QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 2 November 2005
doi: 10.1242/dev.02146

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hittinger, C. T. Articles by Carroll, S. B. Search for Related Content PubMed PubMed Citation Articles by Hittinger, C. T. Articles by Carroll, S. B.

Pleiotropic functions of a conserved insect-specific Hox peptide motif

¹ Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, WI 53706, USA
² Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI 53706, USA
³ Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ 08544, USA
⁴ Laboratory of Molecular Biology, University of Wisconsin-Madison, Madison, WI 53706, USA

View larger version (15K): [in a new window]   Fig. 1. Two-step construction of the UbxQA allele. (A) An X chromosome insertion of the P element construct containing the desired changes to Ubx and the necessary sequences for allelic replacement (Rong et al., 2002) was used to manipulate the endogenous Ubx locus on the 3rd chromosome. Only the 3' end of Ubx, which is also the edge of the BX-C, is shown. The scale is approximate; the targeting construct contained 10,893 bp between the vector cloning sites. At each step, the expressed polypeptide sequence of Ubx following the homeodomain is shown. Each step was catalyzed by crossing to flies containing the appropriate heat-shock transgenes and heat-shocking the progeny (see Materials and methods) (Rong et al., 2002). (B) The first step created a targeted duplication of the 3' end of the Ubx locus that contained one complete and expressed Ubx+ allele and a partial unexpressed UbxQA allele. (C) The second step catalyzed the reduction of this duplication to a single copy, creating both full-length UbxQA alleles and control Ubx+ alleles in independent reduction events. Only the recovery of an UbxQA allele is shown. Black indicates sequences with homology to Ubx; gray indicates vector or unknown sequences; red indicates marker whs gene for screening; blue indicates site-specific recombinase Flp and FRT targets (triangles); purple indicates homing endonuclease I-SceI and I-SceI target (bar); pink indicates homing endonuclease I-CreI and I-CreI targets (bars); green indicates PCR primers (half arrows) and restriction sites (bars) used in preliminary screens of putative targeted duplication and reduction events. Ultimately, the entire region was sequenced from alleles selected for study.

View larger version (139K): [in a new window]   Fig. 2. The QA motif has a limited role in haltere development. Haltere genotypic series for both haltere size and number of ectopic bristles (small blue arrows, single; large blue arrow, large quantity): (A) Ubx+/Ubx+ > (B) UbxQA/UbxQA > (C) Ubx–/Ubx+ > (D) UbxQA/Ubx–. Ubx+/Ubx+ halteres never had bristles, whereas UbxQA/Ubx– always had several bristles. In the WI129 background (shown above), UbxQA/UbxQA flies had 0.86±0.73 bristles per haltere, whereas Ubx–/Ubx+ flies had 2.59±1.04 bristles per haltere (P<10–17; n=90 and 60, respectively). In the Oregon-R background (not shown), UbxQA/UbxQA flies had 0.14±0.25 bristles per haltere, whereas Ubx–/Ubx+ flies had 0.78±0.50 bristles per haltere (P<10–17; n=90 and 60, respectively). Anterior is leftwards, dorsal is upwards.

