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Cell, Vol. 47, 113-122,
October
10. 1988, Copyright
0 1986 by Cell Press
The Localization and Regulation of Antennapedia
Protein Expression in Drosophila Embryos
Sean B. Carroll, Robert A. Laymon:
Michael A. McCutcheon,t
Peter D. Riley,
and Matthew P. Scott
Department of Molecular, Cellular and
Developmental Biology
University of Colorado at Boulder
Boulder, Colorado 80309
Summary
The homeotic Antennapedia (Ant@ gene of Drosophila is required for the normal differentiation of the thoracic segments during embryonic development and
metamorphosis.
Antibodies to a recombinant Anfp
protein were used to localize the protein in whole
mount embryos. Antp is expressed in the nuclei of
cells of the thoracic embryonic epidermis and several
segments of the ventral and peripheral nervous systems. Analysis of Anfp expression in mutant embryos
revealed three levels of Anfp regulation by genes of
the bithorax complex, pleiotropic homeotic loci, and
Anfp itself. The distributions
of the Anfp and the
Ultrabithorax
(Ubx) proteins in doubly-labeled
embryos suggest that the l&x protein may be one direct
negative regulator of Anfp gene expression.
Introduction
The genetic control of body segment diversification of Drosophila melanogaster involves the activity of genes within
two major complexes, the Antennapedia (ANT-C) (Kaufman et al., 1980) and bithorax (BX-C) (Lewis, 1978). Most
loci in these complexes are homeotic, that is, mutations in
them result in the respecification of certain segmental
identities. Homeotic genes in the ANT-C are required in
the head and thorax while BX-C genes are required in
parts of the thorax and abdomen. Previous studies have
shown that homeotic genes control differentiation of segments by being expressed in position-specific patterns:
sites of phenotypic gene function correspond, in general,
to sites of gene expression. Therefore two important questions are: one, how is position-specific expression attained and two, what are the molecular functions of the
gene products. We have used antibody probes for proteins
encoded by the Antennapedia (An@) homeotic locus, one
gene within the ANT-C, to examine issues related primarily to the first of these questions.
Anfp has a major role in the thorax both in promoting
the thoracic pathway and in preventing the development
of antenna1 or head structures (Struhl, 1981a; Kaufman
and Abbott, 1984). Dominant Antp mutations cause adult
’ Present address: Harvard University, Biological Laboratories, Cambridge, Massachusetts 02136.
t Present address: Department
of Biological Sciences, Stanford
University, Palo Alto, California 94305.
flies to develop with antennae transformed into mesothoracic legs. However, the dominant mutations have
been shown to represent abnormal gene function in the
head (Denell, 1973; Duncan and Kaufman, 1975; Struhl,
1981a; Hazelrigg and Kaufman, 1983). Normal head development does not require Antp function (Denell et al.,
1981; Struhl, 1981a; Kaufman and Abbott, 1984). Embryos
homozygous for null alleles of the locus exhibit transformations of the mesothorax and metathorax into hybrid
prothorax-head segments (Wakimoto and Kaufman, 1981;
Struhl, 1983; Sato et al., 1985) a phenotype which has
been difficult to interpret definitively. One interpretation is
that in Antp- embryos, parasegment 4 (posterior Tl plus
anterior T2) is transformed into parasegment 3, and parasegment 5 is transformed into a parasegment of mixed
identity that has characteristics of both thoracic and abdominal segments (Martinez-Arias, 1986). Effects of Antp
gene function on posterior T3 (anterior parasegment 6)
have also been observed (Sat0 et al., 1985). In addition,
in the absence of BX-C function, activity of Antp in the embryonic abdomen can be detected (Duncan, 1982b; Sato
et al., 1985).
Antp is a very large gene, spanning some 103 kb of DNA
(Garber et al., 1983; Scott et al., 1983), but RNA processing results in the production of two size classes of mature
RNA that are only about 3.5 kb and 5.0 kb in length. Sequence analysis of cDNA clones has revealed that only
1.1 kb of each of the RNAs is a protein coding region
(Schneuwly et al., 1986; Laughon et al., submitted). At
least four proteins are encoded; alternative RNA splicing
gives rise to largely identical coding sequences varying
by up to 17 codons (Bermingham, Laughon, and Scott, unpublished data). In situ hybridization of Anrp cDNA probes
to sectioned Drosophila tissue has indicated a complex
spatial pattern of Antp RNA distribution (Levine et al.,
1983; Martinez-Arias, 1986). The large amount of untranslated DNA and RNA in Antp suggests that most of the sequence functions as regulatory elements controlling the
complex pattern of gene expression seen during Drosophila development. There is good correspondence between
the segmental structures influenced by Antp and the
places where the RNA is expressed.
