1 Istituto Pasteur, Fondazione Cenci Bolognetti, Dipartimento di Genetica e
Biologia Molecolare, Università `La Sapienza', 00185 Roma, Italy
2 Dipartimento di Anatomia Patologica e di Genetica (DAPEG), Università
di Bari, 70126 Bari, Italy
* Author for correspondence (e-mail: sergio.pimpinelli{at}uniroma1.it)
Accepted 8 May 2003
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SUMMARY |
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Key words: Drosophila, Fat body, BX-C
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INTRODUCTION |
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Homeotic genes have long been regarded as selector genes whose protein
products bind to and regulate the transcription of downstream genes. Molecular
analyses have shown that DNA binding is mediated by a domain denoted the
`homeodomain' that is shared among the homeotic proteins
(Affolter et al., 1990).
Although the genes of the BX-C were the first of such selector genes to be
described and characterized, the numbers and types of target genes they
regulate have yet to be fully characterized. Hence, a major effort since the
discovery of the homeodomain has been focused on the identification of the
direct targets for both the ANT-C and BX-C genes.
In several studies, immunostaining of Drosophila salivary gland
chromosomes has been used to localize chromosomal binding sites and potential
targets of several chromosomal proteins
(Andrew and Scott, 1994).
Although this is a powerful approach for proteins normally expressed in the
salivary gland, it has limited value for proteins that are not detectably
expressed in this tissue, such as the products of the BX-C. To try to
circumvent this limitation, an inducible transgene has been used to express
Ubx protein in salivary glands. The binding sites of this ectopically produced
protein were then characterized (Botas and
Auwers, 1996
). Although this strategy provides information on the
potential of proteins of interest to bind specific sites, the relevance of the
binding sites should be verified in tissues normally expressing the
protein.
In addition to in vivo chromosomal protein localization studies, salivary
gland chromosomes have also been used to reveal the presence of gene activity.
Most striking has been the observation of chromosome puffs that form when
specific genes are induced developmentally or through treatments such as heat
shock (Ashburner, 1972). There
is also evidence that repressed genes in salivary gland nuclei appear
condensed or are under replicated. This is specifically the case for the BXC
genes that are severely under represented compared to other loci in salivary
gland DNA (Moshkin et al.,
2001
), making the cytological resolution of the BX-C locus
difficult in salivary chromosomes under normal circumstances.
We have investigated the potential of another tissue containing polytene chromosomes, the larval fat body, to address questions relevant to in vivo BX-C expression. We show that despite the lower degree of endoreduplication of fat body as compared to salivary gland chromosomes, fat body cells do provide readable chromosomes. Moreover, they also provide a physiologically relevant environment for studying BX-C gene and protein expression. We also show that the resolution of our cytological studies is sufficient to document chromosomal changes associated with the activation of the BX-C locus and for the identification of chromosomal targets of the Ubx, Abd-A and Abd-B proteins.
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MATERIALS AND METHODS |
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DAPI staining of polytene chromosomes and in situ hybridization
Fat bodies of third instar larvae were dissected in physiological solution
(0.7% NaCl in distilled water), transferred in a drop of ethanol-propionic
acid 3:1 and incubated for 10-20 minutes. The fat bodies were then transferred
in about 6 µl of fixative solution (40% acetic acid, 30% lactic acid, 30%
distilled water) on a siliconized coverslip. After 3-5 minutes, a very clean,
dust-free slide was lowered onto the coverslip, and pressed very lightly with
a finger. Then, the sandwich was reversed and squashed very gently for 1-2
minutes between two sheets of blotting paper. After squashing, the slide was
immersed in liquid nitrogen for about 20 seconds and, after removal of the
coverslip by a razor blade, the slide was immediately immersed in PBS at room
temperature and stained with 0.05 µg/ml of DAPI dissolved in 2xSSC
for 4 minutes. The in situ hybridization assays were performed according to
Pimpinelli et al. (Pimpinelli et al.,
2000).
Antibodies and probes
The antibodies used for immunostaining experiments were: mouse anti-Ubx
monoclonal FP3.38 antibody (White and
Wilcox, 1984); rat anti-Abd-A polyclonal antibody
(Macias et al., 1990
); mouse
anti-Abd-A monoclonal Dmabd-A.1 antibody
(Kellerman et al., 1990
);
mouse anti-Abd-B monoclonal antibody
(Celniker et al., 1989
); rabbit
anti-Polycomb polyclonal antibody affinity purified obtained by R. Paro; and
rabbit anti-Trithorax polyclonal antibody obtained by P. Harte.
