Genome-wide analyses identify transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites

Abstract

Dendrite arborization patterns are critical determinants of neuronal function. To explore the basis of transcriptional regulation in dendrite pattern formation, we used RNA interference (RNAi) to screen 730 transcriptional regulators and identified 78 genes involved in patterning the stereotyped dendritic arbors of class I da neurons in Drosophila. Most of these transcriptional regulators affect dendrite morphology without altering the number of class I dendrite arborization (da) neurons and fall primarily into three groups. Group A genes control both primary dendrite extension and lateral branching, hence the overall dendritic field. Nineteen genes within group A act to increase arborization, whereas 20 other genes restrict dendritic coverage. Group B genes appear to balance dendritic outgrowth and branching. Nineteen group B genes function to promote branching rather than outgrowth, and two others have the opposite effects. Finally, 10 group C genes are critical for the routing of the dendritic arbors of individual class I da neurons. Thus, multiple genetic programs operate to calibrate dendritic coverage, to coordinate the elaboration of primary versus secondary branches, and to lay out these dendritic branches in the proper orientation.

Figure 1.

RNAi effectively reduced target gene expression in class I da neurons. (A) Schematic of the dorsal da neurons of the Drosophila PNS. The class I neurons, ddaD and ddaE, are represented by red and blue ovals, respectively, while the class IV neuron, ddaC, is represented by a green oval. Anterior is left and dorsal is up in all figures. (B,C) Live image of Gal4²²¹, UAS-mCD8GFP embryo injected with buffer control (B) or GFP dsRNA (C). Gal4²²¹ is expressed at higher levels in ddaE than ddaD, sometimes resulting in weak labeling of ddaD. (D,E) Live image of class I dendrites marked by GFP expression driven by Gal4²²¹ in shot³ homozygous mutant (D) and embryo injected with shot dsRNA (E). Bar, 25 μm.

Figure 2.

A subset of group A transcription factors function to promote dendrite arborization. (A–F) Live image and corresponding tracing of ddaD (red) and ddaE (blue) class I dendrites in Gal4²²¹, UAS-mCD8GFP embryos injected with buffer control (A), trh dsRNA (B), pyg dsRNA (C), cg1841 dsRNA (D), hmgD dsRNA (E), or usp dsRNA (F). In C and E, the GFP signal in ddaD is not strong enough to permit unambiguous tracing of ddaD dendrites. Tracings are done to scale. Bar, 25 μm.

Figure 3.

Some group A transcription factors normally limit dendrite arborization. (A–D) Live image and corresponding tracing of GFP expressing class I dendrites in embryos injected with buffer control (A), ab dsRNA (B), Su(z)12 dsRNA (C), or cg5684 dsRNA (D). Bar, 25 μm.

Figure 4.

Group B transcription factors have antagonistic effects on primary dendrite growth and lateral branch extension. (A–F) Live image and corresponding tracing of class I dendrites in embryos injected with buffer control (A), sna dsRNA (B), knirps dsRNA (C), l(3)mbt dsRNA (D), gcm2 dsRNA (E), or pcaf dsRNA (F). In A–D, the cell bodies of ddaE are aligned in the traces below the images, allowing comparison of primary branch length. The neurite projecting ventrally from the dorsal border of the image in B originates from an unidentified dorsally located ectopic neuron rather than a contralateral class I neuron, and is therefore not represented in the trace. Bar, 25 μm.

Figure 5.

Group C transcription factors control the relative placement and orientation of class I dendrites. (A–F) Live image and corresponding tracing of class I dendrites in embryos injected with buffer control (A), cg1244 dsRNA (B), bap55 dsRNA (C), cg9104 dsRNA (D), cg4328 dsRNA (E), or cg7417 dsRNA (F). Bar, 25 μm.

Figure 6.

Some TFs affecting class I da neuron number may also have post-mitotic effects on neuronal morphogenesis. (A–D) Class I dendrites are unaffected by the number of class I neurons. N^ts; Gal4²²¹, UAS-mcd8∷gfp embryos were grown at 25°C (A,C) or grown at 25°C with a 3-h exposure to the nonpermissive temperature (29°C) for 3 h beginning at 4 h AEL to induce neurogenic phenotypes (B,D). Larvae were imaged live using confocal microscopy at 48 h AEL. (B,D) The arborization patterns of dendrites from supernumerary class I neurons (denoted with white asterisks), which overlap extensively with one another, are similar to wild-type dendrites. (B) The dendrites of the adjacent class I neuron is unaffected by the supernumerary neurons. (A,B) The cell body of a class IV da neuron (ddaC) that is also labeled by Gal4²²¹ is indicated with a red arrowhead. (E–G) Live image of class I dendrites in embryos injected with buffer control (E), nerfin1 dsRNA (F), or ci dsRNA (G). ci(RNAi) causes a complete loss of dorsal class I da neurons, but the dorsal class IV da neuron (ddaC; indicated with red arrowhead) that is also labeled by Gal4²²¹ is still present. (H,I) Live image embryo expressing GFP under the control of Gal4^109(2)80, which is expressed in all md neurons, injected with buffer control (H) or ci dsRNA (I). Bar, 25 μm.

Figure 7.

RNAi-based analysis generates reproducible phenotypes. dsRNAs were injected individually (single dsRNAs) or pair-wise (pools) at different concentrations and assayed for effects on dendrite morphogenesis. (A) Following pair-wise injection of dsRNAs (1 mg/mL each) for 730 TFs, dsRNAs were subsequently injected individually from positive pools leading to the identification of 64 candidate genes. (B) For a subset (144) of the 730 TFs, concentrated (5–10 mg/mL) dsRNAs were injected individually leading to the identification of 11 candidate genes (yellow circle), seven of which were also identified by screening the pools (yellow and blue overlap) whereas four were not. Four other genes identified from screening the pools did not yield reliable dendrite phenotypes when tested with injection of concentrated dsRNA, likely as a result of early lethality for at least two of the four genes (see text). (C) For a separate subset (336) of the 730 TFs, dsRNAs (1–2 mg/mL) were injected individually, leading to the identification of 34 candidate genes, of which 26 were previously identified from screening pools (red and blue overlap) and eight emerged only from screening dsRNAs individually.

Figure 8.

RNAi phenocopies loss-of-function dendrite phenotypes for many transcription factors. Representative images of class I neurons marked Gal4²²¹, UAS-mcd8∷gfp for control (A), sna¹⁸ (B), ttk^03A (C), wor¹ (D), nvy^PDfKG-1 (E), and sens^E58 (F) homozygous mutant embryos at 17–18 h AEL. Bar, 25 μm.

Figure 9.

Requirement of certain transcription factors during larval development to maintain proper class I dendritic arbors. (A,B) Live image of GFP-expressing class I dendrites in wild-type (A) and a Mi-2^j3D4 homozygous mutant (B) second instar larva (48 h AEL). (C–E) Live image of GFP-expressing class I dendrites in wild-type (C), Adf1⁰¹³⁴⁹ (D), and E(bx)^ry122 (E) third instar larva. Bar, 50 μm.

Figure 10.

Epistatic interactions between group A and group B transcription factors. (A–E) Live image and corresponding tracing of GFP-expressing dorsal class I dendrites in homozygous mutant sens^E58 embryos injected with buffer control (A), Su(z)12 dsRNA (B), ab dsRNA (C), cg1244 dsRNA (D), or cg1841 dsRNA (E). (F,G) Live image and corresponding tracing of GFP-expressing ventral class I vpda dendrites in homozygous mutant ab^k02807 embryos injected with buffer control (F) or hmgD dsRNA (G). Bar, 25 μm.