It has been proposed that cell fates are determined by
the array of homeotic genes active within each cell (Lewis,
1978). To test this hypothesis it is necessary to determine
where homeotic gene products are expressed. These
questions have been addressed with single cell resolution
only in studies of one homeotic gene, Ultrabithorax (Ubx),
a gene in the BX-C. Ubx protein is expressed at a similar
level in all of the cells of some parasegments, but is expressed at different levels in different cells within other
parasegments (White and Wilcox, 1984; Beachy et al.,
1985). Some of the complexity of the Ubx pattern is due
to regulation by other genes in the BX-C (Struhl and White,
1985). Although it is clear that multiple BX-C homeotic
genes are expressed in some parasegments, the resolution of in situ hybridization studies has not been adequate
Cell
114
b
a
M
C
P10slDMPlosKlM
I
I
Figure 1. Expression
coli and Production
Domains
of 6-Galactosidase-Antp
of Specific Antibodies
PlDslo
Fusion Proteins in E.
to the Amp-Encoded
lgtll (cI*~*~) lysogens containing the Antp inserts were induced at
42°C for 2 hr and cell lysates were prepared for electrophoresis on a
7.5% SDS-polyacrylamide
gel. Part of the gel was stained with Coomassie blue (A), and replicate lanes were transferred to nitrocellulose
and probed with affinity-purified antibodies against 6-galactosidase (B)
and the Antp portion of the hybrid protein (C). The molecular weight
markers in (A) are f%galactosidase (116 kd), phosphorylase b (97 kd),
bovine serum albumin (66 kd), and ovalbumin (45 kd). The dark arrows
indicate the position of the far less abundant full-length products of the
Mntp PlO and lAntp SlO fusions; the open arrows indicate the position of the major products of the fusions that lack the homeodomain
determinants.
to assess the spatial relationship between one homeotic
gene product and another on a cell by cell basis. Immunofluorescence with multiple antibody probes will help
in providing the answers.
Using antibodies against Anrp protein, we observed
that the protein is found predominantly within nuclei and
that the protein distribution during development is very dynamic. Double-label immunofluorescent analysis of wildtype and homeotic mutant embryos with Anrp antibodies
and a monoclonal antibody to the Ubx protein (White and
Wilcox, 1984) support the idea that the expression of
homeotic genes is under multigenic control and that Ubx
and other bithorax complex genes are among the regulators of Anrp expression (Hafen et al., 1984).
Results
Expression of the Antp Protein in E.
In order to generate antibodies directed
protein, gene fusions were constructed
pression vector to produce the protein
and Davis, 1983). Two subclones of the
coli
against the Anrp
in the hgtll exin E. coli (Young
Anrp pDmG1100
cDNA (Scott et al., 1983) were used in gene fusions. One
subclone contained 1,298 bp from the Pstl site to the 3’
end of the cDNA and the other contained 1,816 bp from
the Smal site to the end of the Anrp cDNA clone. Appropriate EcoRl linkers were ligated to these fragments to introduce the DNA in the proper reading frame into hgtll
for expression of f3-galactosidase hybrid proteins. The
phages hAntp PlO and hAntp SlO encode Anrp products
242 and 348 amino acids long, respectively, joined to 114
kd of 8-galactosidase protein.
Lysogens containing the two Anfp fusion phages were
induced to express the hybrid 8-galactosidase-Antpp proteins. Upon thermal induction, fusion proteins migrating
with apparent molecular weights of 135 kd and 150 kd
were produced (Figure la, open arrows) as the major species. These values are in agreement with the protein sizes
predicted from the DNA sequence (Schneuwly et al.,
1986; Laughon et al., submitted). However, immunological
analysis of the proteins produced by the lysogens revealed that the 135 kd and 150 kd bands are in fact not fulllength products. Anti+galactosidase
staining of nitrocellulose blots revealed that much smaller amounts of larger
fusion proteins (approximately 150 and 165 kd) were produced in each case (Figure lb, solid arrows). Antibodies
to the homeodomain, a highly conserved 60 amino acid
protein domain found in the C terminal part of the Anfp
protein (Schneuwly et al., 1986; Laughon et al., submitted)
and in other homeotic protein products (Scott and Weiner,
1984; McGinnis et al., 1984) were useful for determining
whether the C terminal part of the Anrp protein is present
in the fusion proteins. Antibodies against the N terminal
part of the homeodomain (Carroll et al., 1985a) detect the
larger fusion proteins, but not the 135 kd and 150 kd major
bands (data not shown). Thus, the major products accumulating in the hgtll Antp lysogens are not full length
and result from either incomplete synthesis or rapid in vivo
proteolysis of the C terminal parts of the fusion proteins.
The j3-gal-An@ fusion products migrate on gels much
more slowly than predicted from the sequence, probably
because of their high proline (10%) content. Similar behavior has been observed for other proteins with high proline content (e.g., Watt et al., 1985; Carroll and Scott,
1985).
Antibodies against Anfp Protein
The 135 kd Anrp PlO major fusion protein product, which
lacks the homeodomain, was purified by aminophenyl-thiogalactopyranoside
affinity chromatography, anti6-galactosidase immunoaffinity
chromatography, and
preparative SDS gel electrophoresis and then used to immunize rabbits. The An@-specific antibodies were affinitypurified using the hgtll Anrp SlO protein cross-linked to
anti+galactosidase
antibody attached to CNBr-activated
Sepharose 48 (Carroll and Scott, 1985). The specificity of
the final antibody preparation for the Antp-encoded portion of the two hybrid proteins was demonstrated by immunoblotting. The antibody reacts with all forms of the hybrid
proteins containing Anrp determinants but not with 8-galactosidase or other E. co/i proteins (Figure lc). We have
not yet detected Anrp products in extracts from Drosophila
Antennapedia
115
Protein Distribution
Figure 2. Expressionof the
Embryonic
An@ Protein in the
Ectoderm
In all photos, anterior is at the left and ventral
is toward the bottom.
(a) Embryo at the germ band extension stage,
before cell divisions have resumed in the ectoderm. No An@ signal is detectable above background levels.
(b) Embryo at the germ band extension stage.
