Chapter II: Anatomical characterization of AstA-Gal4 transgenic flies
2. Results
An AstA promoter-Gal4 transgenic was made by cloning 2.1 kb of 5’ flanking DNA up to but not including the predicted transcriptional start site (Figure 1a). We obtained five independent insertion lines which we crossed to UAS-mcd8::GFP [7], a membrane-tethered fluorescent reporter, in order to compare and characterize the expression patterns of the independent insertion lines. Immunostaining with an antibody against green fluorescent protein (GFP) revealed no differences in expression patterns between the different independent insertion lines (data not shown). To address the specificity of the Gal4 expression pattern, we double labeled adult tissues with a monoclonal antibody raised against Diploptera punctata AstA that had previously been used to characterize AstA expression in Drosophila [3].
AstA-Gal4 labeled three pairs of neurons in the brain and ~ 30 neurons in each optic lobe. AllAstA-Gal4-expressing neurons co-localized with AstA antibody (Figures 1b–1g, cell bodies are circled in Figures 1b–d). We saw extensive innervation of the subesophageal ganglion (SOG), protocerebrum, and pars intercerebralis (PI) (Figure 2c–2e, brain neural architecture is diagrammed in Figure 2b). In addition, at higher magnification, we observed a descending axon that arborized in the dorsal-most neuromeres of the ventral nerve cord (VNC) (Figure 2a). We were unable to differentiate and trace the axons of the three pairs of brain AstA-Gal4 neurons due to the overlap of the axon tracts and the saturation of the GFP immunostain.
In the last abdominal neuromere of the VNC, Gal4 and AstA co-localized to three pairs of cell bodies (Supplementary Figure 1 shows a higher magnification image that illustrates the number of cell bodies), which send projections dorsally (Figures 2f–2k, indicated with an arrow in Figure 2f–h). These projections innervate the lower midgut, the
hindgut, and the rectum (Figures 3a–3f). Also co-labeled were single neurons in each of two nerves that exit the VNC laterally (Figures 2h–2j, cell bodies are circled).
In the gut, less than half of the AstA-immunoreactive neuroendocrine cells located in the lower midgut expressed Gal4 (Figures 3j–3l), a few of which were only weakly immunoreactive to AstA (In these figures, AstA-immunoreactive neuroendocrine cells are surrounded by squares, Gal4-immunoreactive neuroendocrine cells by circles. Notice the number of squares without circles (AstA+/Gal4-) and the circles without squares (weak AstA+/Gal4+).
Also co-labeled was tissue on both ends of the midgut, adjacent to the proventriculus and at the midgut/hindgut transition (Figures 3a–3c, 3g–3i, indicated using red arrows). In addition, there is speckled immunolabeling that is smaller than the size of neuroendocrine cells and does not appear to be associated with motorneuron projections (Figure 3j–3l). We see this type of labeling in the upper midgut as well (Figure 3g–i). This was not due to precipitates of the fluorescent antibodies, because immunostaining with only secondary fluorescent antibodies did not create this pattern of staining. Instead, we consistently see the highest density of speckling near the midgut transitions, and a gradual decrease in density further from the transitions (Figure 3a–c). The dense and speckled staining is likely staining muscle tissue, but in the lower midgut, it could also be staining varicosities of motor neurons. In the lower midgut, there are many more AstA+/Gal4- speckles than AstA+/Gal4+ speckles (Figure 3a–c, 3j–3l), whereas in the upper midgut, we see AstA+/Gal4-, AstA+/Gal4+ (indicated by arrows), and AstA-/Gal4+ (indicated inside circles) speckles (Figure 3g–i).
Similar to what we observed in adults, AstA-Gal4 is only expressed in a subset of AstA neurons in 3rd instar wandering larvae. Immunostaining the CNS of AstA-Gal4/
UAS-nuclear-lacZ larvae with anti-B-galactosidase, reveals expression of Gal4 in five cell bodies, which all co-express AstA, as assessed by fluorescent in situ hybridization (Figure 4a–4f). The total number and general location of AstA-expressing cell bodies, as assessed by in situ hybridization, was identical to the number and location of neurons previously reported using the D. punctata antibody (Figure 4a–4f, AstA+/Gal4- cell bodies are indicated using arrows and [3]). These results support the specificity of the AstA antibody
for Drosophila AstA, and the conclusion that AstA-Gal4 is only expressed in a subset of AstA-expressing neurons.
Previous reports did not observe AstA immunoreactivity in the foregut or in the muscle tissue at the midgut transitions [7, 8]. In order to determine whether the AstA-Gal4 expression that we observed in these regions was due to auto-fluorescence, we labeled AstA-Gal4,UAS-mcd8::GFP/UAS-Ricin tissues with an antibody against GFP. Ricin promotes cell autonomous death by inhibiting protein synthesis [9]. Expression of Ricin in AstA neurons abolishes GFP immunoreactivity in the gut, indicating that Ricin ablates AstA+/Gal4+ cells and that immunolabelling witnessed in the gut is specific (Figure 4h).
In the brain and VNC, GFP immunolabelling was also abolished in AstA- Gal4,UAS-mcd8::GFP/UAS-Ricin flies (data not shown). This manipulation allowed us to observe the expression pattern of AstA+/Gal4- neurons. Interestingly, the remaining five AstA+/Gal4- neurons in the brain extensively innervate the same regions as the AstA+/Gal4+ neurons: the protocerebrum, the pars intercerebralis, and the SOG (Figure 4g). In addition, expression in the central complex is also seen.
In order to localize potential post-synaptic terminals of AstA neurons, we used UAS-ANF::GFP, which expresses rat atrial natriuretic hormone (ANF) fused to GFP [10].
This neuropeptide is packaged and shuttled similar to Drosophila neuropeptides, thus allowing us to visual neuropeptide trafficking in AstA neurons. Labeling AstA-Gal4/UAS- ANF::GFP fly brains with an antibody against GFP, revealed that potential synaptic terminals occur in the protocerebrum, pars intercerebralis, and SOG (Figure 4i).