- MATLAB (versions 2019b, 2021a, and 2022a were used in creating codes)
- ilastik (versions 1.3.0 and 1.3.2 were used for training)
- FIJI (for image pre-processing)
- the following MATLAB open-source programs: munkres.m, some version of imagesc3D.m
Clone the repository:
git clone https://github.com/Shvartsman-Lab/MaleReconstruction.git
uiopen is a slightly edited version of the MATLAB file that is needed for direct reading of the .h5 files output from ilastik
orb_fusome_select is the main file for reading, reconstructing, assigning proper identity, and measuring the volumes of all fusome pieces. This script runs split_fusome, allowing for the split channels containing information about fusome and ring canal positional information to be used to split the fusome into its component pieces. The script then takes these objects and reassigns their labels to match 16-cell clusters and then measures the volumes of each piece for further analysis. The final portion of this script makes a 3D reconstruction of the input data with color labels for each fusome piece, matching the colors of each cell in the network as in previous work (Imran Alsous et al., Current Biology, 2017). These reconstructions are then overlaid with orb smFISH data, and the correlation between the amount of orb and the amount of fusome between the two central cells of the cyst can then be calculated.
split_fusome uses the input data of the fusome and ring system to recursively split the fusome into its component pieces. In short, the function cuts the fusome piece at each loop at the location of a chosen ring. These pieces are then reformed into 2 separate parts, the ring is removed from the selection pool, and then the function repeats itself on one of the two split fusome portions. This repeats until there are no rings remaining for a given piece of fusome. From here, the function jumps to another branch of the recursive loop until no rings remain. The output of this function for n rings are n+1 objects designating the n+1 fusome pieces.
orb_fusome_plots shows plots of Shot data for the distribution of relative fusome mvolume fractions in each cell across the collected measurements, as well as the correlation between orb fraction and fusome volume fraction between the two central cells, as shown in Figure 10C.