Cell division has begun in the ectoderm. Antp
protein is detectable over about a 28-30 cell diameter broad band. The anterior-most patch
(left arrow) does not extend as far dorsolaterally as the more posterior patch, and is located in the region of the posterior labial and
anterior first thoracic segment. The posterior
patch (right arrow) is three times the size of the
anterior patch and is located in the region of the
primordia for the posterior first thoracic segment through the anterior first abdominal segment. The brighter cells (right arrow) are within
the primordia of parasegment four. The weaker
patch (left arrow) is within parasegment 3.
(c) Higher magnification view of (b).
(d) Embryo undergoing germ band shortening
after segment boundaries
have appeared
(boundaries indicated by white lines). Nuclear
expression of the An@ product extends from
the anterior side of the TV@ boundary (posterior Tl) to the posterior portion of the T3 segment.
(e) Embryo with shortened germ band. Antp
protein levels are diminished in T2p and T3p.
The An@ positive cells in the neurogenic region are visible (arrows) just out of the plane of
focus.
(f) Embryo after head involution. Weak Antp
staining is visible in portions of all three thoracic segments, and is clearest at the TVT2
boundary (arrow). The bright clusters of nuclei
are probably sensory cells (open arrowheads).
The bright region at the upper right of the
micrograph is autofluorescence
from yolk.
tissues using protein blots. Based upon the known exon
composition of all An@ transcripts, the antibody should
recognize all forms of An@ antigen. The lack of homeodomain determinants in the purified fusion protein immunogen eliminated the potential reactivity of the antibody with
other gene products containing the homeodomain.
Localization of Anfp Protein
The Ectoderm: To localize An@ proteins during embryogenesis, fixed whole embryos were stained with anti&@
antibodies as described in Carroll and Scott (1985). Staining of whole mount embryos allows the detection of Antp
protein in many different tissues at all stages of embryogenesis until the cuticle is formed. We have examined
Antp protein distribution in the nervous system and ectoderm, but not in the mesoderm.
After fertilization, the Drosophila zygote undergoes 13
nuclear divisions as a syncytium before cell membranes
form (Foe and Albert% 1983). At thecompletion of cellularization, gastrulation begins as longitudinal and transverse
folds appear (Turner and Mahowald, 1977). The ventrolateral cells along the ventral furrow invaginate to form the
presumptive mesoderm. The ventral germ band then
elongates such that the posterior-most cells move along
the dorsal surface nearly to the point of the transverse
cephalic furrow, with cells moving ventrally around the
sides of the embryo at the same time. During early germ
band extension, when segment primordia are three to four
cells wide, there is no detectable expression of Anfp protein or any reactive antigen within any cells (Figure 2e).
The specificity of the Anfp antibodies is also demonstrated by the absence of staining in Antp- mutant embryos (Figure 3e).
While the germ band is extended, as a cycle of cell divisions is complete in the ectoderm and when the segment
primordia are about eight cells wide, a low level of An@
protein becomes detectable in the presumptive thoracic
region in the anterior ventrolateral region of the embryo
(Figure 2b). The initial pattern of An& expression is not
uniform across the region; it appears to be strongest at the
anterior edges of two separate regions (arrows). The
anterior-most patch of staining does not extend as far dorsolaterally as the more posterior patch (Figure 2~). The
cells within the anterior portion of the second patch are
more heavily stained than the most anterior cells. The position
of the AMP-containing
cells
in the
entire
patch,
as
Cdl
116
Figure 3. Antp Expression
Nervous System
in the Embryonic
(a) Embryonic ventral nervous system at the
germ band shortening stage. Antp expression
extends from the posterior prothoracic segment ganglia to the anterior seventh abdominal
segment ganglia. Note that the levels of protein
in thoracic and abdominal cells at this stage
are comparable.
(b) Higher magnification view of (a).
(c) Ventral nervous system in the process of
condensation. Thoracic levels of Antp are significantly higher than abdominal levels; compare with (b) above.
(d) Ventral nervous system at full condensation. Thoracic levels of Antp are at a maximum,
abdominal levels are much lower. Anfp expression is high in cells of posterior Tl, anterior T2.
and anterior T3. Abdominal staining is specific,
since it extends only to A7. Note the absence
of staining in regions anterior to pT1 and
posterior to anterior A7. The arrows indicate the
positions of four stained nuclei of unknown
identity.
(e) Homozygous Anrpwto embryo. No expression of Anfp product is detectable within any
cells of embryos homozygous for this apparently null allele of the locus.
(f) Embryonic peripheral nervous system An@
expression is visible as thin, discontinuous
strips of nuclei clusters in the thorax and first
seven abdominal segments.
determined by measurement of egg length and cell counting, extends from approximately the posterior compartment of the labial segment through the anterior compartment of the first abdominal segment. This pattern is
surprising since Antp is not known to be required in the
labium or the first abdominal segment. This pattern is
transient (see below) and is also reflected in patterns of
RNA expression when investigated using promoter-specific RNA probes (A. Martinez-Arias, personal communication). The most heavily stained cells are within the
posterior prothorax/anterior mesothorax, a region also designated parasegment 4 (Martinez-Arias and Lawrence,
1985).
As the germ band begins to shorten, after segments
have appeared, the thoracic ectodermal staining becomes stronger ventrolaterally (Figure 2d). It can be seen
that the Anto protein is predominantly nuclear (Figure 2d).
From the segment boundaries visible at this stage, it is apparent that cells in the posterior prothorax (Tl), in all of the
mesothorax (T2), and in all of the metathorax (T3) express
Anto protein. Certain Antp transcripts (from the most 5’
Antp promoter) have been shown to accumulate to the
highest level in parasegments 4 and 5 in the extended
germ band (Martinez-Arias, 1988). We observed that An@
is expressed in these parasegments as well as anterior
parasegment 6 (posterior T3). Reexamination of Anfp
RNA distribution indicates that the region interpreted to
comprise only parasegment 5 may also include posterior
T3 (see Figure 2; Martinez-Arias, 1986).
