Automated Image Analysis Reveals Spatially-Regulated Cell Division Dynamics during Drosophila Axis Elongation

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Axis elongation is a conserved process in which embryos extend their head-to-tail or anterior-posterior (AP) axis. Defective axis elongation results in developmental malformations, including spina bifida and anencephaly. In Drosophila, axis elongation occurs in an epithelial monolayer known as the germband. Cells on the anterior end of the germband divide, but the contribution of anterior cell divisions to axis elongation is unknown. We used spinning disk confocal microscopy to investigate anterior cell divisions during germband extension in Drosophila embryos expressing fluorescently-tagged proteins that label cell outlines. To automate the quantification of cell division dynamics, we developed an image analysis algorithm to segment and track cells from confocal microscopy sequences. One seed point was identified per cell in the first image of the sequence using an adaptive threshold. Seeds were interactively edited when necessary. The watershed algorithm was used to expand seeds until they delineated cells. Seeds were automatically transferred to the next image in the sequence, correcting for cell movement by using the local cross-correlation maximum between consecutive images to shift the seeds. Dividing cells were automatically detected based on their increased area and circularity, and final dumbbell morphology. Seeds corresponding to dividing cells were split into two equidistant seeds along the longest cell axis. We found that 84.3±4.0% of anterior cells immediately adjacent to the ventral midline (ventral cells) divided within 45° of the AP axis. In contrast, anterior cells away from the midline (lateral cells) divided at random orientations. Cell shape analysis revealed that ventral cells rotated 32.5±3.0° before cell division to align their longest axis with the AP axis, while lateral cells did not undergo a similar rotation. Together, our data strongly suggest that the mechanisms that promote oriented cell division are spatially regulated.