Drosophila Eph receptor guides specific axon branches of mushroom body neurons

Abstract

The conserved Eph receptors and their Ephrin ligands regulate a number of developmental processes, including axon guidance. In contrast to the large vertebrate Eph/Ephrin family, Drosophila has a single Eph receptor and a single Ephrin ligand, both of which are expressed within the developing nervous system. Here, we show that Eph and Ephrin can act as a functional receptor-ligand pair in vivo. Surprisingly, and in contrast to previous results using RNA-interference techniques, embryos completely lacking Eph function show no obvious axon guidance defects. However, Eph/Ephrin signaling is required for proper development of the mushroom body. In wild type, mushroom body neurons bifurcate and extend distinct branches to different target areas. In Eph mutants, these neurons bifurcate normally, but in many cases the dorsal branch fails to project to its appropriate target area. Thus, Eph/Ephrin signaling acts to guide a subset of mushroom body branches to their correct synaptic targets.

Fig. 1. Generation of the Eph mutant

(A) The P114 P element lies 3 kb upstream of the Eph transcription start site and ~300 bp upstream of the onecut transcription start site. Below are shown the extents of three deletions generated using P114. Eph^x652 removes the first three exons of Eph, including the transcription and translation start sites; it also removes 5′ sequences from the onecut transcription unit, but does not remove the onecut translation initiation site. Also shown are two deletions extending solely in the direction of the onecut transcription unit, onecut^lx122 and onecut^lx49. Both break within the onecut-coding region. (B) In situ hybridization of Eph antisense RNA to a stage 15 wild-type embryo. Eph expression is restricted to the embryonic CNS and is detected in most, if not all, neurons. (C) Eph expression is eliminated in similarly staged homozygous Eph^x652 embryos. (D) The Ephrin^KG09118 P element insertion is inserted just downstream of the Ephrin transcription start site (Bellen et al., 2004). (E) In situ hybridization of Ephrin antisense RNA to a stage 15 wild-type embryo. Ephrin expression is detected in most if not all neurons within the CNS. (F) Ephrin expression is severely reduced in homozygous Ephrin^KG09118 embryos, suggesting that Ephrin^KG09118 is a hypomorphic loss-of-function mutation. Anterior is leftwards, dorsal is upwards.

Fig. 2. Eph and Ephrin are targeted to distinct neuronal compartments

(A–C) View of approximately five segments of the ventral nerve cord of an unfixed dissected stage 16 embryo double-labeled with the panaxonal BP102 antibody (A) and Ephrin-Fc (B). Ephrin-Fc binds to axons in a pattern that essentially overlaps that of BP102. (D,E) Homozygous Eph^x652 embryo double-labeled with the panaxonal anti-HRP antibody (D) and Ephrin-Fc (E). Homozygous Eph^x652 embryos lack all Ephrin-Fc binding, confirming the specificity of Ephrin-Fc binding and that Eph^x652 is null for Eph function. (F) Ephrin-Fc binding is unaffected in homozygous onecut^lx122 embryos. (G–I) A scrt-Gal4/+;UAS-Ephrin:myc/+ embryo double labeled with the panaxonal anti-HRP antibody (F) and anti-myc antibody(G). scrt-Gal4 drives expression of Ephrin:myc in all neurons where it is targeted to cell bodies. Anterior is upwards.

Fig. 3. Eph and Ephrin can act as a functional receptor/ligand pair in vivo

View of dissected stage 15–16 embryos labeled with anti-HRP to visualize the axon tracts within the CNS, including the anterior (AC) and posterior (PC) commissures. (A) Wild type. In sim-Gal4/+;UAS-Ephrin:myc/+ embryos (B) where Ephrin:myc is ectopically expressed by midline glia, commissural axon tracts are disrupted. In addition, the CNS often appears less compact. This is consistent with an axon repellent activity for Ephrin. (C) Similar disruptions in midline axon crossing are seen in sim-Gal4/+;UAS-Ephrin^ΔIC:myc/+ embryos expressing an Ephrin:myc variant deleted for intracellular sequences, demonstrating that forward signaling through Eph is responsible for producing the guidance defects. (D) Ectopic Ephrin expression in Eph mutant embryos (genotype=sim-Gal4/+;UAS-Ephrin:myc/+;Eph^x652) does not disrupt commissural axons, demonstrating the Ephrin repellent activity is mediated by Eph.

