1 Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, WI
53706, USA
2 Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI 53706,
USA
3 Department of Ecology and Evolutionary Biology, Princeton University,
Princeton, NJ 08544, USA
4 Laboratory of Molecular Biology, University of Wisconsin-Madison, Madison, WI
53706, USA
* Author for correspondence (e-mail: sbcarrol{at}wisc.edu)
Accepted 4 October 2005
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SUMMARY |
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Key words: Ultrabithorax, Distal-less, Limb, Hox, Evolution, Peptide motif, Trichome, Abdominal tergite, Postnotal, Laterotergite, Gene targeting, Allelic replacement, Homologous recombination
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Introduction |
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Several studies have demonstrated strong sequence and functional
conservation of selector proteins across phylum-level evolutionary distances
(Halder et al., 1995;
Malicki et al., 1990
;
McGinnis et al., 1990
;
Zhao et al., 1993
). Highly
conserved regions have tended to be those most relevant for protein function.
For example, in Hox proteins, the DNA-binding homeodomain and the
`hexapeptide' or `YPWM' motif that interacts with the Extradenticle (Exd)
co-factor (Chang et al., 1995
;
Passner et al., 1999
) are both
well-conserved. However, most of the sequence of Hox proteins appears free to
vary across phyla, suggesting that the specific amino acid residues in these
regions contribute less to protein function. In several cases, selector
protein functions have evolved (Galant and
Carroll, 2002
; Grenier and
Carroll, 2000
; Hanks et al.,
1998
; Lamb and Irish,
2003
; Ronshaugen et al.,
2002
; Shiga et al.,
2002
) or evolution has co-opted selectors for derived functions
(Alonso et al., 2001
;
Lohr and Pick, 2005
;
Lohr et al., 2001
;
Stauber et al., 2002
). These
cases have generally involved regions outside of the DNA-binding domain and
have implicated synapomorphic (shared, derived) peptide motifs conserved in
subsets of related taxa (Hsia and
McGinnis, 2003
).
One of the most provocative cases of selector protein evolution correlates
the acquisition of limb repression capacity by the central class Hox selector
protein Ultrabithorax (Ubx) with the reduction of abdominal limb number in
insects (Galant and Carroll,
2002; Grenier and Carroll,
2000
; Ronshaugen et al.,
2002
). Whereas insect Ubx possesses strong limb repression
capacity when ectopically expressed in Drosophila melanogaster,
crustacean (Artemia francisana) and onychophoran (Acanthokara
kaputensis) Ubx do not. Sequences in the C terminus of Ubx are
responsible for much of this functional divergence. A. franciscana
Ubx possesses putative casein kinase II sites that modulate activity, whereas
all insect Ubx orthologs contain the highly conserved C-terminal `QA' motif
required for full Ubx repression activity
(Gebelein et al., 2002
;
Ronshaugen et al., 2002
). This
QA motif is capable of conferring limb repression activity when grafted onto
onychophoran Ubx (Galant and Carroll,
2002
).
The sufficiency of the QA motif to confer limb repression capacity suggests that its acquisition during early insect evolution could have played an important role in the evolution of insects lacking adult abdominal limbs. However, little is known about the role of the QA motif in normal development. For example, is the QA motif required for abdominal limb repression in insects? Is this motif dedicated to limb repression, or is it pleiotropic? What would be the phenotypic consequence of removing such a conserved part of an integral patterning gene?
To characterize the genetic and phenotypic role of the QA peptide motif of
Ubx, we have precisely deleted this motif at the endogenous Ubx locus
via allelic replacement in D. melanogaster
(Rong et al., 2002). The
effects of deleting the QA motif were strong in some tissues but barely
detectable in others. This finding of differential pleiotropy suggests that
peptide motifs in selector proteins can conditionally modulate selector
activity and need not be uniformly pleiotropic across all tissues. We also
find the requirement for the QA motif for limb repression to be dose dependent
and partially redundant with the Abdominal-A (Abd-A) Hox protein, suggesting
that redundancy and the additive contributions of peptide motifs play
important roles in modulating the phenotypic consequences of selector protein
evolution.
