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First published online 20 August 2008
doi: 10.1242/dev.025809

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Polycomb-dependent Ultrabithorax Hox gene silencing induced by high Ultrabithorax levels in Drosophila

Centro de Biología Molecular Severo Ochoa (C.S.I.C.-U.A.M.), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.

View larger version (92K): [in this window] [in a new window]   Fig. 1. Inducing high levels of Ubx eliminates Ubx protein in the haltere disc. Discs are oriented with the anterior compartment towards the left and the ventral compartment upwards. (A) Haltere imaginal disc of a Ubxlac1/TM6B stock, showing lacZ expression in the disc. (B) In a C765-Gal4/UAS-Ubx Ubxlac1 haltere disc, lacZ expression is almost abolished and the size of the haltere is reduced. (C) Wild-type haltere. (D,E) Halteres of E132-Gal4/UAS-Ubx (D) and MS372-Gal4/UAS-Ubx (E) flies. Note the increased size, with respect to C, and the appearance of wing bristles and thricomes. (F) In a MS372-Gal4/UAS-Ubx haltere disc, there are patches lacking Ubx protein expression.

View larger version (84K): [in this window] [in a new window]   Fig. 2. Absence of endogenous (Endo-) and exogenous (Exo-) Ubx when expressing Ubx with certain Gal4 lines. (A-A''') Absence of Ubx protein (in red in A') in UAS-GFP MS372-Gal4/UAS-Ubx Ubxlac1 haltere discs (GFP in green in A) correlates with the absence of Endo-Ubx, detected by the expression of Ubxlac1 (A'', greyscale). Merged image in A'''. The inset indicates a region without GFP, Ubx or Endo-Ubx expression. (B-B'') Exo-Ubx and Endo-Ubx expression in UAS-Ubx-HA/+; MS372-Gal4 UAS-GFP/Ubxlac1 haltere discs. GFP is shown in B (in green), the Exo-Ubx protein is detected with an antibody against the haemagglutinin (HA) epitope (B, in red) and the Endo-Ubx with an antibody against the β-galactosidase protein (greyscale in B'). Merged image in B''. Note the coincidence of Exo-Ubx and GFP expression but not of Endo-Ubx and the absence of Endo-Ubx in many cells, probably owing to the long stability of the β-galactosidase protein. The inset indicates a region without GFP, Exo-Ubx or Endo-Ubx expression. (C) Ubx expression in a tub-Gal80ts/+; dpp-Gal4 UASGFP/UAS-Ubx haltere disc transferred from 17 to 29°C and then back to 17°C during the larval period. Ubx is absent in most of the anterior compartment. Discs are oriented with the anterior compartment towards the left and the ventral compartment upwards.

View larger version (89K): [in this window] [in a new window]   Fig. 3. A transient high amount of Ubx protein permanently represses the endogenous Ubx gene. (A-A''') Haltere disc of an ap-Gal4 UAS-GFP/+; UAS-Ubx Ubxlac1/+ larva, grown at 17°C. GFP (A, in green) and high Ubx protein expression (A', in red) are observed in the dorsal compartment, whereas the expression of the Ubxlac1 reporter (A'', greyscale) is absent in that region (arrow). A''', merged image. In these and subsequent panels, d and v stand for dorsal and ventral compartments, respectively. (B-D') ap-Gal4 UASGFP/+; UAS-Ubx Ubxlac1/tub-Gal80ts haltere discs from larvae grown for 24-48 hours at 17°C, kept for 3 (B,D) or 2 (C) days at 29°C, and then transferred to 17°C for 3 (B, 3d) or 6 (C,D, 6d) days. Note that GFP (in green) and total Ubx (in red) protein expression progressively disappear and that the Endo-Ubx expression (D,D', greyscale and blue) is not restored. (E,F) Comparison of Ubx expression in ap-Gal4 UAS-GFP/+; Df109 UAS-dsUbx/tub-Gal80ts haltere discs from larvae grown for 24-48 hours at 17°C and then at 29°C (E), or reared for 24-48 hours at 17°C, 3 days at 29°C and 4 days (4d) at 17°C (F). The absence of Ubx protein by repression of transcription (B-D) is permanent, whereas the absence by RNA interference allows the recovery of Ubx expression (F). (G) In ap-Gal4/UAS-Ubx flies, the halteres are greatly reduced (arrow). (H) By contrast, in ap-Gal4/UAS-Ubx; tub-Gal80ts/+ adults that were transferred to 17°C after being at 29°C for 2 days during the second and early third larval instars, halteres are strongly transformed into wings (arrow). (I-L) Clones marked by the absence of GFP (in green, I), which expressed Ubx-HA transiently, induced in larvae of the hs-flp; UAS-Ubx-HA/tub-Gal80ts; Ubxlac1/tub>Ubi-GFP, y+>Gal4 genotype. Within the clone, exogenous Ubx protein is no longer present (J, in red) and the endogenous Ubx expression is continuously repressed (K, greyscale). Merged image in L. Discs are oriented with the anterior compartment towards the left and the ventral compartment upwards.

