QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online March 20, 2009
doi: 10.1242/10.1242/dev.031377

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Chiang, A. Articles by Rodrigues, V. Search for Related Content PubMed Articles by Chiang, A. Articles by Rodrigues, V. Social Bookmarking                         What's this?

Neuronal activity and Wnt signaling act through Gsk3-β to regulate axonal integrity in mature Drosophila olfactory sensory neurons

¹ National Centre for Biological Sciences, TIFR, GKVK Campus, Bangalore-65, India.
² Smurfit Institute of Genetics and TCIN, Lloyd Building, Trinity College Dublin, Dublin-2, Ireland.
³ Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA.
⁴ Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai-5, India.

View larger version (64K): [in this window] [in a new window]   Fig. 1. Development is normal, but maintenance of OSN terminals is affected, in Or83b-null mutants. (A) Schematic of the olfactory system in adult Drosophila. The olfactory sensory neurons (OSNs) project to glomeruli within the antennal lobe, where they synapse with projection neurons (PNs) and local interneurons (LNs). Most OSNs send contralateral projections to the opposite antennal lobe, along the antennal commissure. (B-D') Or83b-Gal4-driven GFP expression labels the majority of the OSNs that innervate multiple glomeruli in the antennal lobe at 6 days (B,B'). In 2-day-old animals, the GFP pattern of Or83b-null animals (C,C') is comparable to that of wild type (B,B'), but degenerates in older animals (D,D'). B-D are single optical sections to show glomerular innervation by OSN axon terminals, and B'-D' are projections of five to ten of the anterior-most optical sections to highlight the OSN axons that make up the nerve fiber layer. Arrowheads in D,D' mark degenerating axons, which show characteristic blebbing and beading; the asterisk in B'-D' marks one of the broad nerve fiber tracts, which is severely disrupted in 6-day-old Or83b-null animals (D'). Genotypes: (B,B') w; Or83b-Gal4/UAS-mCD8::GFP; (C-D') w; Or83b-Gal4/UAS-mCD8::GFP; Or83b-/-. Scale bar: 25 µm.

View larger version (62K): [in this window] [in a new window]   Fig. 2. Maintenance of OSNs requires neuronal activity. (A) Antennal glomeruli stained with an antibody against Bruchpilot (Brp). (A') The Or22a-Gal4,UAS-mCD8::GFP line labels a set of 23 OSNs that project to the DM2 glomerulus, shown here alone for clarity. (B-E) Or83b-/- flies with OSNs labeled by expression of GFP driven by Or22a-Gal4. These appear normal for the first 2 days PE (B), but show beading in the axons (arrow) by 4 days (C), which worsens to include the terminals (dotted lines) by 6 (D) and 8 (E) days PE. (F-F'') Conditional targeted expression of Or83b in Or22a-Gal4-expressing OSNs in Or83b-/- animals (Or22a-Gal4, UAS-mCD8::GFP/UAS-Or83b; tub-Gal80ts, Or83b-/tub-Gal80ts, Or83b-) for 2 (F), 4 (F'), or 6 (F'') days PE, and examined at 8 days PE in all cases. (G) Pixel intensities within the terminal arbors (demarcated by dotted lines) were estimated using LabVIEW 6.1 software and plotted as a bar chart (mean±s.e.m., n=5 in each case; for values, see Table S1 in the supplementary material). The letter below each bar indicates the figure in which the genotype being examined is shown. Data were subject to Student's unpaired t-test. *P<0.05; **P<0.005; n.s., not significant (P>0.05). (H) Induction of electrical activity within mutant Or22a OSNs by ectopic expression of Eag-DN and Sh-DN K+ channels. In G, the terminals (within dotted lines in H) were quantified (mean±s.e.m.) in 6-day-old animals; they show significant rescue (compared with D; see Table S2 in the supplementary material); **P<0.005. Genotypes: (A,A') Or22a-Gal4, UAS-mCD8::GFP/+; (B-E) Or22a-Gal4, UAS-mCD8::GFP/+; Or83b-/-; (F-F'') Or22a-Gal4, UAS-mCD8::GFP/UAS-Or83b; tub-Gal80ts, Or83b-/tub-Gal80ts, Or83b-; (H) Or22a-Gal4, UAS-mCD8::GFP/UAS-eag-DN, UAS-Sh-DN; Or83b-/-. Scale bar: 20 µm.

