Cell shape changes during ventral furrow formation

Ventral furrow formation in the fly embryo directly affects an area of 60 cells along the A-P axis and 18-20 cells along the D-V axis (Sweeton, Parks et al. 1991). The cell shape changes that drive the invagination in the ventral furrow occur in a sequence of events that are divided into two phases: a first slow phase and a second rapid phase. In the first phase, the 12 most ventral cells, in the D-V axis along the length of the embryo, flatten their apical sides. Then the flattening expands to an 18-cell width. During flattening the cell membrane probably internalises since there are no blobbing and membrane ruffles visible (Sweeton, Parks et al. 1991). The cytoplasm above the nucleus decreases as the cells flatten. The nucleus then moves from the apical to the basal side of the cell, pushing the cytoplasm basally. This expands the basal side to give a ratio of basal to apical side of 1.6 +/- 0.2 (Kam, Minden et al. 1991). The ventro-lateral cells remain unconstricted till this point.

Ventral cells adopt a wedge-shape conformation by constricting their apical side. The apical cytoskeleton of actin and myosin are thought to play an important role in apical constriction (Parks and Wieschaus 1991). In the mid- ventral cells, the apical constrictions do not occur simultaneously in all cells, but individual cells constrict in a stochastic manner. It is more likely for the more ventral cells to initiate the constrictions. When up to 40% of the cells have constricted then the ventral furrow enters the second phase of events (Sweeton, Parks et al. 1991).

Apical flattening and nuclear movement are active processes that permit the subsequent cell shape changes and do not cause them. In Flightless I

mutant embryos that do not {he cells still flatten and their nuclei migrate basally (Leptin, Casal et al. 1992; Straub, Stella et al. 1996). In addition, the end of cellularisation is not the start signal for gastrulation, since the semi-formed ventral cells in mutant embryos that do not cellularise are able to flatten apically and move their nuclei basally (Leptin, Casal et al. 1992).

The second phase ventral furrow formation is a rapid phase, where the remaining 60% of the cells constrict very quickly and forms membrane blebs. An intermediate stage between 40 and 100% of constriction was not observed even when high numbers of embryos were fixed and analysed. During this stage all the cells are wedge-shaped and form a groove along the A-P axis. The furrow therefore contracts without substantially decreasing its length. As the apical diameter of the cell decreases the blebs continue to increase and the varying widths of cell apices observed indicate that constriction is a gradual process (Sweeton, Parks et al. 1991).

During apical constrictions, the cytoplasm is displaced and contributes to elongate the cells. These cells are reported to elongate from 17% of length/diameter of the embryo at the end of cellularisation to 29% of the egg diameter after the initiation of constrictions. After elongation has finished, the basal sides of the cells increase in surface area. In addition, the nuclei reach their final position 2/3 of the way from the apical surface. All the nuclei in the most ventral part of the embryo (12-cell diameter along the ventral-dorsal axis) have fully descended, by the time that the apical diameter has decreased by 50% (Sweeton, Parks et al. 1991). Nuclei migrate at 2.5 ^m/min, but not always continuously and they do not change orientation during migration (Kam, Minden et al. 1991). This nuclear migration to the basal side occurs approximately 6 minutes after the onset of gastrulation. When the nucleus moves to the basal side, the basal end is wider with a ratio of apical to basal surface area of 0.7 +/- 0.2 (Kam, Minden et al. 1991). The cells that are not in the constricted midventral region retain their nuclei 1/3 of the way from apical surface. Although the cells change their shape dramatically during this process, cell size differences are not observed (Leptin and Grünewald 1990). These cell shape changes lead to the transition from 1®^ to 2"^ phase of ventral furrow formation. The shallow furrow formed invaginates very rapidly, approximately 10 minutes

after the onset of gastrulation. This rapid internalisation is associated with the shortening of the constricting cells of the midventral region. The elongated ventral cells that occupied 29% of the embryo diameter alter to occupy only 15% after shortening. Generally, the cells change their shape from columnar to trapezoid to triangular (Sweeton, Parks et al. 1991).

The ventro-lateral cells behave in a different way to the ventral cells described above. They bend towards the ventral midline during the second phase, their basal surface remains in the original position and orientation, but a force possibly pulls the cells inwards (Sweeton, Parks et al. 1991). These cells have adopted the opposite shape from the midventral cells, where the apical side is expanded, the basal is constricted and their nuclei stay apically (Leptin and Grünewald 1990). After the second rapid phase the ventro-lateral cells move towards the furrow with a rate of 10pm/min and fold over the invaginated central population to form a tube (Fig. 1.17) (Kam, Minden et al. 1991). A possible explanation for this event is that the lateral cells are pulled because of mechanical constrains of the embryo's shape (Sweeton, Parks et al. 1991). The central midventral cells form the tube and the ventro-lateral peripheral cells form the stem of the tube (Leptin and Grünewald 1990).

The initiation of cell shape changes is an autonomous process, since wild type cells in mutant embryos lacking ventral genes are able to invaginate, which shows that the cell shape changes leading to an invagination are independent of the local neighbouring cells (Leptin and Roth 1994). In addition, the size and shape of the furrow depend on the size and shape of the area of cells expressing ventral genes and not on the shape of the embryo (Leptin and Roth 1994). This was demonstrated by injection of wild type cytoplasm in the dorsal side of a dorsalised embryo, which resulted in the formation of a furrow on the dorsal side along the D-V axis and not along the A-P as in wild type embryos (Leptin and Roth 1994). Individual cells therefore initiate the cell shape changes, but the final shape of the invagination depends on the environment in which furrow formation takes place (Leptin and Roth 1994).

In very brief summary the cell shape changes that drive the ventral furrow formation are apical flattening followed by constrictions, nuclear migration and membrane blebbing and when a significant amount of cells have constricted all

the remaining cells constrict very rapidly. The cells initially lengthen after the constrictions and then subsequently shorten with expansion of the basal side. The ventro-lateral cells also change their shape and bend towards the ventral furrow (Fig. 1.17) (Sweeton, Parks et al. 1991).

Figure 1.17: Ventral furrow formation in Drosophila. Cross sections o f successive stages of gastruiating embryos and ceil shape changes occurring at each stage, picture from Leptin 1999.