DC is a morphogenetic process that takes place at the end of Drosophila embryogenesis (Martinez-Arias, 1993) after germ band retraction (GBR) and coincident with head involution (HI) (VanHook and Letsou,2008, for review). At this developmental stage, embryos exhibit a large dorsal hole covered by an extra-embryonic epithelial tissue, the amnioserosa (AS), which lays over the yolk sac and is surrounded by two epithelial sheets (Fig. 4A). During DC, the hole closes by migration of the lateral epithelial sheets toward the dorsal midline and subsequent fusion or zippering (Fig. 4A). Simultaneously, the AS constricts apically and then disappears through basal extrusion of cells and activation of a cell death program, similar to mesenchymal contraction in a wound (Kiehart et al.,2000; Jacinto et al.,2002; Fernández et al.,2007; Gorfinkiel and Arias,2007; Toyama et al.,2008). Defects during DC cause clear phenotypes in embryos, which present a large hole in the dorsal epidermis (Fig. 4B). The use of time-lapse confocal microcopy techniques in transgenic embryos expressing a GFP-Moesin (GFP-Moe) fusion protein (Edwards et al.,1997) allowed the examination of cell and tissue dynamics during DC and revealed it to be a powerful model of epithelial morphogenesis, migration and resealing that recapitulates the process of wound healing (Harden,2002; Jacinto et al.,2002). The resemblance between DC and wound healing was also emphasized with live-imaging techniques to analyze DC and cell dynamics after mechanical perturbation in embryos (laser ablations or incisions) (Kiehart et al.,2000; Hutson et al.,2003; Stramer et al.,2005; Peralta et al.,2008; Rauzi et al.,2008; Rodriguez-Diaz et al.,2008; Toyama et al.,2008; Solon et al.,2009; Gorfinkiel et al.,2009; Gettings et al.,2010; Blanchard et al.,2010). These techniques have provided quantitative information on tissue movements during DC and have led to the development of biophysical models that use mathematical language to represent physical and kinematic properties of the tissues involved in DC (Gorfinkiel et al., 2011, for review).
DC requires coordinated cell shape changes within the AS and the lateral epidermis. At the LE of the two lateral epidermal sheets, both tissues contribute to the formation of an actomyosin cable (Young et al.,1993; Mizuno et al.,2002), similar to that previously observed in wounded chick embryos (Martin and Lewis,1992). It was proposed that the LE generates a contractile force oriented along the length of the cable and perpendicular to the direction of the closure (Hutson et al.,2003; Peralta et al.,2007) to coordinate the dorsal migration of the epithelial sheets toward the dorsal midline and to close the hole (Fig. 4C). This is known as the purse string model (Young et al.,1993). However, laser and genetic ablation experiments followed by physical modeling have suggested that other forces contribute to this process (Kiehart et al.,2000; Hutson et al.,2003; Peralta et al.,2007). Indeed, live-imaging studies using actin-GFP expressing embryos showed the formation of filopodia and lamellipodia structures beyond the LE of the epithelial sheets during DC (Jacinto et al.,2000; Wood et al.,2002; Jankovics and Brunner,2006; Gates et al.,2007; Liu et al.,2008). These structures recognize cells of the same anterior/posterior segment identity on each side of the hole, interdigitate and fuse by AJ formation (Fig. 4D) (Millard and Martin,2008). It has been shown that the fusion of epithelial sheets by actin projections starts first in the posterior and later in the anterior canthi through the formation of contacts modulated by E-Cadherin (E-Cad) and integrins (Danjo and Gipson,1998; Jacinto et al.,2000; Wood et al.,2002; Narasimha and Brown,2004; Fernández et al.,2007; Gorfinkiel and Arias,2007; Gorfinkiel et al.,2009). On the other hand, some evidence suggests that the AS plays an essential role during