Figure 8.

Blocking α-secretase activity increases membrane-associated APPL levels in the EP cells and inhibits their migration. (A) Examples of EP cells in cultured embryos that were treated with different secretase inhibitors and then immunolabeled with a combination of anti-nAPPL (green) and anti-cAPPL antibodies (magenta). (A₁) EP cells in a cultured control preparation. (A₂) EP cells treated with an α-secretase inhibitor showed increased levels of membrane-associated full-length APPL (white). (A₃) EP cells treated with a β-secretase inhibitor showed an increased number of cytoplasmic vesicles containing the holoprotein (arrowheads). (A₄) EP cells treated with a γ-secretase inhibitor showed an apparent increase in C-terminal fragments (magenta). (B) Quantification of the relative amount of membrane-associated APPL (black histograms) in EP cells treated with different secretase inhibitors (normalized to adjacent interband regions in each preparation). Treatment with α-secretase inhibitors caused a noticeable increase in membrane APPL that was significant in a pairwise comparison (^*p < 0.02), but not quite significant after applying the Bonferroni correction for multiple comparisons (p < 0.06). In contrast, none of the other secretase inhibitors affected the relative levels of APP, nor was the intensity of Fas II immunoreactivity altered by any of these treatments (quantified in a separate channel; gray histograms). Statistical comparisons between groups were performed using one-way ANOVA followed by unpaired Student's two-tailed t-tests with the Bonferroni correction to obtain reported p-values. N = 10 per group; histograms show means ± SD. (C) Examples of EP cell migration in cultured embryos (redrawn from camera lucida images of immunolabeled preparations). (C₁) Embryo that was fixed and immunolabeled at experimental onset (57 HPF); at this stage, the pre-migratory EP cells extended short exploratory processes onto the midgut band pathways (“b”) but avoid the adjacent interband regions (“ib”). (C₂) Control preparation that was allowed to develop in culture for 18 h; the EP cells had migrated and extended axons posteriorly along the muscle band pathways (black arrowheads). (C₃) Preparation that was treated with an α-secretase inhibitor; EP cell migration and axon outgrowth were markedly reduced compared to controls, although there was no apparent increase in ectopic migration or neuronal death. (D) Quantification of the extent of EP cell migration (black histograms) and outgrowth (gray histograms) along the midgut band pathways in cultured embryos treated with different secretase inhibitors (distances normalized to controls in each experimental group). Treatment with α-secretase inhibitors caused a significant reduction in both migration (^***p < 0.001) and outgrowth (^**p = 0.002), whereas treatment with β- and γ-secretase inhibitors had no apparent effects on these aspects of EP cell development. Migration and outgrowth distances were normalized to mean values obtained from matched control preparations in each experiment. Statistical comparisons between groups were performed using one-way ANOVA followed by unpaired Student's two-tailed t-tests with the Bonferroni correction to obtain reported p-values. N ≥ 16 per group; histograms show means ± SD. Scale bar in (A) = 5 μm; in (C) = 40 μm.