Comprehensive identification of Drosophila dorsal-ventral patterning genes using a whole-genome tiling array

Abstract

Dorsal-ventral (DV) patterning of the Drosophila embryo is initiated by Dorsal, a sequence-specific transcription factor distributed in a broad nuclear gradient in the precellular embryo. Previous studies have identified as many as 70 protein-coding genes and one microRNA (miRNA) gene that are directly or indirectly regulated by this gradient. A gene regulation network, or circuit diagram, including the functional interconnections among 40 Dorsal target genes and 20 associated tissue-specific enhancers, has been determined for the initial stages of gastrulation. Here, we attempt to extend this analysis by identifying additional DV patterning genes using a recently developed whole-genome tiling array. This analysis led to the identification of another 30 protein-coding genes, including the Drosophila homolog of Idax, an inhibitor of Wnt signaling. In addition, remote 5' exons were identified for at least 10 of the approximately 100 protein-coding genes that were missed in earlier annotations. As many as nine intergenic uncharacterized transcription units were identified, including two that contain known microRNAs, miR-1 and -9a. We discuss the potential functions of these recently identified genes and suggest that intronic enhancers are a common feature of the DV gene network.

Fig. 1.

Identification of Dl targets using a whole-genome tiling array. (a) (Left) The expression patterns of six previously characterized Dl target genes: dpp (dorsal ectoderm); ind, vnd, rho, sog (neuroectoderm); and sna (mesoderm). Embryos are all oriented with anterior to the left, and dorsal is up. (Right) Shown, for each of the six genes, are the RNA signal graphs from three mutant backgrounds as viewed in the Affymetrix Integrated Genome Browser (Affymetrix). The top (blue) graph represents total cellular transcripts from pipe⁻/pipe⁻ mutants; the middle (orange) graph represents transcripts from Toll^rm9/Toll^rm10 mutant embryos; and the bottom (pink) graph represents transcripts in Toll^10B mutant embryos. (b) Classical genetic studies previously characterized ≈30 Dl target genes (small circle, pale green). Microarray analysis identified between 20 and 40 additional targets (intermediate circle, light green; ref. 7). In the present study, the unbiased survey of the entire genome using tiling arrays identified as many as ≈30 additional protein-coding genes (large circle, dark green). (c) In addition to the protein-coding Dl target genes, 23 uncharacterized transfrags were identified that represent previously unidentified 3′ exons (13%; 3 of 23) or 5′ exons (48%; 11 of 23) of known protein-coding genes. The remaining nine transfrags correspond to putative new genes (39%; 9 of 23).

Fig. 2.

Examples of protein-coding genes. Cellularizing embryos are all oriented with anterior to the left and represented in lateral (a, b, e, and f; dorsal is up) or ventral (c and d) views. (a and b) CG13800 is expressed in the dorsal ectoderm in WT embryos (a) and expands along the entire DV axis in pipe⁻/pipe⁻ mutants (b). (c and d) Trim9 is restricted to the neuroectoderm in WT embryos (c) and shows expansion in adjacent territories in Toll^rm9/Toll^rm10 mutants (d). (e and f) CG9005 is present only in the mesoderm in WT embryos (e), but its expression spans the entire DV axis in Toll^10B mutants (f).

Fig. 3.

Uncharacterized transfrags often correspond to novel 5′exon of known protein-coding genes. (a and b) RNA signal graphs from the three mutant backgrounds for the CadN (a) and cv-2 (b) loci, suggesting extended transcription (red double arrows) 5′ of the known transcription start site; both genes are transcribed from the minus strand. (c) Cellularizing embryos hybridized with riboprobes directed against the cDNA (Left) or the recently identified transfrag (Right) of CadN (Upper) and cv-2 (Lower). Expression is detected in the mesoderm and dorsal ectoderm, respectively. All of the embryos are oriented with anterior to the left, and dorsal is up.

Fig. 4.

Examples of noncoding transfrags. Cellularizing embryos are all oriented with anterior to the left and dorsal up. (a and b) transfrag 22/pri-mir-9a is expressed in both the dorsal and neuroectoderm in WT embryos (a) and expands along the entire DV axis in pipe⁻/pipe⁻ (not shown) and Toll^rm9/Toll^rm10 mutants (b). (c and d) transfrag 8/pri-mir-1 is specifically expressed in the mesoderm in WT embryos (c) and shows expansion in adjacent territories in Toll^10B mutant embryos (d). (e and f) Similarly, transfrag 22 is present only in the mesoderm in WT embryos (e) but expands along the entire DV axis in Toll^10B mutant embryos (f).