Adjacent Neuronal Fascicle Guides Motoneuron 24 Dendritic Branching and Axonal Routing Decisions through Dscam1 Signaling

Abstract

The formation and precise positioning of axons and dendrites are crucial for the development of neural circuits. Although juxtracrine signaling via cell-cell contact is known to influence these processes, the specific structures and mechanisms regulating neuronal process positioning within the central nervous system (CNS) remain to be fully identified. Our study investigates motoneuron 24 (MN24) in the Drosophila embryonic CNS, which is characterized by a complex yet stereotyped axon projection pattern, known as 'axonal routing.' In this motoneuron, the primary dendritic branches project laterally toward the midline, specifically emerging at the sites where axons turn. We observed that Scp2-positive neurons contribute to the lateral fascicle structure in the ventral nerve cord (VNC) near MN24 dendrites. Notably, the knockout of the Down syndrome cell adhesion molecule (dscam1) results in the loss of dendrites and disruption of proper axonal routing in MN24, while not affecting the formation of the fascicle structure. Through cell-type specific knockdown and rescue experiments of dscam1, we have determined that the interaction between MN24 and Scp2-positive fascicle, mediated by Dscam1, promotes the development of both dendrites and axonal routing. Our findings demonstrate that the holistic configuration of neuronal structures, such as axons and dendrites, within single motoneurons can be governed by local contact with the adjacent neuron fascicle, a novel reference structure for neural circuitry wiring.

Figure 1:. Neuronal Fascicle Spatially Aligns with the Position of MN24 Dendrite Formation

(A) Top panel shows a schematic of MN24 (black) within the ventral nerve cord of an embryo. The axon stereotypically projects out of the soma, anteriorly along the edge of the longitudinal connective (LC), and away from the midline to target muscle 24 (M24). The bottom panel shows a representative fluorescence image of a lipophilic-dye-labeled MN24. At 15:00h AEL, MN24 form their dendritic processes (magenta dots) at stereotyped positions on the axon routing. For all subsequent images, anterior is to the top, and medial is to the left. AC: Anterior commissure. PC: Posterior commissure. Scale bar, 10 μm. (B) Representative fluorescence images of FasII-positive longitudinal fascicles within the ventral nerve cord. The stereotyped most lateral FasII-positive fascicle structure (arrowhead) provides a frame of reference to characterize the mediolateral position of MN24 dendrites. Gray dashed line depicts the midline. (C) Distribution plots of the mediolateral positions of MN24 dendritic branches (white) (n = 22 neurons) and FasII-positive lateral fascicle (dark gray) (n = 77 hemisegments), where 0 μm indicates the distance from the CNS midline.

Figure 2:. Segment-specific MN24 morphologies in the Wild-Type Background

Representative images depicting the morphology of wild-type MN24 in different abdominal segments are shown. These images show the characteristic dendrites and axon routing, observed in Figure 1A. Notably, the angle of the axon segment projecting towards the muscle varies in a segment-specific manner. Axon routing area (shaded blue) is measured as the area within the loop. For “open” axon routing areas, (left and middle panels), we define the center of the cell body (purple dot) and use the perpendicular line to the soma center as the border for measurement of the axon routing area. Scale bar, 10 μm.

Figure 3:. dscam1 is Required for MN24 Neurite Development

(A) Representative fluorescence images of MN24 within wild-type (top panel) and dscam1^−/− mutant (bottom panel) backgrounds. (B and C) Comparison of mean primary dendritic branch numbers (B) and axon routing areas (C) within wild-type and dscam1^−/− mutant backgrounds; using Mann–Whitney U test. For all graphs, the sample size of neurons is denoted by the number in the parentheses of each genotype unless otherwise specified. For all subsequent statistical analyses, symbols indicate the following: ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns – not significant. (D) Immunofluorescence staining of FasII at 15:00h AEL shows the visual comparison between axon terminals in wild-type (top panel) and dscam1^−/− mutant (bottom panel) backgrounds. Representative image displaying FasII staining in the wild-type and mutant backgrounds exhibits innervation by the SNa nerve branch (open circle). However, in the dscam1^−/− mutant background, the SNa sub-branches have some mild targeting defects (white dots). (E) Quantification of SNa innervation defects within wild-type (n = 76 hemisegments) and dscam1^−/− mutant (n = 67 hemisegments) backgrounds. Data is represented as a percentage – number of hemisegments with innervation defects over the total number of hemisegments observed. SNa innervation defects were characterized as mild (light gray) or severe (dark gray) when the SNa sub-branch had targeting defects or the SNa branch did not exit the nerve cord, respectively. Scale bars, 10 μm in (A and D).

Figure 4:. Longitudinal Fascicles in Wild-Type and dscam1^−/− Mutant Backgrounds

(A) Representative fluorescence images of FasII-positive axon tracts within wild-type (top panel) and dscam1^−/− mutant (bottom panel) backgrounds. Scale bar, 10 μm. (B) Quantification of lateral fascicle defects within wild-type (n = 77 hemisegments) and dscam1^−/− mutant (n = 66 hemisegments) backgrounds. Data is represented as a percentage – length of lateral fascicle defects over total lateral fascicle length. Lateral fascicle defects were characterized as mild or severe when the lateral fascicle was thinning or contained a break, respectively.

