Automatic Segmentation of Drosophila Neural Compartments Using GAL4 Expression Data Reveals Novel Visual Pathways

Abstract

Identifying distinct anatomical structures within the brain and developing genetic tools to target them are fundamental steps for understanding brain function. We hypothesize that enhancer expression patterns can be used to automatically identify functional units such as neuropils and fiber tracts. We used two recent, genome-scale Drosophila GAL4 libraries and associated confocal image datasets to segment large brain regions into smaller subvolumes. Our results (available at https://strawlab.org/braincode) support this hypothesis because regions with well-known anatomy, namely the antennal lobes and central complex, were automatically segmented into familiar compartments. The basis for the structural assignment is clustering of voxels based on patterns of enhancer expression. These initial clusters are agglomerated to make hierarchical predictions of structure. We applied the algorithm to central brain regions receiving input from the optic lobes. Based on the automated segmentation and manual validation, we can identify and provide promising driver lines for 11 previously identified and 14 novel types of visual projection neurons and their associated optic glomeruli. The same strategy can be used in other brain regions and likely other species, including vertebrates.

Keywords: clustering; enhancers; neuroanatomy; vision.

Graphical abstract

Figure 1

Automatic Segmentation of a Brain Region into Domains Sharing Common Enhancer Profiles (A) Thousands of registered confocal image stacks from the Janelia FlyLight and Vienna Tiles projects were used. Within an analyzed brain region (purple outline), a list of driver lines driving expression was compiled for each voxel. Voxel-to-voxel similarity s was computed using the Dice coefficient, a measure of overlap, and k-medoids was used to cluster groups of voxels of putative functional units. These singleton clusters were then agglomerated into a hierarchy. (B) Automatic segmentation of the antennal lobe (AL). The three-dimensional axis scale is 40 μm in lateral (red), dorsal-ventral (green), and anterior-posterior (blue). (C) Individual clusters (left), average images of strongly expressing driver lines with broad driver lines removed (middle), and manually assigned corresponding olfactory glomeruli (right). Scale bars, 20 μm. (D) Automatic segmentation of the optic ventrolateral neuropil (oVLNP). Three-dimensional axis scale, 40 μm. (E) Individual clusters (left), average images of strongly expressing driver lines with broad driver lines removed (middle), and selected driver lines and previously identified visual projection neuron names (right). Scale bars, 50 μm. (F) Selected subtree of the agglomerative clustering of the oVLNP results showing z projections of the singleton clusters (left), dendrogram (middle), and top-level agglomeration (right) of the anterior optic tubercle (AOTU). Scale bars, 25 μm. (A and D–F) Janelia FlyLight data for the oVLNP region defined as the posterior lateral protocerebrum (PLP), posterior ventrolateral protocerebrum (PVLP), and AOTU, run 1, 42,317 voxels, 3,462 driver lines, k = 60. (B and C) Janelia FlyLight data for the right AL, run 1, 23,769 voxels, 3,462 driver lines, k = 60. See also Figure S1.

Figure 2

Automatic Segmentation Reveals Clusters that Correspond to Optic Glomeruli Associated with Newly Identified LC-type Visual Projection Neurons Individual clusters, average images, selected driver lines, 3D segmentations of a particular VPN type, the presynaptic marker (UAS-synaptotagmin::GFP) expressed by a single driver, and 3D segmentation of the presynaptic region to define the optic glomerulus (A–H). Janelia FlyLight data for the oVLNP, run 1, 42,317 voxels, 3,462 driver lines, k = 60. Scale bars, 50 μm. See also Figures S2 and S3.

Figure 3

Automatic Segmentation Reveals Clusters that Correspond to Optic Glomeruli Associated with Newly Identified LPLC-, LPC-, and MC-type Visual Projection Neurons Individual clusters, average images, selected driver lines, 3D segmentations of a particular VPN type, a presynaptic marker (UAS-synaptotagmin::GFP) expressed by a single driver, and 3D segmentation of the presynaptic region to define the optic glomerulus (A–E). Janelia FlyLight data for the oVLNP, run 1, 42,317 voxels, 3,462 driver lines, k = 60. Scale bars, 50 μm. See also Figures S2 and S3.

Figure 4

An Atlas of the Optic Glomeruli Defined by Manual Segmentation of Presynaptic Marker Expression Experiments (A) Three-dimensional rendering of all identified optic glomeruli registered onto a 3D reference brain. Optic glomeruli were segmented from single-driver confocal images expressing a presynaptic marker (UAS-synaptotagmin::GFP). Scale bars, 40 μm in lateral (red), dorsal-ventral (green), and anterior-posterior (blue). (B) Z stack showing the location of each optic glomerulus in a 2D view on the background of an average image of many individual nc82-stained brains. See also Table S1 and Movie S1.

Figure 5

Distribution of Optic Glomeruli within the Lateral Protocerebrum and VPN Axons within Glomeruli (A) VPNs exclusively from the lobula (orange; LC types) project to optic glomeruli distributed throughout the oVLNP, whereas those from the medulla (cyan; MC) and lobula plate (purple; LPLC and LPC) project to restricted areas of the oVLNP. Three-dimensional axis scale, 40 μm. (B) Optic glomeruli that consistently group together across repeated clustering runs with different random initialization seeds. Groups correspond to subtrees in the dendrogram for three of four runs (italics) or four of four runs (non-italics). Three-dimensional axis scale, 40 μm. (C) Dendrogram with consistent hierarchies highlighted. Bold, colored lines correspond to (B) and subtrees in the dendrogram for three of four runs (dashed lines) or four of four runs (non-dashed lines). Dendrogram from Janelia FlyLight data for the oVLNP, run 1, 42,317 voxels, 3,462 driver lines, k = 60. (D) MARCM analysis shows presynaptic varicosities distributed throughout optic glomeruli in single axons of LC04 and LC06 but localized for MC61. Arrowheads denote the glomerulus region. Scale bars, 50 μm. (Genotype for LC06 and MC61: yw, neoFRT19A/hsFLP, tubGAL80, neoFRT19A; UAS-mCD8::GFP/+; VT009855-GAL4/+; genotype for LC04: yw, neoFRT19A/hsFLP, tubGAL80, neoFRT19A; UAS-mCD8::GFP/+; VT046005-GAL4/+.)