The unfulfilled gene is required for the development of mushroom body neuropil in Drosophila

Abstract

Background: The mushroom bodies (MBs) of Drosophila are required for complex behaviors and consist of three types of neurons, gamma, alpha'/beta' and alpha/beta. Previously, roles for transcription factors in MB neuronal differentiation have only been described for a subset of MB neurons. We are investigating the roles of unfulfilled (unf; HR51, CG16801) in MB development. unf encodes a nuclear receptor that is orthologous to the nuclear receptors fasciculation of axons defective 1 (FAX-1) of the nematode and photoreceptor specific nuclear receptor (PNR) of mammals. Based on our previous observations that unf transcripts accumulate in MB neurons at all developmental stages and the presence of axon pathfinding defects in fax-1 mutants, we hypothesized that unf regulates MB axon growth and pathfinding.

Results: We show that unf mutants exhibit a range of highly penetrant axon stalling phenotypes affecting all neurons of the larval and adult MBs. Phenotypic analysis of unfX1 mutants revealed that alpha'/beta' and alpha/beta neurons initially project axons but stall prior to the formation of medial or dorsal MB lobes. unfZ0001 mutants form medial lobes, although these axons fail to branch, which results in a failure to form the alpha or alpha' dorsal lobes. In either mutant background, gamma neurons fail to develop larval-specific dorsal projections. These mutant gamma neurons undergo normal pruning, but fail to re-extend axons medially during pupal development. unfRNAi animals displayed phenotypes similar to those seen in unfZ0001 mutants. Unique asymmetrical phenotypes were observed in unfX1/unfZ0001 compound heterozygotes. Expression of UAS-unf transgenes in MB neurons rescues the larval and adult unf mutant phenotypes.

Conclusions: These data support the hypothesis that unf plays a common role in the development of all types of MB neurons. Our data indicate that unf is necessary for MB axon extension and branching and that the formation of dorsal collaterals is more sensitive to the loss of unf function than medial projections. The asymmetrical phenotypes observed in compound heterozygotes support the hypothesis that the earliest MB axons may serve as pioneers for the later-born MB neurons, providing evidence for pioneer MB axon guidance in post-embryonic development.

Figure 1

unf is required for mushroom body lobe formation. In the adult brain, the mushroom body (MB) is a paired neuropil structure that comprises five axonal lobes, 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 projecting axons medially and dorsally. α' and α axons project dorsally, whereas γ, β', and β axons project medially, forming five distinctive lobes. To visualize the MB lobes, OK107-GAL4 was used to drive expression of the UAS-mCD8GFP transgene in all MB neurons (Kenyon cells) and their axons. Lobes were distinguished by using anti-Fasciclin II (anti-Fas II) to label α and β lobes and anti-Trio to label α', β', and γ lobes. (A) In adult UAS-mCD8GFP;;OK107-GAL4 control animals labeled with anti-Fas II (red), all MB lobes have formed. (B) In unf^X1 mutants, MB axons have formed a peduncle (arrowhead), but have spread out and stalled prior to lobe formation (arrow). (C) In unf^Z0001 mutants, γ, β', and β axons projected medially, but were disorganized. No dorsal lobes were formed (star). (D) This +/Df2426;UAS-unf^RNAi;OK107-GAL4 adult displayed dramatically reduced dorsal lobes in one brain hemisphere (arrow). (E, F) In unf^X1 and unf^Z0001 rescue animals, in which a wild-type unf trangene was expressed in all MB neurons in an otherwise mutant background, all MB lobes were present. It is interesting to note that in rescued flies, MB lobes may have fewer axons, and that some medially projecting axons have extended past the midline (E, arrow). Eb, ellipsoid body; Meb, median bundle; Ped, peduncle. Scale bars = 10 μm.

Figure 2

Summary of unf alleles. The unf^X1 allele disrupts the 5' donor splice site of intron 2, whereas the unf^Z0001 allele has a missense mutation due to a guanine to adenine transition at base 312 of exon 2, resulting in a glycine to arginine substitution (G120R) [25]. The unf^MB05909 line contains a GAL4 insertion in intron 1 of the unf gene (FlyBase). DBD, DNA-binding domain.