As the germ band shortens to return to the ventral sur-
face, the Anto pattern changes in the ectoderm and protein appears in the nervous system. In the ectoderm, Antp
protein expression persists in ventrolateral cells of posterior
Tl and most of T2, but diminishes in posterior T3 (Figure
2e). This correlates well with changes observed in Antp
RNA distributions (Martinez-Arias, 1988). One can also
see the beginnings of Anto expression in the ventral nervous system at this stage (Figure 2e, arrows). The level of
Antp expression in the ectoderm decreases throughout
germ band shortening. Only a small amount of protein is
detected in subsets of thoracic ectodermal cells after the
head segments have involuted, at 14-16 hr of embryogenesis, mostly at the posterior Tllanterior T2 border (solid arrow, Figure 2f). By this time, sensory cells within the thoracic segments express Antp at high levels (open arrows,
Figure 2f). The pattern of disappearance of An@ protein
from the ectodermal cells (Figure 2f) is uneven, in contrast
to the uniformity of staining at an earlier stage (e.g., Figure
2e) suggesting that subcompartmental cell differentiation
has occurred by 14-16 hr.
Antp Protein in the Embryonic
Nervous System
Antp protein expression in the nervous system begins dur-
ing early neurogenesis. While the germ band is beginning
to shorten, ten pairs of patches of staining are visible in
the neurogenic region (Figure 3a, see also Figure 2e, arrows). At this stage, the levels of staining are rather uniform within the nuclei of the thoracic and abdominal neural cells. Antp protein is visible in the ventral nervous
system from the posterior portion of the prothoracic gan-
Antennapedia
117
Protein Distribution
glion to the anterior portion of the seventh abdominal ganglion (Figure 3b). As the ventral nervous system condenses, the level of An@ protein steadily increases in the
posterior prothorax, the anterior mesothorax, and a portion of the anterior metathorax (Figure 3c), leading to a
pattern in which these thoracic cells are intensely labeled
(Figure 3d). The cells in the seven most anterior abdominal segments remain weakly labeled. In segments anterior and posterior to the ten stained segments (including
segment A8) (Figure 3d), no Anfp antigen is detectable in the ventral nervous system. The detectable amount
of An@ antigen is variable from cell to cell, especially in
the abdominal segments (Figures 3b, 4a). Antp protein appears to be.in all of the cells of parasegment 4 but in only
some of the cells of the more posterior compartments. The
specificity of the antibody for Antp proteins can be addressed by examining the level of staining in Antpw’o homozygous embryos. The Anfpw10 mutation behaves genetically as a null allele (Kaufman and Abbott, 1984).
There is no labeling of any cells in the mutant embryos
with the anti-An@ antibody (Figure 3e).
In addition to the ventral nervous system Anfp protein
pattern, there are peripheral nervous system cells that express An@. These are visible as thin discontinuous strips
of cells in thoracic and abdominal regions of the embryo
during germ band shortening (Figure 3f, arrows). The precise number, identity, and arrangement of these peripheral neural cells is presently under study, but it appears
that only a subset of the peripheral neural cells produce
Antp protein at a detectable level.
Since we were able to study Antp expression with clear
resolution in the ventral nervous system, we applied the
antibody probe to the study of Antp protein regulation. We
considered three levels of An@ regulation: one, control
by homeotic genes, such as 6X-C loci; two, control by
pleiotropic regulators of homeosis, such as Polycomb; and
three, autogenous control of Antp. We find evidence for all
three levels of regulation from examining Antp expression
in mutants.
Regulation of Antp Protein Expression
The Spatial Relationship of Antp and Ubx Protein
Distribulions
in the Ventral Nervous System
of Wild-Type and Mutant Embryos
In addition to An@, the U/tmbithorax(Ubx) gene is involved
in controlling thoracic development (Lewis, 1978). It has
been shown that the Ubx protein accumulates at the
highest levels in the posterior metathorax and anterior first
abdominal segment of the ectoderm and ventral nervous
system (White and Wilcox, 1984, 1985; Beachy et al.,
1985). There is also expression of Ubx protein at a low level
in the posterior mesothorax and at a moderate level in the
anterior portions of the second through seventh abdominal segments. It has been proposed that homeotic genes
function in a combinatorial manner, the fate of a segment
depending on the array of genes active within it (Lewis,
1978; Struhl, 1982; Kaufman and Abbott, 1984; Sat0 et al.,
1985). Therefore, an important question is exactly how the
spatial distributions of different homeotic gene products
are related.
To compare the spatial distribution of Antp protein to
that of Ubx protein, we doubly-labeled whole mount embryos with rabbit antidntp antibody and a mouse monoclonal antibody that detects the Ubx proteins (White and
Wilcox, 1984). In the ventral nervous system, Antp is expressed at a high level in posterior Tl and most of T2, and
at a moderate level in part of T3 (Figure 4a). Ubx protein,
examined in the same embryo, is expressed in the
posterior part of T2 at a moderate level, and at a high level
in parasegment 8 (the posterior part of T3 and the anterior
part of Al) (Figure 4b). The parts of the nervous system
where each protein is at high levels do not overlap
(Figures 4c, d); there is generally an inverse relationship
between the levels of the Antp and Ubx proteins. However,
there are clearly cells in which both proteins are expressed. The presence of Ubx protein is correlated with a
reduced amount of An@, but not necessarily with the complete absence of Antp protein. In the more posterior parts
of the abdomen, Ubx expression is largely or completely
confined to the anterior compartments of the abdominal
segments, whereas An@ expression appears to be expressed at a low level within a subset of the nuclei
throughout the segment (Figure 4a).
