Centro de Biología Molecular Severo Ochoa, Universidad
Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
* Present address: Division of Genetics, HHMI, Brigham and Women's Hospital, 20
Shattuck Street, Boston MA 02115, USA
Present address: Instituto de Biologia Molecular e Celular (IBMC), Rua do
Campo Alegre, 823, 4150-180 Porto, Portugal
Author for correspondence (e-mail:
esherrero{at}cbm.uam.es)
Accepted 6 August 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Drosophila, Hox, Abdominal-B, iab regulatory domains
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Abdominal-B (Abd-B) Hox gene of the BX-C specifies
abdominal segments A5 to A9 [parasegments (PS) 10 to 14]
(Casanova et al., 1986;
Sánchez-Herrero et al.,
1985
; Tiong et al.,
1985
). It codes for two proteins that share the C-terminal amino
acid sequence, including the homeodomain, but differ in the N-terminal region:
Abd-B M (or Abd-B I) protein, which is expressed in PS10-13; and Abd-B R (or
Abd-B II) protein, which is present in PS14
(Boulet et al., 1991
;
Celniker et al., 1989
;
DeLorenzi and Bienz, 1990
;
Zavortink and Sakonju, 1989
)
(see Fig. 1A). Mutations that
affect the Abd-B M protein complement those affecting the Abd-B R protein, and
both types of mutations fail to complement Abd-B mutations that eliminate the
two products (Casanova et al.,
1986
; Celniker et al.,
1990
; DeLorenzi and Bienz,
1990
). Abd-B M expression starts in PS13 and is later observed,
successively, in PS12, PS11 and PS10, with increasing levels in more posterior
parasegments (Celniker et al.,
1989
; DeLorenzi and Bienz,
1990
). This indicates that there are regulatory sequences
controlling Abd-B expression in these parasegments.
|
The Abd-B m regulatory region extends over more than 50 kb
3' to the Abd-B transcription unit, and is genetically
subdivided into several infraabdominal (iab) domains, from
iab-5 to iab-8 (Barges et
al., 2000; Galloni et al.,
1993
; Gyurkovics et al.,
1990
; Karch et al.,
1985
; Lewis, 1978
;
Zhou et al., 1999
). Deletions
of these domains cause transformations of the corresponding parasegment into
the next, more anterior one (Galloni et
al., 1993
; Mihaly et al.,
1997
). DNA elements within these domains, when fused to the
lacZ gene, direct ß-galactosidase expression in the embryo with
precise parasegmental anterior limits of expression
(Barges et al., 2000
;
Busturia and Bienz, 1993
;
Zhou et al., 1999
). Similarly,
P-lacZ elements inserted in a specific iab region show
ß-galactosidase signal restricted to particular parasegments
(Barges et al., 2000
;
Bender and Hudson, 2000
;
Galloni et al., 1993
;
McCall et al., 1994
;
Zhou and Levine, 1999
).
Therefore, the iab domains seem to represent autonomous units that
control Abd-B M expression from A5/PS10 to A8/PS13, thus specifying these
metameres (Boulet et al., 1991
;
Celniker et al., 1990
;
Galloni et al., 1993
;
Gyurkovics et al., 1990
;
Mihaly et al., 1997
;
Sánchez-Herrero and Akam,
1989
; Sánchez-Herrero,
1991
).
The iab domains are separated from each other by boundary elements
that isolate them, thus preventing abnormal activation of Abd-B in
more anterior segments (Barges et al.,
2000; Gyurkovics et al.,
1990
; Hagstrom et al.,
1996
; Hagstrom et al.,
1997
; Mihaly et al.,
1997
; Zhou et al.,
1996
; Zhou et al.,
1999
; Zhou and Levine,
1999
). Deletions of these elements cause posteriorward
transformations (Barges et al.,
2000
; Gyurkovics et al.,
1990
; Mihaly et al.,
1997
), owing to the inappropriate expression of Abd-B in
a certain parasegment with the levels and distribution of a more posterior one
(Celniker et al., 1990
;
Galloni et al., 1993
;
Sánchez-Herrero, 1991
).
In some cases, it has been shown that boundary regions are physically close to
sequences that respond to trans-regulators of the Polycomb
type (Polycomb response elements, or PREs), which restrict homeotic
gene expression throughout development
(Brock and van Lohuizen, 2001
).
The combined activity of PRE and boundary elements maintains the particular
Abd-B expression of each parasegment.
Of all the abdominal segments determined by the Abd-B M protein, the
A8/PS13 is clearly different from the rest in the set of structures that
derive from it, both in the embryo or in the adult, such as the posterior
spiracles or the female genitalia (Campos
Ortega and Hartenstein, 1985;
Nöthiger et al., 1977
).
An iab-8 regulatory region, which directs PS13 specific expression in
the embryo, has been identified 3' to the Abd-B transcription
unit (Barges et al., 2000
;
Zhou et al., 1999
)
(Fig. 1A). The iab-8
domain is separated from the iab-7 domain, which is located more
proximally, by a Fab-8 boundary that also contains a PRE sequence
adjacent to it (Barges et al.,
2000
; Zhou et al.,
1999
; Zhou and Levine,
1999
). However, there are no known mutations affecting just the
A8, and rearrangements that affect the iab-8 domain do not transform
this segment (Gyurkovics et al.,
1990
; Hendrickson and Sakonju,
1995
; Hopmann et al.,
1995
; Tiong et al.,
1987
).
