Principal workflow of spatial fly transcriptomics.

(A) Overview of the spatial transcriptomics workflow: adult flies were sectioned, sections were analyzed with Molecular Cartography and data were annotated using cell segmentation, rasterization (i.e. grid) and neighborhood embedding (see Methods). (B, C) Three examples of adult head sections showing various positions in the brain along the anterior-posterior axis (B). Three examples of male whole-body sections taken from the same male (C). mRNAs from each gene are represented in a different color. The combination of colors reveals the different cell types. Scale bars represent 100 µm. Background stain labels DAPI.

Adult body cell types.

(A) Major cell types of adult males identified by marker genes. Scale bars represent 100 µm. Inset for gut shows zoom-ins of different regions (a: apical, b: basal). Inset for flight muscle, shows the percentage of marker gene molecules detected within the outlined area in the section shown. (B) Gene set scores for the main classes of cell types, quantified using 5 µm x 5 µm grid. Scale bar represents 100 µm. The class assignment shown on the right is based on maximum score across classes. Genes used: neurons (elav, Syt1, Sh, acj6, ey, VAChT, Gad1, VGlut, nAChRα7); male reproductive system (Awh, eyg, svp); epithelia (grh, αTry, βTry, hth); heart (tin, Hand); muscle (Mhc); hemocyte (Hml); glia (repo); fat cells (AkhR); oenocytes (FASN2). (C) UMAP showing clustering of 5 µm x 5 µm grid spots. (D) Spatial location of grid clusters. (E) tSNE from male accessory glands from FCA showing expression of marker genes for main gland cells. (F) MC of the main gland cells marker genes highlights several defined populations of cells. Scale bar represents 100 µm. Detailed view shown in F’. Background stain labels DAPI.

Molecular Cartography shows mRNA-specific nuclear enrichment.

(A) Molecular Cartography (MC) visualization of marker genes of muscle subtypes. White boxes mark zoom-in regions shown in (B) and Figure 3 – figure supplement 1. (B) Zoom-in on flight, leg and head muscles. Left, labels are the same as in (A); right, DAPI-labelled nuclei. (C). Density plots show the distance of each mRNA molecule of the indicated genes to its nearest nucleus. Red dotted lines mark the peak density, and black dotted lines the median distance. Scale bars represent 50 µm in (A) and 10 µm in (B). Background stain labels DAPI.

sls mRNA show nuclear enrichment with increased concentration close to muscle-tendon junctions.

(A) Molecular Cartography visualization of sls mRNA (yellow) as pan muscle maker (same section as in Figure 3). White boxes mark zoom-in regions shown in (B) and (C). (B, C) Zoom- in on indirect flight muscles showing colocalization of sls mRNAs and DAPI stained nuclei in anterior (B) or central (C) regions of the flight muscle. (D) HCR-FISH imaging of sls mRNA in an adult thorax. White boxes mark zoom-in regions shown in (E) and (F). (E, F) Zoom-ins on flight muscle at anterior (E) or middle (F) regions. Note the sls mRNA and DAPI co-localization (tracheal cells show background stain in the DAPI channel). Scale bars represent 50 µm in (A) and (D), and 10 µm in (B), (C), (E), and (F). Background stain labels DAPI.

– TpnC4, Act88F, and Mhc mRNAs flight muscle patterning.

(A) Molecular Cartography visualization of TpnC4, Act88F and Mhc mRNAs on an adult fly section (same section as in Figure 3). The white box marks the zoom-in region shown in (B). Zoom-in on the flight muscle showing the striped patterns of TpnC4, Act88F (blue in overlay), and Mhc mRNAs in the indirect flight muscle. (C) HCR-FISH of TpnC4 and Act88F mRNAs in an adult thorax. The white box marks zoom-in the region shown in (D). (D) Zoom- in on the flight muscle of HCR-FISH labelling TpnC4 and Act88F mRNAs (same section as in Figure 4D). (E) HCR-FISH of Mhc mRNAs in an adult thorax. The white box marks the zoom- in region shown in (F). (F) Zoom-in on the flight muscle of HCR-FISH labeling Mhc mRNAs. Scale bars represent 50 µm in (A), (C), (E). and 10 µm in (B), (D), (F). Background stain labels DAPI.

Adult head cell types.

(A) tSNE showing expression of photoreceptor (ninaC), neuronal (para) and glial (repo) markers (left). Molecular Cartography of the same marker genes (right). (B, C) Molecular Cartography of marker genes for olfactory projection neurons (OPN), in an anterior head slice (B) and of perineurial glia of the blood brain barrier (BBB) in a more central brain slice (C). (D, E) Using Molecular Cartography to localized uncharacterized clusters found in scRNA-seq data. (F) UMAP showing clustering of 5 µm x 5 µm grid spots (top). Spatial location of grid clusters in the brain (bottom). (G) Differential expression of central brain (CB), optic lobe (OL) and photoreceptor regions (PR). (H) Molecular Cartography of pros and scro in the brain. (I) tSNE showing split in optic lobe clusters by expression of Wnt4 and Wnt10. Insert shows Molecular Cartography of Wnt4 and Wnt10, spatially localized in ventral and dorsal brain regions, respectively. Scale bars represent 100 µm. Background stain labels DAPI.

Comparison of different techniques for annotating the adult head samples.

(A) Overview of different spatial analysis methods which were used to annotate Molecular Cartography with labels from single-cell RNA-seq: grid-based, neighborhood embedding and nuclei segmentation. Scale bar represents 100 µm. (B) Zoom-in on a high-density region with corresponding segmented nuclei. Scale bar represents 10 µm. (C-F) Comparison of annotation of spatial data with single-cell RNA-seq for three different quantification methods. Grid-based squares/neighborhoods/nuclei are colored based on matching single-cell clusters for (C) glia, (D) optic lobe, (E) central brain and (F) four uncharacterized clusters. In (E) two brain slices are shown at different depths: central (top) and posterior (bottom). NE: neighborhood embedding.