Neural Recognition in Drosophila: Restricted Expression of IrreC-rst is Required for Normal Axonal Projections of Columnar Visual Neurons

Thilo Schneider, Christian Reiter, Eckhart Eule, Brigitte Bader, Beate Lichte, Zhiping Nie, Thorsten Schimansky, Ricardo G.P. Ramos¹ , and Karl-Friedrich Fischbach

Institut für Biologie III, Albert-Ludwigs-Universität Freiburg, Schänzlestr.1, D-79104 Freiburg, Germany

¹ Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21949 Rio de Janeiro, Brazil.

Originally published in: Schneider et al. Neuron 15, 259-271 (1995)

Images (mostly confocal) of IrreC-rst protein expression and mutant phenotypes of the above publication can be accessed by clicking on the thumbnails below. Some unpublished images are displayed as well.

Table of Contents

Abstract
IrreC-rst mediates homophilic adhesion in Schneider cells
Wild type expression pattern of IrreC-rst during optic lobe development
Pupal optic lobe phenotypes of irreC mutants
Global IrreC-rst overexpression during optic lobe development and its effects

Abstract

The 104 kDa IrreC-rst protein, a member of the immunoglobulin superfamily, mediates homophilic adhesion in cell cultures. In larval optic chiasms, the protein is found on recently formed axon bundles, not on older ones. In developing visual neuropils it is present in all columnar domains of specific layers. The number of IrreC-rst positive neuropil stratifications increases until the midpupal stage. Immunoreactivity fades thereafter. The functional importance of the restricted expression pattern is demonstrated by the severe projection errors of axons in the first and second optic chiasms in loss of function mutants and in transformants that express the IrreC-rst protein globally. Epigenesis of the phenotypes can partially be explained on the bases of homophilic IrreC-rst interactions.

IrreC-rst mediates homophilic adhesion in Schneider cells

1. 

   (A, B) In cell aggregation assays, heat shocked S2-Schneider cells, not transfected with pKB256-HB3, do not aggregate (A), while transfected, heat shocked S2-Schneider cells do (B). Bar, 100 µm.

2. 

   (C) When pKB256-HB3 transfected S2 Schneider cells are labelled with RITC (red fluorescent) and mixed with untransfected cells (labelled with FITC, green fluorescent) prior to the aggregation assay, red aggregates form under the exclusion of green cells.

3. 

   (D) In clusters of transfected and heat shocked S2-Schneider cells MAb24A5.1 immunoreactivity accumulates at the sites of cell contact (arrows). Bar, 10µm.

4. 

   (E) Time course of cell aggregation of two independently transfected S2 Schneider cell lines (A and B) in comparison with untransfected control cells. Plotted is the normalized particle number (Nt/N0) as a function of time. The error bars denote the standard deviation between independent experiments (n = 3, controls and cell line B; n = 4; cell line A).

Wild type expression pattern of IrreC-rst during optic lobe development

1. 

   Comparison of irreC-rst mRNA expression and MAb24A5.1 immunoreactivity (conventional micrographs). (C) and (D) show eye imaginal disc and optic lobe mRNA expression in third instar larvae. Details of mRNA expression pattern are reflected in the pattern of MAb24A5.1 immunoreactivity (E and F). mRNA expression slightly precedes protein expression in the eye disc (arrowheads in (C) and (E) point to morphogenetic furrow). Note also the expression in two cell clusters of the lobula complex (arrows in (D) and (F)). MAb24A5.1 labels retinula cell axons in the optic stalk (os). Immunoreactivity is visible in concentric rings of lamina (la), medulla (me) and lobula complex (lo). Bar = 100 µm.

2. 

   Analysis of IrreC-rst expression in the third instar larva and its relation to early optic lobe structure. Anterior is left. (A) Horizontal optical section labeled with MAb24A5.1. IrreC-rst protein is present in the neuropils of lamina (la), distal (dm) and proximal medulla (pm), and lobula (lo). Expression in cortical areas of lamina and medulla (mc) shows a granular distribution; protein does not outline membranes of cell bodies or cell body fibers. In the outer (x1) and inner (x2) chiasms, only few fibres are labelled. Staining in the older medulla neuropil is restricted to two distinct layers that have not yet separated in younger parts. The distal medulla layer shows columnar organization, while this is not so obvious in the proximal medulla layer at this developmental stage. (B) Schematic representation of a horizontal section through the larval optic lobe. Black arrows indicate the gradient of neuropil differentiation. IrreC-rst immunoreactivity is highlighted in red. Note that most projections in the optic chiasms are IrreC-rst negative, only young fascicles are immunoreactive (A). C&T cells (blue) - although IrreC-rst negative - are shown, as posterior lamina fibres take their route to the inner optic chiasm in irreC mutants (indicated by the blue arrow marked with *; see also Figures 6 F,G). lop, lobula plate; lpc, lobula plate cortex; tme, transient medulla neuropil. Bar in A, 25 µm.

3. 