We have made use of a P-element insertion in the 5' region of the Abd-B gene to isolate novel mutations that affect the A8 segment and we have characterized a putative iab-8 region in the 5' region of Abd-B. Our results indicate that iab-8 sequences 5' and 3' to the Abd-B m transcription unit contribute to the regulation of Abd-B in the A8 segment.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Constructs and transformation of embryos
The pRCm1 construct (Busturia and Bienz,
1993) was used in the transformation experiments that gave rise to
the Abd-Blac1 mutation. This vector carries the
rosy gene as a transformation marker, a fragment from the
Abd-B gene and the lacZ gene. The Abd-B fragment is
a 5.6 kb Pst-HindIII fragment from 50,504 to 44,912
(Martin et al., 1995
). This
fragment includes the Abd-B m promoter region, the initiation site of
the Abd-B m (or A) transcript, the leader sequence and the sequence
encoding for the first five amino acids of the Abd-B m protein fused
to the bacterial lacZ gene. This Pst-HindIII fragment is
referred to in the text as the Abd-B proximal promoter, Abd-Bpp
(Busturia and Bienz, 1993
). The
P-element giving rise to the Abd-Blac1 mutation is the
construct pRCm 70.2, and contains, in addition, a HindIII fragment of
the Abd-B 3' regulatory region (from 64,580 to 59,073) inserted
into KpnI-XbaI sites of pRCm1 (see
Fig. 1A).
In the analysis of the 5' Abd-B regulatory region, the following constructs were used to transform embryos.
Transformation of embryos was done as described previously
(Busturia and Bienz, 1993).
Isolation of P-element derivatives
Derivatives of the P-element insertion in Abd-B
(Abd-Blac1) were obtained by several methods. In one of
them, P [ry+ Abd-Blac1]/TM3,
2-3 dysgenic males were crossed to ry506 females and
ry- males obtained from this cross were individually
isolated and balanced. A different set of experiments, intended to isolate
derivatives independently of their having (or not) the ry gene, was
carried out as follows: individual dysgenic males of the genotype st red
sbd2 Abd-Blac1/TM3,
2-3 were
crossed to Abd-BM1/TM6B females. The progeny was checked
for either the presence of an Abd-B phenotype in the
Abd-Blac1/Abd-BM1 progeny, or for the loss of
the haploinsufficient Abd-B phenotype of the
Abd-Blac1 insertion (small seventh tergite and a few
bristles in the sixth sternite) in the Abd-Blac1/TM6B
males. In the appropriate cases, individual males were isolated, and stocks
were established and further characterized by crosses with different
Abd-B alleles. To ascertain if these derivatives were
ry+ or ry-, they were recombined with
ry506 and crossed to ry506 females.
The class II (iab-8,9) mutants described in this work were isolated
by this last method.
In situ hybridization
It was carried out as described by
(Casares and Sánchez-Herrero,
1995). The probe for the m transcript is a genomic
BamHI fragment from 50,702 to 48,864. The probe to detect all the
Abd-B transcripts, referred to as `common probe', is a genomic
HindIII-SalI fragment from 56,556 to 52,989 that includes
the homeobox.
Antibody staining
It was performed as described previously
(Casares and Sánchez-Herrero,
1995). The rabbit anti-ß-galactosidase antibody (Cappel) was
used at a concentration of 1:2000. The Abd-B antibody, used at 1:20
concentration, is monoclonal antibody 1A2E9 (kindly provided by S. Celniker),
and recognizes both Abd-B M and R proteins
(Celniker et al., 1989
). The
anti-En monoclonal antibody (Patel et al.,
1989
) was used at 1:10 concentration, and was kindly provided by
T. Kornberg. Staging of the embryos was determined as described previously
(Campos-Ortega and Hartenstein,
1985
).
Embryonic cuticle analysis
It was carried out as previously described
(Wieschaus and Nüsslein-Volhard,
1986).
DNA techniques
The molecular study of the Abd-Blac1 mutation and of
the different derivatives was carried out by Southern blot analysis using the
following probes: (1) Abd-B DNA not included in the pRCm 70.2
construct; (2) Abd-B DNA included in the construct; and (3) DNA from
the rosy and lacZ genes. The lacZ probe was
obtained from the Bluescript vector. The rosy probe is a 7.2 kb
HindIII fragment from the HZ50PL vector
(Hiromi and Gehring, 1987).
DNA extractions and methods of Southern hybridization were carried out as
described previously (Sullivan et al.,
2000
) with slight modifications.
PCR analysis
Inverse PCR was done as described in FlyBase
(http://www.fruitfly.org/methods).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The fifth line, however, showed a restricted ß-galactosidase
expression, confined to the posterior abdominal segments and resembling the
Abd-B pattern of expression. Males heterozygous for this insert
presented a typical Abd-B haploinsufficient phenotype (some bristles
in the sixth sternite and a small seventh tergite). Therefore, we decided to
characterize this line further. The insertion point of the P-element was
located by inverse PCR at position 48,957
(Martin et al., 1995), 242 bp
upstream the Abd-B m transcription start site
(Zavortink and Sakonju, 1989
).
Genetic tests with Abd-B m and Abd-B r mutations indicated
that the insertion is an amorph for the Abd-B r function and a strong
hypomorph for Abd-B m function. Staining of homozygous embryos with
an anti-Abd-B antibody showed almost absence of expression in PS10-14. These
observations indicate that the P-element has generated a mutation in
Abd-B that we have called Abd-Blac1.
The lacZ expression of Abd-Blac1 embryos differs from that of Abd-B in wild-type embryos in several respects (Fig. 2). In Abd-Blac1 embryos, the lacZ expression starts simultaneously in PS12-14 at stage 9, and this signal is maintained at stage 10 (Fig. 2A) and thereafter (Fig. 2C). Expression in PS10-11 is low or absent and, at late stages, there is a strong PS12 signal in the ventral cord (Fig. 2E). By contrast, Abd-B products are observed only in PS13 and PS14 in stage 10 wild-type embryos (Fig. 2B). Later on, Abd-B expression decreases from PS14 to PS10 both in the epidermis and the ventral cord (Fig. 2D,F). The particular strong lacZ expression in the ventral cord of Abd-Blac1 embryos disappears in Pc3 Abd-Blac1 double mutant embryos (Fig. 2G).
|
We hypothesize that the lacZ expression in Abd-Blac1 embryos may have some characteristics derived from the place of insertion and others due to the Abd-B regulatory region included within the P-element. To delimit each contribution, and to obtain mutations in the region of the Abd-B promoter, we mobilized the P element (see Materials and Methods).
