Combinations of DIPs and Dprs control organization of olfactory receptor neuron terminals in Drosophila

Abstract

In Drosophila, 50 classes of olfactory receptor neurons (ORNs) connect to 50 class-specific and uniquely positioned glomeruli in the antennal lobe. Despite the identification of cell surface receptors regulating axon guidance, how ORN axons sort to form 50 stereotypical glomeruli remains unclear. Here we show that the heterophilic cell adhesion proteins, DIPs and Dprs, are expressed in ORNs during glomerular formation. Many ORN classes express a unique combination of DIPs/dprs, with neurons of the same class expressing interacting partners, suggesting a role in class-specific self-adhesion between ORN axons. Analysis of DIP/Dpr expression revealed that ORNs that target neighboring glomeruli have different combinations, and ORNs with very similar DIP/Dpr combinations can project to distant glomeruli in the antennal lobe. DIP/Dpr profiles are dynamic during development and correlate with sensilla type lineage for some ORN classes. Perturbations of DIP/dpr gene function result in local projection defects of ORN axons and glomerular positioning, without altering correct matching of ORNs with their target neurons. Our results suggest that context-dependent differential adhesion through DIP/Dpr combinations regulate self-adhesion and sort ORN axons into uniquely positioned glomeruli.

Fig 1. Transcriptional profiles of CSR genes in the developing Drosophila olfactory system.

Heatmaps representing normalized log₂ expression values for CSRs. Highly expressed genes are represented in darker colors, and genes with lower expression levels are represented with lighter colors. A) Hierarchical clustering of the developmental expression patterns of in the olfactory system groups genes into clusters. Known regulators of ORN wiring and members of the dpr family are highlighted. B) Schematic showing expression profile of genes in each cluster at the 4 developmental stages. C) Hierarchical clustering of the developmental expression patterns of known regulators of ORN wiring reveals two major expression patterns: high expression at all stages, and low expression at early stages followed by high expression at later stages. Expression patterns of dpr (D) and DIP (E) genes ordered from highest expression across all stages to lowest. Most DIP/dpr genes are expressed primarily at later developmental stages (p40 and Adult).

Fig 2. Combinatorial and ORN class specific expression patterns of DIP and dpr genes.

DIP and dpr-GAL4s were used to drive UAS>STOP>mCD8::GFP in ORNs with eyFLP (green, A-H) to visualize ORN axons with staining for the neuropil (magenta). Each gene is expressed in a particular set of glomeruli and ORN classes. A map of the expression pattern of gene in the antennal lobe is provided in the right panel (A-H). Results for all genes are summarized in Supplemental Fig 1. (I) Hierarchical bi-clustering of DIP/Dpr expression patterns by ORN class reveals that many classes of neurons express a unique combination of DIPs/Dprs. ORN classes highlighted in red neighbor each other in the antennal lobe and are further analyzed in subsequent figures.

Fig 3. Multidimensional scaling clusters ORN classes by DIP/Dpr expression pattern, which groups classes targeting distant glomeruli.

(A) Multidimensional scaling analysis clusters ORN classes based upon DIP/Dpr expression and glomerular distances. (B) K-means clustering was used to determine which ORN classes shared the most similar DIP/Dpr expression profiles in the MDS analysis. Classes that were clustered together were assigned the same color. (C) ORN classes clustered in B tend to target distant glomeruli. Coloring of glomeruli matches clusters in B. (D) Schematic of four glomeruli (Or47b, Or88a, Or83c, and Ir84a) that will be analyzed in subsequent figures. The expression code for each glomerulus is highlighted with each receptor-ligand pair displayed in matching colors.

Fig 4. Cell-autonomous knock down of DIP-η in Or47b ORNs disrupts sorting of Or47b axons from Or88a glomerulus.