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Materials and methods |
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Several lines containing the above construct were crossed to
y1 w; P{ry+t7.2=70FLP}11
P{v+t1.8=70I-SceI}2B nocSco/CyO,
S2 virgin females, and Flp and I-SceI were induced by
heat-shocking 0- to 3-day-old progeny for 1 hour at 38°C
(Rong and Golic, 2000). F1
virgins with mosaic germlines (non-CyO, two or three per vial) were then
crossed to w1118; P{ry+t7.2=70FLP}10 males and
Flp was induced by heat-shocking 0- to 3-day-old progeny. Owing to concerns
about potential difficulty activating whs in the eye from
a location within or near the Bithorax Complex (BX-C)
(Bender and Hudson, 2000
),
developing larvae and pupae were further heat-shocked every 3 days until
eclosion. Progeny were screened for non-mosaic red eyes, which were regarded
as putative insertions (Rong and Golic,
2001
).
Six putative insertions were obtained from 828 vials, four of which mapped
to the 3rd chromosome. Only targeted duplications that retained one complete
copy of Ubx+ and a partial duplication of the
UbxQA allele [a subset of Class
II events (Rong and Golic,
2000
)] were useful for subsequent reduction. Candidate lines were
screened via PCR and restriction digestion for introduction of the novel
AvrII site and loss of the novel XbaI site (which should
have been eliminated during the repair of the double-strand break at the
I-SceI site). Insertions were also screened to ensure the presence of
a copy of Ubx+ with no AvrII site and to ensure
both junctions with vector backbone were intact. Only two independent Class II
insertions fit all these criteria; one insertion line (III65A) was
selected for reduction to single copy (Fig.
1B).
III65A males were crossed to w1118;
P{v+t1.8=hs-I-CreI.R}1A Sb1/TM6 virgin females, and
0- to 3-day-old progeny were heat-shocked for 1 hour at 36°C
(Rong et al., 2002). F1 mosaic
male progeny were crossed to w; TM3, Sb/TM6B,
AntpHu, Tb virgin females. Single F2 males with white eyes
were recovered as TM6B-balanced stocks for analysis; each reduction line was,
thus, isogenous for its 3rd chromosome. Reduction homozygotes were analyzed
via PCR and restriction digestion as above, and only those retaining either
unduplicated Ubx+ or
Ubx
QA alleles were analyzed
further. To confirm that the alleles contained no artifactual mutations, we
mapped the putative crossover points of three Ubx+ (lines
A31, B2 and C3) and four
Ubx
QA (lines A30, B1,
C4 and E7) reduction alleles by sequencing overlapping PCR
products from the entire manipulated region of Ubx and surrounding
sequence (see Table S1 in the supplementary material). This assay was possible
because of the presence of several well-spaced non-coding single nucleotide
polymorphisms between the targeted chromosome and the source of the P1 clone.
As each reduction is easily explained by a small odd number of crossovers, the
alternative single-strand annealing hypothesis
(Dolezal et al., 2003
;
Rong et al., 2002
), where each
difference between strands is retained or lost randomly, may not
mechanistically explain the reduction events. Primer sequences, PCR conditions
and further information are available upon request.
Outcrossing
We introduced X and 2nd chromosomes from Oregon-R (G.
Boekhoff-Falk laboratory stock) and WI129
(Kopp et al., 2003) lines into
several reduction lines and a Sbsbd Ubx6.28 e
line containing a null allele of Ubx to assess the effects of genetic
background in a controlled manner. We used standard genetic manipulations with
balancer chromosomes to prevent recombination and track chromosomes. Thus,
each stock created had X and 2nd chromosomes from either
Oregon-R or WI129 (referred to as genetic background below),
isogenous experimental 3rd chromosomes generated when its Ubx allele
was created, and unknown Y and 4th chromosomes (which were expected to
contribute little to phenotype). The chromosome containing
Sbsbd Ubx6.28 e was treated in the same manner,
except that it was balanced with TM6B, AntpHu, Tb.
