Identification of novel regulatory factor X (RFX) target genes by comparative genomics in Drosophila species

Abstract

Background: Regulatory factor X (RFX) transcription factors play a key role in ciliary assembly in nematode, Drosophila and mouse. Using the tremendous advantages of comparative genomics in closely related species, we identified novel genes regulated by dRFX in Drosophila.

Results: We first demonstrate that a subset of known ciliary genes in Caenorhabditis elegans and Drosophila are regulated by dRFX and have a conserved RFX binding site (X-box) in their promoters in two highly divergent Drosophila species. We then designed an X-box consensus sequence and carried out a genome wide computer screen to identify novel genes under RFX control. We found 412 genes that share a conserved X-box upstream of the ATG in both species, with 83 genes presenting a more restricted consensus. We analyzed 25 of these 83 genes, 16 of which are indeed RFX target genes. Two of them have never been described as involved in ciliogenesis. In addition, reporter construct expression analysis revealed that three of the identified genes encode proteins specifically localized in ciliated endings of Drosophila sensory neurons.

Conclusion: Our X-box search strategy led to the identification of novel RFX target genes in Drosophila that are involved in sensory ciliogenesis. We also established a highly valuable Drosophila cilia and basal body dataset. These results demonstrate the accuracy of the X-box screen and will be useful for the identification of candidate genes for human ciliopathies, as several human homologs of RFX target genes are known to be involved in diseases, such as Bardet-Biedl syndrome.

Figure 1

In vivo observations of reporter constructs in control or dRfx-deficient Drosophila. (a) Schematic of two typical chordotonal organs of the Drosophila leg or antenna. The different segments of the dendrite and of the ciliated ending are shown. Sensory neurons have a single cilium (arrow) extending from their dendrite (arrowhead). (b) Live confocal image of GFP driven expression of osm-1 transgene in a control femur. (c) GFP expression is totally shut down in a dRfx mutant background. (d-i) Confocal imaging of chordotonal neurons labeled with anti-ELAV (red) and anti-GFP (green). oseg1-GFP expression in (d) control flies and (e) a dRfx mutant background. Note that oseg1-GFP expression is not affected in the mutant background. CG3259-GFP expression in (f) control flies and (g) dRfx mutant flies. Reporter construct expression is totally shut down in the mutant background. Johnston's organs from antennae of adult flies carrying CG9227-GFP transgenes in (h) control and (i) dRfx mutant pupae. Note the absence of expression in the mutant background. Scale bar = 10 μm.

Figure 2

Promoter comparisons between Drosophila species. Sequence identities (from 50-100%) between different Drosophila species ranging from D. melanogaster to the most distant D. virilis as calculated and presented in the VISTA interface [91] for two dRfx target genes, CG9595 (osm-6/NDG5) and CG8853 (IFT55/che-13/Hippi). Coding sequences are depicted in dark blue, untranslated regions are in light blue and other conserved regions in pink. Gene orientation is shown by a horizontal arrow. The location of conserved X-boxes for each gene is indicated by numbered vertical arrows. Note that one conserved X-box for osm-6 is in a conserved block of sequence, while others (osm-6 and che-13) are not.

Figure 3

Comparison of the DCBB set of genes with the Ciliary proteome and Ciliome databases. Venn diagram presenting the overlaps between the three datasets: the cilia proteome [46,48]; the ciliome [47,49], and the DCBB (Additional data file 2). Asterisks indicate this study. Note that only 412 common genes are found in the three datasets. The number of genes also found in the 1,462, 412 or 83 X-box gene lists (Table 4), respectively, are noted in parentheses. The numbers of genes selected in the different studies to construct each dataset are given in Additional data file 3.

Figure 4

Reporter GFP expression studies for three X-box containing genes. (a) Stereotypical arrangement of type I sensory neurons in a Drosophila embryo, anterior to the left, stained with the 21A6 antibody with a magnification of the dorsal and lateral neurons of one abdominal segment as visualized in (f,k). The arrowhead indicates the five lateral chordotonal neurons (ch) and the arrows point to the neurons of the external sensory (es) organs. (b) Schematic of two typical chordotonal organs of Drosophila. (c-l) Confocal imaging of GFP expression of transgenic lines carrying the promoter region and coding sequences fused to the GFP for CG6129/rootletin (c-e), CG31036 (f-h) and CG13125/TbCMF46 (k,l). GFP expression is only observed in ciliated sensory neurons of D. melanogaster where the chimeric GFP proteins are localized to the ciliary apparatus. (c) CG6129::GFP reporter expression (green) is observed in embryonic chordotonal organs, mainly along the dendrite from the base of the cilium to the cell body. The 21A6 antibody (red, see Materials and methods) labels the ciliary dilation of the cilium. (d,e) Live GFP imaging of the lateral pentascolopidial chordotonal organs in male third instar larvae of dRfx deficient (e) and control (d) sibs. The elav-RFP expression (red) labels all neurons. CG6129/rootletin is regulated by dRfx as no GFP expression is observed in dRfx deficient larvae. (f-h) CG31036::GFP reporter expression (green) is observed both in the external sensory neurons (arrows in (f)) and the chordotonal neurons in the embryo (arrowhead in (f-h)). The 21A6 antibody (red) labels the ciliary dilation at the tip of the dendrite. CG31036::GFP protein localization appears to be slightly different depending on the fixative used (paraformaldehyde in (g), methanol in (h)). (i-j) Immunodetection of CG31036::GFP expression in leg chordotonal organs of 72-hour pupae in dRfx deficient (j) or control (i) sibs. The anti-ELAV antibody (red) labels all neurons. No CG31036::GFP expression is observed in dRfx deficient pupae (j). (k,l) CG13125::GFP expression is observed by immunodetection in the embryonic chordotonal organs (arrowheads in (k,l)) but also in the external sensory neurons (arrows in (k)). A higher magnification of the lateral chordotonal organs (l) shows that GFP is apposed to the 21A6 immunostaining (red). Scale bar = 10 μm.