Activation of microglia commonly occurs in response to a wide variety of pathological stimuli including trauma, axotomy, ischemia, and degeneration in the CNS. In the retina, prolonged or high-intensity exposure to visible light leads to photoreceptor cell apoptosis. In such a light-reared retina, we found that activated microglia invade the degenerating photoreceptor layer and alter expression of neurotrophic factors such as nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), and glial cell line-derived neurotrophic factor (GDNF). Because these neurotrophic factors modulate secondary trophic factor expression in Müller glial cells, microglia-Müller glia cell interaction may contribute to protection of photoreceptors or increase photoreceptor apoptosis. In the present study, we demonstrate the possibility that such functional glia-glia interactions constitute the key mechanism by which microglia-derived NGF, brain-derived neurotrophic factor (BDNF), and CNTF indirectly influence photoreceptor survival, although the receptors for these neurotrophic factors are absent from photoreceptors, by modulating basic fibroblast growth factor (bFGF) and GDNF production and release from Müller glia. These observations suggest that microglia regulate the microglia-Müller glia-photoreceptor network that serves as a trophic factor-controlling system during retinal degeneration.

To clarify the molecular mechanisms of neural development in vertebrates, we analyzed a novel gene, termed nemp1 (nuclear envelope integral membrane protein 1), which is expressed in the Xenopus anterior neuroectoderm at the neurula stage. Nemp1 has a putative signal peptide and five transmembrane domains, but does not have any other known domains. We show that Nemp1 is localized to the inner nuclear membrane (INM) with its evolutionarily conserved C-terminal region facing the nucleoplasm. Both overexpression and knockdown of Nemp1 in Xenopus embryos reduced the expression of early eye marker genes, rax, tbx3, and pax6, and later resulted mainly in severe eye defects at the tailbud stage. In contrast, the expression of a forebrain/midbrain marker, otx2, and a pan-neural marker, sox2, was largely unaffected. Deletion analysis of Nemp1 showed that nuclear envelope-localization of the C-terminal region is necessary for its eye-reducing activity. Furthermore, nemp1 is coexpressed with baf (barrier-to-autointegration factor) in the eye anlagen, and that Nemp1 interacts with BAF through the BAF-binding site in the C-terminal region and this site is required for Nemp1 activity. These data suggest that Nemp1 is involved in the expression of eye marker genes by functioning at the INM at least partly through BAF.

No abstract

Cell competition is a short-range communication originally observed in Drosophila. Relatively little is known about cell competition in mammals or in non-epithelial cells. Hippo signaling and its downstream transcription factors of the Tead family, control cell proliferation and apoptosis. Here, we established an in vitro model system that shows cell competition in mouse NIH3T3 embryo fibroblast cells. Co-culture of Tead-activity-manipulated cells with normal (wild-type) cells caused cell competition. Cells with reduced Tead activity became losers, whereas cells with increased Tead activity became super-competitors. Tead directly regulated Myc RNA expression, and cells with increased Myc expression also became super-competitors. At low cell density, cell proliferation required both Tead activity and Myc. At high cell density, however, reduction of either Tead activity or Myc was compensated for by an increase in the other, and this increase was sufficient to confer 'winner' activity. Collectively, NIH3T3 cells have cell competition mechanisms similar to those regulated by Yki and Myc in Drosophila. Establishment of this in vitro model system should be useful for analyses of the mechanisms of cell competition in mammals and in fibroblasts.

No abstract

The inner nuclear membrane (INM) protein Nemp1/TMEM194A has previously been suggested to be involved in eye development in Xenopus, and contains two evolutionarily conserved sequences in the transmembrane domains (TMs) and the C-terminal region, named region A and region B, respectively. To elucidate the molecular nature of Nemp1, we analyzed its interacting proteins through those conserved regions. First, we found that Nemp1 interacts with itself and lamin through the TMs and region A, respectively. Colocalization of Nemp1 and lamin at the INM suggests that the interaction with lamin participates in the INM localization of Nemp1. Secondly, through yeast two-hybrid screening using region B as bait, we identified the small GTPase Ran as a probable Nemp1-binding partner. GST pulldown and co-immunoprecipitation assays using region B and Ran mutants revealed that region B binds directly to the GTP-bound Ran through its effector domain. Immunostaining experiments using transfected COS-7 cells revealed that full-length Nemp1 recruits Ran near the nuclear envelope, suggesting a role for Nemp1 in the accumulation of RanGTP at the nuclear periphery. At the neurula-to-tailbud stages of Xenopus embryos, nemp1 expression overlapped with ran in several regions including the eye vesicles. Co-knockdown using antisense morpholino oligos for nemp1 and ran caused reduction of cell densities and severe eye defects more strongly than either single knockdown alone, suggesting their functional interaction. Finally we show that Arabidopsis thaliana Nemp1-orthologous proteins interact with A. thaliana Ran, suggesting their evolutionally conserved physical and functional interactions possibly in basic cellular functions including nuclear transportation. Taken together, we conclude that Nemp1 represents a new type of RanGTP-binding protein.

