Evidence for a Transport-Trap Mode of Drosophila melanogaster gurken mRNA Localization

The Drosophila melanogaster gurken gene encodes a TGF alpha-like signaling molecule that is secreted from the oocyte during two distinct stages of oogenesis to define the coordinate axes of the follicle cell epithelium that surrounds the oocyte and its 15 anterior nurse cells. Because the gurken receptor is expressed throughout the epithelium, axial patterning requires region-specific secretion of Gurken protein, which in turn requires subcellular localization of gurken transcripts. The first stage of Gurken signaling induces anteroposterior pattern in the epithelium and requires the transport of gurken transcripts from nurse cells into the oocyte. The second stage of Gurken signaling induces dorsovental polarity in the epithelium and requires localization of gurken transcripts to the oocyte's anterodorsal corner. Previous studies, relying predominantly on real-time imaging of injected transcripts, indicated that anterodorsal localization involves transport of gurken transcripts to the oocyte's anterior cortex followed by transport to the anterodorsal corner, and anchoring. Such studies further indicated that a single RNA sequence element, the GLS, mediates both transport steps by facilitating association of gurken transcripts with a cytoplasmic dynein motor complex. Finally, it was proposed that the GLS somehow steers the motor complex toward that subset of microtubules that are nucleated around the oocyte nucleus, permitting directed transport to the anterodorsal corner. Here, we re-investigate the role of the GLS using a transgenic fly assay system that includes use of the endogenous gurken promoter and biological rescue as well as RNA localization assays. In contrast to previous reports, our studies indicate that the GLS is sufficient for anterior localization only. Our data support a model in which anterodorsal localization is brought about by repeated rounds of anterior transport, accompanied by specific trapping at the anterodorsal cortex. Our data further indicate that trapping at the anterodorsal corner requires at least one as-yet-unidentified gurken RLE

Eksternt press og intern dynamikk i implementeringen av MaaS: Casestudie av Shanghais kommune

Masteroppgave i Global ledelse (tidl. energiledelse) - Nord universitet 202

Apical-basal distribution of different subtypes of spiral ganglion neurons in the cochlea and the changes during aging.

Sound information is transmitted from the cochlea to the brain mainly by type I spiral ganglion neurons (SGNs), which consist of different subtypes with distinct physiological properties and selective expression of molecular markers. It remains unclear how these SGN subtypes distribute along the tonotopic axis, and whether the distribution pattern changes during aging that might underlie age-related hearing loss (ARHL). We investigated these questions using immunohistochemistry in three age groups of CBA/CaJ mice of either sex, including 2-5 months (young), 17-19 months (middle-age), and 28-32 months (old). Mouse cochleae were cryo-sectioned and triple-stained using antibodies against Tuj1, calretinin (CR) and calbindin (CB), which are reportedly expressed in all type I, subtype Ia, and subtype Ib SGNs, respectively. Labeled SGNs were classified into four groups based on the expression pattern of stained markers, including CR+ (subtype Ia), CB+ (subtype Ib), CR+CB+ (dual-labeled Ia/Ib), and CR-CB- (subtype Ic) neurons. The distribution of these SGN groups was analyzed in the apex, middle, and base regions of the cochleae. It showed that the prevalence of subtype Ia, Ib and dual-labeled Ia/Ib SGNs are high in the apex and low in the base. In contrast, the distribution pattern is reversed in Ic SGNs. Such frequency-dependent distribution is largely maintained during aging except for a preferential reduction of Ic SGNs, especially in the base. These findings corroborate the prior study based on RNAscope that SGN subtypes show differential vulnerability during aging. It suggests that sound processing of different frequencies involves distinct combinations of SGN subtypes, and the age-dependent loss of Ic SGNs in the base may especially impact high-frequency hearing during ARHL

Conservation and predicted secondary structure of the GLS.

<p>(A) Sequence alignment of the <i>gurken</i> transcription unit displayed using the Vista Browser at <a href="http://pipeline.lbl.gov/cgi-bin/gateway2" target="_blank">http://pipeline.lbl.gov/cgi-bin/gateway2</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015448#pone.0015448-Nielsen1" target="_blank">[49]</a>. The estimated years in millions (MYA) of evolution between <i>D</i>. <i>melanogaster</i> and each of the other five species is from Heger and Ponting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015448#pone.0015448-Heger1" target="_blank">[24]</a>. The most highly conserved region is circled and includes the first 39 nt of the GLS. The last 25 nt of the GLS map to the 3′ side of the abutting intron. The arrow indicates the direction of transcription. The red shaded region corresponds to a putative transposable element. The numbers at the bottom of the graph indicate nucleotide position along the chromosome. (B) The 5′ end of the <i>gurken</i> mRNA, where the green dot denotes the translation start site, the red arrows the boundaries of the GLS, and the asterisk the position of the intron. The nucleotides beneath the aligned sequence blocks highlight differences between the <i>D. Willistoni</i> and <i>D. melanogaster</i> sequences. (C) Predicted secondary structure of the GLS, with non-conserved residues shown in red.</p

The GLS is required for <i>gurken</i> RNA localization and gene function.

