unfulfilled interacting genes display branch-specific roles in the development of mushroom body axons in Drosophila melanogaster

Abstract

The mushroom body (MB) of Drosophila melanogaster is an organized collection of interneurons that is required for learning and memory. Each of the three subtypes of MB neurons, γ, α'/β', and α/β, branch at some point during their development, providing an excellent model in which to study the genetic regulation of axon branching. Given the sequential birth order and the unique patterning of MB neurons, it is likely that specific gene cascades are required for the different guidance events that form the characteristic lobes of the MB. The nuclear receptor UNFULFILLED (UNF), a transcription factor, is required for the differentiation of all MB neurons. We have developed and used a classical genetic suppressor screen that takes advantage of the fact that ectopic expression of unf causes lethality to identify candidate genes that act downstream of UNF. We hypothesized that reducing the copy number of unf-interacting genes will suppress the unf-induced lethality. We have identified 19 candidate genes that when mutated suppress the unf-induced lethality. To test whether candidate genes impact MB development, we performed a secondary phenotypic screen in which the morphologies of the MBs in animals heterozygous for unf and a specific candidate gene were analyzed. Medial MB lobes were thin, missing, or misguided dorsally in five double heterozygote combinations (;unf/+;axin/+, unf/+;Fps85D/+, ;unf/+;Tsc1/+, ;unf/+;Rheb/+, ;unf/+;msn/+). Dorsal MB lobes were missing in ;unf/+;DopR2/+ or misprojecting beyond the termination point in ;unf/+;Sytβ double heterozygotes. These data suggest that unf and unf-interacting genes play specific roles in axon development in a branch-specific manner.

Keywords: CG16801; dHR51; neuronal differentiation; nuclear receptor; suppressor screen.

Figure 1

OK107-GAL4 drives expression in the ventral nervous system (VNS). In this ;;UASmCD8GFP;;OK107-GAL4 72-hr pupa labeled with anti-crustacean cardioactive peptide (CCAP), OK107-GAL4-driven GFP is expressed in heterogeneous cells throughout the VNS but not in the CCAP-expressing cells. Scale bar = 200 μm.

Figure 2

Suppressors of the OK107 > unfulfilled (unf)-induced lethality. This schematic maps the third chromosome deficiencies and the 19 candidate genes that suppress the OK107 > unf-induced lethality. *Candidate genes that suppress the lethality and impact mushroom body development in a secondary phenotypic screen. ⁺DopR2 does not suppress the lethality but does impact mushroom body development. 3L, left arm; 3R, right arm. Not to scale.