View larger version (96K): [in this window] [in a new window]   Fig. 3. Effect of TNT-G, Kir2.1 or ShiDN expression on the OSNs in the antennal lobe. (A) Or83b OSNs in the normal Drosophila antennal lobe labeled with anti-GFP. The dotted line demarcates the VA6 glomerulus. (B,C) Conditional expression of Kir2.1 (B) or shiK44A (C) by Or83b-Gal4. (D-E') Or83b-Gal4-driven GFP used to visualize OSNs expressing either IMPTNT-V (D) or TNT-G (E,E'). Disruption of the presynaptic terminals is visible at 3.5 days PE (arrowheads in E), and becomes striking by 6 days PE (E'). (F-G''') Or22a OSNs terminating in DM2 visualized by anti-GFP (F). (G-G''') Kir2.1 is expressed in adult Or22a OSNs. The neurodegeneration is progressive, starting at 3 days PE (G; arrows highlight beaded and fragmenting axons), and is extreme by 5 days PE (G'''). OSN terminals within the glomerulus are demarcated with dotted lines. Insets in G' and G'' show the terminals alone, for greater clarity. Genotypes: (A) w; tub-Gal80ts, tub-Gal80ts/+; Or83b-Gal4, UAS-2xEGFP/+; (B) w; tub-Gal80ts, tub-Gal80ts/+; Or83b-Gal4, UAS-2xEGFP/ UAS-Kir2.1; (C) w; tub-Gal80ts, tub-Gal80ts/+; Or83b-Gal4, UAS-2xEGFP/UAS-shi.K44A; (D) w; tub-Gal80ts, tub-Gal80ts/ UAS-IMPTNT-V; Or83b-Gal4, UAS-2xEGFP/+; (E,E') w; tub-Gal80ts, tub-Gal80ts/UAS-TNT-G; Or83b-Gal4, UAS-2xEGFP/+; (F) Or22a-Gal4, UAS-mCD8::GFP/+; tub-Gal80ts, tub-Gal80ts/+; (G-G''') Or22a-Gal4, UAS-mCD8::GFP/+; tub-Gal80ts, tub-Gal80ts/UAS-Kir2.1. Scale bars: 25 µm in A-E'; 20 µm in F-G'''.

