Glial Cells in the Optic Lobe - Results

E. Eule, S. Tix ¹, and K.-F. Fischbach
Institut für Biologie III, Schänzlestrasse 1, 79104 Freiburg i. Br., Germany. ¹ New address: California Institute of Technology, Pasadena, California 91125, USA

The enhancer trap technique in Drosophila melanogaster facilitates the identification of genetically uniform cell populations by the expression pattern of a bacterial reporter gene. A ß-galactosidase gene (lacZ) is inserted into a mobile DNA element (P-vector) that can be manipulated to move within the Drosophila genome (O'Kane and Gehring 1987; Wilson et al. 1989; Bellen et al. 1989). As the lacZ gene itself does not carry a strong promotor, its expression can only be stimulated by a Drosophila enhancer. The enhancer triggers reporter gene expression according to its own specificity. Consequently, the bacterial lacZ staining can potentially mimic the temporal and spatial expression of a nearby Drosophila gene. Apart from the obvious impact this method has for molecular studies, it is well suited for structural studies by providing histological markers. From a pool of initially 87 enhancer trap lines carrying a nuclear targeted lacZ reporter gene construct, we prepared whole mounts of adult brains. Most of the lines had been preselected for embryonic, nervous system specific ß-galactosidase expression. Those lines showing lacZ staining in distinct populations of potential glial cells in the optic lobe were used for further investigation. We performed semithin sections of Epon embedded X-gal positive brains as well as immuno- histochemistry with the elav antiserum which only recognizes neurons in Drosophila and not glial cells (Robinow and White 1991). The antibody staining procedure was combined with X-gal staining and performed on cryostat sections of adult heads. Using these techniques, it was possible to unequivocally identify glial cells. As ß-Gal activity is restricted to the nucleus and cannot elucidate the entire cell shape, osmium fixed samples were consulted for further structural details. The adult optic lobe consists of 4 neuromeres: the lamina, the medulla, the lobula and the lobula plate. The latter two are also referred to as lobula complex. The general neuronal structure of the optic lobe of Drosophila and other Diptera is well known (e.g. Strausfeld 1976; Fischbach and Dittrich 1989). Ipsilateral connections between the different neuromeres are established via the outer and the inner optic chiasms.

Classification of glial cells in the adult optic lobe

The optic lobe of Drosophila houses many non-neuronal cell types. The optic lobe specific ones are discussed first. More widely distributed cell types are discussed thereafter.

1. Glial cells of the lamina

The first neuromere of the visual system of Drosophila is the lamina ganglionaris (Fig.2, 3). From distally to proximally, it can be divided into three zones: the fenestrated layer underlying the retina, the lamina cortex with the cell bodies of the lamina monopolar cells, and the lamina neuropile, where synaptic interactions between retinula cells and the first order interneurons take place. The neuropile can be subdivided into repetitive elements (cartridges), which correspond in number to the ommatidia. Connectivity between the ommatidia and the cartridges follows the rules of neural superposition eye (Kirschfeld, 1973). Glial cells can be detected in all layers of the lamina (Figs 2, 3).

Fenestrated glia:

Glial cells with discoid shaped nuclei of approximately 5.2 µm length and 1.6 µm width are regularly arranged in an area corresponding to the fen-estrated layer (Figs. 2, 3b, 3c). The fenestrated layer is located proximal to a 8-10 µm wide zone of dense pigmentation, just beneath the basement membrane of the eye. The fenestrated layer can also easily be identified by its wealth of trachea. There are about 250 fenestrated glial cells.

Pseudocartridge glia:

Between the basal membrane of the compound eye and the lamina cortex we found a few round glial nuclei that stand out by their enormous size (6.5 µm length, 5.2 µm width) (Fig. 3a). These nuclei belong to the largest of all lamina cells. According to Saint Marie and Carlson (1983), the cell processes ensheath the pseudocartridges and extend throughout most of the lamina.

Lamina cortex glial cells:

a) distal satellite glia:

Satellite glial cells are closely associated with the round neuronal cell bodies. This gives the cells a cap like appearance. The convex curvatures typical for the cell surface of satellite glial cells is clearly visible in Fig.3b showing blue cells in line 3-74. Given the nuclear localization of ß-galactosidase, the reproducible and specific distribution of label is another indication of intracellular diffusion of the X-gal reaction product. The distal satellite glial cells are about 2.7 µm long and 1.4 µm wide. Their processes form a trophospongium invading cell bodies of monopolar neurons (Saint Marie and Carlson 1983).

b) proximal satellite glia

This glia is situated at the interface between cortex and neuropile. We did not detect any enhancer trap line in which this type of glia was labelled in the lamina. However, osmium fixed specimen allowed a clear identification of this cell type. The cell bodies are situated just on top of the lamina neuropile (Fig. 2, arrowheads) and send processes distally into the neuropile and proximally into the neuronal cell body layer (Saint Marie and Carlson 1983). The flat shaped cell bodies are about 4.3 µm wide and 1.4 µm high.

Lamina neuropile glial cells:

a) epithelial glia:

The elongated nuclei of the approximately 750 epithelial glial cells are about 2.4 µm wide and 5.3 µm high and situated in the distal region of the lamina neuropile (Fig. 3b, 3c). These cells ensheath the lamina cartridges in full length and are therefore comparatively large (up to 30 µm; Saint Marie and Carlson 1983). It is known from electronmicroscopic studies that epithelial glia cells form so-called capitat projections which invade R-cell profiles (Boschek 1971; Meinertzhagen and O'Neil 1991). The function of these specializations is unknown. The epithelial glia cells are clear examples of neuropile glia.

b) marginal glia:

The marginal glia lies at the proximal edge of the lamina neuropile just distal to the outer optic chiasm (Fig. 2c). Their nuclei are relatively small (3.2 µm long, 2.7 µm wide). According to Winberg et al. (1992) these cells have important functions during development. They seem to contain the stop and go signals for short and long retinal axons respectively. Their distal processes invade the neuropile.

2. Glial cells of the optic chiasms

outer chiasm glial cells:

a) outer chiasm small glia:

Unevenly spaced glia cells with small cell bodies are scattered within the outer chiasm between the lamina neuropile and the medulla cortex. They are visible in line T1184 with cytoplasmic targeting of ß- galactosidase (Fig. 4). In addition to the irregular distribution, the cell bodies are variably shaped, often flattened and usually 1.1 µm thick and 5.6 µm long. The number of these cells is about 30. Neighbouring axonal fibres show a thin blue coating probably caused by cytoplasmic lamellae of the small glial cells.

b) outer chiasm giant glia:

On average, 30 almost evenly spaced, huge glial nuclei are forming an arc in ventral to dorsal orientation amidst the crossing fibre bundles of the first optic chiasm (Figs. 5a, 5b, 3c. Table I). Their number varied in wild type flies from 25 - 37. The fat round cell nuclei of 5.7 µm width and 6.7 µm length comprise a big nucleolus. The nuclei are labelled in lines 3-66, 3-74, 3-93, and in 3-97. Interestingly, in all these lines similar cells in the inner chiasm are also labelled.

inner chiasm giant glia:

Just as in the outer chiasm, huge glial cells are arranged in a half-moon shaped manner in the inner chiasm (Figs. 5a, 5c). Their number is on average 33, ranging from 28-38, and their arrangement is the same as that of the outer chiasm glial cells (Table I). The nuclei are more oval than their counterparts in the outer chiasm, with a dimension of 4.7 µm width and 7.6 µm length. Their cell bodies are situated between the planes of visual axon bundles and seem to separate them (Fig. 5c). In counterstaining with Pyronin G, the cytoplasm of the giant glial cell types in both optic chiasms does not appear darker than that of neurons. This is opposed to the behaviour of most other glial cell types described here. But the classification of the giant cells as glia is justified as they do not express the neuronal elav antigen (Fig. 10a; see Robinow and White 1991 for informations about elav).

3. Medulla glial cells

medulla satellite glia:

We find multipolar glial cells of about 4.9 µm length and 3.2 µm width inbetween the neuronal cell bodies of the medulla cortex (Fig. 6b). Again, the marker is accumulated at the surface of the cell bodies, and not in the nuclei of the glial cells. This also suggests that the blue stain in between neighbouring neurons is due to projections of these glial cells.

medulla neuropile glia:

Approximately 240 more or less evenly spaced glial cells per lobe are located at the margin of the medulla neuropile (Table I). Their cell bodies anchor at the surface of the neuropile and they send long (up to 9 µm), regular extensions into deeper layers of the neuropile (Figs. 1, 5a, 6c). The single cells are separated by a distance of 3 to 5 neuron somata equivalents. The cell bodies are 4 µm wide and 2.4 µm long. Some cell bodies could also be identified lateral to the serpentine layer and between medulla and lobula. So, the medulla neuropile glial cells seem to cover the whole medulla neuropile 'surface'. The distribution of the cell population looks fairly irregular, the position of single cells does not seem to be fixed. The MNGl do not show anti-elav immunoreactivity (Fig. 9)

4. Lobula complex glial cells

lobula neuropile glia:

Using our osmium reference preparations, this cell type could be identified at the interface of cortex and neuropile (Fig. 7a). The regularly spread cell bodies are wide and flat (4.2 µm x 2.4 µm), extending finger-like projections into the lobula neuropile. This cell type was not detected in any of our enhancer trap lines. Consequently, we are unable to make a reliable statement about their number.

lobula plate neuropile glia:

Again, this cell type at the interface of lobula plate cortex and neuropile could only be detected in osmium fixed preparations (Fig. 7b). Like the glial cells of the lobula they are evenly distributed and of identical shape (4.2 µm x 2.4 µm). As no lacZ lines specifically labelling these cells were available, we do not know their number. It cannot be ruled out that lobula and lobula plate neuropile glial cells genetically belong to one cell population rather than forming two separate classes.

lobula plate satellite glia:

Multipolar glial cells with nuclei of 3.2 µm height and 4.9 µm width are restricted to the lobula plate cortex (Fig. 7c). They are relatively rare, and we estimate their number to be about 15-20. Similarly to the satellite glia of the medulla, the marker seems to be concentrated at the surface of the cell bodies. It is likely that their thin cytoplasmic lamellae ensheath neighbouring neuronal cell bodies. This could be the origin of the blue label in the entire lobula plate cortex.

5. Glial cells not restricted to the optic lobe

Perineurial cells ensheath the entire central nervous system and constitute the blood-brain barrier in insects (Hoyle 1986, Butt 1991). In our study no enhancer trap line was identified that selectively detects perineurial cells of only the optic lobe (Fig. 8a). Therefore, it is likely that these cells are part of a homogeneous population of perineurial cells covering the central nervous system. The thickness of the brain envelope they form is about 1 µm.

subperineurial glial cells:

Underneath the perineurium there is another class of long, flat glial cells (Fig. 8b). As in the case of the perineurial cells, these cells seem to form a homogeneous cell population throughout the CNS. According to Hoyle (1986), they extend cytoplasmic processes deep into the cortex areas. Although the marker used here is mainly restricted to the nuclei, weak cytoplasmic label discloses the multipolar nature of these glial cells. Their cell bodies are very large. They have the shape of a disc (2.1 µm thick, with a diameter of 8.6 µm). Glial cells of this type are rare; only 8 to 14 could be counted per optic lobe lobe.

tracheal glial cells:

This cell type occurs in close association with trachea. The tracheal glial cells have a highly irregular design and variable sizes (Fig. 8a, arrow). The trachea-associated cells in the CNS and the ones associated with trachea outside the nervous system (arrow head) seem to constitute a genetically homogeneous cell population that is labelled in strains T601 and 3-93. Based on these data it could well be argued that the tracheal cells should not be considered to be glial cells.

Spatial gradients of gene expression in glial cells and neurones

Table I summarizes the characteristics of all the glial cell types mentioned so far.

Table II summarizes the labelled cells in different enhancer trap lines.

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