Immunostaining of whole fat bodies
Whole fat bodies of third instar larvae were dissected in 0.7% NaCl, 1%
Triton solution on a siliconized slide and incubated for 2 min in fixative 1
(3% formaldehyde, 1% Triton in PBS) and then in fixative 2 (45% acetic acid,
3% formaldehyde, 1% Triton) for 8 minutes. After blockage for 30 minutes in
PBS containing 1% nonfat dry milk and1% Triton at room temperature, the
tissues were incubated with primary antibody diluted in PBS/BSA 1% for 1 hour
at room temperature in a humid chamber. After three washes (5 minutes each) in
PBS, the tissues were incubated with secondary antibody diluted in PBS/BSA 1%
for 1 hour at room temperature, washed in PBS for three times (5 minutes each)
in PBS and stained with 0.05 µg/ml of DAPI dissolved in 2xSSC for 4
minutes.
Immunostaining of polytene chromosomes
For immunostaining of polytene chromosomes of fat bodies with antibodies
against Polycomb and Trithorax, the chromosomes were fixed as for DAPI
staining. For immunostaining with antibodies against Ubx, Abd-A and Abd-B, the
chromosomes were fixed according to James et al.
(James et al., 1989). In both
cases, the fixed preparations were stained with 0.05 µg/ml of DAPI
dissolved in 2xSSC for 4 minutes, washed in PBS and examined under the
fluorescence microscope to select satisfactory preparations (slides were
examined in PBS). The selected slides were immersed into PBS containing 1%
Triton X 100 and left for 20 minutes at room temperature, then transferred in
PBS containing 1% nonfat dry milk for 30 minutes at room temperature and then
incubated with primary antibody (diluted in PBS/BSA 1%) for 1 hour at room
temperature and overnight at 4°C in a humid chamber. After incubation with
the primary antibody, the slides were washed three times (5 minutes each) with
PBS containing 0.5% non fat dry milk and incubated with secondary antibody
(diluted in PBS/BSA 1%) for 1 hour at room temperature. The slides were then
washed three times (5 minutes each) in PBS at 4°C. and stained with DAPI
for 4 minutes. After washing in PBS for about 2 minutes, the slides were
finally mounted in anti-fading medium and sealed with nail polish or rubber
cement. Owing to the difficulty in obtaining very good pictures of entire
polytene chromosomes, the mapping of the homeotic proteins has been carried
out by examining about 50 squashed polytene nuclei for each staining.
Southern blot analyses
DNA samples were extracted from different tissues of Drosophila
larvae and adults. The samples were then used for Southern blot hybridization
signals detection according to Moshkin et al.
(Moshkin et al., 2001).
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RESULTS AND DISCUSSION |
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In order to characterize the anterior to posterior expression patterns of the BX-C genes along the length of the fat body, we stained whole tissues with antibodies directed against the Ubx, Abd-A and Abd-B proteins. The patterns of expression of these proteins are shown in Fig. 1A and diagramed in Fig. 1B. We found that Ubx is intensely expressed in a contiguous region, with an anterior limit distal to, but near, the anterior crossbridge in the third thoracic segment (T3). The domain includes the gonad, and the posterior limit falls in a region corresponding approximately to segments A6/A7. The Abd-A protein is expressed anteriorly in a longitudinal line of cells in a region corresponding to the A2 segment. From that point posteriorly it is accumulated in almost all of the cells in a region that is co-extensive with abdominal segments A3-A7. Finally, the Abd-B protein is expressed to the posterior end of the fat body with an anterior limit in the middle of A4.
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The active BX-C genes form visible puffs on polytene chromosomes of
fat body
To determine if the expression of BX-C encoded proteins is correlated with
cytologically visible changes at the BX-C locus, we compared the appearance of
the region corresponding to the 89DE salivary gland chromosome interval in fat
body cells derived from different regions of the fat body. Cells from various
segments along the anterior posterior axis of the fat body were isolated and
polytene chromosomes prepared for DAPI staining. As shown in
Fig. 2B, we found that in
chromosomes from anterior fat body cells the BX-C locus appears highly
condensed as revealed by its intense DAPI bright fluorescent. This appearance
is similar to the BX-C in polytene chromosomes in salivary glands
(Fig. 2A). However, in
chromosomes taken from fat body cells extracted from the mid-posterior part of
the larvae, the 89DE region clearly appears puffed
(Fig. 2C). In situ
hybridization with cDNA probes derived from the Ubx, abd-A and
Abd-B genes clearly shows that although the signals appear tightly
closed in anterior fat body nuclei (Fig.
