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First published online 11 February 2009
doi: 10.1242/dev.026203

Development 136, 813-822 (2009)
Published by The Company of Biologists 2009
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Regulation of the Rac GTPase pathway by the multifunctional Rho GEF Pebble is essential for mesoderm migration in the Drosophila gastrula

Andreas van Impel1, Sabine Schumacher2,*, Margarethe Draga1, Hans-Martin Herz3, Jörg Großhans3 and H. Arno J. Müller1,{dagger}

1 Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
2 Institut für Genetik, Heinrich-Heine Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany.
3 ZMBH, Universität Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.


Figure 1
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  		Fig. 1. Domain organization of Pbl constructs. All constructs are derived from a cDNA encoding the Pbl-A isoform. The extent of the constructs is indicated. The domains from N to C terminus are BRCT (BRCA1 carboxy-terminal domain), NLS (nuclear localization sequence), PEST (rich in proline, glutamic acid, serine and threonine), DH (Dbl homology), PH (Pleckstrin homology domain) and C-term (carboxy-terminal tail). Pbl{Delta}N-term_V531D and PblDH-PH_V531D represent catalytically inactive variants. Scale bar: 100 amino acids.

 

Figure 2
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  		Fig. 2. Rescue potential of mesoderm-spreading defects in pbl mutants by Pbl constructs. (A) Eve is expressed in 11 segmental dorsal mesodermal cell clusters in the wild type (arrowheads). (B) In pbl3 mutants, the number of Eve clusters is strongly reduced (dorsal positions marked by arrowheads). Transgenic UAS::Pbln constructs were expressed in pbl3 mutants using twi::Gal4. (C) Expression of full-length Pbl almost completely rescues pbl3 mutant embryos. (D) Pbl{Delta}BRCT expression. (E) Pbl{Delta}N-term expression; arrows indicate Eve-expressing mesoderm cells. (F) PblDH-PH expression. (G) PblDH-PH_V531D expression. (H) PblDH expression. (I) Pbl{Delta}C-term expression. (J) Quantification of suppression by the various constructs: (pbl3 homozygotes, black; Pbl-A, white; Pbl{Delta}BRCT, yellow; Pbl{Delta}N-term, dark blue; PblDH-PH, red; PblDH-PH_V531D, grey; PblDH, green; Pbl{Delta}C-term, pale blue). The graph depicts the relative proportion of embryos that exhibit eve-positive hemi-segments in the various genotypes indicated (values are shown in Table 1).

 

Figure 3
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  		Fig. 3. Dominant effects of truncated Pbl constructs on mesoderm morphogenesis. (A-R) Embryos were stained with anti-Twi antibody and are depicted either as whole mounts (A-L) or transverse cross-sections [M-R; sections were taken between 30% and 60% embryo length (anterior-posterior axis) at early stage 8 (M,O,Q) and stage 9 (N,P,R)]. Lateral and ventral views are shown as whole mounts at early stage 8 (A,C,E,G,I,K) or late stage 8 (B,D,F,H,J,L). Pbl constructs were expressed in the mesoderm using twi::Gal4; Dmef::Gal4. In comparison with the wild type (A,B,M,N), overexpression of Pbl{Delta}N-term results in embryos in which the mesoderm cells remained at the surface (C,D,O,P). Overexpression of Pbl{Delta}BRCT (E,F) or PblDH (G,H) does not interfere with early mesoderm development. Overexpression of PblDH-PH mainly results in defects during mesoderm spreading (I,J,Q,R). (K,L) The catalytic loss-of-function PblDH-PH_V531D mutant as a control.

 

Figure 4
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  		Fig. 4. Cell cortex localization of Pbl in interphase cells. Embryos were fixed and stained with anti-HA (red in A,D,G,J,M) and anti-Twi (green in B,E,H,K,N), anti-Pbl antibodies (red in P,Q) or DAPI (blue in L). Merged images are shown in C,F,I,L,O. (A-C) Pbl-HA was overexpressed in the mesoderm using the twi::Gal4, Dmef::Gal4 driver. The images represent a z-projection of 47 optical sections in 0.16 µm intervals (7.5 µm total). A 3D reconstruction of a similar data set is provided as Movie 1; note strong localization of Pbl-HA to the nucleus and staining in cell protrusions of the leading edge (A,C). Pbl-HA expressed in mesoderm cells of pbl3 homozygous embryos by twi::Gal4. (D-F) Cell protrusions at the leading edge are stained. Accumulation of HA staining is seen in dividing cells. (G-I) Arrowheads indicate cells in different stages of cytokinesis; note accumulation of staining at cell cortex of dividing mesoderm cells. (J-O) High magnifications of Pbl-HA staining to highlight localization to the cleavage furrow of dividing mesoderm cells; arrowheads indicate Pbl-HA accumulation at the cleavage furrow and the central spindle. (P,Q) Staining of endogenous Pbl protein with anti-Pbl antibodies. Single optical section is depicted in P, note only subtle cortical association (arrowheads) of staining with the antibodies. Projection of z-series (56 sections over 16 µm) in Q demonstrates prominent nuclear localization of Pbl and occasional staining of the cell cortex (arrowheads). (R) Still images (at 20-second intervals) of a time-lapse sequence of Pbl-GFP in haemocytes during late embryogenesis; note that Pbl-GFP localizes to the cell cortex and actin-rich microspikes in a dynamic fashion (see Movie 2 in the supplementary material).

