Nuclear receptor unfulfilled regulates axonal guidance and cell identity of Drosophila mushroom body neurons

Abstract

Nuclear receptors (NRs) comprise a family of ligand-regulated transcription factors that control diverse critical biological processes including various aspects of brain development. Eighteen NR genes exist in the Drosophila genome. To explore their roles in brain development, we knocked down individual NRs through the development of the mushroom bodies (MBs) by targeted RNAi. Besides recapitulating the known MB phenotypes for three NRs, we found that unfulfilled (unf), an ortholog of human photoreceptor specific nuclear receptor (PNR), regulates axonal morphogenesis and neuronal subtype identity. The adult MBs develop through remodeling of gamma neurons plus de-novo elaboration of both alpha'/beta' and alpha/beta neurons. Notably, unf is largely dispensable for the initial elaboration of gamma neurons, but plays an essential role in their re-extension of axons after pruning during early metamorphosis. The subsequently derived MB neuron types also require unf for extension of axons beyond the terminus of the pruned bundle. Tracing single axons revealed misrouting rather than simple truncation. Further, silencing unf in single-cell clones elicited misguidance of axons in otherwise unperturbed MBs. Such axon guidance defects may occur as MB neurons partially lose their subtype identity, as evidenced by suppression of various MB subtype markers in unf knockdown MBs. In sum, unf governs axonal morphogenesis of multiple MB neuron types, possibly through regulating neuronal subtype identity.

Figure 1. unf is required for the proper axonogenesis of the mushroom body neurons.

(A–C) Anti-FasII antibody staining (magenta) revealed the γ and α/β lobe morphology of wild-type (A), EcR- (B) and usp- (C) knockdown adult MBs. The knockdown was induced by GAL4-OK107 dependent induction of miRNAs. Note the EcR- and usp-knockdown MBs contained unpruned γ processes (arrows in [B] and [C]) and lacked a normal-looking γ lobe (e.g. arrowhead in [A]). Scale bar: 20 µm. (D–F) Adult wild-type (D), tll- (E) and unf- (F) knockdown MB, labeled by GAL4-OK107 (green). The knockdown was induced by GAL4-OK107 dependent induction of miRNAs. Note the tll-knockdown MB contained only few neurons (E), and the axon lobes of unf-knockdown MB failed to extend along the correct paths (F).

Figure 2. The MB lobe phenotype was caused by specific knockdown of unf by miRNA.

(A) Illustration of the unf cDNA that contains a 5′ region encoding a putative DNA binding domain (DBD) and a 3′ region encoding a putative ligand binding domain (LBD). *The miRNA target sites for unf miRNA-a. #The miRNA target site for unf miRNA-b. (B–C) unf in MB was knocked down by the GAL4-OK107 induced unf miRNA-b in a wild-type fly (B), or a fly heterozygous for a deficiency Df(2R)ED2426 that covers unf gene (C). Note some MB axons can still project to their correct positions when the unf miRNA-b was induced in the wild-type background (indicated by arrowheads in [B]). Scale bar: 20 µm. (D) Statistic results of the percentage of MB lacking dorsal projection in flies heterozygous for Df(2R)ED2426 (Df), or in flies with OK107-dependent induction of unf miRNA-a(unfi-a), unf miRNA-b (unfi-b), or unf miRNA-b plus one allele of Df(2R)ED2426 (unfi-b/Df).

Figure 3. unf is mainly required for the formation of adult-specific MB lobes.

Wild-type (A, C–F) and unf knockdown (B, G–J) MB at the indicated developmental stages were labeled by GAL4-OK107 (A–J). The insect in [B] is an example of a small portion (12%; n = 75) of the unf -knockdown larval MB that displayed a thinner dorsal lobe phenotype. Brains in [E], [F], [I] and [J] were counterstained with anti-FasII antibody (magenta). unf was knocked down by GAL4-OK107 induced unf miRNA-a. Scale bars: 20 µm.

Figure 4. unf acts in mature γ neurons to govern axon re-extension.