DC. First, it was demonstrated that reduction of the AS surface area is required to generate tension in epithelial sheets (Kiehart et al.,2000). Second, it was shown that AS cells in contact with the lateral epidermis also contain purse string-like structures (Wada et al.,2007). Finally, other experiments showed that removal of the entire AS by genetic ablation produced DC defects caused by disruption of mechanical and molecular signals (Scuderi and Letsou,2005). Taken together, these results suggested that the AS plays structural and signaling roles during DC. Subsequent studies have demonstrated that apical constriction of AS cells is an active process driven by Myosin II (Myo II) (Franke et al.,2005) and that a secreted signal which diffuses from the dorsal-most epidermal (DME) cells to the AS (the morphogen Decapentaplegic (Dpp), a member of the transforming growth factor-β (TGF-β) family) coordinates AS cell contraction and adhesion between AS and epidermal cells (Fernández et al.,2007). Moreover, it has been recently shown that AS cells have the intrinsic capability to pulse during DC through constant contracting and relaxing forces along their surfaces (Fig. 4E) (Solon et al.,2009; Gorfinkiel et al.,2009; David et al.,2010; Blanchard et al.,2010). The pulsed forces created by AS cells are sufficient to transiently displace the flanking epidermal cells dorsally (Solon et al.,2009). Then the epidermal actomyosin cable acts like a cellular ratchet that prevents the relaxation of AS cells during their periodic cycles of contraction and expansion (Solon et al.,2009). However, in the absence of a fully functional actomyosin cable the AS can contract, suggesting that AS cells have the intrinsic capability to contract (Laplante and Nilson,2006; Gorfinkiel et al.,2009). Although the AS is an important tissue during DC, it is not yet clear whether there is an equivalent structure in wound healing. However, it has been suggested that granulation tissue, which is composed of fibroblasts and myofibroblasts, produces contractile movements in wounds to contribute to zippering (Martin and Parkhurst,2004).
DC is primarily regulated by the JNK and Wnt/Wg pathways, similar to wound healing and regeneration in vertebrates, although other factors are involved. Therefore, DC has contributed to the discovery of molecular mechanisms underlying key processes in vertebrates. The JNK pathway is activated at the LE and in AS cells at the end of GBR and downregulated in the AS and maintained at the LE during DC, where it induces the expression of at least two genes: dpp and puckered (puc) (Fig. 3A) (Noselli,1998, for review; Noselli and Agnès,1999; Glise and Noselli,1997; Knust,1997; Martin-Blanco, 1997; Reed et al.,2001; Stronach and Perrimon,2001). It is required for AS cell contraction (Stronach and Perrimon,2001; Fernández et al.,2007) and during actin stress fiber formation, actomyosin cable assembly and filopodia polymerization (Kiehart et al.,2000; Jacinto et al.,2000,2002), formation of focal adhesion complexes in DME cells to regulate adhesion and possibly signal transduction (Ricos et al.,1999; Reed et al.,2001; Kaltschmidt et al.,2002; Homsy et al.,2006), cell-shape changes and ventral epithelial elongation (Fig. 3C) (Glise et al.,1995; Riesgo-Escovar et al.,1996; Sluss et al.,1996; Hou et al.,1997; Kockel et al.,1997; Riesgo-Escovar and Hafen,1997a,b; Zeitleinger et al., 1997; Ricos et al.,1999). Moreover, the JNK cascade has been recently shown to be involved in reprogramming and intercalation of cells at the LE, which helps to relax tissue tension (Gettings et al.,2010). Different assays have been performed to identify new JNK targets during Drosophila DC (Jasper et al.,2001; Thomas et al.,2009). Some of these genes encode cytoskeleton regulators such as Chickadee (Chic) involved in actin polymerization in the LE, cell adhesion molecules (like Myospheroid and