Figure 1.
Relative locations of the CNS midline and trachea within the late Drosophila embyo.
(A) The midline cellular pattern is segmentally repeated throughout the ventral nerve cord at embryonic stage 16. (B) Each segment consists of six neural subtypes and three surviving midline glia whose relative locations within a typical thoracic segment (white box and inset in A) are shown. The midline subtypes include: the MP1 neurons (gray), the H cell (pink), the H cell sib (orange), the ventral unpaired interneurons (iVUMs; purple), the ventral unpaired motorneurons (mVUMs; blue), median neuroblast (MNB) and its progeny (black) and the anterior midline glia (AMG; green); adapted from [24], [108]. (C) By the end of embryogenesis, the trachea form an extensive network that mediates gas exchange throughout the organism. (D) Each tracheal metamere consists of the major dorsal trunk (DT), a dorsal branch (DB), and the visceral (VB), spiracular (SB) and ganglionic (GB) branches and lateral trunk (LT) on the ventral side; adapted from [71]. Lateral views of whole mount embryos stained with anti-GFP (green), anti-sim (red; A) antibodies or monoclonal antibody 2A12 (red; C) and analyzed by confocal microscopy are shown. (A) The embryo contains a reporter gene that expresses GFP in all midline cells.
Figure 2.
CG33275 contains a midline enhancer that is separable and distinct from a nearby tracheal enhancer.
(A) Locations of regions within the fifth intron of CG33275 used to generate the reporter constructs are shown. A scale is indicated on top and the thick lines represent the regions analyzed in (B–T). White boxes represent exons and thin lines represent introns. The indented boxes indicate flanking genes, CG7716 and snoRNA, and arrows indicate the start and direction of transcription. (B) Fragments used to generate reporter constructs are shown. The size of each fragment is indicated below it, vertical lines indicate locations of CMEs and red lines represent sequence blocks conserved in at least 11 Drosophila species. (C–T) Whole mount embryos were double-stained with anti-GFP (green: D, G, J, L and S), anti-sim (red; E, H, K), anti-dys (red; T) antibodies and monoclonal antibody 2A12 (red; M–Q) and analyzed by confocal microscopy. The overlap in expression is shown in yellow in the merge images (C, F, I, M–Q and R). Reporters (C–E) CG33275 ML2544:GFP, (F–H) CG33275 ML1312:GFP and (I–K) CG33275 ML753:GFP drove expression in midline glia. Midline glia can be identified by the overlap in expression of GFP and sim (arrows C–K) and are located on the dorsal side of the nerve chord. Midline neurons are located on the ventral side of the nerve chord and labeled by sim, but do not express CG33275 or any of the CG33275 reporter genes. (L–T) Monoclonal antibody 2A12 labels the tracheal lumen and the anti-dys antibody labels tracheal fusion cells. CG33275 TR1297:GFP is expressed in all trachea, beginning in the tracheal pits at stage 12 (L) and extending to late embryogenesis (M–N), while CG33275 TR265:GFP is expressed only in fusion cells (O–T), indicated by co-localization of GFP and Dys (arrows R–T). Lateral views of stage 16 transgenic embryos are shown; anterior is in the top, left hand corner and ventral is on the left, except (L), which is a dorsal view of a stage 12 embryo.
Table 1.
DNA recognition sequence of PAS heterodimers.
Figure 3.
An esg genomic region contains a midline enhancer that is separable and distinct from two esg tracheal enhancers.
(A) Genomic regions surrounding esg used to generate the reporter constructs and (B) the esg enhancers are shown as above. Gray boxes represent 5′ and 3′ untranslated regions. (C–J) Live larvae were analyzed by confocal and differential contrast microscopy and ventral views of the (C–F) esg TR C1:GFP and (G–J) esg TR C7:GFP reporters are shown. In larvae, the esg TR C1:GFP reporter is expressed sporadically in fusion cells (arrow in E) and the esg TR C7:GFP reporter is expressed in all tracheal branches and sporadically in the dorsal trunks (G–J), but consistently in fusion cells (arrows I). (K–N) Whole mount esg ML C2:GFP reporter embryos were stained with an anti-GFP antibody (green: L), engrailed monoclonal antibody (blue; M) and anti-sim antibody (red; N) and analyzed by confocal microscopy. The overlap in expression is shown in the merge image (K). Anterior midline glia express GFP and sim and are located dorsally within the nerve chord. Posterior midline glia that normally undergo cell death during this time can still be visualized with GFP (three cells surrounding star in N), but not sim or engrailed. The MNB and its progeny express sim, engrailed and GFP and are located ventrally within the nerve chord (arrowheads in K–N). Lateral view of a stage 16 transgenic embryo is shown; anterior is in the top, left hand corner and ventral is on the left.
