Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons

Abstract

Little has been understood about the underlying mechanisms that generate the morphological diversity of dendritic trees. Dendritic arborization neurons in Drosophila provide an excellent model system to tackle this question, and they are classified into classes I-IV in order of increasing arbor complexity. Here we have developed transgenic green fluorescent protein markers for class I or class IV cells, which allowed time-lapse recordings of dendritic birth in the embryo, its maturation processes in the larva, and lesion-induced reactions. The two classes used distinct strategies of dendritic emergence from the cell body and branching, which contributed to differences in their basic arbor patterns. In contrast to the class I cells examined, one cell of class IV, which was a focus in this study, continued to elaborate branches throughout larval stages, and it was much more capable of responding to the severing of branches. We also investigated the cellular basis of field formation between adjacent class IV cells. Our results support the fact that class-specific inhibitory interaction is necessary and sufficient for tiling and confirmed that this intercellular communication was at work at individual dendrodendritic interfaces. Finally, this inhibitory signaling appeared to play a central role when arbors of adjacent cells started meeting midway between the cells and until the body wall became partitioned into abutting, minimal-overlapping territories.

Fig. 1.

Markers for DA neurons. A–D, Multiple dendrite neurons in the dorsal cluster in theDrosophila embryo just before hatching. In this and all subsequent figures, anterior is to the left and dorsal is at the top. Data of other clusters are not shown. A,B, A pan da marker. An image ofVenus-pm/+; IG1–2/+ (A) and schematic representation of cell bodies of ddaA-ddaF and dorsal bipolar dendrite (dbd) neuron (B). C, ddaD and ddaE are highlighted inNP2225/mCD8:: GFP.D, Among DA neurons in the dorsal cluster, only ddaC is reproducibly visualized in NP7028 GFP[S65T]/GFP[S65T]. E, Larva of 45.5–47.5 hr after egg laying (AEL). ddaE is strongly labeled in homozygotes of IG1–1 GFP[S65T] mCD8:: GFP.F, A diagram of the dorsal cluster in a hemisegment, showing a spatial relationship between dendritic arbors of ddaB-E and epidermal denticles (black symbols). For simplicity, cell bodies are illustrated as if they were not clustered. Branches of ddaD and those of ddaE reach the anterior three rows and the most posterior row of denticles, respectively. The space between the most posterior row of a hemisegment and the most anterior one in a posterior neighbor is referred to the neutral zone, where ddaB and C are innervated. Scale bars: A–D, 20 μm; E, 50 μm.

Fig. 2.

Dendritic emergence and branching in ddaC, ddaD, and ddaE in the embryo. A, B, ddaD and ddaE in NP2225/mCD8:: GFP.A, Images 20 min apart are shown of 10 min time-lapse recordings of an embryo at 13–14 hr AEL. Two primary branches are indicated with single and double arrowheads, and one of them was partially in focus in this series (single arrowhead). B, Time-lapse series of another embryo at 15–16 hr AEL show dynamic behavior of lateral branches. C, 10 min time-lapse recordings of ddaC in NP7028 GFP[S65T]/GFP[S65T] (15.5–16 hr AEL) for a total of 140 min and seven images were shown. Bifurcation of a dendritic end is indicated (arrow). Scale bar: A–C, 10 μm.

Fig. 3.

Formation of higher-order branches in ddaC, ddaD, and ddaE in the larva. A, B, ddaD and ddaE in NP2225/mCD8:: GFP.C–F, ddaC in NP7028 GFP[S65T]/GFP[S65T]. Indicated are formation or elongation of lateral branches (double arrowheads), pruning or retraction of branches (arrows), and a dendrodendritic contact (single arrowhead in D). A, B, Two images 15 hr apart starting at 24–25 hr AEL. C–E, Three time-lapse images taken at 23–24 hr AEL, 39.5–40.5 hr AEL, and 63.5–64.5 hr AEL, respectively. E, Dendritic tiling between ddaC and its contralateral homolog and between ddaC and v′ada in the ventral prime cluster are indicated (top bracket and bottom brackets, respectively). F, 15 min recordings of dendritic terminals in a third-instar larva. Scale bar:A–C, F, 20 μm; D, E, 50 μm.

Fig. 4.

Responses of ddaC and ddaE to severing branches.A, B, NP7028 GFP[S65T]/GFP[S65T]. C,D, IG1–1 GFP[S65T] mCD8:: GFP/+. Dendrite shafts (arrows) proximal to cell bodies of ddaC (A, 40–42 hr AEL) and of ddaE (C, 20–22 hr AEL) were severed and observed 2 d later (B and D, respectively). In response to severing of the branches, other isoneuronal branches of ddaC filled extensively in the voided field (arrowheads in A and B and bracket in B), but those of ddaE did not (arrowheads inC and D). In B, another DA neuron, perhaps ddaB, was also weakly labeled. A terminal of posterior adjacent ddaC was indicated (double arrowheads in B).D, ddaE before the severing was shown with the same magnification (inset). Scale bar: A, 50 μm; B, 100 μm; C, D, 20 μm.

Fig. 5.

Observation of dendritic terminals of ddaC and coimaging with muscles. A, B, Images of two larvae of NP7028 GFP[S65T]/GFP[S65T] at 26–29 and 51–53 hr AEL, respectively. Close-up views of dendrodendritic contacts between adjacent ddaC neurons. In the neutral zone (bracket in B), terminals were closely apposed or turned away before crossing each other (arrow) and sometimes it was difficult to identify dendritic tips (arrowheads). C, D,Venus-pm/+; NP7028 GFP[S65T]/MHC-CD8-GFP-Sh in a third instar larva. A muscle attachment site in C (box) was magnified inD. Dendrites passed through splits of muscle bundles (D). Scale bars: A, 20 μm;B–D, 50 μm.

Fig. 6.

Ablation-induced ingrowth of adjacent ddaC.A, ddaC was ablated at 20 hr AEL in the embryo ofNP7028 GFP[S65T]/GFP[S65T] and observed 2 d later. Dendrites of adjacent ddaCs broke through the neutral zones (brackets) and invaded the area that was normally covered by the ablated homolog (arrows). Arrowhead points at an unknown cell.B, C, Positions of dendritic terminals of ddaC were plotted in illustrations of the hemisegment. Each dot represents a single terminal. B, Positions of terminals in the control larva. Orange dots indicate dendritic ends of ddaC in the middle (circle). Violet dots and green dots indicate ends of an anterior adjacent ddaC and a posterior adjacent ddaC, respectively.C, Positions of terminals of adjacent ddaCs when ddaC in the middle (circle with a broken line) was ablated. Violet dots on the left and green ones on the right are as described in B.D, All of the DA neurons in the dorsal cluster except for ddaC were ablated in embryos homozygous for 109(2)80 GFP[S65T] at 16–17 hr AEL, and observed 2 d later. In the middle hemisegment, only ddaC was left. Brackets indicate the neutral zones and dendrites of adjacent hemisegments did not transgress the borders. Scale bars: A, D, 50 μm.

Fig. 7.

Loss of one of the two ddaC terminals, coming close to each other, triggered ingrowth of the other one.A–C, Dorsal clusters in larvae of NP7028 GFP[S65T]/GFP[S65T] just after hatching (20–25 hr AEL). Each green arrow indicates a dendrodendritic contact (A) or a pair of terminals that were getting close to each other (B, C). Yellow arrows represent proximal dendritic roots that were severed.D–F, Images of 16–20 hr after severing of the roots as pointed in A–C, respectively. G–I, Tracings of dendrites of injured neurons (black), those of ipsilateral homologs (green), and those of contralateral ones (purple). The remaining terminals of either ipsilateral or contralateral homologs invaded territories that were normally covered by the detached branches (yellow arrowheads in D, E, G, andH and brackets in F and I). Isoneuronal branches in A and B filled in voided fields as shown in D and E, respectively (blue arrowheads), and some of them covered bounds of the neutral zones (purple arrowheads in D and F). Brackets inD and E indicate the neutral zones. At 31–33 hr AEL between B and E, we confirmed that the branch, which was targeted at 23–25 hr AEL (yellow arrow inB), was actually destroyed (data not shown). Scale bars:A–C, 20 μm; D–F, 50 μm.