Wild-type (A,C,E,G) and unf knockdown (B,D,F,H) MB at the indicated developmental stages, labeled by GAL4-201Y (A–F) or GAL4-MB247 (G,H). Brains in [A], [B], [G] and [H] were counterstained with anti-FasII antibody (magenta). unf was silenced by GAL4-201Y (B,D,F) or GAL4-MB247 (H) induced unf miRNA-a. The arrows indicate the position of normal (A) and truncated γ lobes (B,H). Scale bars: 20 µm.

Figure 5. unf is required for the initial axonal morphogenesis in later types of MB neurons.

Newly generated neurons of wild-type (A,C,E) and unf knockdown (B,D,F) MB labeled by asense-GAL4 (green) at the indicated developmental stages. unf was knocked down by asense-GAL4 dependent induction of unf miRNA-a. The arrowheads in [A]–[C] and [E] indicate the normal axonal bundles of the larval MB. The arrows in [D] and [F] indicate the axonal bundles that failed to extend beyond the MB peduncle. Scale bar: 20 µm.

Figure 6. Direct involvement of unf in later MB morphogenetic processes.

GAL80[ts] was used to control the timing of GAL4-OK107 dependent induction of unf miRNA-a to knock down unf in MB after mid-3rd instar (A–B), after adult eclosion (C–D) or after puparium formation (E–F). MBs were labeled by GAL4-OK107 (green; A,C,E), anti-FasII (magenta; A–F) and anti-Dac (magenta; A,B,E,F) immunostaining. Scale bar: 20 µm.

Figure 7. unf regulates axon pathfinding of MB neurons.

(A–F) GAL4-OK107 labeled flip-out clones (green in [A] and [B]; black in [C]–[F]) induced in a wild-type (A) or a unf knockdown (B–F) MB. Brains in [A] and [B] were co-labeled with anti-FasII Ab (magenta). [C]–[F] are high magnification views of some axons in the MB lobe in [B]. The arrows in [C] and [E] mark the axons with an unusual back turn(C), or even form a loop (E). [D] and [F] are duplicate images of [C] and [E] with the abnormal axonal paths highlighted by black lines for better visualization. Scale bars: 20 µm. (G–H) Wild-type (G) and unf knockdown (H) MARCM clones labeled by GAL4-OK107 (green). unf was knocked down by GAL4-OK107 induced unf miRNA-a. Brains were counterstained with anti-FasII Ab (magenta) to mark MB γ and α/β lobes. The arrow in [H] indicates a misrouted axon. Scale bar: 20 µm. (I–J) The calyx region of a unf knockdown adult MB labeled by GAL4-OK107 (green; I) and anti-FasII immunostaining (magenta; I–J). unf was knockdown by GAL4-OK107 induced unf miRNA-a. The arrows indicate a misrouted FasII-positive axonal bundle. Scale bar: 20 µm. (K–L) A illustration of the axonal projections in wild-type (K) and unf knockdown (L) MB.

Figure 8. unf is continuously expressed in the MB neurons throughout development.

Wild-type brains (A,B,E–P) at the indicated development stages and a 6 hr-old larval brain with GAL4-OK107 dependent induction of unf miRNA-a (C–D) were labeled with GAL4-OK107 (green; A, C, E, G, I, K, M and O) and UNF immunostaining (magenta; A–P). Scale bars: 20 µm.

Figure 9. Subtype-specific markers were lost in unf knockdown adult MBs.

The cell body region of wild-type (A, C, E, G, I and K) or unf knockdown (B, D, F, H, J and L) adult (A–H) or wandering larval (I–L) MBs labeled with various MB specific markers, including Trio (A, B, I and J; magenta), GAL4-NP21 (C and D; green), GAL4-c305a (E and F; green), GAL4-c739 (G and H; green) and EcR-B1 (K and L; magenta). Brains in [A], [B], [I] and [J] were co-labeled with GAL4-OK107. The unf miRNA knockdown in [A], [B] and [I]–[L] was induced by GAL4-OK107, and the unf knockdown in [C]–[H] was induced by asense-GAL4 together with GAL4-NP21 (C and D), GAL4-c305a (E and F), or GAL4-c739 (G and H). Note the Trio expression was normal in unf knockdown larval MB cell bodies (J), but became undetectable in unf knockdown adult MB cell bodies (B). Um8G: UAS-mCD8::GFP. Scale bar: 20 µm.