RalA), proteins involved in ecdysone response (such as ImpE1 and ImpL1), the trypsin-like protease Scarface (Scaf) that negatively regulates JNK activity in the epidermis and the small GTPase Rab 30, which is involved in DC, HI and thorax fusion (see below) through the regulation of intracellular transport of vesicles in the LE (Thomas et al.,2009; Rousset et al.,2010). Other genes encoding JNK regulators or effectors involved in DC are: raw, which is expressed in the AS and influences JNK activity in the LE (Byars et al.,1999), and cabut (cbt) that encodes a transcription factor expressed in the yolk sac, AS, and lateral epidermis, and functions downstream of JNK signaling (Muñoz-Descalzo et al.,2005; Belacortu et al.,2011). Regarding Wnt/Wg signaling, it is required for normal elongation of DME cells, actomyosin cable formation (Kaltschmidt et al.,2002; Morel and Arias,2004), and promotes expression of target genes in the DME cells in collaboration with JNK signaling (McEwen et al.,2000). Arm also contributes to the DC process in a Wg-independent manner through its role in AJ formation (see below) (McEwen et al.,2000). In addition, it has been shown that N is involved in DC through its ICD, specifically regulating patterning of the dorsal epidermis during embryogenesis in a Su(H)-independent manner. Indeed, reduction of N function overactivates the JNK pathway during DC and can rescue bsk and hep mutant phenotypes, thus suggesting that N activity can repress JNK signaling (Zecchini et al.,1999). Cell junctions also contribute to cell shape changes and to transmit the mechanical forces generated during DC. Therefore, different AJ components, such as Canoe (Cno), ZO-1, E-Cad and Arm have been shown to be involved in DC (Brock et al.,1996; Takahashi et al.,1998; McEwen et al.,2000; Tepass et al.,2001; Harden,2002; Gorfinkiel and Arias,2007). Similarly, components of septate junctions (SJ), such as Coracle (Cora), Neurexin IV (NrxIV), Disc large (Dlg), Scrib and Lethal giant larvae (Lgl), are also involved in this process (Lamb et al.,1998; Perrimon,1988; Bilder and Perrimon,2000; Arquier et al.,2001). Proteins involved in intracellular trafficking by vesicles and recycling of AJ components, such as Rab 5, Rab 11 and Rab 30 are also involved in DC, HI and thorax fusion (see below) (Roeth et al.,2009; Sasikumar and Roy,2009; Thomas et al.,2009; Mateus et al.,2011). Other factors such as miRNAs of the 310 family (310/311/12/313/92) have a role in DC, as their silencing causes DC and HI defects in Drosophila embryos (Leaman et al.,2005). Moreover, several lines of evidence support a relationship between ecdysteroid hormones and DC (Fernández et al., 1995; Lamka and Lipshitz,1999; Chávez et al.,2000; Kozlova and Thummel,2003; Petryk et al.,2003; Chavoshi et al.,2010). First, one of the 20-hydroxy-ecdysone (20E) hormone peaks in the Drosophila life cycle occurs during DC. Second, embryos expressing a dominant negative form of the 20E receptor (EcR-DN) and embryos homozygous for the thermosensitive ecdysoneless1 (ecd1) allele, which synthesize low quantities of the hormone, present GBR, CD, and HI defects (Fernández et al., 1995; Lamka and Lipshitz,1999; Kozlova and Thummel,2003; Chavoshi et al.,2010). Moreover, loss of function of genes such as disembodied (dib) and shade (shd) that encode P450 enzymes involved in embryonic ecdysone biosynthesis present DC defects (Chávez et al.,2000; Petryk et al.,2003). In addition, early hormone response genes such as ImpE1 and ImpL1 have been identified as JNK targets during DC (see above) (Jasper et al.,2001). Finally, we have demonstrated in our laboratory that the Cbt transcription factor is probably regulated by ecdysone signaling during DC (Y.B. and N.P., unpublished results), as it occurs during metamorphosis (Beckstead et al.,2005). However, more experiments are necessary to unravel the exact role of ecdysteroids during DC.