Fig. 5: MooSEZ remodeling is mediated by ecdysteroid signaling and required for adult backward walking.
From: A new system for studying neuronal remodeling and its relation to behavior in Drosophila
A–F” Top and middle, expression pattern of MooSEZ expressing CsChrimson.mVenus and EcRDN-B1(W650A) at different developmental time points along pupal development (A–F’). MooSEZ-specific split GAL4 driver line (VT020742-p65.ADՈR60F09-GAL4.DBD) was used to drive UAS-CsChrimson.mVenus and UAS-EcRDN-B1(W650A). Maximum intensity projections of 30–45 confocal sections (1 µm) through anterior (top) and posterior (middle) coronal stacks are presented. Purple arrows indicate VNC dendritic processes undergoing pruning. Scale bar applies to all images. Expression of a dominant negative form of the ecdysone receptor B1 (W650A) in MooSEZ does not eliminate neurite pruning. Nevertheless, the pruning phase in which MooSEZ undergoes dendritic fragmentation is extended compared to wt genotype. Bottom, schematic illustration of MooSEZ morphology at different pupal developmental time points (A”–F”) as presented above. G-K’ Top, expression pattern of adult MooSEZ expressing CsChrimson.mVenus (G) and either EcRDN-B1(W650A) (H), EcRDN-A(W650A) (I), EcRDN-B1(F645A) (J), or EcRDN-A(F645A) (K). MooSEZ-specific split GAL4 driver line (VT020742-p65.ADՈR60F09-GAL4.DBD) was used to drive UAS-CsChrimson.mVenus and one of the UAS-EcR dominant negative constructs (when required) as designated. Maximum intensity projections of 65-100 confocal sections (1 µm) through the central brain are presented. Bottom, three-dimensional reconstructions of MooSEZ morphologies (G’-K’) obtained by tracing MooSEZ neurites in the confocal stacks used for constructing the above projection images. Size scale bar in (G) applies to all projection images (G-K) and three-dimensional reconstructions (G’-K’). Cyan gradient scale bar in (G’) applies to all three-dimensional reconstructions (G’-K’). L–N Analysis of the following quantitative metrics of adult morphology: cable length (L), number of branch points (M) and convex hull size (N) for MooSEZ expressing CsChrimson.mVenus in the absence or presence of either EcRDN-B1(W650A), EcRDN-A(W650A), EcRDN-B1(F645A), or EcRDN-A(F645A). MooSEZ-specific split GAL4 driver line (VT020742-p65.ADՈR60F09-GAL4.DBD) was used to drive UAS-CsChrimson.mVenus and one of the UAS-EcR dominant negative constructs (when required) as designated. MooSEZ displays severe outgrowth defects following expression of either of EcR dominant negative variants (n for each genotype = 10, *p < 0.05, **p < 0.01, ****p < 0.0001 for pairwise comparisons of wt with each experimental group, Kruskal-Wallis test followed by Dunn’s post-hoc tests). O Left, translational velocity ± SEM (shading) of adult flies expressing CsChrimson in the absence or presence of either EcRDN-B1(W650A), EcRDN-A(W650A), EcRDN-B1(F645A), or EcRDN-A(F645A) in MooSEZ following optogenetic activation. MooSEZ-specific split GAL4 driver line (VT020742-p65.ADՈR60F09-GAL4.DBD) was used to drive UAS-CsChrimson and one of the UAS-EcR dominant negative constructs (when required) as designated. The 2-s light pulse is labeled by light red. Expressing either of the EcR dominant negative variants abolishes MooSEZ-mediated adult backward walking. Right, mean translational velocity during the 2-s light pulse obtained from traces on the left. Manipulating ecdysone signaling by disrupting both activation and de-repression (EcRDN-B1(W650A) and EcRDN-A(W650A)), or only activation (EcRDN-B1(F645A) and EcRDN-A(F645A)) of ecdysone downstream genes eliminates MooSEZ-induced adult backward walking (n for VT020742-p65.AD&R60F09-GAL4.DBD > UAS-CsChrimson (II), VT020742-p65.AD&R60F09-GAL4.DBD > UAS-CsChrimson (III), VT020742-p65.AD&R60F09-GAL4.DBD > UAS-CsChrimson (II) UAS-EcRDN-B1(W650A), VT020742-p65.AD&R60F09-GAL4.DBD > UAS-CsChrimson (III) UAS-EcRDN-B1(F645A), VT020742-p65.AD&R60F09-GAL4.DBD > UAS-CsChrimson (III) UAS-EcRDN-A(W650A), VT020742-p65.AD&R60F09-GAL4.DBD > UAS-CsChrimson (III) UAS-EcRDN-A(F645A): 25, 33, 34, 33, 31, 29 respectively, ***p < 0.001, ****p < 0.0001 for pairwise comparisons of experimental groups with respective controls lacking EcRDN expression in MooSEZ, Kruskal-Wallis test followed by Dunn’s post-hoc tests). P Mean translational velocity following optogenetic activation (larvae, 9-s; adults, 2-s) of single larvae and their matched adult flies expressing CsChrimson in the presence (blue) or absence (brown) of EcRDN-B1(W650A) in MooSEZ. MooSEZ-specific split GAL4 driver line (VT020742-p65.ADՈR60F09-GAL4.DBD) was used to drive UAS-CsChrimson and UAS-EcRDN-B1(W650A) dominant negative construct (When required). Individual larvae and adult flies are matched by their identity. Backward crawling larvae expressing EcRDN-B1(W650A) do not persist into backward walking adult flies upon optogenetic activation of MooSEZ (n for each genotype (larva or adult) = 16, **p < 0.01, ****p < 0.0001 for main effect for genotype, and genotype X developmental stage interaction effect, respectively, repeated measures two-way ANOVA). For details of statistical analysis, see Supplementary Data 1.