2B), in chromosomes from the mid-posterior fat body, the signals
appear more distantly positioned relative to each other and show that the
puffed region corresponds to the BXC (Fig.
2C). These results clearly demonstrate that the activity of BX-C
genes is accompanied by visible changes at the chromosomal and locus level. We
investigated the extent to which this change in visible appearance might
reflect changes at the physical level. It has been shown that in polytene
chromosomes of salivary glands the BX-C region is under replicated and that
the under replication seems to exclude the proximal and distal parts of the
complex corresponding to the 3' and 5' ends of the Ubx
and Abd-B genes, respectively
(Moshkin et al., 2001). We
tested whether the observed chromatin changes related to the expression of the
homeotic gene in fat bodies also includes changes in their level of
endoreduplication. To this end, we performed Southern blot experiments, as
described previously (Moshkin et al.,
2001
), on genomic DNA extracted from salivary glands, fat bodies
and diploid cells from adult heads. These blots were hybridized with specific
probes from the 3' and 5' ends of Ubx, from an exon of
abd-A and from the rosy gene as a control. As shown in
Fig. 2D-F, we found that in the
anterior fat body, where the genes are repressed, there is under replication
of BX-C sequences similar to the polytene chromosomes of salivary glands. By
contrast, the same sequences appear amplified relative to controls in fat body
cells where they are actively expressed.
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The BX-C proteins bind multiple mappable sites on polytene
chromosomes of fat body
Given the relatively high resolution of localization we were able to obtain
for the Pc and Trx proteins, we next examined whether the binding sites for
Ubx, Abd-A and Abd-B were discernable in the fat body polytene chromosomes
thereby providing evidence for homeotic gene targets in this tissue. To this
end, we individually stained squashed polytene nuclei from different segments
of the fat bodies with antibodies specific for the three proteins
(James et al., 1989). As shown
in Fig. 3, the staining
patterns reveal that Ubx (Fig.
3A), Abd-A (Fig.
3B) and Abd-B (Fig.
3C) can be found at many sites on the chromosomes. The results of
a detailed analysis of the staining patterns/binding sites of the three
homeotic proteins are reported in Table
1. Interestingly, some of the Ubx binding sites correspond to loci
known as targets of homeotic proteins
(Botas and Awers, 1996
;
Graba et al., 1997
) while other
known target genes map in regions that lack any signal. This suggests the
possibility that the technique is revealing tissue specific regulation of the
expression of different sets of genes. An inspection of the staining patterns
produced by the Ubx, Abd-A and Abd-B antibodies reveals that these proteins
appear to share several targets. Particularly intriguing is the pattern
produced by these proteins along the BX-C itself. In detail, the antibody
against the Ubx protein produces a unique signal correlated with the
Ubx locus (Fig. 3D).
The antibodies against the Abd-A (Fig.
3E) and Abd-B (Fig.
3F) proteins produce overlapping signals where the abd-A
and Abd-B genes are located. The absence of Abd-A and Abd-B staining
at the Ubx locus suggests that these proteins are not involved in
regulating Ubx and provides an explanation for the distribution of
Ubx expressing cells along the AP axis of the fat body (see
Fig. 1). The presence of Ubx on
Ubx does, however, suggest the possibility of positive autoregulation
at that locus. Moreover, the binding patterns of Abd-A and Abd-B suggest that
these proteins may also cooperate in regulating themselves as well as each
other.