Some Abd-Blac1 derivatives constitute a distinct
class of mutations in Abd-B
We obtained different types of derivatives when mobilizing
Abd-Blac1, but most of them could be classified into two
different groups, defined by their phenotype, Abd-B expression and
molecular structure.
Derivatives of the first group (class I; nine alleles isolated) are
ry- but retain the lacZ gene. They present in
heterozygosis an Abd-B haploinsufficient phenotype and complement
Abd-B r (iab-9) mutations. In hemizygosis they show a
transformation of segments A5-A8 (PS10 to PS13) into A4 (PS9)
(Fig. 3A), characteristic of
Abd-B m mutations (Casanova et
al., 1986). Accordingly, in class I homozygous embryos Abd-B
protein or RNA (`common' probe) expression is detected normally in PS14 but
very weakly in PS10-13 (Fig.
3B). ß-galactosidase staining of these embryos shows a strong
signal in PS13 (Fig. 3B). The
phenotype and staining pattern of these mutations resemble the derivatives of
a P-lacZ element, iab-8Plac(+159), inserted very
close to Abd-Blac1
(McCall et al., 1994
).
|
Derivatives of the second group (class II; six alleles isolated) are
ry+, have lost the lacZ gene, and show no
haploinsufficient phenotype. Homozygous adults are not recovered because of a
second-site lethal mutation present in the original
Abd-Blac1 chromosome. In hemizygous condition, these
mutants show a distinct phenotype. In females, the dorsal A8 (eighth tergite)
is covered with bristles and trichomes characteristic of an A6/A7 segment
(Fig. 4B, compare with
DfP9/+ females in Fig.
4A) and the genitalia is variably transformed into a sternite
(Fig. 4F; compare with
wild-type females in Fig. 4E).
The males show an eighth tergite bearing trichomes
(Fig. 4D, compare with
DfP9/+ males in Fig.
4C) and a small eighth sternite with one to six bristles
(Fig. 4H, compare with
wild-type males in Fig. 4G).
These phenotypes indicate there is a partial transformation of A8 to A6/A7. In
both sexes, the A5-A7 segments of class II hemizygous adults only show the
haploinsufficient phenotype that is due to the DfP9. These mutants
lack genitalia and analia, and show occasionally a small ninth segment
(Fig. 4I). Class II mutations
do not affect the dominant phenotype of the Fab-864 and
Fab-8416 mutations, which delete the Fab-8
boundary and transform PS12 into PS13
(Barges et al., 2000). They
also complement mutations like iab-7Sz and
iab-7MX2, which affect A7 and A5-A7 segments, respectively
(Galloni et al., 1983;
Sánchez-Herrero et al.,
1985
), but fail to complement Abd-B r (iab-9)
mutations. In hemizygous embryos, the A8 segment is transformed into a more
anterior one (Fig. 4K, compare
with the wild type in Fig. 4J). In the view of the embryonic and adult phenotypes, we have named these
derivatives iab-8,9 mutations. This is a distinct class of BX-C
mutations that transform the A8 and the A9 but not more anterior segments. The
TabR96 mutation
(Celniker and Lewis, 1987
)
also affects both segments, although the effect in the A8 is very weak.
|
The iab-8,9 phenotype correlates with the Abd-B protein distribution in iab-8,9 homozygous embryos: Abd-B expression is normal in PS10 and 11, reduced in PS13 and almost absent in PS14 (Fig. 5B,D, compare with wild-type expression in Fig. 5A,C). In situ hybridization with either a probe specific for the m transcript or with the `common' probe also shows reduced signal in PS13. In some of these embryos, especially in the germband retracted stage, Abd-B expression in PS12 ectoderm is slightly increased compared with wild-type embryos (Fig. 5D, compare with Fig. 5C). These data suggest that iab-8,9 mutations affect the expression of Abd-B r transcripts in A9/PS14 and of the Abd-B m RNA mainly in the A8/PS13.
|
We have characterized the molecular structure of 3 class I and 2 class II derivatives by Southern blot analysis (see Materials and Methods). The data indicate that both types of mutations are the result of recombination events between the `exogenous' promoter (the Abd-Bpp included in the P element) and the corresponding endogenous sequences. In class I derivatives, the ry, Fab-8 and iab-8 sequences of the P element are lost as a result of this recombination. In iab-8,9 alleles, the lacZ gene is lost (see Fig. 6). We have also defined, by inverse PCR, the location of the defective P-element of the iab-8,9T1J mutation. This location is precisely in the same position as in the Abd-Blac1 mutation.
|
Characterization of iab-8 regulatory sequences in the
5' region of the Abd-B transcription unit
The genetic and molecular characterization of iab-8,9 mutations
suggested that an iab-8 regulatory region could be located upstream
the Abd-B m transcription unit (see
Fig. 6). To test this
prediction, we fused either a 7 kb genomic fragment from 47,992 to 40,968 (X2
fragment) or a 7.3 kb fragment from 40,968 to 33,634 (L14 fragment) to the
basal construct containing the Abd-Bpp, obtaining the BEAbd-BppX2 and
BEAbd-BppL14 constructs, respectively (Fig.
