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doi: 10.1242/10.1242/dev.00297

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Drosophila MBF1 is a co-activator for Tracheae Defective and contributes to the formation of tracheal and nervous systems

Qing-Xin Liu^1,*, Marek Jindra^1,2,*, Hitoshi Ueda¹, Yasushi Hiromi¹ and Susumu Hirose^1,

¹ Department of Developmental Genetics, National Institute of Genetics and Department of Genetics, Graduate University for Advanced Studies, Mishima, Shizuoka-ken 411-8540, Japan
² Institute of Entomology, Czech Academy of Sciences, Ceske Budejovice, 37005 Czech Republic

View larger version (83K): [in a new window]   Fig. 1. Molecular cloning, expression and functional analyses of Drosophila MBF1. (A) Deduced amino acid sequences of fly, silkmoth, human and yeast MBF1. Residues that are identical to those of fly MBF1 are shaded. Thick bar represents the well-structured domain; 1-4 denote four amphipathic helices. (B) Drosophila mbf1 partially rescues sensitivity of yeast mbf1 disruptant to aminotriazole. Indicated Saccharomyces cerevisiae strains were tested for aminotriazole sensitivity. Yeast MBF1 (trp1-1 ura3-52 leu2-P1) and mbf1 (trp1-1 ura3-52 leu2-P1 mbf1::LEU2), and a yeast genomic MBF1 construct pMBF1 have been described previously (Takemaru et al., 1998). pDmMBF1 carries Drosophila mbf1+ cDNA in place of the yeast MBF1 coding region in pMBF1. As a control, these four yeast strains showed essentially the same growth in the absence of aminotirazole (data not shown). (C) Expression of MBF1 as revealed by immunostaining with anti-MBF1 antibody. (1) Syncytial blastoderm stage embryo. (2) Stage 16 embryo. Staining of CNS along the midline and tracheal staining on the margin of the embryo. (3-5) Dissected tissues from a 3rd instar larva: (3) strong staining in both the somatic and germ cells of the developing testis; (4) nuclear staining in the polyploidal salivary gland; and (5) CNS. (D) Expression of a FTZ-F1-dependent reporter gene in mbf1+ and mbf1- homozygous embryos. Expression of a transgene fPE-lacZ carrying the ftz proximal enhancer and the hsp70 minimal promoter fused to lacZ (upper panels) or its mutant derivatives (middle and bottom panels) was analyzed by X-gal staining. Mutation in FTZ-F1-binding sites but not in the mbf1 locus affected the reporter gene expression. The fPE-lacZ is silent in ftz-f1 mutant background (Yussa et al., 2001).

View larger version (49K): [in a new window]   Fig. 3. Co-immunoprecipitation of TDF, MBF1 and TBP. Western blots of nuclear proteins from yw (mbf1+) or mbf1 mutant embryos immunoprecipitated using the anti-TBP (A) and anti-TDF (B) antibodies (IP) were probed with anti-TDF (top), anti-TBP (middle) or anti-MBF1 (bottom) antibodies. Western blots of input proteins are shown in the left columns.

View larger version (35K): [in a new window]   Fig. 4. DNA-binding properties of TDF. (A) Consensus binding sequence of TDF. After five cycles of immunoselection of TDF-bound PCR amplified random oligonucleotides, the recovered DNA was cloned into a T-vector and sequenced. Percentages indicate frequencies at which each nucleotide appeared at a given position from 1 to 14 in the total of 54 sequenced clones. The second line represents the deduced consensus sequence. (B) Electrophoretic mobility shift assay for DNA binding of TDF. Ten fmoles of 32P-labeled DNA probe and 25 ng of His-TDF in 10 µl of binding mixture were incubated at 25°C for 60 minutes. The DNA-TDF complex (b) was separated from the free probe (f) by electrophoresis. Lanes 1 and 2, binding reaction without any competitor; lane 1 received a mock-purified fraction from bacteria carrying an empty vector in place of His-TDF. In lanes 3-8, samples contained fold excess amounts of the unlabeled competitor DNA: either the functional TDS (lanes 3-5), or the mutant TDMS (lanes 6-8) sequence.