Figure 4.
Liprin γ contains a conserved enhancer sufficient to drive expression in both midline glia and trachea.
(A) The genomic regions within the first intron of liprin γ used to generate the reporter constructs and (B) the liprin γ enhancers are shown as in Fig. 1 and previously reported [59]. (C–T) Whole mount embryos were double-stained with an anti-GFP antibody (green: D, G, J, M, P and S) and monoclonal antibody 2A12 (red; E, H, K and N), anti-dys (red; Q) or anti-sim (red; T) and analyzed by confocal microscopy. The overlap in expression is shown in yellow in the merge column (C, F, I, L, O and R). Even though the liprin γ 3141:GFP and liprin γ 889:GFP reporters are expressed in the dorsal trunk (arrows C–E and I–K) and dorsal and ventral branches, liprin γ 1781:GFP is restricted to mostly fusion cells of the dorsal trunk (arrows F–H and O–Q). The liprin γ 182:GFP reporter is expressed in all tracheal cells (L–N), midline glia (arrows R–T) and a few midline neurons in certain segments (arrowheads R–T). Lateral views of stage 16 transgenic embryos are shown; anterior is in the top, left hand corner and ventral is on the left.
Figure 5.
Net contains an enhancer that drives expression in both midline glia and trachea.
(A) A genomic region located between NetA and NetB was used to generate a reporter construct and (B) the Net enhancer is shown. (C–K) Whole mount embryos were double-stained with an anti-GFP antibody (green: D, G and J) and anti-sim (red; E) or monoclonal antibody 2A12 (red; H and K) and analyzed by confocal microscopy. The overlap in expression is shown in yellow in the merge column (C, F and I). The Net797:GFP reporter drove expression in midline glia (arrows C–E; ganglionic branches of trachea are also visible in the image in green) and occasionally midline neurons within some segments (arrowheads C–E). The tracheal expression pattern of this reporter is unique in that GFP is high in tracheal cells, except cells within the dorsal trunk (F–K). Lateral views of stage 16 transgenic embryos are shown; anterior is in the top, left hand corner and ventral is on the left.
Figure 6.
The comm cis-regulatory region contains an enhancer that drives expression in the embryonic midline and larval trachea.
(A) Genomic regions surrounding and within comm were used to generate the reporter constructs and (B) the comm enhancer is shown. The closest gene upstream of comm is CG6244, which is 79,892 bp away. (C–N) Whole mount embryos were double-stained with anti-GFP (green: D, G, J and M) and anti-sim antibodies (red; E, H, K and N) and analyzed by confocal microscopy and the overlap in expression is shown in yellow in the merge column (C, F, I and L). (C–E) comm2575:GFP and (L–N) comm443:GFP are expressed in both midline glia (arrows) and midline neurons (arrowheads), while (F–H) comm693:GFP is restricted to some midline neurons (arrowheads). The comm267:GFP (I–K) and comm737:GFP (data not shown) reporters are not expressed in the midline. Lateral views of stage 16 transgenic embryos are shown; anterior is in the top, left hand corner and ventral is on the left. (O–T) comm443:GFP is also expressed in the tracheal dorsal trunks (arrows in O and S) as well as other tracheal branches (arrow in R). Live larvae containing the comm443:GFP reporter were analyzed by confocal and differential contrast microscopy and dorsal views are shown.
Figure 7.
The moody cis-regulatory region contains a tracheal enhancer that overlaps with a lateral CNS glial enhancer.
(A) The genomic regions surrounding and within moody used to generate the reporter constructs and (B) the moody enhancer is shown. (C–N) Whole mount embryos were double-stained with an anti-GFP antibody (green: D, G, J, and M) and monoclonal antibody 2A12 (red; E, H and K) or anti-repo monoclonal antibody (red; N) and analyzed by confocal microscopy. The overlap in expression is shown in yellow in the merge columns (C, F, I and L). The expression patterns of the (C–E) moody1970:GFP, (F–H and L–N) moody1221:GFP and (I–K) moody608:GFP reporters are shown. Both the moody1970:GFP (not shown) and moody1221:GFP (L–N) reporters also drive expression in lateral glia as indicated by co-localization with repo (arrows). Additionally, moody 1221:GFP is expressed in the dorsal trunk (arrows F–H), while moody1970:GFP is expressed in the dorsal vessel (arrows C and D) and lightly in the dorsal trunk. (C–H) Dorsolateral, (I–K) lateral or (L–N) ventral views of stage 16 transgenic embryos are shown; anterior is in the top, left hand corner.
