– A single, identifiable mechanosensory neuron in Drosophila melanogaster has a hard-wired synaptic targeting pattern and can be isolated for RNA sequencing.

a, The posterior scutellar (pSc) neuron detects movement on the back of the fly and projects into the thoracic ganglion of the central nervous system. b, The pSc bristle is composed of the bristle shaft cell, the bristle socket cell, the mechanosensory neuron, the neuron sheath/glial cell. Scale bar is 20μm. c, After manual dissection and dissociation, a single pSc neuron expressing a red fluorescent protein was isolated based on fluorescence and morphology. Scale bar is 2μm. d, The pSc mechanosensory axonal arbor has a unique and stereotyped wiring pattern within the thoracic ganglion. Scale bar is 50μm. e, The Gr59d chemosensory neuron is found on the forelegs of the fly underneath the f5s sensilla. Underneath each sensillum, there are four gustatory receptor neurons (GRNs), one of which expresses Gr59d. There are two f5s sensilla on each foreleg of the fly and therefore two Gr59d chemosensory neurons per leg, and four total in each animal. For clarity, the diagram of the thoracic ganglion shows only one Gr59d axonal projection (magenta), overlaid with the pSc axonal projection (green). f, The Gr59d axon does not have as elaborate branching as the pSc arbour. The Gr59d axon enters the thoracic ganglion through the ventral prothoracic neuromere (bottom zoomed image), extends towards the midline but does not cross, and extends up to the brain to terminate in the subesophageal zone (circled in top zoomed image). The scale bar is 100µm for the left panel, and 50µm for the zoomed images. The dotted line is the midline.

– Between 50–70 cell surface molecules are significantly differentially expressed between pSc and Gr59d neurons.

a, Principal component analysis (PCA) for adult pSc mechanosensory and adult Gr59d chemosensory neurons. The PCA shows the Gr59d neurons in a tight cluster and away from the pSc neurons. The pSc neurons do not all cluster together and have more variability within themselves. b, Pearson’s correlation heatmap for adult pSc and Gr59d neurons also shows a high intragroup correlation for Gr59d neurons and a larger variability for pSc neurons. The expression levels for all genes using normalized log-transformed counts, with low-count genes filtered out, were used. The scale represents the correlation coefficient, r. c, 74 cell surface or secreted molecules were significantly differentially expressed between the adult pSc and the adult Gr59d neurons, shown in a hierarchical heatmap, padjusted < 0.05, log2 (fold change) of |2|. The scale represents the log2 (fold change). d, 48 cell surface or secreted molecules were significantly differentially expressed between pupal pSc and adult Gr59d neurons, shown in a hierarchical heatmap, padjusted < 0.05, log2 (fold change) of |2|. The scale represents the log2 (fold change). Genes with an asterisk were also identified in the adult pSc versus adult Gr59d comparison (c).

– The highly stereotyped connectivity pattern of the pSc mechanosensory neuron is a result of a precise transcriptome.

a, Three pSc neuron transcriptomes have similar Manhattan Plots. Total reads per gene are shown on the y-axis and genes on the x-axis, arranged alphabetically. The adult aPa transcriptome is shown in the inset for comparison. b, Hierarchical heatmap shows few differences in the expression profile of ten single pSc mechanosensory neuron transcriptomes. The scale represents the log2 (fold change). c, Each mechanosensory neuron has low variability among transcriptomes when compared to each other. The All2All plot displays the correlations and variances between each pSc mechanosensory neuron. The upper right side of the plot shows each log10 normalized scatter plot comparing one pSc to another pSc neuron. The bottom left side of the plot is the Pearson correlation coefficient of each pSc pairing. The diagonal shows the histogram of log10 normalized read counts. Neurons 8 and 9 showed the least similarities.

– Dscam alternative splicing in the pSc neuron is both random and specific.

a, The Dscam gene in Drosophila melanogaster exhibits extraordinary alternative splicing in variable exons 4, 6, and 9. Mutually exclusive splicing of exon alternates in these variable exons allows for different mRNA and protein isoforms that maintain the same overall architecture. b, Single cell RNA sequencing of ten pSc and two aPa mechanosensory neurons shows preferential splicing for specific exon 9 alternates, 9.6, 9.9., 9.13, 9.30, and 9.31. The aPa mechanosensory neuron had a much higher isoform diversity than the pSc neuron. Grey lines represent individual isoform splicing choices for each neuron; thickness of the line represents the number of reads in the sequencing. Splicing of constitutive exons 5, 7, and 8 are not shown for clarity. Samples pSc1 and aPa1 came from the same animal, and pSc2 and aPa2 came from the same animal.