 

Figure 5
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  		Fig. 5. Localization of Pbl constructs. Tagged Pbl constructs were expressed in mesoderm cells by twi::Gal4,Dmef::Gal4. The following antibody staining was used to detect tagged proteins: anti-Myc (red in A,B), anti-HA (red in C-P) and anti-GFP (red in Q,R). Anti-Twi staining (green) marks the mesoderm cells and merged images are shown in D,F,H,J,L,N,P,R. (A,B) Pbl{Delta}BRCT accumulated in the nucleus and low amounts were also detected at the cell cortex (arrows). (C,D) Pbl{Delta}C-term localizes to the nucleus and the cytoplasm with very low cortical protein localization (marked by arrows). (E,F) HA-tagged Pbl{Delta}N-term accumulates prominently at the cell cortex; note that this construct also interferes with cytokinesis (arrows indicate multi-nucleated cells). (G,H) The HA-tagged PblDH-PH is present at the cortex in a punctate fashion (arrows). (I,J) Expression and localization of Pbl{Delta}N-term_V531D.(K,L) Expression and localization of PblDH-PH_V531D. (M,N) PblDH localizes to the cytoplasm. (O,P) PblC-term localizes to the cell cortex. (Q,R) Localization of PblPH-GFP in mesoderm cells; note accumulation at the cell cortex (arrows).

 

Figure 6
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  		Fig. 6. Differential rescue and localization of Pbl{Delta}C-term. Pbl{Delta}C-term was expressed in wild-type (A-F) embryos and stained with anti-HA antibodies (red, A-F) Twi antibodies (green) and DAPI (blue); merged images are shown in B,D,F. (A-F) Accumulation of Pbl{Delta}C-term at the cell cortex in dividing cells (marked by arrows in A,B). (C-F) High magnification of Pbl{Delta}C-term localization at the cleavage furrow of dividing cells (arrows). (G,H) twi::CD2 (red) and Twi (green) in wild-type (G) and pbl3 homozygous (H) embryos; as shown previously cellular protrusions are absent in pbl3 mutants (Schumacher et al., 2004Go). (I,J) Expression of Pbl{Delta}C-term in pbl3 homozygous embryos also expressing twi::CD2. (I) Single optical section indicates multinucleated cells (arrows). (J) z-projection (12 sections at 0.4 µm; 4.9 µm total) of the same embryo as in I showing the leading edge of migrating mesoderm cells; note cellular protrusions at the leading edge (arrows in J).

 

Figure 7
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  		Fig. 7. The tandem DH-PH domain of Pbl interacts with Rho1 and Rac GTPases. (A,B) Expression of PblDH-PH using the eye-specific GMR::Gal4 driver (A) leads to a rough eye phenotype (B). (C) This phenotype depends on the catalytic activity of the DH domain, as PblDH-PH_V531D does not promote a rough eye phenotype. (D) The phenotype is partially suppressed in pbl3 heterozygotes. (E) The PblDH-PH rough eye phenotype is suppressed in flies heterozygous for a loss-of-function mutation in rho1. (F-H) Co-expression of dominant versions of RhoL (dominant active RhoLV20 and dominant negative RhoLN25) (F,G) or lowering the dose of cdc42 (H) has no impact on the phenotype. (I) Reducing the gene doses of all three Drosophila Rac GTPases, Rac1, Rac2 and Mtl, suppresses the eye defects caused by PblDH-PH expression. (J) Co-expression of wild-type Rac1 leads to an enhancement of the phenotype; most flies die as pharate adults and a few escapers hatched and failed to develop any eye structures. (K) Expression of Pbl{Delta}N-term at 18°C leads to pharate adult lethality; animals dissected out of their pupal cases exhibit a strong rough eye phenotype. (L) The lethality caused by expression of Pbl{Delta}N-term is rescued in a Rho1 heterozygous background and the adult flies exhibited a strong rough eye phenotype.

 

Figure 8
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  		Fig. 8. Exchange activity of PblDH in vitro. Results of GEF assays are plotted as relative amounts of 3H-GDP bound to indicated GST-Rho GTPase fusion proteins after a 20-minute incubation at 25°C with GST-DH fusion proteins of Pbl (red) or Trio (yellow) and unlabelled GTP. GST (grey) was used as a control. Note activity of the Pbl DH domain towards GDP exchange for Rho1, Rac1 and Rac2.

 

Figure 9
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  		Fig. 9. Regulation of Rac GTPases is essential for mesoderm migration. (A-D) Ventral views of embryos (during stage 8, germ band extension) stained against Twi. Unlike wild type (A), embryos lacking the maternal and zygotic contribution of Rac1 and Rac2 show strong defects during mesoderm migration; the cells fail to spread out laterally and form a tight aggregate (B). These defects are reminiscent of the phenotype found after loss of both the Htl ligands FGF8-like1 and FGF8-like2 (C). A similar, although slightly weaker, phenotype can be observed after expression of constitutive active Rac1, Rac1V12, in the mesoderm (D). (E-H) Cross-sections of early (G) and late stage 8 (H) embryos expressing Rac1V12 show strongly abnormal mesoderm spreading compared with wild-type embryos (E,F, stage 8 early and late, respectively).

 

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© The Company of Biologists Ltd 2009