View larger version (41K): [in a new window]   Fig. 2. Isolation of MBF1-associated proteins. (A) FLAG-MBF1-associated proteins. Proteins from nuclear extracts of the FLAG-MBF1-expressing transgenic line F (lane 2) were captured on an anti-FLAG affinity column, separated by 10% SDS-PAGE and silver stained. Nuclear extracts of the parental yw line (lane 1) served as a control. Positions of molecular weight markers are shown on the left. (B) Western analyses of the captured proteins. The same samples as in A were resolved on SDS-PAGE, blotted onto membranes and probed with the indicated antibodies. (C) GST pull-down assay for interaction between MBF1 and TDF. Bacterially expressed and purified His-MBF1 (100 ng) was incubated with 200 ng of either GST (lane 2), GST-TDF (lane 3) or the GST-TDF-C56 fusion with the N-terminally truncated TDF (lane 4). The bound His-MBF1 was resolved by 15% SDS-PAGE and detected with the anti-MBF1 antibody. Lane 1 contains 1/10 of input His-MBF1. (D) GST pull-down assay for interaction between MBF1 and TBP. Bacterially expressed and purified His-TBP (100 ng) was incubated with 200 ng of either GST (lane 2) or GST-MBF1 (lane 3). The bound TBP was resolved by 8% SDS-PAGE and detected with the anti-TBP antibody. Lane 1 contains 1/10 of input His-TBP.

View larger version (50K): [in a new window]   Fig. 5. Reporter assay for TDF-dependent transcriptional activation. (A) Schematic illustration of the lacZ reporter constructs driven by either the functional (TDS) or the mutant (TDMS) TDF-binding elements. (B-E) Staining of stage 16 embryos with an anti-ß-galactosidase antibody. (B,C) The reporter gene TDS-lacZ was expressed in the tracheae (Tr), head (Hd), heart precursor cells (Ht) and CNS of a tdf+ line. (D) The mutant reporter TDMS-lacZ was silent in the tdf+ line. (E) TDS-lacZ was not expressed in the tdfP2 mutant. (F-I) Activation of TDS-lacZ upon ectopic expression of TDF in the posterior salivary gland of a third instar larva. Salivary glands from control TDS-lacZ (F, G) or sgP[Gal4]/+; UAS-tdf, TDS-lacZ/+ (H,I) larvae were stained with anti-TDF (F,H) or anti-ß-galactosidase (G,I) antibodies.

View larger version (32K): [in a new window]   Fig. 6. MBF1 participates in the TDF-dependent activation. (A) Colocalization of TDF and MBF1 to the transgenic reporter TDS-lacZ on the polytene chromosome. Salivary gland polytene chromosomes were prepared from a third instar larva of a sgP[Gal4]; UAS-tdf, TDS-lacZ/+; P[FLAG-MBF1]/+ line (4-6) or the same line without TDS-lacZ (1-3) and stained with anti-TDF (2 and 5) and anti-FLAG (1 and 4) antibodies. Blue signals represent DAPI staining. Panels 3 and 6 show the merged signals. The arrow marks the locus harboring the TDS-lacZ transgene. (B) MBF1 is required for full activation by TDF in vivo. Extracts were prepared from stage 16 embryos of the indicated lines and their ß-galactosidase activities were measured. Shown are average specific activities of six independent assays with standard errors.

View larger version (82K): [in a new window]   Fig. 7. CNS and tracheal defects in tdf and mbf1 mutants and genetic interaction between the two genes. CNS and tracheal lumen networks were stained in stage 16 embryos of the indicated genotypes with the mAb BP102 (left) and the mAb 2A12 (right), respectively. Arrowheads represent the defects in the CNS and tracheal network formation. The penetrance of each phenotype (%) is shown in the parentheses. P[MBF1+] is a rescue construct placed on the mbf1 chromosome, providing a wild-type copy of the gene. The tracheal defects in tdfP3 was variable from embryo to embryo. (D) A mild phenotype seen in some embryos. We also observed more severe phenotype in other embryos (data not shown, but see Fig. 1E by Eulenerg and Schuh (Eulenerg and Schuh, 1997). This could be due to variable and partial rescue of the lack of zygotic tdf function by maternally supplied tdf, because the lack of maternal tdf enhances the zygotic tdf phenotype (Eulenberg and Schuh, 1997). Total numbers of examined embryos were: A, 138; B, 136; C, 98; D, 112; E, 89; F, 108; G, 156; H, 98; I, 120; J, 132; K, 118; L, 128; M, 116; N, 121.