View larger version (87K): [in this window] [in a new window]   Fig. 4. The Ubx-Gal4SS.2 line is permanently repressed by high Ubx levels. (A-C) Transformations of haltere to wing (A), metanotum into mesonotum (B) and third leg into second one (C) in Ubx-Gal4SS.2 UAS-Ubx flies. The arrow in B marks mesonotum tissue appearing in the metanotum, and the arrow in C indicates an ectopic apical bristle on the third leg (III). The arrowhead indicates the wild-type apical bristle on the second leg (II). (D) Haltere disc of a Ubx-Gal4SS.2/UAS-GFP larva, showing GFP expression (in green) in the anterior compartment. The posterior compartment is marked by En expression (in red). (E-G) Haltere disc of an UAS-GFP/+; Ubx-Gal4SS.2/UAS-Ubx larva showing the same GFP (E, in green) and Ubx (F, in red) expression in patches of cells, indicating that the endogenous Ubx and the Gal4 driver are coincidentally repressed (G, merged image). (H) Map of the Ubx region showing the position of the Ubx-Gal4M1, Ubx-Gal4M3 (de Navas et al., 2006) and Ubx-Gal4SS.2 insertions. The latter is close to the PRE. Discs are oriented with the anterior compartment towards the left and the ventral compartment upwards.

View larger version (51K): [in this window] [in a new window]   Fig. 5. The Pc group of genes are required for the permanent Ubx repression. (A) Histogram showing the percentage of halteres that are transformed into wings in UbxGal4SS.2 UAS-Ubx animals in different mutant backgrounds. U stands for UbxGal4SS.2 UAS-Ubx. In each pair of columns, we show the control (TM6B; left) and the experimental (right) percentages, indicated over each column. The number of halteres scored is indicated below each column. The heterozygosity for Pc3 decreases the percentage (and the expressivity) of the transformations, whereas the heterozygosity for trx increases the number of transformations. (B) Histogram showing the average percentage of the area in the dorsal compartment of the haltere disc in which Ubx expression is absent. The red and blue columns correspond to average percentages in ap-Gal4 UAS-GFP/+; UAS-Ubx tub-Gal80ts/TM6B and ap-Gal4 UAS-GFP/+; UAS-Ubx tub-Gal80ts/UAS-PclRNAi haltere discs, respectively. The data are from larvae that completed embryonic development at 17°C, were incubated for 2 (left) and 3 (right) days at 29°C, and were then transferred to 17°C. Numbers of halteres studied are above the columns. Representative examples of Ubx staining in the haltere discs of the corresponding days and genotypes are shown below. (C-C'') Haltere disc of a sd-Gal4/hs-flp; UAS-Ubx/tub-Gal80ts; Ubi-GFP FRT2A/hs-CD2 ri PcXT109 FRT2A larva that went through temperature changes to establish Ubx permanent repression (see Materials and methods) and in which several Pc mutant clones (marked by the absence of GFP, in green in C) were induced. There is derepression of Ubx in the clones (in red, C'), showing that Pc is required to maintain permanent repression. The weak derepression in some clones may be related to the position of the clone. There are also cells in which Ubx has not been repressed (asterisk). Merged image in C''. Discs are oriented with the anterior compartment towards the left and the ventral compartment upwards.

