is a valuable tool to produce a repertoire of differentially expressed genes and propose a new physiological role for several identified genes and expressed sequence tags.     

The microtubule destabilizing protein stathmin controls the transition from dividing neuronal precursors to postmitotic neurons during adult hippocampal neurogenesis

The hippocampus is one of the two areas in the mammalian brain where adult neurogenesis occurs. Adult neurogenesis is well known to be involved in hippocampal physiological functions as well as pathophysiological conditions. Microtubules (MTs), providing intracellular transport, stability, and transmitting force, are indispensable for neurogenesis by facilitating cell division, migration, growth, and differentiation. Although there are several examples of MT‐stabilizing proteins regulating different aspects of adult neurogenesis, relatively little is known about the function of MT‐destabilizing proteins. Stathmin is such a MT‐destabilizing protein largely restricted to the CNS, and in contrast to its developmental family members, stathmin is also expressed at significant levels in the adult brain, notably in areas involved in adult neurogenesis. Here, we show an important role for stathmin during adult neurogenesis in the subgranular zone of the mouse hippocampus. After carefully mapping stathmin expression in the adult dentate gyrus (DG), we investigated its role in hippocampal neurogenesis making use of stathmin knockout mice. Although hippocampus development appears normal in these animals, different aspects of adult neurogenesis are affected. First, the number of proliferating Ki‐67+ cells is decreased in stathmin knockout mice, as well as the expression of the immature markers Nestin and PSA‐NCAM. However, newborn cells that do survive express more frequently the adult marker NeuN and have a more mature morphology. Furthermore, our data suggest that migration in the DG might be affected. We propose a model in which stathmin controls the transition from neuronal precursors to early postmitotic neurons. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1226–1242, 2014     

Transducin-like enhancer of Split-1 (TLE1) combines with Forkhead box protein G1 (FoxG1) to promote neuronal survival

Transducin-like enhancer of split-1 (TLE1) plays a critical role in the regulation of neurogenesis by inhibiting the differentiation of neural progenitor cells into neurons. Although TLE1 is also expressed highly in the postnatal brain and through adulthood, its role in postmitotic neurons is not clear. Using cultures of cerebellar granule neurons, we show that expression of TLE1 is reduced in neurons primed to die. Reestablishment of elevated TLE1 levels by ectopic expression protects neurons from death, whereas suppression of TLE1 expression in otherwise healthy neurons induces cell death. These results show that TLE1 is necessary for the maintenance of neuronal survival. Experiments using pharmacological inhibitors as well as expression of point mutants indicate that phosphorylation of TLE1 by casein kinase-2 (CK2) at Ser-239 and Ser-253 is necessary for its survival-promoting activity. TLE1-mediated survival is also inhibited by pharmacological inhibition of PI3K-Akt signaling but not by inhibitors of Raf-MEK-ERK signaling or other molecules, including histone deacetylases, calcium calmodulin kinase, or CK1. The survival-promoting activity of TLE1 depends critically on interaction with FoxG1, another protein involved in the regulation of neurogenesis and shown previously to promote survival of postmitotic neurons. Likewise, the ability of FoxG1 to promote neuronal survival depends on TLE1. Taken together, our study demonstrates that TLE1 cooperates with FoxG1 to promote neuronal survival in a CK2- and PI3K-Akt-dependent manner.