3. Characterization of Cdk9 mutation

3.4. Cdk9 DN expression in imaginal discs affects organ growth and patterning

3.4.1. Imaginal disc expression of Cdk9 DN affects viability and terminal differentiation 84

At the third larval stage, the c701b driver expresses GAL4 in most imaginal discs including the dorsal and ventral meso- and metathoracic discs (Manseau et al., 1997). We first examined the effect of mutant cdk9 expression in these discs on adult viability. The location of this driver on the X chromosome allowed us to obtain, by crosses, a progeny where only the females expressed the UAS transgenes, while the males served as a control for the expected amount of progeny. As control experiment, we quantified the whole progeny coming from crosses between c701b males and w1118 females at 25°C. We crossed in parallel c701b males with UASp-cdk9 and UASp-cdk9DN homozygous females and monitored the proportion of female relative to male progenies. As shown in figure 25A, equivalent percentages of males and females were obtained from control and wild-type cdk9 expressing progenies. In contrast, the female offspring obtained from crosses with mutant cdk9 was reduced to about 10 to 20% depending on the UAS line used. The expression of an additional copy of mutant cdk9 increases the lethality, which occurs at the onset of metamorphosis after puparium formation.

Examination of the cdk9DN female escapers revealed a growth defect of the adult appendages. The most penetrant effect occurred within the ventral discs which give rise to the second and third leg pairs, while the ventral prothoracic discs (1st leg pair) developed normally. Immunostaining of the ventral discs from third-instar larvae expressing a UAS-lacZ reporter indicated that the c701b driver triggered high level of GAL4 in the entire meso- and metathoracic discs, but is restricted to the anterior most region of the prothoracic disc (Fig.

325B). In the Cdk9 DN female escapers, the overall size of the second and third leg pairs

Figure 25. Cdk9DN expression in imaginal discs affects viability and appendages formation. (A) Percentage of the female progeny relative to males obtained from crosses between (1) control, (2) wild-type cdk9 or (3,4) mutant cdk9 females with c701b males at 25°C. Each bar of the graph represents the average number of female progeny obtained from 10 independent crosses. The viability of the female progeny expressing the mutant cdk9 transgene was reduced at about 10% while expression of the wild-type kinase has no significant effect. No female progeny was obtained from crosses with flies encoding 2 copies of the mutant transgene (4). (B) Confocal images of the ventral prothoracic (P1, 1st leg), mesothoracic (P2, 2nd leg) and metathoracic (P3, 3rd leg) discs from third-instar larvae expressing a UAS-lacZ reporter under control of the c701b driver. The discs were stained with an anti-lacZ antibody. The UAS transgene is expressed in the entire meso- and metathoracic discs but is limited to the anterior region of the prothoracic disc. (C) Cuticle preparations of legs from wild-type cdk9 (upper panel) and mutant cdk9 (lower panel) expressing females under control of the c701b driver. The expression of Cdk9DN had no detectable effect on the first leg (P1), but strongly reduced the size of the second (P2) and third (P3) legs. In a few cases we observed fusion of tarsal segments in the third leg (arrow). Co : coxa, tr : trochanter, fe : femur, ti : tibia, ta : tarsa.

became strongly reduced, although the different segments of these appendages (coxa, trochanter, femur, tibia and tarsa) were correctly specified (Fig. 25C). In a few cases however, we observed fusion of some tarsal segments (arrow). This result suggests that Cdk9DN expression in the ventral discs interferes more with cell growth and/or cell proliferation than with cell differentiation. The limited effect of cdk9DN expression on leg patterning might be due to the tardive accumulation of GAL4 protein in the discs during larval growth. The segmentation pattern of the leg is indeed progressively set up during development. Starting from mid-embryogenesis, the leg disc is sequentially subdivided into five domains along the proximodistal axis, and patterning becomes fully established by the late third-instar larvae (reviewed by Kojima, 2004). Therefore, the limited expression of the mutant transgene during the mid-third larval stage might induce only minor effect on leg patterning.

We next asked whether the leg phenotype is caused by a decreased number of cell divisions in the leg discs during the third larval stage. To test this, we fed the larvae with BrdU-containing food for two days at 25°C, and compared the level of BrdU incorporation in discs expressing high levels of GAL4 (ventral meso- and metathoracic) to discs expressing low levels of GAL4 (ventral prothoracic) using an anti-BrdU antibody. No obvious difference of BrdU labeling was detected in one or the other leg discs from control and mutant cdk9 expressing larvae (data not shown). This suggests that Cdk9DN does not strongly affect the rate of cell cycles during the larval growth phase, but interferes with the proper terminal differentiation of the appendages during metamorphosis.

