Academic journal article Genetics

Smurf Downregulates Echinoid in the Amnioserosa to Regulate Drosophila Dorsal Closure

Academic journal article Genetics

Smurf Downregulates Echinoid in the Amnioserosa to Regulate Drosophila Dorsal Closure

Article excerpt

ECHINOID (Ed) is a cell adhesion molecule that is an essential component of adherens junctions (AJs) to mediate cell adhesion/recognition in Drosophila (Wei et al. 2005). Ed participates in multiple developmental processes. For example, Ed negatively regulates the EGFR signaling pathway during eye development, but facilitates Notch signaling during adult sensory bristle patterning (Bai et al. 2001; Ahmed et al. 2003; Escudero et al. 2003; Rawlins et al. 2003a,b; Spencer and Cagan 2003). Moreover, Ed is also involved in the Hippo pathway to mediate organ size control (Yue et al. 2012).

Dorsal closure is a morphogenetic process that occurs from stage 12 to stage 15 of Drosophila embryogenesis; it involves the coordinated migration of two opposing epidermal cells over the underlying amnioserosa, with convergence at the dorsal midline (Young et al. 1993; Kiehart et al. 2000; Harden 2002; Jacinto et al. 2002). Amnioserosa cells are squamous epithelial cells, and exhibit pulsed contraction to gradually constrict the apical area (Solon et al. 2009). At the onset of dorsal closure at stage 12, the dorsal-most epidermal (DME) cells adopt a rectangular shape. Subsequently, at stage 13, DME cells elongate in the dorso-ventral direction and assemble a supracellular actomyosin cable to initiate epidermal cell migration (Young et al. 1993; Kiehart et al. 2000; Hutson et al. 2003). The dorsal movement of epidermal cells is driven by (1) pulsed contraction of amnioserosa cells pulling the flanking epidermal cells dorsally, and (2) the contractile actomyosin cable of DME cells acting as a ratchet to clamp the progressive contraction of the amnioserosa (Hutson et al. 2003; Franke et al. 2005; Solon et al. 2009). However, recent studies have argued that the actomyosin cable cannot drive dorsal closure (Ducuing and Vincent 2016; Pasakarnis et al. 2016). From stage 14 to stage 15, two flanking DME cells extend filopodia and zip together at the dorsal midline to complete dorsal closure. Ed is present in both epidermal cells and amnioserosa before the onset of dorsal closure (Laplante and Nilson 2011). The disappearance of Ed from the amnioserosa at stage 12 generates an asymmetric distribution of Ed that defines the epidermal leading edge (Lin et al. 2007; Laplante and Nilson 2011). This asymmetric Ed expression across the amnioserosa-epidermal cell boundary is required for DME cells to assemble a supracellular actomyosin cable and form a taut leading edge for coordinated cell migration (Lin et al. 2007; Laplante and Nilson 2011). However, the mechanism by which Ed is cleared from the amnioserosa at stage 12 remains unknown. In this study, we found that Ed transcription is repressed in the amnioserosa at stage 9. Subsequently, Smurf degrades the pre-existing Ed in the amnioserosa by the end of stage 12. In Smurf mutant embryos, Ed persisted in the amnioserosa, and actomyosin cable formation was compromised. Thus, both transcriptional and post-translational mechanisms regulate Ed clearance in amnioserosa.

Materials and Methods

Drosophila stocks and genetic crosses

The following stocks were used: Oregon R, c381-Gal4, en-Gal4, Ubi-p63E-shg-GFP, Nedd4GSV1(43350), SmurfGSV1(43483) (Bloomington Stock Center), Smurf5C (Podos et al. 2001), UASSmurfC1029A (Chang et aL 2013), UAS-Ed266-EGFP, UAS-Ed192-EGFP, UAS-Ed81-EGFP, UAS-Ed82-266-EGFP, UAS-Ed82-192-EGFP, UAS-EdPPPF-EGFP, and UAS-EdPPYF-EGFP (this study).

SmurfM/Z mutant embryos lacking both maternal and zygotic Smurf activities were derived from homozygous Smurf150 virgin females mated to Smurf150/Smurf males.

Plasmid construction

The PCR fragments encoding aa 1-1098, 1-1209, and 1-1283 of Ed together with EGFP were subcloned into the pUAST vector to generate UAS-Ed81-EGFP, UAS-Ed192-EGFP, and UAS-Ed266-EGFP, respectively. …

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