Dynamic analysis of filopodial interactions during the zippering phase of Drosophila dorsal closure - PubMed (original) (raw)

Dynamic analysis of filopodial interactions during the zippering phase of Drosophila dorsal closure

Thomas H Millard et al. Development. 2008 Feb.

Abstract

Dorsal closure is a paradigm epithelial fusion episode that occurs late in Drosophila embryogenesis and leads to sealing of a midline hole by bonding of two opposing epithelial sheets. The leading edge epithelial cells express filopodia and fusion is dependent on interdigitation of these filopodia to prime formation of adhesions. Since the opposing epithelia are molecularly patterned there must exist some mechanism for accurately aligning the two sheets across this fusion seam. To address this, we generated a fly in which RFP-Moesin and GFP-Moesin are expressed in mutually exclusive stripes within each segment using the engrailed and patched promoters. We observe mutually exclusive interactions between the filopodia of engrailed and patched cells. Interactions between filopodia from matching cells leads to formation of tethers between them, and these tethers can pull misaligned epithelial sheets into alignment. Filopodial matching also occurs during repair of laser wounds in the ventral epithelium, and so this behaviour is not restricted to leading edge cells during dorsal closure. Finally, we characterise the behaviour of a patched-expressing cell that we observe within the engrailed region of segments A1-A5, and provide evidence that this cell contributes to cell matching.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Expression pattern of _en_-RFP-moesin and _ptc_-GFP-moesin in the dorsal epithelium

A. Schematic illustrating construction of the fly line, transgene expression pattern and dorsal trichome pattern. B,C,F. Images of embryos expressing _en_-RFP-moesin (red) and _ptc_-GFP-moesin (green). B. Dorsal view of embryos at the start (i), midway through (ii), and shortly after completion of DC (iii). C. Time course of the latter stages of DC in a live embryo. Misplaced _ptc_-GFP-moesin expressing cells (indicated by asterisks in Ci) are present at a conserved position at the leading edge of the en domains of all segments shown. D. Graph showing proportion of embryos with misplaced _ptc_-GFP-moesin expressing cells in each segment. E. Dorsal view of stage 16 embryo expressing GFP-moesin (green) constitutively and RFP-moesin (red) under control of _en_-Gal4. Pairs of cells in P compartment not expressing RFP-moesin are indicated by arrowheads. F. Images from movie S1 showing cell rearrangements during DC which lead to the presence of the misplaced ptc cell (indicated by asterisk) in the en domain. Scale bars 10 μm.

Figure 2

Figure 2. Cell matching and realignment during DC is mediated by filopodia

Zippering in _en_-RFP-moesin (red), _ptc_-GFP-moesin (green) expressing embryos. A. Images from movie S2 showing filopodial matching. i. Red and green filopodia protrude from leading edge cells. ii. Contacts are made between red filopodia from opposing epithelia, while at the same time separate contacts are made between green filopodia. iii. Further contacts are made between red filopodia; however, green filopodia in close proximity to these red filopodial contacts do not interact. iii. Green filopodia transiently form contacts between _ptc_-GFP-moesin cells over the top of the fused red cells. B. Images from movie S3 showing realignment of misaligned epithelial sheets by filopodial searching and pulling. i. The two epithelial sheets are initially poorly aligned. A filopodial tether transiently exists between green cells but later breaks. ii. Contacts form between filopodia from _en_-RFP-moesin expressing cells**. iii-iv.** Green filopodia in the lower sheet do not interact with nearby red filopodia and ultimately form contacts with green cells some distance away in the opposing sheet. v. The tethers that result from the contacts made by red and green filopodia pull the sheets into alignment. Panels labelled ′ and ″ show _en_-RFP-moesin and _ptc_-GFP-moesin channels respectively in isolation. The described filopodial interactions are indicated by arrowheads. Scale bars 10 μm.

Figure 3

Figure 3. Matching in embryos with asymmetries between the opposing epithelial sheets

DC zippering in _en_-RFP-moesin (red), _ptc_-GFP-moesin (green) embryos with asymmetries. A. Images from movie S4 showing zippering in an embryo with a spontaneous asymmetry. i. The upper epithelial sheet has two rather than one misplaced ptc cell in the en domain and one of these has associated with the A compartment of the lower sheet blocking matching of en cells. ii. As a result, the blocked en cells make filopodial contacts with en cells of the neighbouring segment. iii. These develop into permanent contacts. iv. Cells which are not associated with matching partners continue to produce filopodia. B. Zippering in an embryo with a laser-induced asymmetry. i. An en stripe has been removed from the leading edge of the lower epithelial sheet by laser ablation (indicated by asterisk) while the opposing en stripe (indicated by arrow) is normal. ii-iii. On contact with the opposing leading edge, the unpartnered en stripe constricts and then withdraws completely from the leading edge. Panels labelled ′ and ″ show _en_-RFP-moesin and _ptc_-GFP-moesin channels respectively in isolation. Scale bars 10 μm.

Figure 4

Figure 4. Filopodial matching occurs during healing of a wound in the ventral epithelium

Images from movie S5 showing healing of a wound across an en stripe in the epithelium of an _en_-RFP-moesin (red), _ptc_-GFP-moesin (green) embryo. A. Filopodia are produced by wound edge cells. B,C. Interactions occur between _ptc_-GFP-moesin cells across the wound. D,E. Eventually a filopodial tether forms between _en_-RFP-moesin cells on either side of the wound and this tether rapidly leads to fusion of the two en cells. Notably, the last part of the wound to heal is at the junction between en and ptc cells. The described filopodial interactions are indicated by arrowheads. Scale bar 10 μm.

Similar articles

Cited by

References

    1. Bokor P, DiNardo S. The roles of hedgehog, wingless and lines in patterning the dorsal epidermis in Drosophila. Development. 1996;122:1083–92. - PubMed
    1. Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118:401–15. - PubMed
    1. Dahmann C, Basler K. Opposing transcriptional outputs of Hedgehog signaling and engrailed control compartmental cell sorting at the Drosophila A/P boundary. Cell. 2000;100:411–22. - PubMed
    1. Edwards KA, Demsky M, Montague RA, Weymouth N, Kiehart DP. GFP-moesin illuminates actin cytoskeleton dynamics in living tissue and demonstrates cell shape changes during morphogenesis in Drosophila. Dev Biol. 1997;191:103–17. - PubMed
    1. Gates J, Mahaffey JP, Rogers SL, Emerson M, Rogers EM, Sottile SL, Van Vactor D, Gertler FB, Peifer M. Enabled plays key roles in embryonic epithelial morphogenesis in Drosophila. Development. 2007;134:2027–39. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources