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First published online 26 November 2003
doi: 10.1242/dev.00896

Development 131, 117-130 (2004)
Published by The Company of Biologists 2004
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Developmental origin of wiring specificity in the olfactory system of Drosophila

Gregory S. X. E. Jefferis1,2, Raj M. Vyas1,*, Daniela Berdnik1,*, Ariane Ramaekers3, Reinhard F. Stocker3, Nobuaki K. Tanaka4,5, Kei Ito4 and Liqun Luo 1,2,{dagger}

1 Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
2 Neurosciences Program, Stanford University, Stanford, CA 94305, USA
3 Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland
4 Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan
5 The Graduate University for Advanced Studies, Japan



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  		Fig. 1. AL organization and development. (A) Organization of the mature Drosophila antennal lobe (AL) with vertebrate counterparts in parentheses. Each colour represents olfactory receptor neurons (ORNs) expressing a particular seven-transmembrane-span receptor or their post-synaptic projection neuron (PN) partners. (B) Three hypotheses for the cellular basis of highly specific wiring between ORN axons and PN dendrites. (Model 1) ORNs pattern the lobe, then PN dendrites grow in. (Model 2) PN dendrites pattern the lobe, serving as a template for ORN axons. (Model 3) The lobe is patterned by the coincident interaction of both cell types. (C) Model equivalent to Model 1 above, illustrating that the order of growth and patterning might be distinct: PNs could ramify extensively through the lobe but remain unpatterned until the arrival of ORNs – models 2 and 3 could be similarly extended. (D) Time course of adult lobe development and larval lobe degeneration. Red structures outline adult lobes; the larval lobes are shown in purple.

 


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  		Fig. 2. Time course of ORN axon development. (A) The approach used to label ORNs – the restricted expression of the eyeless-FLP construct generates GFP-labelled cells only in the antennal disc and eye disc/optic lobe, not in the central brain. (B1-2) Developing antennae at 20 hours APF. (left) GAL4-C155 drives expression of membrane targeted mCD8-GFP (green) in all post-mitotic neurons, whereas a monoclonal anti-Elav antibody stains neuronal nuclei (magenta). (right) An antenna containing MARCM clones generated according to the scheme in (A). Up to half of the cells are randomly labelled. In both cases, the upper group of neurons [second antennal segment (2AS)] are auditory mechanoreceptors of the Johnston's organ, whereas the lower group are the ORNs of the third antennal segment (3AS). (C-F) Time course of ORN invasion of the developing ALs. Note that, at 14 hours APF, no axons touch the lobe (C), whereas, at 16 hours APF (D), axons in the left hemisphere just touch the lobe. C-E are stained for GFP (detecting the mCD8-GFP marker ORN axons, green), N-cadherin (which labels the developing AL in red; an example is outlined in C); In C, nc82 is in blue, whereas, in D and E, blue is FasII (which labels axon tracts). F-H are stained with anti-mCD8 (i.e. the mCD8-GFP marker) and anti-Elav antibody. The developing ALs (C-E) are devoid of Elav staining. Unless indicated otherwise, all images in this and subsequent figures are maximum-intensity z projections of confocal stacks; dorsal is uppermost, with the midline indicated by a dashed white line when within the field of view or located to the left of the panel otherwise.

 


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  		Fig. 3. Single representative specimens of (A1-J1) anterodorsal (adPN), (A2-J2) lateral (lPN) projection neuron neuroblast clone, or (A3-J3) single cell clones of the DL1 class at the indicated developmental stage. Because these are maximum-intensity z projections, structures that are in different z planes sometimes appear to overlap (e.g. B1). Furthermore, because of unavoidable variation in the mounting angle of the brains, there is some variability in the degree of apparent overlap (e.g. compare nc82 staining in B1 and B3). Dotted circles indicate the larval lobe; continuous lines encircle the developing adult lobe. Asterisks in D1 and D2 indicate an area that is innervated by adPN but not lPN dendrites. Green is CD8-GFP, red is FasII and purple is nc82. (G1) The nc82 channel is a single optical section. (I2) Both channels are single optical sections. APF, after puparium formation; L3, wandering third instar larva.

