The Level of C/EBP Protein Is Critical for Cell Migration during Drosophila Oogenesis and Is Tightly Controlled by Regulated Degradation  Pernille Rørth, Kornelia Szabo, Gemma Texido  Molecular Cell  Volume 6, Issue 1, Pages 23-30 (July 2000) DOI: 10.1016/S1097-2765(05)00008-0

Figure 1 Slbo Is Required in Outer Border Cells for Migration (A) Slbo expression during normal border cell migration. Anti-slbo (green and single panels to the right) and anti-β-galactosidase (red) staining of egg chambers from females carrying one copy of the slbo1310 enhancer trap (lacZ reporter gene). ppc indicates posterior polar cells; the anterior polar cells are the two central, smaller nuclei in the migrating border cell cluster. (B) Clonal analysis with the slbo8ex2 null mutant. Lack of the β-galactosidase marker (green, also single channel in enlarged images on the right) indicates homozygous mutant cells. FasIII (red) marks polar cells. The arrows in the middle panels point to the single wild-type border cell; the arrows in the bottom panels point to the single mutant border cell. Scale bars are 20 μM in all panels. In an additional experiment using GFP as clonal marker, 185 border cell clusters with one or more mutant cells were analyzed. In the following, numbers given in parenthesis indicates number of cases observed. In a few cases, APCs and outer border cells (OBCs) could not be clearly assigned. No migration was observed if all cells were mutant (22 cases) or if only one (4) or both (9) APCs were wild type. Conversely, if only one (17) or both (6) APCs were mutant, migration was unaffected. If only one (5) or two (7) OBCs were wild type, these would initiate movement but remained associated with the mutant border cells and moved very little (middle panel). If only one (50) or two (28) OBCs were mutant, these would mostly (57/78) remain associated with the wild-type border cells and be “dragged along” (bottom panel). The mutant OBCs were always in the rear. For 9/28 of clusters with two OBCs mutant and for 7/19 clusters with three OBCs mutant, some or all mutant cells were split (as a cluster) from the front cluster somewhere along the migration path. Only in a minority of such cases (6/16) were cells strictly segregated into wild-type (front) and mutant (rear) clusters. Molecular Cell 2000 6, 23-30DOI: (10.1016/S1097-2765(05)00008-0)

Figure 2 A Ubiquitin Hydrolase Suppresses the slbo Phenotype by Increasing Slbo Protein Levels (A) Map of Ubp64 locus. EP3584 is inserted at position 2777 of genomic sequence with accession AC014815. The 5′ end of the cDNA isolated is at position 3560 and corresponds from +30 to several ESTs (LD26688) in the database. The coding region corresponds to that of Ubp64 (Henchoz et al. 1996). The map of the locus was made by aligning cDNA and genomic sequences. (B) Quantification of border cell migration in slboGAL4,slbo1310/slbo1310; UAS-Ubp64/+ (two different insertion lines) and slboGAL4,slbo1310/slbo1310 control females. Border cells were detected by X-gal staining of ovaries. The percent stage 10 egg chambers with migrated border cells is shown. A minimum of 376 (top), 216 (middle), and 123 (bottom) egg chambers were scored per genotype. (C) Immunodetection of Slbo and β-galactosidase (slbo-lacZ) in border cell nuclei from females of the genotype slboGAL4,slbo1310/slbo1310 (control) and slboGAL4,slbo1310/slbo1310; UAS-UBP64/+. Stainings were done in parallel and all confocal scans made at the same settings. Representative examples are shown. To obtain average nuclear pixel intensity (Slbo protein), nuclei were defined in the anti-β-gal channel, measurements were done in the anti-Slbo channel with background from surrounding tissue subtracted. The slbo-lacZ signal (red) is from lacZ reporter gene in the slbo1310 enhancer trap and was unaffected by Ubp64. Molecular Cell 2000 6, 23-30DOI: (10.1016/S1097-2765(05)00008-0)

Figure 3 Slbo Protein Is Selectively Unstable in Border Cells Females of the genotype +/+; HS-slbo/+ (left panels) or slbo8ex2/slbo1310; HS-slbo/+ (right panels) were subjected to a 1 hr heat shock (37°C), and ovaries were fixed for antibody staining after the indicated amount of recovery time (at room temperature). White arrows indicate border cells; pink arrows indicate other (main body) follicle cells. Because the border cells have not migrated in the slbo mutant, the anterior 2/3 of the egg chambers is shown rather than the posterior 2/3 as for the control. In each case, main body follicle cells at the anterior and posterior parts of the egg chamber showed similar staining. The exact times at which Slbo protein accumulated and disappeared varied in different experiments and in individual egg chambers. However, in +/+ egg chambers, Slbo protein always disappeared from border cells first. In slbo/slbo egg chambers border cells and other follicle cells showed similar Slbo staining at all time points. At 3–4 hr, Slbo protein finally disappeared precipitously from all follicle cells. Molecular Cell 2000 6, 23-30DOI: (10.1016/S1097-2765(05)00008-0)