View larger version (75K): [in a new window]   Fig. 3. Redundancy of the QA motif in larval limb repression. Ventral/lateral views of T2-A2 of first larval stage cuticles with KOs shown in insets (5x, lightened, T3 and A1 only). (A) Ubx+/Ubx+, (B) UbxQA/UbxQA, (C) UbxQA/Ubx–, (D) UbxQA/Ubx– adb-A–, (E) Ubx–/Ubx–, (F) Ubx– abd-A–/Ubx– adb-A–. Each thoracic segment has two three-bristle KOs. Ubx+/Ubx+ (A), UbxQA/UbxQA (B), Ubx–/Ubx+ (not shown) and Ubx– abd-A–/Ubx+ abd-A+ (not shown) larvae never had ectopic A1 KOs. UbxQA/Ubx– (C) larvae had up to two-bristle ectopic A1 KOs. UbxQA/Ubx– adb-A– (D) larvae had up to three-bristle ectopic A1 KOs. Ubx–/Ubx– (E) larvae generally had two-bristle ectopic A1 KOs but occasionally had one-bristle KOs. Ubx– abd-A–/Ubx– adb-A– (F) larvae had full three-bristle ectopic KOs on A1 through A7. Ubx+/Ubx+ larvae (A) have thin, narrow thoracic ventral denticle belts. The A2-A7 denticle belts are broad and trapezoidal, while A1 denticles belts are somewhat less broad and trapezoidal. UbxQA/UbxQA (B), Ubx–/Ubx+ (not shown), Ubx– abd-A–/Ubx+ abd-A+ (not shown) larvae had mild transformations of the A1 denticle belts towards an intermediate T3/A1 identity. UbxQA/Ubx– (C) and UbxQA/Ubx– adb-A– (D) larvae had more severe transformations of the A1 denticle belts to intermediate T3/A1 identity. Ubx–/Ubx– (E) larvae had complete transformations of the A1 denticle belts to T2 identity. Ubx– abd-A–/Ubx– adb-A– (F) larvae had complete transformations of the A1-A7 denticle belts to T2 identity. Anterior is leftwards.

View larger version (92K): [in a new window]   Fig. 4. Redundancy of the QA motif in the repression of limb selector expression. Dll expression in T1-A2 or A3 of stage 12 embryos. (A) Ubx+/Ubx+, (B) UbxQA/UbxQA, (C) UbxQA/Ubx–, (D) UbxQA/Ubx– adb-A–, (E) Ubx–/Ubx–, (F) Ubx– abd-A–/Ubx– adb-A–. All genotypes tested had essentially non-overlapping phenotypes, except for those with no A1 Dll expression: Ubx+/Ubx+ (A), UbxQA/UbxQA (B), and Ubx–/Ubx+ (not shown). At earlier stages, abdominal Dll expression in A1 was inconsistent; some Ubx+/Ubx+ occasionally expressed Dll in one or two cells in A1, while dose or activity reductions enhanced the number of cells affected (not shown). By stage 12, Ubx+/Ubx+ (A), UbxQA/UbxQA (B) and Ubx–/Ubx+ (not shown) embryos did not express Dll in A1, suggesting Dll expression at this stage more accurately marks limb primordia. The Dll expression in A2 and A3 of UbxQA/Ubx– adb-A– embryos (D) was also present in Ubx– adb-A–/Ubx+ abd-A+ embryos (not shown) up to segment A7 and suggests a lag in repression when abd-A dose is reduced as later stages no longer express this ectopic Dll. Anterior is leftwards, dorsal is upwards. (G) UbxQA/Ubx– sub-epidermal A1 leg tissue found in 1.7% of adults and pupae (5/296); an extreme specimen is shown. (H) UbxQA/Ubx– adb-A– sub-epidermal A1 leg tissue found in 6.7% of adults and pupae (13/194); an extreme specimen with a well-developed claw (Cl) and transverse bristle rows (TR) is shown. In addition to producing more well-developed ectopic A1 leg tissue, the frequency difference between UbxQA/Ubx– and UbxQA/Ubx– adb-A– is significant (12 =8.3, P<10–2).

View larger version (72K): [in a new window]   Fig. 5. The QA motif is preferentially required for repression of postnotal tissue. Postnotal genotypic series for T3 laterotergite transformation towards T2 thorax as assessed by number of ectopic bristles (blue arrows): (A) Ubx+/Ubx+ > (B) Ubx–/Ubx+ > (C) UbxQA/UbxQA > (D) UbxQA/Ubx–. The T3 laterotergite (green line) is normally a thin band of tissue separating T2 and A1 but is expanded in mutants lacking the QA motif (surrounded by green lines marking border). The junction of the haltere and the laterotergite is marked with a green `T3' at the edge of the line. Anterior is upwards, lateral is leftwards and the dorsal midline is rightwards. (A) Ubx+/Ubx+ T3 half-laterotergites never had bristles, while UbxQA/Ubx– (D) always had bristles (up to a dozen). In the Oregon-R background (shown above), UbxQA/UbxQA T3 half-laterotergites (C) had 0.972±0.592 bristles, whereas Ubx–/Ubx+ T3 half-laterotergites (B) had 0.017±0.091 bristles (P<10–20; n=90 and 60, respectively). In the WI129 background (not shown), UbxQA/UbxQA T3 half-laterotergites had 0.167±0.290 bristles, whereas Ubx–/Ubx+ T3 half-laterotergites had 0.000±0.000 bristles (P<10–5; n=90 and 60, respectively).

View larger version (130K): [in a new window]   Fig. 6. The QA motif is preferentially required for A1 tergite formation. Deletion of the QA motif had a strong effect similar to that of removing one dose of Ubx. Genotypic series of A1 tergite formation as assessed by number of rows of bristles: (A) Ubx+/Ubx+ > (B) Ubx–/Ubx+ (C) UbxQA/UbxQA > (D) UbxQA/Ubx–. Pooling all data, Ubx+/Ubx+ A1 tergites had 5.32±0.49 rows of bristles, Ubx–/Ubx+ A1 tergites had 4.44±0.44 rows of bristles, while UbxQA/UbxQA A1 tergites had 4.26±0.49 rows of bristles (for either comparison to Ubx+/Ubx+, P<10–22; Ubx–/Ubx+>UbxQA/UbxQA, P=10–2; n=80, 120, and 180; respectively). In the WI129 background (shown above), Ubx–/Ubx+ A1 tergites (B) had 4.68±0.39 rows of bristles, while UbxQA/UbxQA A1 tergites (C) had 4.28±0.59 rows of bristles (P=10–4; n=60 and 90, respectively). In the Oregon-R background (not shown), Ubx–/Ubx+ A1 tergites had 4.20±0.36 rows of bristles, while UbxQA/UbxQA A1 tergites had 4.23±0.51 rows of bristles (P=0.7; n=60 and 90, respectively). Anterior is upwards.

View larger version (89K): [in a new window]   Fig. 7. The QA motif is preferentially required for the repression of leg trichomes. Genotypic series of T2p trichome repression (Oregon-R background shown): (A) Ubx+/Ubx+ > (B) Ubx–/Ubx+> (C) UbxQA/UbxQA > (D) UbxQA/Ubx–. Ubx+/Ubx+ (A) and Ubx–/Ubx+ (B) T2p legs had similarly sized large naked valleys lacking trichomes (bracketed in red). UbxQA/UbxQA (C) and UbxQA/Ubx– (D) T2p naked valleys were highly reduced such that nearly the entire leg had trichomes. Genotypic series of T3p trichome repression: (E) Ubx+/Ubx+ > (F) Ubx–/Ubx+ > (G) UbxQA/UbxQA > (H) UbxQA/Ubx–. Ubx+/Ubx+ (E) and Ubx–/Ubx+ (F) T3p legs had a similarly limited area of trichomes distally (small red arrows), although Ubx+/Ubx+ (E) also had some ventral trichomes (small red arrow) owing to a recessive allele (see text). UbxQA/UbxQA (G) and UbxQA/Ubx– (H) T3p legs had large ventral and distal patches of trichomes (large red arrows) that generally fused. UbxQA/Ubx– T2 legs (D) generally had an eighth row of bristles (cyan arrow), a phenotype of Ubx– clones (D.L.S., unpublished). UbxQA/Ubx– T3 (H) often had ectopic edge bristles (green arrows), a transformation from T3 towards T2 identity, as well as more robust dorsal and proximoventral bristles. Distal is rightwards, dorsal is upwards.