Characterization of the intergenic RNA profile at abdominal-A and Abdominal-B in the Drosophila bithorax complex

Abstract

The correct spatial expression of two Drosophila bithorax complex (BX-C) genes, abdominal-A (abdA) and Abdominal-B (AbdB), is dependent on the 100-kb intergenic infraabdominal (iab) region. The iab region is known to contain a number of different domains (iab2 through iab8) that harbor cis-regulatory elements responsible for directing expression of abdA and AbdB in the second through eighth abdominal segments. Here, we use in situ hybridization to perform high-resolution mapping of the transcriptional activity in the iab control regions. We show that transcription of the control regions themselves is abundant and precedes activation of the abdA and AbdB genes. As with the homeotic genes of the BX-C, the transcription patterns of the RNAs from the iab control regions demonstrate colinearity with the sequence of the iab regions along the chromosome and the domains in the embryo under the control of the specific iab regions. These observations suggest that the intergenic RNAs may play a role in initiating cis regulation at the BX-C early in development.

Fig 1.

Intergenic transcription at the abdA-AbdB locus. (A) Summary of abdA-AbdB locus. The abdA and AbdB transcription start sites are indicated by leftward arrows. The intergenic region is ≈100 kb in length. The iab regions that control expression of the two Hox genes are indicated (IAB2 to IAB8). IAB2, IAB3, and IAB4 (shown in green) regulate expression of abdA. IAB5, IAB6, IAB7, and IAB8 (shown in yellow) direct AbdB expression. The insulator DNAs that separate the different iab regions are indicated (black ellipses). The presumptive Fab6 insulator (gray ellipse) has yet to be identified. Characterized enhancers within the iab regions are shown as blue rectangles. The locations of probes used for in situ hybridization analysis in this study are shown as black bars under the locus. (B–I) Transcription patterns detected with in situ hybridization probes. Embryos are orientated with anterior to the left and dorsal up. Probes against the abdA (B and C) and AbdB (F and G) coding regions detect the expected distribution of transcripts (see text). An APP probe detects transcription at blastoderm stage 5 (D) and later in development (E), unlike a BPP probe, which detects transcription in abdominal segments 8 and 9 only later in embryonic development (H and I).

Fig 2.

Transcription in the iab regions. (A) The location of probes at the abdA-AbdB locus. (B–M) Patterns of sense transcription detected by in situ hybridization RNA probes. At stage 5, transcripts from the different iab regions show distinct distribution patterns along the A–P axis of the embryo. A probe from the iab3 region detects expression (B) extending farther toward the anterior of the embryo than iab4 (E). Expression patterns become increasingly restricted toward the posterior of the embryo in the iab5 region (H) and iab8 region (K). From stage 9 of development, transcription from all iab regions is restricted to the two most posterior abdominal segments, 8 and 9 (C, F, I, and L). This pattern persists through stage 13 of development (D, G, J, and M).

Fig 3.

Temporal activation of sense iab transcripts. (A) At late stage 4 in embryos hybridized with probes against engrailed and iab4s transcripts, expression is detected only in the posterior iab4 domain, before the appearance of anterior engrailed expression. (B) In early blastoderm stage 5 embryos, sense transcripts can be detected in the iab4 domain and the anterior engrailed stripe 2. (C) Colinear distribution of transcripts in the iab regions. Measure of transcript distribution is shown as a percentage of embryo length. The anterior tip of the embryo corresponds to 0 and the posterior tip corresponds to 100. The probes are listed according to their order on the chromosome (see Fig. 1A), with the exception of probes against the two Hox genes, Aexon (green) and Bexon (yellow), which are shown at the bottom and the top of graph, respectively. Colinearity between the chromosomal order of probes and their transcription patterns is observed. The anterior limits of transcription for probes from the iab5, iab6, iab7, and iab8 regions are restricted within the AbdB domain of expression (pale yellow), whereas probes from iab3 and iab4 regions detect patterns extending into the abdA expression domain (pale green). The distribution of the antisense transcripts detected in iab4 and iab6 are also colinear with their chromosomal locations and are shown in black.

Fig 4.

Antisense transcripts in the iab regions. (A–D) Sense transcription pattern at iab4 region during embryonic development detected by probe 4-5. No transcript is detectable at stage 5 (A). Transcription is detectable at stage 8 and, by stage 9, is restricted to abdominal segments 8 and 9. Transcription persists in the two most posterior abdominal segments through stage 13 (D). (E–H) The iab4as transcript (23) also is detected by probe 4-5, although the pattern is distinct from that of sense transcription. At blastoderm stage 5, the antisense transcript is expressed strongly in the iab4 domain (E) and persists in abdominal segments 1–7 through developmental stages 8 (F), 9 (G), and 13 (H), but is excluded from the most posterior segments. The distributions of the iab4as and iab4 sense transcripts appear to be mutually exclusive. (I and J) A novel antisense transcript is detected by probe 6-1. At blastoderm stage 5, the transcript is restricted to the iab6 domain (I) and, by stage 9, is expressed strongly in abdominal segments 4, 5, and 6 and more weakly in 7, 8, and 9 (J). The sense transcription detected by probe 6-1 (data not shown) is mutually exclusive to the iab6as pattern.