Mash1-positive Cells Research Articles

Mash1-expressing cells could differentiate to type III cells in adult mouse taste buds.

The gustatory cells in taste buds have been identified as paraneuronal; they possess characteristics of both neuronal and epithelial cells. Like neurons, they form synapses, store and release transmitters, and are capable of generating an action potential. Like epithelial cells, taste cells have a limited life span and are regularly replaced throughout life. However, little is known about the molecular mechanisms that regulate taste cell genesis and differentiation. In the present study, to begin to understand these mechanisms, we investigated the role of Mash1-positive cells in regulating adult taste bud cell differentiation through the loss of Mash1-positive cells using the Cre-loxP system. We found that the cells expressing type III cell markers-aromatic L-amino acid decarboxylase (AADC), carbonic anhydrase 4 (CA4), glutamate decarboxylase 67 (GAD67), neural cell adhesion molecule (NCAM), and synaptosomal-associated protein 25 (SNAP25)-were significantly reduced in the circumvallate taste buds after the administration of tamoxifen. However, gustducin and phospholipase C beta2 (PLC beta2)-markers of type II taste bud cells-were not significantly changed in the circumvallate taste buds after the administration of tamoxifen. These results suggest that Mash1-positive cells could be differentiated to type III cells, not type II cells in the taste buds.

Netrin-5 is highly expressed in neurogenic regions of the adult brain.

Mammalian netrin family proteins are involved in targeting of axons, neuronal migration, and angiogenesis and act as repulsive and attractive guidance molecules. Netrin-5 is a new member of the netrin family with homology to the C345C domain of netrin-1. Unlike other netrin proteins, murine netrin-5 consists of two EGF motifs of the laminin V domain (LE) and the C345C domain, but lacks the N-terminal laminin VI domain and one of the three LE motifs. We generated a specific antibody against netrin-5 to investigate its expression pattern in the rodent adult brain. Strong netrin-5 expression was observed in the olfactory bulb (OB), rostral migrate stream (RMS), the subventricular zone (SVZ), and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus, where neurogenesis occurs in the adult brain. In the SVZ and RMS, netrin-5 expression was observed in Mash1-positive transit-amplifying cells and in Doublecortin (DCX)-positive neuroblasts, but not in GFAP-positive astrocytes. In the OB, netrin-5 expression was maintained in neuroblasts, but its level was decreased in NeuN-positive mature neurons. In the hippocampal SGZ, netrin-5 was observed in Mash1-positive cells and in DCX-positive neuroblasts, but not in GFAP-positive astrocytes, suggesting that netrin-5 expression occurs from type 2a to type 3 cells. These data suggest that netrin-5 is produced by both transit-amplifying cells and neuroblasts to control neurogenesis in the adult brain.

Neurogenesis in the circumventricular organs of adult mouse brains

The circumventricular organs (CVOs), including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), median eminence (ME), and area postrema (AP), allow parenchyma cells to sense a variety of blood-derived substances and/or secreted peptides into blood circulation. In the present study, we examined continuous neurogenesis in the CVOs of adult mice. The immunohistochemistry of neural progenitor cell (NPC) marker proteins revealed that Math1- and Mash1-positive cells were observed in the discrete regions of CVOs, including the capillary plexus in the OVLT, the internal zone of the ME, and the lateral zone in the AP. A few Mash1- and Math1-positive cells were seen throughout the SFO, and many Math1- but not Mash1-positive cells were observed at the arcuate nucleus. Math-positive cells were often seen to localize in close proximity to the vasculature. Bromodeoxyuridine (BrdU) immunohistochemistry revealed the incorporation of BrdU in a subpopulation of Mash1-, Math1-, HuC/D-, and microtubule-associated protein 2 (MAP2)-positive cells. Mash1- and Math1-positive cells expressed exclusively high level of plasminogen, whereas a subpopulation of HuC/D- and MAP2-positive neurons expressed low or undetectable level of plasminogen. Thus, the present study demonstrates that newborn cells express NPC marker proteins and plasminogen to localize closely at vascular matrix and moreover differentiate into neurons expressing mature neuron marker proteins, indicating that new neurons are possibly generated to integrate into new neural circuits.

Purinergic signaling promotes proliferation of adult mouse subventricular zone cells.