Figure 3

Mushroom body (MB) phenotypes in animals doubly heterozygous for unfulfilled (unf) and single candidate genes. In the adult brain, the MB is a paired neuropil structure composed of three subtypes of MB neurons, γ, α´/β´, and α/β. Each neuron projects dendrites that contribute to a large dendritic field (calyx) and an axon that travels anteroventrally. MB axons fasciculate with other MB axons, forming a peduncle (Ped) before branching and projecting axons medially and dorsally. α´ and α axons project dorsally, whereas the adult γ and the β´ and β axons project medially, forming five distinctive lobes. To visualize the MB lobes, OK107-GAL4 (OK107) was used to drive expression of the UAS-mCD8::GFP (UASmCD8) transgene in all MB neurons and their axons (green). Lobes were distinguished by using anti-Fas II to label α and β lobes (magenta). Note that the OK107 and UASmCD8 transgenes that are present in all control and experimental animals were not included in the genotypes (C−S) due to limited space in the figure. (A, B) In ;UAS-mCD8;;OK107 and ;unf^X1UAS-mCD8;;OK107 control animals, all five MB lobes have formed in each of the two brain hemispheres. (C) In ;UAS-mCD8/+;Fps85D^X21/+;OK107/+ heterozygote controls, all MB lobes are present. (D) In this ;unf^X1UAS-mCD8/+;Fps85D^X21/+;OK107/+ double heterozygote, both β´ and β (medial) lobes are missing in the right hemisphere (star). (E, F) ;UAS-mCD8/+;axn^EY10228/+;OK107 heterozygotes either exhibit the wild type phenotype in which all MB lobes are present, or a mutant phenotype in which β lobes are missing (thin arrow in F). In this case the missing β lobe appears to have misprojected dorsally (thick arrow in F). (G, H) In ;unf^X1UAS-mCD8/+;axn^EY10228/+;OK107 double heterozygotes, β lobes are missing in one or both brain hemispheres (thin arrows in G and H) or β lobes have misprojected dorsally alongside the α (dorsal; magenta) lobe (thick arrow in H). (I) All MB lobes are present in ;UAS-mCD8/+;Tsc1^F01910/+;OK107 heterozygote controls. (J) In this ;unf^X1UAS-mCD8/+;Tsc1^F01910/+;OK107 double heterozygote, the missing β lobe (thin arrow) appears to have misprojected dorsally (thick arrow) in the left brain hemisphere. (K) In ;UAS-mCD8/+;Rheb⁰⁸⁰⁸⁵/+;OK107 heterozygotes, all MB lobes have formed. (L) In this ;unf^X1UAS-mCD8/+;Rheb⁰⁸⁰⁸⁵/+;OK107 double heterozygote, the β (medial; magenta) lobe appears thin in the left hemisphere (thin arrow). (M) In this ;UAS-mCD8/+;msn¹⁰²/+;OK107 heterozygote, all MB lobes have formed. (N) In this ;unf^X1UAS-mCD8/+;msn¹⁰²/+;OK107 double heterozygote, the β´ lobe is thin (thin arrow), and the β lobe is missing (star). (O) In ;UAS-mCD8/+;DopR2^MB05107/+;OK107 heterozygotes, all MB lobes have formed. (P) In this ;unf^X1UAS-mCD8/+;DopR2^MB05107/+;OK107 double heterozygote, both α´ and α (dorsal) lobes are missing (star) in the right brain hemisphere. (Q) In this ;UAS-mCD8/+;Sytβ^PL00192/+;OK107 heterozygote, all MB lobes have formed. (R, S) In ;unf^X1UAS-mCD8/+;Sytβ^PL00192/+;OK107 double heterozygotes, both α´ and α (dorsal) lobes misproject making sharp bends in either direction where they normally should have stopped growing (thick arrow in R and S). Note that medial axons cross the midline in S (arrowhead). Anterior is always up and the midline is in the center with the exception of R and S. Due to the nature of the defect in R and S, only the left brain hemisphere is completely visible. Ped, peduncle; Meb, median bundle. Scale bars = 25 μm.

Figure 4

Double heterozygotes without the UAS-mCD8GFP and OK107-GAL4 transgenes exhibit the same mushroom body (MB) phenotypes as those containing these transgenes. Adult brains of experimental and control animals were labeled with anti-Fas II to visualize only α/β projections. (A) All labeled MB lobes are present in this ;;axn^EY10228/+ heterozygote. (B) In the left hemisphere of this ;unf^X1;axn^EY10228/+ double heterozygote, the β (medial) lobe is missing (star) and the α (dorsal) lobe appears thick (arrow) suggesting that the β axons have misprojected dorsally. In the right hemisphere, the α and β lobes are present, but the β lobe crosses the midline (dotted line) (arrowhead). (C) All labeled MB lobes are present in this ;;Fps85D^X21/+ heterozygote. (D) In this ;unf^X1;Fps85D^X21/+ double heterozygote, the β lobe is missing (star) in the left hemisphere. Eb, ellipsoid body; Meb, median bundle. Scale bars = 25 μm.

Figure 5

fax homozygotes exhibit α (dorsal) axon misprojections. Brains of experimental and control animals were double-labeled with anti-Fas II to visualize α/β neurons, and anti-Trio to visualize γ and α´/β´ neurons. (A) In this ;;fax^M7/+ heterozygote all five mushroom body lobes are present. (B) In this ;;fax^M7/M7 homozygote, the α (dorsal) lobe is missing (star) and two distinct Fas II-positive axon bundles project medially (arrow) alongside the γ and β´ (medial) lobes. The presence of the two Fas II-positive medially projecting bundles suggests that one is the β lobe (thick arrow) and the other is the misprojected α lobe (thin arrow). Ped, peduncle; Eb, ellipsoid body. Scale bars = 10 μm.

Figure 6

Roles for unfulfilled (unf)-interacting genes in the formation of adult-specific branches. This schematic shows that unf negatively regulates the Tsc1/Rheb/Tor/S6K pathway required for adult γ re-extension (Yaniv et al., 2012). The data presented here show that unf-interacting genes have been identified that are involved in both β´ and β lobe formation, β lobe formation only, and both α´ and α lobe formation. This model predicts that there are other unf-interacting genes that specifically control β´ lobe formation, α´ lobe formation, and α lobe formation only.