A cDNA library derived from the anterior neuroectoderm (ANE) of Xenopus lategastrula embryos was systematically screened to isolate novel developmental regulatory genes involved in early brain development. We isolated 1,706 5´ expressed sequence tags (ESTs), which were subdivided into 1,383 clusters and categorized into 19 classes based on predicted functions according to their similarities to other known genes. Of these, 757 clusters that were considered possible novel regulatory genes or unknown genes were subjected to expression pattern analysis using whole-mount in situ hybridization. Genes from 69 clusters (9%) were expressed in the ANE region. Based on their expression patterns and predicted amino acid sequences, 25 genes were selected for further analysis as novel Xenopus genes expressed broadly or region-specifically in the ANE. Eighteen genes were expressed in postulated patterning centers in the neuroectoderm, including the anterior (four genes) and lateral (nine genes) neural ridges, the midbrain-hindbrain boundary region (one gene) and the midline region of the neural plate (two genes), whereas 13 genes were expressed in the eye anlagen. Therefore, early regionalization of the neuroectoderm appears to occur mainly in those neural patterning centers and the eye anlagen. We determined the entire coding regions of p54nrb, Semaphorin 6D and a novel gene designated scribble-related protein 1 (SCRP1). Interestingly, Semaphorin 6D is expressed in the mesoderm with a dorsoventral gradient, as well as in the ectoderm at the gastrula stage, implying a new role for this protein in development other than in axon guidance.

The production of tylosin by Streptomyces fradiae KA-427 in a defined medium was inhibited by ammonium ions and by inorganic phosphate.The production of protylonolide, an early lactonic intermediate of tylosin biosynthesis with the same carbon skeleton as tylosin aglycone, by a mutant of strain KA-427 was also reduced by these two kinds of ions. In contrast, the bioconversion of protylonolide to tylosin by another mutant was less susceptible to ammonium ions but was sensitive to inorganic phosphate.The addition of protylonolide to a culture of S. fradiae KA-427 increased the tylosin yield, suggesting that aglycone synthesis is limiting under the conditions used. When L-valine, L-leucine, L-isoleucine, L-threonine, or the corresponding 2-keto acid was added to the culture medium, the protylonolide titer increased. The addition of [14C]valine gave rise to ['4C]protylonolide.13C NMR spectroscopic analysis revealed that iso-butyrate, which is a valine metabolite, was incorporated into protylonolide at the carbons known to originate from propionate and n-butyrate.Taking account of these findings, the regulation of tylosin biosynthesis in Se fradiae by ammonium ion is discussed in relation to amino acid metabolism.Tylosin, a 16-membered macrolide antibiotic, is produced by strains of Streptomyces. The biosynthesis of tylosin in Se fradiae is subject to carbon regulation and phosphate regulation. Mechanisms for these regulation processes have been proposed at the enzyme level"".Tylosin production is also susceptible to inhibition by ammonium ions. However, the mechanism of action of ammonium ions is not well understood. The biosynthetic pathway to tylosin can be divided into two parts: one is the formation of protylonolide, a precursor of the aglycone (see Fig. 5), and the other involves the synthesis of three sugars (mycaminose, mycinose and mycarose) and their sequential attachment to the aglycone". The aglycone and the sugars are biosynthesized through separate routes, and assembled to form tylosin in a series of reactions','). Assuming that the biosynthetic steps sensitive to the effect of ammonium ions are involved in nitrogen metabolism, two steps in tylosin biosynthesis are possible targets. One is the biosynthesis (or attachment) of the aminosugar mycaminose. The other lies in the pathway for protylonolide formation. The latter notion came from a recent finding that the production of protylonolide was elevated under low ammonium ion conditions created in the presence of ammonium ion-trapping agents such as natural zeolite'). However, nitrogen metabolism was not known to be involved in the biosynthesis of protylonolide, which contains no nitrogen atom. This paper presents evidence that protylonolide biosynthesis is subject to regulation by ammonium Bioconversion and biosynthesis of 16-membered macrolide antibiotics. XXIX. Part XXVIII appeared in ref 5.