A new Prospero and microRNA-279 pathway restricts CO2 receptor neuron formation

Abstract

CO(2) sensation represents an interesting example of nervous system and behavioral evolutionary divergence. The underlying molecular mechanisms, however, are not understood. Loss of microRNA-279 in Drosophila melanogaster leads to the formation of a CO(2) sensory system partly similar to the one of mosquitoes. Here, we show that a novel allele of the pleiotropic transcription factor Prospero resembles the miR-279 phenotype. We use a combination of genetics and in vitro and in vivo analysis to demonstrate that Pros participates in the regulation of miR-279 expression, and that reexpression of miR-279 rescues the pros CO(2) neuron phenotype. We identify common target molecules of miR-279 and Pros in bioinformatics analysis, and show that overexpression of the transcription factors Nerfin-1 and Escargot (Esg) is sufficient to induce formation of CO(2) neurons on maxillary palps. Our results suggest that Prospero restricts CO(2) neuron formation indirectly via miR-279 and directly by repressing the shared target molecules, Nerfin-1 and Esg, during olfactory system development. Given the important role of Pros in differentiation of the nervous system, we anticipate that miR-mediated signal tuning represents a powerful method for olfactory sensory system diversification during evolution.

Figure 1.

Pros mutants exhibit MP CO₂ neurons targeting medial AL glomeruli. A–E, Adult olfactory organs (antenna and MP) and brains stained with anti-GFP (green) or anti-GFP and anti-disc large (magenta) to visualize CO₂ neurons or synaptic projections of CO₂ neurons (Gr21aGAL4, UAS-sytGFP) in the AL, respectively. Note that only mutant cells are marked by GFP. A¹, B¹, C¹, D¹, E¹, CO₂ neurons in MP labeled with Gr21a-GAL,UAS-mCD8GFP in control and mutant palps. A¹, No CO₂ neurons were detected in MPs of control flies. B¹, C¹, D¹, E¹, Ectopic CO₂ neurons were detected in MPs of eyFLP;pros^IG2227, eyFLP;pros^Voila78, eyFLP;miR-279, and eyFLP;pros¹⁷. A², B², C², D², E², CO₂ neurons in the antenna were labeled with Gr21a-mCD8GFP. While CO₂ neuron number appeared normal in pros^IG2227, pros^Voila78, and miR-279 mutants compared with controls, the number was reduced in eyFLP;pros¹⁷ mutants. A³, AL of eyFLP;FRT82B control fly, where CO₂ neurons target a single glomerulus, V-glomerulus. B³, C³, D³, E³, CO₂ neurons labeled with Gr21a-mCD8GFP innervate medial glomeruli (arrowhead) in addition to the V-glomerulus in brains of eyFLP;pros^IG2227, eyFLP;pros^Voila78, eyFLP;miR-279, and eyFLP;pros¹⁷ flies. Note that CO₂ neuron axons project via the labial (arrow) and via the antennal nerve to the V-glomerulus and to medial glomeruli. Axons form contralateral projections. A⁴, B⁴, C⁴, D⁴, E⁴, Similar scenario as in A³, B³, C³, D³, and E³; however, here CO₂ neurons are labeled with Gr21a-sytGFP to label synaptic connections in the AL. A⁵, B⁵, C⁵, D⁵, E⁵, ORNs expressing the Or42a receptor residing on MPs innervate a medial glomerulus (arrowhead) in the AL of eyflp;FRT82B controls (A⁵). B⁵, C⁵, D⁵, E⁵, Or42a neurons in eyFLP;pros^IG2227, eyFLP;pros^Voila78, eyFLP;miR-279, and eyFLP;pros¹⁷ target the V-glomerulus (arrow) in addition to the medial glomerulus. A⁶, B⁶, C⁶, D⁶, E⁶, Or59c neurons reside on the MP. Note that these neurons show the same behavior as Or42a neurons in control and mutants. F, Quantification of CO₂ neurons of antenna and MP for all flies analyzed. Control: n = 15 and 11, pros¹⁷: n = 17 and 45 (8 with ectopic CO₂ ORNs), pros^IG2227: n = 51 and 7, miR-279: n = 37 and 6, Pros^Voila78: n = 9 and 10, for antenna and MP, respectively. Control versus pros^Voila78 antenna, p = 0.07; control versus pros¹⁷ antenna, p = 0.005; pros^Voila78 versus pros^IG2227 antenna, p = n.s.; miR-279 versus pros^IG2227 MP, p = n.s.; pros¹⁷ versus pros^IG2227 MP, p < 0.001. G, Schematic representation of CO₂ neuron projections in wild-type and mutant flies. Ectopic CO₂/MP hybrid neurons are detected in the MPs of miR-279 and pros mutants. While the mutant CO₂ neurons in the antenna show wild-type innervations of the AL (blue), mutant axons from the MP innervate the V-glomerulus and additional medial glomeruli and express the CO₂ receptors and MP ORs (yellow-blue). MP neurons innervate via the labial nerve in both mutants and wild-type flies (yellow). Ant, antenna; pos., positive. Scale bars, 50 μm.

