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. 2010 Mar;184(3):745-58.
doi: 10.1534/genetics.109.112086. Epub 2009 Dec 28.

Alternative splicing modulates Ubx protein function in Drosophila melanogaster

Hilary C Reed  1 , Tim Hoare, Stefan Thomsen, Thomas A Weaver, Robert A H White, Michael Akam, Claudio R Alonso
Affiliations

Alternative splicing modulates Ubx protein function in Drosophila melanogaster

Hilary C Reed et al. Genetics. 2010 Mar.

Abstract

The Drosophila Hox gene Ultrabithorax (Ubx) produces a family of protein isoforms through alternative splicing. Isoforms differ from one another by the presence of optional segments-encoded by individual exons-that modify the distance between the homeodomain and a cofactor-interaction module termed the "YPWM" motif. To investigate the functional implications of Ubx alternative splicing, here we analyze the in vivo effects of the individual Ubx isoforms on the activation of a natural Ubx molecular target, the decapentaplegic (dpp) gene, within the embryonic mesoderm. These experiments show that the Ubx isoforms differ in their abilities to activate dpp in mesodermal tissues during embryogenesis. Furthermore, using a Ubx mutant that reduces the full Ubx protein repertoire to just one single isoform, we obtain specific anomalies affecting the patterning of anterior abdominal muscles, demonstrating that Ubx isoforms are not functionally interchangeable during embryonic mesoderm development. Finally, a series of experiments in vitro reveals that Ubx isoforms also vary in their capacity to bind DNA in presence of the cofactor Extradenticle (Exd). Altogether, our results indicate that the structural changes produced by alternative splicing have functional implications for Ubx protein function in vivo and in vitro. Since other Hox genes also produce splicing isoforms affecting similar protein domains, we suggest that alternative splicing may represent an underestimated regulatory system modulating Hox gene specificity during fly development.

