Diversity of Internal Sensory Neuron Axon Projection Patterns Is Controlled by the POU-Domain Protein Pdm3 in Drosophila Larvae

Abstract

Internal sensory neurons innervate body organs and provide information about internal state to the CNS to maintain physiological homeostasis. Despite their conservation across species, the anatomy, circuitry, and development of internal sensory systems are still relatively poorly understood. A largely unstudied population of larval Drosophila sensory neurons, termed tracheal dendrite (td) neurons, innervate internal respiratory organs and may serve as a model for understanding the sensing of internal states. Here, we characterize the peripheral anatomy, central axon projection, and diversity of td sensory neurons. We provide evidence for prominent expression of specific gustatory receptor genes in distinct populations of td neurons, suggesting novel chemosensory functions. We identify two anatomically distinct classes of td neurons. The axons of one class project to the subesophageal zone (SEZ) in the brain, whereas the other terminates in the ventral nerve cord (VNC). We identify expression and a developmental role of the POU-homeodomain transcription factor Pdm3 in regulating the axon extension and terminal targeting of SEZ-projecting td neurons. Remarkably, ectopic Pdm3 expression is alone sufficient to switch VNC-targeting axons to SEZ targets, and to induce the formation of putative synapses in these ectopic target zones. Our data thus define distinct classes of td neurons, and identify a molecular factor that contributes to diversification of axon targeting. These results introduce a tractable model to elucidate molecular and circuit mechanisms underlying sensory processing of internal body status and physiological homeostasis.SIGNIFICANCE STATEMENT How interoceptive sensory circuits develop, including how sensory neurons diversify and target distinct central regions, is still poorly understood, despite the importance of these sensory systems for maintaining physiological homeostasis. Here, we characterize classes of Drosophila internal sensory neurons (td neurons) and uncover diverse axonal projections and expression of chemosensory receptor genes. We categorize td neurons into two classes based on dichotomous axon target regions, and identify the expression and role of the transcription factor Pdm3 in mediating td axon targeting to one of these target regions. Our results provide an entry point into studying internal sensory circuit development and function, and establish Pdm3 as a regulator of interoceptive axon targeting.

Keywords: Drosophila; axon patterning; interoceptive; pdm3; subesophageal zone; tracheal dendrite.

Figure 1.

td sensory dendrites associate with tracheal branches. A, Schematic of larva showing segmentally repeated td cell bodies (green) relative to the tracheal network. Blue represents only major tracheal branches. B, Schematic of v′td1 and v′td2 neurons in one hemisegment, showing cell bodies (green circles), dendrites (solid green lines), and axons (dashed green line), and association with specific tracheal branches (blue). C, Dual-color labeling of v′td1 (C′; labeled by R35B01-LexA>mCD8::GFP) and v′td2 (C″; labeled by 260-Gal4>mCherry) neurons in segment A3. D, Dendrite lengths and node count quantifications for v′td1 (labeled by R35B01-LexA) and v′td2 (labeled by 260-Gal4) neurons in segments A2–A4. Dendrite length was reduced in v′td2 relative to v′td1 in segment A2 (Bonferroni adjusted *p = 0.012, Mann–Whitney U test, U < 0.0001), but not in segments A3 (Bonferroni adjusted p = 1, U = 17) or A4 (Bonferroni adjusted p = 1, U = 16). Similarly, dendrite nodes were reduced in v′td2 relative to v′td1 in segment A2 (Bonferroni adjusted *p = 0.018, Mann–Whitney U test, U = 1), but not in segments A3 (Bonferroni adjusted p = 1, U = 16.5) or A4 (Bonferroni adjusted p = 0.558, U = 10). n = 6 animals. E, td dendrites project along tracheal branches. Coracle labeling shows septate junctions of tracheal epithelial cells. E′–E‴, Enlarged image of area boxed in E showing enlargements of td dendrites near tracheal fusion cell (yellow arrow). F, Electron micrographs of tracheal tube cross section showing tracheal cell (tc; blue), putative td dendrites (d; green), tracheal lumen (tl), and muscle (ms). F′, Higher-magnification image of boxed area in F, showing two neuronal processes closely associated with the tracheal cell, enclosed by basement membrane. Boxplots represent median (middle line) and 25th to 75th percentile, with whiskers extending to the most extreme data point within 1.5 times the interquartile range of the hinge. Scale bars: C–C″, E, 50 μm; E′–E‴, 10 μm; F, 2 μm; F′, 250 nm.