Antp Expression in Homeotic Mutants
Previous studies showed Antp expression to be under the
influence of the BX-C genes (Hafen et al., 1984; Harding
et al., 1985). The ability to study Antp and Ubx protein distributions simultaneously in whole mount embryos led us
to further investigate the role of BX-C genes in Antp gene
regulation. In Ubx embryos, Antp expression in the ventral nervous system increases in posterior T2, all of T3, and
anterior Al such that the level of Antp protein reaches that
found in wild-type embryos in parasegment 4 (compare
Figures 4a and 4e). Thus, the areas where Ubx is normally
expressed at a high level exhibit increased levels of Antp
protein in the absence of Ubx product(s), in agreement
with the view that Ubx is a regulator of Anfp expression
(Hafen et al., 1984).
Some mutations at the bithoraxoid (bxd) locus cause an
increase in Ubx expression in anterior T3 (compare Figure
4b to Figure 49; Beachy et al., 1985; White and Wilcox,
1985b). If Antp expression is responding to Ubx protein
levels, one would expect that the increased expression of
Ubx in anterior T3 in a bxd- embryo would reduce Antp
expression. This is indeed the case. The expression of
Antp protein in a bxd- mutant in anterior T3 is reduced to
about the level of Antp protein in the abdominal segments
(compare Figure 4f to Figure 4a).
We have examined Antp expression in other BX-C
homeotic mutants. Mutations at the abdominal-A (abd-A or
iab-2) (Lewis, 1978; Karch et al., 1985) locus of the BX-C
transform the second through seventh abdominal segments into segments resembling the first abdominal segment (Sanchez-Herrero et al., 1985; Karch et al., 1985)
and result in an increase in Ubx protein expression
throughout the A2-A7 segments (Struhl and White, 1985).
Antp expression in abdk embryos appears to be essentially unaffected (data not shown). However, the additional
absence of the Ubx gene (i.e., in a Ubx-, abdA- double
mutant) results in high levels of Antp expression from
Cell
115
Figure 4. Spatial Relationship
of the Distribution of the Antp and Ubx Proteins: The Level of Ubx Protein Is Inversely Related to the Antp Protein Level
(a) Wild-type embryonic nervous system labeled with anti-AMP antibody and detected with fluorescein-conjugated
secondary antibody. Antp expression is high in cells of the posterior prothorax, the anterior mesothorax and metathorax, and a smaller subset of anterior metathorax cells.
(b) Same embryo as (a) labeled with a monoclonal antibody to the Ubx protein and detected with a rhodamine-conjugated
secondary antibody. Ubx
expression occurs in posterior cells of the mesothorax, and in anterior cells of the first seven abdominal segments. Note that in the thorax, high
levels of Ubx expression occur within regions where Antp expression is low.
(c) and (d) High magnification views of the embryonic nerve cords shown in (a) and (b) respectively. Inspection of the packing of the respectively
labeled cells indicates that the high levels of each product are in different regions of the thoracic ganglia. The cells of the posterior portion of the
mesothorax (pT2) are indicated.
(e) Antp expression in a homozygous UbfizB embryo. Anfp expression is at high levels over the regions where Ubx is normally at high levels. Antp
expression at high levels extends from posterior Tl through anterior Al.
(f) Antp expression in a homozygous bxd113 embryo. Antp protein expression is greatly reduced in anterior T3, the site of new Ubx expression in
the bxd mutant (see Figure 59).
(g) Ubx expression in a bx@ embryo. Ubx expression increases in anterior T3.
posterior Tl to anterior A7 (compare wild type in Figure 5a to Figure 5b). One interpretation of these observations is that abd-A+ is a negative regulator of both
Anfp and Ubx. In the absence of only abd-A+, both Antp
and Ubx are on in posterior abdominal segments, but Ubx
keeps Antp at a low level. The absence of Abdominal-B
(Abd-8) gene (or iab-z Lewis, 1978; Karch et al., 1985)
function results in transformations of abdominal segments 5-8 into segments resembling the fourth abdominal segment (Sanchez-Herrero et al., 1985) and an
increase in Ubx expression in the eighth abdominal segment (Struhl and White, 1985). Anfp expression is not detectably affected by the absence of the Abd-B gene (data
not shown). The absence of all BX-C functions (i.e., the
Df(3R)P9 deletion which includes Abd-8) affects Anfp expression in the same manner as the absence of just Ubx
and abd-A (Figure 5~). This is surprising because Abd-6
does negatively influence Ubx expression in segment A8
(Struhl and White, 1985). The effects of the BX-C mutations on Antp and Ubx expression in the nervous system
are summarized in Figure 6, and illustrate that Anfp expression is controlled in the posterior embryo by the loci
of the BX-C.
The Sex combs reduced (SW) locus of the ANT-C is required for normal labial and prothoracic segment development (Kaufman et al., 1980; Wakimoto and Kaufman,
1981; Sato et al., 1985). Scr is required in the prothorax
and thus appears to be the next most anteriorly acting
homeotic gene to Anfp (Harding et al., 1985; Kuroiwa et
al., 1985). In embryos homozygous for the strong ScP’17
mutation (Wakimoto and Kaufman, 1981) Antp protein expression is normal. The anterior limit of Antp protein expression remains unchanged (data not shown).
