Quantitative Data

Basic quantitative data, such as border cell migration duration, rate and time, was extracted from live imaging time-lapse movies using ImageJ, a public domain, Java-based image

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Figure 4.1. Shows the characterised stages of Border cell migration in slbo GAL4, UAS mCD8-GFP egg chambers. Images shown are maximum intensity projections of approximately 8-10 optical sections. White arrows indicate protrusions or rounded shape. (A) The first half of migration, characterised by an elongated, polarised cluster. (B) The second half of migration, characterised by a more rounded appearance. The solid white line indicates 15 µm scale bar.

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processing program developed at the National Institutes of Health. Some phenotypes displayed abnormal qualitative behaviour, such as early tumbling in the first half of migration, which were characterised as a percentage of the number of movies or average number of frames per movie (see Chapter 5, Section 5.3.2.).

Duration

The duration of border cell migration was determined by simply counting the number of time-lapse frames it took for the cluster to reach the oocyte, multiplying by the time interval between frames. It was important that the start point and end point were the same in all egg chambers. It was found that the most accurate way to do this was to set guidelines where border cell migration starts and ends. In this thesis, the start point was defined by the point at which the cluster had detached from the epithelium, checking all optical sections through a Z stack of time-lapse images. The end point was defined as the point at which the edge of the cluster had made contact with the oocyte-nurse cell boundary. However, it is important to note that not all egg chambers started and ended in the same way, either as a result of the variations in genotype or simply due to their heterogeneous nature. For instance, during the initial polarised phase of migration some border cell clusters remained attached to the epithelium, causing the cluster to form an elongated shape towards the oocyte. In this case, a decision was made to either exclude the egg chamber from analysis, as no clear start point could be defined, or include the egg chamber and risk a skew in the migration averages. These decisions were guided by examination of other egg chambers of the same genotype to determine what was the normal behaviour for that genotype. If using a genotype already published the findings were compared to the literature. If the egg chamber in question was included in the analysis, a pseudo-start point was designated, which represented the start point in all movies, to ensure all egg chambers were treated

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Direction of Migration

similarly. Figure 4.2 below shows an example of an egg chamber where the cluster has started to migrate but failed to fully detach. Where only a single egg chamber was affected, these data were removed from the analysis to avoid error.

On occasions where the end point of migration was not clear, usually due to the slow tumbling nature of the second half of migration, steps were taken to carefully define an end point. Dorsal migration of the cluster occurs at the end of anterior posterior migration, once the cluster has reached the oocyte (see Introduction 1.5.3.). Frames were not counted once forward migration had stopped and dorsal migration started. Having strict guidelines to adhere to when analysing qualitative elements ensured the data extracted was consistent and accurate throughout.

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Figure 4.2. Shows slbo GAL4, UAS mCD8-GFP egg chambers where there is a delay in the full detachment of the Border cell cluster from the surrounding epithelia. Images are GFP maximum intensity projections of 8-10 Z stacks, taken from the time-lapse imaging of a single egg chamber. White arrows indicate the clusters connection to the epithelium. The pseudo-start point would be positioned at 15 minutes, as seen in the first image, irrespective that it is still attached to the epithelium. This enables a quantitative start point to be categorised. Solid white line indicates 15 µm scale bar.

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In addition to determining the duration of complete migration it was useful to compare the duration of the first half and the second half of migration. To do this, a midway point through migration needed to be determined. Unfortunately, although there are molecular changes in border cells that reflect the changes in behaviour of the border cell cluster during the migration process, no markers exist to easily discriminate between early and late

phases. In the second half of migration there is a gradual transition from PVR to EGFR guidance signalling, which is thought to be responsible for the characteristic change in behaviour of the border cells during the second half of migration, as described above. Using a dominant negative version of the receptor responsible for first half of migration brings about second half behaviour prematurely (Poukkula et al., 2011). Due to the natural heterogeneity of border cell migration, this transition, which is in part determined by the concentration gradient of the ligands (PDGF and EGF) along the egg chamber, does not occur at the same distance throughout migration and can be dependent on the genotype. Using the cluster behaviour to guide when the first half ends and the second half begins was not an option, as the migration behaviour may be affected by the mutants under

investigation. Since egg chambers are generally similar in size irrespective of their border cell genotype, it is reasonable to assume that the distance the cluster needs to travel from start to end point is the same. The length from start to finish consequently is a constant that can be used to determine a specific distance travelled during migration. Using projected images of the egg chamber, the distance from start (the edge of the anterior epithelium) to finish (nurse cell/oocyte boundary) along a straight line was measured (XY measurement), see Figure. 4.3.

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The projected image was found to give a good representation of the entire cluster and enabled a centre point to be determined. Using this approach, the midway point was

determined and the first and second parts identified. The duration of each of the respective parts were calculated when the centre of the cluster reached that point.

Figure 4.3. Shows schematic diagrams of egg chambers to highlight the XY measurements to determine different stages of migration, start and finish. Border cell cluster labelled in green. (A)

Egg chamber with cluster still attached to the epithelium (Early stage 9) where the measurement starts. (B) Egg chamber with cluster at the end point (Stage 10a) where the measurement from the epithelium ends. (C) Egg chamber defining the first half of migration, with the area highlighted in grey. (D) Egg chamber defining the second half of migration, with the area highlighted in grey.

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Rate

The rate of migration was calculated based on basic measurements taken from the time- lapse movies. These rates were either calculated over the complete migration process or specifically in one half. Migration rate was calculated by dividing the distance the cluster had travelled by the time it had taken, averaged over a number of individual movies. Migration rate differences may indicate problems during migration contributing to

abnormal migration behaviour e.g. the second half of migration may take less time than the first as the cluster is migrating at a quicker rate. However, measuring rates of migration 'as the crow flies' (ACF) underestimates the actual distance travelled. By monitoring a central point of the cluster over time it was clear that clusters did not migrate in a straight line.