Figure 5:. Dscam1 Plays a Cell-Autonomous Role for MN24 Neurite Development

(A) Representative fluorescence images of MN24 in wild-type background expressing hh-GAL4 driver (top panel) and dscam1 RNAi expressed under the control of the hh-GAL4 driver (bottom panel). (B and E) Comparison of mean primary dendritic branch numbers (B) and axon routing areas (E) of MN24 in wild-type background expressing hh-GAL4 driver and dscam1 RNAi expressed under the control of the hh-GAL4 driver; using Mann–Whitney U test. (C) Representative fluorescence images of MN24 in dscam1^−/− mutant background expressing hh-GAL4 driver (top panel), and dscam1^−/− mutant background resupplied dscam1 expressed under the control of the hh-GAL4 driver (bottom panel). (D and F) Comparison of mean primary dendritic branch numbers (D) and axon routing areas (F) of MN24 in wild-type background expressing hh-GAL4 driver, dscam1^−/− mutant background expressing hh-GAL4 driver, and dscam1^−/− mutant background resupplied dscam1 expressed under the control of the hh-GAL4 driver; using Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Scale bars, 10 μm in (A and C).

Figure 6:. Scp2-GAL4 Driver Allows Labeling of Lateral Fascicle

Representative images of neuronal fascicles labeled by membrane-bound GFP under the control of Scp2-GAL4 driver (green) or immunostained with anti-FasII antibody (magenta). Scp2-positive fascicles include the medial and lateral fascicles (arrowheads) and exclude the intermediate fascicle. Scale bar, 10 μm.

Figure 7:. Scp2-Positive Lateral Fascicle Provides Non-Cell-Autonomous Dscam1 for MN24 Neurite Development

(A) Representative fluorescence images of MN24 in wild-type background expressing Scp2-GAL4 driver (top panel) and dscam1 RNAi expressed under the control of the Scp2-GAL4 driver (bottom panel). (B and E) Comparison of mean primary dendritic branch numbers (B) and axon routing areas (E) of MN24 in wild-type background expressing Scp2-GAL4 driver and dscam1 RNAi expressed under the control of the Scp2-GAL4 driver; using Mann–Whitney U test. (C) Representative fluorescence images of MN24 in dscam1^−/− mutant background expressing Scp2-GAL4 driver (top panel), and dscam1^−/− mutant background resupplied dscam1 expressed under the control of the Scp2-GAL4 driver (bottom panel). (D and F) Comparison of mean primary dendritic branch numbers (D) and axon routing areas (F) of MN24 in wild-type background expressing Scp2-GAL4 driver, dscam1^−/− mutant background expressing Scp2-GAL4 driver, and dscam1^−/− mutant background resupplied dscam1 expressed under the control of the Scp2-GAL4 driver; using Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Scale bars, 10 μm in (A and C).

Figure 8:. MN24 Soma Position is Medially Shifted in the dscam1^−/− Mutant Background

(A) Representative images of MN24 at 15:00h AEL in wild-type background expressing Scp2-GAL4 driver (green) (top panel) and dscam1^−/− mutant background expressing Scp2-GAL4 driver (bottom panel). Blue and pink bars indicate the distance (μm) from the lateral fascicle and soma, respectively, to the midline. Scale bar, 10 μm. (B) Quantification of lateral fascicle position in wild-type background expressing Scp2-GAL4 driver and dscam1^−/− mutant background expressing Scp2-GAL4 driver; using Welch’s t test. The Scp2-positive lateral fascicle does not have a mediolateral shift in the dscam1^−/− mutant background. (C) Quantification of MN24 soma position in wild-type background expressing Scp2-GAL4 driver and dscam1^−/− mutant background expressing Scp2-GAL4 driver; using Welch’s t test. MN24 soma in the dscam1^−/− mutant background expressing Scp2-GAL4 driver has a more medial shift compared to that of the wild-type background.

Figure 9:. Dscam1 in Both Scp2-Positive Lateral Fascicle and MN24 is Sufficient to Restore MN24 Dendritogenesis and Axon Routing

(A) Representative fluorescence images of MN24 within wild-type background (top panel), dscam1^−/− mutant background with combined Scp2- and MN24-specific expression of membrane-bound GFP (middle panel), and dscam1^−/− mutant background with combined Scp2- and MN24-specific resupply of dscam1 (bottom panel). Scale bar, 10 μm. (B and C) Comparison of mean primary dendritic branch numbers (B) and axon routing area (C) among MN24 in wild-type background, dscam1^−/− mutant background with Scp2- and MN24-specific expression of GFP membrane-bound, and dscam1^−/− mutant background with combined Scp2- and MN24-specific resupply of dscam1; using Kruskal–Wallis test followed by Dunn’s multiple comparisons test.

Figure 10:. Resupplying dscam1 in Scp2-Positive Lateral Fascicle and MN24 Restores Mutant MN24 Soma Position

Quantification of MN24 soma positions in wild-type background with Scp2- and hh-specific expression of membrane-bound GFP, dscam1^−/− mutant background with Scp2- and hh-specific expression of membrane-bound GFP, and dscam1^−/− mutant background with combined Scp2- and hh-specific resupply of dscam1; using Kruskal–Wallis test followed by Dunn’s multiple comparisons test.

Figure 11:. Proposed Model for Fascicle-Mediated MN24 Morphogenesis

(A) Schematic illustrating the proposed model of how the lateral fascicle structure mediates MN24 dendrite outgrowth and soma migration. (B) Electron microscopy (EM) reconstruction of a single MN23/24 in 1st instar larva. Prominent morphological structures such as dendritic outgrowth and axon routing are retained in larval MN24. The backbone is indicated by gray. Blue dots indicate synaptic sites. Scale bar, 20 μm.