<p>(A–B) Wild-type expression patterns of endogenous <i>gurken</i> RNA (A) and protein (B) as revealed by whole mount <i>in situ</i> hybridization and immunofluorescence, respectively. Anterodorsal localization of transcripts and protein is only apparent in the rightmost egg chambers, which are stage 8 and 9, respectively. (C–E) The <i>gurken</i> RNA and protein distribution patterns of <i>gurken</i> null mutants (<i>grk^ΔFRT</i>) carrying the wild-type <i>gurken</i> transgene, <i>grk^wt</i> (C–D) or no transgene (E). (F–H) <i>grk^ΔFRT</i> eggs and egg chambers (from <i>gurken</i> null mothers) carrying the <i>grkGLS^mut</i> transgene. (F) Left panel: representative <i>grk^ΔFRT</i>; <i>grkGLS^mut</i> egg exhibiting a completely ventralized phenotype, i.e., complete loss of dorsal appendage material. Right panel; anterior end of a <i>grk^ΔFRT</i>; <i>grkGLS^mut</i> egg exhibiting a strong, but not complete, ventralized phenotype. Note, for example the short, fused dorsal appendage. (G) <i>grk^ΔFRT</i>; <i>grkGLS^mut</i> ovariole following <i>in situ</i> hybridization with <i>gurken</i> probe. Transcripts are dispersed throughout the germ-line cysts with only slight enrichment in the oocyte and no subcellular localization. (H) <i>grk^ΔFRT</i>; <i>grkGLS^mut</i> ovariole following immunofluorescence using an anti-Grk antibody. The protein is generally dispersed throughout the germ-line cysts, although slight enrichment around the oocyte nucleus is seen in rare stage 10 and 11 egg chambers.</p

Structure of GLS variants.

<p>The wild-type GLS is shown at the left for comparison. The GLS mutant (referred to as <i>grkGLS^mut</i> in Text) contains 12 point mutations (shown in red), which are predicted to disrupt the predicted base pairing pattern of the GLS at five sites (circled). None of the 12 mutations affect the protein coding sequence as shown at the bottom portion of the figure.</p

MiR‐223‐3p alleviates trigeminal neuropathic pain in the male mouse by targeting MKNK2 and MAPK/ERK signaling

Abstract Background Trigeminal neuralgia (TN) is a neuropathic pain that occurs in branches of the trigeminal nerve. MicroRNAs (miRNAs) have been considered key mediators of neuropathic pain. This study was aimed to elucidate the pathophysiological function and mechanisms of miR‐223‐3p in mouse models of TN. Methods Infraorbital nerve chronic constriction injury (CCI‐ION) was applied in male C57BL/6J mice to establish mouse models of TN. Pain responses were assessed utilizing Von Frey method. The expression of miR‐223‐3p, MKNK2, and MAPK/ERK pathway protein in trigeminal ganglions (TGs) of CCI‐ION mice was measured using RT‐qPCR and Western blotting. The concentrations of inflammatory cytokines were evaluated using Western blotting. The relationship between miR‐223‐3p and MKNK2 was tested by a luciferase reporter assay. Results We found that miR‐223‐3p was downregulated, while MKNK2 was upregulated in TGs of CCI‐ION mice. MiR‐223‐3p overexpression by an intracerebroventricular injection of Lv‐miR‐223‐3p attenuated trigeminal neuropathic pain in CCI‐ION mice, as well as reduced the protein levels of pro‐inflammatory cytokines in TGs of CCI‐ION mice. MKNK2 was verified to be targeted by miR‐223‐3p. Additionally, miR‐223‐3p overexpression decreased the phosphorylation levels of ERK1/2, JNK, and p38 protein in TGs of CCI‐ION mice to inhibit MAPK/ERK signaling. Conclusions Overall, miR‐223‐3p attenuates the development of TN by targeting MKNK2 to suppress MAPK/ERK signaling