7A and Materials and Methods). We stained embryos of five
independent lines transformed with each construct with an
anti-ß-galactosidase antibody. The BEAbd-BppX2 construct shows, in four
out of five lines, an increase in staining in lateral epidermal cells of PS13
(A8) compared with embryos transformed with the basal construct
(Fig. 7C; compare with
Fig. 7B), suggesting the
presence of a PS13 regulatory region in the construct. In late stages of
embryogenesis the PS13 signal is not observed, but the embryos show a strong
expression in posterior and anterior spiracles
(Fig. 7F). The BEAbd-BppL14
construct does not give a particular pattern in any segment of the transformed
embryos (not shown).
|
The Abd-Bpp shows, by itself, a strong basal staining that could obscure
some particular signal coming from the tested fragments. To overcome this
problem, and to better define the putative PS13 regulatory region, we fused
the HindIII-EcoRI fragment from 44,912 to 40,968 (B4
fragment) upstream two different basal promoters: The Ubx minimal
promoter (Qian et al., 1991),
generating the construct BEUbxppB4, and the hsp70 minimal promoter
(Hiromi and Gehring, 1987
),
obtaining the construct BEpwHZ50PLB4 (Fig.
7A). Neither the hsp70 minimal promoter nor the
Ubx minimal promoter on their own gives any segment-specific staining
pattern in embryos (Hiromi and Gehring,
1987
; Qian et al.,
1991
). We stained six lines of embryos transformed with the
BEUbxppB4 construct and seven of those transformed with the BEpwHZ50PLB4 one.
None of the two constructs presents a stronger expression in PS13, although
both show a clear signal in both anterior and posterior spiracles in late
embryonic stages (Fig. 7D,G and
data not shown). This suggests that the putative iab-8 sequences
require the presence of an intact Abd-B promoter to direct a PS13
specific expression.
Regulation of expression in the spiracles
The construct containing only the Abd-Bpp shows a strong expression in
anterior and posterior spiracles (Busturia
and Bienz, 1993) (Fig.
1C, Fig. 7E). This
suggests that the Abd-B promoter is specifically activated in both
anterior and posterior spiracles, although gap genes repress Abd-B
expression in anterior spiracles (Harding
and Levine, 1988
; Reinitz and
Levine, 1990
). The same expression in the posterior and anterior
spiracles is observed in embryos transformed with a construct carrying a
BamHI-HindIIII fragment (PS) included within the Abd-Bpp
(see Fig. 7). We note, however,
that the B4 fragment also directs expression in these organs
(Fig. 7G). This suggests that
there is more than a single Abd-B 5' region that could direct
expression in the spiracles.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
This conclusion stems in part from the study of a P-element with
Abd-B regulatory sequences inserted in the Abd-B gene
(Abd-Blac1). This phenomenon, in which a P-element
containing regulatory sequences of a gene is inserted in the same gene is
called `homing' (Hama et al.,
1990) and is particularly frequent in the BX-C
(Bender and Hudson, 2000
;
Fitzgerald and Bender, 2001
;
McCall et al., 1994
). The
analysis of the Abd-Blac1 mutation and of some of its
derivatives has helped to better understand Abd-B regulation.
Class I and Class II (iab-8,9) mutations
Some derivatives from the Abd-Blac1 insertion
(iab-8,9 mutations) transform segments A8 and A9 but not more
anterior segments. Curiously, the transformation of the A8 segment is not to
A7 but, partially, to A6. This suggests that the iab-7 regulatory
sequences are not fully active in the A8 segment, not even when iab-8
activity is reduced. We explain below the effect on Abd-B expression
that causes these transformations.
In iab-8,9 mutations, the defective P-element responsible for the
phenotype retains the ry gene, the 3' iab-8 region and
the Fab-8 exogenous boundary, and is inserted in an intron of the B
and C transcripts (see Fig. 1A
and Fig. 6). We propose that
the ry gene included in the transposon interrupts this transcription,
preventing Abd-B expression in A9/PS14. Such effect on transcription,
which also occurs in Abd-Blac1, has been previously
documented for other P elements inserted in introns of different genes
(Casares et al., 1997;
Horowitz and Berg, 1995
;
McCall et al., 1994
). By
contrast, in iab-8,9 embryos there is no general effect in the
transcription of the Abd-B m transcript. The Abd-B m
promoter is normal, and, accordingly, Abd-B expression and function
is wild type in PS10-12 (except for a slight increase in PS12 Abd-B
expression). Therefore, the transformation of PS13 is due to the specific
inactivation of iab-8 regulatory sequences. To explain this
phenotype, we propose the existence of novel iab-8 regulatory
sequences located upstream the Abd-B m transcription unit and named
by us the 5' iab-8 domain. These sequences would be inactive in
iab-8,9 mutations because of the presence of the exogenous
Fab-8 boundary, placed between them and the Abd-B m promoter
(see Figs 6,
8).
The absence of Abd-B transforms female (A8) and male (A9)
genitalia into leg tissue (Estrada and
Sánchez-Herrero, 2001). In iab-8,9 mutations,
however, the presence of low levels of Abd-B expression in these two
metameres is sufficient to transform them, instead, to abdomen. This
transformation prevents a normal development of the genital disc and
indirectly produces the absence of analia, which derive from the A10 segment
and do not require Abd-B function
(Sánchez-Herrero et al.,
1985
; Tiong et al.,
1985
).
In class I derivatives, the exogenous Fab-8 and 3' iab-8 sequences, as well as the ry gene, are not present. The absence of the exogenous Fab-8 boundary allows for the activity of endogenous iab-8 sequences, and strong lacZ expression in PS13 results. Abd-B expression is reduced in PS10-PS13, as it is in Abd-Blac1 embryos, because the P-element interrupts the Abd-B promoter region. However, as the ry gene is absent, there is complete transcription of Abd-B r RNAs and normal Abd-B expression in PS14 (see Fig. 3B, Fig. 6).
