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. 2011 Jun 7;108(23):9673-8.
doi: 10.1073/pnas.1106386108. Epub 2011 May 23.

Enhancer-driven membrane markers for analysis of nonautonomous mechanisms reveal neuron-glia interactions in Drosophila

Chun Han  1 , Lily Yeh Jan, Yuh-Nung Jan
Affiliations

Enhancer-driven membrane markers for analysis of nonautonomous mechanisms reveal neuron-glia interactions in Drosophila

Chun Han et al. Proc Natl Acad Sci U S A. .

Abstract

Extrinsic factors and the interactions of neurons with surrounding tissues are essential for almost every aspect of neuronal development. Here we describe a strategy of gene expression with an independent enhancer-driven cellular marker (GEEM) for studying roles of cell-cell interactions and extrinsic factors in the development of the Drosophila nervous system. Key to this strategy is robust expression of enhancer-driven transgenic markers in specific neurons. To this end, we have created vectors to achieve bright and even labeling of neuronal processes, easy cloning of enhancer elements, and efficient and flexible generation of transgenic animals. We provide examples of enhancer-driven membrane markers for specific neurons in both the peripheral and central nervous systems and their applications in the study of neuronal projections and connections in the Drosophila brain. We further applied GEEM to examine the wrapping of sensory neuron somas by glia during embryonic and larval stages, and neuron-glia interaction during dendrite pruning in live animals, leading to the discovery that glia play critical roles in the severing and degradation of proximal dendrites. The GEEM paradigm should be applicable to the studies of both cell-autonomous and nonautonomous regulations of any cell type.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Optimization of transgene components and fluorescent membrane markers. (A) Transgenesis based on the pAPIC vector. 5′P and 3′P, P-element sites; In, gypsy insulator. (B) Signal intensity of ppk-CD8-GFP reporters with various transgene components (Table 1). The reporters were integrated to attPVK19 and fluorescent signals from both proximal and terminal dendrites of ddaC neurons were compared. The asterisk indicates nonspecific expression in other classes of da neurons. (C) Signal intensity of CD8- and CD4-fusion reporters. The error bars represent SDs (B and C). (D) Ratios of terminal dendrite signals to proximal dendrite signals in CD8- and CD4-fusion reporter lines. (E–G) Distribution of CD8-GFP (E), CD4-tdGFP (F), and CD4-tdTom (G) in ddaC neurons. The arrowheads point to the somas and the arrows point to terminal dendrites. (H and I) Distribution of CD8-GFP (H) and CD4-tdGFP (I) in DDC neurons in the larval ventral nerve cord. In both images, the main panel shows the z-axis projection; the right panel shows the x-axis projection of the volume within the broken yellow lines; the bottom panel shows the y-axis projection of the volume within the broken blue lines.
Fig. 2.
Fig. 2.
The Hemmar vectors for making enhancer-driven membrane markers. (A) Diagram of enhancer cloning and transgenesis with the Hemmar vectors. (B–E) Comparison of existing C4da reporters and new ones generated with Hemmar vectors. Neurons in B–D were imaged at identical settings. (F–G′) Comparison of expression timing of ppk-CD4-tdGFP (F and F′) and ppk-Gal4 UAS-CD8-GFP (G and G′) in embryos. DdaC neurons in A1-A7 (F and G) and A1-6 segments (F′ and G′) are shown.
Fig. 3.
Fig. 3.
Analysis of neuronal projection and connection with enhancer-driven CNS neuronal markers. (A-B′′) R9D11-CD4-tdTom and TDC2-Gal4 UAS-CD4-tdGFP in the central brain (A) and fan-shaped body (B–B′′) (boxed in A) of the adult. (C) R9D03-CD4-tdTom (magenta) in an adult brain counter stained with NC82 (blue). (D) R9D03-CD4-tdTom (magenta) and ort-Gal4 UAS-CD4-tdGFP (green) in the medulla. (E–E′′) Overlap of R9D03 neurons and ort neurons in the medulla. (F) Projection of horizontal sections showing R9D03-CD4-tdTom (magenta) ramifications and axon terminals of R7/R8 labeled by GMR-Gal4 UAS-CD4-tdGFP (green) in outer medulla layers. The R7 and R8 termination layers are indicated. (G and H) High-magnification views showing R7/R8 axon terminals (green) and R9D03 neurites (magenta) in sections parallel (G) and perpendicular (H) to medulla columns. The broken line in G indicates the proximate position of the section shown in H. (I–K) Colabeling of R9D03 neurites (magenta) and R7/R8 synaptic sites labeled by GMR-Gal4 UAS-Brp-GFP (green) in outer medulla layers. I, J, and K are annotated similar to F, G, and H, respectively. (Scale bars, 5 μm.)
Fig. 4.
Fig. 4.
(A–E′) The wrapping of ddaC somas by glia during embryonic and larval development. Glia are labeled with repo-Gal4 UAS-CD4-tdTom (magenta in merged images) and ddaC neurons are labeled with ppk-CD4-tdGFP (green in merged images). Animals are oriented anterior left, dorsal up. All times indicated are after egg laying. Arrows in C and C′ point to the glial membrane extended from the root of the ddaC axon. Arrows in D and D′ point to the boundary of the incomplete glial wrapping of the soma.
Fig. 5.
Fig. 5.
Correlation of glial wrapping and initial severing of proximal dendrites during dendrite pruning of ddaC. (A–C′) Glial wrapping of proximal dendrites of ddaC in white pupae. Blue arrows indicate the boundaries of complete glial wrapping. Yellow brackets indicate long, thin glial processes. Red brackets indicate glial membrane patches that partially wrap dendrites. (D) Locations of observed first and second dendritic severing points in relation to glial wrapping. (E and E′) Time-lapse images of a ddaC neuron showing the first and second severing events. Blue arrows indicate the first severing at a glial wrapping boundary and the yellow arrowheads indicate the second severing within the glial wrapping. In all panels, the glial membrane is labeled by repo-Gal4 UAS-CD4-tdTom and ddaC neurons are labeled by ppk-CD4-tdGFP.
Fig. 6.
Fig. 6.
Glia are involved in determining the location of initial dendrite severing and degrading the wrapped dendrite segments. (A–B′) Dendrite pruning at 10 h APF in repo-Gal4 (A and A′) and repo-Gal4 UAS-shits (B andB′) after incubation at 29 °C from 3 to 10 h APF. Blue and red arrows indicate the swelling and thinning of proximal dendrites, respectively. The yellow arrowhead indicated a breaking point at an unwrapped dendrite fragment. (C–E′) Dendrite pruning at 16 h APF in repo-Gal4 (C and C′) and repo-Gal4 UAS-EcR-DN (D–E′) at 25 °C. In all panels, the glial membrane is labeled by repo-Gal4 UAS-CD4-tdTom and ddaC neurons are labeled by ppk-CD4-tdGFP.
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