Eph signaling controls mitotic spindle orientation and cell proliferation in neuroepithelial cells.

Mitotic spindle orientation must be tightly regulated during development and adult tissue homeostasis. It determines cell-fate specification and tissue architecture during asymmetric and symmetric cell division, respectively. Here, we uncover a novel role for Ephrin-Eph intercellular signaling in controlling mitotic spindle alignment in Drosophila optic lobe neuroepithelial cells through aPKC activity-dependent myosin II regulation. We show that conserved core components of the mitotic spindle orientation machinery, including Discs Large1, Mud/NuMA, and Canoe/Afadin, mislocalize in dividing Eph mutant neuroepithelial cells and produce spindle alignment defects in these cells when they are down-regulated. In addition, the loss of Eph leads to a Rho signaling-dependent activation of the PI3K-Akt1 pathway, enhancing cell proliferation within this neuroepithelium. Hence, Eph signaling is a novel extrinsic mechanism that regulates both spindle orientation and cell proliferation in the Drosophila optic lobe neuroepithelium. Similar mechanisms could operate in other Drosophila and vertebrate epithelia.

Inhibition of thymidylate synthase affects neural tube development in mice.

Thymidylate synthase (TYMS) is a key enzyme in the de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP) from 2'-deoxyuridine-5'-monophosphate (dUMP). Our aim was to investigate the role of dTMP dysmetabolism via inhibition of TYMS by an inhibitor, 5-fluorouracil (5-FU) in the occurrence of neural tube defects (NTDs). We found that a high incidence of NTDs occurred after treatment with 5-FU at 12.5 mg/kg body weight. TYMS activity was significantly inhibited with decreased dTMP and accumulation of dUMP after 5-FU injection. The proliferation of neuroepithelial cells were markedly inhibited in NTDs compared with control. Expressions of proliferating cell nuclear antigen and phosphohistone H3 were significantly decreased in NTDs, while phosphorylated replication protein A2, P53 and Caspase3 were significantly increased in NTDs compared with control. These results indicated that inhibition of TYMS affected the balance between proliferation and apoptosis in neuroepithelial cells, which might shed some lights on the mechanisms involved in NTDs.

Identification and distribution of neuronal nitric oxide synthase and neurochemical markers in the neuroepithelial cells of the gill and the skin in the giant mudskipper, Periophthalmodon schlosseri.

Mudskippers are amphibious fishes living in mudflats and mangroves. These fishes hold air in their large buccopharyngeal-opercular cavities where respiratory gas exchange takes place via the gills and higher vascularized epithelium lining the cavities and also the skin epidermis. Although aerial ventilation response to changes in ambient gas concentration has been studied in mudskippers, the localization and distribution of respiratory chemoreceptors, their neurochemical coding and function as well as physiological evidence for the gill or skin as site for O2 and CO2 sensing are currently not known. In the present study we assessed the distribution of serotonin, acetylcholine, catecholamines and nitric oxide in the neuroepithelial cells (NECs) of the mudskipper gill and skin epithelium using immunohistochemistry and confocal microscopy. Colocalization studies showed that 5-HT is coexpressed with nNOS, Na+/K+-ATPase, TH and VAChT; nNOS is coexpressed with Na+/K+-ATPase and TH in the skin. In the gill 5-HT is coexpressed with nNOS and VAhHT and nNOS is coexpressed with Na+/K+-ATPase and TH. Acetylcholine is also expressed in chain and proximal neurons projecting to the efferent filament artery and branchial smooth muscle. The serotonergic cells c labeled with VAChT, nNOS and TH, thus indicating the presence of NEC populations and the possibility that these neurotransmitters (other than serotonin) may act as primary transmitters in the hypoxic reflex in fish gills. Immunolabeling with TH antibodies revealed that NECs in the gill and the skin are innervated by catecholaminergic nerves, thus suggesting that these cells are involved in a central control of branchial functions through their relationships with the sympathetic branchial nervous system. The Na+/K+-ATPase in mitochondria-rich cells (MRCs), which are most concentrated in the gill lamellar epithelium, is colabeled with nNOS and associated with TH nerve terminals. TH-immunopositive fine varicosities were also associated with the numerous capillaries in the skin surface and the layers of the swollen cells. Based on the often hypercapnic and hypoxic habitat of the mudskippers, these fishes may represent an attractive model for pursuing studies on O2 and CO2 sensing due to the air-breathing that increases the importance of acid/base regulation and the O2-related drive including the function of gasotransmitters such as nitric oxide that has an inhibitory (regulatory) function in ionoregulation.