   Conventional micrograph of pupal cns wholemount (P7%). IrreC-rst expression (MAb24A5.1 immunoreactivity) in the cns is mainly restricted to the visual system. Abbreviations in magnified image: eid = eye imaginal disc; os = optic stalk; la = lamina; me = medulla; lo = lobula; vg = ventral ganglia. Unpublished preparation by Thilo Schneider.

4. 

   IrreC-rst immunoreactivity in the pupal optic lobe is confined to small field columnar elements with specializations in few neuropil layers. (A-D) Horizontal semithin (A) and optical sections (B,C, D) reveal that IrreC-rst immunoreactivity in the pupa is restricted to certain synaptic layers in the neuropil and to vesicular structures inside a subpopulation of neuronal cell bodies. In (A) the counterstaining of the cell bodies by methylene blue reveals the extent of the neuropils. IrreC-rst immunoreactivity is visible in layers of the distal (dm) and proximal (pm) medulla. Layer specific immunoreactivity is also present at the level of the lobula (lo) and lobula plate (lop) neuropil. Confocal sections (B, C, D) show the developmental dynamics of IrreC-rst expression in the pupal optic lobe. Expression in the distal medulla changes from one (B, P+16%) to two (C, P+31%) and finally to four layers (D, P+43%). Immunoreactivity of the proximal medulla layers becomes organized in a columnar manner during pupal development. This is also true for the immunoreactivity in the lobula which is strongest in the most superficial layer (C, D). Label in the lobula plate (lop) is obvious in late stages (D, P+43%). It is strongest in the posterior half of the neuropil. Dots of immunolabel can be seen at the levels of the lamina (arrowhead in (C)), and in medulla and lobula plate cortices (C). In (C) a very strong staining of the basal lamina is apparent (see also Figure 5B). Note the change in the staining pattern of the lamina neuropil between P+31% (C) and P+43% (D). Bar (A-D) 25µm. Anterior to the left in panels (A-D).
   (E) Layer-specific columnar organization of IrreC-rst immunoreactivity. (E) shows a tangential view of the medulla neuropil, a composite of three confocal planes stepped 1µm apart. This view reveals the columnar organization of the IrreC-rst-positive medulla layers. Bar, 20 µm.

5. 

   Confocal view of pupal irreC-rst expression in the lacZ enhancer trap line 3-66 (this line was obtained from Christian Klämbt and its optic lobe expression pattern was initially characterized by Simone Tix and Eckhart Eule in our laboratory): X-gal staining of the neuronal nuclei in the medulla cortex and of the giant glial cells of the inner chiasm (green) contrasts nicely with anti-irreC-rst immunoreactivity (red). Unpublished preparation by Christian Reiter.

6. 

   Dynamic changes of IrreC-rst protein expression in the lamina
   (A) Superposition of several oblique confocal planes: Club-like terminals of short retinula axons are clearly visible at P37%.
   (B) At P49% the columnar expression of the IrreC-rst protein has been reorganized. Based on their shape and localization, the fine columnar arborizations now displaying the immunoreactivity are probably dendrites of lamina monopolar cells. The transition in the lamina staining pattern occurs between P37% and P43% and is accompanied by similar changes in the staining pattern of the distal medulla (see above). At the base of the lamina neuropil, where the marginal glial cells are situated, strong IrreC-rst immunoreactivity is visible in (B) (arrowhead). It is out of focus in (A). Such glial cells do not form columnar processes. Bar, 20 µm.

Pupal optic lobe phenotypes of irreC mutants

1. 512x213 pixel | 1024x425 pixel | 1969x817 pixel

   (A, B) Optic lobes of wild-type (A) and irreC(1R34) (B) pupae (P16%). In the mutant pupa, fasciclin II-positive fibers in the outer optic chiasm are bundled in an irregular manner (arrow). Bar, 50 µm.

2. 512x522 pixel | 790x805 pixel

   (C) Fasciclin II-positive projections into the medulla of irreC(1R34) pupae (P20%). Arrow indicates termination error, while arrowhead points to projection errors similar to those seen in irreCUB883 (E). Bar in (C), 10 µm.

3. 512x330 pixel | 1024x659 pixel | 1573x1012 pixel

   (D, E) Frontal optical sections through the posterior optic lobes of wild-type (D) and irreC(UB883) (E) pupae (P30%). Dorsal is up. In the mutant, many posterior bundles from the lamina (arrows), not only those at the equator, project into the inner optic chiasm. Bar, 25 µm.

4. 512x428 pixel | 1024x855 | 1212x1012 pixel

   (F, G) Horizontal optical sections through the optic lobes of wild-type (F) and irreC(UB883) (G) pupae (P15%). Arrowhead in (F) points to the cluster of C&T cell bodies. Fasciclin II positive C&T fibers project along the inner face of the medulla (compare with Figure 3B). In the mutant (G), also fiber bundles exiting posterior lamina cartridges project along the inner face of the medulla before they penetrate the medulla neuropil towards their normal target region (arrows). Bar 20 µm.

Global IrreC-rst overexpression during optic lobe development and its effects

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