Homozygotes from lines A31 (Ubx+), B2
(Ubx+), A30
(Ubx
QA), B1
(Ubx
QA) and E7
(Ubx
QA) were quantified in the
WI129 background, and crosses between the above Ubx alleles
and the above Ubx chromosome were used to obtain
Ubx/Ubx+ and
Ubx
QA/Ubx
adults for quantification. Lines B2 (Ubx+),
C3 (Ubx+), A30
(Ubx
QA), B1
(Ubx
QA) and E7
(Ubx
QA) were quantified in the
Oregon-R background, and crosses between the above Ubx
alleles and the above Ubx chromosome were used to
obtain Ubx/Ubx+ and
Ubx
QA/Ubx
adults for quantification. All flies were raised on sugar food at 25°C
with 70-80% humidity on a 12-hour light cycle.
|
Embryonic and larval phenotypic characterization
First larval stage cuticles were prepared essentially as described
previously (Stern and Sucena,
2000). Denticle belts were photographed using dark-field
microscopy, and KOs were photographed using phase-contrast microscopy. Embryos
were stained for Distal-less (Dll) as described previously
(Panganiban et al., 1995
).
Df(3R)Ubx109 (Lewis,
1963
), a deficiency deleting all of Ubx and
abd-A (Bender et al.,
1983
), was used in Ubx
abd-A assays.
Sub-epidermal adult leg tissue
Adults and dead pupae
(UbxQA/Ubx
and especially
Ubx
QA/Ubx
abd-A often died as pupae) were dissected, and their A1
cavity was examined for sub-epidermal leg tissue. Only growths with clearly
identifiable bristles were counted as leg tissue and were removed and
photographed using bright-field microscopy
(Stern and Sucena, 2000
). Rare
(1.6%; 8/490) cases where the positions of legs were shifted towards the
posterior were not counted as producing ectopic A1 leg tissue because no
additional leg tissue was formed.
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Results |
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The scheme for creating the targeted mutation is shown in
Fig. 1. The targeting construct
was first randomly inserted into the genome on the X chromosome
(Fig. 1A). In the first
`targeting' step, Flp and I-SceI catalyzed the excision and subsequent
insertion of the construct into the Ubx locus via homologous
recombination. Rarely, this produced a partial duplication of the Ubx
locus with one complete wild-type Ubx+ copy and the
3' end of a mutant UbxQA
copy [specifically, a targeted `Class II'
(Rong and Golic, 2000
)
insertion that retained the introduced mutations only in the 3'
duplicated copy; Fig. 1B]. Two
such insertions were recovered after screening 828 vials.
In the second `reduction' step, I-CreI efficiently created a double-strand
break between the duplicated regions of Ubx, which was then repaired
via homologous recombination to leave either a single unaltered
Ubx+ allele or a single mutant
UbxQA allele with the premature
stop codon deleting the QA motif (Fig.
1C). After verification by PCR, restriction digestion and
sequencing, one insertion meeting the criteria shown in
Fig. 1B was reduced to several
single-copy Ubx+ and
Ubx
QA alleles
(Fig. 1C). We screened and
confirmed the molecular identity and fidelity of several alleles using a PCR
and restriction digestion assay. Ultimately, the entire region covered by the
targeting construct was sequenced for at least three independently generated
reduction lines for both the Ubx+ and
Ubx
QA alleles, and only those
with the desired changes were analyzed further.
The UbxQA allele is pleiotropic
While the QA motif was originally characterized for its role in limb
repression (Galant and Carroll,
2002; Ronshaugen et al.,
2002
), analysis of the homozygous
Ubx
QA/Ubx
QA
phenotype revealed the QA motif to be highly pleiotropic and involved in
several Ubx functions. As will be described below in detail, we found a strong
requirement for QA function in some tissues and much less of a requirement in
others.