Fig. 4. Eph^x652 mutant embryos lack obvious axon guidance defects in the embryonic CNS

View of the ventral nerve cord of stage 15–16 embryos in wild type (A,C,E) and Eph^x652 mutants (B,D,F). (A,B) Embryos labeled with BP102 to visualize all axons. No obvious defects in axon guidance are detected in Eph^x652 mutant embryos (B). (C,D) Staining with anti-Fas2 antibody, which labels five distinct axon bundles running anteroposteriorly on each side of the midline, three of which are in the plane of focus, is normal in Eph^x652 mutants (D). (E,F) Embryos double-labeled for Fas2 (red) and for β-galactosidase (green) driven by the expression of apC-tau-lacZ to visualize axonal projections of Ap neurons. Ap neurons extend axons (arrows) normally along the medial Fas2 bundle in Eph^x652 mutants (F). Genotypes: (E) apC-tau-lacZ/+; Eph^x652/+; (F) apC-tau-lacZ/+;Eph^x652/Eph^x652. Anterior is upwards for all panels.

Fig. 5. Eph is expressed on axons of the developing and adult MB

(A) Diagram of MB development, showing the left hemisphere. Broken line indicates the midline. MB neuroblasts are shown in yellow, the axonal projections of γ neurons in black, α′/β′ neurons in green and α/β neurons in red (see text for details). (B–P) Eph expression, as assayed by Ephrin-Fc binding, during MB development. A high-power view of the left hemisphere of a late embryonic/early 1st instar larval brain (B–D), stained with anti-Fas2 (B) and Ephrin-Fc binding (C). Eph is present throughout the neuropil at this stage, including the MB γ neurons and their bifurcated axons (D, dorsal lobe; M, medial lobe; Ped, penduncle). (E–G) A 3rd instar larval MB from an individual carrying the γ neuron driver 201Y-Gal4 driving the expression of UAS-mCD8:GFP stained for GFP (E) and Ephrin-Fc binding (F). Eph is present at low levels throughout the γ neurons and their bifurcated axons. (H–J) An early pupal MB from an individual carrying the MB-specific driver OK107-Gal4 driving the expression of UAS-mCD8:GFP stained with anti-GFP (H) and Ephrin-Fc binding (I). Eph is expressed throughout the MB, including α′/β′ neurons (arrows). (K–M) Late pupal stage MB stained for Fas2 (K) and Ephrin-Fc binding (L). Eph expression is restricted to primarily α/β lobes, here identified by double labeling with Fas2, with only low level Ephrin-Fc staining present within the terminal region of the γ lobe (arrow in I). (N–P) An adult MB double stained for Fas2 (N) and Ephrin-Fc (O). Eph expression is maintained within the α/β lobes, with the highest levels of staining restricted to the terminal regions of the γ lobe (arrow in O). Fas2 expression at this stage also labels the fan-shaped body (arrowhead in N) lying directly beneath the medial lobes. (Q,R) A pupal MB from an individual carrying the OK107-Gal4 driver plus UAS-mCD8:GFP and UAS-Ephrin:myc stained for GFP (green) and myc (red). Ephrin-myc is concentrated within MB cell bodies (arrows) and absent from axons within the peduncle (Ped, arrowhead in N). (S) One hemisphere of a late 3rd instar larval brain double labeled to visualize Ephrin mRNA expression (blue) by in situ hybridization and anti-GFP (brown) to detect mCD8:GFP driven by the MB-specific OK107-Gal4 driver. Ephrin RNA expression is widespread within the brain at this time, but is detected within the MB neuron cell bodies, two clusters of which are in the focal plane (arrows). Co-labeled MB cell bodies project axons within the peduncle (arrowhead indicates the middle of peduncle), here labeled by mCD8:GFP. Genotypes: (E–G) 201Y-Gal4/+; UASmCD8:GFP/+; (H-J,S) OK107-Gal4/+;UAS-mCD8:GFP/+; (Q,R) OK107-Gal4/+; UASmCD8:GFP/UAS-Ephrin:myc.

