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Drosophila p27Dacapo expression during embryogenesis is controlled by a complex regulatory region independent of cell cycle progression

Claas A. Meyer, Ina Kramer*, Rainer Dittrich, Sandra Marzodko, Jan Emmerich and Christian F. Lehner{dagger}

Department of Genetics, University of Bayreuth, 95440 Bayreuth, Germany
* Present address: Abt. für Zellbiologie, Biozentrum, Universität Basel, Klingelbergstr. 70, 4056 Basel, Switzerland



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  		Fig. 1. The role of cell cycle progression in the control of dap expression. dap expression was analyzed by in situ hybridization in wild-type embryos (A,B), mutant embryos lacking both CycA and CycB (C,D), and embryos expressing UAS-CycE under the control of prd-GAL4 (E,F). Cells in the dorsolateral epidermis arrest in G1 of cycle 17 in wild type, prematurely in G2 of cycle 15 in CycA CycB double mutants, or too late after an extra division cycle 17 in the UAS-CycE expressing segments of prd-GAL4 UAS-CycE embryos. These variations in the cell proliferation program do not affect the program of dap transcription during stage 11 (compare A,C,E). The high-magnification views (B,D) illustrate that the nuclear density in the CycA CycB double mutants (D) is half of that in wild type (B) because of the premature cell cycle arrest. The high magnification view of two epidermal segments at stage 12 (F) illustrates that the disappearance of dap transcripts is delayed in the prd-GAL4 UAS-CycE expressing segments (right side).

 


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  		Fig. 2. Regulators of tracheal cell fate control dap and stg expression independently. Wild-type (A,B), vvl (C,D) and trh (E,F) embryos before the onset of mitosis 16 were hybridized in situ with a probe for either dap (A,C,E) or stg (B,D,F) followed by detection of the probe by fluorescence.

 


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  		Fig. 3. dap expression in the early central nervous system. Wild-type embryos (A-C) and prospero mutant embryos (D) were double labeled with antibodies against p27DAP (green) and Prospero (red), and two complete segments of the developing nervous system are shown. At stage 10, co-expression of p27DAP and Prospero is readily detected in the MP2 neuroblasts (arrowheads) but never in the first GMCs and neurons, which are labeled by anti-Prospero. A merged image is shown in A and the corresponding p27DAP labeling in B. p27DAP is also detected in MP2 of prospero mutant embryos (D). At stage 11, a merged image reveals co-expression of p27DAP and Prospero in additional cells of the central nervous system in wild-type embryos, as well as cells expressing either p27DAP or Prospero (C).

 


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  		Fig. 4. Analysis of the dap regulatory region. The dap genomic regions of D. melanogaster and D. virilis were isolated, sequenced and compared. The coding sequences are indicated by black boxes. Red boxes indicate blocks of high sequence similarity within the non-coding regions. The putative transcriptional start site in the D. melanogaster gene is indicated by an arrow. Distances (kb) from this start site are given by negative numbers. The green box indicates the extent of the deletion present in dap9. Black lines indicate the sequences that were present in the various transgene constructs of the dap-g and dap-gm series (1gm, 2g, 3gm, 4gm, 5gm, 6gm). Blue lines indicate the sequences that were present in the lacZ reporter constructs of the dap-l series (1l to 15l, and 15l-A8). The region present in the transgene dap-5l and the transgenes used for the dissection of this region are shown at a higher resolution. Gray boxes indicate distinct cis-regulatory elements driving expression in the interstitial cell precursors (ICP), tracheal pits (TP), peripheral nervous system (PNS), central nervous system (CNS), epidermis, gut and in the prospective posterior spiracle region in the abdominal segment 8 (A8). The table summarizes the results of the dissection of the 5l region in more detail. Expression of the relevant transgenes (dap-6l to dap-15l and dap-15l-A8) in epidermis, gut and prospective posterior spiracles region (A8) was scored as either particularly strong (++), strong (+) or absent (–). The D. melanogaster and D. virilis sequences present in the conserved block required for high level expression in the prospective posterior spiracle region (A8) is shown at the bottom. Red arrows indicate potential Abdominal B-binding sites, and the positions matching the Abdominal B-binding site consensus (TTTATGGC) (Ekker et al., 1994) are written with red letters. Other positions of identity between D. melanogaster and D. virilis are written in green letters. For details see text.

