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First published online November 10, 2005
doi: 10.1242/10.1242/dev.02138

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Evolutionarily conserved domains required for activation and repression functions of the Drosophila Hox protein Ultrabithorax

¹ Section in Cell and Developmental Biology, Division of Biology, University of California, San Diego, La Jolla, CA 92093, USA
² Howard Hughes Medical Institute, Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI 53706, USA

View larger version (79K): [in a new window]   Fig. 1. The repression of larval limbs and Dll transcription is highly dependent on Ubx concentration. (A) The repression of Keilin's organs (in black) and the sensory hairs (red) of Keilin's organs as a function of ectopic Ubx concentration in the thorax. Each data point represents a different ectopic expression experiment, within which at least 120 larval limb phenotypes were scored and then averaged. Error bars: standard error of the mean for the limb repression values and 95% confidence intervals for Ubx concentration and Dll repression values. (B) The repression of sensory hairs (red) and Dll transcriptional repression (blue) plotted against Ubx concentration. (C) Dll transcriptional repression as a function of ectopic Ubx levels in the anterior (red) and posterior (blue) compartments of the thoracic segments. (D-F) Ubx protein expression in mid-stage 11 embryos of the following genotypes: (D) wild type, (E) ectopically expressed Ubx at 76% of endogenous levels and (F) ectopically expressed Ubx at 32% of endogenous levels. Ubx protein was detected by staining with FP3.38 anti-Ubx antibody. White ovals indicate the positions where Ubx protein levels were measured. (G,H) Transcripts of Dll (red) and wg (green) in the limb fields of (G) wild-type embryos, and (H) embryos expressing ectopic Ubx at 32% of endogenous levels. In all figures, anterior is towards the left and dorsal is upwards.

View larger version (78K): [in a new window]   Fig. 2. The Ubx N-terminal region deletions. (A) Diagram of Drosophila UbxIa and the eight deletions covering its N-terminal arm. All deletions, except for 252-280 (the optional intron region), contained regions highly conserved between Ubx proteins from other arthropod species (see Fig. S1 in the supplementary material). The deletion breakpoints were placed between the conserved regions. (B) Examples of ectopically expressed wild-type Ubx and Ubx deletion mutant proteins (produced at 75-80% of the levels of endogenous Ubx), detected with anti-HA staining in mid-stage 11 embryos.

View larger version (25K): [in a new window]   Fig. 3. The YPWM and the 20-61 regions are quantitatively required for Ubx limb repression function. (A) The limb repression values of Ubx deletion mutants when expressed at the level of endogenous Ubx. (B) The limb repression activity of wild-type Ubx, Ubx20-61 and UbxYPWM deletion mutants as a function of protein concentration. The UbxYPWM protein exhibits a flatter concentration-dependence curve of repression activity than wild-type Ubx.

View larger version (80K): [in a new window]   Fig. 4. The UbxYPWM protein is a defective transcriptional repressor of Dll and Antp. (A-F) In situ hybridization of mid-stage 11 embryos, hybridized with Dll (green) and Antp (red) antisense probes. The broken white lines in A,C,E indicate the posterior boundary of wg expression, which was detected in the same embryos (not shown). Dll and Antp P1 transcripts shown in the thoracic segments of (A,B) a wild type embryo, (C,D) an embryo ectopically expressing wild type Ubx and (E,F) an embryo ectopically expressing UbxYPWM protein. (G) A dose-response plot of Dll repression as a function of the logarithm of the protein concentration of wild-type Ubx and UbxYPWM.

View larger version (85K): [in a new window]   Fig. 5. The N-terminal region of Ubx is required for ectopic activation of dpp in visceral mesoderm. (A-E) dpp transcripts in the visceral mesoderm of stage 13 embryos. (A) In wild-type embryos, dpp transcripts are detected in parasegments 4 and 7 (arrowheads). (B) Ectopic wild-type Ubx activates dpp transcripts anterior to and posterior to parasegment 7 (arrows). (C) Ectopic Ubx2-19 protein barely activates ectopic dpp in parasegments 5 and 6, and represses endogenous dpp in parasegments 4 and 7 (arrowheads). (D) Ectopic Ubx20-61 protein activates dpp transcripts anterior to, but not posterior to parasegment 7. (E) Ectopic UbxYPWM protein activates dpp transcripts in a pattern and amount indistinguishable to wild-type Ubx. (F) Quantitation of the ectopic Ubx protein levels and dpp transcripts in parasegments 5 and 6. Error bars: 95% confidence intervals.

View larger version (72K): [in a new window]   Fig. 6. The N-terminal region of Ubx protein is required for the activation of tsh transcripts in the head epidermis. (A-E) Shown are the head and anterior thorax of late stage 11 embryos, hybridized with a tsh antisense probe. (A) In wild-type embryos, tsh is transcribed in the epidermis of parasegment 3 (as well as in parasegments 4-13, not shown). (B) Ectopic wild-type Ubx induces tsh transcripts in the clypeolabrum (cl), the procephalon (pc), and the mandibular (Md) and maxillary (Mx) segments. (C) Ectopic Ubx2-19 activates very low levels of tsh transcripts in the pc and cl, and in only a few cells of the Md and Mx segments. (D,E) Ectopic Ubx20-61 and UbxYPWM proteins activate tsh transcripts at similar levels and in similar pattern to wild-type Ubx, but with less uniformity. (F) Quantitation of ectopic protein expression levels and tsh transcripts, averaged over the entire head region. Error bars: 95% confidence intervals. (G) Alignment of the N termini of the Ubx proteins from Drosophila melanogaster (Dm), Tribolium castaneum (Tc), Porcellio scaber (Ps) and Artemia franciscana (Af). Ten out of the 18 amino acid residues eliminated in the Ubx2-19 mutant are identical in the four Ubx homologs.

View larger version (65K): [in a new window]   Fig. 7. The conserved N-terminal region of Scr is required for the activation of fkh and CrebA expression. (A) Alignment of the N-termini of insect Scr proteins [Drosophila melanogaster (Dm), Anopheles gambiae (Ag), Tribolium castaneum (Tc) and Bombyx mori (Bm)]. In the region deleted in the ScrSSY mutation (bracket), 12 out of 17 amino acid resides are identical. (B) Expression levels of ectopic wild-type Scr (Scr) and the ScrSSY mutant (SSY) in ventral parasegment 1 (ps 1), compared with the levels of the endogenous Scr protein (wt) in ventral parasegment 2 (ps 2). Error bars: 95% confidence intervals. (C-E) Anterior regions of mid-stage 11 embryos, hybridized with a fkh transcript antisense probe. (C) In wild-type embryos, fkh is activated in ventral parasegment 2. (D) Ectopic wild-type Scr activates fkh transcripts in ventral parasegment 1, the anterior mandibular segment (asterisk) and in the procephalon. (E) Ectopic ScrSSY protein activates fkh transcripts in only a few cells of parasegment 1. (F-H) Mid-stage 11 embryos stained with anti-CrebA antibody. (F) In wild-type embryos, CrebA expression is limited to ventral parasegment 2. (G) Ectopic wild-type Scr activates CrebA in parasegment 1 and the procephalon. (H) Ectopic ScrSSY protein activates CrebA in only a few cells of parasegment 1. (J) Alignment of the N termini of human (Homo sapiens), mouse (Mus musculus), sea urchin (Strongylocentrotus purpuratus) and fly (Drosophila melanogaster) Hox proteins. In all of these proteins, the N terminus conserves an SSYF motif or a subtle variant. Asterisks indicate Hox proteins in which a requirement of the N-terminal region for transcriptional activation in embryos has been demonstrated.

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