The 5' iab-8 regulatory domain of Abd-B
The existence of the 5' iab-8 domain is supported by the
analysis of transformants carrying fragments of this region. The PS13
lacZ expression of embryos with constructs containing the X2 fragment
is enhanced. Sequences from the distal part of this region (B4 fragment, not
included in the Abd-Bpp), however, do not reproduce this particular expression
when fused to heterologous minimal promoters (hsp70 or Ubx
promoters), suggesting that they require the Abd-B promoter for their
proper activity. The need of enhancers to have their own promoter for a
wild-type activity has been previously reported in Drosophila
(Li and Noll, 1994;
Merli et al., 1996
;
Ohtsuki et al., 1998
;
Schier and Gehring, 1992
).
The preceding data, combined with previous results from the literature,
help to delimit the putative 5' iab-8 domain. The mutation
tuh-3 maps around 34,000 and affects the A9 but not the A8 segment
(Casanova et al., 1986;
Mack et al., 1997
) (see
Fig. 8), thus setting the
distal limit of the putative iab-8 region. In the more proximal
region, the Abd-B48 mutation, located at about 45,700,
causes a strong reduction of Abd-B expression in PS10-PS13
(Celniker et al., 1990
;
Karch et al., 1985
;
Kuhn et al., 1992
) (see
Fig. 8), indicating that this
breakpoint interrupts the Abd-B promoter region required for correct
PS10-13 expression (Sipos et al.,
1998
). By contrast, the Abd-B65 mutation,
which breaks at about 43,700, only mildly affects PS10-PS12
(Boulet et al., 1991
;
Celniker et al., 1990
) (B. E.,
F. C., A. B. and E. S.-H., unpublished), but affects a bit more strongly PS13
(data not shown), suggesting the mutation breaks near the region that
separates the Abd-B m upstream regulatory region common for PS10-PS13
from the 5' iab-8 domain (see
Fig. 8). All these results
confine the 5' iab-8 domain to the region indicated in
Fig. 8A.
Models to explain the PS12 lacZ expression in
Abd-Blac1 embryos
The lacZ gene in Abd-Blac1 shows an early and
strong signal in PS12 when compared with the Abd-B wild-type
expression. The 3' iab-8 sequences included in the P-element
are unlikely to be responsible for this particular staining for three reasons:
first, they are separated from the lacZ gene by the Fab-8
boundary, which should prevent or attenuate the lacZ transcription
directed by these sequences (Barges et al.,
2000; Zhou et al.,
1999
; Zhou and Levine,
1999
) (see Figs 1,
8). Second, the 3'
iab-8 sequences direct expression in PS13 and not in PS12
(Barges et al., 2000
;
Zhou et al., 1999
). Finally,
embryos carrying this transposon outside the Abd-B gene or in the
Ultrabithorax region of the BX-C do not show this PS12 expression
(Fig. 1B,C and data not shown).
Rather, we think that the Fab-8 and PRE exogenous sequences included
in the P-element are the cause of this particular staining. We have observed
that the strong PS12 ß-galactosidase expression in the ventral cord of
Abd-Blac1 embryos disappears in a Pc mutant
background. This suggests that PREs are instrumental in maintaining this PS12
signal.
A PRE is found distal to the Fab-8 boundary
(Barges et al., 2000;
Zhou et al., 1999
), and
therefore included in the Abd-B 3' regulatory region of the
P-element (see Fig. 8A). As
PREs frequently pair (Fauvarque and Dura,
1993
; Kassis,
1994
; Muller et al.,
1999
; Sigrist and Pirrotta,
1997
), it is possible that the exogenous and endogenous PREs may
pair in Abd-Blac1, creating a loop in the DNA. The
Fab-8 boundaries may also contribute to this pairing
(Hogga et al., 2001
), as is
the case with other types of boundary regions [such as the su
(Hw) insulators (Cai and Shen,
2001
; Muravyova et al.,
2001
)]. Based on this pairing mechanism, we propose two models to
explain the early and strong signal in PS12 of Abd-Blac1
embryos.
In the first model (Fig.
8B), the pairing takes place between the endogenous and exogenous
Fab-8/PRE sequences (iab-8 sequences could also contribute
to it). This would both approximate the iab-7 sequences (which direct
expression in PS12) to the lacZ gene and stabilize the looping of
these sequences, resulting in a strong expression in PS12. Supporting this
model, P-lacZ elements inserted in the iab-7 domain show a similar
strong ß-galactosidase expression in PS12 in the ventral cord
(Barges et al., 2000;
Galloni et al., 1993
). This
suggests that the iab-7 sequences are driving lacZ
expression in PS12 of Abd-Blac1 embryos. This model could
also explain the early lacZ expression in PS12 for the following
reason. In the wild type, the m transcript is detected consecutively
and with decreasing amount of expression in PS13, PS12, PS11 and PS10
(Boulet et al., 1991
;
Kuziora and McGinnis, 1988
;
Sánchez-Herrero and Crosby,
1988
). The outcome is that stage of development in which
transcription is detected and the amount of expression are related
(Crosby et al., 1993
). The
increased lacZ expression in PS12 of Abd-Blac1
embryos may result in an `earlier' ß-galactosidase expression in PS12. A
similar case has been described for a P-lacZ insertion in the
iab-7 domain: in this bluetail mutation, lacZ
expression starts in PS12 at stage 8, before Abd-B expression is
detected in PS12 of wild-type embryos
(Galloni et al., 1993
). The
model requires the iab-7 sequences to bypass the Fab-8
boundary, although this also takes place in the wild type.