The activities of the homeotic genes of the ANT-C and
BX-C are regulated in part by a number of pleiotropic
regulatory loci (Lewis, 1978; lngham and Whittle, 1980;
Duncan and Lewis, 1982; Duncan, 1982a; Struhl, 1982;
Ingham, 1984; Dura et al., 1985; Struhl and White, 1985).
These loci affect the fate of segments normally under the
control of both complexes. Mutations at the maternal ef-
Antennapedia
119
Protein Distribution
Figure 5. Regulation of Antp Expression by
Other Homeotic Genes
(a) Wild-type embryo stained with antiffnfp antibody. Expression of Antp extends from posterior Tl to anterior A7. High levels of protein are
in posterior Tl, anterior T2, and anterior T3.
(b) Homozygous Ubx109 (Ubx-, ab&A-) embryo. High levels of Anfp expression now extend from posterior Tl to anterior A7.
(c) Homozygous Df(3R)P9 (Ubr, ab&A-, Abd6-) embryo. High levels of Anfp expression
still extend to anterior A7 in the absence of
abd-6.
(d) Homozygous Pb/ycombR1 embryo. Antp expression is at low levels throughout the CNS,
including the brain (large arrow) and AB (eighth
small arrow).
(e) Embryo from es&esc1° parental cross.
Antp expression is at low levels throughout the
CNS; note pairs of large nuclei visible in each
segment (small arrows).
feet extra sex combs (esc) locus and in the pblycomb (PC)
locus result in embryos with all segments resembling the
eighth abdominal segment (Struhl, 1981a; Lewis, 1978;
Duncan and Lewis, 1982; Denell and Frederick, 1983).
Antp protein expression is depressed in all segments of
the nervous system in PC (Figure 5d) and esc- (Figure
5e) embryos, to a weaker than wild type abdominal segment level of expression. An@ expression also extends
into the presumptive head segments (Figure 5d). These
results are in agreement with the previously reported effects of Flc on Antp transcript distribution (Wedeen et al.,
1988).
Mutations of the Antp Locus
The Antp gene has two promoters, one (Pl) for a 103 kb
transcription unit and one (P2) for an overlapping (nested)
36 kb transcription unit. Transcripts initiating at both promoters contain the same protein-coding exon sequences,
but have different untranslated 5’ leader sequences
(Schneuwly et al., 1986; Laughon et al., submitted). In situ
hybridizations to sectioned embryos with transcription
unit-specific probes have revealed that the promoters are
expressed with similar, but slightly different, spatial patterns during embryogenesis
(A. Martinez-Arias et al., unpublished data). The Pl- and PBderived transcripts are
found in roughly equal amounts in embryo RNA (Laughon
et al., submitted). The antibody probe used here does not
discriminate between Antp proteins derived from one tran-
scription unit or the other.
Many Antp alleles cause lethality at late embryogenesis
or larval stages. Included in this group of alleles are null
alleles such as An&Y0
(an apparent
point mutation, see
with a chro-
Figure 48) and Antps’, which is associated
Figure 6. Summary of the Spatial Regulation of Antp Expression in
the Ventral Nervous System
Schematic diagram of the patterns of Antp and Ubx protein distribution
in wild-type and mutant embryonic nerve cords. Segments are indicated on the left, parasegments on the right. The protein products
diagrammed are indicated at the top of the figures, the genotypes studied are shown at the bottom. Black areas represent high levels of expression, medium shading represents moderate levels of expression,
and the lighter areas weak expression. Ubx and abdd regulate the
level of Anfp in the abdominal segments. bxd modulation of Ubx expression leads to repression of Antp in anterior T3.
mosome inversion that interrupts the Pl transcription unit,
but leaves the smaller P2 transcription unit and the coding
exons apparently intact. The An@@ breakpdint is about
35 kb upstream of the internal P2 promoter (Scott et al.,
1983; Laughon et al., submitted). Therefore, in An@
homozygotes we would expect to observe only the Antp
protein that is encoded by transcripts from the P2 transcription unit. Surprisingly, no Antp protein can be detected in Antps2 homozygous embryos. An@ protein is
also undetectable in embryos homozygous for five other
Cdl
120
alleles: Anfp73b, AntpB, Antpw, AntpCB, and Antpctx (data
not shown), all of which are dominant alleles associated
with chromosome rearrangements that interrupt the Pl
transcription unit and leave the P2 unit apparently intact. Heterozygotes carrying the dominant mutations have
head-to-thorax transformations. Flies homozygous for the
dominant mutations die as embryos or larvae, and have
T2 to Tl transformations, like homozygous Antps2 or
Antpwl’J flies (Wakimoto and Kaufman, 1981). The breakpoint of the AntpCB inversion is just within the Pl unit,
about 85 kb upstream of the P2 promoter (Scott et al.,
1983). Thus, a lesion in the Pl unit, even at a considerable
distance from the P2 promoter, prevents both the Pl and
P2 units from producing protein at detectable levels.
the spatial patterns of Antp RNA and protein. The RNA described as being in parasegments 4 and 5 is probably also
in anterior parasegment 6, judging from the relative widths
of the two parasegment signals (Martinez-Arias, 1985), in
agreement with our results. However, the time at which the
protein was detectable was much later than when transcripts have been observed. Antp protein was not detectable until the germ band extension stage of embryogenesis, like Ubx protein, whereas transcripts are detectable at
the cellular blastoderm stage (Levine et al., 1983). The discrepancy is likely to reflect the time needed to process the
very long Antp transcripts, and possibly a system of translational control as well. The large 5’ leaders of Antp
mRNAs (Schneuwly et al., 1986; Laughon et al., submitted), may be involved in control of translation.