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Conclusions |
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Taken together, these data provide an important basis for identifying specific downstream targets of the BX-C encoded proteins, and for identifying new chromosomal proteins that regulate the BX-C locus. Recent technological advances have increased the sensitivity of microarray analyses allowing investigators to perform experiments on small numbers of cells. Hence, it should now be possible to compare the transcriptional profile of different domains of the larval fat body using this technique. A comparison of microarray data with the results described in this report should help identify candidate genes that are likely to be direct, as opposed to indirect, targets of each of the BX-C genes. Finally, on a practical note, it should be pointed out that in addition to the fat body and salivary gland, other tissues in Drosophila have polytene chromosomes that might be suitable for the types of approaches described in this report.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Affolter, M., Schier, A. and Gehring, W. J. (1990). Homeodomin proteins and the regulation of gene espression. Curr. Opin. Cell Biol. 2, 485-495.[Medline]
Andrew, D. J. and Scott, M. P. (1994). Immunological methods for mapping protein distributions on polytene chromosomes. Methods Cell Biol. 44,353 -370.[Medline]
Ashburner, M. (1972). Puffing patterns in Drosophila melanogaster and related species. In Developmental Studies on Giant Chromosomes (ed. W. Beermann), pp. 101-151. New York, NY: Springer-Verlag.
Bate, M. and Martinez Arias, A. (1993).The Development of Drosophila melanogaster . Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Botas, J. and Auwers, L. (1996). Chromosomal binding sites of Ultrabithorax homeotic proteins. Mech. Dev. 56,129 -138.[CrossRef][Medline]
Celniker, S. E., 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]
Chinwalla, V., Jane, E. P. and Harte, P. J. (1995). The Drosophila trithorax protein binds to specific chromosomal sites and is co-localized with Polycomb at many sites. EMBO J. 14,2056 -2065.[Abstract]
Gonzales-Reyes, A., Urquia, N., Gehring, W. J., Struhl, G. and Morata, G. (1990). Are cross-regulatory interactions between homoeotic genes functionally significant? Nature 344, 78-80.[CrossRef][Medline]
Graba, Y., Aragnol, D. and Pradel, J. (1997). Drosophila Hox Complex downstream targets and the function of homeotic genes. BioEssays 19,379 -388.[Medline]
James, T. C., Eissenberg, J. C., Craig, C., Dietrich, V., Hobson, A. and Elgin, S. C. R. (1989). Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila. Eur. J. Cell Biol. 50,170 -180.[Medline]
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 -362.[Medline]
Kellerman, K. A., Mattson, D. M. and Duncan, I. (1990). Mutations affecting the stability of the fushi tarazu protein of Drosophila. Genes Dev. 4,1936 -1950.[Abstract]
Kennison, J. A. (1993). Transcriptional activation of Drosophila homeotic genes from distant regulatory elements. Trends Genet. 9, 75-79.[CrossRef][Medline]
Lewis, E. B. (1978). A gene complex controlling segmentation in Drosophila. Nature 276,565 -570.[Medline]
Macias, A., Casanova, J. and Morata, G. (1990). Expression and regulation of the abd-A gene of Drosophila. Development 110,1197 -1207.[Abstract]
Moshkin, Y. M., Alekseyenko, A., Semeshin, F. V., Spierer, A.,
Spierer, P., Makarevich, G. F., Belyaeva, E. S. and Zhimulev, I. F.
(2001). The Bithorax Complex of Drosophila melanogaster:
Underreplication and morphology in polytene chromosomes. Proc.
Natl. Acad. Sci. USA 98,570
-574.
Paro, R. (1990). Imprinting a determined state into the chromatin of Drosophila. Trends Genet. 6,416 -421.[CrossRef][Medline]
Pimpinelli, S., Bonaccorsi, S., Fanti, L. and Gatti, M. (2000). Preparation and analysis of mitotic chromosomes of Drosophila melanogaster. In Drosophila: A Laboratory Manual (ed. W. Sullivan, M. Ashburner and S. Hawley), pp1 -24. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Pirrotta, V. (1997). Pc-G complexes and chromatin silencing. Curr. Opin. Genet. Dev. 7, 249-258.[CrossRef][Medline]
Rizki, T. M. and Ritzki, M. R. (1978). Larval adipose tissue of homoeotic bithorax mutants of Drosophila. Dev. Biol. 65,476 -482.[Medline]
Valentine, J. W., Erwin, D. H. and Jablonski, D. (1996). Developmental evolution of metazoan bodyplans: the fossil evidence. Dev. Biol. 173,373 -381.[CrossRef][Medline]
White, R. A. H. and Wilcox, M. (1984). Protein products of the Bithorax Complex in Drosophila. Cell 39,163 -171.[Medline]
Zink, B. and Paro, R. (1989). In vivo binding pattern of a trans-regulator of homoeotic genes in Drosophila melanogaster. Nature 337,468 -471.[CrossRef][Medline]