Experimental investigations of eukaryotic enhancers suggest that multiple binding sites and trans-acting regulatory factors are often required for wild-type enhancer function. Genetic analysis of the stripe 2 enhancer of even-skipped (eve), an important developmental gene in Drosophila, provides support for this view. Given the importance of even-skipped expression in early Drosophila development, it might be predicted that many structural features of the stripe 2 enhancer will be evolutionarily conserved, including the DNA sequences of protein binding sites and the spacing between them. To test this hypothesis, we compared sequences of the stripe 2 enhancer between four species of Drosophila: D. melanogaster, D. yakuba, D. erecta and D. pseudoobscura. Our analysis revealed a large number of nucleotide substitutions in regulatory protein binding sites for bicoid, hunchback, Kruppel and giant, as well as a systematic change in the size of the enhancer. Some of the binding sites in D. melanogaster are either absent or modified in other species. One functionally important bicoid-binding site in D. melanogaster appears to be recently evolved. We, therefore, investigated possible functional consequences of sequence differences among these stripe 2 enhancers by P-element-mediated transformation. This analysis revealed that the eve stripe 2 enhancer from each of the four species drove reporter gene expression at the identical time and location in D. melanogaster embryos. Double staining of native eve protein and transgene mRNA in early embryos showed that the reporter gene mimicked native eve expression and, in every case, produced sharply defined stripes at the blastoderm stage that were coincident with eve stripe 2 protein. We argue that stripe 2 eve expression in Drosophila evolution can be viewed as being under constant stabilizing selection with respect to the location of the anterior and posterior borders of the stripe. We further hypothesize that the stripe 2 enhancer is functionally robust, so that its evolution may be governed by the fixation of both slightly deleterious and adaptive mutations in regulatory protein binding sites as well as in the spacing between binding sites. This view allows for a slow but continual turnover of functionally important changes in the stripe 2 enhancer.

REFERENCES

Ahringer
J.
(
1996
)
Posterior patterning by the Caenorhabditis elegans even-skipped homolog vab-7.
Genes Dev
10
,
1120
1130
Akashi
H.
(
1995
)
Inferring weak selection from patterns of polymorphism and divergence at ‘silent’ sites in Drosophila DNA.
Genetics
139
,
1067
1076
Arnone
M. I.
,
Davidson
E. H.
(
1997
)
The hardwiring of development: organization and function of genomic regulatory systems.
Development
124
,
1851
1864
Arnosti
D.
,
Barolo
S.
,
Levine
M.
,
Small
S.
(
1996
)
The eve stripe 2 enhancer employs multiple modes of transcriptional synergy.
Development
122
,
205
214
Bastian
H.
,
Gruss
P.
(
1990
)
A murine even-skipped homologue, Evx1, is expressed during early embryogenesis and neurogenesis in a biphasic manner.
EMBO J
9
,
1839
1852
Fujioka
M.
,
Jaynes
J. B.
,
Goto
T.
(
1995
)
Early even-skipped stripes act as morphogentic gradients at the single cell level to establish engrailed expression.
Development
121
,
4371
4382
Goto
T.
,
Macdonald
P.
,
Maniatis
T.
(
1989
)
Early and late periodic patterns of even-skipped expression are controlled by distinct regulatory elements that respond to different spatial cues.
Cell
57
,
413
422
Gray
S.
,
Szymanski
P.
,
Levine
M.
(
1994
)
Short-range repressionpermits multiple enhancers to function autonomously within a complex promoter.
Genes Dev
8
,
1829
1838
Hanes
S. D.
,
Riddihough
G.
,
Ish-Horowicz
D.
,
Brent
R.
(
1994
)
Specific DNA recognition and intersite spacing are critical for action of the bicoid morphogen.
Mol. Cell Biol
14
,
3364
3375
Harding
K.
,
Hoey
T.
,
Warrior
R.
,
Levine
M.
(
1989
)
Autoregulatory and gap response elements of the even-skipped promoter of Drosophila.
EMBO J
8
,
1205
1212
Jeffs
P. S.
,
Holmes
E. C.
,
Ashburner
M.
(
1994
)
The molecular evolution of the alcohol dehydrogenase and alcohol dehydrogenase-related genes in the Drosophila melanogaster species subgroup.
Mol. Biol. Evol
11
,
287
304
Jiang
J.
,
Kosman
D.
,
Ip
Y. T.
,
Levine
M.
(
1991
)
The dorsal morphogen gradient regulates the mesoderm determinant twist in early Drosophila embryos.
Genes Dev
5
,
1881
1891
Jiang
J.