Fig. 6. Eph^x652 mutants exhibit defects in dorsal lobe formation throughout MB development

(A,B) Adult MBs from wild-type (A) and Eph^x652 (B) individuals carrying UAS-mCD8:GFP driven by elav-Gal4 double-labeled with anti-GFP (green) and anti-Fas2 (red). Eph^x652 mutant has missing dorsal projections of both α′/β′ (green) and α/β (yellow) MB neurons. (C,D) Pupal MBs from wild type (C) and Eph^x652 (D) stained with anti-Fas2 showing severely reduced dorsal lobe projections (arrow) in the Eph^x652 mutant. (E,F) Third instar MB from wild-type (E) and Eph^x652 (F) individuals carrying 201Y-Gal4 driving UAS-mCD8:GFP (green), which labels cell bodies of γ neurons (arrowhead in E) and their dorsal and medial axonal projections. γ Neurons show reduced dorsal lobe projections (arrow) in the Eph^x652 mutant (F). Reduction of dorsal lobe projections is accompanied by an increase in medial lobe projections (arrowhead in F). (G,H) Late stage embryos stained with anti-Fas2. In wild type (G), the developing MB γ neurons and their dorsal and medial projections are labeled (D, dorsal lobe; M, medial lobe; Ped, penduncle). (H) Eph^x652 mutant showing loss of dorsal lobe projections (asterisk). (I) MB of an Eph^x652/onecut^lx122 adult stained with anti-Fas2. Eph^x652/onecut^lx122 MBs are indistinguishable from wild type, showing no dorsal lobe defects. (J) MB of an Ephrin^KG09118 adult stained with anti-Fas2, showing defects in dorsal branch formation similar to Eph^x652. (K) Anti-Fas2 staining of an adult MB overexpressing Eph using OK107-Gal4 driving a UAS-Eph transgene. Similar to Eph^x652 loss-of-function mutants, dorsal lobes are absent, and medial β projections are thicker (arrows), often appearing fused at the midline (arrowhead). (L) MB from an Eph^x652 adult stained with anti-Fas2 showing loss of dorsal α lobes and thicker β medial lobes (arrow), which are fused at the midline (arrowhead). Genotypes: (A) elav-Gal4 (X)/+ or Y;UAS-mCD8GFP/+; Eph^x652/CiD; (B) elav-Gal4 (X)/+ or Y;UAS-mCD8GFP/+;Eph^x652/Eph^x652; (C,G) Eph^x652/CiD; (D,H,K) Eph^x652/Eph^x652; (E) 201Y-Gal4/UAS-mCD8GFP;Eph^x652/CiD; (F) 201Y-Gal4/UAS-mCD8GFP;Eph^x652/Eph^x652; (I) Eph^x652/onecut^lx122; (J) Ephrin^KG09118/Ephrin^KG09118; (K) OK107-Gal4/+;UAS-Eph/+.

Fig. 7. Eph is not required for branch formation but for guidance of axon branches within the dorsal lobes of the MB

Axonal projections of labeled α/β neurons generated by MARCM (Lee and Luo, 2001) labeled with UAS-mCD8:GFP. Images of one half of the MB, double-labeled with anti-GFP (green in A,C,E,G and shown alone in B,D,F,H) and anti-Fas2 (red in A,C,E,G) to visualize the projections of α/β neurons. (A,B) In wild type, a single-labeled α/β neuron bifurcates, sending one projection dorsally in the α lobe and one medially in the β lobe. Inset in A shows the axon projection (arrowhead) within the peduncle of this singly marked clone. (C,D) In Eph^x652 mutant MBs where the dorsal α lobe is absent, a single-labeled α/β neuron bifurcates as in wild type, but both projections extend along the medial projecting β lobe (arrows in D). Unlike wild type, extra branches can be seen along the length of both branches in the mutant (arrowheads in D). Inset in C shows the axon projection (arrowhead) within the peduncle of this singly marked clone. (E,F) In Eph^x652 mutant MBs where the dorsal α lobe is present, but reduced multiple labeled α/β neurons show fewer axons within the α lobe compared with the medial β lobe. (G,H) A single-labeled α/β neuron in an Eph^x652mutant where the overall morphology of the MB is unaffected, showing normal bifurcation and branching. Genotypes: (A,B) elav-Gal4, UAS-mCD8GFP, hs-FLIP (X)/+ or Y;FRT^G13/FRT^G13 Gal80;Eph^x652/CiD; (C–H) elav-Gal4, UAS-mCD8GFP, hs-FLIP (X)/+ or Y;FRT^G13/FRT^G13 Gal80;Eph^x652/Eph^x652.