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Figures

F<sc>igure</sc> 1.—
Figure 1.—
Alternative spliced modules in Drosophila Hox proteins. For the four proteins Ultrabithorax (Ubx), Antennapedia (Antp), Proboscipedia (Pb), and Labial (Lab), differential splicing modifies the structure of the linker region (red) that separates the DNA-binding module (green) from the hexapeptide, an Exd interaction motif (purple). The inset details the protein isoforms generated from the Ubx gene through the inclusion/exclusion of alternative exons, i.e., the “b” element (b) and microexons 1 (M1) and 2 (M2) that separate the hexapeptide (Hx) from the homeodomain (HD). Each isoform has a specific pattern of expression that is conserved across evolutionarily distant Drosophila species (Bomze and Lopez 1994). The diagram is approximately to scale, but in the inset, the size of the microexons is exaggerated.
F<sc>igure</sc> 2.—
Figure 2.—
Function of ectopically expressed Ubx proteins in the mesoderm. Embryos were stained for β-gal produced from a dpp-lacZ construct, which by stage 15 outlines the forming gut tube. (A) Wild-type embryo displaying the three embryonic midgut constrictions (C1–C3, red arrowheads). (B) 24B-Gal4/UAS-UbxIa embryo. Note that the formation of the anterior midgut constriction is abnormal (asterisk, blue bracket) while the second and third constrictions are normal (C2 and C3). All Ubx protein isoforms from all our transgenic lines produced the same morphological distortion. (Embryos are shown in dorsal view, with anterior end to the left.)
F<sc>igure</sc> 3.—
Figure 3.—
Isoform-specific functions of Ubx proteins during embryonic development. β-Gal immunostaining is shown of stage 11 (A–G) and stage 13 (H–K) fly embryos carrying a dpp-lacZ reporter construct. (A and H) Wild-type control embryos where dpp-lacZ is activated in a subregion of the midgut corresponding to parasegment 7 (PS7, bracket). In stage 13 embryos (H), signal can also be seen in the primordium of the gastric caeca (visceral mesoderm PS4, labeled PS4) and in the faint rings of PS9 (PS9). (B–G) Effects of Ubx protein expression driven by 24B-Gal4 on the activation of the dpp-lacZ reporter. Isoforms Ia (B), IIa (C), Ib (E), and IIb (F) display similar abilities to activate dpp throughout the midgut visceral mesoderm from parasegment 2 to 7 and in two rather weak rings located approximately on PS9. Notably, isoforms IVa (D) and less strongly, isoform IVb (G) are able to activate dpp-lacZ throughout the midgut visceral mesoderm in a region that extends from parasegment 2 posteriorly to at least parasegment 12 (see dotted lines). By stage 13, isoforms I and II continue to activate the dpp reporter in the midgut visceral mesoderm (I and J, respectively) while isoforms IV (K) are also able to activate the lacZ construct in the somatic musculature (arrows). The square bracket indicates the approximate region of reporter expression in control embryos. Labels at the bottom left corner of each panel indicate the Ubx isoform expressed in each case. I–K show the effects of isoforms “a” only. Variants “b” for each isoform yield comparable results to their “a” counterparts (not shown). [Embryos are shown in lateral view (A–G) or dorsal view (H–K) always with anterior end to the left.]
F<sc>igure</sc> 4.—
Figure 4.—
Regulation of the endogenous dpp gene by Ubx splicing isoforms in the visceral mesoderm. (A) Wild-type expression of dpp mRNA as judged by RNA in situ hybridization (stage 13 embryos). A discrete domain of dpp expression is detected in the visceral mesoderm PS7 (represented along the antero-posterior axis by an orange rectangle). (B) Regulation of dpp mRNA expression following generalized expression of UbxIa in the mesoderm as driven by twist-Gal4. Note (i) the existence of an anteriorly extended expression domain of dpp mRNA in this condition (blue rectangle) and (ii) the discrete posterior boundary of the UbxIa-induced dpp expression domain with no detectable dpp mRNA signal posterior to PS7. (C) dpp mRNA expression after UbxIVa expression in the mesoderm (driven by twist-Gal4). Note (i) the extended domain of dpp expression anterior to PS7 (blue rectangle) and (ii) a patchy but significant upregulation of dpp mRNA in the visceral mesoderm posterior to PS7 (gray rectangle). (All embryos are oriented with their anterior end to the left; embryos on the top are shown in dorsal view, and bottom embryos are in lateral view.)
F<sc>igure</sc> 5.—
Figure 5.—
UbxMX17 phenotype in somatic mesoderm demonstrates specific in vivo functions of the Ubx isoforms. (A and B) Schematic of the pattern of relevant Connectin-expressing muscles in (A) wild-type third thoracic (T3) segment and in (B) the abdominal segments (A1–A7) (adapted from Nose et al. 1992 and Bate 1993). LT, lateral transverse; DT, dorsal transverse; VA, ventral acute. (C and D) Immunolabeling of Connectin in stage 16 embryo flatpreps. (C) Wild type. The abdominal-specific muscles are indicated; asterisks in A1–A7 indicate muscle DT1 and the arrowhead in A1 indicates VA2. (D) UbxMX17. A1 and A2 show transformation toward thoracic identity with loss of DT1 and thinner thoracic-like LT muscles; asterisks indicate DT1 muscles in A3 and A4. The transformation affects specific muscles as VA2 (arrowhead in A1) is still present in A1 and A2. (E and F) In situ hybridization for slouch mRNAs on stage 14 embryo whole mounts. (E) Wild type. Expression corresponding to the dorsal muscle DT1 is present from A1 to A7 (arrowhead in A3). (F) UbxMX17. A1 and A2 (open arrowheads) lack the DT1 slouch expression (arrowhead in A3). In this embryo, ventral cells expressing slouch are also in focus. (Embryos are shown in lateral view, anterior end to the left.)
F<sc>igure</sc> 6.—
Figure 6.—
Ubx in vitro interaction with Exd is isoform specific. (A) Equal amounts of different Ubx proteins were incubated in the absence (lanes 1–4) or presence of Exd (lanes 5–8). Ubx proteins alone displayed very low binding activity for the element analyzed (lanes 1–4). Exd was able to interact with all Ubx protein forms although the degree of these interactions was notably different (see below). Doubling the amount of Ubx protein in the reaction (lanes 9–12) proportionally increased the signal detected in ternary complexes, indicating that the differential interaction observed among Ubx proteins represents intrinsic properties of the proteins rather that some other experimental limiting factor. Exd alone is not able to bind to this element in a stable manner (lane 13). The quantification of the experiment is shown to the right. (B) Wild-type UbxIVa/Exd bind the DNA probe more strongly than UbxIa/Exd. Similar amounts of UbxIa and UbxIVa were incubated in the presence of Exd and the relative amount of protein–DNA complexes was quantified (see chart). (C) Ubx “hexapeptide-independent” interaction with Exd on DNA is isoform specific. A similar experiment to that in B shows that for isoform UbxIVa, the elimination of the hexapeptide decreases the interaction with Exd. (D) UbxIa lacking the hexapeptide is able to interact with Exd in the presence of a DNA target. Similar amounts of UbxIa wild type (lanes 1–6) and UbxIa Ala (lanes 7–12) display a similar Exd interaction profile in the presence of a Hox-Exd element, suggesting that UbxIa possesses areas of interaction with Exd other than the hexapeptide. When compared to C, these results also indicate that the linker region is able to affect the strength of Ubx–Exd protein interactions on the DNA element under study. Note that the levels of relative binding in the plots shown in C and D have been normalized to the level of binding of wild-type proteins in experiment B (for further details please see materials and methods).
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