Formation of the median bundle and mACT (GAL4 strain Mz 699 x UAS-tau)

Original images that appeared in Fig. 4 of:
Kei Ito, Heinz Sass, Joachim Urban, Alois Hofbauer and Stephan Schneuwly (1997)
GAL4-responsive UAS-tau as a tool for studying the anatomy and development of the Drosophila central nervous system
Cell and Tissue Research 290:1-10

Contents:

Another approach for observing the whole-mount preparations is to use thin capillaries. With the thickness of only 150-200 um, Drosophila brains can be kept in thin capillaries (outer diameter 0.2 to 0.35 mm), which can be rotated freely under oil-immersion objectives to be observed at any desired angle. For presentation, the preparations are subjected to digital montage from two perpendicular viewing angles (horizontal and frontal). By comparing each pair of photographs, the complicated three-dimensional structures of the labelled neurones can be resolved.

The advantages of this approach over the combination of fluorescence and confocal microscopy (see Fig. 3) are:
1: By using Nomarski optics, the morphology of the labelled cells can be observed in the context of the unlabelled background structure.
2: The stained preparations can be kept almost indefinitely.
3: The focal plane and the viewing angle can be changed interactively during observation.
4: Expensive confocal microscopes and workstations are not required.

The disadvantages are:
1: It is impossible to make 3-D stereographs.
2: It takes patience and long time (usually several hours) to make a single montage.

By using the GAL4 strains with continuous enhancer activity during development, it is possible to trace the morphological differentiation of the labelled neurones. We demonstrate the development of identified neuronal structures labelled with the GAL4 strains Mz 699 (this page) and Mz 423 (Fig. 5). The consistency of labelling was confirmed by staining staged specimens at 12-24 hour intervals from larval hatching to adult.

The strain Mz 699 labels five different sets of neurones, termed arbitrarily as #1 to #5, most of which can be traced throughout the postembryonic development. Here we concentrate on the development of two neurone groups that contribute to the median bundle (m bdl) and the medial antennocommissural tract (mACT).

Click each thumbnail image to see the original figure. This picture corresponds to Fig. 3A.

The strain Mz 699 labels the following neurones: #1 cells in the suboesophageal ganglion (sog) that send fibres via the median bundle (m bdl) #2 cells in the deuterocerebrum that arborise in the antennal lobe (a l), form the mACT, and innervate the lateral horn (l ho) #3 cells in the ventral part of the lateral cortex (the cortex region between the central brain and the optic lobe) that arborise in the ventro-lateral protocerebrum (v-l pr), contact both sides via the great commissure (g c), and innervate the lateral horn (l ho via the posterior lateral fascicle (p l fasc) #4 cells in the posterior cortex that innervates the v-l pr #5 cells above the superior protocerebrum that send fibres to the superior protocerebrum and to the median bundle (m bdl) Other abbreviations oes: oesophagus, la: lamina, me: medulla, lo: lobula, lo p: lobula plate, e b: ellipsoid body, f b: fan shaped body Note that not all the labelled cells appear in each photograph, although the depth of focus is considerably enlarged by the digital montage.

(Bar = 50 um)

In larvae just after hatching, the cell bodies of the median bundle neurones (#5) lie in the anterior cortex of each hemisphere. They send fibres to the anteriormost region of the brain neuropile and connect with both sides via a central commissure (arrowhead). The medial antenno-comissural tract (mACT) is formed by a subset of unilateral ACT relay interneurones (uACTI: Fig. 13 of Stocker et al., 1990). The tract and its arborsation in the lateral horn area can already be seen in this preparation. In the posterior cortex, the cell bodies of the neurons #4 are seen.

(Bar = 50 um)

In late larvae (72 hrs after larval hatching: ALH), the relative position of the #5 cell bodies shifts medially, as the lateral optic lobe primordium enlarges during the second and third larval instar. The cells contribute to the fibres of the central commissure (arrowheads) and to the putative median bundle tracts (m bdl in C) that run on both sides of the oesophagus foramen (oes). The mACT cell bodies lie in the deuterocerebrum (#2 in C) and the fibre terminals arborise in the putative lateral horn region (l ho in B). Until puparium formation, the mACT neurones have no apparent arborisation between the cell bodies and the terminals (see C).

(Bar = 50 um)

Brain in the early pupa (17 hrs after puparium formation: APF). On the first day of the pupal metamorphosis, the brain experiences a major morphogenic movement. During this process, the two clusters of the #5 cell bodies on both brain hemispheres move closer to the midline. The central commissure connecting the two clusters (arrowheads) becomes obscured during this fusion process. The mACT neurones have no apparent arborisation between the cell bodies (#2) and the terminals (l ho) in the larval brain. In early pupae, however, small branches appear at the position of the antennal lobe (a l). This corresponds to the formation of the antennal lobe glomeruli (Tolbert and Oland, 1990).

Brain in the late pupa (82 hrs APF). The two clusters of the #5 cell bodies fuse to form a single cluster in the dorsal cortex (shown only in G). The median bundle tracts (m bdl) on both sides are not completely fused. From the cell bodies (#2), the fibres of the mACT run lateral to the antennal lobe (a l) and bend inwards. They again bend posterodorsally to run along with the inner ACT (iACT, not labelled; also called the antenno-glomerular tract, see Stocker et al. 1990). In front of the fan-shaped body (f b in F) the mACT fibres bend laterally to separate from the iACT. They finally bend posterodorsally to circumvent the fan-shaped body and reach the lateral horn (l ho). It is notable that this complex bending pattern remains essentially the same throughout postembryonic development, even during metamorphosis. In the antennal lobe (a l), the arborisation of the mACT grows during late pupal stages to cover numerous glomeruli. The terminal arborisation in the lateral horn (l ho) also becomes extensive in late pupae. The projection of the neurones #3 from the ventro-lateral protocerebrum (v-l pr) via the posterior lateral fascicle (p l fasc) also contributes to the growth of the lateral horn neuropile (l ho).

Brain in the mature adult (10 days after eclosion). The cell bodies of the #5 neurons are scattered near the midline in the dorsal cortex. The tracts of the adult median bundle are also fused in the dorsal region, but remain separate in the more ventral part. The arborisation in the antennal lobe (a l, by neurons #2) and lateral horn (l ho, by neurons #2 and #3) are even more extensive than in late pupae. For more information about the labelled neurones, see the same figures on top of this page.

Methods

References

• Prokop A, Technau GM (1993) Cell transplantation. In: Hartley DA (ed) Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford/New York/Tokyo, pp 33-57
• Stocker RF, Lienhard MC, Borst A, Fischbach K-F (1990) Neuronal architecture of the antennal lobe in Drosophila melanogaster. Cell Tissue Res 262:9-34

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