Analysis of an even-skipped rescue transgene reveals both composite and discrete neuronal and early blastoderm enhancers, and multi-stripe positioning by gap gene repressor gradients - PubMed (original) (raw)

Analysis of an even-skipped rescue transgene reveals both composite and discrete neuronal and early blastoderm enhancers, and multi-stripe positioning by gap gene repressor gradients

M Fujioka et al. Development. 1999 Jun.

Abstract

The entire functional even-skipped locus of Drosophila melanogaster is contained within a 16 kilobase region. As a transgene, this region is capable of rescuing even-skipped mutant flies to fertile adulthood. Detailed analysis of the 7.7 kb of regulatory DNA 3' of the transcription unit revealed ten novel, independently regulated patterns. Most of these patterns are driven by non-overlapping regulatory elements, including ones for syncytial blastoderm stage stripes 1 and 5, while a single element specifies both stripes 4 and 6. Expression analysis in gap gene mutants showed that stripe 5 is restricted anteriorly by Krüppel and posteriorly by giant, the same repressors that regulate stripe 2. Consistent with the coregulation of stripes 4 and 6 by a single cis-element, both the anterior border of stripe 4 and the posterior border of stripe 6 are set by zygotic hunchback, and the region between the two stripes is 'carved out' by knirps. Thus the boundaries of stripes 4 and 6 are set through negative regulation by the same gap gene domains that regulate stripes 3 and 7 (Small, S., Blair, A. and Levine, M. (1996) Dev. Biol. 175, 314-24), but at different concentrations. The 3' region also contains a single element for neurogenic expression in ganglion mother cells 4-2a and 1-1a, and neurons derived from them (RP2, a/pCC), suggesting common regulators in these lineages. In contrast, separable elements were found for expression in EL neurons, U/CQ neurons and the mesoderm. The even-skipped 3' untranslated region is required to maintain late stage protein expression in RP2 and a/pCC neurons, and appears to affect protein levels rather than mRNA levels. Additionally, a strong pairing-sensitive repression element was localized to the 3' end of the locus, but was not found to contribute to efficient functional rescue.

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Figures

Fig. 1

Fig. 1

A deletion analysis of the eve 3′ region. Numbers along the top show distance (in kb) to the transcription start site of the eve locus. Regions of the sequence included in each construct are indicated by thick lines, and deleted regions by thin lines. All fragments were placed downstream of an eve promoter-lacZ reporter in a pCaSpeR-based vector (see Materials and Methods). Five to ten independent transgenic lines were analyzed for each construct. Boxes at the bottom indicate minimal elements required to drive the following lacZ expression: _ftz_-like, 7 stripes in the even-numbered parasegments; EL, EL neurons in the central nervous system; CQ, GMC 7-1a and CQ neurons; early APR, early anal plate ring; late APR, late anal plate ring; 4/6, blastoderm stripes 4 and 6; muscle, mesodermal precursors; 1, blastoderm stripe 1; 5, blastoderm stripe 5; RP2,a/pCC, GMCs 4-2a and 1-1a, and neurons RP2, aCC and pCC. PSR indicates the region causing pairing-enhanced repression of the mini-white gene.

Fig. 2

Fig. 2

Elements for early stripes 1, 5 and 4+6 are negatively regulated by gap genes. In situ hybridization was performed for lacZ mRNA (blue), followed by staining with anti-Eve antibody (orange). (A) lacZ expression driven by the region +6.6 to +7.4 kb; (B) by the region +7.4 to +8.4 kb; (C) by the region +6.6 to +8.4 kb; (D) by the region +4.0 to 5.2 kb. The transgenic lines in C and D were crossed into several gap gene mutant backgrounds and stained as above. Staining patterns in these mutant embryos are shown below the corresponding wild-type pattern: hb (E,F), Kr (G,H), kni (I,J), gt (K,L) and tll (M,N). A summary of the apparent gap gene regulatory influences on stripes 1, 4, 5 and 6 is diagrammed at the bottom.

Fig. 3

Fig. 3

Isolated elements drive various components of tissue-specific expression. In situ hybridization was performed for lacZ mRNA (blue), followed by staining with anti-Eve antibody (orange). (A) lacZ expression in GMC 7-1a progeny at early stage 11 driven by the region + 3.5 to +4.3 kb. (B) lacZ expression in CQ neurons at stage 15 in the same transgenic line as in A. (C) lacZ expression in neurons RP2, aCC, and pCC at stage 11 driven by the region +7.9 to +9.2 kb. Note that at this stage lacZ expression is strong. (D) lacZ expression in RP2, aCC and pCC neurons at stage 15 in the same transgenic line as in C. Overall expression of lacZ at this stage is faint. (E) lacZ expression in EL neurons at stage 15 driven by the region +1.9 to +3.2 kb. (F) lacZ expression in the anal plate ring at stage 12 driven by the region +2.6 to +4.8 kb. (G) lacZ expression in muscle precursor cells at stage 11 driven by the fragment +5.8 to +6.6 kb. (H) lacZ expression in the anterior portion of even-numbered parasegments driven by the region +1.5 to +2.6 kb.

Fig. 4

Fig. 4

The eve 3′ UTR is required for high level protein expression in RP2 and aCC/pCC neurons at later stages. (A-D) Wild type; (E-H) transgenic line carrying lacZ construct driven by the +7.9 to + 9.2 kb fragment, with the α-tubulin 3′ UTR (See Materials and Methods); diffuse ectopic expression due to a position effect is visible. (I-L) Transgenic line carrying lacZ construct driven by the same +7.9 to + 9.2 kb fragment, but with the eve 3′ UTR; again, there is diffuse ectopic expression, particularly at stage 11. (A,C,E,G,I,K) mRNA expression visualized by in situ hybridization with appropriate probe; (B,D,F,H,J,L) protein expression visualized by staining with appropriate antibodies. (A,C) eve mRNA; (B,D) Eve protein; (E,G,I,K) lacZ mRNA; (F,H,J,L) β-galactosidase protein. Expression in RP2, aCC and pCC neurons, at stage 11 (E,F,I,J); at stage 15 (G,H,K,L). Arrows, a representative neuron in a representative line, showing increased protein staining at stage 15 with the eve 3′ UTR (in L) relative to that with the α-tubulin 3′ UTR (in H).

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