AB5726 Sigma-AldrichAnti-Dlx2 Antibody

Despite their similarities to bone marrow precursor cells (PC), human umbilical cord blood (HUCB) PCs are more immature and, thus, they exhibit greater plasticity. This plasticity is evident by their ability to proliferate and spontaneously differentiate into almost any cell type, depending on their environment. Moreover, HUCB-PCs yield an accessible cell population that can be grown in culture and differentiated into glial, neuronal and other cell phenotypes. HUCB-PCs offer many potential therapeutic benefits, particularly in the area of neural replacement. We sought to induce the differentiation of HUCB-PCs into glial cells, known as aldynoglia. These cells can promote neuronal regeneration after lesion and they can be transplanted into areas affected by several pathologies, which represents an important therapeutic strategy to treat central nervous system damage. To induce differentiation to the aldynoglia phenotype, HUCB-PCs were exposed to different culture media. Mononuclear cells from HUCB were isolated and purified by identification of CD34 and CD133 antigens, and after 12 days in culture, differentiation of CD34+ HUCB-PCs to an aldynoglia phenotypic, but not that of CD133+ cells, was induced in ensheathing cell (EC)-conditioned medium. Thus, we demonstrate that the differentiation of HUCB-PCs into aldynoglia cells in EC-conditioned medium can provide a new source of aldynoglial cells for use in transplants to treat injuries or neurodegenerative diseases.

Acquiring resistance against transforming growth factor β (TGFβ)-induced growth inhibition at early stages of carcinogenesis and shifting to TGFβ\'s tumour-promoting functions at later stages is a pre-requisite for malignant tumour progression and metastasis. We have identified the transcription factor distal-less homeobox 2 (Dlx2) to exert critical functions during this switch. Dlx2 counteracts TGFβ-induced cell-cycle arrest and apoptosis in mammary epithelial cells by at least two molecular mechanisms: Dlx2 acts as a direct transcriptional repressor of TGFβ receptor II (TGFβRII) gene expression and reduces canonical, Smad-dependent TGFβ signalling and expression of the cell-cycle inhibitor p21(CIP1) and increases expression of the mitogenic transcription factor c-Myc. On the other hand, Dlx2 directly induces the expression of the epidermal growth factor (EGF) family member betacellulin, which promotes cell survival by stimulating EGF receptor signalling. Finally, Dlx2 expression supports experimental tumour growth and metastasis of B16 melanoma cells and correlates with tumour malignancy in a variety of human cancer types. These results establish Dlx2 as one critical player in shifting TGFβ from its tumour suppressive to its tumour-promoting functions.

Most plants have two types of H(+)-translocating inorganic pyrophosphatases (H(+)-PPases), I and II, which differ in primary sequence and K(+) dependence of enzyme function. Arabidopsis thaliana has three genes for H(+)-PPases: one for type I and two for type II. The type I H(+)-PPase requires K(+) for maximal enzyme activity and functions together with H(+)-ATPase in vacuolar membranes. The physiological role of the type II enzyme, which does not require K(+), is not clear. We focused on the type II enzymes (AtVHP2;1 and AtVHP2;2) of A. thaliana. Total amounts of AtVHP2s were quantified immunochemically using a specific antibody and determined to be 22 and 12 ng mg(-1) of total protein in the microsomal fractions of suspension-cultured cells and young roots, respectively, and the values are approximately 0.1 and 0.2%, respectively, of the vacuolar H(+)-PPase. In plants, AtVHP2s were detected immunochemically in all tissues except mature leaves, and were abundant in roots and flowers. The intracellular localization of AtVHP2s in suspension cells was determined by sucrose density gradient centrifugation and immunoblotting. Comparison with a number of marker proteins revealed localization in the Golgi apparatus and the trans-Golgi network. These results suggest that the type II H(+)-PPase functions as a proton pump in the Golgi and related vesicles in young tissues, although its content is very low compared with the type I enzyme.