The second model (Fig. 8C)
proposes that Fab-7/PRE sequences pair with the exogenous
Fab-8/PRE DNA. Pairing between different Fab sequences have
been previously proposed to occur in normal development
(Hogga et al., 2001). We
assume there is a competition between endogenous and exogenous
Fab-8/PRE sequences to pair with Fab-7/PRE and that the
pairing with the exogenous ones is somehow preferred. As a result, the
3' iab-8 sequences would be within the same domain as the
iab-7 DNA and both would activate the lacZ gene in PS12.
This model would explain the similar PS12 and PS13 expression in
Abd-Blac1 embryos. Supporting this model, the
lacZ signal in germ band extended Abd-Blac1
embryos is similar to that of Fab-864 embryos. This
mutation is a derivative of a P-lacZ element inserted in the
iab-7 domain, which retains the lacZ gene; in this mutant,
the Fab-8 boundary is deleted and, as a result, both the
iab-7 and iab-8 regulatory sequences activate the
lacZ gene (Barges et al.,
2000
). However, the model does not explain the absence of strong
PS13 lacZ expression in the nervous system of
Abd-Blac1 embryos. It is possible that the pairing between
Fab/PRE sequences changes throughout development. If so, the
lacZ pattern of Abd-Blac1 embryos could be
explained by a combination of both models.
The two models predict an increase of Abd-B protein expression in PS12 of Abd-Blac1 and iab-8,9 embryos. In the first model, iab-7 interaction with the Abd-B m promoter is reinforced. In the second one, 3' iab-8 sequences could now be active in PS12. In Abd-Blac1 embryos, this effect could be difficult to detect, as Abd-B transcription is strongly reduced. However, it should be visible in iab-8,9 embryos, because the exogenous and endogenous Fab-8/PRE sequences could also pair and the Abd-B promoter is intact. We have observed a slight increase in Abd-B expression in PS12 of iab-8,9 homozygous embryos, but this effect is insufficient to cause a clear phenotype in either iab-8,9 homozygous embryos or iab-8,9 heterozygous females.
Abd-B regulation in PS13
Our data, and previous results (Barges
et al., 2000; Zhou et al.,
1999
), support the existence of both 3' and 5'
iab-8 domains. The 3' domain seems dispensable for a wild-type
A8/PS13 development if the 5' domain is intact, as in the
Fab-7R59 and iab-7S10 mutations, but
it is not clear if the opposite is true. In iab-8,9 mutations, the
5' iab-8 domain is inactive (determined by the presence of the
exogenous Fab-8 boundary), but, according to the first model, the
pairing of exogenous and endogenous 3' iab-8 sequences may also
reduce the activity of the 3' iab-8 domain. Thus, in these
mutations the activity of both 5' and 3' iab-8 sequences
could be compromised. This interpretation suggests that either the 3' or
the 5' iab-8 domains could determine Abd-B protein distribution
in A8/PS13. Alternatively, both domains should be necessary for A8/PS13
development, but the 5' domain alone could direct A8/PS13 development if
the 3' domain is absent. In any case, these results underline the
complexity of Abd-B regulation.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barges, S., Mihaly, J., Galloni, M., Hagstrom, K., Muller, M.,
Shanower, G., Schedl, P., Gyurkovics, H. and Karch, F.
(2000). The Fab-8 boundary defines the distal limit of
the bithorax complex iab-7 domain and insulates iab-7 from
initiation elements and a PRE in the adjacent iab-8 domain.
Development 127,779
-790.
Bender, W. and Hudson, A. (2000). P element
homing to the Drosophila bithorax complex.
Development 127,3981
-3992.
Bender, W., Akam, M., Karch, F., Beachy, P. A., Peifer, M., Spierer, P., Lewis, E. B. and Hogness, D. S. (1983). Molecular genetics of the bithorax complex of Drosophila.Science 221,23 -29.
Brock, H. W. and van Lohuizen, M. (2001). The Polycomb group-no longer an exclusive club? Curr. Opin. Genet. Dev. 11,175 -181.[CrossRef][Medline]
Boulet, A. M., Lloyd, A. and Sakonju, S. (1991). Molecular definition of the morphogenetic and regulatory functions and the cis-regulatory elements of the Drosophila Abd-B homeotic gene. Development 111,393 -405.[Abstract]
Busturia, A. and Bienz, M. (1993). Silencers in Abdominal-B, a homeotic Drosophila gene. EMBO J. 12,1415 -1425.[Abstract]
Cai, H. N. and Shen, P. (2001). Effects of
cis arrangement of chromatin insulators on enhancer-blocking
activity. Science 291,493
-495.
Campos-Ortega, J. A. and Hartenstein, V. (1985). The Embryonic Development of Drosophila melanogaster. Berlin: Springer-Verlag.
Casanova, J., Sánchez-Herrero, E. and Morata, G. (1986). Identification and characterization of a parasegment specific regulatory element of the Abdominal-B gene of Drosophila. Cell 47,627 -636.[Medline]
Casares, F. and Sánchez-Herrero, E.
(1995). Regulation of the infraabdominal regions of the
bithorax complex of Drosophila by gap genes.
Development 121,1855
-1866.
Casares, F., Bender, W., Merriam, J. and Sánchez-Herrero,
E. (1997). Interactions of Drosophila Ultrabithorax
regulatory regions with native and foreign promoters.
Genetics 145,123
-137.