 


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  		Fig. 4. PN dendrites occupy discrete regions of the lobe at 18 hours APF. (A) Two DL1 single cell clones in the same brain; both occupy a similar region of the dorsolateral corner of the developing AL. The green (CD8-GFP) and blue (TOTO-3-stained DNA) channels are from a single optical section about 12 µm into the lobe, whereas the red channel (FasII) is a maximum-intensity z projection of 46 1-µm sections. (B) Two anterodorsal neuroblast clones imaged at the same depth in the developing AL. Arrowheads indicate an example of a bilaterally symmetric focal concentration of dendrites, probably VA1d/VA1lm; an arrow indicates probable VA3 dendrites. (C-E) Single cell clones generated 72 hours after larval hatching can be divided into three morphological classes. (C1-C3) Class I have dendrites to the lateral edge of the lobe. (D1-D3) Class II have dendrites in the dorsomedial corner of the lobe. (E1-E2) Examples of single cell clones with two main dendritic branches. (F) A pair of single cell clones in the same hemisphere, with two distinct zones of innervation.

 


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  		Fig. 5. Development of VA1d and DA1 PNs revealed by GAL4-Mz19. Green is Mz19-driven CD8-GFP, red is FasII and magenta is nc82. White arrowheads indicate dendritic arborizations of neighbouring VA1d and DA1 PNs before they can be clearly resolved. Blue arrowheads indicate VA1d and red indicates DA1 areas once they are distinguishable.

 


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  		Fig. 6. Development of DL1 projection neuron axons. (A-J) Maximum intensity z projections of confocal stacks of representative DL1 single cell clones at successive developmental time points. Asterisks in C indicate diffuse outgrowths from the existing axon in the mushroom body calyx (blue) and lateral horn (red) areas; arrowheads in D-I indicate collaterals in these same areas. Green is mCD8-GFP, red FasII and magenta nc82.

 


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  		Fig. 7. Relationship between larval and adult ALs in early pupal stages. (A-C) Images of GAL4-GH146 driving membrane-targeted GFP. Each panel is a single confocal section through the developing AL stained with anti-GFP antibody (green), anti-N-cadherin antibody (red) and nc82 (blue). Dotted lines indicate the larval area of the lobe; solid lines indicate the adult-specific zone. Notice that, in C4, nc82 is extremely weak in spite of an increase in laser power from 3% to 10% (A4 and B4, respectively). (D-F) z projections of confocal images of GAL4-MJ94 driving mCD8-GFP in larval ORNs. Asterisks indicate the ORN axons of the antennal nerve entering the larval AL; the arrow in F indicates the `stump' of remaining ORN axon terminals and the arrowheads indicate examples of nc82-positive densities that seem to lie along the few remaining ORN processes. Dotted lines indicate the extent of the larval AL. Green is mCD8-GFP; (D,F) magenta is nc82; (E) red is N-Cadherin and blue is nc82.

 


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  		Fig. 8. Candidate cellular components of the developing adult AL. (A1-A5) Time course of repo-GAL4 driving mCD8-GFP expression, marking glial cell membrane. Each panel is a single optical section through the developing AL. (B1-B3) Comparison of repo-GAL4-driven marker expression, and anti-Repo antibody. Repo-GAL4 strongly labels all glia but has weak background staining in all cells. (B4) Location of Repo-positive nuclei in relation to GH146-positive dendrites. (B5) Triple stain of Repo, Elav and TOTO-3 (a DNA stain, most prominent in nucleoli) showing that Repo- and Elav-stained cells (glia and neurons, respectively) account for all nuclei in the vicinity of the AL. (C1-C5) Time course of GAL4-C155 driven UAS:mCD8-GFP (single sections); C3 and C5 correspond to the green channel of C2 and C4, respectively.

 

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© The Company of Biologists Ltd 2004