,
Hoey
T.
,
Levine
M.
(
1991
)
Autoregulation of a segmentation gene in Drosophila: Combinatorial interaction of the even-skipped homeo box protein with a distal enhancer element.
Genes Dev
5
,
265
277
Kenyon
C.
(
1994
)
If birds can fly, why can't we? Homeotic genes and evolution.
Cell
78
,
175
180
Kimura
M.
(
1981
)
Possibility of extensive neutral evolution under stabilizing selection with special reference to non-random usage of synonymous codons.
Proc. Natl. Acad. Sci. USA
78
,
5773
5777
Langland
J. A.
,
Carroll
S. B.
(
1993
)
Conservation of regulatory elements controlling hairy pair-rule stripe formation.
Development
117
,
585
596
Lawrence
P. A.
,
Johnston
P.
,
Macdonald
P.
,
Struhl
G.
(
1987
)
Borders of parasegments in Drosophila embryos are delimited by the fushi tarazu and even-skipped genes.
Nature
328
,
440
442
Ludwig
M. Z.
,
Kreitman
M.
(
1995
)
Evolutionary dynamics of the enhancer region of even-skipped in Drosophila.
Mol. Biol. Evol
12
,
1002
1011
Ludwig
M. Z.
,
Tamarina
N. A.
,
Richmond
R. C.
(
1993
)
Localization of sequences controlling the spatial, temporal, and sex-specific expression of the esterase 6 locus in Drosophila melanogaster adults.
Proc. Natl. Acad. Sci. USA
90
,
6233
6237
Ma
X.
,
Yuan
D.
,
Diepold
K.
,
Scarborough
T.
,
Ma
J.
(
1996
)
The Drosophila morphogenic protein Bicoid binds DNA cooperatively.
Development
122
,
1195
1206
Nusslein-Volhard
C.
,
Kluding
H.
,
Jurgens
G.
(
1985
)
Genes affecting the segmental subdivision of the Drosophila embryo.
Cold Spring Harbor Symp. Quant. Biol
50
,
145
154
Patel
N. H.
,
Ball
E. E.
,
Goodman
C. S.
(
1992
)
Changing role of even-skipped during the evolution of insect pattern formation.
Nature
357
,
339
342
Patel
N. H.
,
Condron
B. G.
,
Zinn
K.
(
1994
)
Pair-rule expression patterns of even-skipped are found in both short-and long-germ beetles.
Nature
367
,
429
434
Robertson
H. M.
,
Preston
C. R.
,
Phillis
R. W.
,
Johnson-Schlitz
D. M.
,
Benz
W. K.
,
Engels
W. R.
(
1988
)
A stable genomic source of P element transposase in Drosophila melanogaster.
Genetics
118
,
461
470
Ruiz i Altaba
A.
,
Melton
D. A.
(
1989
)
Involvement of the Xenopus homeobox gene Xho3 in pattern formation along the anterior-posterior axis.
Cell
57
,
317
326
Small
S.
,
Blair
A.
,
Levine
M.
(
1992
)
Regulation of even-skipped stripe 2 in the Drosophila embryo.
EMBO J
11
,
4047
4057
Small
S.
,
Arnosti
D. N.
,
Levine
M.
(
1993
)
Spacing ensures autonomous expression of different stripe enhancers in the even-skipped promoter.
Development
119
,
767
772
Small
S.
,
Blair
A.
,
Levine
M.
(
1996
)
Regulation of two pair-rule stripes by a single enhancer in the Drosophila embryo.
Dev. Biol
175
,
314
324
Sommer
R.
,
Tautz
D.
(
1991
)
Segmentation gene expression in the housefly Musca domestica.
Development
113
,
419
430
Stanojevic
D.
,
Small
S.
,
Levine
M.
(
1991
)
Regulation of a segmentation stripe by overlapping activators and repressors in the Drosophila embryo.
Science
254
,
1385
1387
Thummel
C. S.
,
Boulet
A. M.
,
Lipshitz
H. D.
(
1988
)
Vectors for Drosophila P element-mediated transformation and tissue culture transfection.
Gene
74
,
445
456
Williams
J. A.
,
Paddock
S. W.
,
Vorwerk
K.
,
Carroll
S. B.
(
1994
)
Organization of wing formation and induction of a wing-patterning gene at the dorsal/ventral compartment boundary.
Nature
368
,
299
305
Warrior
R.
,
Levine
M.
(
1990
)
Dose-dependent regulation of pair-rule stripes by gap proteins and the initiation segment polarity.
Development
110
,
759
767
This content is only available via PDF.