– An RNAi screen against cell surface receptor genes to characterize their specific function in axonal targeting.

a, Axonal targeting errors were categorized into six grades of severity. RNAi knockdown occurred only within the pSc mechanosensory neuron and experiments and analysis were performed unaware of genotype. Representative examples of pSc axonal arbor phenotypes for each grade are shown. Red arrows point to ectopic branches and red arrowheads indicate missing branches. b, Frequency distribution of axonal arbor severity for 213 cell surface receptor genes, from least severe (Grade 0) to most (Grade 5). Black asterisks indicate a gene that was detected in pSc neurons in the RNA sequencing data; white asterisks indicate genes that were not detected in the RNA sequencing. c, Loss of function of differentially expressed genes that were significantly higher in the pSc than in the aPa severely disrupted axonal targeting of the pSc neuron. Representative images of RNAi knockdown mutants of Eph, Appl, Tequila, Lar, and Tsp42Ed are shown. Red arrows point to ectopic branches, red arrowheads point to missing branches. Schematic on the right depicts the aPa mechanosensory neuron. d, Manipulating cell surface receptor expression within the pSc neuron of differentially expressed genes between the pSc and the Gr59d chemosensory neuron shifted the axonal targeting pattern towards that of the Gr59d neuron (right schematic). RNAi knockdown and mis-expression of differentially expressed cell surface receptors decreased axonal branch length and complexity and increased axon guidance errors (magenta arrows) of the pSc neuron. Quantification of axonal arbours is shown in Supplemental Figure 9. Top row shows RNAi knockdown (left images) and mis-expression (right image, UAS-kuz) of differentially expressed genes between the pupal pSc and adult Gr59d. Bottom row shows representative images of RNAi knockdown of differentially expressed genes between the adult pSc and adult Gr59d. The pSc axon exited the thoracic ganglion similar to the Gr59d neuron in nearly all gene manipulations. Red arrows point to ectopic branches, red arrowheads point to missing branches, magenta arrows point to axon guidance errors. Dashed line represents the midline; scale bars are 50μm.

– RNAi knockdown within pSc neurons of cell surface receptors required for synaptic targeting reduces the grooming reflex.

a, A grooming response from the rear legs of the Drosophila can be elicited by stimulating the pSc bristle. b, The frequency distribution of animal response rate for grooming versus gene targeted by RNAi is shown. Genes with highly penetrant axonal targeting errors (“severe errors”) in RNAi knockdown (e.g., Beat-Ic, Nrm, Robo1, and Klingon) also significantly impaired the grooming reflex when knocked down. Knockdown of Beat-Ib selectively in the pSc neuron significantly reduced the grooming response but did not impair axonal arbor morphology. Single asterisk represents p < 0.05, double asterisk is p < 0.01.

– Rewiring the Gr59d neuron.

a, The mutually exclusive alternative splicing of the Drosophila Dscam gene is the basis for the poly-transgene expression system (PXGS). Dscam variable exon 4 is shown. b, The PXGS system allows for expression of 12 different transgenes by replacing each Dscam exon 4 alternate with a gene of interest (GOI). Conditional expression is achieved by placing a UAS regulatory sequence upstream of the PXGS. Only the last 300 bases of exon 3 and the first 40 bases of exon 5 are kept from the Dscam gene, and remain as untranslated regions (UTRs) in the mRNA. A 6×His tag is included at the 3’ end for ease of molecular biology verification and is also not translated into protein. c, After multiple rounds of PXGS transcription due to overexpression, Drosophila cells express all PXGS transgenes. Transgenic flies containing UAS-PXGS constructs with different genes in different alternate positions were used to verify that all possible alternates were expressed. The brains of transgenic flies containing UAS-PXGS_iRFPnols4.1-COX8::mScarlet4.2-BFPnols4.3- mCD8::mNeonGreen4.4-iRFPnols4.5-COX8::mScarlet4.6-mCD8::mNeonGreen4.8-BFPnols4.9-iRFPnols4.10-BFPnols4.12 driven by nSyb-Gal4 are shown. iRFP is a near-infrared fluorescent protein, nols is a nucleolar localization signal, COX8 is a mitochondrial localization domain, mScarlet is a red fluorescent protein, BFP is a blue fluorescent protein, mNeonGreen is a green fluorescent protein, and mCD8 is a membrane localization domain. White arrowheads in the zoomed in merge image (bottom right) point to cells that express all four fluorophores. d, Overexpression of cell surface receptors differentially expressed from the pSc neuron into Gr59d neurons using PXGS resulted in ectopic axonal branch formation (yellow arrows) within the thoracic ganglion. Representative images of overexpression of dpr12, dpr8, kek1, kirre, tutl, Toll-6, Bsg, and sli within the Gr59d neuron using UAS-PXGS are shown. The frequency of occurrence of two error types, ectopic branching before the midline and midline posterior extension is shown in the bottom right. A schematic for each error is shown below the x-axis, where blue branches indicate ectopic branches. n > 6 for all genotypes. Statistical significance compared to control is indicated directly above the bar representing each genotype. Single asterisk represents p < 0.05, quadruple asterisks is p < 0.0001. The dotted white line is the midline. Scale bar is 5µm in the zoomed in images, and 50µm for all other images.