Specification of Dendritogenesis Site in Drosophila aCC Motoneuron by Membrane Enrichment of Pak1 through Dscam1

Abstract

Precise positioning of dendritic branches is a critical step in the establishment of neuronal circuitry. However, there is limited knowledge on how environmental cues translate into dendrite initiation or branching at a specific position. Here, through a combination of mutation, RNAi, and imaging experiments, we found that a Dscam-Dock-Pak1 hierarchical interaction defines the stereotypical dendrite growth site in the Drosophila aCC motoneuron. This interaction localizes the Cdc42 effector Pak1 to the plasma membrane at the dendrite initiation site before the activation of Cdc42. Ectopic expression of membrane-anchored Pak1 overrides this spatial specification of dendritogenesis, confirming its function in guiding Cdc42 signaling. We further discovered that Dscam1 localization in aCC occurs through an inter-neuronal contact that involves Dscam1 in the partner MP1 neuron. These findings elucidate a mechanism by which Dscam1 controls neuronal morphogenesis through spatial regulation of Cdc42 signaling and, subsequently, cytoskeletal remodeling.

Figure 1. GFP::Cdc42^V12 accumulation spatially corresponds with the aCC dendritogenesis site

(A) In each half-segment of an embryo, the aCC motoneurons begin to develop their dendrites at a stereotyped position within the CNS where its axon intersects with the longitudinal connective. (B) The distribution FWHMs for Cdc42 activation, GFP::Cdc42^V12 accumulation as well as for the primary dendritic branches’ positions. The sample size (n) is the number of aCCs examined in abdominal segments from A2 to A5. (C) Representative images of lipophilic-dye-labeled aCC aligned to the CNS midline (dashed line) from 11:00 to 15:00 in a wild-type strain. The distribution of primary dendritic branches is also plotted (bottom). (D) The mean number of dendritic tips from 11:00 to 15:00. (E) GFP::Cdc42^V12 is localized at the base of the primary branches (inset). The mean of relative GFP::Cdc42^V12 fluorescence at 10 to 30 μm from the midline is shown. Their concentrations are normalized to the average concentrations in the aCC cell body. GFP::Cdc42 localization at 15:00 was used as a control. (F) Quantification of the GFP::Cdc42^V12 amount at 10 to 30 μm from the midline at the indicated hours versus at 15:00. Error bars: SEM. See also Figure S1

Figure 2. Pak1 is required for GFPCdc42^V12 accumulation as well as aCC dendritogenesis

(A) Images of GFP::Cdc42^V12 and GFP::Cdc42^V12C40 localization in aCC at 15:00. As opposed to GFP::Cdc42^V12, GFP::Cdc42^V12C40 failed to accumulate at the aCC dendritogenesis site. We quantified the amount of GFP::Cdc42^V12 with dsRNA injection. Compared to sham-operated control (an empty vector injection), GFP::Cdc42^V12 accumulation at the site is reduced (asterisk, p < 0.005 by two-tailed t-test). (B) Expression of RNAi constructs or injection of mixed RNAi constructs caused significant reduction of the number of dendritic tips in the aCC at 15:00 (asterisk, p < 0.001 compared with eve’-GAL4/+ (control) alone by two-tailed t-test). (C) Loss-of-function mutants of pak1−/− reduce dendritic tip number in aCC. The phenotype is rescued by resupplying wild-type pak1 gene to aCC (asterisk, p < 0.005 compared with the one in a wild-type background at 15:00 by two-tailed t-test). (D) Representative images of Pak1 localization in aCC at 11:00 and 15:00. Pak1 is pre-localized where the dendrites normally start to sprout at 11:00. Pak1 localization at 11:00 was not affected even in cdc42−/− mutants. Bottom: Quantification of the Pak1 amount at 10 to 30 μm from the midline in the indicated hours and genotypes versus the one at 15:00. Error bars: SEM. See also Figure S2.

Figure 3. Pak1 is enriched at the ventral membrane of the aCC dendritogenesis site

(A) 3D SIM of aCC expressing membrane marker, its cross-sectional view, and averaged, normalized slices along the Anterior-Posterior axis (A-P) and Dorsal-Ventral axis (D-V) (shades represent standard deviations, n = 8). The membrane marker is stained with both Alexa Fluor 488 (green) and Alexa Fluor 555 (magenta). Full colocalization in the cross sections demonstrates our capability to precisely align 2-color SIM images. (B) 3D SIM of Pak1 prior to aCC dendritogenesis at 11:00 (n = 10), showing cross-sectional views at 11μm, 13 μm and 15 μm from the midline. Pak1 accumulates at the ventral-proximal half of the juxta-membrane at 13 μm, but not at 11 or 15 μm. The two separated color channels were also shown in Figure S2D. (C) 3D SIM of aCC dendrites and its cross-sectional view at 13 μm from the midline at 15:00 in wild-type embryos. Radial distribution of primary dendrites, heavily biased to the ventral side, is measured from the center of the axonal cross section to the base of primary dendrite branches.

