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doi: 10.1242/10.1242/dev.00587

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Differential expression of the Drosophila BX-C in polytene chromosomes in cells of larval fat bodies: a cytological approach to identifying in vivo targets of the homeotic Ubx, Abd-A and Abd-B proteins

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

View larger version (70K): [in a new window]   Fig. 1. Expression patterns of the Ubx, Abd-A and Abd-B proteins on the AP axis of the fat body. (A) Patterns produced by antibodies directed against the BX-C encoded proteins on the AP axis of Drosophila larval fat body. Green arrows indicate the larval gonads. (Top) The Ubx pattern in the fat body of a third instar male larva. The expression of the Ubx protein starts from the anterior bridge (white arrows) and ends in a region situated posterior to the gonad (white arrows) while the anterior of the fat body (large arrowheads) and the salivary glands (small arrowheads) appear devoid of signal. (Middle) Expression pattern of Abd-A in the fat body of a third instar female larva. This protein is accumulated more posteriorly than Ubx (arrows) but anteriorly partially overlaps the Ubx domain. Note that Abd-A is not expressed in all nuclei within its domain. (Bottom) The expression pattern of Abd-B in the fat body of a third instar female larva. This protein is the most posteriorly expressed of the three (arrows). However, its anterior limit of expression does partially overlap with the expression of the other two proteins. As with Abd-A, the protein is not expressed in all the nuclei of its domain. Note that the same immunopatterns were observed in both male and female fat bodies. However, the male gonads showed a low level of immunostaining with all the antibodies, while the female gonads lack any staining. At present, it is not possible to determine if this difference is due to a sex specific function of the homeotic proteins in the gonadal mesoderm or to the difference in size of the gonads in the two sexes. (B) The topological relationship of the Ubx, Abd-A and Abd-B staining patterns with the cuticular ectodermal segments (top). The most anterior expression of the Ubx corresponds approximately to T3 while the posterior border is approximately situated in the A6 segment. The Abd-A protein is expressed in a region co-extensive with segments A3-A7. At its anterior limit it is expressed along two external lines of cells in a region corresponding to segment A2. The Abd-B protein is expressed from a region corresponding to the middle of A4 to the posterior end of the fat body. Note that while Ubx seems to be expressed homogeneously along its domain, Abd-A and Abd-B are expressed only in subsets of cells within their domains of accumulation. Another point of interest is that the immunopatterns seem to suggest that the three proteins could be simultaneously expressed in most nuclei in a region corresponding to segments A4-A6. This point deserves to be better analyzed by tripe labelling when appropriate primary antibodies become available.

View larger version (75K): [in a new window]   Fig. 2. The expression of the BX-C genes in the fat body is related to cytologically visible changes of the 89DE BX-C region of the polytene chromosomes. (A) The 89DE region appears strongly condensed in polytene chromosomes of the salivary glands. (B) The 89DE region also appears condensed in polytene chromosomes in cells from the anterior parts of the fat body (big arrows in Fig. 1A). In situ hybridization with probes corresponding to the Ubx, abd-A and Abd-B genes clearly shows that these genes are tightly physically linked. (C) The 89DE region in polytene chromosomes of mid-posterior fat body cells appear strongly puffed and, by in situ hybridization with the same probe as in B, the three genes appear more physically distinct. (D-F) The BX-C genes that are active in fat body cells are also amplified relative to cells in which they are quiescent. (D) The structure of the Ubx and abd-A genes. The gray boxes indicate the exons. The black blocks above indicate the probes used to determine the degree of amplification of the corresponding genomic sequences. (E) The panel shows a Southern blot of genomic DNA from adult heads (a.h.), mid fat body (m.f.b.) and salivary glands (s.g.) probed with a DNA fragment corresponding to the rosy gene and two other fragments corresponding to the 5' and 3' regions of the Ubx gene. As has been shown (Moshkin et al., 2001), a comparison of the two Ubx signals with the rosy signal between adult heads and the salivary glands demonstrates that the 3' end of the Ubx gene is amplified while the 5' end is under replicated. We observed the same pattern in anterior fat body where the Ubx gene is repressed (not shown). However, in mid fat body, where the Ubx gene is active, both the 3' and 5' ends appear amplified. (F) The panel shows a Southern blot of genomic DNA from adult heads (a.h.), anterior fat body (a.f.b.), posterior fat body (p.f.b.) and salivary glands (s.g.) probed with a DNA fragment corresponding to rosy and a fragment corresponding to a region of abd-A. Again abd-A appears under replicated in salivary glands and anterior fat body where this gene is repressed, while it appears amplified in posterior fat body where abd-A is expressed. (G-L) Patterns of localization of the Polycomb (Pc) and Trithorax (Trx) proteins in the 89DE region of polytene chromosomes from salivary glands and mid-posterior fat body. (G,H) Staining of polytene chromosomes from salivary glands with Pc and Trx antibodies respectively. As has been shown, Pc is strongly accumulated on the BX-C locus (arrow). Trx is also located in the same region (arrow) although the fluorescence intensity suggests that is accumulated at much lower levels than Pc. (I,L) Staining of polytene chromosomes from mid posterior fat body with Pc (I) and Trx (L) antibodies. Pc is not detectable on strongly puffed 89DE region (large arrowhead) where the BX-C genes are active, while the same region shows a strong accumulation of Trx (arrow). Small arrowhead indicates a PC signal outside the 89DE region.

View larger version (82K): [in a new window]   Fig. 3. Patterns of localization of Ubx, Abd-A and Abd-B on polytene chromosomes of the fat body. (A-C) The patterns after staining with Ubx (A), Abd-A (B) and Abd-B (C) antibodies. All the antibodies give numerous signals along the length of all of the chromosomes with the exception of the chromocenter. (D) Enlarged picture of the BX-C region showing that Ubx is located in the proximal part of the region corresponding to the Ubx locus. (E) Enlarged picture of BX-C region sequentially stained with Abd-A antibodies and in situ hybridized with probes derived from Ubx, abd-A and Abd-B cDNAs. The merged figure clearly shows that the Abd-A signal overlaps with both the abd-A and Abd-B genes, while the Ubx gene appears devoid of signal. (F) Enlarged picture of the BX-C region showing the Abd-B staining pattern. Similar to the Abd-A protein, we observed that Abd-B is located on the abd-A and Abd-B genes but is excluded from Ubx. X, X chromosome; 2L and 2R, left and right arms of the second chromosome; 3L and 3R, left and right arms of the third chromosome; chr, chromocenter.