 


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  		Fig. 5. Distinct cis-regulatory elements are required for dap expression in epidermis and nervous system. Embryos with either the transgenes dap-1gm (A,C,E) or dap-4gm (B,D,F), which drive expression of p27DAP fused to six Myc epitope tags at the C terminus, were labeled with a DNA stain (A,B) and anti-Myc (C-F) at stage 11 (A-D) or stage 13 (E,F). High-magnification views of the epidermis are shown. (A,B) The DNA staining of the regions shown in C,D, respectively. While dap-1gm is expressed in all epidermal cells during G2 of the final division cycle (C) like the endogenous dap locus (de Nooij et al., 1996; Lane et al., 1996), dap-4gm fails to drive epidermal expression (D). However, dap-4gm is expressed in the peripheral nervous system (F) in a subset of the cells that express dap-1gm (E). Wild-type embryos (G) and homozygous dap9 embryos (H) were labeled with anti-DAP at stage 13. dap expression in the nervous system is almost completely eliminated by the dap9 deletion.

 


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  		Fig. 6. The dap regulatory region contains a complex array of independent elements. Embryos with the lacZ reporter constructs dap-2l (A-C), dap-3l (D-F), dap-4l (G-I), dap-5l (J-L), dap-12l (M), dap-15l (N,Q), and dap-15l-A8 (O,R) at late stage 11 (A,B,D,E,G,H,J,K,M-R) or stage 14 (C,F,I,L) were labeled with anti-ß-galactosidase antibodies (A-O,Q,R, green in C,F,I,L) and double labeled with anti-Futsch/22C10 (red in C,F,I,L) to identify cells in the peripheral nervous system. The merged images (C,F,I,L) show a region from two abdominal segments with the lateral chordotonal organs (lch). The regulatory region present in dap-2l drives expression in the epidermis (A), in the anterior and posterior midgut (amg, pmg) and the interstitial cell progenitors (icp) (B, amg, pmg, icp) and in some cells of the peripheral nervous system (C, lch). The three subfragments of this region introduced into dap-3l, dap-4l and dap-5l drive different aspects of the dap-2l expression pattern. dap-3l is expressed in the peripheral nervous system (D,F), dap-4l primarily in the icp (H) and dap-5l in epidermis (J) and midgut (K). Global expression in the epidermis is driven by an element present in dap-12l (M). An element with ABD-B-binding sites (conserved A8 block, see Fig. 4) present in dap-15l (N) and deleted in dap-15l-A8 (O) results in early and high level expression in the prospective posterior spiracle region (ps, arrowheads in J,M-O) which is specified by Abd-B. prd-GAL4 driven ectopic expression of UAS-Abd-B does not induce dap-15l-A8 expression (R), while it readily induces ectopic expression of dap-15l (Q, stars) and of the endogenous dap gene (P, stars) as detected by in situ hybridization.

 


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  		Fig. 7. ABD-B binds to the dap regulatory region in vitro. EMSA was performed after incubating radiolabeled double stranded oligonucleotides with either buffer (none) or reticulocyte lysate that contained ABD-B protein (ABD-B) or which did not contain ABD-B protein (mock). ABD-B binds to the wild-type sequence present in the A8 block of the dap gene of Drosophila virilis (Dv-wt) and Drosophila melanogaster (Dm-wt). However, ABD-B does not bind when the sequences corresponding to the ABD-B core binding motif are mutated within this region (Dm-mut). The specificity of the binding to Dm-wt was further confirmed by experiments in which a ten- or one hundredfold excess of unlabeled oligonucleotides were added to the binding reaction as competitors (comp). Dm-wt but not Dm-mut acted as a competitor.

 

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