Figure 4. Membrane-tethering of Pak1 determines the position of dendrite outgrowth

(A) Compared with eve’-GAL4/+ (control), the Pak1 mutations caused irregular distribution of primary dendritic branches in aCC (the representative images of aCC in a wild-type and a pak1−/− mutant background are shown in Figure 1B and the Figure 2C, respectively). Although continuous over-expression of pak1 induces no change from the control, myristoylated Pak1 (pak1^myr) expression induces expansion of dendrite outgrowth region with normal dendritogenesis timing. (B) Summary of the distribution FWHMs for Cdc42 activation, GFP::Cdc42^V12 accumulation and Pak1 accumulation, as well as for the dendrite positions in a wild-type, a pak1 overexpression, a membrane tethered pak1^myr overexpression and a pak1−/− mutant background. Error bars: SEM.

Figure 5. Dscam1 and Dock are localized at the aCC dendritogenesis site and are important for aCC dendrite outgrowth

(A) Images of aCC in indicated genotypes at 15:00. We also quantified the positions of primary dendritic branches, the numbers of dendritic tips and the distribution FWHMs for the primary dendritic position in indicated genotypes at 15:00 (asterisk, p<0.005 by one-way ANOVA). (B-C) Images of Dscam1 and Dock distributions in aCC. Before aCC dendritogenesis starts at 11:00, Dock (B) and Dscam1 (C) are pre-localized at the aCC dendritogenesis site. The amounts of Dock and Dscam1 at 10 to 30 μm from the midline were also quantified from 11:00 to 15:00 and were normalized to the ones at 15:00. (D) Comparison of the distribution FWHMs for Dscam1, Dock and Pak1. Error bars: SEM. See also Figure S4 and S5.

Figure 6. Dscam1 via Dock localizes Pak1 at aCC dendritogenesis site

(A) Images and quantifications of Dscam1, Dock and Pak1 localized at the aCC dendritogenesis site in various mutant and knock-down backgrounds (asterisk, p<0.005 compared with their amounts in a wild-type background at 15:00 by two-tailed t-test). (B-C) Images of aCC in indicated genotypes at 15:00. We also quantified the positions of primary dendritic branches, the numbers of dendritic tips and the distribution FWHMs for the primary dendritic position in indicated genotypes at 15:00 (asterisk, p<0.005 by one-way ANOVA). Error bars: SEM. Control data (eve’-GAL4/+) from Figure 2B and 4A are re-plotted. See also Figure S6.

Figure 7. Dscam1 in MP1 neuron is necessary and sufficient in signaling to Dscam1 in aCC neuron during aCC dendritogenesis

(A) Morphology of aCC (green) and neurons expressing membrane-bound mCherry (magenta) under the control of an R23E04-GAL4 driver. mCherry-positive neurons appear in a patchy fashion in a small subset of neurons in the CNS. Among these neurons, MP1 has an axon positioned at close apposition of the aCC dendritogenesis site. MP1 projects its axon and crosses at the ventral side of aCC where normally dendrites emerge (cross-section). The position of MP1 axon at the crossing site as well as the MP1-aCC center-to-center distance was unaffected by dscam1 RNAi in MP1. (B) Correlation between presence of dscam1-RNAi-expressing MP1 and the loss of dendritic processes in the aCC. The dscam1-RNAi-expressing MP1 has been labeled by mCherry (magenta) within the CNS of otherwise non-labeled and R23E04-GAL4/+ animals. (C) Rescue of Dscam1 mutant phenotype by resupplying dscam1 to both MP1 and aCC. The dscam1-expressing MP1 was labeled by mCherry (magenta). We quantified the positions of primary dendritic branches, the numbers of dendritic tips and the distribution FWHMs for the primary dendritic position in indicated genotypes at 15:00. n indicates the number of aCC analyzed in abdominal segments from A2 to A5. Error bars: SEM. See also Figure S7.

Figure 8. Dscam1 in MP1 is responsible for the localization of Dscam1 in aCC

(A) Representative images of Dscam1 localized in MP1. The fluorescence intensity profiles were aligned and averaged at the aCC-MP1 intersection (dashed line). (B) Representative images of Dscam1 in aCC in dscam1 RNAi driven by the R23E04-GAL4 driver. (C) Quantification of Dscam1 accumulation in aCC (purple) displays a bimodal distribution, with the two populations matching that of the no RNAi control and aCC dscam1 RNAi (black), respectively. n indicates the number of aCC analyzed in abdominal segments from A2 to A5. Error bars: SEM. (D) Model for the spatial-temporal control of aCC dendritogenesis.