View larger version (80K): [in this window] [in a new window]   Fig. 4. Maintenance of OSNs requires autonomous neuronal activity. (A-F'') Or47b-Gal4-driven GFP expression labels a subset of OSNs (50) that innervate the VA1v glomerulus. Clones generated by MARCM label a subset of Or47b-Gal4-expressing OSNs. In B,D,F, clonal OSNs express TNT-G (and GFP, green in all panels), whereas the rest of the OR47b OSNs are unmarked and normal. Controls (A,C,E) and experimental (B,D,F) samples showing similar clone sizes were examined at 1 (A,B), 3 (C,D) and 7 (E,F) days PE. Samples were stained using anti-GFP (green; A-F) and anti-Brp (A'-F'). A''-F'' are three-dimensional reconstructed images of clonal cells (Amira 4.1 and 5.0). GFP-marked regions from consecutive confocal sections were selected and merged. Genotypes: (A,C,E) FRT19A/FRT19A, tubGal80, hsFlp; Or47b-Gal4(15.7), UAS-mCD8::GFP/+; (B,D,F) FRT19A/FRT19A, tub-Gal80, hsFlp; Or47b-Gal4(15.7), UAS-mCD8::GFP/UAS-TNT-G. Scale bar: 20 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 5. The state of Gsk-3β activation influences OSN stability. (A) Or22a-Gal4,UAS-mCD8::GFP-marked OSNs in Or83b-null flies at 6 days PE. The control image shown in Fig. 2D is reproduced in A for ease of comparison. (B) Co-expression of a sgg DN.A81T transgene in mutant OSNs. (C) OSNs are rescued in mutants fed for 6 days with 10 mM LiCl. OSN terminals in a DM2 glomerulus (dotted lines; B,C) show reduced degeneration upon Gsk-3β inactivation as compared with age-matched controls (A). Arrowheads in B,C indicate gaps in the glomerular innervation of Or22a-Gal4 expressing OSNs at the DM2 glomerulus. (D) Pixel intensities in footprints (mean±s.e.m.) of preparations shown in B and C (n=5 each) are compared with A (n=5) and with normal Or22a-Gal4,UAS-mCD8::GFP animals as shown in E (n=5). **P<0.005. (E,E') Or22a-Gal4,UAS-mCD8::GFP (E) and with conditional expression of constitutively activated Gsk-3β (UAS-sgg CA.S9A) (E'). (F,F') Or47b-Gal4,UAS-mCD8::GFP (F) and with adult-specific expression of constitutively activated Gsk-3β (UAS-sgg CA.S9A) (F'). (G,G') Or83b-Gal4,UAS-2xEGFP (G, the control image shown in Fig. 3A is reproduced in G for ease of comparison) and with constitutively activated Gsk-3β (UAS-sgg CA.S9A) (G'). Animals in E-G' are 6 days PE. (H,H') Or22a-Gal4,UAS-mCD8::GFP-marked OSNs in Or83b-null animals (H) and co-expressing Dsh (H'). The terminal arbors were assessed by measuring the pixels within the DM2 glomerulus (plotted in D, **P<0.005). Samples in H were prepared in parallel (see Table S4 in the supplementary material). Genotypes: (A,C) Or22a-Gal4, UAS-mCD8::GFP/+; Or83b-/-; (B) Or22a-Gal4, UAS-mCD8::GFP/UAS-sgg DN.A81T; Or83b-/-; (E) Or22a-Gal4, UAS-mCD8::GFP/+; tub-Gal80ts, tub-Gal80ts/+; (E') Or22a-Gal4, UAS-mCD8::GFP/+; tub-Gal80ts, tub-Gal80ts/UAS-sgg CA.S9A; (F) w; Or47b-Gal4(15.7), UAS-mCD8::GFP/+; tub-Gal80ts, tub-Gal80ts/+; (F') w; Or47b-Gal4(15.7), UAS-mCD8::GFP/+; tub-Gal80ts, tub-Gal80ts/UAS-sgg CA.S9A; (G) w; tub-Gal80ts, tub-Gal80ts/+; Or83b-Gal4, UAS-2xEGFP/+; (G') w; tub-Gal80ts, tub-Gal80ts/+; Or83b-Gal4, UAS-2xEGFP/UAS-sgg CA.S9A; (H) Or22a-Gal4, UAS-mCD8::GFP/+; Or83b-/-; (H') Or22a-Gal4, UAS-mCD8::GFP/+; UAS-dsh-myc,Or83b-/Or83b-.