Ubx most strongly influences the development of metathoracic (T3)
and first abdominal segment (A1) structures. Ubx loss-of-function
mutations transform these tissues towards more anterior identities
(Bender et al., 1983;
Lewis, 1963
;
Lewis, 1978
), while ectopic
expression of Ubx in anterior tissues transforms them toward T3 or A1 identity
(Mann and Hogness, 1990
). In
dipterans, Ubx is required to sculpt the T3 hindwing into a reduced,
balloon-shaped haltere. Modest reductions in Ubx activity result in
increased haltere size and ectopic bristles, making this tissue the most
obvious phenotypic readout of reduced Ubx activity. Reduction of
Ubx to a single genetic dose in heterozygotes for null alleles
(Ubx/Ubx+) results in halteres that are
about twice the volume of wild type and often have multiple ectopic bristles
(Fig. 2A,C).
|
The QA motif plays a partially redundant role in limb repression
Previous studies have suggested that the QA motif played an important role
in limb repression (Galant and Carroll,
2002; Gebelein et al.,
2002
; Ronshaugen et al.,
2002
). However,
Ubx
QA/Ubx
QA
adults had a normal complement of limbs. This result suggested the QA motif
was not strictly required for limb repression and seemed to agree with
previous ectopic expression assays that found that deleting the QA motif
caused only a slight reduction in the ability of Ubx to repress thoracic limb
primordia and the associated sensory Keilin's organs (KOs)
(Gebelein et al., 2002
;
Ronshaugen et al., 2002
). It
was possible that the
Ubx
QA/Ubx
QA
adult phenotype was milder than embryonic or larval phenotypes because the
limb repression developmental program was more robust at later stages or
because phenotypically extreme animals died early in development. Although
Ubx
QA/Ubx
QA
larvae had mild A1 denticle belt transformations towards intermediate T3/A1
identity, ectopic A1 KOs were never detected
(Fig. 3A,B). Similarly,
Ubx
QA/Ubx
QA
embryos never produced ectopic limb primordia, as assessed by examination of
the expression pattern of the appendage selector and marker protein Dll
(Cohen, 1990
)
(Fig. 4A,B). Thus, the
phenotype of
Ubx
QA/Ubx
QA
adults and embryos were both wild type with respect to abdominal limb
repression.
Ectopic expression of Ubx is sufficient to repress thoracic limbs and
transform segments to A1 identity (Mann
and Hogness, 1990). However, the requirement of Ubx to
pattern A1 is partially masked by the expression of its Hox paralog
abd-A in the posterior compartment
(Karch et al., 1990
). For
example, Ubx
abd-A/Ubx abd-A
larvae formed full three-bristle ectopic KOs on A1
(Fig. 3F), but
Ubx/Ubx larvae formed only
partial two-bristle ectopic KOs on A1 (Fig.
3E) (Lewis, 1978
).
Similarly, complete ectopic limb primordia formed in A1 of
Ubx abd-A/Ubx
abd-A embryos (Fig.
4F) (Simcox et al.,
1991
; Vachon et al.,
1992
), but only incomplete ectopic limb primordia formed in A1 of
Ubx/Ubx embryos
(Fig. 4E). Moreover, fully
formed ectopic A1 adult legs have been recovered only from individuals
carrying strong bithoraxoid (bxd) loss-of-function
regulatory alleles in Ubxbxd/Ubx
abd-A adults where the genetic doses of both
Ubx and abd-A were reduced
(Bender et al., 1983
;
Lewis, 1963
). We wondered
whether the lack of strict necessity for the QA motif in limb repression might
be due to the additive contribution of other peptide motifs within Ubx and/or
redundancy with Abd-A.
|
|
Similarly, comparison of Ubx/Ubx+ and
UbxQA/Ubx
QA
embryos with
Ubx
QA/Ubx
embryos (Figs 3,
4) suggests that other peptide
motifs within Ubx contribute to the repression of Dll at normal expression
levels, which has also been inferred in previous studies
(Gebelein et al., 2002
;
Ronshaugen et al., 2002
).
