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First published online 3 July 2006
doi: 10.1242/dev.02449

Development 133, 2915-2924 (2006)
Published by The Company of Biologists 2006
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The PDZ-GEF Dizzy regulates cell shape of migrating macrophages via Rap1 and integrins in the Drosophila embryo

Sven Huelsmann*, Christina Hepper, Daniele Marchese, Christian Knöll and Rolf Reuter{dagger}

Interfakultäres Institut für Zellbiologie, Abteilung Genetik der Tiere, Fakultät für Biologie, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany.


Figure 1
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  		Fig. 1. Genomic structure of the dizzy locus and embryonic dizzy expression. (A) The dizzy locus comprises eight exons (UTRs dark gray). The P-elements of dizzyEP and of dizzyP are inserted into exon 0 at positions +39 and +46, respectively. The various alleles dizzy{Delta}n have been obtained by imprecise excision of dizzyEP and delete the transcription start site or the translation start site. The inset shows neighboring genes of dizzy. (B) All conserved domains of the PDZ-GEF Dizzy are encoded by exon 3: cNMP, cyclic nucleotide binding domain; RasGEFN, N-terminal Ras-GEF domain; PDZ, PDZ domain; RA, Ras association domain; RasGEF, Ras-GEF domain. The deletions of the alleles dizzy{Delta}3 and dizzy{Delta}10 predict the expression of truncated Dizzy proteins lacking the cNMP domain (gray triangles: predicted translation starts); dizzy{Delta}12 might lead to the expression of a protein without cNMP, RasGEFN and PDZ domains. (C) dizzy RNA is ubiquitously expressed during embryogenesis at relatively low levels. (D) Embryos homozygous or hemizygous for one of the alleles associated with a deletion of the transcription start site (here dizzy{Delta}8 homozygous) show no expression: the signal is indistinguishable from a signal obtained by the sense probe (not shown).

 

Figure 2
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  		Fig. 2. dizzy is required for proper cell migration and cell shape of embryonic macrophages. (A,C,E,G) Hemizygous dizzy{Delta}n mutant embryos; (I,K) homozygous dizzy{Delta}1 mutant embryos; (B,D,F,H,J,L) embryos, wild-type for dizzy; (M) embryo carrying srph-Gal4 UAS-ds.dizzy. (A,E,I,K) Mutations in dizzy affect the migration of macrophages. (A) At stage 14, the posterior part of the ventral nerve cord (VNC) lacks macrophages in dizzy mutants (between red arrowheads), whereas in wild-type embryos (B), the VNC is completely surrounded by macrophages. (E) In the dizzy mutants the gap eventually disappears toward stage 15. (F) Wild-type embryos have an even distribution of macrophages around the VNC at this stage. (C,D,G,H) In addition, dizzy mutant macrophages form smaller protrusions than wild-type cells, here shown for macrophages migrating along the dorsal edge of the epidermis at stage 14 (C,D) and at lateral position at stage 15 (G,H). At this stage there are fewer macrophages in lateral positions in mutant embryos (G) than in wild-type (H). (I,K) In dizzy mutants, macrophages become trapped beneath the amnioserosa (white arrowhead, dorsal view). The hindgut primordium is devoid of macrophages (arrow, I, dorsal view; K, sagittal view). (J,L) In wild-type embryos, macrophages effectively enter the posterior end and leave the space between amnioserosa and yolk. A fraction of them surrounds the developing hindgut (arrow, J, dorsal view; L, sagittal view). (M) dizzy function is required in macrophages for proper cell migration. In embryos, expressing a fragment of dizzy dsRNA in macrophages, these cells do not populate the posterior nervous system at stage 14 and leave a gap (between red arrowheads), which is not entirely closed later. Like in the mutants, macrophages become trapped between yolk and amnioserosa (white arrowhead). Embryos with srph>cd2 and stained for CD2 to visualize macrophages; anterior to the left and dorsal up. For A-H, each picture consists of serveral merged confocal images representing a slice of about 12 µm thickness.

 

Figure 3
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  		Fig. 3. The length of the cellular protrusions of macrophages depends on dizzy. Cellular protrusions per macrophage have been measured for the given genotypes in fixed and immunostained embryos beneath the dorsolateral epidermis at stage 14. In dizzy mutants and in embryos expressing ds.dizzy in macrophages the average length of cellular protrusions per cell is about half of the length seen in wild-type macrophages. Overexpression of dizzy (dizzyh.EP) or of dominant-active Rap1V12 in macrophages leads to an increase of total length per cell by a factor of about four and three, respectively. As the protrusions of macrophages in these embryos span from cell body to cell body, standard deviations are here based on the lengths of individual protrusions rather than on the overall length per cell. The increase in protrusion length depends in both cases, dizzyh.EP and Rap1V12, on the zygotic expression of ßPS integrin mys. In zygotic mys mutants the length per cell is similar to wild-type even when dizzy or dominant-active Rap1V12 is expressed in the macrophages.