In adult mammalian brains, neural stem cells (NSCs) exist in the subventricular zone (SVZ), where persistent neurogenesis continues throughout life. Those NSCs produce neuroblasts that migrate into the olfactory bulb via formation of transit-amplifying cells, which are committed precursor cells of the neuronal lineage. In this SVZ niche, cell-cell communications conducted by diffusible factors as well as physical cell-cell contacts are important for the regulation of the proliferation and fate determination of NSCs. Previous studies have suggested that extracellular purinergic signaling, which is mediated by purine compounds such as ATP, plays important roles in cell-cell communication in the CNS. Purinergic signaling also promotes the proliferation of adult NSCs in vitro. However, the in vivo roles of purinergic signaling in the neurogenic niche still remain unknown. In this study, ATP infusion into the lateral ventricle of the mouse brain resulted in an increase in the numbers of rapidly dividing cells and Mash1-positive transit-amplifying cells (Type C cells) in the SVZ. Mash1-positive cells express the P2Y1 purinergic signaling receptor and infusion of the P2Y1 receptor-specific antagonist MRS2179 decreased the number of rapidly dividing bromodeoxyuridine (BrdU)-positive cells and Type C cells. Moreover, a 17% reduction of rapidly dividing BrdU-positive cells and a 19% reduction of Mash1-positive cells were observed in P2Y1 knock-out mice. Together, these results suggest that purinergic signaling promotes the proliferation of rapidly dividing cells and transit-amplifying cells, in the SVZ niche through the P2Y1 receptor.

The dynamic expression of Mash1 in the hippocampal subgranular zone after fimbria-fornix transection

Mash1, a member of the basic helix-loop-helix (bHLH) transcription factor family, has previously been considered essential for neuronal differentiation and specification in the nervous system. In this study, we investigated the expression of Mash1 in the hippocampus after fimbria-fornix (FF) transection. Western blot showed that protein of Mash1 increased significantly and peaked at day 7 after FF transection. Immunofluorescence indicated that after FF transection, more newborn cells differentiated into Mash1 positive cells in the deafferented side than that in the normal side, and we investigated that in the neurogenic area, subgranular zone (SGZ), a part of Mash1 positive cells were NeuN positive, and more Mash1/NeuN double positive neurons were identified in the deafferented side than that in the normal side. Additionally, the number of Mash1/NeuN double positive neurons in SGZ increased significantly and peaked at day 7 after FF transection. In vitro, immunofluorescence revealed that extracts of the deafferented hippocampus promoted neuronal differentiation to a greater extent than extracts from normal hippocampus. Deafferented extracts also enhanced Mash1 expression in MAP-2 positive neurons. This study concludes that after FF transection, Mash1 expression in the deafferented hippocampus increased and might play an important role in inducing local progenitors to differentiate into neurons.

Features and a possible role of Mash1-immunoreactive cells in the dentate gyrus of the hippocampus in the adult rat

Neurogenesis occurs throughout life in both the subventricular zone (SVZ) and subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus in the adult brain. In the SVZ, it has been demonstrated that transit-amplifying neural progenitor cells, which appear between neural stem/progenitor cells (NSPCs) and neuroblasts during the neuronal differentiation process, express mammalian achaete–scute homolog 1 (Mash1), which regulates differentiation during neurogenesis. Although Mash1-positive cells (Mash1+ cells) are observed in the SGZ, the importance of Mash1 in hippocampal neurogenesis is not sufficiently understood. In the present study, using immunohistochemical techniques, we examined whether Mash1+ cells in the SGZ act as transit-amplifying neural progenitor cells, and whether chronic treadmill running can induce alterations of the Mash1+ cells in the SGZ of the DG. The present results indicated that Mash1 immunoreactivity is detected in proliferative cells, and that astrocytes or NSPCs and neuroblasts express Mash1. A quantitative analysis of Mash1-positive astrocytes or NSPCs and Mash1-positive neuroblasts indicated that approximately 90% of Mash1+ cells did not belong to astrocytic and neuronal cells. Furthermore, chronic treadmill running induced an increase in the number of proliferating Mash1+ cells. The present study suggests that the majority of the Mash1+ cells in the SGZ may be transit-amplifying neural progenitor cells. In addition, the proliferation of Mash1-positive transit-amplifying neural progenitor cells may contribute to the exercise-induced neurogenesis that is associated with the improvement of learning and memory function.

Differential Regulation of Basic Helix-Loop-Helix Factors Mash1 and Olig2 by β-Amyloid Accelerates Both Differentiation and Death of Cultured Neural Stem/Progenitor Cells

Despite increased neurogenic differentiation markers in the hippocampal CA1 in Alzheimer disease, neurons are not replaced in CA1 and the neocortex in the disease. β-Amyloid (Aβ) might cause deterioration of the brain microenvironment supporting neurogenesis and the survival of immature neurons. To test this possibility, we examined whether Aβ alters the expression of cell fate determinants in cerebral cortical cultures and in an Alzheimer disease mouse model (PrP-APPSW). Up-regulation of Mash1 and down-regulation of Olig2 were found in cerebral cortical cultures treated with Aβ-(1-42). Mash1 was expressed in nestin-positive immature cells. The majority of Mash1-positive cells in untreated cortical culture co-expressed Olig2. Aβ increased the proportion of Olig2-negative/Mash1-positive cells. A decrease in Olig2+ cells was also observed in the cerebral cortex of adult PrP-APPSW mice. Cotransfection experiments with Mash1 cDNA and Olig2 siRNA revealed that overexpression of Mash1 in neurosphere cells retaining Olig2 expression enhanced neural differentiation but accelerated death of Olig2-depleted cells. Growth factor deprivation, which down-regulated Olig2, accelerated death of Mash1-overexpressing neurosphere cells. We conclude that cooperation between Mash1 and Olig2 is necessary for neural stem/progenitor cells to develop into fully mature neurons and that down-regulation of Olig2 by Aβ in Mash1-overexpressing cells switches the cell fate to death. Maintaining Olig2 expression in differentiating cells could have therapeutic potential.

