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First published online 5 May 2004
doi: 10.1242/dev.01149

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The RhoGEF Pebble is required for cell shape changes during cell migration triggered by the Drosophila FGF receptor Heartless

Institut für Genetik, Heinrich Heine Universität, Düsseldorf, Germany

View larger version (115K): [in a new window]   Fig. 1. HTL is required for cell shape changes during mesoderm migration. Embryos expressing twi::CD2 were stained with anti-CD2 antibodies (red) and anti TWI antibodies (green) and either visualized as whole mounts after optical sectioning by confocal microscopy (D-F,J,K,L,N) or cross-sectioned after plastic embedding (A-C,G-I). (M) Wild-type embryo stained against NRT (red) and TWI (green). Scale bar: 20 µm for A-L. (A-F) Wild-type embryos. (A,D) Phase 1. (B,E) Phase 2; long cellular protrusions are marked with arrows in E. (C,F) Phase 3. (G-L) Embryos homozygously mutant for htlAB42; (G,J) phase 1; note that some mesoderm cells are out of the focal plane, because they are not attached to the ectoderm (J). (H,K) Phase 2; note that cells remain rounded and no cell shape changes occur. (I,L) Phase 3; some cells extend protrusions (arrow in L). (M) Cross-section through the migrating mesodermal aggregate of a wild-type embryo during phase 2. The cells adopted a polarized morphology, with the front attached to the ectoderm and the rear attached to the neighboring cells. (N) htlAB42 mutant embryo after mesoderm migration (stage 10). Note the formation of cellular protrusions of cells attached to the ectoderm (arrowheads).

View larger version (121K): [in a new window]   Fig. 2. HTL is required for the attachment of the mesoderm to the ectoderm. Embryos (stage 7; phase 1 of migration) were fixed and stained with anti-NRT (red) and anti-TWI (green) antibodies (A,C) or with anti-CD2 antibodies (B,D). (A) In the wild type, the mesodermal tube is attached to those ectodermal cells that lie adjacent to the basal cells of the tube (arrowheads). (B) The invaginated mesoderm cells are aligned along the midline of the embryo, indicated with arrowheads. (C) In embryos homozygously mutant for htlAB42, the basal cells of the mesodermal tube fail to adhere to the ectoderm. (D) Misalignment of mesoderm cells in htl mutants with respect to the ventral midline (arrowheads in B,D). (B,D) Projections of a stack of confocal images covering a total of 25 µm from the ventral surface of the embryo to the interior.

View larger version (120K): [in a new window]   Fig. 3. Identification of pbl as novel zygotic factor required for mesodermal cell migration. (A-C) Embryos at extended germband stages carrying a synthetic chromosomal deletion produced by C(3)se females crossed to T(3;2)C309/TM3 males (uncovering genomic segment 61 to 68) were stained with anti-EN (brown) and anti-TWI (black) antibodies. The reduction of EN stripes is due to deletion of the hairy locus in this genomic interval and was used as independent marker for the genotype. (A,B) Lateral (A) and ventral (B) views of whole-mount staining. (C) Cross-section demonstrates that the mesoderm cells fail to migrate in these embryos. Wild-type embryos (D) and pbl3 homozygous embryos (E) at stage 11 were stained for EVE (blue) and TWI (brown) protein. In pbl mutants the number of EVE-positive hemisegments is strongly reduced. (F-H) Cell shape changes were analyzed by examining twi::CD2 expression in pbl3 homozygotes; cell shape changes are blocked in pbl mutant embryos. (I,J) F-actin staining (red) of wild-type (I) and pbl3 homozygous embryos. TWI staining is seen in green. F-actin-rich protrusions are indicated with arrowheads in I.

View larger version (126K): [in a new window]   Fig. 4. The function of PBL in cell migration is independent of its function in cytokinesis. (A-C) Embryos at stage 10 stained for EVE (blue) and TWI and ßGal (brown). In the wild-type (A) and stg7M homozygous embryos (B), eleven eve-positive hemisegments are stained on either side of the embryo. In pbl3, stg7M double mutant embryos (C), the number of eve-positive hemisegments is strongly reduced, reminiscent of pbl single mutant embryos. (D-F) Cell shape changes in twi::CD2; pbl3/pbl3 (D), twi::CD2; stg7M/stg7M (E), and twi::CD2; pbl3,stg7M/pbl3,stg7M double mutant (F) embryos as visualized with anti CD2 (red) and anti TWI (green) staining. Protrusive activity in phase 2 is absent in pbl and pbl,stg double mutant embryos, while it can still be observed in stg single mutants. Note bi-nucleated cells are present in pbl mutants (arrows in D), but are absent in stg and pbl,stg double mutants (arrows in E,F).

View larger version (95K): [in a new window]   Fig. 5. Different requirements for PBL and RHO1 in cell migration and cytokinesis. (A-C) eve expression in the wild type (A) and in embryos homozygously mutant for pbl3 (B) or pbl11D (C). Although a strong reduction of EVE-positive cells is seen in the pbl3 mutants (B), pbl11D mutants (C) exhibit a significantly higher number of EVE-positive hemisegments (see also Table 1). (D,E) Cytokinesis defects in pbl3 (D) and pbl11D (E) homozygous embryos. Ventral view of embryos at extended germband stage (stage 11) stained with antibodies against EVE. Segmental expression of eve in the central nervous system can be seen as described before (Cui and Doe, 1995; Weigmann and Lehner, 1995). The EVE-positive cells represent products of an incomplete cytokinesis of neuroblast 7-1. Inserts show confocal sections of anti-EVE and anti-NRT stained embryos to label cell outlines (both antibodies in red); note the presence of two nuclei in the neuroblasts. (F) Expression of Rho1N19 in the mesoderm affects cytokinesis, but not migration. twi::Gal4; UAS::Rho1N19 embryo stained with anti-EVE (blue) and anti-TWI (brown). Embryos expressing UAS::Rho1N19 exhibit normal mesoderm differentiation indicated by the normal pattern of EVE-positive cells. Formation of cellular protrusions in twi::Gal4; UAS::Rho1N19 embryos is normal (G; twi::CD2 (red), anti-TWI (green)). (H) Cytokinesis in such embryos is blocked as binucleated cells (arrowheads) can be seen in a different focal plane of the embryo shown in (G). (I,J) EVE expression in twi::Gal4; Dmef2::Gal4/UAS::Rho1N19 embryos at stage 10 (I) and 11 (J). Note some irregularity in the arrangement of EVE-expressing cells (arrowhead in J).

View larger version (85K): [in a new window]   Fig. 6. The function of PBL in mesoderm migration is mesoderm autonomous. Embryos hemizygous for pbl3, expressing twi::Gal4, UAS::pbl3.2 were stained with anti-NRT (red) and anti-TWI (green) antibodies and sectioned. Embryos are shown in phase 1 (A), phase 2 (B) and phase 3 (C) of migration. A complete rescue of cell shape change defects in pbl mutants is seen. Note that cytokinesis is occurring normally in the mesoderm while still blocked in the ectoderm (insert in B; the asterisks mark nuclei). (D,E) Differentiation of EVE-positive mesoderm cells is rescued by expression of UAS::pbl3.2 with twi::Gal4 in pbl3 hemizygously mutant embryos. (D) Stage 11 embryo containing at least one wild-type copy of the pbl gene; the blue staining indicates presence of the TM3 (ftz::lacZ) chromosome. (E) Stage 11 embryo of the genotype twi::Gal4/UAS::pbl3.2; pbl3/Df(3L)pblNR.

View larger version (118K): [in a new window]   Fig. 7. Requirement of PBL for HTL-triggered cell shape changes. (A,C,E) Sections of embryos at stage 7 stained with anti-NRT (red) and anti TWI (green) antibodies. The contacts of the mesoderm with the ectoderm cells are marked with arrowheads in A,C. (B,D,F) Lateral surface views of embryos at stage 11 stained with anti-EVE antibodies. (A,B) htlAB42 homozygous embryos overexpressing full-length HTL protein (twi::Gal4; htlAB42, UAS::htlM/htlAB42). (C,D) htlAB42 homozygous embryos overexpressing constitutively activated HTL (twi::Gal4; htlAB42, UAS::htl/htlAB42). (E-G) pbl3 hemizygous embryos overexpressing the activated form of HTL (twi::Gal4; pbl3, UAS::htl/Df(3L)pblNR); embryo in G is at stage 9. Arrowhead in E indicates the failure of mesoderm cells to contact the ectoderm. (H,I) Cross-sections of wild-type (H) and pbl3 homozygous mutant (I) embryo at stage 8 stained with anti-dpERK antibodies. Arrowheads indicate mesoderm cells at the leading edge that stain for dpERK.