 

Figure 4
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  		Fig. 4. The overexpression of dizzy in macrophages changes their cell shape. Macrophages overexpressing dizzy from a single copy of the allele dizzyEP under the control of srph-gal4 (A,C,E,G,I; hereafter named dizzyh.EP) have a significantly different cell shape from macrophages of wild-type embryos (B,D,F,H,J). (A,B) At stage 11, dizzyh.EP macrophages migrate in a similar way to wild-type cells. (C) During and after migration through the embryo, dizzyh.EP macrophages form long protrusions that contact each other and the substrate. These protrusions span dorsoventrally through the entire VNC (arrowhead). (D) In wild-type embryos, the protrusions are much smaller. (E) The change in cell form is maintained also in late embryos affecting the clearance of the VNC from macrophages (arrowhead). (F) In a wild-type embryo, macrophages have left the inner region of the VNC at stage 16. (G) In dorsolateral positions, the dizzyh.EP macrophages form a network with their large cellular extensions. (H) In wild-type embryos, the cells are smaller and do not touch each other. (I,J) Magnifications of areas indicated in G and H. (K) Expression of dizzy from two copies of dizzyEP also affects migration of macrophages, most strongly at stage 14.

 

Figure 5
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  		Fig. 5. Macrophages overexpressing dizzy leave long stable protrusions behind during their migration. The panels show time series of pictures taken from embryos expressing actin::gfp in the macrophages, at stage 13; numbers are minutes lapsed after the start of the series. The cells are located on the ventral side of the midline of the VNC (indicated by black line; anterior is to the left). (Top row) dizzyh.EP embryo; one of the cells (below asterisk) moves to the left edge of the VNC and maintains contact with the cells at the midline by a long cellular extension. (Bottom row) Wild-type embryo; one of the cells (asterisk) is followed along the midline and from there to the left edge of the VNC. Although the cell occasionally forms a short tail (20' panel), it does not maintain contact to cells it has passed on its path (pictures from Movies 1 and 2 in the supplementary material. Each panel represents 70 µm in width.

 

Figure 6
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  		Fig. 6. Dizzy acts via Rap1 during the migration of macrophages. (A,B) The migration of macrophages is only slightly disturbed in zygotic rap1 mutant embryos. (A) In wild-type embryos, the macrophages are already found in the entire midline after stage 13. (B) The macrophages of zygotic, homozygous rap1 mutants fail to completely surround the midline of the VNC at stage 14 (arrowhead). (C) Confocal section of an embryo, highlighting the dorsal edge as shown in D-K. (D-K) Macrophages migrating along the dorsal edge. (D,E) Macrophages are not significantly affected in zygotic rap1 mutants. (F,G) Dizzy fails to induce cell shape changes in a rap1 mutant background. (H,I) Similarly, the even stronger cell shape phenotype induced by overexpression from two copies of dizzyh.EP is rescued in rap1 mutants. (J) The expression of dominant-active Rap1V12 induces a phenotype similar to 2xdizzyh.EP. (K) Overexpression of wild-type Rap1 fails to induce cell shape changes in migrating macrophages. Embryos at stage 14.

 

Figure 7
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  		Fig. 7. Dizzy behaves like a Rap1-GEF in vivo. Embryos solely expressing dominant-negative rap1N17 (A,C) compared with embryos simultaneously expressing rap1N17 and dizzyEP (B,D) in macrophages. (A) The expression of rap1N17 severely disrupts the migration of macrophages along the VNC. (B) This effect of rap1N17 is rescued by the simultaneous expression of dizzy, presumably by substituting for GEFs sequestered by Rap1N17. (C,D) Similarly, the effect of Rap1N17 on macrophages migrating along the dorsal edge (C) is rescued by the coexpression of dizzy (D). A,B: sagittal optical sections; C,D: magnifications of similar areas as indicated by the rectangle in Fig. 6C.

 

Figure 8
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  		Fig. 8. Dizzy- and Rap1-induced cell adhesion require integrin function. Dizzy (A) and Rap1V12 (E) can induce similarly strong cell shape changes in macrophages. (B,F) These changes depend on the function of ßPS integrins, as both Dizzy (B) and RapV12 (F) fail to show the effect in a mys mutant background. (C,D) Macrophages at the dorsal edge of a wild-type (C) and a mys mutant (D) embryo. The latter (lacking only the zygotic mys contribution) is indistinguishable from wild type concerning macrophage shape and motility. Embryos at stage 14; field of view as in Fig. 6D-K.

 

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© The Company of Biologists Ltd 2006