Figure 2.

td axons project to the VNC and the SEZ. A, Schematic of CNS showing axon trajectories of typical abdominal mechanosensory neurons (A), and td neurons (A′). A′, Dashed line and question mark indicates unresolved terminal trajectory of td axons. Purple band indicates T3 neuromere. B, td axons labeled by R31D10-LexA>GFP fasciculate and project anteriorly in the CNS (outlined in white). Roman numerals and dotted cyan lines indicate position of cross sections on the right, showing that td axons initially travel anteriorly along the ventrolateral fascicle (Bi), then shift to the dorsomedial fascicle (Biv). C, Two subtypes of td axon projections to the VNC and the SEZ. A subset of td neurons labeled by R31D10-LexA>mCD8::GFP targets the SEZ (C). Another subset of td neurons labeled by R22C07-Gal4>mCherry targets the VNC (C′). Tsh-Gal80 was used to suppress VNC expression in the R22C07-Gal4 driver. R22C07-Gal4 also labels other sensory neurons from the head that project axons to the SEZ (asterisk). D, VNC- and SEZ-targeting axons from the same segment initially fasciculate with each other as they enter the VNC (Di) but segregate into separate bundles in the ventrolateral tract (Dii). Roman numerals and dotted cyan lines indicate position of cross sections on bottom. E, SEZ-targeting td axons (green) terminate in close proximity to axon terminals of gustatory sensory neurons (magenta). F, G, Presynaptic sites of VNC-targeting td axons (F) and SEZ-targeting td axons (G) labeled by brp.short.cherry reveal en passant synapses along the td axon bundle tract (labeled by GFP in green). Scale bars: B, C–C″, F, G, 50 μm; D, E, 20 μm.

Figure 3.

GR and IR GAL4 reporters label td neurons. A–F, CNS images showing expression of Gr-Gal4 and Ir-Gal4 lines that label td neurons. A′–F′, Same images dual-labeled with R31D10-LexA, which labels a subset of td neurons. In addition to td neurons, most of the GAL4 lines labeled gustatory neurons from the head (A–D, F), and a few lines labeled terminal sensory neurons (A, D), cIV sensory neurons (B), external sensory neurons (F), or CNS neurons (B, E). Scale bars, 50 μm.

Figure 4.

td axon projections correlate with cell identity and segment of origin. A–M, Individual central axon projections of all 13 td sensory neurons. Labeled single td neurons were generated using the MCFO technique. Magenta asterisks indicate the approximate location of the T3 neuromere. Arrowhead indicates axon termination. For all image panels, axon of interest is shown on the left side of the CNS. Blue dashed lines outline the CNS. G, White arrow indicates collateral branches from the A7 v′td2. N, O, Schematic summarizing the axon projections (N) of SEZ-targeting td neurons (green; SEZ-TDs) and VNC-targeting td neurons (red; VNC-TDs), and the segment of origin and identity of these neurons in the larva body (O). Magenta asterisks indicate the approximate location of the T3 neuromere, as in A–M. Scale bars: A–M, 50 μm.

Figure 5.

Pdm3 is expressed in SEZ-targeting td neurons. A, Expression of OK282-Gal4>UAS-mCD8::GFP in td neurons (arrow) and a nearby external sensory neuron in the periphery (arrowhead). Image spans three abdominal hemisegments. B, OK282-Gal4 is inserted upstream of the pdm3 locus. C, D, OK282-Gal4 expression pattern matches anti-Pdm3 labeling in td neurons (C) and in CNS (D). Yellow arrows indicate some areas of the CNS with high overlap between the two expression patterns. E, Schematic summarizing expression pattern of Pdm3 in td neurons, as determined by pdm3^OK282-Gal4 expression pattern and anti-Pdm3 labeling. Pdm3 (P3) is expressed in most of the SEZ-targeting td neurons (green circles), but not in the VNC-targeting td neurons (red circles). Scale bars: A, D, 50 μm; C, 20 μm.

Figure 6.

