MicroRNA processing pathway regulates olfactory neuron morphogenesis

Abstract

The microRNA (miRNA) processing pathway produces miRNAs as posttranscriptional regulators of gene expression. The nuclear RNase III Drosha catalyzes the first processing step together with the dsRNA binding protein DGCR8/Pasha generating pre-miRNAs [1, 2]. The next cleavage employs the cytoplasmic RNase III Dicer producing miRNA duplexes [3, 4]. Finally, Argonautes are recruited with miRNAs into an RNA-induced silencing complex for mRNA recognition (Figure 1A). Here, we identify two members of the miRNA pathway, Pasha and Dicer-1, in a forward genetic screen for mutations that disrupt wiring specificity of Drosophila olfactory projection neurons (PNs). The olfactory system is built as discrete map of highly stereotyped neuronal connections [5, 6]. Each PN targets dendrites to a specific glomerulus in the antennal lobe and projects axons stereotypically into higher brain centers [7-9]. In selected PN classes, pasha and Dicer-1 mutants cause specific PN dendrite mistargeting in the antennal lobe and altered axonal terminations in higher brain centers. Furthermore, Pasha and Dicer-1 act cell autonomously in postmitotic neurons to regulate dendrite and axon targeting during development. However, Argonaute-1 and Argonaute-2 are dispensable for PN morphogenesis. Our findings suggest a role for the miRNA processing pathway in establishing wiring specificity in the nervous system.

Figure 1. pasha and Dicer-1 are required for dendrite targeting of olfactory projection neurons

(A) Overview of miRNA processing pathway. After transcription, pri-miRNA hairpin structures are cleaved by the RNase III enzyme Drosha into a pre-miRNA of about ~70-nt length. Drosha requires the dsRNA binding protein Pasha for this processing step in the nucleus. The pri-miRNA is exported into the cytoplasm by Exportin-5 where it is cleaved into a ~21-nt long miRNA duplex by the RNase III enzyme Dicer-1. The mature single-stranded miRNA is subsequently loaded into the Argonaute containing RNAi-induced silencing complex (RISC) that binds to complementary mRNAs to regulate translation. (B) Genomic organization of the pasha and Dicer-1 gene. Black bars represent coding and gray bars non-coding exons while the lines represents introns. Red triangles indicate the insertion sites of the piggyBac transposons LL03660 and LL06357. The insertion in pasha is in the 5′UTR 515bp upstream from the start codon. The insertion in Dicer-1 is in the first exon 2220bp 3′ of the start codon. (C) WT adPNs (C₁), lPNs (C₂), and vPNs (C₃) target dendrites to stereotypical sets of glomeruli; VA1d and VA1lm adPNs are encircled (C₁), so are DA1 and VA1lm vPNs (C₃). (D) pasha^−/− adPNs (D₁), lPNs (D₂), and vPNs (D₃) exhibit severe dendrite targeting defects, with a general reduction in dendritic mass (compare encircled glomeruli in 1D₁ and 1D₃ to WT in 1C₁and 1C₃) and spilling of dendrites into inappropriate areas (arrows in 1D_1–2). (E) Dicer-1^−/− PNs show very similar targeting defects like pasha^−/− PNs. (F and G) PN mutant phenotypes can be rescued completely by expressing either UAS-pasha-HA in pasha^−/− mutant PNs (F) or UAS-Dicer-1 in Dicer-1^−/− PNs (G). Pasha-HA localizes to the nucleus (F₂), whereas Dicer-1 is enriched in the cytoplasm (G₂) of PNs. Green is mCD8-GFP labeled MARCM clones, red is the presynaptic marker nc82, and blue is anti-HA (F₁) or anti-Dicer-1 (G₁), respectively. Scale bar represents 20μm. All images are z-projections of confocal stacks except in F₁, F₂, G₁, and G₂, which are single sections.

Figure 2. pasha and Dicer-1 mutants cause dendrite targeting defects in specific PNs

(A) WT adPN single cell clone densely innervates DL1, a dorsolateral, posterior glomerulus (arrowhead). (B) pasha^−/− DL1 single neurons typically innervate DL1 more sparsely, and also mistarget to five additional glomeruli (VA7m, VC2, VA6, DL5, and DL2d) which are only partially innervated (arrowheads). (C) Similar DL1 singe neuron phenotypes are seen in Dicer-1^−/− PNs (arrowheads). (D) Overexpression of Pasha-HA in WT DL1 single neurons results in normal dendrite targeting. (E and F) Expressing either Pasha-HA in pasha^−/− DL1 single neurons or Dicer-1 in Dicer-1^−/− DL1 single neurons rescues the DL1 mistargeting phenotype completely. (G) In WT MARCM clones, Mz19⁺ adPNs innervate the glomeruli VA1d (asterisk) and DC3 (posterior to VA1d). (H) In pasha^−/− Mz19⁺ adPNs, the dendritic mass in VA1d and DC3 is markedly reduced and additional glomeruli are innervated, such as DA1, VM7, and VA2 (arrowheads). (I) Dicer-1^−/− Mz19⁺ adPNs sparsely innervate the VA1d glomerulus and mistarget dendrites to incorrect glomeruli, such as DA3, D, and DA2 (arrowheads). The innervation pattern targeting additional glomeruli varies for pasha^−/− and Dicer1^−/−adPNs but is always restricted to the dorsal half of the antennal lobe. (J) In WT, Mz19⁺ lPN MARCM clones innervate a single dorsolateral glomerulus, DA1 (arrowhead). (K) In pasha^−/− and (L) Dicer-1^−/− Mz19⁺ lPNs the dendritic density in DA1 is equal to WT and no additional glomeruli are innervated in most samples examined. Green is mCD8-GFP labeled PNs and their dendrites generated by MARCM using either Gal4-GH146 or -Mz19, red is the presynaptic marker nc82. Insets in (D) and (E) represent anti-HA, and in (F) anti-Dicer-1 stainings in corresponding PNs, respectively. Scale bar represents 20μm for color images and 5μm for insets in D, E, and F. Color images are z-projections of confocal stacks, insets are single confocal sections of cell bodies.

Figure 3. Pasha and Dicer-1 mutants affect axon termination in the lateral horn

(A) WT DL1 PNs project their axons in a stereotypical pattern to the mushroom body calyx (MBC), where they form collateral branches, and to the lateral horn (LH) with a characteristic dorsal (arrow) and main lateral (arrowhead) branch. (B and C) pasha^−/− and Dicer-1^−/− DL1 axons project into the LH but the main branches do not reach the lateral edge of the LH (arrowheads). Moreover, the dorsal branch is shorter or absent (arrow). (D) All mutant phenotypes in axons can be fully rescued by expressing Dicer-1 in Dicer-1^−/− DL1 single neurons. Green is mCD8-GFP labeled PN axons, red is the presynaptic marker nc82. Scale bar represents 20μm. All images are z-projections of confocal stacks.

Figure 4. pasha^−/− dendrite targeting defects are manifested during development

(A) WT adPNs (A₁), LPNs (A₂), and DL1 single neurons (A₃) already exhibit significant dendritic elaboration at 18 hours (h) after puparium formation (APF) in the pupal proto-antennal lobe (encircled). (B) In pasha^−/− PNs the dendritic mass is strongly reduced in adPNs (B₁) and LPNs (B₂). Dendrites of DL1 single neurons fail to elaborate on the dorsolateral area where the future DL1 glomerulus will form (compare arrowheads in A₃ to B₃). (C) At 50h APF, WT adPNs (C₁) and LPNs (C₂) already show the stereotypic glomerular innervation of the respective lineages, VA1lm and VA1d being encircled. Dendrites of DL1 single neurons are restricted to a single, dorsolateral glomerulus (arrowhead in C₃). (D) In pasha^−/− adPNs and LPNs, the dendritic mass appears strongly reduced (encircled glomeruli in D₁) and dendrites target to non-specific glomeruli (arrowheads in D_1–2). The DL1 mistargeting phenotype to additional glomeruli like VC2, VA6, DL5, and DL2d is already manifested at 50hAPF in pasha mutants (arrowheads in D₃). Note that in addition to the lPN neuroblast clones, the antennal lobe in C₂ shows vPNs and a DL1 single neuron (asterisks mark cell bodies). Their dendrites are either masked by overlaying ones in the z-stack (DL1) or weaker in intensity (vPNs). Green is mCD8-GFP labeled MARCM clones, red is either anti-Ncadherin labeling the proto-antennal lobe at 18h APF (in A and B) or the presynaptic marker nc82 at 50h APF (in C and D). Scale bars represent 20μm. All images are z-projections of confocal stacks.

Figure 5. Normal PN dendrite targeting in the absence of AGO-1, AGO-2 or both

(A and B) AGO1^l(2)k08121 adPNs (VA1lm and VA1d are encircled) and DL1 single neurons (arrowhead) exhibit WT PN targeting. (C and D) AGO2⁴¹⁴ adPNs and DL1 single neurons target glomeruli as in WT. (E and F) AGO1^l(2)k08121; AGO2⁴¹⁴ double mutant adPNs and DL1 single neurons (arrowhead) cause no defect in glomerular target selection. Green is mCD8-GFP labeled MARCM clones, red is the presynaptic marker nc82. Scale bar represents 20μm. All images are z-projections of confocal stacks.