Celniker, S. E. and Lewis, E. B. (1987). Transabdominal, a dominant mutant of the Bithorax Complex, produces a sexually dimorphic segmental transformation in Drosophila. Genes Dev. 1,111 -123.[Abstract]
Celniker, S., Keelan, D. J. and Lewis, E. B. (1989). The molecular genetics of the bithorax complex of Drosophila: characterization of the products of the Abdominal-B domain. Genes Dev. 3,1424 -1436.[Abstract]
Celniker, S. E., Sharma, S., Keelan, D. J. and Lewis, E. B. (1990). The molecular genetics of the bithorax complex of Drosophila: cis-regulation in the Abdominal-B domain. EMBO J. 9,4277 -4286.[Abstract]
Crosby, M. A., Lundquist, E. A., Tautvydas, R. M. and Johnson,
J. J. (1993). The 3' regulatory region of the
Abdominal-B gene: genetic analysis supports a model of reiterated and
interchangeable regulatory elements. Genetics
134,809
-824.
DeLorenzi, M. and Bienz, M. (1990). Expression of Abdominal-B homeoproteins in Drosophila embryos. Development 108,323 -329.[Abstract]
Estrada, B. and Sánchez-Herrero, E.
(2001). The Abdominal-B Hox gene of Drosophila
antagonizes appendage development. Development
128,331
-339.
Fauvarque, M.-O. and Dura, J.-M. (1993). polyhomeotic regulatory sequences induce developmental regulator-dependent variegation and targeted P-element insertions in Drosophila. Genes Dev. 8,1508 -1520.
Fitzgerald, D. P. and Bender, W. (2001).
Polycomb group repression reduces DNA accesibility. Mol.
Cell. Biol. 21,6585
-6597.
FlyBase (1999). The Flybase database of the
Drosophila genome projects and community literature.
Nucleic Acids Res. 27,85
-88.
Galloni, M., Gyurkovics, H., Shedl, P. and Karch, F. (1993). The bluetail trasposon: evidence for independent cis-regulatory domains and domain boundaries in the bithorax complex. EMBO J. 12,1087 -1097.[Abstract]
Gyurkovics, H., Gausz, J., Kummer, J. and Karch, F. (1990). A new homeotic mutation in the Drosophila bithorax complex removes a boundary separating two domains of regulation. EMBO J. 9,2579 -2586.[Abstract]
Hagstrom, K., Muller, M. and Schedl, P. (1996). Fab-7 functions as a chromatin domain boundary to ensure proper segment specification by the Drosophila bithorax complex. Genes Dev. 10,3203 -3215.
Hagstrom, K., Muller, M. and Schedl, P. (1997).
A Polycomb and GAGA dependent silencer adjoins the Fab-7
boundary in the Drosophila bithorax complex.
Genetics 146,1365
-1380.
Hama, C., Ali, Z. and Kornberg, T. B. (1990). Region-specific recombination and expression are directed by portions of the Drosophila engrailed promoter. Genes Dev. 4,1079 -1093.[Abstract]
Harding, K. and Levine, M. (1988). Gap genes define the limits of Antennapedia and Bithorax gene expression during early development in Drosophila. EMBO J. 7, 205-214.[Abstract]
Hendrickson, J. E. and Sakonju, S. (1995).
Cis and trans interactions between the iab
regulatory regions and abdominal-A and Abdominal-B in
Drosophila melanogaster. Genetics
139,835
-848.
Hiromi, Y. and Gehring, W. J. (1987). Regulation and function of the Drosophila segmentation gene fushi tarazu. Cell 50,963 -974.[Medline]
Hogga, I., Mihaly, J., Barges, S. and Karch, F. (2001). Replacement of Fab-7 by the gypsy or scs insulator disrupts long-distance regulatory interactions in the Abd-B gene of the Bithorax complex. Mol. Cell 8,1145 -1151.[Medline]
Hopmann, R., Duncan, D. and Duncan, I. (1995).
Transvection in the iab-5,6,7 region of the bithorax complex of
Drosophila: homology independent interactions in trans.Genetics 139,815
-833.
Horowitz, H. and Berg, C. A. (1995). Aberrant
splicing and transcription termination caused by P element insertion into the
intron of a Drosophila gene. Genetics
139,327
-335.
Karch, F., Weiffenbach, B., Peifer, M., Bender, W., Duncan, I., Celniker, S., Crosby, M. and Lewis, E. B. (1985). The abdominal region of the bithorax complex. Cell 43, 81-96.[Medline]
Kassis, J. A. (1994). Unusual properties of
regulatory DNA from the Drosophila engrailed gene: three
`pairing-sensitive' sites within a 1.6 kb region.
Genetics 136,1025
-1038.
Kaufman, T. C., Seeger, M. A. and Olsen, G. (1990). Molecular and genetic organization of the Antennapedia gene complex of Drosophila melanogaster. Adv. Genet. 27,309 -361.[Medline]
Kuhn, D. T., Sawyer, M., Packert, G., Turenchalk, G., Mack, J.
A., Sprey, Th. E., Gustavson, E. and Kornberg, T. B. (1992).
Development of the D. melanogaster caudal segments involves
suppression of the ventral regions of A8, A9 and A10.
Development 116,11
-20.
Kuziora, M. A. and McGinnis, W. (1988). Different transcripts of the Drosophila Abd-B gene correlate with distinct genetic sub-functions. EMBO J. 7,3233 -3244.[Abstract]
Lewis, E. B. (1978). A gene complex controlling segmentation in Drosophila. Nature 276,565 -570.[Medline]
Li, X. and Noll, M. (1994). Compatibility between enhancers and promoters determines the transcriptional specificity of goosberry and gooseberry neuro in the Drosophila embryo. EMBO J. 13,400 -406.[Abstract]
Mack, J. A., Smith, R. D. and Kuhn, D. T.
(1997). Mobile element 297 in the Abd-B gene of
Drosophila melanogaster, not Delta 88, is responsible for
the tuh-3 mutation. Genetics
147,679
-688.