Discussion
The Antp Protein Is Located in the Nucleus
In all of the tissues examined, the Antp protein is localized
in the nuclei of Antp-expressing cells. The molecular function of the Antp proteins is unknown, but it has been
hypothesized that by virtue of their homeodomains they
may be DNA-binding proteins (Laughon and Scott, 1984;
Laughon et al., 1985). The nuclear location of Antp protein
is consistent with this hypothesis and extends previous
observations on the localization of homeodomain-containing products. The antigens recognized by antibodies
against the U/trabithorax(White and Wilcox, 1984; Beachy
et al., 1985), engrailed (DiNardo et al., 1985), and fushi
tarazu (Carroll and Scott, 1985) protein products, each of
which contains a homeodomain, are all located in the
nuclei of the cells in which they are expressed.
The Dynamics of Anfp Protein Expression and
Its Correlation with Sites of Gene Function and
RNA Expression
In the ectoderm, the Antp protein distribution changes
rapidly. Initially, Antp expression is detected over an eight
compartment region extending from the posterior labial to
the anterior first abdominal segment. Later, as segments
form, Antp proteins are found in five compartments from
posterior Tl to posterior T3; the proteins then gradually
disappear from the ectoderm. The initial distribution of
Antp protein was unexpected, as no function of Antp has
been established for the development of the posterior
labium, anterior Tl, or anterior Al compartments. The
longer lasting, higher level of expression in posterior Tl
through posterior T3 correlates with the compartments
where Antp+ is known to be required (Wakimoto and
Kaufman, 1981; Struhl, 1983; Sato et al., 1985; Struhl,
1981a, 1982; Kaufman and Abbott, 1984). In addition to
the thoracic ectodermal expression of Antp, there is a high
level of Antp expression in thoracic ganglia of the ventral
nervous system. Although the complexities of cell divisions and movements within the nervous system make observations difficult, it appears that the nervous system
pattern of Antp expression does not exactly correspond to
the initial ectodermal pattern in that cells within the
posterior T2 ganglia and T3 ganglia do not express Antp
at high levels.
There is no clear indication of a discrepancy between
The Regulation of Anlennapedia
Expression
Examination of Antp expression in the nervous system in
embryos homozygous for a variety of mutations has revealed three main features of Antp regulation: one, the
level of Antp expression is negatively regulated by the
more posteriorly active genes Ubx and abd-A; two, Antp
expression is influenced by the more globally acting extra
sex combs and hlycomb loci; and three, normal embryonic expression of Antp protein depends upon the integrity of elements up to 65 kb upstream of the smaller
transcription unit and the coding exons.
Antp protein levels are modulated by the activity of BX-C
genes, and exhibit an inverse relationship to the level of
Ubx expression (summarized in Figure 6). The level of
Antp in the more posterior abdominal segments is also influenced by the abd-A gene, as can be seen by the dramatic effects of removal of abd-A product in a Ubx, abd-A
double mutant. It is surprising that the Abd-B locus has
no effect on Antp expression, since it does affect Ubx expression in segment A8 (Struhl and White, 1985). Other
genes, outside of the BX-C, must act to repress Antp expression in A8.
In the absence of esc or Pblycomb products, Antp and
Ubx (Beachy et al., 1985; White and Wilcox, 1985b; Struhl
and White, 1985) patterns of expression degenerate to an
improper spatial pattern with low levels of expression extending through most segments of the nerve cord, including the head (Figure 5d). Thus, the esc and PC products are
required in all segments (not just the more posterior
regions) for proper homeotic gene activity. It has been
found that Antp RNA is initially derepressed in most cells
in PC- embryos (Wedeen et al., 1986). The low level of
Antp protein observed in esc- and PC- embryos may be
due to the eventual repression of Antp expression by the
derepressed expression of Ubx, abd-A, and possibly other
negative regulators of Antp (Struhl and White, 1985).
Proper embryonic expression of Antp protein depends
upon the integrity of elements far upstream from the coding exons of the locus. We could not detect protein expression in embryos homozygous for the six chromosome
rearrangement mutations analyzed, even though the P2
unit is apparently intact in each case. The five dominant
alleles must, at some later point in development, express
Antp product(s), since the alleles do affect head development. Two hypotheses are suggested by these results.
Antennapedia
121
Protein Distribution
One is that cis-acting elements required by the smaller P2
transcription unit are located as far as 65 kb upstream of
the P2 promoter. The second is that Antp protein may itself
be required for initiating or maintaining P2 transcription
unit activity, and that protein (or RNA) made using the Pl
promoter stimulates protein production by the P2 unit. In
the absence of a functional Pl unit, in cis or in trans, the
P2 unit would fail to produce protein. A less likely possibility is that the P2 unit transcripts are not translated into protein in either wild-type or mutant embryos.
The enormous size and complex pattern of expression
of the An@ locus suggest that there are multiple mechanisms through which other genes may control the time,
place, and level of An@ activity. Transcription and RNA
processing are clearly both important levels of control that
affect Antp protein production. Having learned of some of
the genes, such as Ubx and abd-A, that control Antp in
tram, it will be important to find out the molecular mechanisms that underlie the regulatory interactions among the
genes.
