Fig 1.
Fusome formation, structure, and patterns of division.
A) Schematic of cyst formation during oogenesis and spermatogenesis in Drosophila melanogaster. A founder cell gives rise to a germline cyst whose 16 cells are connected through ICBs or ring canals. During oogenesis, one cell becomes the oocyte while the remaining cells serve a supporting role. During spermatogenesis, two subsequent meiotic divisions with incomplete cytokinesis generate 64 haploid spermatids. In both sexes, germline cysts contain a fusome, which permeates the cysts through the ring canals. B) Schematic of the four rounds of synchronous cell divisions that give rise to the female germline cyst, comprising 16 cells (nodes) connected by 15 ring canals (edges). The fusome (cyan) derives from the spectrosome and grows with each cell division; association of the fusome with one of the two mitotic centrosomes orients the planes of cell division, ensuring the stereotypical and maximally branched pattern in female cysts. C) Projection of a 3D image showing the fusome (α-Spectrin, Spec) permeating the cyst through the ring canals (Pavarotti, Pav) in cysts of various sizes (2-, 4-,8- and 16-cell cysts, and a Stage 1 egg chamber (EC)). Scale bar = 5 μm. D) Schematic for the ‘predetermination’ model for oocyte selection, which posits that the initial bias in fusome volume determines which cell becomes the oocyte; as such, oocyte identity is determined at the 1-cell stage. E) Projection of a 3D image of developing cysts during spermatogenesis, where the fusome (Adducin) and ring canals (Pav) are shown. Scale bar = 20 μm. F) Schematic showing that cell divisions can occur in one of two ways based on the orientation that the internal cells (green) divide. If all internal cells divide to form new branches, the cyst will remain maximally branched; however, internal cells can also divide without forming new branches, thus creating in-line divisions and paving the way for topologies that deviate from the maximally branched pattern.
Fig 2.
Pipeline for 3D fusome reconstructions in developing germline cysts.
(A) Starting from 3D confocal images of fusomes with fluorescently-labeled ring canals and fusome, training in a machine learning program and processing in MATLAB as described in [44, 45] produced a 3D reconstruction of the fusome and ring canal system at successive stages of germline cyst formation (Fig 1A). From here, ring canals at the junction between sister cells were used to segment the fusome into its component parts (multicolored cells) and, in the case of oogenesis, produce an adjacency matrix representing intercellular connections within the cyst to compare with the known female germline cyst network topology, permitting unambiguous cell label assignments to the individual fusome fragments. These fusome fragments were then labeled according to their position relative to the presumptive oocyte. (B) Male cysts underwent the same procedure of collective adjacency information, but as no oocyte is present, no distinction was made between cells. Scale bars = 2 μm (A) and 10 μm (B).
Fig 3.
A model for fusome growth and inheritance during oogenesis.
A) Starting with a spectrosome of volume v0 and fusome plug at the first division of volume v1, the volume fraction f1,2 of the 2-cell female cyst can be represented as shown in Eq 1, where the volume fraction in cell 1 at this stage is α and the asymmetry in sharing of the newly formed fusome component between mother and daughter cell is β. B) At the 2-to-4-cell transition, two new fusome plugs are added in the same manner as in A, with each contributing volume v2. C) Divisions occur again to form 8- and 16-cell cysts, where the total fusome volume can be tracked throughout cyst formation, and expressions for volume fractions of fusome within each cell at each cyst size to be derived. D) Error calculations for ranges of the model parameters α and β. For each set of parameters, model volume fractions for each cyst size were calculated and compared with measured data by summing over the squared residuals between measured and derived volume fractions over each cell at all cyst sizes. The minimum error using this metric was found at the value α = 0.700, β = 0.515, corresponding to a scenario where fusome plugs are added evenly between the mother and daughter cells at each successive division. Explanation of model assumptions and equations can be found in the Materials and Methods. Comparison with measured data can be found in S1 Fig.
Fig 4.
Fusome fragmentation during spermatogenesis and loss of maximal branching.
A) Starting with n = 62 reconstructed fusomes, a range of cyst sizes can be inferred by identifying the largest connected portions of fusome within each sample. These fusome portions, and their corresponding ring canals, can be used to identify the number of cells connected by each fusome fragment (fusome-connected cells). In approximately half of these, the number of fusome-connected cells was a power of 2 (n = 31). B) Two examples of fusomes connecting 16 and 15 cells, respectively, whose topology cannot be embedded onto that of a maximally branched network. C) Projections of 3D images of dividing cysts with labeled fusome (Adducin, gray), ring canals (Pav, red), and mitotic chromosomes (Phosphohistone 3, cyan), showing examples of a 2-cell (left), 4-cell (middle), and 8-cell cyst (right) during mitosis. In at least one location in the 8-cell cyst, cells are dividing without forming a new branch; as such, the resulting 16-cell tree will not possess a maximally-branch topology. In all examples, yellow dotted lines denote the likely plane of division for actively dividing cells, while yellow arrows depict clear bundles of aggregated or migrating chromosomes. In addition, nodes representing the locations of existing cells, as well as the likely positions of newly-formed cell based on the plane of division are shown. Scale bars = 10 μm.