However, these motifs are not sufficient for full limb repression when both
the QA motif is removed and the genetic dose is reduced in
Ubx
QA/Ubx
embryos. In the accompanying manuscript
(Tour et al., 2005
), the
quantitative contributions of two motifs, the YPWM motif and the highly
conserved YRXFPLXL motif, are demonstrated. Additionally, our loss-of-function
data are in agreement with the steep sigmoidal curves proposed
(Tour et al., 2005
) for Ubx
activity and limb repression.
In rare instances, ectopic A1 limb primordia survived through metamorphosis
and produced sub-epidermal adult leg tissue in A1
(Fig. 4G,H). In
UbxQA/Ubx
abd-A flies, the leg tissue could undergo a great deal
of differentiation such that most bristles had bracts and diverse
morphologies, such as claws and transverse bristle rows
(Fig. 4H). The extensive
differentiation achieved suggests that this genotype allows the production of
nearly complete abdominal legs.
The QA motif is preferentially required in several tissues
Ectopic leg tissues may have failed to evert and remained sub-epidermal
because they were blocked from doing so by the A1 pleurum, which formed
normally in
UbxQA/Ubx
abd-A flies. By contrast, the A1 ventral histoblast
nests that form the ventral and lateral pleural epidermis
(Madhavan and Madhavan, 1980
)
are deleted in Ubxbxd/Ubx
abd-A larvae capable of producing A1 legs
(Frayne and Sato, 1991
). The
ability of
Ubx
QA/Ubx
abd-A flies to form normal A1 pleural tissue, even as
they failed to repress A1 limb formation, suggests that the QA motif might be
preferentially required for a subset of tissues or target genes under Ubx
control.
We reasoned that comparing the phenotype of
UbxQA/Ubx
QA
and Ubx/Ubx+ adults in several tissues
could provide a rigorous test of this hypothesis. The haltere phenotype
indicated a clear genotypic series for Ubx activity in the haltere
with respect to both size and bristle number:
Ubx+/Ubx+>Ubx
QA/Ubx
QA>Ubx/Ubx+>Ubx
QA/Ubx
(Fig. 2). If the QA motif were
uniformly pleiotropic and similarly required across all tissues and Ubx
targets, this genotypic series would hold true for all tissues examined.
However, if the QA motif were differentially pleiotropic and preferentially
required in a subset of tissues, the placement of
Ubx
QA/Ubx
QA
in the genotypic series might differ among tissues. This was, in fact, the
case.
In addition to its role in reducing the dipteran T3 hindwing to a haltere,
Ubx represses the formation of other tissues derived from the dorsal T3 disc,
such that the adult dorsal mesothoracic (T2) structures nearly abut the adult
dorsal A1 structures (anterior histoblast-derived tergite) with only a thin
band of T3 laterotergite separating them
(Fig. 5A). This function is
most clearly illustrated by the phenotype of the four-winged fly, which has
nearly all of its dorsal thorax duplicated because of the complete
transformation of the dorsal T3 disc to dorsal T2 identity
(Lewis, 1963). More moderate
laterotergite transformations with extensive ectopic `postnotal' tissue (named
for the dorsal and medial position of the ectopic tissue relative to the notum
of the haltere; not named for the postnotum, which is T2 tissue) are
characteristic of homozygotes and hemizygotes for strong
Ubxpbx and Ubxbxd alleles but have not
been described in Ubx/Ubx+ flies
(Bender et al., 1983
;
Lewis, 1963
). We found that
Ubx/Ubx+ adults only very rarely
developed limited postnotal tissue (Fig.
