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First published online 6 October 2004
doi: 10.1242/dev.01427

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Morphogenesis in the absence of integrins: mutation of both Drosophila ß subunits prevents midgut migration

Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Anatomy, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK

View larger version (57K): [in a new window]   Fig. 1. Mutation of the gene that encodes the ß integrin subunit. (A) ß genomic region. The transposon insertion EP(2)2030 was used to create imprecise excisions that partially delete the ß locus. The ß1 deletion removes the first 69 amino acid codons while ß2 removes the first eight and 2 kb upstream regulatory sequence. Neither allele affects surrounding genes. (B) In situ hybridization against ß transcripts in wild-type and ß2 embryos demonstrates that ß2 mutants do not make any detectable transcript. (C) Adult flies homozygous for ß1 and ß2 are viable, and have no obvious morphological defects.

View larger version (122K): [in a new window]   Fig. 2. Removal of ß enhances the defects in midgut morphogenesis caused by the absence of ßPS. (A,C,E) Longitudinal confocal sections of stage 17 embryos are labelled with phalloidin (inverted, in black), which highlights the somatic musculature and visceral muscle surrounding the midgut. Autofluorescence from the yolk is shown in blue. (A) The wild-type midgut is highly convoluted and is surrounded by a thin layer of visceral muscle. (C) The midgut forms primary constrictions but fails to elongate in embryos lacking zygotic ßPS. (E) Midgut constrictions are lost in embryos lacking zygotic ßPS and maternal and zygotic ß. (B,D,F) High magnification of visceral muscle labelled with rhodamine phalloidin. (B) The visceral muscle (vm) in wild type, stage 16 embryos is a single layer of flattened cells that completely surrounds the columnar midgut epithelium (en, endoderm). (D) In embryos lacking zygotic ßPS the visceral muscle does not flatten, but still remains attached to the midgut epithelium. (F) In embryos lacking both ß subunits, as in E, the visceral muscle is highly disorganized and detaches from the underlying endoderm.

View larger version (150K): [in a new window]   Fig. 3. Integrins are required to maintain epithelial polarity in the midgut. Embryos were stained for DE-cadherin to mark the apicolateral surface of the midgut epithelium (A'-D' and green in A-D) and laminin to mark the basal side (magenta in A-D). (A'-C') At stage 15, DE-cadherin is distributed apically and laterally in the midgut epithelium in wild type, ßPS- and ßPS- ß- mutant embryos. (D,D') By stage 16, the midgut epithelium has lost its polarity and become highly disorganized in the absence of zygotic ßPS and maternal and zygotic ß.

View larger version (125K): [in a new window]   Fig. 4. Midgut migration is entirely dependent on integrins. Wild-type and mutant embryos were stained with anti-filamin 1 (Cheerio) antibodies to mark the migrating midgut endoderm (A'-H' and magenta in A-H) and anti-Fasciclin 3 antibodies to label the visceral mesoderm palisade (green in A-H) upon which the midgut cells migrate. Genotypes are labelled above and stages are indicated on the left. All mutant embryos lack both the maternal and zygotic contributions of the indicated gene products. (A,B) While the midgut primordia have nearly met in the centre of stage 12 wild-type embryos, migration is delayed in embryos lacking ßPS. (F) ßPS- mutant embryos eventually recover and midgut migration is complete by stage 13. (G) Embryos lacking ßPS and ß do not recover, however, and midgut migration is completely blocked. (D) Midgut migration is delayed in embryos lacking talin but, like ßPS- mutants, migration is complete by stage 13 (H).

View larger version (68K): [in a new window]   Fig. 5. Early expression of ß in the midgut does not rescue the delay in migration caused by the absence of ßPS. ß was expressed earlier than normal in the midgut primordium of embryos lacking ßPS using the 48YGal4 driver, which drives expression in the endoderm and some mesodermally derived tissues (arrow in D). ß levels and endoderm morphology were detected by in situ hybridization against ß transcripts. (A) ß is not normally expressed at stage 9 but visible at stage 12 (C). (B) ß driven by 48YGal4 was detectable from stage 9 onwards. (D) Midgut migration was still delayed when ß was precociously expressed in ßPS- mutant embryos (compare C and D with wild type embryo in Fig. 4A).