Taste bud contains both short-lived and long-lived cell populations

Taste bud cells undergo continual turnover even in adulthood, and their average lifespan has been estimated as approximately 10 days. However, it is not clear whether this figure can be applied to all the different cell types contained in a taste bud. Here, we describe the age and life cycle of taste bud cells in rat circumvallate papillae, and indicate that the lifespan is heterogeneous, ranging from 2 days to over 3 weeks. Taste bud cells were incorporated from the basal proliferative layer in 1–2 days after birth. After incorporation, approximately half of the cells were eliminated within 2–3 days, and the remaining half were maintained with gradual decrease, suggesting that there are at least two types of cells; short-lived cells and long-lived cells. Moreover, above 10% of the incorporated cells were maintained at 3 weeks. In order to gain information about the relationship between the cell functions and the cell age, we carried out double-labeling experiments using 5-bromo-2′-deoxyuridine and each of two markers for in situ hybridization: mammalian achaete-scute homolog 1 (Mash1) and phospholipase C beta 2 (PLCβ2) as markers of early differentiation and functional taste signaling, respectively. Mash1 expression began immediately after the incorporation and reached a maximum at 5–6 days after birth. Fewer but distinct Mash1-positive cells were still observed after 3 weeks. PLCβ2 expression was observed from day 5, reached a maximum at day 12, and continued over 3 weeks. Taken together, a taste bud contains both short-lived and long-lived cells: the short-lived cells are eliminated in a time course similar to the surrounding epithelial cells, and the long-lived cells including taste receptor cells have a lifespan longer than the previous estimation.

Mash1 specifies neurons and oligodendrocytes in the postnatal brain

Progenitors in the telencephalic subventricular zone (SVZ) remain mitotically active throughout life, and produce different cell types at embryonic, postnatal and adult stages. Here we show that Mash1, an important proneural gene in the embryonic telencephalon, is broadly expressed in the postnatal SVZ, in progenitors for both neuronal and oligodendrocyte lineages. Moreover, Mash1 is required at birth for the generation of a large fraction of neuronal and oligodendrocyte precursors from the olfactory bulb. Clonal analysis in culture and transplantation experiments in postnatal brain demonstrate that this phenotype reflects a cell-autonomous function of Mash1 in specification of these two lineages. The conservation of Mash1 function in the postnatal SVZ suggests that the same transcription mechanisms operate throughout life to specify cell fates in this structure, and that the profound changes in the cell types produced reflect changes in the signalling environment of the SVZ.

Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium.

To clarify the mechanisms that regulate neuroendocrine differentiation of fetal lung epithelia, we have studied the expression of the mammalian homologs of achaete-scute complex (Mash1) (Ascl1 - Mouse Genome Informatics); hairy and enhancer of split1 (Hes1); and the expression of Notch/Notch-ligand system in the fetal and adult mouse lungs, and in the lungs of Mash1- or Hes1-deficient mice. Immunohistochemical studies revealed that Mash1-positive cells seemed to belong to pulmonary neuroendocrine cells (PNEC) and their precursors. In mice deficient for Mash1, no PNEC were detected. Hes1-positive cells belong to non-neuroendocrine cells. In the mice deficient in Hes1, in which Mash1 mRNA was upregulated, PNEC appeared precociously, and the number of PNEC was markedly increased. NeuroD (Neurod1 - Mouse Genome Informatics) expression in the lung was detected in the adult, and was enhanced in the fetal lungs of Hes1-null mice. Expression of Notch1, Notch2, Notch3 and Notch4 mRNAs in the mouse lung increased with age, and Notch1 mRNA was expressed in a Hes1-dependent manner. Notch1, Notch2 and Notch3 were immunohistochemically detected in non-neuroendocrine cells. Moreover, analyses of the lungs from the gene-targeted mice suggested that expression of Delta-like 1 (Dll1 - Mouse Genome Informatics) mRNA depends on Mash1. Thus, the neuroendocrine differentiation depends on basic helix-loop-helix factors, and Notch/Notch-ligand pathways may be involved in determining the cell differentiation fate in fetal airway epithelium.