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First published online 5 May 2004
doi: 10.1242/dev.01150

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The epithelial-mesenchymal transition of the Drosophila mesoderm requires the Rho GTP exchange factor Pebble

Centre for the Molecular Genetics of Development and Research School of Biological Sciences, The Australian National University, Canberra, ACT, 0200, Australia

View larger version (78K): [in a new window]   Fig. 8. PBL is not required for activation of the HTL/MAPK pathway. Ventrolateral view of stage 8 embryos heterozygous (A) and homozygous (B) for pbl2. Activation of the MAPK pathway, visualised using an antibody specific to dpERK, the dual-phosphorylated form of the MAPK (Gabay et al., 1997a; Gabay et al., 1997b), is seen in the cells at the leading front (arrowheads) of the mesoderm during dorsal migration.

View larger version (75K): [in a new window]   Fig. 1. pbl expression in the mesoderm is developmentally regulated. Whole-mount in situ hybridisation of wild-type embryos with a pbl RNA antisense probe. (A) During early stage 5, pbl transcripts are restricted to the pole cells. (B-F) Ventral view. (B) At late stage 5, pbl transcripts are observed on either side of the presumptive mesoderm. (C) At the onset of gastrulation, pbl transcripts are evident in the presumptive mesoderm (arrow). (D) This expression becomes restricted as stage 6 progresses (arrow). (E) pbl transcripts accumulate in a band of cells at the edge of the invaginating mesoderm (arrows). (F) As invagination proceeds, pbl transcripts are concentrated in the ventral furrow (arrow).

View larger version (56K): [in a new window]   Fig. 2. pbl mutant embryos have reduced numbers of EVE-positive mesodermal cells. (A,B) Anti-Fasciclin 3 stains and (C,D) anti-EVE stains of wild-type (A,C) and pbl2/pbl3 mutant embryos (B,D). (A) At stage 12 in a wild-type embryo, the visceral mesoderm is seen as a band of cells running along the anteroposterior axis (arrow). (B) In a similarly staged pbl2/pbl3 mutant embryo, the visceral mesoderm is evident, although it is less organised (arrow). (C) At stage 11 in a wild-type embryo, EVE is expressed in segmentally repeated clusters of dorsal mesodermal cells. The EVE-expressing neuroblasts are out of focus in this image. (D) In a similarly staged pbl2/pbl3 mutant embryo, the majority of EVE-positive mesodermal cells fail to form. One EVE-positive hemisegment can be seen in this embryo (arrow). Based on their position and morphology, the remainder of the EVE-expressing cells are neuroblasts (arrowheads).

View larger version (27K): [in a new window]   Fig. 3. The number of EVE-positive hemisegments is reduced in pbl mutant embryos. (A-F) Histograms of the number of EVE-positive hemisegments in wild-type (A), pbl2/pbl3 (B), pbl5/pbl5(C), UAS-myc-pbl, pbl3/prd-GAL4, pbl2 (D), UAS-pblBRCT, pbl3/prd-GAL4, pbl2 (E) and UAS-pblDH; pbl3/prd-GAL4, pbl2 (F) embryos. (G) The mean number of EVE-positive hemisegments in all genotypes examined. Error bars indicate the standard error of the mean (s.e.m.).

View larger version (129K): [in a new window]   Fig. 4. pbl mutant embryos have a mesodermal cell migration defect. (A,B) Transverse sections of in situ hybridisations with a twi antisense RNA probe. (A) In late stage 10 wild-type embryos, the mesodermal cells have dissociated and migrated dorsally to form a uniform layer beneath the ectoderm (arrows indicate dorsalmost mesodermal cells, which lie adjacent to the dorsalmost epidermal cells). (B) In a similarly staged pbl2/pbl3 mutant embryo, the mesodermal cells appear aggregated and have failed to complete dorsolateral migration (arrows indicate the dorsalmost epidermal cells). (C) A stage 10, wild-type embryo stained for F-Actin. Mesodermal cells have formed a monolayer beneath the ectoderm. (D,E) Equivalently staged pbl2/pbl3 embryos, in which mesoderm spreading is defective. (F) A stage 10 pbl2/pbl3 embryo expressing GFP-Actin driven by twi-GAL4 visualised with an anti-GFP antibody. Similar to D,E, the spreading of the mesoderm is defective. (G) A stage 10, pbl2/pbl3 embryo co-expressing wild-type PBL and GFP-Actin with twi-GAL4 visualised with an anti-GFP antibody. The dissociation and migration of the mesodermal cells has been rescued, such that the mesodermal cells form a uniform layer beneath the ectoderm similar to wild type. (G') The same embryo showing multinucleate cells in the ectoderm (arrows).