Figure 8.
The Ect3 cis-regulatory region contains a midline enhancer sensitive to context.
(A) The genomic region upstream of Ect3 (and within the first intron of Tk) used to generate the reporter constructs and (B) the Ect3 enhancers are shown. (C–L’) Whole mount embryos were stained with anti-sim (red; F, L, R, X, D’ and J’), anti-odd (blue; G, M, S, Y, E’ and K’) and anti-GFP (green: H, N, T, Z, F’ and L’) antibodies and analyzed by confocal microscopy. The overlap in expression is shown in the merge columns: all three antibodies (C, I, O, U, A’ and G’), sim and GFP (D, J, P, V, B’ and H’) and GFP and odd (E, K, Q, W, C’ and I’). Odd is expressed only in MP1 midline neurons. Both Ect3 1955:GFP (C–H) and Ect3 1456:GFP (I–N) drive expression in the all midline cells, with the exception of a single iVUM. Ect3 2311:GFP (O–T) and Ect3 517:GFP (U–Z) are restricted to some midline glia, MP1 neurons, the H cell and H cell sib. Ect3 572:GFP (A’–F’) is expressed in midline glia, some MP1 neurons and some of the MNB and its progeny. Finally, Ect3 1071:GFP (G’–L’) drives only spotty midline expression. Lateral views of stage 16 transgenic embryos are shown; anterior is in the top, left hand corner and ventral is on the left. The midline expression pattern of each reporter is shown schematically on the right.
Table 2.
Ten identified midline and tracheal enhancers.
Table 3.
Proximal CME context in midline and tracheal enhancers.
Table 4.
Sequence of synthetic reporter constructs.
Figure 9.
Proximal sequence context flanking the CME contributes to the midline and tracheal expression pattern.
Whole mount transgenic embryos containing one of the multimerized synthetic reporter constructs were labeled with anti-GFP (green; B, D, F, H, J, L, N, P, R, T, V, X, Z, B’, D’, F’, H’, J’, L’, N’, P’, R’, T’, V’, X’, Z’, B’’ and D’’) and anti-sim (red; C, G, K, O, S, W, A’, E’, I’, M’, Q’, U’, Y’ and C’’) antibodies and analyzed by confocal microscopy. The overlap in expression between GFP and sim is yellow (merge; A, E, I, M, Q, U, Y, C’, G’, K’, O’, S’, W’ and A’’). Midline GFP expression is driven by the (A–D) synth 1:GFP, (E–H) synth 2:GFP (I–L) synth 3:GFP, (M–P) synth 4:GFP and (Q–T) synth 5:GFP, whereas the (U–X) Sox:GFP and (Y–B’) synth 6:GFP reporters are not expressed in either the midline or trachea. The (C’-F’) Toll:GFP, (G’-J’) synth 7:GFP, (K’-N’) synth 8:GFP, (O’-R’) synth 9:GFP and (S’-V’) synth 10:GFP synthetic reporters are expressed in both the midline and trachea, whereas the expression pattern of the (W’-Z’) synth 11:GFP and (A’’-D’’) synth 12:GFP reporters are restricted to trachea only. The expression patterns of the Toll:GFP and Sox:GFP reporters were previously reported [59]. Note that midline GFP expression driven by the (A–D) synth 1:GFP, (M–P) synth 4:GFP, (Q–T) synth 5:GFP, (C’-F’) Toll:GFP, (G’-J’) synth 7:GFP, (O’-R’) synth 9:GFP and (S’-V’) synth 10:GFP reporters is in both neurons and glia, whereas expression driven by the (E–H) synth 2:GFP reporter is restricted to midline glia and expression of the (I–L) synth 3:GFP and (K’-N’) synth 8:GFP reporters is restricted to midline neurons. The immediate CME context within each synthetic sequence is indicated to the left of the images and the entire sequence of each synthetic reporter construct is listed in Table 4. Arrows indicate midline glia and arrowheads indicate midline neurons. Lateral or ventrolateral views of stage 16 transgenic embryos are shown; anterior is in the top, left hand corner and ventral is bottom, left. Four copies of each synthetic sequence were tested within the reporter constructs.
Figure 10.
Motifs overrepresented in midline and tracheal enhancers identified with MEME.
MEME [61] was used to identify motifs overrepresented in midline and tracheal enhancers. The expected number of motifs one would find in a similarly sized set of random sequences (E-value) and the number of times each site was found within the enhancers are indicated in Table 5. Each motif was identified using two, related data sets (see text).
Table 5.
Midline and tracheal motifs identified with MEME.