Figure 3

unf^X1/unf^Z0001 compound heterozygotes display a range of aberrant mushroom body phenotypes, suggesting that unf^X1 and unf^Z0001 alleles interact. OK107-GAL4 was used to drive expression of the UAS-mCD8GFP transgene in all MB neurons and their axons (green), and anti-Fas II (red) to label the α and β axons. (A) In this unf^X1/unf^Z0001 compound heterozygote all MB axons stall (arrows), similar to the unf^X1 mutant phenotype. (B) In this unf^X1/unf^Z0001 heterozygote only medial lobes are present, similar to the unf^Z0001 mutant phenotype; a thin fascicle of α' axons is present in the right hemisphere (star). (C) In this unf^X1/unf^Z0001 heterozygote, left hemisphere β' and β axons extend medially beyond the midline (arrow), whereas γ axons appear to stall; right hemisphere β and β' axons misproject ventrally (arrowhead) and γ axons are highly disorganized; only a few α' dorsal axon projections are present in either hemisphere (stars). (D) In this unf^X1/unf^Z0001 heterozygote α and α' dorsal axon projections are present in the left hemisphere, whereas only a thin fascicle of α' axons is present in the right hemisphere (star); β axons are present in the right hemisphere and appear to stall (arrow), whereas they are completely absent in the left hemisphere. (E) In this unf^X1/unf^Z0001 heterozygote β', β, and γ axons project medially and cross the midline (arrow), but dorsal axons are missing in the left hemisphere; in the right hemisphere γ axons project medially and α and α' axons project dorsally but appear to stall (arrowhead). (F) In this unf^X1/unf^Z0001 heterozygote, α and α' dorsal axon projections are present in the left hemisphere, whereas only α' dorsal axons are present in the right hemisphere; medial axon projections are disorganized and stall before reaching the midline in both right and left hemispheres (arrows). Eb, ellipsoid body; Meb, median bundle; Ped, peduncle. Scale bars = 25 μm.

Figure 4

unf impacts all mushroom body neurons early during development. OK107-GAL4 was used to drive expression of the UAS-mCD8GFP transgene in all MB neurons and their axons (white). (A) In UAS-mCD8GFP;;OK107-GAL4 late third instar larvae controls, γ neurons project larval-specific axons medially and dorsally. (B) In unf^X1 mutant late third instar larvae, γ neurons project medially only; dorsal axons are missing (star). (C) Medial and dorsal γ axons are present in unf^X1 rescue third instar larvae. (D) In UAS-mCD8GFP;;OK107-GAL4 control pupae at 18 hours APF, γ axons have been pruned back to the peduncle and only axon fragments are visible; some β' and α' axons project medially and dorsally, respectively. (E) In unf^X1 mutant pupae at 18 hours APF, γ axons have been pruned with some fragments visible (arrow); β' and α' axons are completely missing (stars). (F) All MB lobes are visible in UAS-mCD8GFP;;OK107-GAL4 control pupae at 48 hours APF. (G) All MB lobes are absent in unf^X1 mutant pupae at 48 hours APF (stars). Scale bars = 10 μm.

Figure 5

201Y-GAL4 and c739-GAL4 driven GFP expression is unf-dependent. The UAS-mCD8GFP reporter was used with three enhancer trap constructs to visualize MB neurons (Kenyon cells (KCs), green). Fluorescently labeled phalloidin (red) was used to visualize actin-rich structures, including the dendritic fields (calyces) of the MBs and the protocerebral bridge (Pb) that lies in the same plane. (A, B) The c747-GAL4 transgene drives the expression of GFP in MB neurons in UAS-mCD8GFP/c747-GAL4 controls and in unf^X1,UAS-mCD8GFP/Df2426,c747-GAL4 mutants. (C, D) The 201Y-GAL4 transgene drives the expression of GFP in a subset of the MB neurons in UAS-mCD8GFP/201Y-GAL4 controls (C), but unf^X1,UAS-mCD8GFP/Df2426,201Y-GAL4 mutants do not express 201Y-GAL4-driven GFP in MB neurons (D). (E, F) UAS-mCD8GFP/c739-GAL4 controls express GFP in MB neurons (E). GFP expression is greatly reduced in unf^X1,UAS-mCD8GFP/Df2426,c739-GAL4 mutants (F). The calyx and Pb are positively labeled with phalloidin in all mutants and controls. Scale bars = 10 μm.