Experimental
Procedures
Gene Fusions
The Antj~ cDNA clone pDmGllO0 has been described previously (Scott
et al., 1983) and completely sequenced (Laughon et al., submitted). A
1298 bp Pstl-EcoRI fragment and a 1616 bp Smal-EcoRI fragment
were purified, single-stranded ends were made blunt with T4 DNA polymerase, and EcoRl IO-mer linkers that had been phosphorylated with
T4 polynucleotide kinase were ligated to the fragments. After ligation,
the DNA was digested with EcoRl and the fragments were repurified.
The fragments were subcloned into pUC6 and cloned plasmid DNA
was digested with EcoRi to recover and purify the linkered fragments.
Each fragment was ligated into the unique EcoRl site of Igtll at the
3’ end of the /acZ gene (Young and Davis, 1963). Ligated DNA was
packaged and the phage were plated on RYlO90 and scored for colorless plaques. The DNA from phages yielding colorless plaques was
purified and analyzed for inserts of the Antp fragments in the proper
orientation. The construction was checked by Allen Laughon for correct reading frame usage by DNA sequencing insert junction frag
ments.
Expression and Purification
of &Galactosidase-http
Hybrid
Proteins in E. coli
Lysogens of kgtll Antp PI0 and Igtll Antp SIO were established in
RY1089 (Young and Davis, 1983). Hybrid proteins were induced, extracted, and purified essentially as described elsewhere (Carroll and
Scott, 1985).
Production,
Purification,
and Assay of Antibodies
Rabbits were immunized with 500 pg of soluble AntpPlO antigen in
complete Freunds adjuvant on day 0 and again with 200 pg of antigen
on days 14 and 21 (and monthly thereafter) in incomplete adjuvant. Ail
injections were subcutaneous at multiple sites. Immunizations with gel
purified SDS-denatured
Antp PI0 antigen (200 vg each) were intramuscular in incomplete Freund’s adjuvant. Rabbits were bled on
day 28 and about monthly thereafter until the final exsanguination.
Antisera were absorbed on p-galactosidase-Sepharose
48 as described (Carroll and Scott, 1965). The flow-through fraction from the
P-galactosidase column of the anti-An@ PI0 serum was affinity-purified on a column containing the Antp SIO hybrid antigen crosslinked
to anti-bgaiactosidase
antibody that was coupled to CNBr-activated
Sepharose 48. Affinity columns containing the Antp PI0 antigen did
not yield antibody that was useful for immunofluorescence.
The procedures for constructing the affinity columns are described elsewhere,
as are the immunobiotting procedures for assaying antibody specificity
(Carroll and Scott, 1985).
lmmunotluorascence
Whole Drosophila embryos
were dechorionated,
permeabilized
in
heptane, fixed in formaldehyde, and devitellinized in heptane/methanol as described (Karr and Alberts, 1986). Embryos were stained with
primary antidntp antibody and monoclonal anti-Ubx antibody (White
and Wilcox, 1984) and secondary fluorescein-conjugated
goat antirabbit IgG and rhodamineconjugated
goat anti-mouse Ig as described
for other antibodies (Carroll and Scott, 1985). UbxQa is a null mutant
that does not produce detectable Ubx protein (Beachy et al., 1986).
abd-A- mutant embryos were generated from either the ab&-AM1 stock
(Sanchez-Herrero
et al., 1985) or as progeny of DpPlO; DflO9ITM3
parents. AM-Bembryos were derived from the Abd-P1
stock
(Sanchez-Herrero et al., 1985) Ubx. abdk
double mutants were derived from the Df(3R)l09 stock, which is a deletion of the B&C into the
iabd region (Karch et al., 1985). It is possible that Df(3R)l09 has some
negative cis effect on abd-6. esc- embryos were derived from a cross
of esc2/esc70 frans-heterozygotes
generated as described (Struhl and
White, 1985). Embryos mutant for the bxd, Ubx, al&A, a/&B, & and
esc loci were unambiguously identified on the basis of their altered Ubx
staining and then inspected for theirAn@ pattern. The zygotic mutants
(and the zygotic effect of R) were constructed by crosses of heterozygotes. For the Scrw” mutation, several hundred stained embryos
were inspected, all of which exhibited the wild-type pattern.
Acknowledgments
Dr. Allen Laughon provided information on the structure of the Antp
cDNA, which was essential to the analysis of the gene fusions and hybrid proteins. Vern Twombly provided expert technical assistance. We
thank Drs. Mike Levine and Alfonso Martinez-Arias for valuable discussions, and Dr. Martinez-Arias
for communication
of unpublished
results. Drs. Chris Doe and Corey Goodman provided helpful information about the nervous system patterns. We thank Dr. Robert White for
the monoclonal anti-Ubx antibody, and Drs. Gary Struhl, Ian Duncan,
Phil Beachy, and Jim Kennison for mutant stocks. We are grateful to
Dr. Michael Klymkowsky for generous access to fluorescent microscopes. We thank Drs. Allen Laughon, Anne Boulet, Paul Mains, and
John Tamkun for review of the manuscript, and Cathy lnouye and Karen Brown for its preparation.
This work was supported by National Institutes of Health grant
HDXl8163 to M. P. S., a Hoosier fellowship to R. A. L., and Damon
Runyon-Walter Winchell Postdoctoral Fellowship DRG-659 and NIH
postdoctoral fellowship GM-09756 to S. B. C. M P. S. also gratefully acknowledges a Basil O’Connor grant from the March of Dimes and a
Searle Scholar Award.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 USC. Section 1734
solely to indicate this fact.
Received June IO. 1986; July 24, 1986
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