5B), but
Ubx
QA/Ubx
QA
adults had moderate transformations of the T3 laterotergite towards T2
identity with up to three ectopic bristles per half-laterotergite
(Fig. 5C). This result suggests
a specific requirement for the QA motif in this region of the dorsal T3 disc,
a requirement further supported by the extensive postnotal tissue in
Ubx
QA/Ubx
adults with up to a dozen ectopic bristles per half-laterotergite
(Fig. 5D). The reversal of the
order of the
Ubx
QA/Ubx
QA
and Ubx/Ubx+ genotypes in the haltere
and postnotal region (compare Fig.
2B,C with Fig.
5C,B) was consistent in different genetic backgrounds and, thus,
allows us to reject the null hypothesis that the QA motif plays uniform
pleiotropic roles in favor of the differential pleiotropy hypothesis.
|
|
The small ventral patch, unusually high amount of distal trichomes present
on our Ubx+/Ubx+ T3p, and the slight
quantitative reductions in the size of the naked valley on our
Ubx+/Ubx+ T2p relative to previously studied
`wild-type' lines (Stern,
1998) suggests that the 3rd chromosome targeted in our study
contains other genetic variation affecting trichome patterning. Most, if not
all, of this variation was recessive. Despite this variation, the loss of the
QA motif had a strong effect on both T2p and T3p trichome patterning whether
the recessive variation in our targeted chromosome was homozygous
(Ubx+/Ubx+ versus
Ubx
QA/Ubx
QA;
compare Fig. 7A,E with
Fig. 7C,G) or heterozygous
(Ubx/Ubx+ versus
Ubx
QA/Ubx;
compare Fig. 7B,F with
Fig. 7D,H). Moreover, the
Ubx
QA/Ubx
QA
phenotype is more severe than is ever seen among numerous
Ubx null alleles when heterozygous (D.L.S.,
unpublished) (Stern, 1998
).
These data support the conclusion that removing the activities of the QA motif
had a greater effect on the capacity of Ubx to repress leg trichomes than
reducing Ubx activity by half.
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Discussion |
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|
Hox selector proteins, such as Ubx, may accomplish their diverse genetic
and regulatory functions by using distinct peptide motifs for the regulation
of subsets of target genes. Ubx is expressed throughout development in many
tissue types (White and Wilcox,
1984) and engages in both direct activation and repression of
multiple target genes (Beachy et al.,
1988
; Capovilla et al.,
1994
; Galant et al.,
2002
; Hersh and Carroll,
2005
; Krasnow et al.,
1989
; Vachon et al.,
1992
), suggesting that distinct activation and repression motifs
exist. In the accompanying study, Tour et al. describe at least three motifs
that quantitatively and differentially affect the expression of specific
target genes when ectopically expressed, suggesting that Ubx contains several
differentially pleiotropic peptide motifs that influence the expression of Ubx
target genes. The YPWM motif interacts with Exd
(Chang et al., 1995
;
Passner et al., 1999
) and is
differentially pleiotropic at least in part because nuclear Exd is not present
in all regions where Ubx is active and required
(Aspland and White, 1997
).
Detailed studies on the derived Hox protein Fushi Tarazu (Ftz) have also
demonstrated that beetle (Tribolium castaneum) Ftz has distinct
homeotic and segmentation functions that are differentially mediated by a YPWM
motif and a nuclear receptor box or `LXXLL' motif, respectively
(Lohr and Pick, 2005
;
Lohr et al., 2001
). By
contrast, use of different peptide motifs on different targets may not be a
necessary feature of selector proteins dedicated to one cell type, such as the
mouse photoreceptor selector Crx (Livesey
et al., 2000
), or dedicated to either activation or repression,
such as the posterior compartment selector Engrailed (En)
(Courey and Jia, 2001
;
John et al., 1995
;
Smith and Jaynes, 1996
).
