Flybrain

Optic Lobe Development
in 3rd Instar and Early Pupal Stages

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Christian Reiter, Annette Sulzbacher and Karl-Friedrich Fischbach
Institute of Biology III, Schänzlestr.1, D-79104 Freiburg i. Brsg., Germany


Table of Contents

Abstract
Abbreviations
Introduction
Materials, Methods and Software Requirements
Available Resources
Literature


Abstract

The resources provided here are intended to enable researchers to compare enhancer trap or gene expression patterns to reference stainings and to clarify the complex topology of the early optic lobe. The developing lamina is the projection area of the ingrowing photoreceptors. The actual lamina neuropil is only a very thin layer in the larval stage. The lamina surrounds the inner cell plug, which derives from the inner optic anlage. The lamina itself is proliferated from the outer optic anlage (OOA). The Bolwig nerve connects the larval photoreceptors with the larval optic neuropil, which is situated at the base of the medulla. The inner optic anlage (IOA) is the proliferation region which creates neurons of the lobula and proximal medulla. The distal medulla neuropil is the projection area of long retinula cells R7 and R8 and of lamina monopolar neurons. Here connections are made to dendrites of transmedulla neurons derived from the OOA. The proximal medulla neuropil contains the aborizations of neurons formed by the IOA as well as branches of transmedulla neurons that project to the lobula complex. Those cells that project from the lobula complex neuropils to central brain regions are derivatives of the IOA. The inner cell plug built by the IOA contains the population of T and C cells.


Abbreviations used

bn, bolwig nerve; GFP, green fluorescent protein; inner (second) chiasm; IOA, inner optic anlage;la, lamina; lo, lobula; lc, lobula cortex; me, medulla; mc, medulla cortex; OOA, outer optic anlage; T/C, projections of T2/T3 and C2/C3 cells; os, optic stalk; x1, outer (first) chiasm; tmn, transient medulla neuropil; x2, inner (second) chiasm


Introduction

Compound eyes of holometabolic insects are structures of the adult animal. They develop from the eye imaginal discs (see review of Wolff and Ready, 1993) which consist of undifferentiated cells that are put aside during embryonic development. It is not earlier than in the third instar larva of flies that retinula cells differentiate and send their axons into the optic lobe anlagen which are not part of the imaginal disc, but two crescent-shaped clusters of neuroblasts in the lateral part of both globular brain hemispheres. These clusters of neuroblasts are called the outer and inner optic anlagen (OOA and IOA respectively). The ring-like OOA underlies the surface of the lateral brain hemisphere, while the IOA is situated more deeply. The spatial relationship of the anlagen changes continuously in the third instar due their proliferative activity (White and Kankel, 1978; Hofbauer and Campos-Ortega, 1990; Meinertzhagen and Hanson, 1993). In the third instar larva and early pupa most cells (neurons and glia) of the adult optic lobe are born. Lamina development is completely dependent on the ingrowth of retinal axons, as differentiation of lamina precursor cells is triggered by this process (Selleck and Steller, 1991; Selleck et al., 1992). A lamina is never formed when innervation from the retina is missing. Development of medulla and lobula complex is partially independent from retinal innervation (Power, 1943; Fischbach, 1981). Not unexpectactly, as a rule the more proximal the neuropil, the more independent it is from retinal innervation.

In contrast to the adult visual system the structure of the larval and pupal neuropils and in particular the structure of individual neurons at these developmental stages is not well known. As a step towards a better understanding of the changing spatial relationships of visual neuropils during development, we here have collected confocal serial images of (mainly) late third instar larval optic lobes expressing the green fluorescent protein (GFP-S65T, a gift of Kei Ito) under the control of Gal4 enhancer trap lines (Brand and Perrimon, 1993). We used several antibodies with known specificity to characterize the expression patterns of the Gal4-lines. It is shown that GFP is well suited to visualize neural structures during development and the use of other Gal4 lines which target GFP expression to different cell types is encouraged.