(A, B) Knock down of DIP-η with the Or47b-GAL4 driver, also used to drive UAS-syt::GFP (green), causes Or47b axons to expand and invade a neighboring glomerulus. (C-E’) Simultaneous labeling of Or47b (red) and Or88a (green) axons reveals that knock down of DIP-η causes Or47b ORN axons to invade the Or88a ORN target glomerulus, while Or88a ORN axons retain their ability to coalesce into a glomerulus despite intermingling with Or47b axons. (F-F’) Co-labeling of Or47b axons (red) and MZ19 expressing PNs (green). During knock down of DIP-η, MZ19 expressing PNs do not invade the VA1v glomerulus. (G) Schematic of axon sorting phenotypes in DIP-η knock down. When DIP-η is specifically ablated from Or47b ORNs (black X), Or47b ORN axons (red) invade the Or88a ORN target glomerulus (white arrows) and intermingle with Or88a ORN axons (red/green striping). (H-J’) Developmental analysis of DIP-η RNAi phenotype with Or47b axons labeled in green and N-Cadherin in magenta, at 45–47 hrs APF (H-H’), 50–52 hrs APF (I-I’), and 55–57 hrs APF (J-J’). Disruptions to VA1v glomerular morphology (arrowheads) can be detected as early as 45–47 hrs APF (H’) and larger expansions, as seen in the adult, can be observed by 55–57 hrs APF (J’). (K-K’) Knock down of DIP-η using the Bar-GAL4, which expresses in Or88a, Ir84a, and Ir75d ORNs [33]. Anterior sections of the antennal lobe are shown, and disruption to the VA1d glomerulus can be observed (K’).

Fig 5. Combinatorial knock down and overexpression of DIPs causes cell non-autonomous defects in VA1v glomerular organization.

(A, B) Knockdown of DIP-η and depletion of DIP-δ protein using the deGradFP system causes the splitting of specifically the Or47b glomerulus, but not the Gr21a or Or47a glomeruli, when driven with the peb-GAL4. (C) Penetrance of the DIP-η/DIP-δ knockdown phenotype. 50% of individuals displayed a split of the Or47b glomerulus in one or both antennal lobes. (D-F) Overexpression of DIP-δ (E) and DIP-γ (F) in Or47b ORNs (green). Overexpression of either gene does not disrupt the VA1v glomerulus. (G-I) When DIP-δ and DIP-γ were over expressed in all ORNs using the peb-GAL4 driver, no disruptions were observed in the target glomeruli for Or47b, Or47a or Gr21a ORNs (green). (J-M) OR-GAL4s were used to drive expression of UAS-DIP-δ (K), UAS-DIP-γ (L) and UAS-syt::GFP (green), and both UAS-DIP-δ and UAS-DIP-γ simultaneously (M). Mis-expression of DIP-δ in Or47b ORNs caused the target glomerulus to deform and split (K), while other ORNs mis-expressing DIP-δ were unaffected. Over expression of DIP-γ in Or47b ORNs caused their axons to partially invade the Or88a target glomerulus (L), like knock down of DIP-η (Fig 4). In contrast, other glomeruli that over expressed DIP-γ were unaffected. Surprisingly, overexpression of both DIP-δ and DIP-γ returned the shape of the VA1v glomerulus to its wildtype morphology (M).

Fig 6. dpr1 overexpression leads to changes in glomerular organization via adhesive interactions.

(A, B) Overexpression of dpr1 was in Or47b ORNs (green) did not perturb the VA1v glomerulus. (C, D) A subset of Or47b neurons (red) intermingle with all Or47b axons (green) in wildtype (C) and dpr1 overexpressing flies (D). (E-J) Overexpression of dpr1 with the peb-GAL4 caused a split of the VA1v glomerulus (E-H). Overexpression of dpr1 with this driver also caused mistargeting/splitting of some Or47a ORN axons (E-F, I-J), creating a highly reproducible, ectopic glomerulus. (K, L) Overexpression of dpr1 in four classes of ORNs with Or-GAL4 drivers caused a split Or47b glomerulus.

Fig 7. Dpr10 controls ORN wiring in the antennal lobe.

Mutation of dpr10 causes disruption of DM3 (A, B), V (C, D) and DL1 (E, F) glomeruli but not the VA1v (G, H) glomerulus as visualized with OR reporters diving syt::GFP (green). Disruptions to these glomeruli included splitting of the glomerulus (B, D), mistargeting (B and F, arrow) and expansion (D, arrow). Penetrance and full summary of these phenotypes are described in S5 Fig. (I-O) ORN cell bodies were visualized in heterozygous and mutant flies for Gr21a (I, J), Or47a (K, L), and Or88a (M, N) ORNs using Or-GAL4 driven UAS-mCD8::GFP. A statistically significant decrease was observed for Or47a ORNs (p<0.001, O) but not for Gr21a or Or88a ORNs (p>0.05, O).