Gastrulation is composed of several morphogenetic events that require coordinated cell shape changes to generate the embryonic germ layers. The first morphogenetic event during Drosophila
gastrulation is ventral furrow formation (VFF) where a swath of columnar epithelial cells become wedge-shaped and drive the invagination that produces the mesoderm (Leptin and Grunewald, 1990
; Kam et al., 1991
; Sweeton et al., 1991
). Genetic analysis of VFF has revealed many components of the signaling pathway responsible for the initiation of the shape changes (Thisse et al., 1988
; Moussian and Roth, 2005
; Padash Barmchi et al., 2005
). VFF cell-shape changes can be broken down into four morphogenetic components: (1) apical-membrane flattening, (2) apical-to-basal nuclear migration, (3) apical constriction and (4) apical-to-basal cell shortening (Leptin and Grunewald, 1990
). These highly dynamic cell-shape changes take place primarily along the apicobasal cell axis.
Two of the most widely used methods to analyze cell-shape changes during VFF include fixed preparations of transversely sectioned embryos (Leptin and Grunewald, 1990
; Dawes-Hoang et al., 2005
), and three-dimensional time-lapse microscopy (Kam et al., 1991
; Oda and Tsukita, 2001
; Padash Barmchi et al., 2005
). These approaches have been indispensable for characterizing the roles of the signaling molecules responsible for initiating VFF and describing the associated cell-shape changes. While transversely sectioned embryos provide adequate resolution to study these apicobasal cell-shape changes, the temporal sequence of events of this dynamic process must be inferred from numerous different embryos. Time-lapse imaging of ventrally-mounted embryos provides precise temporal information, but gathering sufficient apicobasal image data at high resolution by optical sectioning is limited by the axial resolution of the microscope and the imaging rate, which is slow relative to the rate of VFF.
To understand the connection between the signaling events and the mechanical mediators that drive VFF cell-shape changes, we developed a technique to capture time-lapse images of transversely mounted embryos. We refer to this new technique as “end-on imaging” because the embryos are imaged through their posterior ends. With end-on imaging, all apicobasal cell-shape changes along the dorsoventral axis can be imaged simultaneously. This technique provides the optimal perspective to obtain the information needed to fill the gaps in our understanding of how the signaling molecules that determine a cell's fate impinge on the cytoskeletal regulators that change the cell's shape.
In this report, we show that end-on imaging provides new insights into the events leading up to the invagination of the ventral furrow. First, a wave of syncytial nuclear divisions that predicts the location of the ventral furrow was observed using embryos that express nuclear-localized GFP (nGFP). Using GFP-tagged myosin II regulatory light chain (Spaghetti squash-GFP, Sqh-GFP), we measured the interval between the disappearance of myosin from the basal surfaces of cells completing cellularization and its reappearance at the apical surfaces of ventral cells. The duration of this interval indicates the same pool of myosin may be used for both processes. Lastly, using intervitelline injection of quantum dots, we show a decrease in the space between the apical surfaces of the ventral cells and the vitelline membrane during apical flattening, suggesting new models for the mechanism underlying this first step in VFF.