View larger version (89K): [in this window] [in a new window]   Fig. 6. Effect of the Abd-A and Abd-B proteins on Ubx permanent repression in the haltere disc. (A) Haltere disc of a MS372-Gal4/UAS-abd-A larva. Ubx is repressed in a large area of the disc. (B) Ubx-Gal4SS.2/UAS-abd-A adults, showing a strong transformation of the haltere into wing. (C) In UAS-Abd-B/+; MS372-Gal4/+ flies, there is barely an increase in haltere size and only the occasional appearance of some bristles or a small amount of wing tissue. (D-G) An ap-Gal4 UAS-GFP/UAS-Abd-B; tub-Gal80ts/+ haltere disc, grown for 3 days at 29°C. GFP (green, D) and Abd-B (red, E) are expressed in the dorsal compartment, whereas Ubx (greyscale, F) is repressed. Merged image in G. (H) In ap-Gal4 UAS-GFP/UAS-Abd-B; tub-Gal80ts/+ haltere discs from larvae that were grown at 17°C after 3 days at 29°C, the repression of Ubx is only occasionally maintained. (I) In most UAS-UbxHX/+; UbxGal4SS.2/+ adults, there are transformations of haltere into wing (arrowhead). (K) By contrast, in UbxGal4SS.2/UAS-UbxUbdA flies, the halteres are abnormal but they are not transformed into wings. These transformations correlate with Ubx expression in discs that went through the standard treatment. (J,L) In ap-Gal4 UAS-GFP/+; tub-Gal80ts/UAS-UbxHX haltere discs (J), there is repression of Ubx in the dorsal compartment but such repression is never observed in ap-Gal4 UAS-GFP/+; tub-Gal80ts/UAS-UbxUbdA haltere discs (L) under the same experimental conditions. (M) In ap-Gal4 UAS-GFP/UAS-UbxUbdA; Ubxlac1/+ haltere discs, grown at 29°C for 5 days, there is strong repression of the lacZ gene in most dorsal cells. d, dorsal compartment. Magnification in I and K is the same, but different from that in B and C. Discs are oriented with the anterior compartment towards the left and the ventral compartment upwards.

View larger version (60K): [in this window] [in a new window]   Fig. 7. Permanent repression of the engrailed gene induced by high Engrailed levels. (A-C) Posterior to anterior transformations in the mesonotum (A) and wing (B) of en-Gal4/UAS-en flies. See the duplicated notum (arrow in A) and the presence of anterior bristles in the posterior wing (C, detail of the square in B). A and P indicate anterior and posterior, respectively. (D) En expression in a wild-type wing disc. (E) En expression in an en-Gal4/UAS-en wing disc is confined to small groups of cells and the posterior compartment is enlarged. (F,G) GFP (F) and En (G) expression in an en-Gal4 UAS-GFP/+; UAS-en/+ wing disc is coincidentally repressed. (H-K) ap-Gal4 UASGFP/en-lacZ; UAS-en/tub-Gal80ts wing disc from larvae grown for 5 days at 29°C and transferred to 17°C for 3 days. GFP signal (H, green) is still present (though reduced) in the apterous (dorsal) domain of the disc, and both En (I, in red) and β-galactosidase (J, greyscale) expression disappear in the posterior dorsal compartment (arrows). There is ectopic β-galactosidase signal in the dorsoventral boundary (arrowhead in J). Merged image in K. d and v indicate dorsal and ventral compartments, respectively. Discs are oriented with the anterior compartment towards the left and the ventral compartment upwards.

View larger version (14K): [in this window] [in a new window]   Fig. 8. Model of how an excess of Ubx may trigger permanent Ubx repression. (A) In the wild type, Ubx protein expression downregulates, but does not suppress, Ubx transcription in the haltere disc. Ubx transcription through the PRE prevents the function of Pc-group complexes either by reducing binding of some proteins to the PRE or by preventing the assembly of such complexes. (B) When Ubx protein levels are highly raised, Ubx transcription is repressed and this leads to the binding and/or activation of the Pc-group complexes at the PRE. (C) Pc-group activity prevents the expression of the Gal4 gene in the Ubx-Gal4SS.2 insertion and, eventually, of the Ubx gene. The UbdA domain of the decaying Ubx protein may help in this role.

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