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Sensory neurons of the Atonal lineage pioneer the formation of glomeruli within the adult Drosophila olfactory lobe

¹ Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005 India
² National Centre for Biological Sciences, TIFR, GKVK PO, Bangalore 560065, India

View larger version (137K): [in a new window]   Fig. 1. Effect of sense organ specificity on olfactory lobe patterning. The olfactory neurons in wild type and mutants were traced using the SG18.1-Gal4;UAS-GFP strain (green). (A-C) Wild-type antennal lobes; arrowheads mark entry of the antennal nerve and arrows indicate the inter-antennal commissure. (A) Adult lobe. (B) 60 hour APF lobe stained with anti-Fas II. (C) 48 hour APF lobe at the same magnification as (B) showing several well formed glomeruli (*). (D) Adult SG18.1 UAS GFP; ato1/Df(3R)p13 lobes at the same magnification as A. Antennal nerves enter the lobe (arrowheads) but no glomeruli can be discerned. The expected position of the inter-antennal commissure is indicated by (?). (E) 60 hour APF mutant lobe stained with anti-FasII; no glomeruli can be distinguished (compare with B, at the same magnification). (F) 48 hour APF SG18.1 UAS GFP; ato1/Df(3R)p13 lobe. Most afferents are stalled immediately upon entry of the antennal nerve (arrowhead); only a few invade the lobe (arrow). (G-I) Adult wild-type (G) and ato– mutant (H,I) lobes stained with anti-Dachshund (blue in G,H) and anti-Repo (red in G,I). Dorsal (D) and lateral (L) clusters of interneurons are demarcated with dotted lines in G,H. Arrows in G,I indicate glial cells at the lobe periphery. (J) Adult SG18.1-Gal4 UAS GFP/+ (K) and lz3; SG18.1-Gal4 UAS GFP/+ lobes. Well-formed glomeruli are indicated by *, # and o.; white dots indicate glomeruli in the wild type that are poorly innervated in the mutant. (L) 60 hours APF lobe stained with anti-FasII shows presence of several well-developed glomeruli. Scale bars: 25 µm in A,B; 20 µm in K. G-L are at the same magnification.

View larger version (144K): [in a new window]   Fig. 2. Ectopic expression of Ato does not rescue the glomerular phenotype of ato1/Df(3R)p13. (A,B) The SG18.1-Gal4 line was used to drive UAS-Ato and UAS-GFP in the SG18.1 UAS GFP/UAS-Ato; ato1/Df(3R)p13 genotype. The adult lobe morphology is similar to ato1/Df(3R)p13 (see Fig. 1D). Inter-antennal commissure is absent (?). Arrowheads mark the entry of sensory neurons. The left lobe in A is enlarged in B for clarity. Dotted line marks lobe boundary. (C,D) Third antennal segments from 22 hours APF pupae. Sensory neurons from the third segment and the arista (ar) have differentiated and leave the antenna in three distinct fascicles (1, 2, 3). (C) Staining with anti-Ato (red) shows no immunoreactivity in the third segment (III). Cells of the Johnston’s organ (arrows) in the second segment (II) express Ato (D). (E,F) RNA in situ hybridization on horizontal sections of the adult antenna with digoxygenin-labeled Or22b probe. Wild-type (E) and ato1/Df(3R)p13 (F) antennae show the odorant receptor gene expression in a subset of large basiconica (arrows). The altered morphology of the ato– antenna makes the location of the sensilla appear somewhat different, but we verified that the same number of expressing cells were present in wild-type and ato1/Df(3R)p13 antennae. Scale bars: 25 µm in A,B; in D, 25 µm in C,D.