Epigenetic activation of mouse ganglioside synthase genes: implications for neurogenesis.

The quantity and expression pattern of gangliosides in mammalian brain change drastically during development and are mainly regulated through stage-specific expression of ganglioside synthase genes. Despite extensive investigations in the past, it remains largely unclear how the transcriptional activation of the genes encoding glycosyltransferases is regulated. Here, we show that in the neuronogenic cultures of mouse embryonic brain-derived neuroepithelial cells, histone modifications including acetylated histone H3 and histone H4, but not histone H3 trimethylation at lysine 27 of two genes encoding two key regulatory GTs, namely, N-acetylgalactosaminyltransferase I and sialyltransferase II, were extensively and gradually enhanced, respectively. As a consequence, the level of each GT mRNA was increased correspondingly. Hyperacetylation of histones on the GalNAcT promoter resulted in recruitment of the trans-activation factors Sp2 and AP-1 when cellular histone deacetylases 1 and 2 were knocked down with RNA interference or inhibited by treatment with valproic acid. Moreover, epigenetic activation of GalNAcT was also detected, as accompanied by a pronounced induction of neural differentiation in primary neuroepithelium culture responding to an exogenous supplement of ganglioside GM1, a downstream product of the gene's encoding enzyme. Our findings thus provide direct evidence of novel pathways for ganglioside expression via the epigenetic up-regulation of ganglioside synthase genes during neural development.

Cell-type-specific profiling of gene expression and chromatin binding without cell isolation: assaying RNA Pol II occupancy in neural stem cells.

Cell-type-specific transcriptional profiling often requires the isolation of specific cell types from complex tissues. We have developed "TaDa," a technique that enables cell-specific profiling without cell isolation. TaDa permits genome-wide profiling of DNA- or chromatin-binding proteins without cell sorting, fixation, or affinity purification. The method is simple, sensitive, highly reproducible, and transferable to any model system. We show that TaDa can be used to identify transcribed genes in a cell-type-specific manner with considerable temporal precision, enabling the identification of differential gene expression between neuroblasts and the neuroepithelial cells from which they derive. We profile the genome-wide binding of RNA polymerase II in these adjacent, clonally related stem cells within intact Drosophila brains. Our data reveal expression of specific metabolic genes in neuroepithelial cells, but not in neuroblasts, and highlight gene regulatory networks that may pattern neural stem cell fates.

The small GTPase RhoA is required to maintain spinal cord neuroepithelium organization and the neural stem cell pool.

The regulation of adherens junctions (AJs) is critical for multiple events during CNS development, including the formation and maintenance of the neuroepithelium. We have addressed the role of the small GTPase RhoA in the developing mouse nervous system using tissue-specific conditional gene ablation. We show that, in the spinal cord neuroepithelium, RhoA is essential to localize N-cadherin and ß-catenin to AJs and maintain apical-basal polarity of neural progenitor cells. Ablation of RhoA caused the loss of AJs and severe abnormalities in the organization of cells within the neuroepithelium, including decreased neuroepithelial cell proliferation and premature cell-cycle exit, reduction of the neural stem cell pool size, and the infiltration of neuroepithelial cells into the lumen of the ventricle. We also show that, in the absence of RhoA, its effector, mammalian diaphanous-related formin1 (mDia1), does not localize to apical AJs in which it likely stabilizes intracellular adhesion by promoting local actin polymerization and microtubule organization. Furthermore, expressing a dominant-negative form of mDia1 in neural stem/progenitor cells results in a similar phenotype compared with that of the RhoA conditional knock-out, namely the loss of AJs and apical polarity. Together, our data show that RhoA signaling is necessary for AJ regulation and for the maintenance of mammalian neuroepithelium organization preventing precocious cell-cycle exit and differentiation.