GnRH, the central regulator of reproductive function, is produced by only approximately 800 highly specialized hypothalamic neurons. Previous studies identified a minimal promoter [GnRH minimal promoter (GnRH-P)] (-173/+1) and a neuron-specific enhancer [GnRH-enhancer (E)1] (-1863/-1571) as regulatory regions in the rat gene that confer this stringent specificity of GnRH expression to differentiated GnRH neurons. In transgenic mice, these two elements target only GnRH neurons but fail to drive expression in the entire population, suggesting the existence of additional regulatory regions. Here, we define two novel, highly conserved, upstream enhancers in the GnRH gene termed GnRH-E2 (-3135/-2631) and GnRH-E3 (-4199/-3895) that increase neuron-specific GnRH expression through interactions with GnRH-E1 and GnRH-P. GnRH-E2 and GnRH-E3 regulate GnRH expression through similar mechanisms via Oct-1, Msx1, and Dlx2, which bind both GnRH-E2 and the GnRH-E3 critical region at -3952/-3895. Overexpression of Dlx2 increases transcription through GnRH-E2 and GnRH-E3. Remarkably, these novel elements are contained within the 3' untranslated region of the neighboring upstream gene, yet are marked endogenously by histone modification signatures consistent with those of enhancers. Thus, GnRH-E2 and GnRH-E3 are novel regulatory elements that, together with GnRH-E1 and GnRH-P, confer the specificity of GnRH expression to differentiated and mature GnRH neurons.

The discoveries of neural (NSCs) and brain tumor stem cells (BTSCs) in the adult human brain and in brain tumors, respectively, have led to a new era in neuroscience research. These cells represent novel approaches to studying normal phenomena such as memory and learning, as well as pathological conditions such as Parkinson's disease, stroke, and brain tumors. This new paradigm stresses the importance of understanding how these cells behave in vitro and in vivo. It also stresses the need to use human-derived tissue to study human disease because animal models may not necessarily accurately replicate the processes that occur in humans. An important, but often underused, source of human tissue and, consequently, both NSCs and BTSCs, is the operating room. This study describes in detail both current and newly developed laboratory techniques, which in our experience are used to process and study human NSCs and BTSCs from tissue obtained directly from the operating room.

The neuronal loss associated with Alzheimer's disease (AD) affects areas of the brain that are vital to cognition. Although recent studies have shown that new neurons can be generated from progenitor cells in the neocortices of healthy adults, the neurogenic potential of the stem/progenitor cells of AD patients is not known. To answer this question, we compared the properties of glial progenitor cells (GPCs) from the cortices of healthy control (HC) and AD subjects. The GPCs from AD brain samples displayed reduced renewal capability and reduced neurogenesis compared with GPCs from HC brains. To investigate the mechanisms underlying this difference, we compared beta-catenin signaling proteins in GPCs from AD versus HC subjects and studied the effect of amyloid beta peptide (Abeta, a hallmark of AD pathology) on GPCs. Interestingly, GPCs from AD patients exhibited elevated levels of glycogen synthase kinase 3beta (GSK-3beta, an enzyme known to phosphorylate beta-catenin), accompanied by an increase in phosphorylated beta-catenin and a decrease in nonphosphorylated beta-catenin compared with HC counterparts. Furthermore. we found that Abeta treatment impaired the ability of GPCs from HC subjects to generate new neurons and caused changes in beta-catenin signaling proteins similar to those observed in GPCs from AD patients. Similar results were observed in GPCs isolated from AD transgenic mice. These results suggest that Abeta-induced interruption of beta-catenin signaling may contribute to the impairment of neurogenesis in AD progenitor cells.

Distinct olfactory bulb (OB) interneurons are thought to become specified depending on from which of the different subregions lining the lateral ventricle wall they originate, but the role of region-specific transcription factors (TFs) in the generation of OB interneurons diversity is still poorly understood. Despite the crucial roles of the Dlx family of TFs for patterning and neurogenesis in the ventral telencephalon during embryonic development, their role in adult neurogenesis has not yet been addressed. Here we show that in the adult brain, Dlx 1 and Dlx2 are expressed in progenitors of the lateral but not the dorsal subependymal zone (SEZ), thus exhibiting a striking regional specificity. Using retroviral vectors to examine the function of Dlx2 in a cell-autonomous manner, we demonstrate that this TF is necessary for neurogenesis of virtually all OB interneurons arising from the lateral SEZ. Beyond its function in generic neurogenesis, Dlx2 also plays a crucial role in neuronal subtype specification in the OB, promoting specification of adult-born periglomerular neurons (PGNs) toward a dopaminergic fate. Strikingly, Dlx2 requires interaction with Pax6, because Pax6 deletion blocks Dlx2-mediated PGN specification. Thus, Dlx2 wields a dual function by first instructing generic neurogenesis from adult precursors and subsequently specifying PGN subtypes in conjunction with Pax6.

Neural progenitor cells (NPCs) are found in the subventricular zone (SVZ) of the adult brain, a specialized neurogenic niche that might provide a substrate for brain repair after injury. The incomplete knowledge of how NPCs in the niche respond to local signals limits the use of cultured NPCs in the development of cell transplantation strategies. We show that neurospheres obtained from the SVZ of the adult mouse expressed functional mGlu1 and mGlu5 metabotropic glutamate receptors. Pharmacological blockade of mGlu5 receptors promoted the apoptotic death of progenitors undergoing differentiation into neurons (PSA/NCAM+ cells for the most part), whereas blockade of mGlu1 receptors reduced the proliferation rate of NPCs, and promoted their differentiation towards the neuronal lineage. We conclude that endogenous activation of mGlu5 receptors might support specifically the survival of neuronal-restricted precursors, whereas endogenous activation of mGlu1 receptors might sustain the proliferation of earlier progenitors. Moreover, mGlu1 receptor antagonists increased the survival of NPCs, suggesting that endogenously activated mGlu1 receptors might play a role in the natural cell loss regulating the number or the type of progenitors.

Insulin-like growth factor-I (IGF-I) promotes bone formation by stimulating proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP), is thought to function in the initial mineralization of bone, is selectively expressed by differentiated osteoblast. To determine the molecular mechanism of IGF-I regulation of osteogenesis, we analyzed the effects of IGF-I on the expression of BSP in osteoblast-like Saos2 and in rat stromal bone marrow (RBMC-D8) cells. IGF-I (50 ng/ml) increased BSP mRNA levels at 12 h in Saos2 cells. In RBMC-D8 cells, IGF-I increased BSP mRNA levels at 3 h. From transient transfection assays, a twofold increase in transcription by IGF-I was observed at 12 h in pLUC3 construct that included the promoter sequence from -116 to +60. Effect of IGF-I was abrogated by 2-bp mutations in either the FGF2 response element (FRE) or homeodomain protein-binding site (HOX). Gel shift analyses showed that IGF-I increased binding of nuclear proteins to the FRE and HOX elements. Notably, the HOX-protein complex was supershifted by Smad1 antibody, while the FRE-protein complex was shifted by Smad1 and Cbfa1 antibodies. Dlx2 and Dlx5 antibodies disrupted the formation of the FRE- and HOX-protein complexes. The IGF-I effects on the formation of FRE-protein complexes were abolished by tyrosine kinase inhibitor herbimycin A (HA), PI3-kinase/Akt inhibitor LY249002, and MAP kinase kinase inhibitor U0126, while IGF-I effects on HOX-protein complexes were abolished by HA and LY249002. These studies demonstrate that IGF-I stimulates BSP transcription by targeting the FRE and HOX elements in the proximal promoter of BSP gene.

Gonadotropin-releasing hormone (GnRH) is the central regulator of the hypothalamic-pituitary-gonadal axis, controlling sexual maturation and fertility in diverse species from fish to humans. GnRH gene expression is limited to a discrete population of neurons that migrate through the nasal region into the hypothalamus during embryonic development. The GnRH regulatory region contains four conserved homeodomain binding sites (ATTA) that are essential for basal promoter activity and cell-specific expression of the GnRH gene. MSX and DLX are members of the Antennapedia class of non-Hox homeodomain transcription factors that regulate gene expression and influence development of the craniofacial structures and anterior forebrain. Here, we report that expression patterns of the Msx and Dlx families of homeodomain transcription factors largely coincide with the migratory route of GnRH neurons and co-express with GnRH in neurons during embryonic development. In addition, MSX and DLX family members bind directly to the ATTA consensus sequences and regulate transcriptional activity of the GnRH promoter. Finally, mice lacking MSX1 or DLX1 and 2 show altered numbers of GnRH-expressing cells in regions where these factors likely function. These findings strongly support a role for MSX and DLX in contributing to spatiotemporal regulation of GnRH transcription during development.