View larger version (137K): [in a new window]   Fig. 3. Peripheral glia direct fasciculation of olfactory neurons in the antenna but do not influence patterning of the lobe. (A-C) Pupal antennae stained with mAb 22C10 (blue) to visualize sensory neurons; glia are labeled with anti-Repo antibodies (red). In the wild type (A) at 25 hours APF, Repo-positive glial cells associate with the axons of the sensory neurons in three fascicles (1, 2, 3). Expression of GFP driven by MZ317-Gal4 GAL4 shows glial cell bodies and their processes wrapping each of the fascicles (green). (B) In 36 hour APF ato1/Df(3R)p13 antennae, there is a reduction of glia ( 35 Repo-labeled cells in ato hypomorphs compared with 100 in the wild type) and the three fascicles (1*, 2* and 3*) merge into a single bundle. (C) 25 hours APF; MZ317-Gal4 UAS-GFP/UAS-DNCdc42; ato1/Df(3R)p13 antenna. The MZ317-Gal4 enhancer trap line drives expression of DN-Cdc42 as well as GFP in glia. Glial cells (red) are reduced in number; remaining cells are aggregated in the proximal region of the antenna. Only two exiting fascicles (1* and 2*) can be recognized. Inset shows glial cells stained with anti-Repo; cell bodies are demarcated by dotted lines. Dying cells show a fragmented staining. (D-F) Olfactory lobe morphology of the genotype described in (C). (D) 36 hours APF; a single glomerulus shows Fas II staining; lobe associated glia (green) are present normally surrounding the lobe (dotted line). (E,F) 60 hours APF; glomerular formation proceeds normally and several glomeruli can be recognized (* in E). Ectopic expression of DN-Cdc42 in the glial cells associated with the lobe does not appear to affect their morphology and processes can be seen entering lobe (arrowheads in F). (G,H) Ectopic expression of constitutively active form of Rac in sensory neurons in SG18.1-Gal4 UAS GFP/+; UAS RacV12/+ animals. (G) 36 hour APF antenna stained with mAb22C10 (blue) and anti-Repo (red); the SG18.1-Gal4 GFP is shown in green. Sensory neurons exit normally in three fascicles (1, 2, 3) (ar, arista). (H) Adult antennal lobe from genotype described in G. The entry of sensory neurons into the antennal lobe is shown by arrowheads, but neurons within the lobe are completely disorganized. The inter-antennal commissure (?) fails to form. Scale bars: in A, 30 µm in A-C; in D, 30 µm in D-F; in H, 20 µm.

View larger version (122K): [in a new window]   Fig. 4. A subset of coeloconic sense organs which form in weak hypomorphic mutants are sufficient to pattern the antennal lobe. (A-C) Region of the adult antenna showing coeloconic sensilla (arrowheads). Numbers given below are based on counts from at least 10 antenna. Similar numbers were obtained from the canton-S wild-type strain (A) as well as ato1/+ and Df(3R)p13/+ heterozygotes. (B) ato1/Df(3R)p13. (C) ato1/ato2. (D,E) GFP expression in the sensory neurons using SG18.1 in ato1/ato2 background. (D) Adult antennal lobe showing almost normal looking lobe with identifiable glomeruli and presence of commissure (arrow) (compare with Fig. 1A). (E) A single lobe enlarged to show the presence of distinct glomeruli (*). Arrowhead marks entry of antennal nerve to the lobe. Scale bars: in A, 10 µm in A-C; in D, 20 µm in D,E.

View larger version (187K): [in a new window]   Fig. 5. Formation of glial processes and dendritic arborization of interneurons within the lobe are influenced by sensory inputs. (A-C) MZ317-Gal4 driven expression of GFP in the adult antennal lobes. Glial cell bodies lie at the periphery of the lobe (red arrowheads). (A) Wild type; processes extend into to lobe and ensheath individual glomeruli (small arrows). (B) Glial cells in ato1/Df(3R)p13 lobes show excessive branching. (C) ato1/ato2; glial organization is comparable with wild type. (D-I) GH146 expression in pupal lobes from wild-type (D,E,G,I) and ato1/Df(3R)p13 (F,H) lobes. (D-F) Late pupae (90 hours APF). Dotted blue lines demarcate the lateral cluster of projection neurons cell bodies in the wild-type (D) and mutant (F) pupae. Projection neurons enter the lobe (arrow in D) and terminate in well defined glomeruli (* in D,E). (F) In ato1/Df(3R)p13, projection neurons are present within the lobe (boundaries indicated by dotted yellow lines); but these are unpatterned. (G,H) Mid-pupal (45 hours APF) lobes from wild type (G) and mutant (H). At this time, wild-type projection neurons have arborized within well-defined glomeruli (* in G). No such organization is visible in the mutant. (I) 14 hour APF projection neurons invade the lobe anlage (dotted yellow lines), although patterning has not yet occurred. Scale bars: in A, 20 µm for A-C; in D, 20 µm in D-I.

View larger version (28K): [in a new window]   Fig. 6. Antennal lobe development in Drosophila. In the wild type, the projection neurons (blue) enter the lobe at the pupal stages before sensory afferents arrive in the lobe (A). (B) Ato-lineage neurons (green) arrive at the lobe at 20 hours APF and remain at the periphery in close association with glial cells (red). (C) Other sensory neurons (purple) arrive later. (D) Between 30 and 60 hours APF, glomerular formation occurs. Sensory neurons invade the nascent lobe; projection neurons arborize at glomerular sites and glial processes ensheath the developing glomeruli. (E) In ato1/Df(3R)p13 animals, the residual sensory neurons enter the lobe but do not pattern glomeruli. These interneurons, although present in the lobe, do not target specific glomeruli and the glial cells produce ectopic branching within the lobe.