Redundancy with Abd-A
The QA motif is not strictly necessary for limb repression in A1 at any
stage of development because of the additive roles played by other peptide
motifs in Ubx and because it is partially redundant with the Hox protein Abd-A
(Figs 3,
4). We observed extensive limb
derepression in A1 in embryos and adults when both the QA motif was absent and
when the Ubx and abd-A doses were reduced but not when
either was manipulated singly. The partial redundancy of the Ubx and Abd-A in
limb repression is mechanistically explained by their direct repression of the
Dll limb primordia enhancer through the same binding site
(Gebelein et al., 2004;
Vachon et al., 1992
). The
absence of ectopic limb primordia or limbs on the more posterior abdominal
segments of
Ubx
QA/Ubx
abd-A flies suggests that the higher level and broader
expression of Abd-A (Karch et al.,
1990
) are sufficient to repress limb formation in more posterior
segments (A2-A7).
Differential pleiotropy and redundancy may facilitate selector protein evolution
Compared with the relatively rapid turnover of cis-regulatory
elements, the evolution of selector protein function appears to be a rare
occurrence, owing, at least in part, to the pleiotropic consequences of
mutations in protein coding regions
(Carroll, 2005;
Mann and Carroll, 2002
). By
contrast, many cis-regulatory elements have a modular architecture
and mutations in these elements can more easily adjust the expression of a
single gene in a single tissue. Analogously, the differential pleiotropy we
observed for the QA motif may provide a degree of modularity to some selector
proteins. If natural selection can quantitatively alter a specific trait by
modifying selector protein sequence and accrue minimal pleiotropic fitness
trade-offs in other tissues, this route might be taken if the fitness gains
are great compared with any offsets, if genetic suppressors arise, or if it is
the most readily available path.
Redundancy may further limit the number of functions subject to intense
purifying selection. For example, if a selector protein performs n
functions but n1 are redundant with the function of other
selectors, natural selection may be free to modulate the nth function
through coding changes with limited effects on the other traits. The two most
extreme cases of the evolution of Hox protein function have involved Hox genes
that were co-opted for other regulatory functions
(Alonso et al., 2001;
Lohr and Pick, 2005
;
Lohr et al., 2001
;
Stauber et al., 2002
). The
ancestral Hox3 and Ftz expression domains both overlapped with multiple Hox
proteins (Hughes and Kaufman,
2002
), suggesting they were at least partially redundant with
neighboring Hox genes during their co-option. We propose that the rare
instances of the evolution of selector protein function tend to be facilitated
when a combination of redundancy and the differential pleiotropy of peptide
motifs alleviates the constraints on selector protein evolution.
The power of purifying selection
There is an intuitive but misleading contradiction between the
UbxQA/Ubx
QA
phenotype and the macroevolutionary time-span over which the motif has been
conserved. The QA motif has been conserved in all insects, but the phenotype
we observed in
Ubx
QA/Ubx
QA
D. melanogaster affected traits that vary between insects, not between
insects and other arthropods. This suggests that, as Ubx has acquired
different genetic targets in different insect lineages
(Tomoyasu et al., 2005
;
Weatherbee et al., 1999
), so
has the QA motif. We have shown that some of the phenotypic effects of
deleting the QA motif are mitigated by the contributions of other motifs,
redundancy with Abd-A, and differential pleiotropy. Yet, we have also argued
that these same forces could facilitate the evolution of selector function
under the right combination of circumstances. Why, then, is the QA motif still
present in all insect orders studied?
Sudden variation in all of the traits governed by the pleiotropic QA motif
would probably not be tolerated in a natural, competitive environment. Even
though
UbxQA/Ubx
QA
flies are viable and fertile and have a modest phenotype from a developmental
perspective, natural selection acts on genetic variation that has a selection
coefficient as small as the inverse of twice the effective population size
(Li, 1997
;
Wright, 1931
). For insects,
which are likely to have effective population sizes of 105 to
106 (Lynch and Conery,
2003
), the difference between the production of an average of one
fewer offspring out of a million literally makes the difference between
variation that is tolerated and that which is selected against. Therefore,
despite a turnover of targets and traits governed, pleiotropic peptide motifs
that subtly modulate selector protein function can experience consistent
purifying selection that preserves them across vast periods of time.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/23/5261/DC1
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REFERENCES |
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