View larger version (105K): [in a new window]   Fig. 6. ßPS is required for talin localization in the midgut. Wild-type and mutant embryos deficient for both maternal and zygotic ß subunits were stained with anti-talin and anti-filamin 1 antibodies. (A) Talin (A' green) and filamin 1 (A'' red) are concentrated at the interface between the midgut endoderm (en) and the visceral mesoderm (vm). Filamin 1 is expressed in the endoderm while talin is expressed in both tissues. (B,C) In the absence of maternal and zygotic ßPS, talin is diffuse throughout the cytoplasm in both the endoderm and mesoderm (B') and is not altered by the additional absence of ß (C'). Filamin 1 localization to the basal surface of the endoderm is not altered by the absence of ßPS (B'') or by the absence of both ßPS and ß (C'').

View larger version (149K): [in a new window]   Fig. 7. ß-dependent cell-surface expression of PS3 indicates that ß makes a heterodimer with PS3. Embryos were labelled with antibodies against the PS2 subunit (green) and the PS3 subunit (magenta). (A) The PS2 subunit is expressed in the somatic and visceral muscles, and localizes to sites of attachment between adjacent muscles in wild-type stage 16 embryos (arrows). The PS3 subunit is expressed in the midgut endoderm and is localized cortically (A'). (B) In stage 16 embryos lacking both maternal and zygotic ßPS, PS2 failed to be transported to the surface of muscle cells and remained trapped in the endoplasmic reticulum (arrow). However, the plasma membrane expression of PS3 was retained in midgut endodermal cells (B'). (C) In the absence of both ß and zygotic ßPS, the PS3 subunit failed to be transported to the surface of endodermal cells (C').

View larger version (121K): [in a new window]   Fig. 8. Integrins are not required for other migration events. (A,B) Embryos were stained for the Vasa protein, which labels the migrating primordial germ cells (green). The mesoderm surrounding the germ cells was labelled with antibodies against talin (magenta). (A) Wild-type germ cells have migrated to their final position near the posterior of the embryo and have nearly coalesced at late stage 13. (B) Germ cells migrate properly and coalesce in embryos that lack the maternal and zygotic contributions of both ß subunits despite defects in germ-band retraction. (C) Migration of the border cells. Clones of cells lacking ßPS were generated by mitotic recombination in ß2 homozygous larvae. A recombination event yields two cells: one homozygous mutant and one homozygous wild type, which is marked with GFP. ßPS-;ß2 mutant border cells (lacking GFP, broken outline) do not lag behind or otherwise stray from wild-type border cells (GFP positive). Filamentous actin is shown in magenta, GFP in green.

View larger version (99K): [in a new window]   Fig. 9. Loss of ß does not enhance other ßPS mutant phenotypes. (A,B) Embryos lacking maternal and zygotic ßPS or both ß subunits were stained with anti-filamin 1 antibodies to label somatic muscles. The muscle phenotypes of single and double ß integrin mutants are identical. (C) Cuticles from embryos lacking ßPS or both ß subunits exhibit an equal frequency of the dorsal hole and tail-up phenotypes, owing to the failure of dorsal closure and germ band retraction, respectively.

View larger version (79K): [in a new window]   Fig. 10. Integrin adhesion is not required for cell proliferation or establishment of epithelial polarity. (A) ßPS- clones were induced by mitotic recombination in the wing disks of ß2/ß1 larvae. Non-GFP expressing cells lack ßPS and ß. Bright green cells are the progeny of the wild type sibling cell from the mitotic recombination. Double integrin mutant clones proliferate as well as their twin spots, as seen by their similar size and cell number. (B,C) ßPS- clones were induced in the follicular epithelium of ß2/ß1 female egg chambers. The apicolateral surfaces of follicle cells are labelled with antibodies against DE-cadherin, and mutant cells lack GFP. Large clones lacking GFP are generated, indicating that follicle cell clones lacking both ß integrin subunits are able to proliferate. (B,B') Surface view of an egg chamber. DE-cadherin is localized to the cell cortex in double ß integrin mutant follicle cells. (C,C') Longitudinal confocal section through an egg chamber. Eliminating integrins from follicle cells causes multi-layering and cell shape defects. However, in mutant cells that have not yet completely rounded up, DE-cadherin is properly localized to the apicolateral surfaces.

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