3.4.2. Cdk9DN expression in the wing discs affects cell growth and wing patterning.

To further investigate the requirement for Cdk9 in imaginal discs, we tested two other GAL4 drivers known to be active earlier during development. The en-GAL4 driver is ubiquitously expressed during embryogenesis and subsequently restrained to the posterior compartment of the larval imaginal discs (Perrimon, 1995) (Fig. 26A). The MS1096 driver specifically expresses GAL4 in the wing discs during larval growth. Its expression is restricted to the dorsal compartment of the wing pouch in early to mid third-instar larvae, and it becomes expressed in the ventral compartment, albeit more weakly than in the dorsal compartment, in late third-instar wing discs (Capdevila and Guerrero, 1994 ; Milan et al., 1998). The expression of cdk9DN by en-GAL4 induced wing malformations ranging from vein patterning defects to blade size reduction in the posterior compartment (Fig. 26D,E).

Disruption of the L5 vein and/or emergence of ectopic veins along the L5 and the

posterior-cross vein (pcv) was frequently observed (arrowhead). Similarly, expression of cdk9DN in the entire wing disc under control of the MS1096 driver dramatically altered wing formation (Fig.

26J). The wing blade became shorter and the vein patterning was highly disrupted. Such phenotypes have been reported for mutations in several genes involved in transcription and cell cycle regulation including Cdk7/CyclinH, cdc2/CyclinE and Brahma (Leclerc et al., 2000

; Merino et al., 2002 ; Marenda et al., 2004). Therefore, within the context of wing development, the ectopic veins associated with cdk9DN expression reveal a role for P-TEFb in regulating vein patterning differentiation in intervein cells. As previously observed in the eye discs, we found that co-expression of wild-type and mutant Cdk9 using either driver rescued the wing patterning (Fig.426C and E), indicating that these phenotypes are due to specific alteration of Cdk9 function.

The reduction of the posterior compartment of wings expressing cdk9DN driven by en-GAL4 led us to ask whether the cell cycle rate is affected in growing imaginal discs or is due to a cell growth defect during wing differentiation. To test whether there are less cell divisions, we performed a BrdU incorporation experiment in wing imaginal discs. Third instar larvae expressing wild-type or mutant Cdk9 proteins were fed with BrdU-containing food for 2 days at 25°C, and the discs were probed with an anti-BrdU antibody. We were not able to detect significant differences of BrdU labeling in the posterior compartment compared to the anterior one in wild-type or mutant cdk9 expressing discs (data not shown). This result suggests that an overexpression of wild-type Cdk9 kinase or a partial disruption of P-TEFb activity in imaginal discs has no detectable effects on cell proliferation. Consistently, by

Figure 26. Cdk9DN expression in wing imaginal discs affects wing patterning. (A) Confocal image of a wing imaginal disc from control third-instar larvae (en-GAL4/+) that was stained with DAPI (blue) and with an anti-en antibody (green). The engrailed expression pattern is restricted to the posterior compartment of the disc. (B-F) Cuticle preparation of a wing from (B) control, (C) wild-type cdk9, (D,F) mutant cdk9 and (E) wild-type plus mutant cdk9 expressing flies under control of the en-GAL4 driver. The green area in (B) indicates the posterior compartment of the wing where en is expressed. Expression of Cdk9DN induced the formation of ectopic veins along the L5 and the pcv (arrowhead). The phenotype was further aggravated by expression of 2 copies of cdk9DN. In addition, the anterior compartment also became affected and resulted in a decreased size of the entire wing (F).

By contrast, the co-expression of wild-type and mutant cdk9 transgenes rescued the cdk9DN phenotype (E). acv : anterior-cross vein, pcv : posterior-cross vein. (G-J) Cuticle preparation of a wing from (G) control, (H) wild-type cdk9, (J) mutant cdk9 and (I) wild-type plus mutant cdk9 expressing flies under control of the MS1096 driver. The wing phenotype caused by Cdk9DN expression can be rescued by co-expressing the wild-type kinase (compare J and I). The vein defects seen in the rescue experiment (interruption of the pcv and ectopic vein : arrowheads) were not frequent and also detected in control animals due to MS1096 (Milan et al., 1998). (A,B) : GAL4/+ ; (C) : GAL4/UASp-cdk9 ; (D) : en-GAL4/+ ; UASp-cdk9DN ; (E) : en-GAL4/UASp-cdk9 ; UASp-cdk9DN/+ ; (F) : en-GAL4/UASp-cdk9DN ; UASp-cdk9DN/+ ; (G) : MS1096/+ ; (H) : MS1096/+ ; UASp-cdk9/+ ; (I) : MS1096/+ ; UASp-cdk9/+ ; UASp-cdk9DN/+ ; (J) : MS1096/+ ; UASp-cdk9DN/+.

counting the number of cells in selected areas from the anterior and posterior compartments of the adult wings, we did not detect an effect of Cdk9DN expression on the rate of cell cycles in the posterior compartment. Instead, we noticed that the cells expressing Cdk9DN were slightly smaller (approximately 10%) than their control counterpart. Taken together, these observations indicate that mutant P-TEFb rather interferes with cell growth than with cell proliferation during wing development.

Dans le document The transcription elongation factor P-TEFb in 'Drosophila melanogaster' : characterization of a mutation of the Cdk9 kinase and of the different activities of two co-factors (Page 85-90)