Figure 2.

Analysis of pros and miR-279 mutants reveals specific and general effects on ORN number and targeting. A, MP ORNs were quantified using the general OR marker Or83b-GFP (Or83b-GAL4, UAS-mCD8GFP). The number of ORNs in pros^IG2227 clones was increased compared with control clones. This increase correlated with the number of ectopic CO₂ neurons in the mutants. The number of ORNs in pros¹⁷ clones was decreased by ∼10 ORNs on average compared with control clones. B, eyFLP mosaic analysis was used to analyze ORN projection patterns in the AL of several classes of ORNs. miR-279 and pros mutants show very mild mistargeting in few classes of non-CO₂ receptor ORNs. In general, mistargeting was mild and random. Pros mutants display a reduction of Or22a and Or47a neurons, as shown by the reduced innervations of the AL glomerulus. C, Ectopic CO₂ neurons on the MP coexpress Or42a and Or59c. Examples of whole-mount in situ hybridization on control and mutant MP clones expressing the Gr21a-mCD8GFP reporter are shown. Scale bars: B, 50 μm; C, 10 μm.

Figure 3.

Hybrid neurons respond to CO₂ and MP key odors. A, Representative single sensillum recording traces from the MP basiconic sensilla of control, miR-279, and pros^IG2227 flies. Control trace (top) shows spikes of two neurons. In both miR-279 (middle) and pros^IG2227 (bottom) traces, spikes of an extra neuron are visible. During a 500 ms CO₂ stimulation (black bar), the spike frequency of the B neuron in both miR-279 and pros^IG2227 increased significantly. No response was observed in control MPs. B, Spike frequency of B neurons to CO₂ stimulation in control, miR-279, and pros^IG2227 flies are represented in the graph (n = 10 recordings for each genotype, ±SEM). C, Representative single sensillum recording traces from the MP basiconic sensilla of control (n = 5), miR-279 (n = 6), and pros^IG2227 (n = 6) flies stimulated with two key ligands of pb3 sensilla, iso-amyl acetate, and 3-octanol.

Figure 4.

Prospero and miR-279 are coexpressed in the MP. A, Schematic drawing of a fully differentiated wild-type MP sensillum with two neurons. B, Developing MP at 6 h APF stained with anti-Elav (green, B¹) and anti-Pros (pink, B²). Note the almost complete overlap of expression. C, Developing MP at 25 h APF stained with anti-Elav (C¹) and anti-Pros (C²). Low and high Pros-expressing cells are indicated with arrow and arrowhead, respectively. Cells with low Pros expression seem to also express Elav. D, MP of elav-GAL4,UAS-mCD8GFP fly at 45 h APF stained with anti-GFP (D¹) and anti-Pros (D²). Elav-positive cells (arrow) do not express Pros anymore. Two Elav-expressing cells are located next to one Pros-expressing cell (arrowhead). E, Developing MP of miR-279-Gal4,UAS-nlsGFP at 6 h APF stained with anti-GFP (E¹ and E⁴), anti-Elav (E⁵), and anti-Pros (E²). miR-279 starts to be expressed and colabels with Pros (arrow and arrowhead). The weaker Pros-positive cell is also Elav-positive (arrow). F, Developing MP of miR-279-GAL4,UAS-mCD8GFP at 30 h APF stained with anti-GFP (F¹) and anti-Pros (F²). Many cells express miR-279-GFP, some of which are also Pros-positive (arrowheads) G, MP from miR-279-GAL4,UAS-nlsGFP at 42 h APF stained with anti-GFP (G¹) and anti-Pros (G²). miR-279 remains coexpressed with Pros (arrowhead) in some cells. H, MP from miR-279-GAL4,UAS-nlsGFP at 42 h APF stained with anti-GFP (H¹) and anti-Elav (H²) miR-279-positive cells (arrowhead) do not colabel with Elav (arrow). I, MP, miR-279-GAL4,UAS-mCD8GFP in an eyFLP control clone at 70 h APF stained with anti-GFP (I¹) and anti-Prospero (I²). Arrowhead shows a miR-279-positive cell, which is expressing Pros. J, Model of the divisions of the cells of the inner (pIIb) lineage. Respective expression of Pros, Elav, and miR-279 are indicated. SOP, Sensory organ precursors; n, neuron; sh, sheath cell; L3, larval stage 3. Scale bars, 5 μm.

Figure 5.

Pros and miR-279 suppress CO₂ neuron formation in MP sensilla. A–F, MPs at 45 h APF (A–D) or pharate adults (E, F), stained with anti-GFP (turquoise), anti-Elav (yellow), and anti-Pros (pink). Cells belonging to one sensillum are circled with a dashed line. A, Wild-type showing two Elav-GAL4-positive cells (arrows, A³) costained with anti-Elav (arrows, A¹) and one adjacent Pros-expressing cell (arrowhead, A²). B, miR-279 MP clone (circled area) showing three Elav-GFP cells (arrow, B³), which are positive for Elav (arrows, B¹). Pros-positive cell is seen next to the cluster (arrowhead). C, Two examples of pros^IG2227 clones (circled areas) with three neurons positive for Elav (arrows, C¹). Notably, Pros labeling is reduced compared with wild-type clones (arrowhead, C²). D, Similar example of a pros¹⁷ clone of three Elav-GAL4-positive cells (arrow). The adjacent neurons outside the circled area belong to different sensilla in close vicinity. Pros expression is not detectable in pros¹⁷-null mutant. E, miR-279 clone with single Gr21a-positive cell (arrow, E³), which costains with anti-Elav (arrow, E¹) but not anti-Pros (arrowhead, E²). F, Similar example of a pros^IG2227 clone with a GR21a-positive cell (arrow, F³), which is positive for anti-Elav (arrow, F¹). G, Schematic model of wild-type sensillum housing two neurons (N1 and N2) and one sheath cell (sh). Mutant sensillum with three neurons and one sheath cell is depicted. The additional neuron is a hybrid neuron (HN) and expresses the CO₂ receptors and either Or42a or 59c. Scale bars, 5 μm.

Figure 6.

Pros and miR-279 define olfactory neuron number. MPs at 45 h APF stained with anti-GFP (turquoise), anti-Elav (yellow), and anti-Pros (pink). A, In wild-type sensilla, two Elav-GAL4-positive cells costain with anti-Elav (arrows) and one Pros-expressing cell resides next to them (arrowhead). B, pros¹⁷ clone with single Elav-positive cells (arrows). Pros expression is not detectable in the pros¹⁷-null mutation. C, pros¹⁷ clones of Elav-GAL4-positive cells (arrow), which failed to maintain the expression of Elav protein. D, Pros activity is required at distinct steps during lineage formation. pros¹⁷ mutants show an early phenotype during pIIb to pIIa specification. The pIIb lineage partly converts to the pIIa lineage in these mutants. This results in a partial or complete loss of ORNs, giving rise to sensilla with only one neuron (31%) or clusters of undifferentiated (undiff.) cells (15.5%). These scenarios are also seen rarely in pros^IG2227 (10%) and miR-279 mutants (4%). n(pros¹⁷) = 13, n(pros^IG2227) = 19, and n(miR-279) = 22 MPs. Hypomorphic alleles of pros^IG2227 and pros^Voila78 uncover a second phase of Pros requirement during definition of sensilla neuron number. In these mutant palps and in 54% of pros¹⁷ mutant palps, a third neuron was formed within a sensillum. This neuron expressed Gr21a receptor (CO₂ receptor) starting at late pupal stages. While Pros is involved in pIIb versus pIIa specification, miR-279 is specifically required downstream of Pros within the already determined neuronal lineage to define the number of ORNs in certain MP sensilla. Scale bars, 5 μm.

Figure 7.

miR-279 and Pros interact in vitro and in vivo. A, Expression of UAS-pros as well as UAS-miR-279 in the eyFLP;pros^IG2227 mutant background rescued the phenotype almost fully (100%, n = 25 and 80%, n = 81, respectively), while expression of UAS-pros in the eyFLP;miR-279 mutant background only led to a partial rescue of 37% (n = 25). Overexpression of Pros or miR-279 in wild-type clones did not result in a detectable phenotype in Gr21a-expressing neurons (n = 20). B, Scheme of the putative enhancer region of miR-279 depicting the relative positions of the predicted Pros binding sites (P1–P4). The binding sites include either the consensus motif TWAGVYD or CWYNNCY. The primary transcript starts ∼1200 bp upstream of miR-279. Sequences of putative binding sites and sequences of mutated sites within the consensus sequence are shown. C, Gel shift assay using purified homeodomain of Pros. Radiolabeled oligos carrying either P1 or P4 sites were assayed for direct Pros binding. Pros homeodomain binds to P4, which results in a strong shift (lane 3). The shift is strongly reduced when mutated P4 oligos were tested (lane 4). D, S2 cell miR-279 promoter reporter assays with wild-type and mutated versions of the promoter. Note that RNAi against Pros strongly reduced miR-279 reporter expression, while Pros overexpression did not significantly enhance reporter expression of the wild-type and mutated promoter constructs (*p < 0.05). E, Quantification of percentage PCR product of immunoprecipitated (IP) chromatin compared with the input. Anti-FLAG to pull down Pros-FLAG was compared with mouse IgG plus chromatin and anti-FLAG antibody without chromatin. The PCR was conducted with a primer set amplifying the region around P4. The sequence of the PCR product is shown.

Figure 8.

miR-279 expression is strongly reduced in Pros mutant MPs. A, B, Representative expression patterns of miR-279-GAL4 (turquoise) combined with anti-Pros (pink) and anti-Elav (yellow) staining in control and mutant clones in the developing MP at 6 h APF. Pros and miR-279 coexpressing cells are indicated with arrows. We detected colabeled cells in 73% (n = 15) and 66% (n = 18) of control and miR-279 MPs, respectively. In pros^IG2227 mutant clones, only 14% (n = 14) of the MPs showed cells coexpressing miR-279 and Pros. C, Expression of miR-279-GAL4, UAS-mCD8GFP in adult MPs of wild-type and mutant clones. miR-279-GFP expression is strongly reduced in 59% and 52% of MP of pros¹⁷ and pros^IG2227 mutants compared with controls, respectively (n = 117, 37, 112). miR-279-GFP expression is only slightly reduced in miR-279 MP (n = 107, 92% compared with controls). Note that efficiency of FLP recombination was not different among the genotypes (data not shown). Scale bars, 5 μm.

Figure 9.

Escargot and Nerfin-1 are shared target of miR-279 and Prospero. A, Quantification of S2 cell assays using nerfin-3′UTR reporter with and without overexpression of miR-279 or miR-315. Experiments were performed in triplicates and data from four repetitions are pooled. B, Quantification of S2 cell assays with esg 3′UTR and overexpression of miR-279 or miR-315. Overexpression of miR-279 reduced esg-3′UTR reporter expression to 51 ± 9%. C, MPs of different developmental stages: LacZ (blue) staining and anti-βGal (turquoise) immunoreactivity reflects expression of esg (P[lacZ]esg). At 6 h APF, esg-lacZ is seen strongly in cells of the antennal disc, including the future MP (circled area). At 25 h APF, esg-lacZ is expressed at much lower levels in the MP. No expression was detected in MPs at 30 or 48 h APF (arrow). D, MPs at 6 h APF from control and mutant flies carrying an esg-lacZ. Esg expression was visualized using anti-βGal (esg-lacZ), expression of Elav was monitored with anti-GFP, and Pros was labeled with anti-Pros. Coexpression of Pros with esg was analyzed in the area of control or mutant clone (Elav-GFP, yellow). In the control case, only 16% of MPs have double-labeled cells (n = 12). In the absence of miR-279 or Pros, repression of esg expression in the developing MP is released. In miR-279 and pros^IG2227 mutants, 90% (n = 11) and 61% (n = 13) of the MPs, respectively, counted show cells with Pros-esg coexpression. Scale bars, 5 μm.

Figure 10.

Increased expression of Nerfin-1 and Escargot is required and sufficient to induce CO₂ neuron formation and mistargeting on the MP. A, Genetic interaction of pros^IG2227 and two target genes nerfin-1 and esg. The number of ectopic Gr21a-positive cells in pros^IG2227 mutant MPs missing either one allele of esg, nerfin-1, or both esg and nerfin-1 is reduced by 55% (n = 22), 49% (n = 30), and 40% (n = 20) compared with controls (n = 37), respectively. B, Quantification of the genetic interaction of miR-279 and esg showing a reduction by 44% of the ectopic Gr21a neurons after removal of one copy of esg in the miR-279 mutant background (n = 30) compared with miR-279 mutant only (n = 30). The removal of one copy hb9 (exex) did not result in a reduced number of ectopic CO₂ neurons (n = 24). C, nerfin-1-RNAi expression in the background of the miR-279 and the pros^IG2227 background rescues the phenotype by 100% (n = 35) and 80% (n = 29), respectively. Nerfin-1 RNAi in control flies induced no phenotype (n = 20). D, Overexpression of targets in the wild-type background. Single overexpression of Nerfin-1 and Esg did not result in Gr21a medial mistargeting. In contrast, overexpression of both Esg plus Nerfin-1 phenocopied the phenotype of miR-279 and pros^IG2227 in 40% (n = 24) of all fly brains analyzed. Overexpression of the apoptosis inhibitor p35 (n = 20) and the cell cycle gene CyclinE (n = 25) did not result in a miR-279-like phenotype. E, Mechanistic model of a network of Pros, miR-279, Nerfin-1, and Escargot: In wild-type MPs, Pros directly binds and represses the expression of at least two target genes, nerfin-1 and escargot. In a parallel pathway, Pros directly activates the expression of miR-279, which in turn represses nerfin-1 and esg on a posttranscriptional level. Our genetic data indicate that indirect repression via miR-279 post-transcriptionally is more powerful compared with direct Pros repression on the genomic level (indicated by line thickness). This regulation represses the formation of MP CO₂ neurons. In pros or miR-279 mutants, the repression of Nerfin-1 and Esg expression directly and/or indirectly is lost. As a consequence, an excess of Nerfin-1 and Esg leads to the formation of ectopic CO₂ neurons within Or42a and Or59c olfactory sensilla on the MP. Scale bars, 20 μm.