Materials, Methods and Software requirements

Antibodies used: mAb 22C10 (gift of Seymour Benzer), mAb 24A5 directed against IrreC-rst, mab 5A6 directed against DPTP69D (gift of Kai Zinn). Gal4 lines Mz1407 and Mz1369 are a gift of Joachim Urban and Gerhard Technau (Mainz).

All images were acquired on a Leica TCS 4D laser scanning microscope. 3D rendering, projection and VRML reconstructions were done with Bitplane Imaris. Most images were originally recorded in 1024x1024 resolution and subsequently downsized for web presentation.

Movie files require the Quicktime Plug-In, VRML1 and VRML2 files require appropriate viewers. The Hemisphere Browser requires browsers that understand frames, e.g. Netscape 2.0 or higher or MS Internet Explorer > 2.0. All this software is freely available on the Internet.


Available resources:

  1. GFP expression in larval optic lobe driven by Gal4(Mz1369)
    Gal4(Mz1369) targets strong Gal4 expression to the inner optic anlage (IOA). Counterstained by mab24A5 (anti-IrreC-rst). Three confocal stacks sectioned in orthogonal orientations are provided in the form of Quicktime movie files or as consecutive series of projections. 3D surface renderings of the IOA and surrounding neuropils (VRML1/VRML2) can be viewed individually or together with the confocal stacks.

    -completed-Projections from the stacks
    -completed-Confocal stacks as movie files
    -completed-3D reconstructions

  2. Hemisphere browser of larval optic lobe using Gal4(Mz1369) data
    This is a frame based page that lets the viewer select from the projection data sets of all orientations and browse through them, while simultaneously comparing the projections with selectable movie files of all orientations. Annotations can be toggled on and off, and 3D reconstructions can be accessed.
  3. GFP expression in larval optic lobe driven by Gal4(Mz1407), mab22C10 and mab5A6 stainings
    Here additional views of the neuropils and axonal tracts within the developing optic lobe are provided.

    -completed-Frontal sections
    -completed-Horizontal sections
    -completed-Sagittal, oblique frontal sections
    -completed-3D projections volume rendered from confocal stack

  4. Early pupal optic lobe 3D surface rendering (VRML reconstruction)


References

Brand, A.H. and Perrimon, N. (1993). Targeted gene expression as means of altering cell fates and generating dominant phenotypes. Development 118, 401-415

Fischbach K.-F. (1983). Neural cell types surviving congenital sensory deprivation in the optic lobes of Drosophila melanogaster. Dev. Biol. 95, 1-18

Hofbauer A. and and Campos-Ortega J.A. (1990). Proliferation pattern and early differentiation of the optic lobes in Drosophila melanogaster. Roux's Arch. Dev. Biol. 198, 264-274

Meinertzhagen I.A. and Hanson T.E. (1993). The development of the optic lobe. In The development of Drosophila melanogaster. Volume II. Eds. Michael Bate and Alfonso Martinez Arias. Cold Spring Harbor Laboratory Press, p. 1363-1491

Power M.E. (1943). The effect of reduction in numbers of ommatidia upon the brain of Drosophila melanogaster. J. exp. Zool. 94, 33-71

Selleck S.B. and Steller H. (1991). The influence of retinal innervation on neurogenesis in the first optic ganglion of Drosophila. Neuron 6, 83-99.

Selleck S.B., Gonzales C., Glover D.M., and White K. (1992). Regulation of the G1-S transition in postembryonic neuronal precursors by axon ingrowth. Nature 355, 253-255

White K. and Kankel D.R. (1978). Patterns of cell division and cell movement in the formation of the imaginal nervous system in Drosophila melanogaster. Dev. Biol. 65, 296-321

Wolff T. and Ready D.F. (1993). Pattern formation in the Drosophila retina. In The development of Drosophila melanogaster. Volume II. Eds. Michael Bate and Alfonso Martinez Arias. Cold Spring Harbor Laboratory Press, p. 1277-1325


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