View larger version (178K): [in a new window]   Fig. 5. The morphology of mesodermal cells is defective in pbl mutant embryos. Control (A-C,E) and pbl2/pbl3 mutant (D,F) stage 8 embryos in which GFP-Actin was expressed in mesodermal cells using the twist-GAL4 driver and visualised with an anti-GFP antibody. (A) A UAS-GFP-Actin/twist-GAL4 control embryo typical of the stage used in this morphological analysis, between the first two waves of mitosis in the mesoderm. (B) Cross-section of a control embryo expressing GFP-Actin in mesodermal cells. Embryos were oriented so that the leading mesodermal cells were parallel to the plane of the microscope. The white line indicates the plane of focus seen in C,D. The black line indicates a deeper plane of focus seen in E,F. (C,D) Projections of 1 µm optical sections of mid-stage 8 control (C) and pbl2/pbl3 (D) embryos showing the morphology of migrating mesodermal cells at the leading front. (C) Cells in a control embryo exhibit numerous protrusions (arrows) in the direction of migration and appear dissociated from each other. (D) Cells in a pbl2/pbl3 embryo extend far fewer protrusions (arrow) and appear more tightly adhered to their mesodermal neighbours. (E) Mesodermal cells in a control embryo appear more rounded with numerous intercellular gaps (arrowhead) present. (F) Cells in a pbl2/pbl3 embryo appear more tightly packed and are less rounded, with fewer intercellular gaps. Scale bars: 10 µm.

View larger version (93K): [in a new window]   Fig. 6. The defects in the mesodermal epithelial-mesenchymal transition and the subsequent loss of the EVE-positive mesodermal cells in pbl mutant embryos are independent of the cytokinetic defect. (A,C,E,G) UAS-myc-pbl, pbl3/prd-GAL4, pbl2 embryos. (B,D,F,H) UAS-pblBRCT, pbl3/prd-GAL4, pbl2 embryos. (J-L) UAS-GFP-Actin/twist-GAL4;UAS-pblBRCT, pbl3/pbl2 embryos. (A,B) Stage 11 embryos stained with anti-EVE and visualised using an alkaline phosphatase assay. (C,D) Stage 14 embryos stained with anti-EVE and visualised using fluorescence microscopy (white). (E-H) Anti-Spectrin (green) and Hoechst 33258 DNA stain (blue). (G,H) Merge of the fluorescent images above. Anti-EVE (red), Anti-Spectrin (green) and Hoechst 33258 DNA stain (blue). (A,C,E,G) Expression of a wild-type copy of a pbl cDNA in a pbl mutant background rescues the EVE-positive mesodermal cell formation and the cytokinetic defect. (A) EVE-positive mesodermal cells are evident in embryos rescued with the wild-type pbl cDNA. (C,E,G) EVE-positive mesodermal cells are evident at a higher magnification (C,G) and rescue of cytokinesis is seen in epidermal cells in alternating stripes corresponding to the prd-GAL4 induced wild-type pbl expression (E,G). (D,F,H) PblBRCT expression rescues the EVE-positive mesodermal cell defect (D,H) but fails to rescue the cytokinetic phenotype in pbl mutants (F,H). Note that the EVE-positive mesodermal cells in C,D are imaged at a different focal plane than the epidermis shown in E,F. (I) Schematic representations of the PBL constructs used in the EVE-positive hemisegment rescue assay. (J-L) Expression of PBLBRCT using the twist-GAL4 driver alters the cellular phenotype of GFP-Actin-expressing mesodermal cells. (J) Cells extend more protrusions (arrows) in the direction of migration. (K) Cell bodies in the central mass of mesodermal cells appear more rounded (compare with Fig. 5F) and intercellular gaps (arrowhead) are present. (L) Single optical slice of cells in J showing binucleate cells (arrowhead) more clearly. Scale bars: 10 µm.

View larger version (158K): [in a new window]   Fig. 7. The GEF function of PBL is required for the transition of the mesoderm from an epithelium to migratory cells during Drosophila gastrulation. (A,B) The majority of EVE-positive hemisegments fail to form in UAS-pblDH; pbl3/prd-GAL4, pbl2 (A) and pbl5/pbl5 embryos (B). (C) Cross-section of a stage 10 pbl5/pbl5 embryo stained for F-actin showing a typical failure of the mesoderm to disaggregate and spread dorsally. (D,E) UAS-GFP-Actin/twist-GAL4; pbl5/pbl5 embryos show similar morphology to UAS-GFP-Actin/twist-GAL4; pbl2/pbl3 embryos (compare with Fig. 5D,F). Scale bars: 10 µm.