Homophilic Dscam interactions control complex dendrite morphogenesis

Abstract

Alternative splicing of the Drosophila gene Dscam results in up to 38,016 different receptor isoforms proposed to interact by isoform-specific homophilic binding. We report that Dscam controls cell-intrinsic aspects of dendrite guidance in all four classes of dendrite arborization (da) neurons. Loss of Dscam in single neurons causes a strong increase in self-crossing. Restriction of dendritic fields of neighboring class III neurons appeared intact in mutant neurons, suggesting that dendritic self-avoidance, but not heteroneuronal tiling, may depend on Dscam. Overexpression of the same Dscam isoforms in two da neurons with overlapping dendritic fields forced a spatial segregation of the two fields, supporting the model that dendritic branches of da neurons use isoform-specific homophilic interactions to ensure minimal overlap. Homophilic binding of the highly diverse extracellular domains of Dscam may therefore limit the use of the same "core" repulsion mechanism to cell-intrinsic interactions without interfering with heteroneuronal interactions.

Figure 1. Dscam loss-of function-results in excessive iso-neuronal self-crossing in Class I neurons

(A) Schematic representation of the receptive fields of two class I neurons, ddaD and ddaE. (B-G) MARCM was used to generate wildtype (B, E) and Dscam-null (C-D, F-G) single-cell clones of class I neurons (scale bar, 50μm). (H-K) Ig1-1 Gal4 was used to visualize ddaE neurons in 2^nd instar larvae. (H) Dscam-null larvae (Dscam²¹/Dscam³³) have disorganized dendrites compared to wildtype (I). This phenotype is rescued by the expression of either Dscam^1.30.30.1 (J) or Dscam^11.31.25.1 (K) in an otherwise Dscam-null background (scale bar, 10μm). (L) A single Dscam-null ddaE neuron was selected for quantitative analysis (scale bar, 20μm). (I, J) A 5X zoom of the region of interest marked in panel (M) was used to quantify the number of iso-neuronal self-crossings in both a confocal stack (N) and a single z-section (J) (scale bar, 4μm). Arrows indicate points of self-crossing between sister-branches.

Figure 2. Quantification of Dscam’s null phenotype in ddaE neurons

(A) The average number of iso-neuronal self-crossings for wildtype, Dscam²¹ and Dscam³³ is graphed (P <0.001). (B) The probability distribution of each genotype is plotted as a histogram. (Wildtype N= 21, Dscam²¹ N = 14, Dscam³³ N = 9). Neither the number of dendritic termini per cell (C) nor the total dendritic area (D) was significantly changed by the loss of Dscam, indicating further that Dscam does not significantly alter the cell-fate determination of ‘da’ neurons.

Figure 3. Dscam loss-of-function results in excessive iso-neuronal self-crossing in Class II neurons

(A) Schematic representation of the receptive field of ddaB, the only Class II neuron within the dorsal cluster. (B-D) MARCM was used to generate wildtype (B) and Dscam-null (C,D) single cell clones of ddaB neurons (scale bar, 20μm). Arrows mark the cell bodies of ddaB neurons. The area of ddaB receptive fields was unchanged by Dscam loss-of-function (E) although there was a modest increase in the total number of dendritic termini (P < 0.05) (F). Similar to Class I neurons, the frequency of iso-neuronal self-crossing was dramatically increased in Dscam-null neurons (P < 0.001) (G). (Wildtype N = 8, Dscam-null N = 7).

Figure 4. Dscam loss-of-function results in excessive iso-neuronal self-crossing in Class III neurons

(A-F) MARCM was used to generate wildtype (A, B) and Dscam-null (C-F) single-cell clones of class III neurons. Arrows mark the cell bodies of ddaA and ddaF neurons. Neither the area of the receptive field of ddaF neurons (G) nor the dorsal length of ddaF neurons (H) was noticeably altered by Dscam loss-of-function. (Wildtype N = 7, Dscam-null N = 7).

Figure 5. Loss of exon 4 diversity does not significantly disrupt the morphology of Class I neurons

(A-B) C161-Gal4 was used to drive expression of CD8-GFP in ddaE neurons in wildtype (A) and Dscam^C22-1 (B) larvae. ddaE neurons were traced in red in photoshop to aid visualization. Loss of exon 4 diversity in Dscam^C22-1 larvae had no noticeable effect on the frequency of iso-neuronal self-crossing (C) the number of dendritic termini (D) or the overall area of ddaE receptive fields (E). (Wildtype N = 10, Dscam^C22-1 N = 10).

Figure 6. Dscam loss-of-function does not disrupt hetero-neuronal tiling between neighboring Class III neurons

(A, B) MARCM was used to generate labeled clones of Class III neurons. In a minority of larvae, two neighboring Class III neurons are labeled, permitting the analysis of hetero-neuronal tiling. In (A) two wildtype Class III neurons strictly avoid each other’s receptive field. Similarly, in Dscam-null neurons (B), the individual dendrites generally respect the boundary of the neighboring cell’s receptive field, although iso-neuronal avoidance is severely disrupted (scale bar, 50μm). Arrows mark the cell bodies of Class III neurons. (C,-F) Traces of the dendritic arborizations of wildtype (C) and Dscam-null (D-F) neurons; in these examples, the neuron projecting from the dorsal side is labeled in red, the ventral neurons are labeled in blue.

Figure 7. Mis-expression of a single isoform of Dscam in neighboring Class I and Class III neurons causes inappropriate repulsion between dendrites

(A-F) C161-Gal4 was used to drive expression of CD8-GFP in Class I (ddaE) and Class III neurons (ddaF, ddaA). (A, C, D) Class I and Class III neurons overlap extensively in wildtype animals (marked by white arrowheads). (B, E, F) In contrast, mis-expression of a single isoform of Dscam (1.30.30.1 or 11.31.25.1) prohibits Class I and Class III neurons from sharing the same region of the hemi-segment (scale bar A, B - 20μm, C, D - 10μm). These data are quantified in panel (G) (P<0.01) (Wildtype N=7, Dscam mis-expression 1.30.30.1, N=5, Dscam mis-expression 11.31.25.1, N = 12). In A-B, ddaE neurons have been pseudocolored and traced in red.

Figure 8. Dscam does not signal through Pak in Class I dendrites

(A-B, D-E) IG1-1 Gal4 was used to drive expression of CD8-GFP in ddaE neurons. (A-B) The morphology of Class I neurons from 2^nd instar larvae was examined in wildtype (A) and Pak-null (B) animals (scale bar, 20μm). (C) Loss of Pak had no significant effect on either iso-neuronal self-crossing or the complexity of dendritic arborizations (Wildtype, N = 12, Pak-null, N=13). Over-expression of myristoylated Pak (E) caused excessive branching of ddaE neurons compared to wildtype cells (D) (scale bar, 20μm), precisely opposite the phenotype of mis-expressing Dscam (Figures 3, 4). These data are quantified in panel (F) (P<0.001) (Wildtype N=48, UAS-Pak N=20).