End-on imaging: a new perspective on dorsoventral development in Drosophila embryos

Abstract

Drosophila ventral furrow formation has frequently been used as a model to study developmentally-regulated cell-shape changes. However, a technique to follow all cellular changes during this process within a single living embryo has been lacking. We describe a novel technique, called "end-on imaging", to collect time-lapse images of transversely mounted living embryos. End-on imaging revealed several new features of dorsoventral development. First, we observed a wave of syncytial nuclear divisions predicting the location of the ventral furrow. Second, we determined that there is a 5-min gap between the end of cellularization and the start of ventral furrow formation, suggesting that the two processes may share the same pool of cytoskeletal components. Lastly, we show that apical-membrane flattening, the first step in ventral furrow formation, is due to the ventral cells pushing against the vitelline membrane, rather than flattening the dome-shaped, apical surfaces of these cells by a pulling or constriction motion.

Fig. 1

Diagram illustrating the end-on imaging setup. A prefabricated polyacrylamide gel holds the embryos on their posterior ends. This shows the setup for an inverted microscope, however the setup can be inverted for an upright microscope.

Fig. 2

Wave of syncytial nuclear divisions predicts VF location. A-L: Frames from a time-lapse movie of syncytial nuclear divisions in a nGFP-expressing embryo. Syncytial divisions occur below arrowheads in B-E, nuclei return from syncytial divisions below arrowheads in H-J, an arrow marks the pole cells in the amnioproctodeal invagination, and VF indicates the ventral furrow. A: Nuclei during interphase between syncytial divisions 12 and 13. B: Nuclei on the ventral surface are the first to enter syncytial division 13. C-F: The wave of nuclear divisions gradually progresses toward more lateral and dorsal nuclei. G: Dorsal nuclei are the last to divide. H-K: When mitosis is complete, the GFP is relocalized back to the nuclei. L: Ventral furrow formation occurs at the location to first enter mitosis. Images were collected at 23°C on a laser scanning confocal microscope with single photon light. These images are of a single optical section focused 50-60 μm in from the posterior end of the embryo using a 63× glycerin objective. Dorsal is up. Time is in minutes. Scale bar = 25 μm.

Fig. 3

Syncytial nuclear divisions in Toll^10B ventralized embryos do not predict VF location. A-H: Frames from a time-lapse movie of syncytial nuclear divisions in a nGFP-expressing embryo laid by a Toll^10B female. The presumptive dorsal is up. Syncytial divisions occur above arrowheads and an arrow marks the pole cells in the amnioproctodeal invagination. A-D: Ventral nuclei are the last to divide, with the other nuclei dividing mostly synchronously. E-G: Most nuclei return from nuclear divisions at the same time. H: The amnioproctodeal invagination formed where nuclei first divided. Images were collected at 23°C on a laser scanning confocal microscope with single photon light. These images are of a single optical section focused 50-60 μm in from the posterior end of the embryo using a 63× glycerin objective. Time is in minutes. Scale bar = 25 μm.

Fig. 4

Myosin II relocalizes between cellularization and VFF. A-O: Frames from a time-lapse movie of a Sqh-GFP-expressing embryo, shown in 1 minute intervals starting 15 minutes prior to VF sealing. Dorsal is up. Arrowheads mark the pole cells in the amnioproctodeal invagination, VF is in L-N. Scale bar = 25 μm. P. Kymograph of ventral cells, with 60 seconds between each frame. Notice that there are several minutes between myosin's disappearance basally and its reappearance apically, bracketed by the arrows. Images were collected at 23°C on a laser scanning confocal microscope with single photon light. These images are of a single optical section focused 60-70 μm in from the posterior end of the embryo using a 40× oil objective.

Fig. 5

Nuclear migration and apical flattening in wild-type VFF. A-O: Selected confocal images from an in vivo time series of embryo gastrulation, shown in 1 minute intervals at 23°C starting 15 minutes prior to VF sealing. Nuclei are green. Quantum dots (red) fill the intervitelline space. Dorsal is up. Arrows mark the intervitelline space, arrowheads mark the amnioproctodeal invagination and a white box outlines the ventral region in A-O. Scale bar = 25 μm. P. Kymograph of ventral cells, with 30 seconds between frames. The space between ventral cells and the vitelline membrane disappears immediately before VFF. Q. Kymograph of lateral cells, with 60 seconds between frames. There is little change in the amount of space between lateral cells and the vitelline membrane until the ventral furrow is invaginating. The first arrow marks the beginning of apical flattening and nuclear drop, and the second arrow marks the beginning of apical constriction and furrow formation in P and Q. Images were collected on a laser scanning confocal microscope with single photon light. These images are of a single optical section focused 60-70 μm in from the posterior end of the embryo using a 40× oil objective.