Martin, C. H., Mayeda, C. A., Davis, C. A., Ericsson, C. L., Kafels, J. D., Mathog, D. R., Celniker, S. E., Lewis, E. B. and Palazzolo, M. J. (1995). Complete sequence of the bithorax complex of Drosophila. Proc. Natl. Acad. Sci. USA 92,8398 -8402.[Abstract]
McCall, K., O'Connor, M. B. and Bender, W. (1994). Enhancer traps in the Drosophila bithorax complex mark parasegmental domains. Genetics 138,389 -399.
Merli, C., Begstrom, D. E., Cygan, J. A. and Blackman, R. K. (1996). Promoter specificity mediates the independent regulation of neighbouring genes. Genes Dev. 10,1260 -1270.[Abstract]
Mihaly, J., Hogga, I., Gausz, J., Gyurkovics, H. and Karch,
F. (1997) In situ detection of the Fab-7 region of
the bithorax complex into a chromatin domain boundary and a
Polycomb-response element. Development
124,1809
-1820.
Muller, M., Hagstrom, K., Gyurkovics, H., Pirrotta, V. and
Schedl, P. (1999). The Mcp element from the
Drosophila melanogaster bithorax complex mediates long-distance
regulatory interactions. Genetics
153,1333
-1356.
Muravyova, E., Golovnin, A., Gracheva, E., Parshikov, A.,
Belenkaya, T., Pirrotta, V. and Georgiev, P. (2001). Loss of
insulator activity by paired su (Hw) chromatin insulators.
Science 291,495
-498.
Nöthiger, R., Dübendorfer, A. and Epper, F. (1977). Gynandromorphs reveal two separate primordia for male and female genitalia in Drosophila melanogaster. Roux' Arch. Dev. Biol. 181,367 -373.
Ohtsuki, S., Levine, M. and Cai, H. N. (1998). Different core promoters possess distinct regulatory activities in the Drosophila embryo. Genes Dev. 15,547 -556.
Patel, N. H., Martín-Blanco, E., Coleman, K. G., Poole, S. P., Ellis, M. C., Kornberg, T. B. and Goodman, C. S. (1989). Expression of engrailed proteins in arthropods, annelids and chordates. Cell 58,955 -968.[Medline]
Qian, S., Capovilla, M. and Pirrotta, V. (1991). The bx region enhancer, a distant cis control element of the Drosophila Ubx gene and its regulation by hunchback and other segmentation genes. EMBO J. 10,1415 -1425.[Abstract]
Reinitz, J. and Levine, M. (1990). Control of the initiation of homeotic gene expression by the gap genes giant and tail-less in Drosophila. Dev. Biol. 140, 52-72.
Sánchez-Herrero, E. (1991). Control of the expression of the bithorax complex genes abdominal-A and Abdominal-B by cis-regulatory regions in Drosophila embryos. Development 111,437 -449.[Abstract]
Sánchez-Herrero, E. and Akam, M. (1989). Spatially ordered transcription of regulatory DNA in the bithorax complex of Drosophila. Development 107,321 -329.[Abstract]
Sánchez-Herrero, E. and Crosby, M. A. (1988). The Abdominal-B gene of Drosophila melanogaster: overlapping transcripts exhibit two different spatial distributions. EMBO J. 7,2163 -2173.
Sánchez-Herrero, E., Vernós, I., Marco, R. and Morata, G. (1985). Genetic organization of the Drosophila Bithorax complex. Nature 313,108 -113.[Medline]
Schier, A. F. and Gehring, W. J. (1992). Direct homeodomain-DNA interaction in the autoregulation of the fushi tarazu gene. Nature 356,804 -807.[CrossRef][Medline]
Sigrist, C. J. A. and Pirrotta, V. (1997).
Chromatin insulator elements block the silencing of a target gene by the
Drosophila Polycomb response element (PRE) but allow trans
interactions between PREs on different chromosomes.
Genetics 147,209
-221.
Sipos, L., Mihaly, J., Karch, F., Schedl, P., Gausz, J. and
Gyurkovics, H. (1998). Transvection in the Drosophila
Abd-B domain: extensive upstream sequences are involved in anchoring
distant cis-regulatory regions to the promoter.
Genetics 149,1031
-1050.
Sullivan, W., Ashburner, M. and Hawley, R. S. (2000). Drosophila Protocols. Cold Spring Harbor Laboratory Press. New York.
Tiong, S., Bone, L. M. and Whittle, J. R. (1985). Recessive lethal mutations within the bithorax complex in Drosophila. Mol. Gen. Genet. 200,335 -342.[Medline]
Tiong, S., Whittle, J. R. S. and Gribbin, M. C. (1987). Chromosomal continuity in the abdominal region of the bithorax complex of Drosophila is not essential for its contribution to metameric identity. Development 101,135 -142.[Abstract]
Wieschaus, E. and Nüsslein-Volhard, C. (1986). Looking at embryos. In Drosophila: A Practical Approach (ed. D.B. Roberts), pp.199 -227. Washington: IRL Press.
Zavortink, M. and Sakonju, S. (1989). The morphogenetic and regulatory functions of the Drosophila Abdominal-B gene are encoded in overlapping RNAs transcribed from separate promoters. Genes Dev. 3,1969 -1981.[Abstract]
Zhou, J. and Levine, M. (1999). A novel cis-regulatory element, the PTS, mediates an anti-insulator activity in the Drosophila embryo. Cell 99,567 -575.[Medline]
Zhou, J., Barolo, S., Szymanski, P. and Levine, M. (1996). The Fab-7 element of the bithorax complex attenuates enhancer-promoter interactions in the Drosophila embryo. Genes Dev. 10,3195 -3201.[Abstract]
Zhou, J., Ashe, H., Burks, C. and Levine, M.
(1999). Characterization of the transvection mediating region of
the Abdominal-B locus in Drosophila.Development 126,3057
-3065.