View larger version (41K): [in this window] [in a new window]   Fig. 6. Perturbation of Wg/Wnt signaling leads to OSN degeneration. (Aa-Ag) Or22a-Gal4,UAS-mCD8::GFP-marked OSNs target to DM2. (Ba-Bg) Or83b-Gal4,UAS-2xEGFP-labeled OSNs (the control image shown in Fig. 3A is reproduced in Ba for ease of comparison). The tub-Gal80ts system was used to express GPI-fz (Ab,Bb), arrow C (Ac,Bc), DN-dsh (Ad,Bd), Axin (Ae, Be), activated arm.S10 (Af,Bf) and DN-dTCF (Ag,Bg). All perturbations, except Arm.S10 and DN-dTCF, show neurodegeneration. Genotypes: (Aa) w; Or22a-Gal4, UAS-mCD8::GFP/+; tub-Gal80ts, tub-Gal80ts/+; (Ab) w; Or22a-Gal4, UAS-mCD8::GFP/UAS-GPI-fz+; tub-Gal80ts, tub-Gal80ts/+; (Ac) w; Or22a-Gal4, UAS-mCD8::GFP/UAS-arrow C; tub-Gal80ts, tub-Gal80ts/+; (Ad) w; Or22a-Gal4, UAS-mCD8::GFP/UAS-dsh DN.DIX; tub-Gal80ts, tub-Gal80ts/+; (Ae) w; Or22a-Gal4, UAS-mCD8::GFP/UAS-Axn-GFP; tub-Gal80ts, tub-Gal80ts/+; (Af) w; UAS-arm.S10; Or22a-Gal4, UAS-mCD8::GFP/+; tub-Gal80ts, tub-Gal80ts/+; (Ag) w; Or22a-Gal4, UAS-mCD8::GFP/UAS-pan.dTCFN; tub-Gal80ts, tub-Gal80ts/+; (Ba) w; tub-Gal80ts, tub-Gal80ts/+; Or83b-Gal4, UAS-2xEGFP/+; (Bb) w; tub-Gal80ts, tub-Gal80ts/UAS-GPI-fz+; Or83b-Gal4, UAS-2xEGFP/+; (Bc) w; tub-Gal80ts, tub-Gal80ts/UAS-arrow C; Or83b-Gal4, UAS-2xEGFP/+; (Bd) w; tub-Gal80ts, tub-Gal80ts/UAS-dsh DN.DIX; Or83b-Gal4, UAS-2xEGFP/+; (Be) w; tub-Gal80ts, tub-Gal80ts/UAS-Axn-GFP;Or83b-Gal4, UAS-2xEGFP/+; (Bf) w; UAS-arm.S10; tub-Gal80ts, tub-Gal80ts/+; Or83b-Gal4, UAS-2xEGFP/+; (Bg) w; tub-Gal80ts, tub-Gal80ts/UAS-pan.dTCFN; Or83b-Gal4, UAS-2xEGFP/+.

View larger version (45K): [in this window] [in a new window]   Fig. 7. Intersection of neuronal activity, Gsk-3β and Wg signaling in conferring stability on adult OSNs. (A-B') Adult Drosophila antennal lobes stained with anti-Wg antibody; the DM2 glomerulus is circled with a dotted line. Control brains (A) and brains depolarized by spaced 5x K+ exposure (B). Levels of immunoreactivity are represented by the colors of the heat map (A',B'). Asterisks highlight the glomerulus neighboring DM2, which also shows a significant increase in Wg levels compared with the control. (C) Mean pixel intensity ±s.e.m. in control (A) and stimulated (B) DM2 glomeruli (identified by Or22a>GFP expression) was estimated and plotted (**P<0.005). (D) Or22a OSNs labeled in the Or83b-/- mutants. The image shown in Fig. 5H is shown here again for ease of comparison. (E) Conditional targeted expression of Wg in the Or22a neurons results in rescue from degeneration. (F) Pixel intensities of the terminal arbors of controls (D) and experimental samples (E) show a significant difference (mean±s.e.m.; **P<0.005) (see Table S4 in the supplementary material). (G) In normal adult OSNs, Or83b-dependent neuronal activity leads to a release of Wg, which activates signaling leading to inhibition of Gsk-3β. Gsk-3β acts through a non-transcriptional pathway to stabilize axons/synapses. Neuronal activity could also impinge on Gsk-3β through Wg-independent pathways (represented by dotted lines). Genotypes: (A-B') w; Or22a-Gal4, UAS-mCD8::GFP/+; (D) Or22a-Gal4, UAS-mCD8::GFP/+; Or83b-/-; (E) Or22a-Gal4, UAS-mCD8::GFP/UAS-wg; Or83b-/-. Scale bar: 15 µm in A-B'.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter