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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02666

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Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology

Wesley B. Grueber^1,*,, Bing Ye¹, Chung-Hui Yang¹, Susan Younger², Kelly Borden², Lily Y. Jan^1,2 and Yuh-Nung Jan^1,2,

¹ Departments of Physiology and Biochemistry, University of California, San Francisco, Rock Hall, Room GD481, 1550 4th Street, San Francisco, CA 94143, USA.
² Howard Hughes Medical Institute, University of California, San Francisco, Rock Hall, Room GD481, 1550 4th Street, San Francisco, CA 94143, USA.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Sensory axon morphology is correlated with peripheral dendritic morphology. (A) Schematic representation of the abdominal PNS (magenta box) and sensory axon projections to the CNS (green box). Sensory neurons are arranged in dorsal, lateral, ventral' and ventral clusters. Segments A2-A6 are shown. (B) A representative dorsal view of the ventral nerve cord with sensory axons (large arrowheads) and a few central neurons (small arrowheads) labeled by the MARCM system (left, green box). Schematic of the PNS sensory neurons with dendritic arborization (da) neurons indicated by diamonds (right, magenta box). Classes of each neuron are indicated by color key: class I, green; class II, yellow; class III, tan; class IV, blue. (C) Top: dendritic morphologies of the four distinct classes of da neurons. Class I and II neurons have simple dendrites, with the class I dendrites directed towards the segment border. Class III neurons have numerous short dendritic protrusions extending from their main trunks. Class IV neurons have extensive and highly branched dendrites. Below: individual axon morphologies of the different classes of da neurons. Class II neurons each show collateral branches (arrowheads). Class III neurons (three of five are shown here) have simple axons without collateral branches. Dorsal and ventral class IV neurons cross the midline, and showed elaborate tufts of branches where the axon meets its anteroposterior fascicle. Arrows mark the location of axons relative to the CNS midline. Genotype: tubP-Gal80, hsFLP, FRT19A/yw FRT19A; Gal4109(2)80, UAS-mCD8::GFP/+. Scale bars: 25 µm (B, axons in C). d, dorsal; l, lateral; v, ventral; v', ventral'.

View larger version (94K): [in this window] [in a new window]   Fig. 2. Dorsoventral distinctions in axon position. (A) Transverse section of a wild-type ventral nerve cord labeled with Fas II antibody. Fascicle nomenclature according to Landgraf et al. (Landgraf et al., 2003). Dorsal is up in this and all other transverse sections. (B-E) FLP-out analysis of da neuron axon position along the DV axis. Top are dorsal views, bottom are transverse sections. Sections correspond to the location of the yellow lines. Note the position of the dbd terminal next to the DM fascicle (arrowheads in D and E). (B) The terminations of vpda and ddaE, both class I neurons, are likewise immediately next to the DM fascicle. (C) The class II neuron ddaB terminates in ventral neuropil next to VMv (arrow) and forms a collateral branch at VL (arrowhead). (D) The class III neuron v'pda terminates ventrally, adjacent to VMv (arrows). Notice that the dbd neuron next to DM (arrowheads). (E) The class IV neuron, vdaB, terminates ventrally, immediately next to VMv (arrows). (F) Schematic drawing of one half of a CNS section as in A, showing the positions of major fascicles and the approximate locations of da axons (green). Arrow indicates distinct lateral branch of class II neurons. Arrowheads indicate the midline. Genotypes: hsFLP/+; Gal4109(2)80/+; UAS>CD2>mCD8::GFP/+. Scale bar: 25 µm. C, central; D, dorsal; I, intermediate; L, lateral; M, median; V, ventral.

View larger version (68K): [in this window] [in a new window]   Fig. 3. Axons of different classes of da neurons form a laminar pattern. (A) Dorsal view of axon scaffold in ppk-Gal4, UAS-mCD8::GFP ventral nerve cord. A strongly labeled set of axons (arrow in transverse section `1') is flanked by a weakly labeled set of terminals (arrowheads). Transverse section `2' shows the thickened clumps of axons at each segment. (B) FLP-out analysis shows that class III axons (arrowheads) terminate immediately lateral to the scaffold of class IV axons (arrows). Top is dorsal view, bottom is transverse section. Notice that some class IV axons show expression of mCD8::GFP due to FLP-out and, thus, the left portion of the CNS contains marked class III and IV neurons. (C) Image of CNS labeled with mCD8 (green) and Fas II (magenta). Section (yellow) shows the class III axon of v'pda (arrowhead) terminating just medial to the class II axon of ldaA (arrow). (D) FLP-out clones made with ppk-eGFP in the background to label class IV axons. Arrows indicate the class II axon. The ppk-eGFP scaffold is marked (IV). Notice that the class II axon terminates lateral to the class IV scaffold (arrowhead) and all other da axons (which are labeled by CD2). (E) Relative terminal positions of class II, III and IV neurons. The cell body position is indicated along with the class of the neuron. Genotypes: (B) hsFLP/+; ppk-Gal4/ppk-Gal4; UAS>CD2>mCD8::GFP/+; (C) hsFLP/+; Gal4109(2)80/+; UAS>CD2>mCD8::GFP/+. (D) hsFLP/+; Gal4109(2)80/+; UAS>CD2>mCD8::GFP/ppk-eGFP; (E) tubP-Gal80, hsFLP, FRT19A/yw FRT19A; Gal4109(2)80, UAS-mCD8::GFP/+. Scale bars: 25 µm. D, dorsal; L, lateral; V, ventral; V', ventral'.

View larger version (112K): [in this window] [in a new window]   Fig. 4. Embryonic origin of laminar pattern. (A,A') Expression of ppk-eGFP in larval class IV sensory cell bodies, dendrites and axons, and expression of photoconverted Kaede protein under the control of Gal4109(2)80 to label all da neurons. Labeled oenocytes are encircled. (B) Dorsal view of stage 17 embryonic scaffold of class IV axons (arrowhead) flanked on both sides by other da axons expressing red Kaede protein (arrows). (C) The laminar pattern of class IV neurons (arrowhead) and other da neuron (arrow) in early larval stages. Scale bars: 50 µm in A,A'; 10 µm in B,C.

View larger version (66K): [in this window] [in a new window]   Fig. 5. Study of Robo family members in sensory axon patterning. (A) Dorsal view of ppk-eGFP pattern in robo, robo2 and robo3 mutants show abnormal commissure formation or longitudinal branching. (B) Wild-type class IV axon scaffold in dorsal view (left) and Robo3 misexpression under the control of the pan-da neuron driver Gal4109(2)80 (right). Robo3 induces ectopic collateral branch formation at the VL fascicle (arrows) and loss of some midline crossing by class IV neurons. Neurons are visualized using ppk-eGFP.(C) Robo2 misexpression in single neurons using MARCM does not cause disruptions in axon terminal layering or axon morphology (left panels). Shown are two class III neurons that terminate immediately next to the class IV axon scaffold, and two class II neurons that are separated from the class IV scaffold by a narrow space (arrows point to the collateral branches of the class II neurons). Robo3 misexpression causes defects in axon morphology in class II, III and IV neurons (right panels). The class II neuron ldaA truncates its arbor before reaching its usual position (arrow). Class III and class IV neurons show ectopic collateral branching (small arrowheads). The class IV neuron v'ada reaches a medial position comparable to wild-type class IV axons, but projects laterally in the next anterior segment when forced to express Robo3 (large arrowhead). Genotypes: (B) yw;Gal4109(2)80/+ or Gal4109(2)80/UAS-robo3; ppk-eGFP/+; (C) yw/hsFLP; UAS-robo2 or robo3/Gal4109(2)80, UAS-mCD8::GFP; ppk-EGFP th st FRT2A/tubP-Gal80, FRT2A. Scale bars: 25 µm in B,C.

View larger version (76K): [in this window] [in a new window]   Fig. 6. A mutagenesis screen identifies sensory axon and dendrite arborization phenotypes. (A) Wild-type class IV embryonic axon scaffold visualized by ppk-eGFP. (B-D) Defects in longitudinal branch formation shown by lines isolated from the screen. Arrowheads in B-D indicate missing longitudinal tracts. (E) l(3)2332 mutants show loss of most commissural branches. (F) boojum (bum) mutants show loss of commissural axon branching in the CNS and highly reduced dendritic branching (G,H). Arrowheads in E-F indicate missing commissural branches. Arrows in H indicate stunted dendritic arbors. (I) Labeling with the axonal marker BP102 shows that despite the lack of sensory axon commissural branches, commissural tracts seem to develop normally but undergo a successive loss of labeling or integrity during development. Temporal progresssion is inferred by gut morphology and VNC length. Arrows indicate grossly intact areas of the VNC; arrowheads indicate areas that are disrupted. (J-O) Mutations disrupting axon tract coherence or position. Lines l(3)11534 (J) and l(3)1025 (K) lack a coherent longitudinal axon tract (arrowhead). (L-M) Mutations in brahma (brm), a chromatin remodeling factor, show defasciculation or thinning of longitudinal tracts. (N-O) Axon phenotype of two boa alleles. Note that axons generally branch lateral to, rather than between, longitudinal branches (arrowheads). (P) boa mutants show no obvious abnormalities in dendrite morphology. (Q) Staining of boa2 mutants with the axonal marker BP102 reveals the presence of longitudinal (arrows) and commissural (arrowhead) tracts but relatively weak labeling. Anterior is up in CNS images. Dorsal is up in PNS images. Animals are stage 17 except E,P, which are hatchling larvae. Scale bars: 25 µm in I,Q.