The shaping of the embryo during development is driven by large-scale movements of epithelial cell sheets as they bend, spread, and fuse with each other. One such major, well-defined morphogenetic event is dorsal closure, which occurs during the early stages of Drosophila embryogenesis. It involves the closing of an elliptical gap at the dorsal midline of the embryo by the convergence and fusion of epithelial cell sheets on either side of the embryo. The dorsal gap is underlined by the amnioserosal tissue that is made up of squamous epithelial cells and the lateral epithelial sheets contract and pull over the amnioserosal cells in a mechanism that closely resembles wound healing .
In live fly embryos, dorsal closure is observed to commence as soon as germ-band retraction starts. Germ-band retraction is the preceding event in Drosophila development in which, a germ-band that extended from the ventral and posterior side of the embryo to its dorsal side during the gastrulation stage, now retracts back to the posterior end of the embryo. This exposes the dorsal amnioserosa, which causes the anterior-most lateral epithelial leading edges to move in and seal regions of the dorsal gap. Following this, closure continues progressively from the anterior and posterior ends towards the center of the amnioserosa. The precise sequence of cellular events that take place during dorsal closure can be described in four stages, which are initiation, epithelial sweeping, zippering, and termination .
Initiation: Although the exact cellular events that occur during the onset of dorsal closure are not well known, a combination of signals from preceding morphogenetic events such as dorsoventral patterning and germ-band retraction, are known to trigger the initiating steps by activating the Jun N-terminal Kinase (JNK) signaling pathway. During the initial stages of dorsal closure, JNK activity is upregulated in the leading edge cells of the lateral epithelia while it is down regulated in the amnioserosal cells . At this stage, there is minimal internal tension in the lateral epithelial cells as they do not yet contain any actin-based contractile structures and the cells assume an irregular, scalloped shape. The leading edge is not clearly defined and it advances very slowly, with no net progression over the amnioserosa. However, the amnioserosal cells undergo active pulsatile contractions, which leads to a significant reduction in their apical surface area.
Epithelial sweeping: A short time after the initiation of dorsal closure, there is an accumulation of actin filaments at the leading edge of each cell of the epithelial cell sheet. The actin filaments organize into contractile units and these individual units are linked through intercellular junctions to form a continuous, supracellular acto-myosin cable that contracts and generates intrinsic tension . The leading edge cells stiffen under the influence of this tension and change their appearance from a scalloped shape to a tightly organized, regular row of cells. They also become polarized as they elongate along the dorsoventral axis and start advancing over the amnioserosa in sweeping movements. In conjunction with amnioserosal cell contractions, the sweeping movement of the lateral epithelia causes the dorsal gap to shrink in size significantly during this stage.
Zippering: Towards the end of the epithelial sweeping stage, the leading edge starts to put forth numerous filopodial protrusions, as well as a few lamellipodial protrusions. These actin-rich membranous protrusions facilitates establishment of contacts with the opposing leading edge, as soon as the leading edges from the two lateral epithelial sheets come close enough at the anterior and posterior ends of the gap. Once the protrusions touch one another, they seal together the remaining gap in a zippering mechanism to form a tight seam at the midline. In addition to the zippering function, the filopodia also play a key role as specialized sensors for pairing up the correct embryonic segments from both sides of the embryo. This ensures that the free lateral edges fuse in a coordinated manner and the general patterning within the epithelium is conserved. In mutant flies lacking Cdc42, a major activator of the assembly of actin-based protrusions, the opposing cells on the lateral edges failed to fuse properly and form a tight seam at the midline . Although zippering forces dominate at this stage, the contractility of the actin cable and the amnioserosal cells are still contributing to the final stages of dorsal closure .
Termination: Upon the establishment of contacts between the opposing leading edges via filopodial protrusions, certain ‘stop’ signals are activated that lead to the disassembly of actin-based structures and prevent further advancement of the leading edges. These signals also activate biochemical pathways that are essential for the reinforcement of the initial, temporary contacts between epithelial cells into permanent, tight adherens junctions. This mechanism recapitulates contact inhibition, and has been used as an ideal platform to gain insights into this fundamental biological process and in particular, its relevance in cancer progression . With the formation of tightly sealing adherens junctions, the process of dorsal gap closure is considered to be completed.
Mechanical control of dorsal closure. In addition to protrusive forces that favor the forward movement of the leading edge, there also exist counter-acting or opposing forces that are generated by the active spreading of the bulk of the lateral epidermis. The balancing effects of these various forces acting at the interface of the leading edge and the amnioserosal tissue enable the complete closure of the dorsal gap .
Within a short time following the onset of dorsal closure, intrinsic tension is generated in the leading edge of the lateral epithelia by the assembly of a supracellular actin cable that contracts like a purse-string . These contractile forces provide structural integrity to the apical surfaces of the leading edge cells and promote their advancement over the amnioserosal cells. Concomitant with the purse-string contraction of the lateral epithelium, the amnioserosal cells also undergo non-synchronous, pulsed contractions at their apical surfaces. Such contractions introduce shape changes into these flat, squamous epithelial cells, as they push the bulk of their cytoplasm into the embryo . This causes a reduction in the surface area of the extra-embryonic amnioserosal tissue and a progressive narrowing down of the gap at the midline. The non-muscle myosin protein, MyoII, plays an essential role in generating contractile forces in both these cell types. While MyoII forms the contractile purse-string in the leading edge cells, it occurs at much lower concentrations in the cortex of both amnioserosal as well as lateral epidermal cells. In mutant Drosophila embryos that lack MyoII heavy chains, the process of closure is completely abolished .
Ratchet-like mechanism driving dorsal closure
Recent studies analyzing force distribution within cells during dorsal closure have put forth a ratcheting mechanism that coordinates the contractions within the lateral epithelial cells and the amnioserosal cells, that drives the forward movement of the leading edge. Real-time fluorescence imaging used to track the position of a fluorescently-labeled apical cell circumference marker has revealed that individual amnioserosal cells start contracting in a pulsatile manner before the onset of dorsal closure. During this stage, the leading edge cells also undergo oscillatory movements, as they get pulled dorsally and then retract back ventrally, synchronous with the pulsatile contractions of the first row of amnioserosal cells immediately surrounding the gap. However, the initiation of dorsal closure is marked by the formation of an actin cable around the apical surfaces of the leading edge cells and the purse-string contractile activity of the cable functions like a ratchet that prevents the ventral relaxation of the leading edge cells, resulting in their net dorsal displacement. As the pulsatile contractions in the first row of amnioserosal cells gets dampened soon after, the ratcheting mechanism acts on the next row of amnioserosal cells and the dorsal movement of the leading edge sequentially progresses towards the center of the gap .
The relative significance of each of these independent forces in driving dorsal closure has been thoroughly investigated using laser ablation studies, in which either the actin cable or the amnioserosa are repeatedly laser-cut at various sites in order to release tension from these tissues. Although the mechanical integrity of each of these tissues seemed to be disrupted due to multiple cuts, dorsal closure progressed to completion in both cases, suggesting that neither of the two forces (purse-string vs. amnioserosal contractions) are solely responsible for mediating dorsal closure. However, when both of these tissues were ablated at the same time, dorsal closure was completely abolished. More interestingly, ablations to the lateral epidermis caused the leading edge to rapidly advance over the amnioserosa, as it released the counter-acting forces from the bulk of the lateral epidermis. Such findings reinforce the existence of cellular mechanisms that regulate the magnitude of individual forces and ensure that the closure of the gap progresses normally .