AstA (magenta) and GFP (green) immunolabeling of nervous systems and guts of adult AstA³⁴>GFP (A–D, F–H) and tsh-Gal80; AstA³⁴>GFP (E, I) flies.

<p><b>(A)</b> GFP expression is detectable in two paired groups of brain neurons. In each hemisphere, one group with two somata in the posterior lateral protocerebrum (PLP cells, solid arrowheads in <b>A'''</b>) and a second group with two to four somata in the lateral cell body rind (LCBR cells, open arrowheads in <b>A'''</b>) are labelled (see also <b>C</b>). The LCBR neurons are anti-AstA-negative. Some of the AstA-IR medulla neurons also express GFP. In the abdominal TAG, the six AstA-IR DLAa cells show strong anti-GFP staining and project through the median abdominal nerve towards the gut (asterisk in <b>A'''</b>). Four peripheral cells located on nerves that exit the TAG dorso-laterally also exhibit co-labeling (arrows in <b>A'''</b>). Inset in <b>A'''</b>: Single optical sections showing double-labelled PLP cells of both hemispheres. <b>(B)</b> Close-up of the PLP neurons (solid arrowheads in <b>B',B''</b>) from <b>A</b>, maximum projection of the horizontal sections between the level of central body and calyx. The extensive arborisations in the superior protocerebrum are visible. <b>(C)</b> The GFP signal in brains of different individuals illustrates the variability of expression intensity among PLP, LCBR and medulla neurons. The PLP cells (solid arrowhead in <b>C'</b>) innervate the superior protocerebrum, while the ramifications of the LCBR neurons (open arrowhead in <b>C'</b>) lie mainly within the posterior lateral protocerebrum and the posterior slope. <b>C'</b> and <b>C''</b> show a strong and moderate GFP-expression intensity, respectively. The arrow in C''' marks a strongly stained neuron in the medulla, which occurred only in a few preparations <b>(D)</b> Detail of the abdominal TAG. Three pairs of AstA-IR DLAa neurons co-express GFP and run through the median abdominal nerve to innervate the hindgut and posterior midgut (see <b>F</b>). The membrane-targeted GFP distinctly marks the projections of these neurons. Two single optical sections (insets in <b>D'''</b>) reveal six co-labelled cell bodies. <b>(E)</b> AstA34-Gal4 expression in the TAG is absent with <i>tsh-Gal80</i>. <b>(F)</b> AstA and GFP labeling are present in neuronal processes at the hindgut, which extend onto the posterior midgut (Malpighian tubules have been removed during dissection). <b>(G)</b> The rectal part of the hindgut is likewise innervated by double-labelled neurons. <b>(H)</b> GFP is expressed in most of the AstA-producing EECs that are scattered within the epithelium of the posterior midgut. The maximum intensity projection of a single EEC in the inset of <b>H'''</b> illustrates that the main GFP signal is restricted to the narrow apical portion of the cells. <b>(I)</b> GFP expression is absent from gut neurons in individuals carrying <i>tsh-Gal80</i>, but remains in the AstA EECs. (Malpighian tubules have been removed during dissection.) Scale bars: in A-C and E–H 50 μm; in D 10 μm; in I 100 μm. BR brain, CB central brain, CC cervical connective, IL ileum, MG midgut, OL optic lobe, PV pyloric valve, R rectum, RV rectal valve, GNG gnathal ganglia, TAG thoracico-abdominal ganglion.