Figure 4 Tribbles Downregulates Slbo Protein Level In Vivo In all pictures, anterior is to the left; border cells would migrate to the right. (A–D) X-gal staining of stage 10 egg chambers from females of the genotype slboGAL4,slbo1310/+ plus the indicated transgenes. Red arrows indicate border cells; black arrows indicate centripetal cells that normally migrate during stage 10. In slboGAL4,slbo1310/+, UAS-src64, the centripetal cells do not migrate between nurse cells and oocyte as they should, but usually remain on the surface of the egg chamber. (E and F) Anti-slbo (green and single channel to the right) and anti-β-galactosidase (red) staining of late stage 9 border cells from slboGAL4,slbo1310/+ (E) or slboGAL4,slbo1310/+; UAS-tribbles/+ (F) females. The β-galactosidase signal is from the lacZ reporter gene in the slbo1310 enhancer trap and is unaltered by tribbles expression. (G–I) Slbo protein in tribbles1119 mutant border cells. The tribbles1119 mutant is a severe loss-of-function allele (Mata et al. 2000). Clones were produced using the FLP/FRT technique (Xu and Rubin 1993) in heterozygous FRT80,tribbles1119 females. Lack of the clonal marker (red) indicates a homozygous mutant cell (arrows). The egg chambers were early stage 9 (G), midstage 9 (H), and stage 10A (I). Molecular Cell 2000 6, 23-30DOI: (10.1016/S1097-2765(05)00008-0)

Figure 5 Tribbles Stimulates Slbo Ubiquitination and Proteolysis (A) tribbles counteracts the effect of overexpressing slbo in ectopic locations. Wings from female flies of the indicated genotype. (B) Total cell extracts from cells transfected with vectors encoding HA-slbo alone (control) or with HA-slbo and T7-tribbles (tribbles). (C) Northern blots of mRNA from Schneider cells transfected as in (B). Induced transcripts are mark with arrowheads. tribbles is expressed endogenously at a low level (small arrow). (D) Transfection as in (B), lactacystin (+) or solvent (−) was added to cells for 6 hr before harvest. (E) Transfection as in (B), but including a vector expressing His-tagged ubiquitin. Loading was adjusted to give equal amount of total HA-Slbo in each lane (see short exposure in lower panel). Higher molecular weight forms (bracket) are presumptive ubiquitinated forms of HA-Slbo. (F) HA-slbo, but not a negative control HA-nle (Royet et al. 1998), coimmunoprecipitated with T7-tribbles from transfected cells. (B), (D), (E), and (F) are Western blots probed with HA antibody; for (B), T7 antibody was also included. Equal amounts of Mt-HA-slbo and Mt-T7-tribbles (or empty vector control) expression vectors were used, except in (E) where vectors encoding His-tagged ubiquitin, HA-slbo, and T7-tribbles (or empty vector) were used in 5:2:1 ratio. Molecular Cell 2000 6, 23-30DOI: (10.1016/S1097-2765(05)00008-0)

Figure 6 Tribbles Does Not Affect Another bZIP Protein, DJun (A–C) Eye disc from UAS-tribbles/+; sevenless-GAL4/+ third instar larvae, double-stained with anti-DJun ([B], and green in [A]) and anti-tribbles ([C], and red in [A]). (D) Western blots of extracts from cells transfected with DJun expression vector alone or with tribbles expression vector. Molecular Cell 2000 6, 23-30DOI: (10.1016/S1097-2765(05)00008-0)

Figure 7 Increased Slbo Activity Perturbs Border Cell Migration (A–D) X-gal staining of stage 10 egg chambers. (A) and (B) show egg chambers from females of the genotype slboGAL4,slbo1310/+ plus the indicated transgenes, raised at 25°C. In (C) and (D), slboGAL4,slbo1310/slbo1310; UAS-slbo-control/+ (C) or slboGAL4,slbo1310/slbo1310; UAS-slbo-LZmut/+ (D) females were raised at 18°C, where GAL4 induced expression is lower. (E and F) Antibody staining of stage 10A egg chambers from females of the genotype slboGAL4,slbo1310/+; UAS-slbo/+ (control) (E) or slboGAL4,slbo1310/+; UAS-slbo-LZmut/+ (F), raised at 28°C. White arrowheads mark the most posterior position of stretch follicle cells, which in wild type corresponds to the position of border cells along the anterior–posterior axis. (G) Schematic drawing illustrating the effect of the leucine-to-proline mutation in the leucine zipper (Slbo-LZmut), details are given in Experimental Procedures. Molecular Cell 2000 6, 23-30DOI: (10.1016/S1097-2765(05)00008-0)