Misexpression of Pdm3 in VNC-targeting td neurons promotes SEZ targeting. A–C, td axons in control (A, B) and upon ectopic Pdm3 expression (C) conditions. Arrowhead indicates termination of td axons. Below each confocal image are keys showing SEZ-targeting td neurons labeled by R31D10-LexA (A; green) or VNC-targeting td neurons labeled by R22C07-Gal4 (B; red). P3, Pdm3 expression. Ectopic Pdm3 expression using R22C07-Gal4 switches td axon targeting from the VNC to the SEZ (C). D, Quantification of the length of the axon bundle beyond the axons' anterior turn at T3. Kruskal–Wallis test showed a significant difference of means (H₍₂₎ = 24.40, p < 0.0001). Post hoc pairwise comparisons showed that VNC-targeting td neurons ectopically expressing Pdm3 have increased axon lengths (***p < 0.0001, Mann–Whitney U test, U < 0.0001), which did not differ significantly from the length of SEZ-targeting td neurons (p = 0.332, Mann–Whitney U test, U = 48). n = 12 animals per condition. E, Ectopic expression of Pdm3 in R22C07-Gal4 neurons promotes presynaptic structures in the overextended axon segments. F, G, Ectopic expression of Pdm3 in non-td sensory neurons (cIV nociceptive neurons) causes defasciculation and axon wandering. Boxplots represent median (middle line) and 25th to 75th percentile, with whiskers extending to the most extreme data point within 1.5 times the interquartile range of the hinge. Scale bars, 50 μm.

Figure 7.

Pdm3 is required for correct axon targeting within the SEZ. A–C, td axons in control and Pdm3 loss-of-function conditions. In control condition (A), td neurons labeled by R31D10-LexA project axons to the SEZ, where the axons turn slightly laterally away from the midline. In pdm3 mutant (B) and pan-neuronal Pdm3 RNAi knockdown conditions (C), td axon terminals extend toward the SEZ but incorrectly contact the midline (yellow arrowheads). D, Quantification of midline contacts by td axon terminals. Increased axon midline contacts were observed in pdm3 mutant (χ² (1, N = 26) = 19.07, ***p < 0.0001) and pan-neuronal Pdm3 RNAi knockdown (χ² (1, N = 26) = 19.07, ***p < 0.0001) conditions. n = 13 animals per condition. E, Confirmation of Pdm3 knockdown in td neurons. F, The VNC is abnormally elongated in pdm3 mutant larvae, but not upon pan-neuronal knockdown of Pdm3. Kruskal–Wallis test showed significant difference of means (H₍₂₎ = 9.89, p = 0.007). Post hoc pairwise comparisons showed increased VNC lengths in pdm3 mutants (***p < 0.0001, Mann–Whitney U test, U < 0.0001), but not pan-neuronal Pdm3 RNAi knockdown animals (p = 0.590, Mann–Whitney U test, U = 7.5). n = 5 animals per condition. G, Loss of Pdm3 does not lead to morphological defects in td dendrites as measured by total dendrite lengths (p = 0.602, Mann–Whitney U test, U = 10) and node counts (p = 0.190, Mann–Whitney U test, U = 6.5). Analysis of A5 v′td1. n = 5 animals per condition. Boxplots represent median (middle line) and 25th to 75th percentile, with whiskers extending to the most extreme data point within 1.5 times the interquartile range of the hinge. Scale bars: A–C, 50 μm; E, 10 μm.

Figure 8.

Misexpression of Pdm3 in VNC-tds suppresses Gr33a-QF reporter expression. A, B, Summary of GR/IR reporter expression in wild-type, pdm3 mutant (A), and ectopic Pdm3 expression (B) conditions. Green and red cells represent SEZ- and VNC-targeting td neurons, respectively. +, Cells with expression (100% of cell labeled, n > 3 animals); −, cells with reduced expression (n > 3 animals). B, Cells with blue outline indicate td neurons misexpressing Pdm3 through R22C07-Gal4>UAS-pdm3. Gr33a-QF labeled v′td2 in A4–A6 with 100% frequency (n = 7 animals, 42 total cells examined) in control condition and only 31% frequency (n = 7 animals, 42 total cells examined) in ectopic Pdm3 condition. C, Expression of Gr33a-QF>QUAS-mtdTomato in td neurons in control and ectopic Pdm3 expression conditions. Gr33a-QF also labels glial cells, near td and adjacent nerves. Anti-Elav labels td nuclei. Images show td neurons in A5. Scale bars, 10 μm.